WO2022187725A1

WO2022187725A1 - Cell culture system and methods of using

Info

Publication number: WO2022187725A1
Application number: PCT/US2022/019052
Authority: WO
Inventors: Jean-Charles F. NEEL
Original assignee: Sensorium Bio Inc.
Priority date: 2021-03-05
Filing date: 2022-03-05
Publication date: 2022-09-09

Abstract

The present invention is related to a cell culture system and method of culturing cells. More particularly, the present invention is related to providing a system and method for culturing cells at ambient temperature without incubating the cells at 37°C in the presence of 5% CO₂.

Description

CELL CULTURE SYSTEM AND METHODS OF USING

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/157,445, filed March 5, 2021. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[2] This invention relates to the field of biologic detection systems. The cells of such systems possess receptors and reporters that provide a detectable signal when contacted by a compound that binds to the cell receptors. The invention therefore relates to detection of molecules of interest, including molecules that are volatile.

BACKGROUND

[3] In the United States, lung cancer (LC) accounts for 135,000 deaths or 25% of cancer deaths annually. Early detection of LC is strongly linked to improved 5-year survival rates. Unfortunately, nearly 70% of LC patients are diagnosed after their cancer has progressed to a stage where curative treatment is no longer an option. Current approaches for cancer diagnostic screening are low-dose CT and tissue biopsy. CT scans are expensive and expose patients to ionizing radiation, which can be dangerous even at low doses. Fine-needle tissue biopsies are often inconclusive and more invasive biopsies require surgical intervention. In addition, both imaging and surgical pathology require post-processing and analysis that may take hours to days to provide results. Other approaches rely on indirect analysis of volatile organic compounds (VOCs) using expensive and time-consuming methods, making them unsuitable for widespread clinical use. Thus, a non- invasive rapid early detection method to screen for cancer in time to provide life-saving curative treatment is needed.

[4] Cell cultures and systems of storing and using cell cultures have utility in the life sciences. Traditional cell cultures have limited functionality and lifespan, thus, limiting their utility in producing a detectable signal. There is a need in the field of biotechnology for improved cell cultures and systems to obtain viable cell cultures with a prolonged lifespan that are also useful in producing a detectable and reliable signal.

SUMMARY

[5] Disclosed herein are cell culture systems for culturing cells at ambient temperature, the systems comprising: (a) a cultureware for culturing the cells, wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (b) cells adhered to the bottom of the cultureware, wherein the bottom of the cultureware is optically transparent; (c) a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (d) a cell culture medium covering the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture.

[6] Disclosed herein are methods of culturing cells at ambient temperature, the methods comprising: (a) depositing cells in a cultureware, wherein the cultureware has a top, a bottom, an inner side and an outer side and is gas impermeable and wherein the bottom of the inner side of the cultureware has a coating of a synthetic polymer, wherein the cells passively adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer in the absence of carbon dioxide; (b) adding non-biodegradable cytocompatible hydrogel around the adhered cells, wherein the cells are immobile within the hydrogel; and (c) adding a cell culture medium to the cells in step b), wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and (d) Culturing the cells at ambient temperature without incubating the cells at 37°C and 5% CO2.

[7] The methods for culturing cells according to the invention provide for maintaining viable and functional cells outside of a controlled temperature environment without having supplemental carbon dioxide provided to the cell’s surrounding environment typically found in a cell culture incubator. Moreover, the cells cultured according to the invention are able to be maintained as viable and functional outside of an incubator and without temperature control or supplemental carbon dioxide for periods of at least 3 days. The cells cultured according to the invention may be used in the devices and detection methods disclosed herein. As such, the invention disclosed herein provides a closed system in which cells remain viable and functional without supplemental carbon dioxide and wherein the amount of medium present with the cells after sealing is an amount sufficient to maintain viability and functioning of the cells without further supplementation. In some aspects, the system may remain closed without such supplemental nutrients until cells are used for detecting a target molecule, including volatile organic molecules. [8] Disclosed herein are methods of detecting one or more volatile organic molecules in a fluid sample, the methods comprising: a) contacting a cell culture with a fluid sample, wherein the fluid sample comprises one or more volatile organic molecules, wherein the cell culture comprises cells in a cultureware having a top, an optically clear bottom, an inner side and an outer side and is gas impermeable, wherein the cells are adhered to the bottom of the inner side of the cultureware, are functional at room temperature, and present in a cell culture medium that is buffered by a high buffering capacity carbon dioxide-independent buffer; wherein the cells comprise one or more G- protein coupled receptors capable of binding to the one or more volatile organic molecules, wherein the one or more G-protein coupled receptors comprises a reporter; b) exposing the cell culture to a light; and c) detecting the presence of a fluorescence emitted by the reporter after binding of the one or more G-protein coupled receptors to the one or more volatile organic molecules, an increase in brightness of the fluorescence, or a combination thereof.

[9] Disclosed herein are cell culture systems for culturing cells at a temperature from about 15°C to about 30°C, the systems comprising: a cultureware for culturing the cells, wherein the cultureware comprises a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and wherein the cultureware has at least one access region to add or remove contents within the cultureware, and wherein the access region is sealable to be gas impermeable; cells adhered to the bottom of the cultureware; a layer of non- biodegradable cytocompatible hydrogel, wherein said hydrogel sufficiently surrounds the adhered cells to maintain the cells in a position within the hydrogel; and a cell culture medium covering the adhered cells, wherein the cell culture medium is buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide.

[10] Disclosed herein are methods of culturing anchorage dependent cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients and wherein said cells remain adhered to a cultureware surface of said culture device for at least 5 days, said method comprising: a) maintaining cells in a cell culture device wherein the culture device comprises a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware of said culture device is gas impermeable and wherein the culture device has at least one access region to add or remove contents within the culture device, and wherein the access region is sealable to be gas impermeable; b) maintaining the cells adhered to the bottom of the cultureware for at least 5 days; and wherein a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel; and c) covering the cells in the culture device with a culture medium buffered to maintain a pH of from about 7 to about 8 in in the absence of supplemental carbon dioxide and wherein the cells remain viable in said device for at least 5 days.

[11] Disclosed herein are cell culture devices for culturing anchorage dependent, target molecule detecting cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients, and wherein said cells remain adhered to a cultureware surface of said culture device for at least 5 days, said cell culture device comprising: (a) a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware of said cell culture device is gas impermeable and wherein the cell culture device has at least one access region to add or remove contents within the cell culture device, and wherein the access region is sealable to be gas impermeable; (b) target molecule detecting cells adhered to the bottom of the cultureware for at least 5 days; and wherein a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel; and (c) a culture medium buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide and wherein the culture medium is present in the device in an amount sufficient for the cells to remain viable in said device for at least 5 days in the absence of controlled temperature or supplemental carbon dioxide; wherein said cells comprise a surface G protein- coupled receptor, an intracellular G-protein and an intracellular reporter that provides a detectable signal when said cells contact the target molecule.

[12] In some aspects, the systems and methods for culturing cells disclosed herein can be performed at ambient temperature and in ambient air without incubating the cells at 37°C and 5% CO2. The cell culture system and methods described herein provides a prolonged longevity and functionality of the cells. In some aspects, the cells cultured using the systems and methods disclosed herein can be used in a cell-based assay to detect molecules of interest, including but not limited to metabolites from a living organism.

BRIEF DESCRIPTION OF THE DRAWINGS

[13] FIG. 1 is a schematic diagram of a cross section of the present invention showing a cultureware having multiple wells with the top of the cultureware open and the cultureware is totally covered and tightly sealed with a gas impermeable film. [14] FIGS. 2A-B show the effects of temporal changes in osmolality on cellular morphology. FIG. 2A shows the average circularity measurements of HEK cells post exposure to CO2I10 for 1 to 8 days in vitro. Circularity = 4 (area/perimeter^A2) As the value approaches 1.0, it is indicative of a perfect circle. As the value approaches 0, it indicates an increasingly elongated polygon. Media was prepared and conditioned separately in polystyrene 96-well plates for 8 days, filtered, and added to respective experimental wells and allowed to incubate for 30 minutes at 37°C, 5% CO2, and 95% humidity. For each condition, n > 20 cells/well were assayed for n = 3 wells (n > 60 cells per condition). Data is represented as mean +/- STD and between group differences measured via a two-tailed t-test. *P < 0.05, **P < 0.01, and ***P < 0.001. FIG. 2B shows the maximum ligand- induced fluorescence in cells expressing a surface receptor.

[15] FIG. 3 shows the average area (pm²) measurement of cells post exposure to carbon dioxide independent media + 10% FBS + gentamicin (CO2I10) for 1 to 8 days in vitro.The gradual decrease in average cellular area indicative of a loss of cellular spreading onto the substrate. This is indicative of anchoring points decreasing over a period of 7 days.

[16] FIG. 4 shows the effects of temporal changes in osmolarity on cellular morphology. Phase micrographs of cells exposed to carbon dioxide independent media + 10% FBS + gentamicin (CO2I10) for 1 to 8 days in vitro. HEK cells were seeded at a density of 20,000 cells/well and exposed to conditioned media evaporated for 1 day and 8 days to assess the effect of osmolarity. Media was prepared and conditioned separately in polystyrene 96-well plates for 8 days, filtered, and added to respective experimental wells and allowed to incubate for 30 minutes at 37°C, 5% CO2, and 95% humidity.

[17] FIG. 5 shows the functional response profile of cells cultured from day 3 to day 12 in carbon dioxide independent media. HEK cells expressing a GPCR were challenged with propylbenzene and cytosolic calcium increase was measured via a genetically-encoded calcium indicator. The mean fluorescence intensity was acquired by imaging 20X fields of cells via an epifluorescence microscope. Images were acquired every minute for a total of 15 minutes and data are represented as a fold induction normalized to the baseline value prior to ligand challenge.

[18] FIG. 6 shows the effect of ambient air on pH of DMEM and carbon dioxide independent media.

[19] FIGS. 7A-B show the effect of temporal changes in osmolarity on cellular morphology. FIG. 7A shows the average area (pm²) measurement of HEK cells post exposure to carbon dioxide independent media + 10% FBS + gentamicin (CO2I10) for 1 day and 8 days in vitro. FIG. 7B shows the average circularity measurements of cells post exposure to CO2I10 for 1 day and 8 days in vitro. Media was prepared and conditioned separately in polystyrene 96-well plates for 8 days, filtered, and added to respective experimental wells and allowed to incubate for 30 minutes at 37°C, 5% CO2, and 95% humidity. For each condition, n > 20 cells/well were assayed for n = 3 wells (n > 60 per condition). Data is represented as mean +/- STD and between group differences measured via a two-tailed t-test. *P < 0.05, **P < 0.01, and ***P < 0.001.

[20] FIG. 8 shows the influence of ambient air on pH of DMEM and carbon dioxide independent media. In the control measurements, media was procured directly from commercial bottle (n= 3). In the experimental conditions, DMEM and C02I were equilibrated (n= 3). Data is represented as mean +/- STD and between group differences measured via a two-tailed t-test.

