RU2260046C2 - Cellular microarray and using thereof in living cell investigations - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: claimed microarray represents ensemble of gel microcells on substrate made of glass, polymer, ceramic or composite material. Microcells contain immobilized procariotic or eucariotic cells. Microcells with immobilized cells are prepared using gel-forming solution including glycerol. Cellular microarray is used in living cell investigation. In this purpose cellular microarray is incubated in presence of marker, signal (e.g. cell fluorescence) is detected and according to signal level cell living function in microarray is evaluated.

EFFECT: simplified living cell investigation method without losses of cell viability.

12 cl, 5 dwg, 3 ex

Description

Technical field

The invention relates to the field of molecular biology, microbiology and biotechnology and relates to a method for the manufacture of microchips based on cells immobilized in its elements. Such microchips can be used in molecular and cellular biology, biotechnology, microbiology, in determining the antimicrobial activity of antibiotic drugs in experimental pharmacology, in environmental studies, in medicine, and for other applications. A method for manufacturing a microchip is described, in the gel elements of which prokaryotic or eukaryotic cells are immobilized. A microchip is made by polymerizing a gelling solution containing immobilized cells. Moreover, gelling solutions before polymerization are applied to the substrate in the form of microdrops.

State of the art

A number of methods are known for analyzing hereditary traits, patterns of growth and development, and other properties of prokaryotic and eukaryotic cells. In particular, there is a group of methods for monitoring the vital functions of cells under the influence of various factors on them.

To obtain data on the development of a bacterial population, the most common methods are:

1. registration of the optical density of the bacterial suspension;

2. colony counting on solid medium [C.E. Clifton. Introduction to bacterial physiology. 1957];

3. counting the number of cells in a Petroff-Hauser chamber [C.E. Clifton. Introduction to bacterial physiology. 1957];

4. flow cytometry [H.B.Steen. Flow cytometry of bacteria: glimpses from the past with a view to the future. J. of Microbiological Methods 42 (2000) 65-74].

However, methods 1,2 and 3 are rather laborious, require significant researcher participation, often do not allow parallel analysis of several samples, have low potential for automation, therefore, are time and cost consuming. Flow cytometry is a highly automated method that allows you to quickly obtain data with minimal effort from the researcher. However, in addition to the fact that flow cytometers and consumables are expensive, this method is designed more for multi-parameter screening than for observing one or more parameters in dynamics.

Cells, as multicomponent biocatalytic systems, are used in biosensors for environmental analysis [Simonian A.L. et al. A biosensor for L-proline determination by use of immobilized microbial cells. / Appl. Biochem, Biotechnol. 1992 Sep; 36 (3): 199-210; Rainina E.I. The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins. / Biosens. Bioelectron 1996; 11 (10): 991-1000]. However, methods using such biosensors make it possible to simultaneously determine an insignificant number (often one) of environmental pollutants. Enzyme-based biosensors [Simonian A.L. et al. A tryptophan-2-monooxygenase based amperometric biosensor for L-tryptophan determination: use of a competitive inhibitor as a tool for selectivity increase. / Biosens. Bioelectron 1997; 12 (5): 363-71] for the most part have the same drawback.

A method for determining the activity of antimicrobial agents in the domestic pharmacopeia is the method of diffusion of antibiotics into agar by comparing the sizes of zones of inhibition of growth of test microbes [State Pharmacopoeia of the USSR: Issue 2. General methods of analysis. Medicinal plant material / Ministry of Health of the USSR. - M .: Medicine, 1989]. It has the drawbacks mentioned above for methods 1, 2, and 3.

A well-known method for detecting the sensitivity of microorganisms to antibiotics - the method of serial dilution - consists in recording the presence (or absence) of culture growth in a number of tubes with a liquid nutrient medium containing a series of antibiotic concentrations. [Bryan L.E. Bacterial resistance and susceptibility to chemotherapeutic agents. Cambridge University press. Cambridge, London, New York, New Rochelle, Melbourne, Sydney, 1982]. Despite the high information content, this method has not found wide application in laboratory diagnostics for the combination of the following disadvantages:

- requires a test separately for each studied culture;

- requires a large number of consumables (test tubes, dies, antibiotics, etc.);

- the result of the analysis can be obtained after 18-20 hours.

