CN106609242B - Cell bearing device and cell culture system - Google Patents

Cell bearing device and cell culture system Download PDF

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Publication number
CN106609242B
CN106609242B CN201510699990.7A CN201510699990A CN106609242B CN 106609242 B CN106609242 B CN 106609242B CN 201510699990 A CN201510699990 A CN 201510699990A CN 106609242 B CN106609242 B CN 106609242B
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cell
cells
elastic membrane
culture system
membrane
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CN106609242A (en
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朱国栋
蔡坤成
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Taisheng International Polytron Technologies Inc
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Taisheng International Polytron Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract

A cell bearing device comprises an elastic membrane, a supporting structure and at least one pore channel, wherein the elastic membrane is made of polyurethane. The elastic membrane is used for bearing cells. The supporting structure is fixed on the periphery of the elastic membrane. The at least one pore channel is communicated with the bottom of the elastic membrane to apply stress to the elastic membrane. The invention also provides a cell culture system, which comprises the cell carrier device and the attached cells attached to the elastic membrane of the cell carrier device. The cell bearing device can provide good cell growth environment and cell attachment effect without additional chemical modification or protein coating, so as to be suitable for observing attachment cells.

Description

Cell bearing device and cell culture system
Technical Field
The present invention relates to a cell supporting device, and more particularly, to a cell supporting device including a polyurethane elastic membrane, a supporting structure and at least one pore channel, and a cell culture system including the cell supporting device.
Background
Since the location in an organism where many cells are grown is in a dynamic environment, for example: the walls of organs such as heart, lung or stomach expand and contract with pressure, and the muscle or skin surface stretches with pressure, so that when cells are observed in vitro, a dynamic environment conforming to the organism needs to be simulated to carry and culture the cells.
The existing device for providing dynamic environment to observe cells uses an elastic silica gel membrane, and applies external force through machinery or fluid to make the cells loaded on the silica gel membrane receive dynamic stimulation. However, the silicone membrane often reduces the durability of the device due to elastic fatigue after long-term use.
In addition, the device for observing the adherent cells (adherent cells) needs to provide a good cell growth environment and have a good cell adhesion effect, and thus, additional chemical modification or protein coating is generally required to enhance the cell adhesion effect.
Disclosure of Invention
The invention aims to provide a cell bearing device which is suitable for observing attachment cells.
The cell bearing device comprises an elastic membrane, a supporting structure and at least one pore channel, wherein the elastic membrane is made of polyurethane. The elastic membrane is used for bearing cells. The supporting structure is fixed on the periphery of the elastic membrane. The at least one pore channel is communicated with the bottom of the elastic membrane for applying stress to the elastic membrane.
Another object of the present invention is to provide a cell culture system, which comprises the cell carrier as described above, and adherent cells attached to the elastic membrane of the cell carrier.
The polyurethane elastic membrane of the cell bearing device has good elasticity and durability, and the cell adhesion effect can be improved without additional chemical modification or protein coating.
The present invention will be described in detail below:
preferably, the support structure is fixed to two opposite sides of the periphery of the elastic membrane.
In a first embodiment of the present invention, the cell-supporting device further comprises a base spaced apart from the bottom of the elastic membrane, and the at least one hole penetrates through the base.
In a second embodiment of the present invention, the cell supporting device further includes a supporting column disposed at the bottom of the elastic membrane and spaced apart from the supporting structure, and the elastic membrane is partially attached to the supporting column.
Preferably, the polyurethane is obtained by reacting a polyether polyol with an aliphatic polyisocyanate, followed by reaction with a chain extender. More preferably, the polyether polyol is selected from polyethylene glycol (PEG), polypropylene glycol (PPG), or a combination thereof. More preferably, the molar ratio of PEG to PPG is 3: 7 or more. The aliphatic polyisocyanate avoids the toxicity risks that aromatic compounds may have. More preferably, the aliphatic polyisocyanate is selected from isophorone diisocyanate (IPDI), 1, 6-Hexamethylene Diisocyanate (HDI), or a combination thereof. The chain extender is selected from 1, 4-butanediol (1,4-BD), ethylene glycol or combinations thereof.
More preferably, the molar ratio of the aliphatic polyisocyanate, the polyether polyol and the chain extender is in the range of 2: 1: 1-3: 2: 1 to obtain a polyurethane having excellent elasticity (high elongation) and durability (high strength).
In accordance with the cell culture system of the present invention, the adherent cells include those readily available to those skilled in the art (from domestic or foreign depositories) or those isolated and purified from natural sources by cell isolation methods customary in the art. Preferably, the adherent cells are selected from the group consisting of: mesenchymal stem cells (mesenchyme stem cells), fibroblasts (fibroplasts), epithelial cells (epithelial cells), endothelial cells (endotelial cells), stellate cells (astrocytes), kidney cells (kidney cells), hepatocytes (hepatocytes), epidermal cells (epiermai cells), corneal cells (comealcells), and combinations thereof. More preferably, the adherent cell is selected from the group consisting of a placenta-derived mesenchymal stem cell (e.g., human placenta-derived mesenchymal stem cell), an embryonic kidney cell (e.g., human embryonic kidney cell line HEK293), an embryonic fibroblast cell (e.g., mouse embryonic fibroblast cell line 3T3), or a combination thereof.