****p < 0 0001_.

[21] FIG. 9 shows the basal mean fluorescence intensity per cell per well as a function of time. Day 1 is significantly lower compared to other experimental time points. No significant difference was found in the baseline level of fluorescence from Day 2 - 9. Each data point is an average value of at least n = 3 wells and are represented as mean +/- SEM. A one way ANOVA was performed with a post hoc Tukey test to assess between group differences. HEK cells were cultured in C02I +10% FBS + G for 9 days at ambient temperature

[22] FIGS. 10A-B show the distribution of baseline fluorescence across a healthy adherent cell population. FIG. 10A shows that nearly 70% of cells have an initially low baseline of GCaMP7s fluorescence. FIG. 10B shows the distribution of cellular assay responsiveness as a function of baseline fluorescence. In healthy cultures, 70% of cells have a very low baseline fluorescence and these cells perform best in a fluorescence induction assay as determined by the ligand-induced fluorescence fold induction. Cells that have a higher baseline fluorescence do not register a proportionally increased fluorescence upon stimulation with a ligand as assessed by an imaging system. As a result, maintaining lower baseline fluorescence allows for a larger response range.

[23] FIG. 11 shows an example of a microarray with adjustable dimensions. Left image shows microfabricated microarrays on a coverslip. Right image shows an electron micrograph of the same 400 pm by 400 pm array taken with an Apreo 2. Right image shows fluorescent cells adhering to non-fluorescent protein spot arrays. [24] FIG. 12 shows HEK cells sedimenting and adhering in the nano-wells. The left panel shows fluorescent cell size polymer microbeads dispensed into 150 pm nano-well. The right panel shows untransfected cells seeded at confluence into microarray nano-wells.

DETAILED DESCRIPTION

[25] The disclosure can be understood more readily by reference to the following detailed description of the invention, the figures and the examples included herein.

[26] Before the present methods and compositions are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[27] Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

[28] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

DEFINITIONS

[29] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[30] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list. [31] When values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[32] As used herein, the term “ambient temperature” refers to the actual temperature of the air in any particular place. It can vary from place to place. In some aspects, the ambient temperature can be from about 15°C to about 25°C or even to about 30°C. In some aspects, the ambient temperature can be about 10°C to about 35°C. In some aspects, the ambient temperature can be from about 20°C to about 22°C. In some aspects, the ambient temperature can be from about 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C.

[33] As used herein, the term “adherent cells” refers to a cell or a cell line or a cell system, that remain(s) associated with, immobilized on, or in certain contact with the inner surface of a substrate (e.g., the bottom of the inner side of a cultureware). In some aspects, the adherent cells are anchorage dependent, and, thus, need solid support for its growth.

[34] Disclosed herein are cell culture systems and methods of culturing cells. Also disclosed herein are synthetic biology methods using living cells to detect and report the presence of diagnostic VOCs for early screening of cancer (e.g., lung cancer). For example, modified mammalian or non-mammalian cells that express or modified to express specific olfactory G protein-coupled receptors (GPCRs) selected to both detect and report the presence of specific VOCs that can be used as a diagnostic tool. In some aspects, engineered cells are deposited onto an optically clear substrate through which intracellular Ca²⁺ levels can be monitored as a readout of VOC-induced GPCR activation. The systems and methods disclosed herein can provide a higher degree of selectivity and sensitivity than existing technologies and are significantly less expensive to manufacture. Accurate real-time actionable output and a low price point make a point-of-care solution for early cancer (e.g., lung cancer) screening.

[35] In some aspects, primary cells are cells that are directly extracted from a living tissue and have not been adapted to be cultured in the laboratory. Primary cells have to undergo adaptation to culture to establish a cell line from the living tissue to allow for long-term study of the tissue. The adaptation to culture of the primary cells in the laboratory can be such that the cells continue to divide to allow studies to be conducted. A difference between a primary cell culture and a cell line is the number of passing times each of them possesses. The primary cell culture is directly isolated whereas a cell line is prepared by passing a primary cell culture for several times thereby selecting for cells best adapted for in vitro culture.

[36] Primary cells grown in a laboratory initially retain intrinsic features. For example, primary cells typically divide a finite number of times before becoming senescent and dying while cells of a cell line divide nearly indefinitely. Primary cells experience contact inhibition while cells of a cell line typically do not. Contact inhibition of primary cells occurs when the primary cells grow and divide until they run out of space to grow into which causes them to stop dividing. Contact inhibition is a self-regulating mechanism of cell division, a feature that is lost in most cells when primary cells become an ever-dividing cell line. Cells that are adherent to the culture substrate typically continue to divide until the surface of the substrate to which they adhere is fully occupied or covered by the cells. Cells that are contact inhibited will cease dividing and growth is inhibited at confluence. Cells will continue to divide when there is still unoccupied surface area, and the cells are sub-confluent.

[37] Cell lines are typically maintained in a growth medium with sufficient density at 37°C and 5% CO2 in a humidified incubator. Some cell lines can be maintained at temperatures other than 37°C and with gas supplementation other than 5% CO2 (i.e., some cells grow best under slightly hypoxic conditions where the incubator provides 5% CO2, 85% N2 and 10% O2). A commonly used cell growth medium is the Minimum Eagle’s Medium (MEM) supplemented with 5% to 10% Fetal Bovine Serum (FBS) which provides many growth factors. MEM is commercially available (e.g., Sigma-Aldrich, St. Louis, MO). The base cell growth medium provides salts, sugars, vitamins and amino acids, which can vary in compositions. Other base cell growth media include but are not limited to Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI (including RPMI 1640), F12 and McCoy. Under these conditions, cells in the cell line divide until they reach confluence. Certain cell lines do not stop trying to divide when there is no space and will often undergo cellular stress leading to apoptosis of the cells which limits the longevity of the cell line. In some aspects, the carbon dioxide independent media can further comprise one or more antibiotics. In some aspects, the one or more antibiotics can be gentamicin, penicillin, streptomycin, amphotericin or a combination thereof. [38] The base cell growth media disclosed herein are carbonate-buffered media that use exogenous CO2 provided by the incubator to maintain appropriate medium pH. When the cell line is removed from the incubator and kept in ambient conditions, the cells rapidly lose pH-sensitive active anchorage, such as integrin-mediated anchorage, from the cultureware and adopt a rounded morphology. When the medium is removed from the 5% CO2 incubator, the dissolved CO2 degases out of the medium according to the reaction below:

HCO3- + H⁺ H2CO3 H2O + C0_2, thereby causing the pH to increase to a more basic condition and the cells gradually lose their ability to actively attach to the cultureware resulting in cellular stress and limiting the longevity of anchorage-dependent cells.

[39] Thus, cells cultured under normal and standard conditions, for example, being incubated at 37°C and 5% CO2 are under severe stress and do not grow well when incubated at temperatures below 37°C (such as ambient temperature) and/or without exogenous CO2. Examples of evidence of stress may include, but not be limited to, reduction of anchorage to the substrate, cell death, altered metabolism, reduced division and changes in expression of particular phenotypic characteristics or functionality.

[40] In some aspects, it is desirable to culture the cells at temperatures lower than 37°C and in the absence of exogenous CO2. For example, when the cell culture needs to be transported from one location to another, it is desirable that it takes place at ambient temperature while the cells are still maintaining the health and functionality particularly the ability of the cells to register an increase in cellular signaling as measured by a reporter activity upon stimulation by the appropriate stimulus; and, for example, when the cells are used in a cell-based assay to detect one or more molecules of interest, including but not limited to a metabolite from a living organism. Such assays are often carried out at ambient temperatures, such as 20-22°C and without 5% CO2 supplementation. As described herein, the cells cultured using the systems and methods disclosed herein maintain their function and remain alive for an extended period of time. If the cells are alive but unable to perform their signaling function involving for example a ligand-induced increase in intracellular Ca²⁺ which can be measured by a fluorescent reporter, for example, or in another example if the cell signaling pathway fails to deliver an expected readout, the cells are not functional and are described as non-functional. [41] Disclosed herein are systems and methods for culturing cells at ambient temperature without incubating the cells at 37°C and 5% CO2 such that the cells have a prolonged longevity and functionality by limiting one or more causes of cellular stress(es).

[42] As described herein, the systems and methods disclosed herein are superior to and distinguishable from the standard cell culture system or method by: (1) altering the surface coating where the cells initially bind; (2) altering the environment in which the cells are in immediate in contact; (3) altering the temperature in which the cells operate; and (4) the insulation of the system.

CELL CULTURE SYSTEMS

[43] Disclosed herein are cell culture systems for culturing cells at ambient temperature. The cultured cells remain viable and functional outside of a controlled temperature environment without carbon dioxide supplementation for at least 3 days. Further, throughout the process of culturing the cells, the cells are not supplemented with carbon dioxide nor are the cells incubated. The cell culture systems and devices disclosed herein can be a closed system in which cells remain viable and functional without supplemental carbon dioxide and wherein the amount of medium present with the cells after sealing is an amount sufficient to maintain viability and functioning of the cells without further supplementation. In some aspects, the system may remain closed without such supplemental nutrients until cells are used for detecting a target molecule, including volatile organic molecules.

[44] In some aspects, the cell culture system comprises: a cultureware for culturing the cells, wherein the cultureware has a top, a bottom, an inner side and an outer side. In some aspects, the cultureware is gas impermeable and can be tightly sealed. In some aspects, the cell culture system comprises cells adhered to the bottom of the cultureware. In some aspects, the bottom of the cultureware is transparent. In some aspects, a layer of non-biodegradable cytocompatible hydrogel can be placed around, or over, the cells, wherein the cells remain in a stationary position within the hydrogel. In some aspects, the cells are sufficiently stationary to be locked in position within the hydrogel. In some aspects, the cell culture system comprises a cell culture medium covering the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide- independent (generally speaking a non-volatile) buffer to maintain a physiologically relevant pH of from about 7 to 8 in the medium throughout the culture.

[45] In some aspects, disclosed herein are cell culture systems for culturing cells at ambient temperature, wherein the cells can be used in a cell-based assay. In some aspects, the system comprises: (1) a cultureware for culturing the cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (2) a coating of synthetic polymer at the bottom of the inner side of the cultureware wherein the synthetic polymer having a positive surface electrostatic charge to allow passive adhesion of the cells; (3) cells adhered to the bottom of the inner side of the cultureware on top of the coating of synthetic polymer wherein the cells having a surface G-protein coupled receptor, an intracellular G-protein and an intracellular reporter; (4) a layer of optically clear non-biodegradable cytocompatible hydrogel around of the cells locking the cells in position within the hydrogel; and (5) a cell culture medium covering the hydrogel and submerging the cells wherein the cell culture medium has a high buffer capacity to maintain pH of from about 7 to about 8 in the medium throughout the extended culture duration.

[46] Further disclosed herein are cell culture systems for culturing cells at a temperature from about 15°C to about 30°C, orlO°C to about 35°C. In some aspects, the systems can comprise: a cultureware for culturing the cells. In some aspects, the cultureware comprises a top, an optically transparent bottom, an inner side and an outer side. In some aspects, the cultureware is gas impermeable. In some aspects, the cultureware has at least one access region to add or remove contents within the cultureware, and the access region is sealable to be gas impermeable. In some aspects, the systems comprise cells adhered to the bottom of the cultureware. In some aspects, the systems comprise a layer of non-biodegradable cytocompatible hydrogel. In some aspects, the hydrogel sufficiently surrounds the adhered cells to maintain the cells in a position within the hydrogel. In some aspects, the systems comprise a cell culture medium covering the adhered cells. In some aspects, the cell culture medium is buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide.

