KR102146467B1 - Micro gravity culture apparatus - Google Patents

Abstract

The present invention relates to a microgravity culture apparatus and method, and more particularly, in a microgravity tissue culture apparatus, by culturing cells in a completely weightless state, in culturing cells or tissues, by more completely reproducing the zero gravity state It relates to a method and apparatus for culturing. The microgravity culture device according to an embodiment of the present invention is a gravity generating device that artificially generates gravity to form a zero-gravity environment, a culture section composed of a membrane membrane and filled with a culture solution, and fixing the culture section and generating the gravity. Disclosed is a configuration including a connection part connected to the device and a support body on which cells are attached and cultured, which are included so as to be suspended in a culture solution inside the culture part.

Description

Micro-gravity culture device {MICRO GRAVITY CULTURE APPARATUS}

The present invention relates to a microgravity culture apparatus and method, and more particularly, in a microgravity tissue culture apparatus, by culturing cells in a completely weightless state, in culturing cells or tissues, by more completely reproducing the zero gravity state It relates to a method and apparatus for culturing.

Humans are exposed to various natural environments. There are not only differences in temperature such as hot or cold, but also humidity, jet lag, elevation, and underwater, and recently, air conditions are also included in the variables of the natural environment. Among these diverse environments, gravity is expected to affect almost all biological systems, and this mechanism has not yet been fully elucidated. Therefore, research on the effect of gravity on biological systems is a key topic, and many papers have been experimented using microgravity recently, and various types of systems have been developed to mimic microgravity on the Earth's surface. . Among them, 3D clinostat has been commonly used in experiments in the field of life science. This Clinostat equipment has two axes, and it is possible to set the environment of various conditions by applying a constant RPM, and it can set the environment of various conditions rather than one condition.

The biggest problem in the treatment of cancer is not death by early tumors, but more important by metastasis of cancer cells. Therefore, many studies have been conducted on changes in proliferation and migration of cancer cells in order to devise new strategies for cancer treatment and to effectively inhibit metastasis. An important part of cancer cell invasion and migration is the degradation of proteins in the extracellular matrix (ECM) and basement membrane, which cancer cells cannot normally pass through. Cancer cells secrete many types of proteolytic enzymes to degrade these protein membranes. In particular, there is a matrix metalloproteinase (MMP), which is important for decomposing the main components of the extracellular matrix and the basement membrane. Therefore, MMP inhibitors are focusing their research as a new target that can effectively prevent the proliferation and migration of cancer. In a recent study, the expression and activation of MMP, which is important for cancer metastasis, and the change of tissue inhibitor of metalloproteinase (TIMP), an inhibitor of MMP, were investigated in human lung cancer cells using culture bag and clinostet equipment. Check and study how microgravity affects cancer cell metastasis.

However, the conventional microgravity cell culture apparatus (DCC plate) has the disadvantage of not knowing the exact effect of gravity because it cannot create a perfect weightless state because cells are cultivated by attaching and growing cells to the membrane membrane ( Biological Sciences in Space, Vol. 28, 1-5, 2014).

Therefore, it is necessary to create a perfect zero-gravity state in order to know the effect of gravity on cells.

Accordingly, an object of the present invention is to provide a microgravity culture apparatus capable of providing a more complete zero gravity environment.

The microgravity cell culture apparatus according to an embodiment of the present invention for solving the above problem is a gravity generating device that generates artificial gravity to form a zero-gravity environment, a culture unit composed of a membrane membrane and filled with a culture solution, the It may include a connection part for fixing the culture part and connecting to the gravity generating device, and a support body which is included so as to be suspended in a culture liquid inside the culture part, and in which cells are attached and cultured.

According to an embodiment of the present invention, the support may have a disk shape.

According to an embodiment of the present invention, the support may be a three-dimensional structure.

According to an embodiment of the present invention, the gravity generating device,

According to an embodiment of the present invention, the gravity generating device may artificially generate gravity by rotating the culture unit to form a zero gravity environment.

According to an embodiment of the present invention, it further includes an exchange tube for exchanging material with the inside of the culture part, the connection part has a tube formed therein, and the exchange tube may be inserted into the inner tube of the connection part.

According to an embodiment of the present invention, a sealing portion for sealing the entrance of the culture portion so that the culture medium of the culture portion does not escape; may further include.

According to an embodiment of the present invention, the connection part may adjust the tilt of the rotation axis of the culture part.