Drawings
Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments with reference to the accompanying drawings, in which:
FIG. 1 is a sectional view illustrating the structure of a cell carrier device according to a first embodiment of the present invention;
FIG. 2 is a sectional view showing the structure of the cell support device according to the first embodiment under a vertical vibration stress;
FIG. 3 is a sectional view showing the structure of a cell carrier device according to a second embodiment of the present invention; and
FIG. 4 is a sectional view showing the structure of the cell support device according to the second embodiment under a tensile stress applied in the horizontal direction.
Detailed Description
Before the present invention is described in detail, it should be noted that in the following description, like elements are represented by like reference numerals.
The invention will be further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the practice of the invention.
< preparation of elastic film >
IPDI, PEG1000 (weight average molecular weight 1,000), and PPG1000 (weight average molecular weight 1,000) were mixed at a ratio of 2: 0.5: 0.5, reacting at 70 ℃ for 2 hours to obtain a prepolymer, adding 1,4-BD (as a chain extender, the molar ratio of the chain extender to IPDI is 1: 2), reacting at 110 ℃ for 24 hours, dissolving in Dimethylacetamide (DMAC) at a solid content of 20%, coating on a piece of release paper, drying to remove DMAC, and tearing off the release paper to obtain a polyurethane elastic film with the thickness of about 0.025 mm.
< test of elastic Membrane >
The polyurethane elastic film obtained above was subjected to an elongation (elongation) test using ASTM D5034, and the result was 800%, showing that the polyurethane elastic film obtained above had a considerably high elongation.
Human placenta-derived mesenchymal stem cells (experimental group) were cultured using the polyurethane elastic membrane prepared above, and a commercial culture dish was used as a control group at 37 ℃ with 5% CO2After culturing in the environment of (1) for 7 days, the viable cell density (viable cell density) measured in the experimental group and the control group was 8.48X 10, respectively4/cm2And 5.76X 104/cm2Compared with the control group, the living cell density of the experimental group is greatly increased by 47%, and the polyurethane elastic membrane prepared by the method can effectively promote the growth of the adherent cells.
< cell supporting device and cell culture system >
Referring to fig. 1 and 2, a first embodiment of a cell-supporting device 1 according to the present invention comprises a flexible membrane 2, a supporting structure 3, four channels 4 and a base 5. The elastic membrane 2 is the polyurethane elastic membrane prepared by the method, and the adhesive cells 9 are attached to the elastic membrane 2, so that the dynamic growth environment of the cells on the walls of organs such as heart, lung or stomach and the like which are frequently expanded and shrunk can be simulated.
The support structure 3 is fixed to two opposite sides of the periphery of the elastic membrane 2 so that the elastic membrane 2 is fixed and does not move.
The pore channel 4 is communicated with the bottom of the elastic membrane 2, and an external vacuum device (not shown) is used for pumping air through the pore channel 4, so that the part of the elastic membrane 2 bearing the adherent cells 9 is stressed in the vertical direction and is sunken downwards; when the air-exhaust is stopped, the elastic membrane 2 will return to the non-concave state before the air-exhaust. By changing the pressure and frequency of the pumping, the elastic membrane 2 can vibrate between a concave state and a non-concave state, thereby providing a vertical vibration stress to the adherent cells 9 on the elastic membrane 2.
The bases 5 are spaced at the bottom of the elastic membrane 2, and the holes 4 penetrate the bases 5 to control the elastic membrane 2 to receive uniform vertical stress during air-extraction.
Referring to fig. 3 and 4, the second embodiment of the cell-supporting device 1 of the present invention comprises a flexible membrane 2, a supporting structure 3, a channel 4 and a supporting column 6. The elastic membrane 2 is the polyurethane elastic membrane prepared by the method, and the adhesive cells 9 are attached to the elastic membrane 2, so that the dynamic growth environment of the simulated cells on the surface of frequently stretched muscles or skin can be provided.
The support structure 3 is fixed to two opposite sides of the periphery of the elastic membrane 2 so that the elastic membrane 2 is fixed and does not move.
The pore channel 4 is communicated with the bottom of the elastic membrane 2 and surrounds the carrying column 6, and an external vacuum device (not shown) is used for pumping air through the pore channel 4, so that the part of the elastic membrane 2 carrying the attachment type cell 9 is stressed in the horizontal direction and extends outwards; when the evacuation is stopped, the elastic membrane 2 returns to the non-stretched state before evacuation. By varying the pressure and frequency of the evacuation, the elastic membrane 2 can be stretched between a stretched state and a non-stretched state, thereby providing a horizontal tensile stress to the adherent cells 9 on the elastic membrane 2.
The bearing column 6 is arranged at the bottom of the elastic membrane 2 and is arranged at an interval with the supporting structure 3 through the pore passage 4, and the elastic membrane 2 is partially attached to the bearing column 6 so as to control the attached part to be stressed in the horizontal direction during air exhaust.
In summary, the cell-bearing device 1 of the present invention can provide a good cell growth environment and cell adhesion effect without additional chemical modification or protein coating by the elastic membrane 2 with good elasticity and durability, so as to be suitable for observing the adhesion type cell 9, thereby achieving the object of the present invention.
The above description is only an example of the present invention, and the scope of the present invention should not be limited thereby, and the invention is still within the scope of the present invention by simple equivalent changes and modifications made according to the claims and the contents of the specification.