[47] Also disclosed herein are cell culture devices for culturing anchorage dependent, target molecule detecting cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients, and wherein said cells remain adhered to a cultureware surface of said culture device for at least 3 days. In some aspects, said cell culture device can comprise: a top, an optically transparent bottom, an inner side and an outer side. In some aspects, the cultureware of said cell culture device is gas impermeable. In some aspects, the cell culture device has at least one access region to add or remove contents, including a sample comprising a target molecule, within the cell culture device. In some aspects, the access region is sealable to be gas impermeable. In some aspects, said cell culture device can comprise target molecule detecting cells adhered to the bottom of the cultureware for at least 3 days. In some aspects, a layer of non- biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel. In some aspects, the cell culture device can comprise a culture medium buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide and wherein the culture medium is present in the device in an amount sufficient for the cells to remain viable in said device for at least 3 days in the absence of controlled temperature or supplemental carbon dioxide. In some aspects, said cells comprise a surface G protein-coupled receptor, an intracellular G-protein and an intracellular reporter that provides a detectable signal when said cells contact the target molecule.

[48] In some aspects, the cell culture systems described herein can be used for culturing cells at ambient temperature. In some aspects, the system comprises: (1) a cultureware for culturing the cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (2) cells adhered to the bottom of the cultureware; (3) a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (4) a cell culture medium covering the cells wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to 8 in the medium throughout the culture.

[49] In some aspects, the ambient temperature can be 15, 20, 25, 30°C, 35°C or any temperature in between. In some aspects, the ambient temperature can be 20, 21, or 22°C. In some aspects, the ambient temperature can be between 10°C to about 35°C. In some aspects, the ambient temperature can be between 15°C to about 30°C. In some aspects, the ambient temperature can be between 20°C to about 25°C. In some aspects, the ambient temperature can be from about 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C. In some aspects, the cell can be cultured at a first temperature or temperature range, but adherent, viable, and functional at a second temperature (e.g., room temperature, 37°C). In some aspects, the cells can be cultured at a first temperature (e.g., between 15°C and 30°C) to reduce metabolic rate and limit cellular division while preserving cellular viability and adherence and ability of the cells to respond in the presence of molecules that bind to the GPCR and generate a detectable reporter response. [50] In some aspects, the cells can be any cells. In some aspects, the cells can be primary cells. In some aspects, the cells can be derived from a cell line. In some aspects, the cells can be prone to apoptosis. For example, the cells can include, but not limited to, animal cells (such as cells derived from salmon), human cells, plant cells, insect cells, etc. In some aspects, the cells can be animal cells. In some aspects, the cells can be mammalian cells. In some aspects, the cells can be human embryonic kidney cells. In some aspects, the cells can be fish cells. In some aspects, the cells can be any fish cell. In some aspects, the fish cells can be from freshwater, marine or brackish water fish. Examples of fish cells include but are not limited to salmon, trout, minnow, catfish, bluegill, flounder, sea bream, cobia, grouper, snakehead, sea bass, pompano, and the like. The cells can be primary cells or cells derived from cell lines such as, but not limited to, tumor cells, hybridoma cells, etc. In some aspects, the cell is an immortalized human embryonic kidney (HEK) cell. In some aspects, the animal cells can be mammalian cells, including by not limited to bovine, canine, feline, hamster, mouse, porcine, rabbit, rat, or sheep. In some aspects, the mammalian cells can be cells of primates, including but not limited to, monkeys, chimpanzees, gorillas, and humans. In some aspects, the animals cells can be rodent cells (e.g., mouse cells). Animal cells include, for example, fibroblasts, epithelial cells (e.g., renal, mammary, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, hematopoietic cells. The animal cells can be adult cells (e.g., terminally differentiated, dividing or non-dividing) or stem cells. Mammalian cell lines can also be used. In some aspects, the cell lines can be derived from Chinese hamster cells, Human kidney cells, or Monkey kidney cells. The cell lines disclosed at the web-sites for ThermoFisher, ATCC, and Charles River Laboratories are incorporated by reference in their entirety for all purposes. In some aspects, the fish cells can be coho salmon-derived cell lines. Examples of salmon-derived cell lines include but are not limited to Salmon Head Kidney- 1, Anterior Salmon Kidney (ATCC Cat# CRL-2747™), and Atlantic salmon gill cells ASG-10 and ASG-13. Other cell lines that can be used include ATTC CRL-1681 (CHSE-241, chinook salmon embryo); ATTC CCL-55 (RGT-2, rainbow trout gonad); epithelioma papulosum cyprinid; ATTC CCL-42 (fathead minnow); ATTC CCL-59 (brown bullhead); ATTC CCL-91 (bluegill fry); WSSK-1 (white sturgeon skin); WSS-2 (white sturgeon spleen); CCO (channel catfish ovary); ATTC CRL-2747 (ASK, atlantic salmon kidney); and SHK-1 (salmon head kidney).

[51] In some aspects, the cells can be cancerous cells, non-cancerous cells, tumor cells, non tumor cells, healthy cells or any combination thereof. [52] In some aspects, the cells can be transformed cells. In some aspects, the cells can be immortal. In some aspects, the cells lose contact inhibition. In some aspects, the cells have a cancer cell phenotype.

[53] In some aspects, the cells are adherent at around 20°C. In some aspects, the cells can be genetically modified to be adherent at around 20°C. In some aspects, the cells are adherent around 12°C to 22°C. In some aspects, the cells are adherent around 22°C to 38°C. In some aspects, the cells are adherent at around 15°C to about 25°C or even to about 30°C. In some aspects, the cells are adherent at around 10°C to about 35°C. In some aspects, the cells are adherent at around 20°C to about 22°C. In some aspects, cells are adherent at around 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C.

[54] In some aspects, plant cells can be cells of monocotyledonous or dicotyledonous plants, including, but not limited to, alfalfa, almonds, asparagus, avocado, banana, barley, bean, blackberry, brassicas, broccoli, cabbage, canola, carrot, cauliflower, celery, cherry, chicory, citrus, coffee, cotton, cucumber, eucalyptus, hemp, lettuce, lentil, maize, mango, melon, oat, papaya, pea, peanut, pineapple, plum, potato (including sweet potatoes), pumpkin, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, tobacco, tomato, turnip, wheat, zucchini, and other fruiting vegetables (e.g. tomatoes, pepper, chili, eggplant, cucumber, squash etc.), other bulb vegetables (e.g., garlic, onion, leek etc.), other pome fruit (e.g. apples, pears etc.), other stone fruit (e.g., peach, nectarine, apricot, pears, plums, etc.), Arabidopsis, woody plants such as coniferous and deciduous trees, an ornamental plant, a perennial grass, a orage crop, flowers, other vegetables, other fruits, other agricultural crops, herbs, grass, or perennial plant parts (e.g., bulbs; tubers; roots; crowns; stems; stolons; tillers; shoots; cuttings, including un-rooted cuttings, rooted cuttings, and callus cuttings or callus-generated plantlets; apical meristems etc.). As used herein, the term “plants” refers to any of the physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage and fruit.

[55] In some aspects, the cells described herein can be used in cell-based assays to detect a target molecule. In some aspects, the cells are capable of detecting one or more target molecules. In some aspects, the cells are capable of detecting one or more target molecules in a cell-based assay. In some aspects, the target molecule can be a metabolite from a living organism. In some aspects, the metabolite can be a volatile organic compound (VOC). As used herein, “VOC” can refer to a molecule having a weight of less than 1000 g/mol. In some aspects, the VOC can be a compound that can bind to an odorant receptor or a modified odorant receptor. In some aspects, the term “compound” or “target molecule” refers to a molecule that can produce a detectable signal in a cell. In some aspects, the VOC or target molecule is capable of acting as a ligand at the GPCR. In some aspects, the signal can be a fluorescent signal.

