WO2016052472A1 - 三次元細胞集合体の作製方法 - Google Patents
三次元細胞集合体の作製方法 Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3826—Muscle cells, e.g. smooth muscle cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/10—Petri dish
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
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- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0661—Smooth muscle cells
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- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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- C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces
Definitions
- the present invention relates to a method for producing a three-dimensional cell aggregate.
- Patent Document 1 improves the above problems.
- Patent Document 1 discloses a technique of using a “temperature-responsive cell culture device” to recover cultured cells as a cell sheet simply by changing the culture temperature. Since the obtained cultured cells are not subjected to the enzyme treatment, there is an advantage that they can be collected as one sheet without breaking the protein between the cells.
- An object of the present invention is to provide a means that makes it possible to produce a thick cell aggregate by a simple process without an operation of peeling and superposing cells.
- the inventors of the present application can obtain a thick sheet-like cell aggregate simply by applying pressure to a cell suspension or a medium containing cells, and further, applying pressure by adding cells. It was found that a cell aggregate in which cells are adhered and assembled in a sheet shape with sufficient strength can be obtained by repeating the above several times, and the present invention has been completed.
- the present invention provides a method for producing a three-dimensional cell aggregate, which includes a cell accommodation step of containing a cell suspension in a cell storage vessel, and a pressure application step of applying pressure to the cells in the vessel. .
- this invention provides the cell sheet containing the three-dimensional cell aggregate produced by the method of the said invention.
- the present invention provides a cell container that contains a cell suspension, and the cells in the cell container are continuously, periodically, intermittently, or a combination thereof.
- An apparatus for producing a three-dimensional cell assembly comprising a pressure applying means for applying.
- a thick and highly robust cell assembly can be obtained by a simple operation of applying pressure to a cell suspension or a medium containing cells.
- a sheet-like cell aggregate can be produced using a system having a simple configuration without using special culture equipment as in the technique described in Patent Document 1. Since the operation of superposing a plurality of cell sheets is unnecessary, the damage to the cells is extremely small and the cells can be kept in a good state, which is advantageous when used as a transplanted tissue piece.
- the cell aggregate obtained by the method of the present invention can be used in various fields such as drug discovery such as drug efficacy evaluation and drug screening, and food such as artificial meat (cultured meat). Application in the field is expected.
- FIG. 1 It is a schematic diagram of an example of the system for enforcing the method of this invention. It is the result of incubating a human smooth muscle cell suspension with or without pressure treatment for 15 hours.
- A Cells subjected to pressure treatment
- B Cells not subjected to pressure treatment
- C Section stained image of cells subjected to pressure treatment
- D Section stained image of cells not subjected to pressure treatment. It is the result of incubating rat heart-derived myoblast H9C2 for 17 hours with or without pressure treatment. It is the result of incubating mouse embryo-derived fibroblasts NIH3T3 for 17 hours with or without pressure treatment.
- FIG. 6 is a photograph of a cell aggregate obtained by a production method in which addition of cells and application of pressure shown in FIG. 5 are repeated. It is a figure which shows the time course of the production method which repeated the addition of a cell and the addition of a pressure which were performed in the Example, and only the addition of a cell without applying a pressure.
- 8 is an HE-stained image of a cell aggregate obtained by the production method shown in FIG. It is the result of immunofluorescent staining of F-actin and cell nucleus of umbilical artery smooth muscle cells applied with pressure in a microfluidic chip.
- the method for producing a three-dimensional cell assembly includes a cell accommodation step for accommodating a cell suspension in a cell accommodation vessel, and a pressure application step for applying pressure to the cells in the vessel.
- the pressure application step may be performed immediately after the cell suspension is placed in the container, or may be performed immediately after the cells in the suspension sink to the bottom of the container after a certain amount of time. Application may be performed. That is, in this invention, the non-pressurization process which maintains a cell suspension in non-pressurization may be included between a cell accommodation process and a pressure application process.
- the length of the non-pressurization time is not particularly limited, and can be set as appropriate according to the amount and type of cells used. Usually, if the reaction is performed for about 30 minutes to several hours or more, most of the cells in the cell suspension settle on the bottom of the container, but it may be about 12 hours or more, for example, about 20 hours or more.