According to an embodiment of the present invention, the gravity generating device may further include a gravity sensor capable of detecting a direction and a size in which gravity is applied to the culture unit.

According to an embodiment of the present invention, the artificial gravity generated in the culture section and the inclination of the culture section may be adjusted according to the direction and magnitude of the gravity detected by the gravity sensor.

According to an embodiment of the present invention, the microgravity culture device may be a cell culture for 24 hours or more.

According to the present invention, cells can be cultured in a more complete zero gravity environment than in a microgravity cell culture apparatus.

In addition, it is possible to provide a device capable of forming a zero gravity environment in response to changes in the direction and size of gravity during cell culture by adjusting the inclination of the culture unit.

Meanwhile, the effects of the present invention are not limited to the above-mentioned effects, and various effects may be included within a range that will be apparent to a person skilled in the art from the contents to be described below.

1 is an example of a conventional cell culture device.
2 is an example of a culture unit of an existing cell culture device.
3 is a perspective view of a cell culture apparatus according to an embodiment of the present invention.
4 is an example of a cell culture apparatus according to an embodiment of the present invention.
5 is a perspective view of a culture unit according to an embodiment of the present invention.
6 is an example of a support included in the culture unit according to an embodiment of the present invention.
7 and 8 are results of comparing the cell growth according to the cell culture device according to an embodiment of the present invention and the conventional device.
9 to 11 are graphs of results of confirming cell growth according to the cell culture apparatus according to an embodiment of the present invention and the existing apparatus at the RNA level.

Hereinafter, a'fine gravity culture apparatus and method' according to the present invention will be described in detail with reference to the accompanying drawings. The described embodiments are provided so that those skilled in the art can easily understand the technical idea of the present invention, and the present invention is not limited thereto. In addition, matters expressed in the accompanying drawings are schematic drawings for easy explanation of embodiments of the present invention and may be different from the actual implementation form.

Meanwhile, each component expressed below is only an example for implementing the present invention. Accordingly, in other implementations of the present invention, other components may be used without departing from the spirit and scope of the present invention.

In addition, each component may be implemented with purely hardware or software, but may be implemented with a combination of various hardware and software components that perform the same function. In addition, two or more components may be implemented together by one piece of hardware or software.

In addition, the expression'including' certain elements is an expression of'open type' and simply refers to the existence of the corresponding elements, and should not be understood as excluding additional elements.

1 is an example of a conventional cell culture device.

Referring to FIG. 1, in the conventional cell culture apparatus, the cells were cultured by filling the culture solution in a culture unit made of a hard material and attaching the cells to the outer wall. The culture unit is capable of exchanging substances with the outside, and the cells are fixed to be cultivated stably, but there is a disadvantage in that the cells are fixed outside the culture unit even if a zero gravity environment is created, so that a zero gravity environment cannot be completely configured.

2 is an example of a culture unit of an existing cell culture device.

Referring to FIG. 2, the culture unit of the conventional cell culture apparatus may include a membrane membrane 201 capable of gas exchange. The membrane layer 201 may form an outer wall of the culture unit. The membrane layer 201 may form an outer wall of the culture unit and a culture solution 203 may be injected therein. The culture unit of the conventional cell culture apparatus may be cultured by attaching the cells 202 to the membrane membrane 201 therein. The cells 202 are not floating and can be grown only when they are attached and supported at a specific location. Therefore, when the cells 202 attach to the membrane membrane 201 and take root, the gas generated while the cells grow and the gas required are exchanged through the membrane membrane 201 to grow. Therefore, even if the weightless state was created by the artificial gravity generated in the culture unit, the effect could not be fully applied.

3 is a perspective view of a cell culture apparatus according to an embodiment of the present invention.

Referring to FIG. 3, the microgravity culture apparatus according to an embodiment of the present invention may include a culture unit 301, a connection unit 302, a support 303, and an exchange tube 304.

Cell culture may be performed in the culture unit 301. The culture unit 301 may be injected with a culture solution therein, and may be sealed or sealed while the culture solution is contained therein. The culture unit 301 may be formed of a membrane membrane. The culture unit 301 may be partially or entirely composed of a membrane membrane. The culture unit 301 may be formed of a membrane membrane on the slope, and material exchange with the outside may be performed. The material to be exchanged may include gas. The gas may include oxygen and carbon dioxide, but is not limited thereto, and may include any gas that may be generated or required in cell culture. The culture unit 301 may culture cells therein. The culture unit 301 is connected to a gravity generating device and may be affected by artificial gravity artificially generated by the gravity generating device. The culture unit 301 may culture cells in a state in which gravity is controlled under the influence of artificial gravity. The culture unit 301 may culture cells in a microgravity state under the influence of artificial gravity. The culture unit 301 may culture cells in a zero gravity state under the influence of artificial gravity.

