JP2012105587A - Cell support, method for producing cell sheet, and cell sheet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly producing a cell sheet at a point of use.SOLUTION: There is provided a cell support having a polymer membrane, comprising a temperature-responsive polymer having a 0-80°C upper critical dissolution temperature or lower critical dissolution temperature to water, and holding an antibody against the cell. The method for producing the cell sheet comprises a membrane-forming step S1 of forming a polymer membrane by coating the surface of a substrate with the temperature-responsive polymer, an antibody-holding step S2 of bringing the formed polymer membrane formed in the membrane-forming step S1 to hold the antibody against the cell, a sowing step S3 of sowing cells on the substrate, where the antibody is held on the polymer membrane in the antibody-holding step S2, at a temperature equal to or lower than the upper critical dissolution temperature or equal to or upper than the lower critical dissolution temperature, and, after the sowing step S3, a peeling step S4 of peeling the cells from the substrate at a temperature equal to or higher than the upper critical dissolution temperature or equal to or lower than the lower critical dissolution temperature.

Description

  The present invention relates to a cell support, a cell sheet production method, and a cell sheet.

  Conventionally, cells were cultured on a cell culture support whose surface was coated with a polymer having a critical dissolution temperature in water, and then the temperature was changed to dissolve the polymer in water to form a sheet. A cell culturing method for recovering an aggregate of cells (hereinafter referred to as a cell sheet) as it is is known (for example, see Patent Document 1).

JP-A-5-192138

  However, in the case of Patent Document 1, in order to produce a cell sheet, it is necessary to wait for cells to grow to a sufficiently dense state by culturing the cells for a certain period after seeding the cells on a cell culture support. There is. Therefore, when cells are separated from tissues collected from the patient's body, and cell sheets are produced from the separated cells on the spot and transplanted to the patient, sufficient time for culturing the cells on the cell culture support is sufficient. There is a problem that it is difficult to manufacture a cell sheet because it cannot be secured.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a cell support, a cell sheet manufacturing method, and a cell sheet that can quickly manufacture a cell sheet on the spot where it is used. And

In order to achieve the above object, the present invention provides the following means.
The present invention provides a cell support comprising a polymer membrane that comprises a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water and that holds antibodies against cells.

According to the present invention, after seeding a sufficient number of cells at a temperature below the upper critical solution temperature or above the lower critical solution temperature on a substrate whose surface is coated with a polymer film, the temperature is set higher than the upper critical solution temperature or By dissolving the temperature-responsive polymer in water at a temperature lower than the lower critical solution temperature, cells that have formed aggregates in a planar shape on the substrate are detached from the substrate while maintaining the shape to produce a cell sheet. be able to.
In this case, since the cells rapidly bind to the polymer membrane via the antibody to form an aggregate, culture is unnecessary, and the cell sheet can be rapidly produced on the spot.

In the above invention, the antibody may be an anti-CD90 antibody.
In this way, when adipose-derived cell groups containing cells of different types and states separated from adipose tissue are seeded, fat with high therapeutic effect expressing CD90 from among the adipose-derived cell groups The cell derived from a fat-derived cell group with a high therapeutic effect can be manufactured by selectively binding the derived cell to the polymer membrane.
Moreover, in the said invention, it is preferable that the said polymer film has exposed the said antibody to the surface.
By doing so, cells can be more efficiently bound to the polymer membrane.

  The present invention also includes a film forming step of forming a polymer film by coating the surface of a substrate with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water; An antibody holding step for holding an antibody against cells in the polymer film formed in the film forming step, and the upper critical solution temperature or lower on the substrate on which the antibody is held on the polymer film in the antibody holding step or A seeding step in which the cells are seeded at a temperature equal to or higher than a lower limit lysis critical temperature; and after the seeding step, the cells are transferred from the substrate at a temperature higher than the upper limit critical lysis temperature or lower than the lower limit lysis critical temperature. A cell sheet manufacturing method comprising a peeling step for peeling.

  According to the present invention, after seeding cells in the seeding step on the substrate on which the polymer film has been formed in the film forming step, the temperature-responsive polymer is dissolved in the stripping step, and the cells are detached from the substrate as aggregates. Thus, a cell sheet can be produced. In this case, cells quickly bind to the polymer membrane via the antibody retained in the polymer membrane in the antibody retention step to form an aggregate, so there is no need to culture after seeding the cells, and the cell sheet can be rapidly Can be manufactured.