- the upper limit is not particularly limited, but is usually about several days or less, for example, about 72 hours or less.
- the cell accommodation step and the pressure application step can be repeated twice or more. That is, it is possible to repeatedly perform two or more sets, where the cell containing step ⁇ the pressure applying step is one set.
- the content of the pressurization process may be the same pressurization pattern throughout each pressure application step, or different pressurization patterns. May be.
- the number of repetitions when repeating two or more sets is not particularly limited, and the cell accommodation step and the pressure application step may be repeated many times over a period of several weeks or longer.
- a non-pressurization step may be included between the cell accommodation step and the pressure application step, but the time of this non-pressurization step may be the same length throughout each time. It can be changed.
- the cell used in the present invention is an animal cell, preferably a mammalian cell such as a human cell.
- the cell type is not particularly limited, and smooth muscle cells, cardiomyocytes, fibroblasts, myoblasts, chondrocytes, osteoblasts, and the like can be used.
- Cells prepared by differentiating pluripotent cells other than human embryonic stem cells, such as induced pluripotent stem cells and stem cells or progenitor cells of various tissues, may also be used.
- a cell suspension When preparing a cell aggregate for use as a tissue to be transplanted into a human patient, a cell suspension may be prepared by culturing, proliferating, and differentiating cells collected from the patient or another person as necessary. According to a conventional method, after monolayer culture of cells in a culture container such as a petri dish, treatment with an enzyme such as trypsin separates the monolayer cells, disperses the cells, and after centrifugation, the supernatant is removed to obtain an appropriate cell density. The adjusted one can be used as a cell suspension.
- an enzyme such as trypsin
- a medium for suspending cells a medium generally used for cell culture or a buffer such as HEPES buffer (4- (2-HydroxyEthyl) -1-PiperazineEthaneSulfonic acid) and phosphate buffered saline is used. be able to.
- HEPES buffer (2-HydroxyEthyl) -1-PiperazineEthaneSulfonic acid
- phosphate buffered saline phosphate buffered saline
- the appropriate cell density may vary depending on the shape and size of the cell container and the size of the cell itself.
- the cell suspension may contain an amount of cells that exceeds the amount capable of forming a single layer with respect to the bottom area in the gravity direction of the cell container when pressure is applied.
- the bottom area in the direction of gravity is the inner area of the lower half of the laid cylinder.
- a cell density of 300,000 cells / mL or more is sufficient for preparing a three-dimensional cell aggregate.
- a cell suspension of 1000 or more, for example, about 100,000 or more, or about 250,000 or more per square centimeter.
- the upper limit of the cell density is not particularly limited, but is usually 10 million or less per square centimeter.
- the cell suspension may be prepared with only one type of cell, or may be prepared by mixing two or more types of cells.
- a cell suspension may be prepared by mixing cardiomyocytes and vascular endothelial cells.
- you may change the kind of cell added in a cell accommodation container. For example, it is possible to construct an artificial tissue by stacking different cells, such as fibroblasts for the first two layers, smooth muscle cells for the next four layers, and endothelial cells for the uppermost layer.
- the cell container is not particularly limited as long as it is a material that does not adversely affect living cells and has a strength that can withstand pressure treatment.
- the shape is not particularly limited, and may be a cylindrical shape such as a syringe or a tube, a shape such as a flask, or a flat container such as a culture dish.
- the cell container may be, for example, a non-sealing container having an opening that communicates with a gas phase outside the container. In the case of using a container with a lid that is not airtight, a gap between the lid and the container body serves as an opening, and serves as a communication port for the gas phase outside the container.
- a lid is not essential for the cell container.
- a cell container is accommodated in a pressure container that can withstand high pressure, and pressure is applied.
- pressure can be applied to the cells (medium containing cells) inside the cell storage vessel.
- the pressurization method using compressed air has a simple system configuration and the shape of the cell container. The degree of freedom of selection is high and more preferable.
- the surface treatment for cell adhesion (collagen coating, polylysine coating, etc.) is not necessarily required on the inner wall of the cell container.