The connection part 302 may fix the culture part 301. The connection part 302 may be included in a single or plural number. The connection part 302 may connect the culture part 301 to a gravity generating device. The connection part 302 may allow the culture part 301 to rotate. The connection portion 302 may be coupled to one side or both sides of the culture portion 301 to fix the culture portion 301. The connection part 302 may be combined with the culture part 301 to rotate the culture part 301. The connection part 302 may include a joint part. The connection part 302 may individually include a plurality of joint parts, and the angle and direction may be freely adjusted or changed by moving the joint part. The connection part 302 may adjust the angle, direction or inclination of the culture part 301. The connection part 302 may adjust the three-dimensional arrangement by adjusting the angle, direction or inclination of the culture part 301.

The support 303 may be included in the culture unit 301. The culture unit 301 may include an inlet, and the support 303 may be inserted into the culture unit 301 through the inlet. Cells to be cultured may be attached to the support 303. The support 303 may be attached to the cells to be cultured and may be suspended in the culture medium inside the culture unit 301. The support 303 may have a two-dimensional structure or a three-dimensional structure. The support 303 may have a two-dimensional disk shape. The support 303 may have a three-dimensional structure shape. The two-dimensional disk-shaped support 303 may be configured in the form of a disk, and cells may be attached to not only both sides but also arcs to grow. The support 303 in the form of a three-dimensional structure may have a frame shape in which only edges of a hexahedron are connected. The support 303 in the form of a three-dimensional structure may be formed in a form in which a plurality of corner frames of a hexahedron are combined. The support 303 in the form of a three-dimensional structure may be cultured with cells attached to all positions of the skeleton.

The exchange tube 304 may be connected to the culture unit 301. The exchange tube 304 may be used for material exchange of the culture unit 301. The exchange tube 304 may inject or discharge a substance into the culture unit 301. The exchange pipe 304 may be made of an elastic material. The exchange pipe 304 may rotate together when the culture unit 301 rotates. The exchange pipe 304 may be formed as an empty space inside the connection part 302, or the connection part 302 may be formed as an empty pipe and inserted into the connection part 302. The exchange pipe 304 may be included in plural. The exchange tube 304 may be used individually for each material to be exchanged. The exchange tube 304 rotates together when the culture unit 301 rotates, so that the plurality of exchange tubes 304 may rotate without being entangled.

4 is an example of a microgravity cell culture apparatus according to an embodiment of the present invention.

Referring to FIG. 4, the microgravity cell culture apparatus according to an embodiment of the present invention may fix the culture unit 301 to the gravity generating device through the connection unit 302. The inclination, angle, and direction of the culture unit 301 may be adjusted by moving the connection unit 302. The gravity generating device may artificially generate gravity in the culture unit 301. The gravity generating device may generate artificial gravity inside the culture unit 301 by rotating the culture unit 301, and is not limited thereto, and generates artificial gravity in any manner capable of generating artificial gravity. I can make it. The connection part 302 attached to the gravity generating device may include at least one joint, and the cultivation part 301 may determine an angle, direction, or inclination made by the earth's gravitational acceleration by moving the joint. The connection part 302 may move the joint so that the direction in which the artificial gravity is applied to the culture part 301 when the gravity generating device is generated is applied in a direction opposite to the acceleration of the earth's gravity.

The microgravity cell culture apparatus according to an embodiment of the present invention may include a gravity sensor (not shown). The gravity detection sensor may detect the direction and magnitude of gravity applied to the culture unit 301. The gravity generating device may generate artificial gravity according to the magnitude and direction of the gravity when the gravity detection sensor detects the magnitude and direction of gravity applied to the culture unit 301. The gravity generating device may generate artificial gravity in a direction opposite to the direction of gravity applied to the culture unit 301. The gravity generating device may generate artificial gravity in the same size as the gravity applied to the culture unit 301. The gravity generating device may generate artificial gravity in the same direction as the direction of gravity applied to the culture unit 301. The gravity generating device may generate artificial gravity in a direction perpendicular to a direction of gravity applied to the culture unit 301. The gravity generating device may generate artificial gravity at a specific ratio of the size of gravity applied to the culture unit 301. The specific ratio may be determined according to the user's setting.