In the above invention, the cells may be a group of adipose-derived cells separated from adipose tissue, and the antibody may be an anti-CD90 antibody.
Moreover, in the said invention, it is preferable that the said antibody holding step hold | maintains the said antibody on the surface of the said polymer membrane.
Moreover, this invention provides the cell sheet manufactured by the cell sheet manufacturing method in any one of the said.

  According to the present invention, there is an effect that the cell sheet can be rapidly manufactured on the spot where it is used.

It is a flowchart which shows the cell sheet manufacturing method which concerns on one Embodiment of this invention. It is a whole block diagram of the cell support body manufactured at the antibody holding step of FIG. It is a figure which shows the cell sheet peeled from the board | substrate in the peeling step of FIG.

Hereinafter, a cell support 1, a cell sheet manufacturing method, and a cell sheet 10 according to an embodiment of the present invention will be described with reference to the drawings.
The cell sheet manufacturing method according to the present embodiment is a method for manufacturing a cell sheet 10 of an adipose-derived cell group, and as shown in FIG. 1, a film forming step for coating the surface of a substrate 2 with a temperature-responsive polymer A. S1, antibody holding step S2 for holding the anti-CD90 antibody B on the polymer film, seeding step S3 for seeding the fat-derived cell group on the substrate 2, and exfoliating the fat-derived cell group in the form of a sheet on the substrate 2 Peeling step S4.

  In the film forming step S1, the substrate 2 and the temperature-responsive polymer A or a solution containing the monomer constituting the temperature-responsive polymer A are brought into contact with each other, and these are irradiated with electrons, γ-rays, ultraviolet rays, and plasma treatment. , By performing a corona treatment or by causing an organic polymerization reaction. As a result, the temperature-responsive polymer is bonded to the substrate surface, and the substrate surface is covered with the polymer film made of the temperature-responsive polymer. The film formation step S1 may be performed by applying or physically adsorbing a temperature-responsive polymer onto the substrate 2.

  The substrate 2 only needs to be able to stably hold the temperature-responsive polymer A on the surface thereof, and is usually made of glass, plastics such as polystyrene or polymethacrylate, or ceramics metals used for cell culture. It is done.

  As the temperature-responsive polymer A, one having an upper critical solution temperature or a lower critical solution temperature of 0 to 80 ° C., more preferably 20 to 50 ° C., is used. Thereby, at a temperature equal to or higher than the upper critical solution temperature or lower than the lower critical solution temperature, the polymer film is solid-phased and exhibits hydrophobicity, and the cells easily adhere to the polymer film. On the other hand, at a temperature lower than the upper critical solution temperature or higher than the lower critical solution temperature, the polymer film dissolves in water and exhibits hydrophilicity, and the cells are difficult to adhere to the polymer film.

If the upper or lower critical solution temperature of the temperature-responsive polymer A is lower than 0 ° C. or higher than 80 ° C., the influence on the cells in the seeding step S3 or the peeling step S4, which will be described later, is not preferable.
Examples of the temperature-responsive polymer A include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. A polymer obtained by polymerizing one or more kinds as monomers is used.

  The antibody holding step S2 is performed by binding the anti-CD90 antibody B to the polymer membrane via a water-soluble spacer. For example, biotin C and streptavidin D are used as spacers, both anti-CD90 antibody B and temperature-responsive polymer A are biotinylated, and anti-CD90 antibody B is bound to temperature-responsive polymer A via streptavidin D. Thereby, the anti-CD90 antibody B is exposed on the surface of the polymer film without being buried in the polymer film in a state where the polymer film is solid-phased.

  As shown in FIG. 2, the above-described film formation step S1 and antibody holding step S2 make the cell according to the present embodiment formed of a polymer film that covers the surface of the substrate 2 and holds the anti-CD90 antibody B on the surface. The support 1 is manufactured.

  The seeding step S3 is performed by seeding the fat-derived cell group on the substrate 2 coated with the cell support 1 at a temperature not lower than the lower critical solution temperature of the temperature-responsive polymer A or not higher than the upper critical solution temperature. . The adipose-derived cell group is separated from the adipose tissue by centrifuging a suspension of the adipose-derived cell group obtained by digesting adipose tissue collected from the living body with a digestive enzyme. The separated adipose-derived cell group includes vascular endothelial cells, fibroblasts, hematopoietic stem cells, hematopoietic cells, adipose-derived stem cells, and the like.