- the surface treatment for cell adhesion is not necessarily required on the inner wall of the cell container.
- a surface treatment for cell adhesion may be applied to the inner wall of the cell container.
- a sheet-like cell aggregate that is thicker and more robust can be prepared.
- the cell aggregate after preparation can be easily removed from the container by manually peeling it off.
- atmospheric pressure is applied to the cell suspension or the medium containing the cells.
- “applying pressure to the cells in the container” means that the medium containing the cells is subjected to a pressure exceeding atmospheric pressure.
- a pressure of about 100 kPa to about 101 kPa is referred to as “atmospheric pressure”.
- a state in which the cell suspension or the medium containing cells is only subjected to atmospheric pressure may be expressed as “non-pressurized”.
- the pressure applied to the cell suspension or the medium containing cells may be about 102 kPa or more, preferably about 110 kPa or more, for example, about 150 kPa or more, or about 180 kPa.
- the upper limit of the pressure is not particularly limited and can be appropriately selected depending on the cell type, but is usually 100 MPa or less, for example, about 1000 kPa or less, about 500 kPa or less, about 400 kPa or less, or about 300 kPa or less.
- the time for the pressure treatment is not particularly limited, and can be appropriately set according to the type of cells used, the number of cells, and the like. Usually, even with a pressure treatment of several tens of minutes, the cells are sufficiently assembled and adhered, and a three-dimensional cell aggregate can be obtained.
- the pressure treatment time in one pressure application step can be, for example, about 30 minutes or more, about 90 minutes or more, about 3 hours or more, or about 5 hours or more.
- the upper limit of the pressure treatment time in one pressure application step is not particularly limited, but is usually about several days or less, for example, about 72 hours or less, or about 48 hours or less.
- the upper limit of one pressurizing treatment time is not limited to the above, and a period exceeding several days, for example, 1 to 2 It may be performed for a period of about a month.
- replacement of the cell suspension medium is not essential, but may be applied during pressure treatment or other suitable as needed to maintain a favorable environment for the cells.
- the medium may be changed as appropriate at the timing.
- the pressurization pattern is not particularly limited, and may be continuous pressurization or periodic pressurization.
- the pressure of the same strength may be continuously applied throughout the pressurizing time, or the periodic pressurizing process may be performed by changing the pressure applied every several seconds to several minutes or hundreds of seconds.
- a pressurization pattern (for example, 300 kPa ⁇ atmospheric pressure ⁇ 150 kPa ⁇ atmospheric pressure) combining three or more pressures may be employed.
- periodic pressurization of the same cycle may be continued, or the pressurization pattern may be changed during the process.
- a suitable pressure pattern can be appropriately selected according to the type of cells, the number of cells used, and the like.
- a pressure of 180 kPa for 90 minutes in addition to applying a pressure of 180 kPa to cells in the medium continuously for 90 minutes, a pressure of 180 kPa and a lower pressure are applied.
- Repeating pressure or non-pressurization (atmospheric pressure) for several seconds to several hundred seconds is also included, for example, applying 180 kPa for 10 seconds ⁇ atmospheric pressure for 10 seconds for 90 minutes is also included.
- the temperature during the pressure treatment is appropriately set according to the type of cells used. Usually, the temperature conditions used when culturing the cells to be used are employed. The temperature normally used for animal cells is about 30 ° C to 40 ° C, and for human somatic cells, it may be about 35 ° C to 38 ° C. However, the temperature is not particularly limited, and the temperature may be lowered to a room temperature or a low temperature (about 15 ° C.) that does not freeze. When the non-pressurization step is included, the non-pressurization step may be the same temperature as the pressure application step, but a different temperature may be used.
- cell suspension refers to a liquid in which cells are dispersed throughout the medium, and cells in the medium settle downward, resulting in uneven cell density. Also included are liquids in the wet state.
- the pressure applying means for applying pressure to the cells in the cell storage container includes a compressor 8 and a regulator 6.
- a pressure vessel 4 that accommodates the cell accommodation container 2 and a compressor 8 are connected via a regulator 6, and these are accommodated in an incubator 10 as a closed space.