5 is a perspective view of a culture unit according to an embodiment of the present invention.

Referring to FIG. 5, the culture unit according to an embodiment of the present invention may include a membrane membrane 501. The membrane film 501 may constitute the entire outer surface of the culture unit. The membrane film 501 may form a part of the outer surface of the culture unit. The membrane membrane 501 may allow the culture unit to exchange gas with the outside. The membrane film 501 may include at least one outlet port. The material contained in the culture unit may be injected, injected, or discharged through the outlet.

The culture unit according to an embodiment of the present invention may include a culture solution 502 inside the membrane film 501. The culture solution 502 may be included in the membrane film 501 through the outlet port. The culture medium 502 may contain a material necessary for cell culture. The culture medium 502 may include water, which is indispensable for the survival and development of living organisms, as well as a large amount of nutrients and a trace element. The culture solution 502 may contain all inorganic or organic compounds except that it is obtained in a gaseous phase. The inorganic or organic compounds may include all compounds necessary for nutritional types, such as autotrophic and dependent nutrition. The culture medium 502 may contain a compound such as a carbon source, a nitrogen source, an inorganic salt, and a growth factor (vitamins). In particular, it may include a natural medium (天然培地) whose main body is one having a relatively complex composition extracted from living organisms in relation to growth factors. The culture medium 502 may contain meat juice and serum in the case of bacterial culture, and malt extract in the case of mold culture. The culture medium 502 may contain only inorganic salts or a synthetic medium having a clear composition in which a carbon source and a nitrogen source having a certain structure are added. The culture medium 502 may include a liquid medium and a solid medium.

The culture unit according to an embodiment of the present invention may include a sealing part 503. The sealing part 503 may completely block the inside of the membrane layer 501 from the outside. The sealing part 503 may contact or couple a part of the membrane film 501 of the culture part to completely block the inside of the membrane film 501 from the outside. The sealing part 503 may block the movement of the material through the outlet of the membrane film 501. The sealing part 503 may be configured in the form of tongs. The sealing part 503 may pick up the outlet of the membrane film 501 to block the inflow and outflow of the material. The sealing part 503 may have a zipper lock shape. The sealing part 503 may have a form of a separated forceps, and may seal the membrane film 501 by combining the separated forceps into one. The sealing part 503 may have a heat sealing form. The sealing part 503 may seal the membrane layer 501 by combining two membrane membranes made of a polymer material through which gas passes through heating.

6 is an example of a support included in the culture unit according to an embodiment of the present invention.

Referring to Figure 6 (a), the support according to an embodiment of the present invention may have a two-dimensional disk shape. The two-dimensional disk-shaped support 303 may be configured in the form of a disk, and cells may be attached to not only both sides but also arcs to grow.

Referring to FIG. 6B, the support 303 may have a three-dimensional structure. The support 303 in the form of a three-dimensional structure may have a frame shape in which only edges of a hexahedron are connected. The support 303 in the form of a three-dimensional structure may be formed in a form in which a plurality of corner frames of a hexahedron are combined. The support 303 in the form of a three-dimensional structure may be formed in a spherical shape in which at least one through hole is formed. The support 303 in the form of a three-dimensional structure may be cultured with cells attached to all positions of the skeleton.

7 and 8 are results of comparing the cell growth according to the cell culture device according to an embodiment of the present invention and the conventional device.

7 and 8, microgravity culture of the present invention under conditions of 37 degrees and 5% CO2 using a medium in which 10% FBS and 1% aniti-anti were added to RPMI 1640 medium with A549 and H1703 human lung cancer cells. The results of culturing by attaching cells to the outer wall of the culture unit of the device and the conventional microgravity culture device can be confirmed.

To give microgravity, seeding of 1.7 x 10^5 cells/dish and 2 x 10^5 cells/dish respectively for A549 and H1703 using 3D Clinostat equipment, Confocal dish (SPL), and culture bag (SPL). After proceeding, the slip was removed from the confocal dish, 50 ml of RPMI 1640 with 10% FBS, and 1% anti-anti were added, sealed, and fixed to the clinostat equipment.