In addition, existing routine methods for determining bacterial resistance to antibiotics have the common drawbacks noted above for methods 1,2 and 3.

The identification of the activity of cytostatic and other pharmaceutical preparations in relation to animal cells at the stages of development, production (current control) and application (individual selection) is carried out using the following methods: MTT test [Carmichael, J .. et al. (1987) Cancer Research 47: 936}, flow cytometry, ArrayScan ™ cytometry. One of the serious drawbacks of the MTT test is the high frequency (up to 30%) of unsuccessful experiments, the results of which cannot be correctly interpreted. The disadvantage of the other two methods is the high cost of equipment and supplies.

Despite the existence of a large number of methods for analyzing the properties of cells and studying various environmental objects using cells, the need to develop new, more advanced ones remains.

The objective of the invention is to develop a method that would allow:

1. simplify the analysis of signs, patterns of growth and development and other properties of prokaryotic and eukaryotic cells;

2. to simplify the monitoring of cell activity under the influence of various factors on them;

3. expand the capabilities of analysis of environmental objects (water, soil, etc.) for the presence of certain chemicals in them;

4. simplify the determination of the activity of antibiotic, cytostatic and other pharmaceutical preparations in relation to prokaryotic and eukaryotic cells; which will lead to a decrease in the cost of the described studies, a reduction in the time of their carrying out and labor input.

We see the solution of the tasks in the application of the technology of biological microchips. Namely, a biological microchip based on cells (or the so-called cell microchip). Currently known methods of manufacturing biological microarrays based on DNA [RF patent No. 2157385] and proteins [Arenkov P., Kukhtin A., Gemmell A., Voloshchuk S., Chupeeva V., Mirzabekov A. Protein Microchips: Use for Immunoassay and Enzymatic Reactions. Analytical Biochemistry 278, 123-131 (2000)]. However, the exact transfer of the existing technology for their manufacture to create a cell microchip is not possible, because the above technology involves the use of processes that are detrimental to eukaryotes and most prokaryotes. This is polymerization under the influence of ultraviolet radiation, contact with an organic solvent, etc.

The immobilization of cells in various gels is widely reported in the scientific literature. Methods of immobilization of prokaryotic and eukaryotic cells in agarose, alginate [D. Roger, A. David, H. David (1996) Plant Physiol. 112: 1191-1199], acrylamide and other gels [N.S. Egorov, N.S. Landau, E.A. Borman, I. B. Kotova (1984) Applied Biochemistry and Microbiology, vol. XX, issue 5 , p. 579-592]. Almost any kind of cell can be immobilized [D. Roger, A. David, H. David (1996) Plant Physiol. 112: 1191-1199].

Thus, the tasks are solved as a result of using a cell microchip in the research method.

SUMMARY OF THE INVENTION

One aspect of the invention is a cellular microchip, which is a substrate containing gel elements in which cells are immobilized. The base of the gel elements is selected from the group consisting of acrylamide, alginate, carrageenan, collagen, fibrin, agar, agarose, gelatin, polyvinyl alcohol, silicic acid derivatives (e.g., silica gel), etc. The gel element of the cell microchip has a pore size sufficient for the penetration of nutrients and substances, the interaction of which with the cells must be investigated. The substrate material of the cell microchip is selected from the group consisting of glass of various grades, metals, plastic materials, ceramic materials, polymer carriers (polymethyl methacrylate, polystyrene, polypropylene, etc.), any composite materials, etc. The substrate is a plate made and processed in such a way as to hold the gel elements of the microchip on its surface. The cell microchip substrate may also be a porous plate made and processed in such a way as to retain the gel elements of the microchip in its volume. Cells immobilized in the elements of the cell microchip can be representatives of both eukaryotes and prokaryotes. A cell microchip can be used in the field of creating biosensors, testing nutrient media, determining the activity of antibacterial and cytostatic drugs, for monitoring the development of a population of microorganisms under the influence of various factors, identifying the antibiotic sensitivity of microorganisms, screening clones for creating genomic libraries, gene diagnostics of infections of various nature, as well as other applications.