- the regulator 6 is connected to a computer 12 outside the incubator 10, and the intensity and pressure pattern of the pressure applied to the cell suspension 14 in the cell container 2 are controlled by commands from the computer 12.
- the pressure applying means and the pressure container including the cell storage container are accommodated in the closed space.
- at least the cell storage container is accommodated in the closed space. It only has to be.
- the closed space here refers to a space in which the composition and temperature of the gas inside can be controlled separately from the outside.
- the pressure vessel 4 has an appropriate internal shape according to the cell storage vessel 2 to be used.
- the cell container 2 accommodated in the pressure container 4 is a container without airtightness (in the example of FIG. 1, the cell container 2 is a syringe, and the tip of the syringe is opened and is not sealed). ),
- the cell suspension 14 communicates with the outside world (the gas phase in the incubator 10) via the regulator 6, and pressure treatment is performed by sending the air in the incubator 10 compressed by the compressor 8 into the pressure vessel 4. Is done.
- the pressure setting should be about 100 kPa to 101 kPa.
- the system It is also possible to intermittently perform continuous pressurization, periodic pressurization, or a combination thereof. That is, in the system, the computer 12 can command the pressure to be applied continuously, periodically, intermittently, or a combination of these.
- the CO 2 concentration in the enclosed space (in the incubator) may be any concentration that is used when cells to be used are cultured as usual. In the case of human somatic cells, the CO 2 concentration is generally 5.0%.
- the CO 2 concentration in the incubator 10 may be adjusted so that the pH of the cell suspension is appropriately maintained. That is, in the example system shown in FIG. 1, the CO 2 concentration in the closed space (incubator), i.e. may be provided with means for adjusting the CO 2 concentrations of the air fed into the pressure vessel. However, even when pressure is applied at intervals of several minutes to a non-pressurization cycle, the pH of the cell suspension can be appropriately maintained by keeping the CO 2 concentration in the incubator 10 constant without fluctuation.
- the CO 2 concentration in the incubator 10 is continuously maintained at 5.0% while the pressure application process is performed on human somatic cells. It's okay.
- adjustment range of the CO 2 concentration in the pressure treatment, cell type, can be appropriately selected according to the strength or the like of the pressure is not particularly limited, a human somatic cells
- the CO 2 concentration during the pressure application process may be controlled within a range of about 3.0% to 5.0%, for example, about 3.5% to 5.0%.
- the CO 2 concentration during non-pressurization may be adjusted to 5.0%, and the CO 2 concentration during pressurization may be adjusted to approximately 3.5%.
- the CO 2 concentration may be kept constant at a slightly lower concentration than in normal cell culture in the pressure application step.For example, human somatic cells are used, and the non-between the cell containing step and the pressure application step.
- the CO 2 concentration of about non pressurizing step to 5.0% may be maintained at a constant concentration of CO 2 pressure application step is less than 3.0% and 5.0% (e.g. 3.5% before and after).
- a container and a pump for storing a medium are arranged instead of the compressor 8, and a liquid supply line is provided for connecting these to the inside of the cell storage container 2 stored in the pressure container 4. It is good also as a structure which pressurizes by sending a medium in the cell storage container 2 over.
- a porous membrane having pores with a size that prevents cells contained in the container from passing through the connecting portion between the liquid feeding line and the cell container is disposed outside the cell container by reverse flow or the like. Cell escape can be prevented.
- a cell container that can withstand high pressure, in which the pressure container and the cell container are integrated, may be used.
- the system configuration may be such that the medium can be changed automatically or manually as necessary.
- the medium can be replaced at the same time by removing a part of the medium after the pressurization and adding a new cell suspension. Can be done.
- a waste liquid line that can discard the medium in the cell storage container 2 outside the container, and a medium on the line
- Pressure adjusting means such as a regulator for adjusting the hydrostatic pressure may be provided so that the medium can be exchanged at a timing other than when the cell suspension is added.
- a porous membrane may also be disposed at the connection portion between the waste liquid line and the cell storage container 2 in the same manner as the connection portion between the liquid supply line and the cell storage container 2. However, even when processing is performed over a long period of about one month, it is not necessary to frequently change the medium, and it is possible to replace a part of the medium when adding the cell suspension.