In order to measure migration, A549 and H1703 are cultured in O/N with 1.7 x 10^5 cells/dish and 2 x 10^5 cells/dish seeding, respectively, in a confocal dish, wound with a yellow tip, and attached to a confocal dish. Remove the slip and put it in a culture bag, put RPMI 1640 with 10% FBS, 50 ml of 1% anti-anti medium, and seal it. After culturing for 24 h and 48 h, the Wound healing assay was confirmed with a confocal microscope.

In order to find out the effect of microgravity on the migration of A549 and H1703, microgravity was cultured for two hours for 24h and 48h, and then the width of the wound was measured and confirmed by a cell migration experiment that was determined through wound healing assay. After microgravity was applied, it was found that migration occurred much better over time compared to the control group. That is, it can be seen that the microgravity cell culture apparatus according to an embodiment of the present invention has a large cell culture effect.

9 and 10 are graphs of results of confirming cell growth according to the cell culture apparatus according to an embodiment of the present invention and the existing apparatus at the RNA level.

9 and 10, in order to compare the amount of RNA through RNA extraction, RT-PCR and real-time PCR, cells cultured for 24h and 48h were removed from the culture bag and transferred to a 35 pie dish to extract RNA. For this purpose, 1 ml of Trizol solution was dispensed each, collected in an EP tube using a cell lifter, and 200 ul of chloroform was added and vortexed. After incubation at 37 degrees for 5 minutes, centrifugation at 12000 rpm 4 degrees for 20 minutes, 400 ul of sample and 2-propanol (400 ul) were added 37 After centrifugation for 5 minutes at 12000rpm 4°C for 20 minutes, discard the supernatant, add 600ul of 75% ethanol, centrifuge at 7500rpm 4°C for 5 minutes, discard the supernatant and air dry for 5 minutes. After adding 20ul of RNase free water, heating the heating block at 65°C for 5 minutes, the sample was quantified using a nano drop.

RT-PCR was performed based on 2ug, and according to the quantitative value, 1ul of RNA and Primer oligo(dt), 1ul of 10mM DNTP mix, 4ul of 5x RT buffer, 1ul of Ribosafe RNase inhibitor, and 1ul of Tetro Reverse Transcripase were added, and the total volume was 20ul. RT PCR was performed using a PCR machine at 45 degrees 30 minutes, 85 degrees 5 minutes, and 4 degrees O/N.

Real-time PCR was performed with RT PCR DNA, SYBR 6.25ul, Primer 1ul, cDNA 1ul, H20 4.25ul, and Real-time PCR. (Here primers are MMP2, MMP9, TIMP1, TIMP2, GAPDH)

When exposed to A549 and H1703 under microgravity, it was confirmed that migration and proliferation were promoted. Real-timp PCR was performed to confirm the change in RNA level. As a result of checking at two times of 24h and 48h, it was confirmed that the expression of MMP2 and MMP9 was increased at 48h of A549, and the increase of MMP2 and MMP9 at 48h of H1703 was also confirmed.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the technical field to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (10)

A gravity generating device that generates an artificial gravity to form a zero gravity environment;
A culture unit composed of a membrane membrane and filled with a culture solution therein;
A connection part fixing the culture part and connecting it to the gravity generating device; And
Microgravity cultivation apparatus comprising a; is included so as to be suspended in the culture medium inside the culture unit, a support for culturing by attaching cells. The method of claim 1,
The support,
Microgravity culture device in disk form. The method of claim 1,
The support,
Microgravity culture device, a three-dimensional structure The method of claim 1,
The gravity generating device,
Microgravity culture device for creating a zero gravity environment artificially generating gravity by rotating the culture unit. The method of claim 1,
Further comprising; an exchange tube for exchanging substances with the inside of the culture unit,
The connection part has a tube formed therein,
The exchange tube is a microgravity culture device inserted into the inner tube of the connection part. The method of claim 1,
Microgravity culture apparatus further comprising a; sealing portion for sealing the entrance of the culture portion so that the culture medium of the culture portion to escape. The method of claim 1,
The connection part,
Microgravity culture device capable of adjusting the tilt of the rotation axis of the culture unit. The method of claim 1,
The gravity generating device,
Microgravity culture apparatus further comprising a; gravity sensor capable of detecting the direction and size of gravity is applied to the culture unit. The method of claim 8,
Microgravity culture device for adjusting the artificial gravity generated in the culture section and the inclination of the culture section according to the direction and magnitude of the gravity sensed by the gravity sensor. The method of claim 1,
The microgravity culture device,
Microgravity culture apparatus, characterized in that the cell culture for more than 24 hours.

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