[56] In some aspects, the target molecule can be a lung cancer metabolite. In some aspects, the lung cancer metabolite can be present in breath of a subject. In some aspects, the lung cancer metabolite can be a lung cancer metabolite VOC. In some aspects, the lung cancer metabolite VOC can be 2-decanone, 1 -butanol, limonene, 2-undecanone, n-octane, isopropylamine, methyl cyclopentane, 1,2,4-trimethylbenzene, 2-nonanone, 2-methylpentane, decane, propylbenzene, ethylbenzene, pentane, heptane, 1-hexanol, 1,2,3-trimethylbenzene, acetophenone, 2,2,4,6,6-pentamethylheptane, 3-methylhexane, 2-methylhexane, 2- pentadecanone, nonadecane, pentanal, octanal, nonanal, 4-heptanone, thiophene, 2,3-butadione, or a combination thereof. In some aspects, the lung cancer metabolite VOC can be acetone, 2- butanone, n-propanol, hexane, 2-methylpentane, trimethyl heptane, isoprene, benzene, toluene, ethylbenzene, cumene, trimethyl benzene, alkylbenzene, styrene, naphthalene, 1- methylnaphthalene, propanal, acetone, 2-butanone, phenol, benzaldehyde, acetophenone, nonanal, ethyl propanoate, methyl isobutanoate, dichloromethane, dichlorobenzene, trichloroethane, trichlorofluoromethane, tetrachloroethylene, styrene, 2,2,4,6,6-pentamethylheptane, 2- methylheptane, decane, n-propylbenzene undecane, methyl cyclopentane, l-methyl-2- pentyl cyclopropane, trichlorofluoromethane, benzene, 1,2,4-trimethylbenzene, 3-methyloctane, 1- hexene, 3-methylnonane, 1-heptene, 1,4-dimethylbenzene, 2,4-dimethylheptane, hexanal, cyclohexane, 1 -methyl ethenylbenzene, heptanal, butane, 3-methyltridecane, 7-methyltridecane, 4- methylctane, 3-methylhexane, heptane, 2-methylhexane, pentane, 5-methyldecane, 2- methylpentane, pentane, xylenes, trimethylbenzene, toluene, benzene, heptane, decane, styrene, octane, pentamethyl heptane, l,5,9-trimethyl-l,5,9-cyclododecatriene, 2, 2, 4-trimethyl- 1,3- pentanediol tributyrate, ethyl 4-ethoxybenzoate, 2-methyl- propanoic acid, (1,1 -dimethyl ethyl)-2- methyl-l,3-propanediyl ester, 10,1 l-dihydro-5H-dibenz-(b,f)-azepine, 2,5-2,6-bis(l,l- dimethylethyl)-cyclohexadiene-l,4-dione, 1,1-oxybi-benzene, 2, 5 -dimethyl -furan, 2,2-diethyl- 1, 1-biphenyl, 2,4-dimethyl-3-pentanone, trans-caryophyllene, 2,3-dihydro-l,l,3-trimethyl-3- phenyl-lH-indene, 1-propanol, 4-methyl-decane, 1,2-benzenedicarboxylic acid, diethyl ester, 2,5- dimethyl-2,4-hexadiene, formaldehyde, isopropanol, isoprene, acetone, methanol, 2-butanone, benzaldehyde, 2,3-butanedione, pentanal, hexanal, octanal, p-cymene, toluene, dodecane, 3,3- dimethylpentane, 2,3,4-trimethylhexane, (1 -phenyl- l-butenyl)benzene 1,3-dimethylbenzene, 1- iodononane, [(1,1 -dimethyl ethyl) thiol] acetic acid, 4-(4-propylcyclohexyl)-40 -cyano[l,10 - biphenyl]4-yl ester benzoic acid, 2-amino-5-isopropyl-8-methyl-l-azulenecarbonitrile, 5-(2- methylpropyl)nonane, 2,3,4-trimethyldecane, 6-ethyl-3-octanyl 2-(trifluoromethyl)benzoate, p- xylene, and 2,2-dimethyldecane, 3 -hydroxy-2 -butanone, isoprene, acetone, 2-butanone, cyclohexanone, dimethyl sulfide, acetonitrile, ethanol, isopropanol, acetaldehyde, propanal, butanal, pentanal, hexanal, octanal, 2-propenal, 2-butenal, propane, butane, pentane, hexane, heptane, 2-methylbutane, 2-methylpropanal, 2,2-dimethylbutane, 2,3-dimethylbutane, 2- methylpentane, 3-methylpentane, 2,2-dimethylpentane, 2,4-dimethylpentane, 3,3- dimethylpentane, 2-methylhexane, benzene, toluene, chlorobenzene, 1,2-dimethylbenzene, 1,2- dichlorobenzene, carbon disulfide, dimethyl formamide, 2,5-dimethylfuran, propane, carbon disulfide, ethylbenzene, isopropyl alcohol, ethanol, acetone, butane, dimethyl sulfide, isoprene, 2- pentanone, furan, o-xylene, ethylbenzene, pentanal, hexanal, nonane, 1-octene, 2,4,6- trimethyloctane, 2-methyldodecane, 2-tridecanone, 2-pentadecanone, 8-methylheptadecane, 2- heptadecanone, nonadecane, eicosane, butanal, ethyl acetate, ethylbenzene, 2-propanol, 3- methyldodecane, 1 -butanol, 2-methylbutylacetate/2-hexanol/nonanal isopropylamine, ethylbenzene, hexanal, 3-methyl-l-butanol, caprolactam, propanoic acid, 2-methyl-5- propylnonane, butylated hydroxytoluene, 2,6,11-trimethyl-dodecane, hexadecanal, 8- hexylpentadecane, ethanol, 2-butanone, thiophene, 4-heptanone, butanoic acid, , acetic acid, cyclohexanone, 2,2,-dimethyl-hexanal, 1,1-di ethoxy-3 -methylbutane; 1-(1 -ethoxy ethoxy)- pentane, 2,2,6-trimethyloctane, 2-ehtyl-l-hexanol, undecane, thymol, 2-methyl- 1-decanol, 3,7- dimethyl-decane, pentane, 2-methylpentane, hexane, benzene, ethylbenzene, trimethylbenzene, heptane, pentamethyl heptane, toluene, total xylenes, styrene, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, trans-2-hexenal, trans-2-heptenal, trans-2-nonenal, p-cresol, eicosenamide, 1-hexadecylindane and cumyl alcohol, propanal, butanal, decanal, butanal, ethylbenzene, hydrogen cyanide, acetonitrile, isoprene, or a combination thereof. In some aspects, the lung cancer metabolite VOC can be dimethyl sulphide, 2-hydroxyacetaldehyde, 4- hydroxyhexenal, ethylbenzol, n-dodecane, isopropyl benzene, p-xylene + m-xylene, 5-(2-methyl- )propyl-nonane, 2,6,-ditertbutyl-4-methyl-phenol, 4-hydroxy-2-hexenal, hydroxyacetaldehyde, 4- hydroxy-2-nonenal, n-hexane, n-nonanal, diethyl ether, isothiocyanatocyclohexane, hydrogen isocyanide, or a combination thereof. Examples of lung cancer metabolite VOC that can be detected using the cells and/or methods disclosed herein are described in Jia, et ah, Metabolites (2019). Vol. 9, No. 52; Ratiu, et al. (2021) J. Clin. Med., Vol. 10, No. 32; and Dent et al. (2013) J. Thorac Dis Vo. 5(S5): S540-S550.

[57] In some aspects, the cells can be adherent or adhered cells. In some aspects, cells adhere to the bottom of the inner side of the cultureware.

[58] In some aspects, the cells can be a modified cell. In some aspects, the cells can be a genetically modified cell. In some aspects, the modified cell can comprise one or more cell-surface receptors. In some aspects, the modified cell can comprise a modified cell-surface receptor. In some aspects, the modified cell-surface receptors can be modified to increase or decrease their ability to bind to a variety of compounds. In some aspects, modified cell-surface receptors can be modified to increase or decrease their ability to bind to a specific compound. In some aspects, the modified cell can comprise a deletion of one or more endogenous cell-surface receptors.

[59] In some aspects, the cells can comprise one or more surface G-protein-coupled receptors (GPCRs). In some aspects, the cell can comprise a GPCR, an intracellular protein and an intracellular reporter. In some aspects, the cells express or can be modified to express a GPCR, an intracellular G-protein and an intracellular reporter.

[60] GPCRs are a large family of related receptors that are cell surface receptors that interface the cell with the outside world by detecting molecules outside the cell and activating a cellular response to couple with the intracellular G-protein followed by generation of an intracellular signal. The G-protein signaling pathway can comprise G-protein-mediated activation of adenylate cyclase with the resultant production of cAMP as a second messenger. In some aspects, the cAMP can interact with a cAMP activated cation channel.

[61] The G-protein can be comprised of three subunits the Ga subunit (e.g., Uniprot P38405), GP subunit (e.g., Uniprot P62879) and Gy subunit (e.g., Uniprot P63218). In some aspects, the adenylate cyclase and the G protein can be from the same species. In some aspects, the adenylate cyclase and the G protein can be from different species. In some aspects, the G protein subunits can be from the same or from different species. In some aspects, the intracellular reporter can be cAMP. In some aspects, the intracellular G proteins can interact directly with the intracellular reporter to produce a detectable signal, e.g., adenylate cyclase can produce cAMP. In some aspects, the cAMP molecule itself can be detected. The G proteins can interact with adenylate cyclase to induce a reporter. In some aspects, the G proteins can interact with adenylate cyclase to produce a first signal, and a second system amplifies the first signal when the reporter responds to the first signal.

[62] In some aspects, the GPCRs can comprise one or more reporters. In some aspects, a heterologous gene encoding a reporter protein can be introduced into the cells disclosed herein so that the cells express the reporter, and the G-protein activates the reporter when an appropriate interaction (e.g., binding event) occurs at the GPCR. In some aspects, the cells described herein can be engineered or genetically modified to express a single reporter. In some aspects, different cells can each express a different reporter, and can be used to enhance signal detection. In some aspects, the cells described herein can be engineered to express two or more reporter products, for example by using a single vector construct encoding two or more reporters.

[63] In some aspects, the reporter or reporters can be one or more of a fluorescent reporter, a bioluminescent reporter, or a combination thereof. Examples of fluorescent reporters include, but are not limited to, green fluorescent protein from Aequorea victoria or Renilla reniformis , and active variants thereof (e.g., blue fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, etc.); fluorescent proteins from Hydroid jellyfishes, Copepod, Ctenophora, Anthro50 zoas, and Entacmaea quadricolor, and active variants thereof; and phycobiliproteins and active variants thereof. Other fluorescent reporters include, for example, small molecules such as CPSD (Disodium 3-(4-methoxyspiro {l,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.137] decan} -4-yl) phenyl phosphate, ThermoFisher Catalog #T2141). Bioluminescent reporters include, but are not limited to, aequorin (and other Ca²⁺ regulated photoproteins), luciferase based on luciferin substrate, luciferase based on Coelenterazine substrate (e.g., Renilla, Gaussia, and Metridina), and luciferase from Cypridina, and active variants thereof. The bioluminescent reporter can be, for example, North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metrida luciferase, OLuc, and red firefly luciferase. For example, see, U.S. Patent No. 6,670,449 and U.S. Patent No. 5,625,048, which are incorporated herein by reference for describing mutants and modified green fluorescent proteins, respectively.

[64] In some aspects, the reporter can be a fluorogenic reaction that can participate in the quenching or suppressing of fluorescence or luminescence. Changes in light intensity or wavelength can be used to optically detect the presence or activation of the reporter. Such reporter activity can result from ionic changes including but not limited to pH changes, quenching, presence or absence of enzyme substrates, or ability to bind a Second reagent, such as an antibody conjugate, which itself participates in generating an optically detectable signal.

[65] In some aspects, light-generating reporters typically are two-component systems, where light emitted by one component undergoes either a spectral or an intensity change due to a physical interaction of the first component with the second component.

[66] As used herein, the term “signal” can refer to a signal in response to a binding event. For example, a signal can be generated by a compound binding to a cell-surface receptor of a cell, and activating adenylate cyclase which in turn activates cAMP that leads to the generation of a signal that can be visually detected. In some aspects, the signal can be a signal measured over a period of time. In some aspects, the signal can be a measurement of amplitude, period or a frequency or a combination thereof. In some aspects, the signal is an intracellular change, for example, emission of light or change of pH that activates a detectable signal such as fluorescence. Detailed description of an example of this G-protein coupling pathway and a chemical calcium reporter is disclosed by Zhuang, H. and Matsunami, H. (J. of Bio. Chem. Vol. 282, No. 20, p. 15284-15293; 2007) and a genetically-encoded calcium indicator is described in Dana H. et al. (Nat Methods 16, 649-657; 2019). U.S. Patent No. 7,879,565 is incorporated herein by reference for disclosing G-protein coupling pathways linked to reporters (or reporter agents) for detecting GPCR activation.

[67] In some aspects, the signal can be measured in real-time. In some aspects, the reporter can emit light or produce a molecule that can be detected with an optical sensor. Real time measurements can be obtained through the cultureware comprising the cells disclosed herein by recording the change in light emission over time as the cells comprising the GPCR interacts with a sample and wherein a sample comprising a volatile organic compound or a target molecule will cause the activation of light as a detectable signal. In some aspects, the real time measurements can be used to quantify the binding interaction by an absolute measurement or a relative measurement. In some aspects, in the absolute measurement, the real time signal can be compared to a standard to determine the binding activity of the GPCR. Known amounts of the volatile organic compound or a target molecule for the GPCR can be used to generate a standard binding curve for receptor occupancy versus reporter gene output. Binding of a sample comprising a target molecule can then be compared to the standard curve to quantify interaction of the target molecule at the GPCR. For example, the amount of signal (e.g., light) produced correlates with the amount of calcium present in the cell(s) upon GPCR activation such that the light intensity is proportional to the concentration of the target molecule (e.g., VOC). In some aspects, in the relative measurement, the cells disclosed herein can include internal references that allow differences in interactions at the GPCR to be compared. In some aspects, a reference GPCR can be included in any of the cells disclosed herein, and a known amount of the reference ligand can be added to the reference receptor to act as a standard. The reference receptor can be coupled to a different reporter, e.g., a reporter that provides a different optical signal from the GPCR reporter. The reference and test receptors can be coupled to different fluorescent proteins such as green fluorescent protein (GFP), and red fluorescent protein (RFP). In some aspects, the ratio of green fluorescence to red fluorescence can be compared for different target molecules at the same GPCRs, or to compare binding of the same test ligand to different GPRCs. GFP’s sensitive to different pH changes may also be used. See, U.S. patent 6,670,449.