  The separated adipose-derived cell group is suspended in a medium or a buffer solution on the spot and seeded on the substrate 2, and then, for example, for several minutes to several tens of minutes, the lower limit critical lysis of the temperature-responsive polymer A is performed. It is allowed to stand while maintaining the temperature above the temperature or below the upper critical solution temperature. As a result, the cells settle on the substrate 2, and cells expressing CD90 on the cell membrane in the adipose-derived cell group bind to the surface of the cell support 1 via the anti-CD90 antibody B. At this time, by seeding a fat-derived cell group containing a sufficient number of cells so that the surface of the cell support 1 is densely covered with cells, the cells that are in close contact on the cell support 1 are horizontal to each other. Adhering in the direction, a sheet-shaped aggregate composed of adipose-derived cells is formed.

  In the peeling step S4, the temperature of the substrate 2 is set lower than the lower critical solution temperature of the temperature-responsive polymer A or higher than the upper critical solution temperature to dissolve the polymer film in the surrounding water, and the cell support 1 surface This is performed by peeling the aggregates in the form of a sheet. Thereby, as FIG. 3 shows, the cell sheet 10 which concerns on this embodiment is manufactured.

  Thus, according to the present embodiment, cells efficiently bind to the polymer membrane via the anti-CD90 antibody B, and a cell aggregate is rapidly formed. Therefore, the culture period of several days to about 1 week, which was conventionally required for spontaneously adhering the cells to the cell support 1 and the cells, becomes unnecessary. Thus, for example, after separating a fat-derived cell group from adipose tissue collected from a patient, the fat-derived cell group is produced on a cell sheet in a sufficiently short time on the spot and used for treatment. There is an advantage that a cell sheet can be rapidly produced even in the field.

  The separated fat-derived cell group includes a plurality of types of cells, and the adhesion to the cell support 1 varies depending on the type of cells. In addition, adhesion of cells to the cell support 1 is likely to be inhibited by adipose tissue residues or blood cells remaining in the adipose-derived cell group. However, there is an advantage that various cells can be efficiently bound to the cell support 1 by using an antibody having high specificity and strong binding force. In addition, by adopting anti-CD90 antibody B as the antibody, among cells contained in the adipose-derived cell group, cells that are considered to have a high therapeutic effect are selectively bound to the cell support 1 and the adipose-derived cell group There is an advantage that the cell sheet 10 having a high therapeutic effect can be produced.

Next, examples of the cell support, the cell sheet manufacturing method, and the cell sheet according to the above-described embodiment will be described.
The film forming step uses a polystyrene dish as a substrate, and contains a monomer solution containing 99 mol% poly-N-isopropylacrylamide (IPAAm) and 1 mol% 2-carboxyisopropylacrylamide (CIPAAm). This was performed by irradiating with an electron beam.

  As a result, IPAAm and CIPAAm are polymerized at a molar ratio of IPAAm: CIPAAm = 99: 1 to produce a temperature-responsive polymer, and the amino group end of the temperature-responsive polymer is covalently bonded to the bottom surface of the dish. A polymer film fixed on the bottom surface was formed. The produced temperature-responsive polymer had a lower critical solution temperature of 32 ° C. in water.

  Next, the antibody retention step was performed by binding an anti-CD90 antibody to the free end of the temperature-responsive polymer on the carboxyl group side. Specifically, 5- (biotinamide)-in which 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy-sulfosuccinimide (sulfo-NHS) were added to the dish. A solution of pentylamine was accommodated and reacted at 25 ° C. for 16 hours to bind biotin to the carboxyl group at the free end of the temperature-responsive polymer.

  Next, the solution in the dish was replaced with a solution of streptavidin and reacted at 25 ° C. for 1.5 hours to bind streptavidin to biotin. Next, the solution in the dish was replaced with a biotinylated anti-CD90 antibody solution and reacted at 25 ° C. for 1.5 hours to bind the anti-CD90 antibody to streptavidin. After each reaction, the dish was thoroughly washed with a buffer solution or the like to remove the reagent, and then replaced with a solution for the next reaction.

