JP2014143970A - Method of producing sheet-like cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing stably a sheet-like cell culture.SOLUTION: The invention is a method of producing a sheet-like cell culture, the method including the steps of covering a cell culture substrate with vitronectin, culturing a cell on the cell culture substrate covered with vitronectin and forming a sheet-like cell culture of the cell, and separating the sheet-like cell culture from the cell culture substrate.

Description

  The present invention relates to a method for producing a sheet-like cell culture, and a sheet-like cell culture produced by the production method.

Regeneration using various cells (iPS cells, pluripotent stem cells, adult stem cells, somatic cells, etc.) for the purpose of regenerating and restoring the cells, tissues, and organs of the body lost due to accidents and diseases in recent years Medical care is attracting attention.
As a method for adapting desired cells to damaged tissues and organs, there is a method in which cells are directly injected into a cell suspension. However, in this method, the expected therapeutic effect cannot be realized due to problems such as outflow of transplanted cells from the tissue, obstacle due to puncture of the recipient tissue, and difficulty in extensive tissue repair.
As a method for solving these drawbacks, development of a sheet-shaped cell culture (also referred to as a cell sheet) in which desired cells are cultured in a sheet shape has been underway. Sheet-like cell cultures are very useful because they can transplant large numbers of desired cells to the site of injury and transplant a moderately organized cell population according to the characteristics of the recipient tissue. It is a simple treatment means.

  A sheet-shaped cell culture is obtained by culturing desired cells on the surface of a culture substrate and forming a layered structure of cells from the culture substrate so as not to break the structure. Manufactured by peeling. In general, in order to form a sheet-like structure by cell culture, cell culture is performed on the surface of a culture substrate coated with a component having a function of adhering cells to the substrate. Examples of such components include collagen, fibronectin, laminin, heparan sulfate proteoglycan, cadherin, gelatin, fibrinogen, fibrin, integrin, poly L lysine, hyaluronic acid, platelet-rich plasma, polyvinyl alcohol, and an antibody that recognizes a specific antigen. Etc. have been reported (Patent Documents 1 to 6). In addition, serum (heterologous serum, allogeneic serum, autologous serum, etc.) is also used as one containing a plurality of components having cell adhesiveness.

  Furthermore, in order to obtain a sheet-like cell culture that can be used for transplantation, it is necessary to peel the culture grown in the form of a sheet as a whole from the culture substrate without damaging the structure. A culture dish having a polymer layer of poly-N-isopropylacrylamide, which is a temperature-sensitive polymer, has been developed as a culture container for facilitating separation of a sheet-shaped culture from a culture substrate (patent) Reference 7).

  However, even in the case where the above-described components for adhering cells to a substrate or a culture dish having a temperature-sensitive polymer layer on the surface of a culture substrate are used, the sheet is formed from the step of forming a sheet-like cell culture and the culture substrate. It is difficult to reliably achieve the step of detaching without damaging the structure of the dendritic cell culture. As described above, although there are various reports so far, an important component that determines whether or not a sheet-shaped cell culture can be formed has not yet been elucidated, and a sheet-shaped cell culture used to treat damage to living tissue. There remains a problem to be solved regarding a method for stably producing a product.

JP2012-105587 JP2011-224398A JP 2009-178050 A JP2009-5639A JP 2006-238841 A JP 3-87174 JP2000-212144A

  The present invention provides a method for stably producing a sheet-shaped cell culture. In particular, the present invention provides a method for producing a sheet-shaped cell culture, comprising a step of easily peeling without damaging the structure of the sheet-shaped cell culture formed on a culture substrate.

  As a result of intensive research on the method for producing a sheet-shaped cell culture, the inventors cultured cells on a culture substrate coated with vitronectin to form a sheet-shaped cell culture. Thus, the present inventors have found that the sheet-like cell culture can be easily peeled off and can be recovered without impairing the sheet-like structure, and the present invention has been completed.