[68] The GPCR, G-protein or intracellular reporter can be already present, e.g., endogenous, in the cells, or one or more of them can be genetically engineered to be expressed by the cells. Any one or all of them can be naturally occurring or they can be genetically engineered (referred to as synthetic). In some aspects, the G-protein or the intracellular reporter is artificial and engineered to be expressed by the cell. In some aspects, the GPCR, the G protein or the reporter can be natural (e.g., endogenous) or synthetic (e.g., genetically engineered). In some aspects, the artificial G- protein or intracellular reporter is also known as a synthetic G-protein or synthetic intracellular reporter, respectively.

[69] In some aspects, the cells can be genetically modified to express one or more GPCRs. In some aspects, the cells also can be genetically modified to express the human G protein subunits Ga, Gp, and Gy. The genes encoding the human Ga, Gp, and Gy subunits can be placed under the control of appropriate control sequences (promoters, enhancers, translation start sequences, polyA sites, etc.) for the desired cell, and these constructs for the human Ga, Gp, and Gy subunits can be placed into the desired cell. The human G-protein also can be associated with adenylate cyclase. The gene for an appropriate adenylate cyclase can be placed under the control of appropriate control sequences for the desired cell, and this construct can be placed into the desired host cell. In some aspects, the Ga subunit can be a Gq protein alpha subunit. In some aspects, the cell can express or can be genetically modified to express Gq protein alpha subunit. Gq proteins couple to GPCRs to activate beta-type (phospholipase C-beta) enzymes. PLC-beta in turns hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacyl glycercol (DAG) and inositol triphosphate (IP3), which activates Ca²⁺ . In some aspects, the reporter activity can result from changes (increases) in intracellular Ca²⁺ levels. In some aspects, the reporter activity can be detected by a fluorescent or bioluminescent reaction and/or changes in light intensity or wavelength. In some aspects, the reporter can be a genetically-encoded Ca²⁺ biosensor. In some aspects, the reporter can be GCaMP7s. In some aspects, the reporter can be a resonance energy transfer pairs or self- quenching fluorophores. In some aspects, the report can be fluorescence resonance energy transfer (FRET) or a bioluminescence resonance energy transfer (BRET) pair. Examples of FRET pairs include but are not limited to CFP and YFP; ECFP and EYFP ; EBFP and GGFP; mCerulean and mVenus; SBFP2 and EBFP2; EBFP2 and mEGFP, MiCy and mKO; TFP1 and mVenus; EGFP and mCherry; Venus and mCherry; Venus and TdTomato; and Venus and mPlum. Examples of BRET pairs are not limited to RLuc and Topaz; RLuc and GFP; Aequorin and GFP; Firefly luciferase and red fluorescent protein; and Firefly luciferase and D-luciferin.

[70] In some aspects, the GPCRs can detect one or more VOCs. In some aspects, the cells can express a single type of compound-sensing receptors or a combination of compound-sensing receptors.

[71] In some aspects, a single VOC can bind to different GPCRs with different binding affinities. In some aspects, the binding of a single VOC to a GPCR can activate a signaling pathway within the cell.

[72] Disclosed herein are cultureware that is gas impermeable and tightly sealed to prevent the evaporation of the cell culture medium from the cell culture during the culture. Evaporation leads to osmotic stress, which is deleterious to cell health, and minimizing, reducing or preventing evaporation can allow the cells to achieve prolonged longevity and functionality. In some aspects, a gas impermeable foil can be applied to seal the cultureware. Such application with the gas impermeable foil can prevent osmolarity drift. Under increased osmotic conditions, cells lose their active anchorage and adopt a rounded morphology and detach from the cultureware.

[73] In some aspects, the cultureware can be in the form of cassette. In some aspects, the cassette can be inserted, for example, into a device and positioned to be in contact with a sample comprising one or more VOCs. FIG. 14 shows an example of a microfabricated microarray on a coverslip. FIG. 15 shows that nano-wells can be individually addressable, by dispensing 10 pm fluorescent polymer microbeads using a Nanoject micro injector under stereotactic control. Horizontal lines in the empty wells are microfabrication photopolymerization artifacts. Cells were seeded cells onto a microarray, and were sedimented and adhered in the nano-wells (see, FIG. 15). The microarrays can be imaged by a fluorescence microscope.

[74] In some aspects, the lifespan of the cells within the cultureware can be at least 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days or longer. In some aspects, the lifespan of the cells within the cultureware can be at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer. In some aspects, the cells can be functional within the cultureware for at least at least 3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 20 days or longer. In some aspects, the cells can be functional within the cultureware for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer. In some aspects, the cells remain viable and adherent to the substrate for at least at least 3 days, 4 days, 5 days without supplemental CO2. In some aspects, the cells remain viable for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer without supplemental CO2.

[75] In some aspects, the cells within the cultureware can be adherent for at least 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days or longer. In some aspects, the cells within the cultureware can be adherent for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer. In some aspects, the cells within the cultureware can be adherent for at least at least 3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 20 days or longer. In some aspects, the cells within the cultureware can be adherent for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer. In some aspects, the cells remain viable and adherent to the substrate for at least 3 days without supplemental CO2. In some aspects, the cells remain viable and adherent to the substrate for at least at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer without supplemental CO2. In some aspects, the cells remain viable and adhered to the bottom of the cultureware for at least 3 days without supplemental CO2. In some aspects, the cells are adherent for at least 12 days at ambient temperature without CO2 supplementation. In some aspects, the cells remain viable and adherent to the substrate indefinitely without supplemental CO2

[76] In some aspects, the cultureware can be a single chamber, such as a culture flask. In some aspects, the cultureware can be a plate with a plurality of chambers or wells. For example multi well plates can be used include but are not limited to the 96-Well Black/Clear Bottom Plate or White/Clear Bottom Plate available from ThermoFisher (Chicago, IL). In some aspects, the cultureware has a plurality of chambers that are in the form of wells with the top of the cultureware open and wherein the cultureware is entirely sealed on the outer side with a gas impermeable film to provide gas impermeable chambers. In some aspects, the gas impermeable film can function as a reversible sealable opening to allow one or more substances to be added or removed from the cultureware. In some aspects, the one or more substances can be a target molecule.

[77] Gas impermeability of the cultureware can be achieved by constructing the cultureware with a gas impermeable material. In some aspects, the gas impermeable material is glass or metal. In some aspects, the gas impermeable material is transparent. In some aspects, the gas impermeable material is a UV-transparent substrate. In some aspects, the UV-transparent substrate can be made of optically clear plastic polymers. For example, the optically clear plastic polymer can be cyclic olefin copolymers (COC). In some aspects, the gas impermeable material is such that the adhered cells remain in the focal plane for the imaging system to capture the signal (e.g., fluorescent response). In the embodiment of a single chamber cultureware such as a culture flask, the cultureware can be tightly sealed by capping the flask with a cap. In some aspects, the flask contains a one-way opening that can be used to allow passage into the flask of ambient gas from the environment so that VOC present in the gas may dissolve in the cell medium so that the VOC’s may contact the cells comprising one or more GPRCs. In some aspects, the cultureware can be plastic. In some aspects, the cultureware can be polystyrene.

[78] In some aspects, the cultureware is a plate with a plurality of wells, the top of the wells is open for passing the materials into the wells. In some aspects, the plate can be made gas impermeable by covering the entire outer side of the cultureware with a gas impermeable film. In some aspects, the gas impermeable film can provide a gas impermeable seal to the cultureware. In some aspects, the gas impermeable film is light insulating. In some aspects, the gas impermeable film is light insulating when the culture medium contains light sensitive components such as L- glutamine or other aromatic components. In some aspects, the gas impermeable film is an aluminum film. For example, the Gas Impermeable Aluminum Adhesive (Cat# A2350, Sigma, St. Louis, MO) can be used. This adhesive provides gas impermeability and a tight seal while also being light insulating.

[79] The cells described herein adhere to the bottom of the inner side of the cultureware. The adhesion can be “active” or “passive”. In an “active” adhesion, the cells sediment to the bottom of the inner side of the cultureware and secrete an extracellular matrix to allow the cells to adhere. The binding of the cells to the secreted extracellular matrix proteins may be temperature-, osmolarity- or pH-dependent and therefore prone to failure when environmental conditions change over time. “Actively” adhered cells tend to lose adhesion within a few days when kept at ambient temperature, limiting their longevity.

[80] In some aspects, the adhesion is at least in part “passive”. “Passive” adhesion does not involve any cellular activities of the cells for the adhesion. “Passive” adhesion can be accomplished by providing a positive surface electrostatic charge to the bottom of the inner side of the cultureware. This positive surface electrostatic charge can be provided by a coating of a synthetic polymer having the charge. In some aspects, the bottom of the inner side of the cultureware can have a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware. In some aspects, the positive surface electrostatic charge is provided by a coating of a synthetic polymer with a positive surface electrostatic charge. The cell surface proteins are mainly negatively charged under physiological conditions allowing the cells to adhere electrostatically to the positively-charged coating. Commercially available synthetic polymers can be used to provide this function. Examples of these polymers include, but not limited to, poly-D-lysine, poly-L-lysine, poly-D-omithin or polyetherimide. In some aspects, the synthetic polymer is poly-D-lysine (e.g., Cat# 7886, Sigma, St. Louis, MO). Spontaneous loss of adhesion by “passively” adhered cells tends to be substantially delayed even at ambient temperature. The coating of positive surface electrostatic charge can be supplied by the vendor with the cultureware, or it can be applied to the cultureware before the culture by the user. In some aspects, the cultureware or substrate can be pre-coated with the synthetic polymer. In some aspects, the synthetic polymer can be a polycationic polymer. In some aspects, the polycationic polymer can be polylysine, polyomithine or polyethyleneimine. The application of the synthetic polymer can limit the lowered temperature impact (e.g., about 22°C instead of 37°C). For example, the applied synthetic polymer (e.g., hydrogel) can lock the cells in place because the synthetic polymer (e.g., hydrogel) is applied as a liquid and then turns into a conformal solid upon gelation. The cells become trapped by the hydrogel so if they lose attachment to the substrate, they will remain in the same focal plane and remain surrounded by their neighboring cells, limiting cellular stresses which can cause an increase in baseline Ca²⁺ which would otherwise limit the functionality of the cells. Example 2 in the Examples Section describes an example of coating the bottom of the inner side of the cultureware with poly-D-lysine.

[81] Passive adhesion of the cells may require incubating the cells in MEM and 37°C for a period of time. This allows the freshly seeded cells to gradually sediment to the bottom of the cell suspension until the cells adhere to the coating.