- a three-dimensional scaffold such as a porous scaffold made of a material such as a polymer or a biomolecule, which has been conventionally used in the production of a three-dimensional cell assembly.
- a cell tissue (cell aggregate or cell aggregate) consisting essentially of cells only (cells and substances produced from the cells) can be produced.
- tissue engineering for regenerative medicine research and development of a three-dimensional tissue using a biodegradable polymer three-dimensional scaffold, and a cell sheet not using a three-dimensional scaffold are underway.
- the adhesion between cells can be promoted only by the pressure treatment, and a thick cell mass can be obtained.
- any scaffold material may be used depending on the intended use of the cell aggregate.
- the three-dimensional cell aggregate obtained by the method of the present invention can be used as it is as a cell sheet.
- the conventional cell sheet manufacturing method when manufacturing a multilayered cell sheet having a large thickness, an operation of overlapping a plurality of single-layer cultured cells is required.
- the method of the present invention since there is no operation of sequentially stacking the prepared monolayer cells, the production process is simple and the damage to the cells is greatly reduced. Since the size and shape of the cell aggregate can also be influenced by the shape of the cell container used, the size and shape of the cell aggregate can be controlled by selecting the cell container.
- FIG. 300 ⁇ L of a human smooth muscle cell suspension having a cell density of 1 million cells / mL prepared as described above was placed in a 1 mL syringe, and the syringe was housed in a pressure vessel in an incubator.
- the inside of the incubator was 5% CO 2 and 37 ° C., and the CO 2 concentration was controlled between 3.6% and 5.0% during the pressurizing process.
- the pressure treatment was repeated from 180 kPa for 10 seconds to 110 kPa for 10 seconds.
- the state of the cells in the syringe was observed at the time of the pressure treatment time of 90 minutes and 15 hours, the cells were assembled and adhered in a sheet form after the pressure treatment time of 90 minutes.
- FIG. 2A is a cell mass after 15 hours of pressure treatment.
- Fig. 2B shows cells without pressure treatment.
- 2C and 2D are images obtained by HE-staining the obtained cell mass sections. Although the cells are concentrated to some extent even without pressure treatment because of the high-density cell suspension, as shown in the HE-stained image, the robustness of the cell mass is low, the thickness is thin, and it is used as a cell sheet. It was not the level to get ( Figure 2B, 2D).
- the pressure treatment was performed, most of the cells in the suspension gathered, and a cell mass having a thickness of 100 ⁇ m or more was obtained (FIGS. 2A and 2C). Nuclei were also observed inside the cell mass, and the cells gathered closely, resulting in a highly robust cell mass. When the cell mass was observed after culturing at 37 ° C. for a maximum of 24 hours, the cells did not loosen and collapsed, and the shape as a sheet-like cell aggregate was maintained.
- FIG. 3 is a photograph of a cell after pressurizing a suspension of rat heart-derived myoblast H9C2 (right) and a cell after incubation without pressurization (left).
- FIG. 4 is a photograph of cells after the suspension treatment of mouse embryo-derived fibroblast NIH3T3 (right) and after incubation without pressure treatment (left). It was confirmed that even in these cells, a sheet-like cell mass having high robustness can be obtained as in the case of smooth muscle cells.
- the density of the prepared cell suspension is 1.95 million cells / mL for rat heart-derived myoblasts H9C2, 3.15 million cells / mL for mouse embryo-derived fibroblasts NIH3T3, and all suspensions contained in syringes. 300 ⁇ L, pressure treatment was repeated from 180 kPa for 10 seconds to 110 kPa for 10 seconds, and the treatment time was 17 hours.
- cell aggregates were prepared by a method of repeatedly adding cells to the cell container and applying pressure (FIG. 5, (1) to (3) below).
- a square or circular glass culture dish was used as the cell container.
- the culture dish was coated with fibronectin.
- the pressurizing pattern was a cycle of 110 kPa for 250 seconds ⁇ 180 kPa (0.002 Hz) for 250 seconds.
- the temperature in the incubator was 37 ° C., and the CO 2 concentration was maintained at 5% including the pressure application step.