The invention also proposes a method for studying living cells on a cellular microchip, including incubating the microchip, recording a fluorescence signal due to the property being studied, followed by its evaluation in comparison with the control.

The method involves the use of a cellular microchip disclosed in this invention.

The method assumes that the basis of the gel element of the cell microarray is selected from the group consisting of acrylamide, alginate, carrageenan, collagen, fibrin, agar, agarose, gelatin, polyvinyl alcohol, silicic acid derivatives (e.g., silica gel), etc.

In addition, the method uses a microchip in which the gel element has a pore size sufficient for the penetration of nutrients and substances, the interaction of which with the cells must be investigated.

Further, in the method, the substrate material of the cell microchip is selected from the group consisting of glass of various grades, metals, ceramic materials, polymer carriers (polymethyl methacrylate, polystyrene, polypropylene, etc.), any composite materials, etc., and the substrate a cell microchip is a plate made and processed so as to hold the gel elements of the microchip on its surface, or the substrate of the cell microchip is a porous plate made and processed by kim way to keep in volume gel elements of the microchip.

The method is characterized in that the cells to be immobilized are selected from the group of eukaryotes or from the group of prokaryotes, the cells being not necessarily in a lyophilized state.

In addition, the invention discloses a method of manufacturing a microchip based on cells immobilized in a gel prepared by polymerizing a gel-forming solution containing immobilized cells. Moreover, gel-forming solutions containing a suspension of cells are applied to the substrate in the form of microdrops, after which polymerization is carried out.

List of figures

The figure 1 schematically presents the incubation chamber and the numbers denote: 1 - plugs; 2 - quartz glass; 3 - gasket; 4 - microchip.

The figure 2 graphically shows the kinetics of signal growth from cells of a bacterial microchip incubated in LB medium with ampicillin (50 μg / ml), and where the curves are indicated by numbers: 1 - curve from cells with E. coli resistant to ampicillin; 2 and 3 - curves from cells with E. coli sensitive to ampicillin.

The figure 3 presents the result of incubation of an acrylamide microchip in a medium with an antibiotic.

The figure 4 presents the results of incubation of the bacterial microchip in an environment with an antibiotic, where the numbers 1 and 2 indicate sensitive strains, and the numbers 3 and 4 - resistant strains.

The figure 5 shows the gel elements of the microchip after incubation in a nutrient medium with an antibiotic (A is a cell with a sensitive culture, and B is a cell with a stable culture).

Detailed Disclosure of Invention

1. The surface of the substrate is pre-treated so that the microdroplets of the gel after application and polymerization are firmly held on it.

2. Prepare a series of gelling solutions containing the appropriate culture of microorganisms.

3. The application procedure is carried out manually, by an automatic microdoser or otherwise. The gel microdroplets of the same volume are applied from each other at a distance equal to 1/2 of their diameter or greater.

4. The polymerization reaction is carried out properly after application.

5. The fabricated microchip can be used immediately or canned.

A. An example of short-term preservation is the storage of a microchip in a sterile isotonic solution at 4 ° C.

B. In the case of long-term storage of a microchip based on prokaryotes or yeast, freeze-drying or cryopreservation at -50 ° C, -70 ° C of the finished microchip can be used, or the use of lyophilized microorganisms at the stage of manufacture of the microchip.

The use of polyacrylamide gel in the manufacture of microchips based on animal cells is unacceptable, because causes the death of 100% of the cells. Alginate seems to be the most suitable for this purpose. The problem of fixing alginate gel elements to the surface of the substrate is solved as follows. A thin layer (5-10 μm) of polyacrylamide gel (similar to [Arenkov P., Kukhtin A., Gemmell A., Voloshchuk S., Chupeeva V., Mirzabekov A. Protein Microchips: Use) forms on the surface of Bind-Silane-treated glass. for Immunoassay and Enzymatic Reactions. Analytical Biochemistry 278, 123-131 (2000)], but instead of a mask, transparent quartz glass is used) onto which microdroplets of alginate with cells are applied and polymerized in CaCl ₂ . In this case, the gel elements are firmly fixed to the substrate. A more technological solution is the application of microdrops on a chemically modified glass surface. For specialists in the field of chemistry, it will not be difficult to find a way to chemically sew alginate to the glass surface.