[82] Disclosed herein are hydrogels. In some aspects, the hydrogels are non-biodegradable and cytocompatible. In some aspects, the non-biodegradable cytocompatible hydrogel can be optically clear. Hydrogel is formed when the monomer of the hydrogel polymerizes in the presence of an appropriate catalyst. Water (or more generally medium) is trapped within the polymer during polymerization to form the hydrogel. What is meant by “cytocompatible” is that neither the resulting hydrogel, nor the monomer nor the catalyst causes either short-term or long-term toxicity to the cells. While some hydrogels are non-cytotoxic, the monomer and/or catalyst used can be cytotoxic. For example, copper is used as a catalyst for the formation of some thiol-based hydrogels and is cytotoxic. It is important that the hydrogel be cytocompatible since the hydrogel is formed in the cultureware in the presence of the cells. The polymerization process may not fully use up all the monomers and/or the catalyst. The residual monomer and/or catalyst may still be in the culture and they should not cause any cytotoxicity to affect the health of the cells. In some aspects, the hydrogel-entrapped cells can be incubated in a wash solution to allow for passive diffusion of catalyst and/or monomers to exit the hydrogel. Suitable hydrogels for the present invention are commercially available. Examples of hydrogels include but are not limited to the QGel Hydrogel from QGel SA (Cat# NS22-A; Lausanne, Switzerland) and the TruGeBD™ (Sigma, St. Louis, MO). The components for making the hydrogel come as a kit and the hydrogel is prepared according to the manufacturer instruction.

[83] In some aspects, the cells are used for cell-based assays and the intracellular signal is emission of light. In some aspects, the cells are imaged to detect the emitted light. In some aspects, the hydrogel is optically clear. An example of an optically clear cytocompatible hydrogel is the QGel Hydrogel. [84] The hydrogel can be formed inside the cultureware in the presence of the adhered cells so that the hydrogel is conformal around the cells locking the cells in position within the hydrogel. Locking the cells in position within the hydrogel prevents the cells from movement and further limiting division which otherwise leads to eventual apoptosis, thereby prolonging the longevity and functionality of the cells. In some aspects, the hydrogel is non-biodegradable so that it can maintain its structural integrity during the entire culture. In some aspects, the culture system can be used for cell-based assays. In some aspects, the hydrogel is receptor ligand permeable so that the ligand can penetrate the hydrogel to reach the cells. Hydrogels can be ligand permeable due to mesh size of the hydrogel being larger than molecular size. In some aspects, the hydrogel can be sufficiently permeable to allow nutrients and waste to diffuse in and out of the cells.

[85] Formation of the hydrogel inside the cultureware around the cells can be accomplished by incubating the culture at 37°C for a period of time. Example 3 in the Examples Section provides an example of forming the hydrogel inside the cultureware in the presence of the adhered cells.

[86] In aspects, wherein the cells are used for cell-based assays, and wherein the cells are imaged to detect the emitted light, the cells can be locked in position in the focal plane by limiting their movement allows better quality of the images.

[87] Locking or maintaining the adhered cells in a stationary position by the hydrogel can be applicable to cells that are prone to apoptosis. In primary cells, the cells grow and divide until they are in contact with each other (known as contact inhibition) then the resulting senescence precedes initiation of apoptosis events leading to cell death. With the cells locked in position, contact inhibition is prohibited and thus delaying the apoptosis process. Cells from cell lines do not exhibit contact inhibition but they grow and divide indefinitely and eventually undergo apoptosis and cell death. When the cells are locked in position in the hydrogel, their growth is mechanically controlled to delay apoptosis and cell death. Cells in cell cultures that are normally cultured optimally at 37°C can detach from the cultureware and undergo apoptosis due to loss of anchorage when they are exposed to lower temperatures, such as ambient temperature. By locking the cells in position within the hydrogel, the cells no longer detach from the cultureware at the lower temperatures to delay the apoptosis process. Yet in anchorage-dependent cells, they undergo anoikis when they detach from the surroundings. Anoikis is a form of programmed cell death. Thus, these anchorage-dependent cells can benefit from being locked in position by the hydrogel. [88] Disclosed herein is a cell culture medium that can be in the cell culture. In some aspects, the medium is carbon dioxide-independent. In some aspects, the pH of the medium is independent of the presence of carbon dioxide. This is in contrast to standard cell cultures that require incubating the culture in presence of 5% CO2 in the incubator. Reduction in the level of CO2 in the culture causes the pH of the culture to rise and induces stress to the cells. The culture medium as described herein is buffered by a high buffering capacity medium which is independent of the presence of carbon dioxide. Thus, the culture is not incubated in the presence of 5% CO2 as required in many standard cell culturing methods. In some aspects, the high buffering capacity of the buffer maintains a pH of about 7 to about 8 for the culture medium during the entire culture. In some aspects, the high buffering capacity of the buffer can prevent pH drift. Suitable buffers include, but not limited to, MOPS. HEPES, MES, BICINE or phosphate. Commercially available culture media with high buffering capacity and carbon dioxide-independent include but are not limited to carbon dioxide- independent medium from ThermoFisher (Cat# 18045088; Chicago, IL), Hibemate™-E Medium and Leibovitz’s L-15 Medium from Sigma (both from ThermoFisher, Chicago, IL). PCT Publication No. 1991/000451 by VisticaD. T. etal. discloses a carbon dioxide- independent growth medium for maintenance and propagation of cells.

[89] In some aspects, the high buffering capacity of the cell culture medium can be accomplished by using an increased amount of the medium, such as topping the cultureware with the medium. In some aspects, the high buffering capacity of the cell culture medium can be achieved by having an excessive amount of the medium.

[90] In some aspects, the cell culture medium can comprise growth factors. In some aspects, the cell culture medium can comprise Fetal Bovine Serum (FBS). In some aspects, the FBS can be in the range of about 1% to about 20%.

[91] Disclosed herein are cell culture systems. For example, FIG. 1 is a schematic diagram of a cross-section of cultureware that can be useful in the methods described herein. For example, the cultureware can be a plate having multiple wells with the top of the wells open for passage of materials into the wells. The cell culture system 10 has multiple wells 20. A coating of synthetic polymer 30 provides a positive surface electrostatic charge to allow passive adhesion of the cells 40 to the bottom of the inner side of the cultureware. A layer of non-biodegradable cytocompatible gel 50 surrounds the cells 40 locking the cells 40 into position. The well 20 of the cultureware is filled with a culture medium 60 buffered with a high buffering capacity and carbon dioxide independent buffer. The entire cultureware is covered tightly with a gas impermeable light insulating film 70 to prevent the evaporation of the culture medium 60 during the culture.

[92] Disclosed herein are cell culture systems for culturing cells that can be used in a cell-based assay at ambient temperature. In some aspects, the system comprises: (1) a cultureware for culturing the cells, wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; (2) a coating of synthetic polymer at the bottom of the inner side of the cultureware wherein the synthetic polymer having a positive surface electrostatic charge to allow passive electrostatic adhesion of the cells; (3) cells adhered to the bottom of the inner side of the cultureware on top of the coating of synthetic polymer wherein the cells having a surface G-protein coupled receptor, an intracellular G-protein and an intracellular reporter; (4) a layer of optically clear hydrogel around of the cells locking the cells in position within the hydrogel; and (5) a cell culture medium covering the cells wherein the cell culture medium is buffered by a high buffer capacity to maintain pH of from about 7 to about 8 in the medium throughout the culture.

METHODS OF CULTURING CELLS

[93] Also disclosed herein are methods for culturing cells at ambient temperature. The methods described herein provide a means for culturing cells such that the cultured cells remain viable and functional outside of a controlled temperature environment without supplemental carbon dioxide for at least 3 days. Further, the cultured cells use the methods described herein. Additionally, the methods used to culture cells include a sufficient amount of medium that after sealing the cells in the system or device disclosed herein, the cultured cells remain viable and functioning without further supplementation.

[94] In some aspects, the method comprises the steps of: (1) providing a cultureware for culturing cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the bottom of the inner side of the cultureware having a coating with a synthetic polymer to provide a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware; (2) depositing the cells to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (3) allowing the cells to adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (4) providing a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (5) covering the cells with a cell culture medium wherein the cell culture medium is buffered by a high capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and (6) culturing the cells at ambient temperature without incubating the cells at 37°C and 5% CO2. In some aspects, the ambient temperature can be from about 15°C to about 25°C or even to about 30°C. In some aspects, the ambient temperature can be from about 20°C to about 22°C. In some aspects, the ambient temperature can be from about 15°C to about 30°C, about 20°C to about 28°C, about 20°C to about 25°C or about 20°C to about 24°C. In some aspects, the cell can be cultured at a first temperature or temperature range, but adherent, viable, and functional at a second temperature (e.g., room temperature, 37°C). In some aspects, the cells can be cultured at a first temperature (e.g., between 15°C and 30°C) to reduce metabolic rate and limit cellular division while preserving cellular viability and adherence and ability of the cells to respond in the presence of molecules that bind to the GPCR and generator a detectable reporter response. In some aspects, the cells remain viable and adherent to the substrate for at least 3 days without supplemental CO2.

[95] Also disclosed herein are methods for culturing cells at ambient temperature. In some aspects, the methods comprise the steps of: (1) providing a cultureware for culturing cells wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the bottom of the inner side of the cultureware having a coating with a synthetic polymer to provide a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware; (2) depositing the cells to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (3) allowing the cells to adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer; (4) providing a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and (5) covering the cells with a cell culture medium wherein the cell culture medium is buffered by a high capacity carbon dioxide- independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and (6) culturing the cells at ambient temperature without incubating the cells at 37°C and 5% C0₂.

[96] Also disclosed herein are methods of culturing cells at ambient temperature, the method comprising: depositing cells in a cultureware, wherein the cultureware has a top, a bottom, an inner side and an outer side and is gas impermeable and wherein the bottom of the inner side of the cultureware has a coating of a synthetic polymer. In some aspects, the methods can comprise adding non-biodegradable cytocompatible hydrogel around the adhered cells, wherein the cells are immobile within the hydrogel; and adding a cell culture medium to the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and culturing the cells at ambient temperature without incubating the cells at 37°C and 5% CO2.

[97] Also disclosed herein are methods of culturing anchorage dependent cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients and wherein said cells remain adhered to a cultureware surface of said culture device for at least 3 days. In some aspects, said method can comprise: maintaining cells in a cell culture device, wherein the cell culture device comprises a top, an optically transparent bottom, an inner side and an outer side. In some aspects, the cultureware of said cell culture device is gas impermeable. In some aspects, the cell culture device has at least one access region to add or remove contents within the cell culture device. In some aspects, the access region is sealable to be gas impermeable. In some aspects, the methods can comprise maintaining the cells adhered to the bottom of the cultureware for at least 3 days. In some aspects, a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel. In some aspects, the methods can comprise covering the cells in the culture device with a culture medium buffered to maintain a pH of from about 7 to about 8 in in the absence of supplemental carbon dioxide and wherein the cells remain viable in said device for at least 3 days.