- 0.4 mL of a cell suspension of 750,000 cells / mL was added to a square glass culture dish (0.8 square centimeter) and placed at atmospheric pressure for 1 day, followed by 1 day of pressure treatment. 0.4 mL of the cell suspension was added to the culture dish, placed at atmospheric pressure for 1 day, and then pressurized for 2 days.
- (2) 0.4 mL of a cell suspension of 500,000 cells / mL was added to a round glass culture dish (0.7 square centimeters), and placed at atmospheric pressure for 1 day, followed by pressure treatment for 2 days.
- 0.4 mL of the cell suspension was added to the culture dish, placed at atmospheric pressure for 1 day, and then pressurized for 1 day.
- FIG. 6 shows a photograph of the cell aggregate prepared as described in (1) to (3) above. When the sheet was pulled with tweezers, it stretched to about 1.8 times the length without breaking. A robust cell sheet could be prepared.
- FIG. 8 shows an HE-stained image.
- the pressurized cell assembly (1) four or more layers of cells were arranged in layers.
- the cell aggregate of (2) where no pressure was applied almost no cells were laminated, and no layered arrangement was observed.
- FIG. 12 is an image of the obtained cell aggregate section stained with Elastica Wangison.
- the scale bar was 100 ⁇ m, and the thickness of the cell aggregate exceeded 200 ⁇ m.
- viable cells were counted by trypan blue staining, the viable cell ratio was 90% or more.
- elastic fibers were also formed in a layered manner, and more than doubled progress was seen in the tensile test. This is a property comparable to human small-diameter blood vessel walls, and the obtained cell aggregate was confirmed to be extremely robust.
- a human umbilical artery smooth muscle cell suspension with a cell density of 1 million cells / mL cultured in a single layer in a petri dish is pressurized for 90 minutes (periodic pressure of 110kPa 10sec-180kPa 10sec), then usually Incubated at atmospheric pressure for 24 hours. Thereafter, RNA was collected from the cells, and mRNA of the extracellular matrix was measured by real-time PCR. The results are shown in FIG. The expression level of any extracellular matrix was increased by pressurization.
- Actin filaments not only determine the skeleton of cells, but are also involved in cell-cell adhesion. It is considered that the increase in actin filaments and various extracellular matrixes generated in cells subjected to pressure application contributes to the collective adhesion of cells in a short time.
- fibrillin1, fibrillin2, fibulin4, fibulin5, LOX, LOX-like1 are elastic fiber-related genes
- collagen I, collagen III are the major collagen subtypes that support the vascular wall