Accordingly, the present invention includes the following (1) to (5).
(1) A method for producing a sheet-shaped cell culture comprising the following steps (a) to (c).
(A) coating the culture substrate with vitronectin;
(B) culturing cells on a culture substrate coated with vitronectin to form a sheet-like cell culture of the cells;
(C) Step of peeling the sheet-shaped cell culture from the culture substrate (2) The cell culture substrate is coated with a cell adhesion component and / or a cell adhesion inhibitory component other than vitronectin. The method according to (1) above.
(3) The method according to (1) or (2) above, wherein the culture surface of the cell substrate is made of a temperature-responsive material or a non-temperature-responsive material.
(4) The method according to any one of (1) to (3) above, wherein the cell is a skeletal myoblast.
(5) A sheet-shaped cell culture produced by the method according to any one of (1) to (4) above.

According to the method of the present invention, a cell layer can be formed on a culture substrate, and the sheet-shaped cell culture can be collected without impairing the formed structure.
Therefore, a sheet-like cell culture can be obtained with a high recovery rate.

The present invention is a method for producing a sheet-shaped cell culture comprising the following steps (a) to (c).
(A) coating the culture substrate with vitronectin;
(B) culturing cells on a culture substrate coated with vitronectin to form a sheet-like cell culture of the cells;
(C) Step of peeling off the sheet-shaped cell culture from the culture substrate In the present invention, the “sheet-shaped cell culture” refers to a culture of cells in which cells are combined in a sheet shape. It may be composed of a cell layer or may be composed of two or more cell layers.
The present invention is characterized in that it includes the steps of coating the surface of the culture substrate cells with vitronectin, culturing the cells on the culture surface, and peeling the formed sheet-shaped cell culture. .
Here, the “culture substrate” may be any material as long as the cells can form a sheet-shaped cell culture on the surface, and at least a flat portion where the cells can adhere is used. It is typically a cell culture dish or cell culture bottle (or flask), and a commercially available culture dish or the like can be used, and the material is not particularly limited. Examples of the culture substrate material include polyethylene, polypropylene, polyethylene terephthalate, and the like.
The culture surface of the “culture substrate” is made of a material whose physical properties change due to a temperature change or the like (temperature responsive material), or the culture surface of the culture substrate is layered by the temperature responsive material. It may be coated. Here, the “temperature-responsive material” refers to a material that changes the adhesion of cells to the surface of a substrate depending on a change in temperature. Examples thereof include acrylamide derivatives such as poly-N-isopropylacrylamide. (See JP 2000-212144, for example).

The present invention includes a step of coating the cell culture surface of the culture substrate with vitronectin (covering the surface of the culture substrate uniformly with vitronectin).
Vitronectin is a cell adhesion factor that exists in serum and has a function of adhering cells to a culture substrate. The human-derived vitronectin has been clarified in amino acid sequence, and mature vitronectin is composed of 463 amino acids (the amino acid sequence of human-derived vitronectin is described in Suzuki et al., EMBO 4: 2519-2524 1985). This document is incorporated herein by reference). Vitronectin is produced mainly in the liver and exists in plasma, and is involved in the control of blood coagulation as well as cell adhesion. Integrin is known as a receptor for vitronectin, and functions as cell adhesion in cooperation with vitronectin. The vitronectin used in the present invention is preferably a polypeptide consisting of the amino acid sequence described in Suzuki et al., EMBO 4: 2519-2524 1985, but it is not necessarily a sequence that completely matches the amino acid sequence. There is no need, and as long as the function of adhering cells to the culture substrate is maintained, amino acid substitution, deletion, addition, and the like may be included. For example, in the amino acid sequence described in Suzuki et al., EMBO 4: 2519-2524 1985, one or several (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 A protein having an amino acid sequence in which (˜5) amino acids have been deleted, substituted or added and having a function of adhering cells to a culture substrate can also be used as vitronectin of the present invention.
In addition, it may be a naturally occurring vitronectin or a recombinant produced by a genetic engineering technique, and even if it has undergone post-translational modifications such as phosphorylation, the cells are allowed to adhere to the culture substrate. It can be used as long as it has a function. Furthermore, commercially available standards (for example, Becton, Dickinson and Company, etc.) may be used.