The application of gelling solutions is carried out as described in the patent of the Russian Federation No. 2157385. It is preferable to use a feeding device for applying the solution in the form of microdrops, which allows to distribute at high speed the microvolumes of a large number of solutions on a flat surface.

The proposed method allows in one step to apply a microchip with a high density of matrix elements, which is necessary for parallel manipulations with a large number of cells. The proposed method makes it possible to use cell microarrays as a biosensor for analyzing environmental elements (water, soil, etc.) for the presence of a wide range of pollutants in them, monitoring the development of the microorganism population under the influence of various factors on them, and screening for clones genomic libraries, diagnosis of infections of various nature, testing the activity of antimicrobial and cytostatic agents, the effect of drugs on the intestinal microflora (in case of immobilization tion representatives of the intestinal microflora in the microchip elements), isolated from a patient resistance to antibiotics crops, etc.

As a substrate for a microchip, a slide can be used. It is treated with a reagent that modifies the surface of the glass with unsaturated groups. Such a reagent may be, for example, H- [3- (triethoxysilyl) propyl] acrylamide, allyl triethoxysilane and others. For the manufacture of a cell microchip based on bacteria or yeast, it is preferable to use acrylamide as one of the monomers.

The following are examples of the manufacture and use of cell microarrays based on E. coli. The description of these examples should not be used to limit the claims of this patent, it only illustrates the possibility for specialists in this field to implement the invention.

To demonstrate the fundamental possibility of studying the properties of cells using a microchip, model experiments were performed to determine the antibiotic resistance of various E. coli strains.

Example 1

The following strains are used in the experiment: JM-109; DH5a, pBR 322, amp ^r ; BL21 (DE3) pLysS, chm ^r ; BL21 (DE3) pLysS, pET23b, chm ^r amp ^r . A gelling mixture of the following composition is prepared: 5% acrylamide-N, N'-methylene bisacrylamide 19: 1, 1 × PBS (pH 7.2), 60% glycerol, 0.5% ammonium persulfate and 10% (v / v) night suspension culture of E. coli (reduced to optical density OD ₆₀₀ = 2 OE) in a nutrient medium LB. Application to the glass surface is carried out manually or on a robot (Arrayer GMS 417, Genetic Microsystems) with a pin ⌀500 or 375 μm. Respectively. Glass is pretreated with Bind-Silane. The formed matrix of solutions is placed in a vessel of known volume and TEMED is added to it at the rate of 0.1-1 μl / 1 dm ³ of vessel volume.

Stand for 3 minutes. The obtained microchip is placed in an incubation chamber (Fig. 1), which is filled with LB liquid nutrient medium containing one of the antibiotics and a nucleic acid marker in SYTO 9 living cells. During the experiment, the temperature in the chamber is maintained at 37 ° C and every 5 min a fluorescent signal from the microchip cells is recorded. The results are reflected in the form of curves of I _norms (t) (figure 2), where I _norms - normalized intensity of the fluorescent signal. There is a bright signal from cells containing strains resistant to the antibiotic of bacteria, and a signal close to the background from cells with strains of sensitive bacteria, which is reflected in figure 3.

A characteristic exponential growth of the signal occurs as a result of the multiplication of antibiotic-resistant bacteria in the microchip cells and their accumulation of the fluorescent dye contained in the nutrient medium (curve 1 in FIG. 2). In this case, separate colonies are formed in the gel cell. The signal from cells with antibiotic-sensitive microorganisms practically does not increase (curves 2,3 in FIG. 2). Figure 3 presents the image of the microchip at the end of the experiment. To register the signal, equipment consisting of a luminescent microscope, a CCD camera and a personal computer with the appropriate software is used [1. A-D. Mirzabekov (1992) Bioorganic chemistry Vol. 32, No. 10-11, pp. 1361-1374; 2. A.D. Mirzabekov (1994) Tibtech January (vol. 12)]. This example shows the possibility of using acrylamide gel for the manufacture of a microchip based on prokaryotes.