In addition, as an antibody retention step, the anti-CD90 antibody was bound to a temperature-responsive polymer using the following two types of fixation methods instead of the above-described fixation method using biotin and streptavidin.
The first immobilization method is a method using polyethylene glycol (PEG) as a linker for binding a temperature-responsive polymer and an anti-CD90 antibody. That is, a solution to which EDC and sulfo-NHS were added was placed in the dish and reacted at 25 ° C. for 16 hours to activate the side chain carboxyl group of the temperature-responsive polymer on the dish surface.

  Next, the solution in the dish was replaced with a solution of PEG having an amino group and a carboxyl group at both ends, respectively, and reacted at 37 ° C. for 16 hours to bind PEG to the activated carboxyl group. Next, the solution in the dish was replaced with a solution of EDC and sulfo-NHS and reacted at 25 ° C. for 16 hours to activate the carboxyl group at the PEG end. Next, the solution in the dish was replaced with an anti-CD90 antibody solution and reacted at 25 ° C. for 16 hours to bind the anti-CD90 antibody to the activated carboxyl group at the PEG end. After each reaction, the dish was thoroughly washed with a buffer solution or the like to remove the reagent, and then replaced with a solution for the next reaction.

The second fixing method is a method in which an anti-CD90 antibody is directly bound to a temperature-responsive polymer. That is, a solution to which EDC and sulfo-NHS were added was placed in the dish and reacted at 25 ° C. for 16 hours to activate the side chain carboxyl group of the temperature-responsive polymer on the dish surface. Next, the solution in the dish was replaced with an anti-CD90 antibody solution and reacted at 25 ° C. for 16 hours to bind the anti-CD90 antibody to the free end of the temperature-responsive polymer. After each reaction, the dish was thoroughly washed with a buffer solution or the like to remove the reagent, and then replaced with a solution for the next reaction.
Using the above three types of fixation methods, a cell support according to this example, which was made of a polymer membrane held with an anti-CD90 antibody exposed on its surface, was produced.

  The seeding step was performed by seeding a fat-derived cell group having a sufficient number of cells at 37 ° C. in a dish whose bottom surface was covered with the cell support according to the present example. After sowing, the dish was allowed to stand at 37 ° C. for 30 minutes. As a result, the adipose-derived cell group is precipitated in the dish, the cells expressing the CD90 antibody on the cell membrane are bound to the anti-CD90 antibody on the polymer membrane, and the cells that are in close contact in the planar direction on the bottom surface A sheet-shaped aggregate of adipose-derived cell groups was formed by adhering to each other.

  The exfoliation step was performed by lowering the dish temperature to 20 ° C. and sufficiently dissolving the polymer film in water, and then exfoliating the cell aggregates in the form of a sheet. Thereby, a cell sheet according to this example was obtained.

  As described above, according to the cell support and the cell sheet manufacturing method according to the present example, after seeding the adipose-derived cell group in the dish, the cells are collected into a sheet-like aggregate only by allowing the dish to stand for a sufficiently short time. The time required for production can be drastically shortened as compared with the conventional method for producing cell sheets.

DESCRIPTION OF SYMBOLS 1 Cell support 2 Substrate 10 Cell sheet A Temperature-responsive polymer B Anti-CD90 antibody C Biotin D Streptavidin S1 Membrane formation step S2 Antibody retention step S3 Seeding step S4 Peeling step

Claims (7)

  A cell support comprising a polymer membrane made of a temperature-responsive polymer having an upper critical solution temperature or a lower critical solution temperature of 0 to 80 ° C in water and holding an antibody against the cells.   The cell support according to claim 1, wherein the antibody is an anti-CD90 antibody.   The cell support according to claim 1, wherein the polymer membrane exposes the antibody on the surface thereof. A film forming step of forming a polymer film by coating the surface of the substrate with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water;
An antibody holding step for holding the antibody against the cell in the polymer film formed in the film forming step;
A seeding step of seeding the cells on the substrate on which the antibody is held in the polymer film in the antibody holding step, at a temperature equal to or lower than the upper limit critical lysis temperature or lower limit critical lysis temperature;
A cell sheet manufacturing method comprising, after the seeding step, a peeling step of peeling the cells from the substrate at a temperature higher than the upper critical solution temperature or lower than the lower critical solution temperature. The cell is a group of adipose-derived cells separated from adipose tissue;
The cell sheet production method according to claim 4, wherein the antibody is an anti-CD90 antibody.   The cell sheet production method according to claim 4, wherein the antibody holding step holds the antibody on the surface of the polymer film.   The cell sheet manufactured by the cell sheet manufacturing method in any one of Claims 4-6.

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