METHODS OF USING CULTURED CELLS

[98] Disclosed herein are methods of detecting one or more target molecules, ligands, metabolites or volatile organic molecules using the cultured cells described herein. In some aspects, the methods can comprise contacting a cell culture with a fluid sample. In some aspects, the fluid sample comprises one or more volatile organic molecules. In some aspects, the cell culture can comprise cells in a cultureware having a top, an optically clear bottom, an inner side and an outer side and is gas impermeable. In some aspects, the cells are adhered to the bottom of the inner side of the cultureware. In some aspects, the cells are functional at room temperature. In some aspects, the cells are present in a cell culture medium that is buffered by a high buffering capacity carbon dioxide-independent buffer. In some aspects, the cells comprise one or more G-protein coupled receptors capable of binding to the one or more volatile organic molecules. In some aspects, wherein the one or more G-protein coupled receptors comprises a reporter. In some aspects, the methods can comprise exposing the cell culture to a light. In some aspects, the methods can comprise detecting the presence of a fluorescence emitted by the reporter after binding of the one or more G-protein coupled receptors to the one or more volatile organic molecules, an increase in brightness of the fluorescence, or a combination thereof. In some aspects, the cells in the cultureware are non-mammalian cells. In some aspects, the non-mammalian cells can be fish cells. In some aspects, the fluid sample can be a gaseous sample from an exhaled breath of a mammalian subject. In some aspects, the cells can be maintained in the culture for at least 3 days in an uncontrolled temperature environment and without supplemental carbon dioxide.

EXAMPLES

[99] Example 1: Preparation of coating on bottom of the inner side of the cultureware using poly-D-lysine

[100] The synthetic polymer, poly-D-lysine (PDL), can be used to coat the bottom of the inner side of the cultureware . Other synthetic polymers that can be used include but are not limited to poly-L-lysine (PLL) and poly-L-omithin (PLO), or polyetherimide (PEI). These synthetic polymers can be used interchangeably. Described herein is a method of coating the bottom of the inner side of the cultureware using a synthetic polymer (e.g., poly-D-lysine). Any suitable synthetic polymers can be used.

[101] An aliquot of frozen stock solution of poly-D-lysine (1 mg/ml) in pure water stored at - 20°C was retrieved from a freezer and allowed to thaw. The poly-D-lysine solution was then diluted in ultrapure water before the resulting diluted working solution was applied onto the inner side of the cultureware to be coated. The cultureware was allowed to incubate while surfaces were covered with the poly-D-lysine working solution for coating to take place. The coating solution was removed and the excess unbound poly-D-lysine was washed away by applying phosphate buffered saline (PBS) to the surface and removing the PBS. This step was repeated several times. If the poly-D-lysine coated cultureware was to be used immediately, the cells could be deposited at this stage. If the poly-D-lysine coated cultureware was to be used later, the inner side of the cultureware could be rinsed with deionized water before being stored at 4°C to avoid crystallization of salts from PBS.

[102] Example 2: Depositing the cells onto the bottom of the inner side of the cultureware coated with poly-D-lysine

[103] If the cells are deposited into a cultureware not coated with the synthetic polymer, the cells sediment to the bottom of the cultureware due to gravity and roll on the bottom until they secrete enough natural extracellular matrix proteins to coat the cultureware to allow their adhesion to the cultureware. The coating of the cultureware with synthetic polymers speeds up the initial adhesion of the cells to the cultureware.

[104] HEK cells were dispensed in suspension into the poly-D-lysine coated cultureware. After the cells were allowed to passively sediment to the bottom of the suspension to the poly-D-lysine coated inner side of the cultureware, the cells no longer moved independently as floating cells. They moved in unison once they were adhered to the bottom of the cultureware. When the side of the cultureware was lightly tapped, no cell continued to move with inertia as cells were now locally adhered to the surface of the inner side of the cultureware following sedimentation.

[105] This process of depositing the cells is termed herein as “passive adhesion” which does not involve the cell’s internal process.

[106] Cells per unit area under the experimental conditions described herein is about 5,000/mm². This number is dependent on the cell type as larger cells would be over-crowded at the density of 5,000/mm². This number is for standard size cells like Human Embryonic Kidney (HEK) or Chinese Hamster Ovary (CHO).

[107] Example 3: Application of hydrogel onto the cell layer

[108] Hydrogel is formed inside the cultureware in the presence of the adhered cells according to manufacturer instructions. QGel Hydrogel (from QGel SA, Lausanne, Switzerland) was used.

[109] The lyophilized monomer is mixed with a HEPES solution (0.3 M) until the monomer dissolves entirely. Culture medium is then added (1 part of culture medium to 4 parts of hydrogel monomer in HEPES solution). The polymerization reaction starts immediately and the QGel monomer solution is dispensed onto the adhered cells before it gels. The optically clear hydrogel can then be allowed to form around the cells at 37°C for one hour in a conformal way thereby locking the cells in position. The hydrogel is not biodegradable so the cells are limited in their movement.

[110] Example 4: Effect of temporal changes in osmolality on cellular morphology

[111] FIGs. 2 A and 2B show the effect of temporal changes in osmolality on cellular morphology. FIG. 2A shows the average area (pm²) measurement of HEK cells post exposure to carbon dioxide independent media + 10% FBS + gentamicin (carbon dioxide-independent medium supplemented with 10% FBS, abbreviated as CO2I10) for 1 to 8 days in vitro. FIG. 2B shows the average circularity measurements of cells post exposure to CO2I10 for 1 to 8 days in vitro. Circularity = 4 (area/peri meter²). As the value approaches 1.0, it is indicative of a perfect circle. As the value approaches 0, it indicates an increasingly elongated polygon. Media was prepared and conditioned separately in polystyrene 96-well plates for 8 days, filtered through a 0.22 pm pore size filter, and added to respective experimental wells and allowed to incubate for 30 minutes at 37°C, 5% CO2, and 95% humidity. For each condition, n > 20 cells/well were assayed for n = 3 wells (n > 60 cells per condition) Data is represented as mean +/- STD.

[112] Example 5: Protective effect of the microenvironment on cell-based assay stability

[113] FIG. 3 shows the response profile of biosensor (e.g., cells cultured using the disclosed methods) detecting 1 mM propylbenzene with a 2.5-fold increase of fluorescence following a 15- minute exposure compared to baseline fluorescence. Over 12 days at room temperature outside the laboratory setting, the propylbenzene response of cells maintained in the engineered microenvironment remains steady.

[114] At each time point registered on the graph, 1 mM propylbenzene was added in the cell environment. As the metabolite binds to the cell surface receptor, the receptor-mediated cellular changes indirectly lead to an intracellular increase in calcium ion concentration which are detected by the calcium indicator. Upon binding to the calcium, the indicator experiences changes in optical properties. By tracking the amount of 517 +/- 23 nm emitted green light under 480 +/- 17 nm excitation light, the amount of calcium in the cell can be estimated and the amount of propylbenzene that activates the cell surface receptor can be measured.

[115] Example 6: Effect of temporal changes in osmolality on cellular morphology

[116] FIG. 4 shows phase micrographs of HEK cells exposed to carbon dioxide independent media + 10% FBS (CO2I10) allowed to evaporate for different amounts of time hereby referred to as the different conditions. Cells were seeded at a density of 20,000 cells/well where they were given 24 hours at 37°C, 5% CO2_, 95% humidity to adopt the reference morphology. Cells were then exposed to conditioned media evaporated for 1 day and 8 days to assess the effect of osmolality. CO2I10 was prepared and conditioned separately in polystyrene 96-well plates for 8 days, sterilized by 0.22 pm filtration after collection, and 50 pL were added to respective experimental wells and allowed for 30 minutes at 37°C, 5% CO2_, 95% humidity.

[117] Example 7: Functional response profile of cells cultured from day 3 to day 12 in carbon dioxide-independent media [118] FIG. 5 shows HEK cells expressing a GPCR were challenged with propylbenzene and cytosolic calcium increase was measured via a genetically-encoded calcium indicator. The mean fluorescence intensity was acquired every minute for a total of 15 minutes and data are represented as a fold induction normalized to the baseline value prior to ligand challenge. By tracking the amount of 517 +/- 23 nm emitted green light under 480 +/- 17 nm excitation light, the amount of calcium in the cell can be estimated and the amount of propylbenzene that activates the cell surface receptor can be measured.

[119] Example 8: The influence of ambient air on pH of DMEM and carbon dioxide independent-media

[120] FIG. 6 shows in the control measurements, media was procured directly from commercial bottles. In the experimental conditions tested, DMEM and C02I were gas equilibrated to ambient air, n = 3 measurements were acquired. Data is represented as mean +/- STD and between group differences measured via a two-tailed t-test ***P < 0,0001.

[121] Example 9: Microenvironment conditions for maintaining functionality of adherent cells for 12 days at ambient temperature without the need for CO2 supplementation

[122] To identify the causes of failure of standard culture practices for long-term ambient temperature cell-based assays, adherent HEK cells were cultures in standard laboratory conditions using high glucose DMEM (Gibco Cat# 11965118) supplemented with 10% FBS. The cells were cultured using a humidified CO2 incubator. In the absence of a 5% excess of incubator CO2, dissolved buffering CO2 immediately degassed and the pH increased within minutes to toxic levels as shown in FIG. 8. The pH rapidly increased from pH 7.4 to pH 8.0. Next, the culture medium was replaced from DMEM to CCh-independent medium to remove the acute pH drift. After culturing the cells out of the incubator for 8 days, cell morphology was tracked daily and a gradual change was observed. As shown in FIGS. 3 and 2A, respectively, the cell morphology changed over time with a decrease in observed cell area from about 450 pm² to about 200 pm² as well as an increase in circularity from 0.45 to 0.90. These changes were not associated with a corresponding change in cell viability as assessed with an ATP assay. These findings show that gradual osmolarity changes were leading the cells to lose anchoring points and adopt a round morphology. The increase in cell circularity was associated with an increase in cytoplasmic Ca²⁺ concentrations indicative of cellular stress. Next, to assist with cell adhesion and limit evaporation, the substrate was pre-coated with polycationic polymers (e.g., poly-L-lysine) to limit evaporation by insulating the culture. As shown in FIG. 4, it was observed that cells retained their morphology over many days and cytoplasmic Ca²⁺ levels no longer increased as a function of time. The mean baseline cell GCaMP7s fluorescence intensity stayed constant over the course of 7 days at 15-20 arbitrary units of fluorescence (FIG. 9). GCaMP7 is genetically-encoded, high-performance GFP- based calcium reporter for imaging activity in cell populations and microcompartments. As shown in FIG. 10, it was observed that at the time assay a higher GCaMP7s fluorescence baseline was negatively correlated with cell assay responsiveness. The adherent cells were further locked in place by overlaying a thin layer of a custom non-degradable star PEG-based hydrogel (e.g., Qgel). FIG. 5 shows the functional response profile of cells cultured from day 3 to day 12 in carbon dioxide independent media, by limiting passive pH increase, osmolarity increase and assisting passive cell adhesion with the use of polycationic polymers. The results show a sustained cell assay performance over a 12 day period. The cells were excited with 418/17 nm and read at 517/23nm. The ZOE imager (BioRad) was used.

[123] Materials and Methods. A 96-well plate (Greiner Bio-One Cat# 655809) was pre-treated overnight at 37°C and 5% CO2 with 50 pL per well of Poly-D-lysine (Sigma Cat# P6407) at 75 ng/mL in UltraPure water (Invitrogen Cat# 10977023). After an initial coating step, the excess Poly-D-lysine coated microwells were rinsed twice with 100 pL ambient temperature PBS at pH7.4 (Gibco Cat#10010072) and once with lOOpL UltraPure water. Coated microwells were then seeded with HEK cells dissociated with Trypsin-EDTA 0.05% (Gibco Cat# 25300062) at a viability of >97% as assessed diluting cell suspension to an equal volume of 0.4% Trypan Blue (BioRad Cat# 1450021) and using automated cell counting with a TC20 (BioRad). After seeding, cells were allowed to sediment and adhere at 37°C under 5% CO2 for 24 hrs. After confirming adherent cell morphology, the overlaying medium was replaced with non-HEPES buffered CO2- independent medium (Gibco Cat# 18045088) supplemented with 10% FBS and gentamicin (Gibco Cat# 15710064). After replacing the culture medium, plates were sealed with gas impermeable foil and stored at ambient temperature outside of the CO2 incubator.