Claims (7)

1. A cell support apparatus, comprising:
a sheet of elastic membrane for carrying cells;
a support structure secured to opposite sides of the periphery of the flexible membrane;
at least one pore channel communicated to the bottom of the elastic membrane for applying stress to the elastic membrane; and
a bearing column arranged at the bottom of the elastic membrane and arranged at an interval with the supporting structure through the pore passage, and part of the elastic membrane is attached to the bearing column,
wherein, the material of the elastic membrane is polyurethane.
2. The cell carrier of claim 1, wherein: the polyurethane is obtained by reacting a polyether polyol with an aliphatic polyisocyanate, followed by reaction with a chain extender.
3. The cell carrier of claim 2, wherein: the aliphatic polyisocyanate is selected from isophorone diisocyanate, 1, 6-hexamethylene diisocyanate, or a combination thereof.
4. The cell carrier of claim 2, wherein: the chain extender is selected from 1, 4-butanediol, ethylene glycol or combinations thereof.
5. A cell culture system comprising a cell support device according to claim 1 and adherent cells attached to the flexible membrane of the cell support device.
6. The cell culture system of claim 5, wherein: the adherent cell is selected from the group consisting of: mesenchymal stem cells, fibroblasts, epithelial cells, endothelial cells, stellate cells, kidney cells, liver cells, epidermal cells, corneal cells, and combinations thereof.
7. The cell culture system of claim 6, wherein: the adherent cells are selected from human embryonic kidney cell line HEK293, mouse embryonic fibroblast cell line 3T3, or combinations thereof.
CN201510699990.7A 2015-10-26 2015-10-26 Cell bearing device and cell culture system Active CN106609242B (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220177823A1 (en) * 2019-01-04 2022-06-09 Biotech Foods S.L. Bioreactor and method for the production of adherent cell cultures employing said bioreactor
CN110484444B (en) * 2019-08-15 2022-04-01 广州迈普再生医学科技股份有限公司 Biological culture assembly and biological culture system

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2818484Y (en) * 2005-07-15 2006-09-20 中国人民解放军第四军医大学 Cell tractive tension controller
CN101475908A (en) * 2009-01-16 2009-07-08 中国人民解放军第三军医大学 Cultivation apparatus and method for implementing continuous tension stress action to cell
CN101842474A (en) * 2007-08-30 2010-09-22 哈佛学院董事会 Compliant surface multi-well culture plate
CN102676446A (en) * 2012-05-04 2012-09-19 华中科技大学 Method and device for loading cell fluid stress on deformable curved surface and experimental platform
CN103146572A (en) * 2011-12-07 2013-06-12 清华大学 Apparatus and method for realizing homogeneous growth of cell colony
CN103477222A (en) * 2010-09-29 2013-12-25 麻省理工学院 Device for high throughput investigations of cellular interactions

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2818484Y (en) * 2005-07-15 2006-09-20 中国人民解放军第四军医大学 Cell tractive tension controller
CN101842474A (en) * 2007-08-30 2010-09-22 哈佛学院董事会 Compliant surface multi-well culture plate
CN101475908A (en) * 2009-01-16 2009-07-08 中国人民解放军第三军医大学 Cultivation apparatus and method for implementing continuous tension stress action to cell
CN103477222A (en) * 2010-09-29 2013-12-25 麻省理工学院 Device for high throughput investigations of cellular interactions
CN103146572A (en) * 2011-12-07 2013-06-12 清华大学 Apparatus and method for realizing homogeneous growth of cell colony
CN102676446A (en) * 2012-05-04 2012-09-19 华中科技大学 Method and device for loading cell fluid stress on deformable curved surface and experimental platform

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