- fibronectin is the major vascular wall gene Extracellular matrix, a component of elastic fibers.
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Abstract
Description
ヒト臍帯動脈平滑筋細胞等の細胞をシャーレで単層培養したのち、トリプシンで処理して単層細胞を剥離し、細胞を10%ウシ血清入りDMEM培地に分散させた。遠心分離後に上清を除去して、100万個/mL~数百万個/mLの適当な細胞密度に調整した。
ヒト平滑筋細胞を用いて、細胞収容容器内への細胞の追加と圧力印加を繰り返す方法(図5、下記(1)~(3))で細胞集合体を作製した。細胞収容容器として、四角形又は円形のガラス製培養皿を用いた。培養皿にはフィブロネクチンによるコーティングを行なった。加圧パターンは、250秒間110kPa→250秒間180kPa(0.002Hz)のサイクルとした。インキュベータ内の温度は37℃とし、CO2濃度は圧力印加工程も含め5%で維持した。
(2) 50万個/mLの細胞懸濁液の0.4mLを円形ガラス製培養皿(0.7平方センチメートル)に加え、1日間大気圧で置いた後に2日間の加圧処理を行なった。細胞懸濁液0.4mLを培養皿に追加し、1日間大気圧で置いた後に1日間の加圧処理を行なった。
(3) 75万個/mLの細胞懸濁液の0.4mLを円形ガラス製培養皿(0.7平方センチメートル)に加え、1日間大気圧で置いた後に2日間の加圧処理を行なった。細胞懸濁液0.4mLを培養皿に追加し、1日大気圧で置いた後に1日間の加圧処理を行なった。さらに細胞懸濁液0.4mLを培養皿に追加し、1日間大気圧で置いた後に2日間の加圧処理を行なった。
ヒト平滑筋細胞を用いて、細胞追加と加圧処理を繰り返して細胞集合体を作製した場合と、加圧処理を行なわずに細胞の追加のみを行なって大気圧で放置することで細胞集合体を作製した場合との比較を行なった。細胞収容容器として、四角形ガラス製培養皿(0.8平方センチメートル)を用いた。培養皿にはフィブロネクチンによるコーティングを行なった。CO2濃度は5%一定、温度は37℃とした。
ラット新生児大動脈平滑筋細胞100万個をガラス製培養皿内で110-180kPa, 0.002Hzで24時間加圧し、翌日、培養皿内に細胞100万個を追加した。処理中のCO2濃度は5%一定、温度は37℃とした。この工程を15回繰り返し、1ヶ月後に15層積層した細胞集合体を得た。図12は得られた細胞集合体の切片をエラスチカワンギーソン染色した像である。スケールバーは100μmであり、細胞集合体の厚みは200μmを越えるほどとなった。生細胞をトリパンブルー染色でカウントしたところ、生細胞率は90%以上であった。また、弾性線維も層状に著明に形成されており、引っ張り試験で2倍以上の進展が見られた。これは、ヒトの小口径血管壁に匹敵する性状であり、得られた細胞集合体は極めて頑強性が高いことが確認された。
(1) シリンジポンプを使用してマイクロ流体チップ内で単層ヒト臍帯動脈平滑筋細胞に圧力印加(180kPa)し、細胞レベルで加圧の影響を調べた。F-アクチン及び細胞核を免疫蛍光染色して観察したところ、圧力印加しない場合(図9右)と比べて、圧力印加した細胞(図9左)ではアクチンフィラメントが劇的に増加することが確認された。
4 圧力容器
6 レギュレータ
8 コンプレッサ
10 インキュベータ
12 コンピュータ
14 細胞懸濁液
Claims (9)
- 細胞収容容器内に細胞懸濁液を収容する細胞収容工程、及び前記容器内の細胞に圧力を印加する圧力印加工程を含む、三次元細胞集合体の作製方法。
- 前記圧力印加工程は、多孔性の三次元足場材の非共存下で行われる、請求項1記載の方法。
- 印加する圧力が102kPa~100MPaである、請求項1又は2記載の方法。
- 前記細胞が動物細胞である、請求項1ないし3のいずれか1項に記載の方法。
- 前記細胞収容工程及び前記圧力印加工程が2回以上反復して行なわれる、請求項1ないし4のいずれか1項に記載の方法。
- 細胞収容工程及び圧力印加工程の間に、細胞懸濁液を非加圧で維持する非加圧工程を含む、請求項1ないし5のいずれか1項に記載の方法。
- 請求項1ないし6のいずれか1項に記載の方法により作製された三次元細胞集合体を含む細胞シート。
- 細胞懸濁液を収容する細胞収容容器と、該細胞収容容器内の細胞に連続的に、周期的に、若しくは間欠的に、又はこれらのうちの複数の組み合わせによって圧力を印加する圧力印加手段とを含む、三次元細胞集合体の作製装置。
- 細胞収容容器内に多孔性の三次元足場を含まないことを特徴とする、請求項8記載の装置。
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US11479792B2 (en) | 2017-07-13 | 2022-10-25 | Upside Foods, Inc. | Compositions and methods for increasing the efficiency of cell cultures used for food production |
WO2023219142A1 (ja) * | 2022-05-12 | 2023-11-16 | 国立大学法人 東京医科歯科大学 | 三次元メカノシグナル細胞培養系を用いて作製した腱/靱帯様人工組織 |
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CN108559700B (zh) | 2018-01-30 | 2021-10-08 | 京东方科技集团股份有限公司 | 细胞片层转移装置和细胞片层处理系统 |
EP4334436A1 (en) | 2021-05-06 | 2024-03-13 | Yeda Research and Development Co. Ltd | Method for inducing hypertrophic muscle fibers for industrial meat production |
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