Example 2

Microdroplets of a gel-forming mixture at 37 ° C and containing 3% low-melting agarose, 25% glycerol, and 5% (v / v) suspension of E. coli overnight culture were applied to a glass substrate coated with a thin layer (20 μm) of polyacrylamide gel and cooled up to 0 ° С. In this case, agarose gel elements were formed that were firmly attached to the substrate. The further course of the experiment is similar to that described in Example 1. The results are shown in figure 4. This example illustrates the possibility of using agarose as a gel base for microchip elements.

Example 3

The example given illustrates the possibility of using a cell microchip as an alternative to the serial dilution method [Bryan L.E. Bacterial resistance and susceptibility to chemotherapeutic agents. Cambridge University press. Cambridge, London, New York, New Rochelle, Melbourne, Sydney, 1982].

A gelling mixture of the following composition is prepared: 5% acrylamide-N, N'-methylenebisacrylamide 19: 1, 1 × PBS (pH 7.2), 60% glycerol, 0.5% ammonium persulfate. Bacterial cultures isolated from patients are pre-grown on solid medium. All colonies of interest are selected by loop and introduced into separate tubes with a gel-forming solution, carefully suspended. An automatic microdoser selects 0.1-0.2 μl from each tube and, in a specific order, is applied to a Bind-Silane-treated glass slide. Polymerized in TEMEDa vapors (see Example 1). The number of manufactured microchips is determined by the number of antibiotics, the sensitivity of which must be checked and the number of dilutions of these antibiotics. A drop (covering all elements) of LB liquid medium containing a certain concentration of one of the antibiotics is applied to each microchip. Microchips are placed in a humid chamber and incubated for 3-4 hours at 37 ° C. The registration of the results is as follows. The microchips are washed with water, placed under a microscope (400 ^x ) and the presence of colonies in each cell of the microchip is evaluated (Fig. 5A). The absence of colonies in the cell (Fig.5B) indicates the effectiveness of this antibiotic at a given concentration in relation to this culture.

As follows from the description of the application, specialists in this field can easily find improvements to the proposed invention, which also fall within its scope, which is reflected in the claims.

Sources and publications listed in the description are an integral part of it, as if all their contents were included in the description.

Claims (12)

1. A cell microchip, which is a set of gel microcells on a substrate, inside which are immobilized living cells, while microcells with immobilized living cells are formed by applying microdrops of a gel-forming solution containing glycerin to the substrate. 2. The cell microchip according to claim 1, characterized in that the gel-forming solution is prepared on the basis of acrylates, in particular acrylamide, alginate, carrageenan, collagen, fibrin, agar, agarose, gelatin, polyvinyl alcohol. 3. The cell microchip according to claim 1, characterized in that the gel microcells have a pore size that ensures the penetration of the necessary substances to the immobilized cells. 4. The cell microchip according to claim 1, characterized in that the substrate is a glass, metal, ceramic material, a polymeric carrier or a composite material. 5. The cell microchip according to claim 4, characterized in that the polymer carrier is polymethyl methacrylate, polystyrene or polypropylene. 6. The cell microchip according to claim 1, characterized in that the substrate is a plate chemically modified to fix gel microcells on its surface. 7. The cell microchip according to claim 1, characterized in that the cells to be immobilized are eukaryotic or prokaryotic cells, 8. The cell microchip according to any one of claims 1 to 7, characterized in that it is used for the manufacture of biosensors, testing nutrient media; determining the activity of antibacterial drugs, monitoring the development of a population of microorganisms under the influence of various factors on them. 9. A method for studying living cells, including incubating a cell microchip according to any one of claims 1 to 8 in the presence of a marker, registering a signal due to the studied property of the cells, and evaluating the received signal. 10. The method according to claim 9, characterized in that the marker is a fluorescent marker, and the recorded signal is due to the fluorescence of microchip cells. 11. The method according to claim 9, characterized in that the recorded signal is due to the fluorescence of proteins expressed by microchip cells. 12. The method according to claim 9, characterized in that the results are evaluated visually or using software.

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