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[125] While the present invention is described in connection with what is presently considered to be the most practical and preferred embodiments, it should be appreciated that the invention is not limited to the disclosed embodiments, and is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the claims. Modifications and variation as defined in the claims. The appended claims should be construed broadly and in a manner consistent with the spirit and scope of the invention herein. It is understood that, given the above description of the embodiments of the invention, various modifications may be made by one skilled in the art. Such modifications are intended to be encompassed by the claims below.

Claims

CLAIMS What is claimed is:

1. A cell culture system for culturing cells at ambient temperature, the system comprising: a) a cultureware for culturing the cells, wherein the cultureware having a top, a bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and can be tightly sealed; b) cells adhered to the bottom of the cultureware, wherein the bottom of the cultureware is optically transparent; c) a layer of non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and d) a cell culture medium covering the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture.

2. The cell culture system of claim 1, wherein the cells are animal cells.

3. The cell culture system of claim 1, wherein the cells are mammalian cells.

4. The cell culture system of claim 1, wherein the cells are human embryonic kidney cells.

5. The cell culture system of claim 1, wherein the cells are fish cells.

6. The cell culture system of claim 1, wherein the cells are capable of detecting a target molecule in a cell-based assay.

7. The cell culture system of claim 6, wherein the cells comprise a surface G protein-coupled receptor, an intracellular G-protein and an intracellular reporter.

8. The cell culture system of claim 7, wherein the G protein-coupled receptor, the G protein or the reporter is endogenous or genetically engineered to be expressed by the cells.

9. The cell culture system of claim 6, wherein the target molecule is a product of a living organism.

10 The cell culture system of claim 6, wherein the target molecule is a volatile organic compound.

11. The cell culture system of claim 1, wherein the cells are primary cells.

12. The cell culture system of claim 1, wherein the cells are derived from a cell line.

13. The cell culture system of claim 1, wherein the cells are prone to apoptosis.

14. The cell culture system of claim 1, wherein the gas impermeability of the cultureware is achieved by covering the outer side of the cultureware with a gas impermeable film to provide a gas impermeable seal to the cultureware.

15. The cell culture system of claim 1, wherein the cultureware has a single chamber.

16. The cell culture system of claim 1, wherein the cultureware contains a plurality of chambers.

17. The cell culture system of claim 16, wherein the cultureware has a plurality of chambers in the form of wells with the top of the cultureware open and wherein the cultureware is entirely sealed on the outer side with a gas impermeable film to provide gas impermeable chambers.

18. The cell culture system of claim 17, wherein the gas impermeable film is light insulating.

19. The cell culture medium of claim 18, wherein the gas impermeable film is an aluminum film.

20. The cell culture system of claim 1, wherein the bottom of the inner side of the cultureware having a positive surface electrostatic charge to allow passive adhesion of the cells to the bottom of the cultureware.

21. The cell culture system of claim 20, wherein the positive surface electrostatic charge is provided by a coating of a synthetic polymer with a positive surface electrostatic charge.

22. The cell culture system of claim 21, wherein the synthetic polymer is poly-D-lysine, poly- L-lysine, poly-D-omithin or polyetherimide.

23. The cell culture system of claim 22, wherein the synthetic polymer is poly-D-lysine.

24. The cell culture system of claim 1, wherein the non-biodegradable cytocompatible hydrogel is optically clear.

25. The cell culture system of claim 1, wherein the buffer is MOPS. HEPES, MES, BICINE or phosphate.

26. The cell culture system of claim 1, wherein the high buffering capacity of the cell culture medium is achieved by having an excessive amount of the medium.

27. The cell culture system of any one of claims 1-26, wherein the cells remain viable and adhered to the bottom of the cultureware for at least 3 days without supplemental carbon dioxide.

28. The cell culture system of claim 27, wherein the cells remain viable for at least 3 to 20 days.

29. A cell culture system for culturing cells used in a cell-based assay at ambient temperature, the system comprising: a) a cultureware for culturing the cells, wherein the cultureware having a top, a bottom, an inner side and an outer side, wherein the cultureware is gas impermeable and can be tightly sealed, and wherein the bottom of the cultureware is optically transparent; b) a coating of synthetic polymer at the bottom of the inner side of the cultureware wherein the synthetic polymer having a positive surface electrostatic charge to allow passive adhesion of the cells; c) cells adhered to the bottom of the cultureware on top of the coating of synthetic polymer, wherein the cells having a surface G protein-coupled receptor, an intracellular G protein and an intracellular reporter; d) a layer of optically clear non-biodegradable cytocompatible hydrogel around the cells locking the cells in position within the hydrogel; and e) a cell culture medium covering the cells, wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture.

30. The cell culture system of claim 29, wherein the G protein-coupled receptor, the G protein or the reporter is naturally existing in the cells or expressed by the cells through genetic engineering and wherein the G protein-coupled receptor, the G protein or the reporter is natural or synthetic.

31. A method of culturing cells at ambient temperature, the method comprising: a) depositing cells in a cultureware, wherein the cultureware has a top, a bottom, an inner side and an outer side and is gas impermeable and wherein the bottom of the inner side of the cultureware has a coating of a synthetic polymer, wherein the cells passively adhere to the bottom of the inner side of the cultureware on the coating of synthetic polymer in the absence of carbon dioxide; b) adding non-biodegradable cytocompatible hydrogel around the adhered cells, wherein the cells are immobile within the hydrogel; c) adding a cell culture medium to the cells in step b), wherein the cell culture medium is buffered by a high buffering capacity carbon dioxide-independent buffer to maintain a pH of from about 7 to about 8 in the medium throughout the culture; and d) culturing the cells at ambient temperature without incubating the cells at 37°C and 5% C0_2.

32. The method of claim 31, wherein the cultureware has a single chamber or has a plurality of wells and wherein the cultureware with a plurality of wells having the top open.

33. The method of claim 31, further comprising sealing the cultureware with a gas impermeable film.

34. The method of claim 33, wherein the gas impermeable film is an aluminum film.

35. The method of claim 31, wherein the coating of the synthetic polymer provides a surface electrostatic charge allowing passive adhesion of the cells to the bottom of the cultureware.

36. The method of claim 31, wherein the synthetic polymer is poly-D-lysine, poly-L-lysine, poly-D-omithin or polyetherimide .

37. The method of claim 36, wherein the synthetic polymer is poly-D-lysine.

38. The method of claim 31, wherein the ambient temperature is between 10°C and 35°C.

39. The method of claim 31, wherein the ambient temperature is between 15°C and 30°C.

40. The method of claim 31, wherein the ambient temperature is between 20°C and 25°C.

41. The method of claim 31, wherein the cells are animal cells, human cells, plant cells or insect cells.

42. The method of claim 41, wherein the animal cells are fish cells.

43. The method of claim 31, wherein the cells are adherent for at least 12 days at ambient temperature without CO2 supplementation.

44. A method of detecting one or more volatile organic molecules in a fluid sample, the method comprising: a) contacting a cell culture with a fluid sample, wherein the fluid sample comprises one or more volatile organic molecules, wherein the cell culture comprises cells in a cultureware having a top, an optically clear bottom, an inner side and an outer side and is gas impermeable, wherein the cells are adhered to the bottom of the inner side of the cultureware, are functional at room temperature, and present in a cell culture medium that is buffered by a high buffering capacity carbon dioxide-independent buffer; wherein the cells comprise one or more G-protein coupled receptors capable of binding to the one or more volatile organic molecules, wherein the one or more G- protein coupled receptors comprises a reporter; b) exposing the cell culture to a light; and c) detecting the presence of a fluorescence emitted by the reporter after binding of the one or more G-protein coupled receptors to the one or more volatile organic molecules, an increase in brightness of the fluorescence, or a combination thereof.

45. The method of claim 44, wherein the cells in the cultureware are non-mammalian cells.

46. The method of claim 45, wherein the non-mammalian cells are fish cells.

47. The method of claim 44, wherein the fluid sample is a gaseous sample from an exhaled breath of a mammalian subject.

48. The method of any of claims 44-47, wherein the cells are maintained in the culture for at least 3 days in an uncontrolled temperature environment and without supplemental carbon dioxide.

49. A cell culture system for culturing cells at a temperature from about 15°C to about 30°C, the system comprising: a) a cultureware for culturing the cells, wherein the cultureware comprises a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware is gas impermeable and wherein the cultureware has at least one access region to add or remove contents within the cultureware, and wherein the access region is sealable to be gas impermeable; b) cells adhered to the bottom of the cultureware; c) a layer of non-biodegradable cytocompatible hydrogel wherein the hyrdogel sufficiently surrounds the adhered cells to maintain the cells in a position within the hydrogel; and d) a cell culture medium covering the adhered cells, wherein the cell culture medium is buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide.

50. A method of culturing anchorage dependent cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients and wherein said cells remain adhered to a cultureware surface of said culture device for at least 3 days, said method comprising: a) maintaining cells in a cell culture device, wherein the cell culture device comprises a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware of said cell culture device is gas impermeable and wherein the cell culture device has at least one access region to add or remove contents within the cell culture device, and wherein the access region is sealable to be gas impermeable; b) maintaining the cells adhered to the bottom of the cultureware for at least 3 days; and wherein a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel; and c) covering the cells in the culture device with a culture medium buffered to maintain a pH of from about 7 to about 8 in in the absence of supplemental carbon dioxide and wherein the cells remain viable in said device for at least 3 days.

51. A cell culture device for culturing anchorage dependent, target molecule detecting cells in a sealable culture device in the absence of controlled temperature and supplemental carbon dioxide or nutrients, and wherein said cells remain adhered to a cultureware surface of said culture device for at least 3 days, said cell culture device comprising: a) a top, an optically transparent bottom, an inner side and an outer side and wherein the cultureware of said cell culture device is gas impermeable and wherein the cell culture device has at least one access region to add or remove contents within the cell culture device, and wherein the access region is sealable to be gas impermeable; b) target molecule detecting cells adhered to the bottom of the cultureware for at least 3 days; and wherein a layer of non-biodegradable cytocompatible hydrogel sufficiently surrounds the cells to maintain the cells in a position within the hydrogel; and c) a culture medium buffered to maintain a pH of from about 7 to about 8 in the absence of supplemental carbon dioxide and wherein the culture medium is present in the device in an amount sufficient for the cells to remain viable in said device for at least 3 days in the absence of controlled temperature or supplemental carbon dioxide; wherein said cells comprise a surface G protein-coupled receptor, an intracellular G-protein and an intracellular reporter that provides a detectable signal when said cells contact the target molecule.

52. The cell culture system of any of claims 1 to 30 or 49, the method of any of claims 31 to 48 or 50, or the cell culture device of claim 51, wherein the cells are HEK cells.