In order to coat the culture surface of the culture substrate with vitronectin, treatment may be performed so that the vitronectin is uniformly present on the culture surface. For example, a solution containing vitronectin is added to the culture surface (or contacted). It can also be achieved by leaving it to stand for an appropriate time. The time for adding and leaving the vitronectin solution is not particularly limited as long as the vitronectin is necessary to adhere to the culture surface of the cell culture substrate. It may be allowed to stand for about 72 hours or about 12 to 72 hours. Further, the temperature at which the solution of vitronectin is added and allowed to stand is not particularly limited. For example, 0 to 60 ° C, 30 to 40 ° C, 35 to 38 ° C, preferably about 37 ° C. Good. In order to coat the culture surface of the cell culture substrate, an appropriate amount of vitronectin solution can be added to the culture surface.
In addition, the step of coating the culture substrate with vitronectin involves mixing cells and vitronectin solution (which may contain other adhesive components) without previously coating the culture surface of the culture substrate with vitronectin alone. After the suspension, coating may be performed by seeding the suspended cells on the culture surface on the cell culture substrate. In this case, the vitronectin solution and the cells are suspended and then allowed to stand (for example, 0 to 50 ° C., preferably about 30 to 40 ° C. for about 0 to 24 hours), and then the suspended cells are seeded. May be.
When cells are seeded after previously covering the culture substrate with vitronectin (or vitronectin and cell adhesion component and / or cell adhesion inhibitory component), a solution containing no vitronectin and other cell adhesion components before seeding ( For example, the cells may be washed with a culture solution, PBS, physiological saline, or the like).
By culturing cells on the culture surface of a culture substrate coated with vitronectin, a sheet-like cell culture that can be detached from the culture surface can be obtained.

  In the present invention, a cell culture substrate whose culture surface is coated with vitronectin is used. On the culture surface of this cell culture substrate, there are cell adhesion components and / or cell adhesion inhibitory components other than vitronectin. May be present. The cell adhesive component other than vitronectin may be any component that is commonly used for adhering cells to the culture surface in cell culture technology. For example, collagen, fibronectin, laminin, heparan sulfate proteoglycan, cadherin Gelatin, fibrinogen, fibrin, poly-L-lysine, hyaluronic acid, platelet-rich plasma, polyvinyl alcohol and the like. As the cell adhesion-inhibiting component other than vitronectin, any component that is usually used for adhering cells to the culture surface in the cell culture technique may be used, and examples thereof include albumin and globulin. When the cell surface of the cell culture substrate is coated with a cell adhesion component or cell adhesion inhibitory component other than these vitronectins, it can be basically performed by the same method as the method of coating with vitronectin. However, since the concentration of the solution used to coat the culture surface differs depending on each component, the solution concentration for coating each component can be determined by methods that can be easily studied by those skilled in the art, such as preliminary experiments. Can be determined.

The method for producing the sheet-shaped cell culture of the present invention can be used for cells derived from any animal species and tissue. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, mesenchymal stem cells, embryonic stem cells, iPS cells, synovial cells, epithelial cells ( Examples thereof include corneal epithelial cells, oral mucosal epithelial cells), endothelial cells, hepatocytes, pancreatic cells, periodontal ligament cells, skin cells, and the like. In addition, these cells were produced through a process of isolating desired cells from tissues or biological fluids collected from the subject, a process of growing the isolated cells, and a process of differentiating the expanded cells into specific cells. Or a commercially available cell line already established.
Only one type of cell may be used for forming the sheet-shaped cell culture, but two or more types of cells may be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a cell culture, the most cell ratio (purity) is 65% or more, preferably 70% or more at the end of cell culture production. Preferably it is 75% or more.
The process of culturing cells to produce a sheet-shaped cell culture can be performed by a normal culture method suitable for each cell. Moreover, it can seed | inoculate at the cell density in which a sheet-like culture is formed, without proliferating a cell. The seeding density is preferably such that the cells do not shift to a differentiated state, and is in the range of about 3.0 × 10 ⁵ cells / cm ^{2 to} 3.5 × 10 ⁶ cells / cm ² . The number of cells after forming the sheet-shaped cell culture is, for example, 300% or less, preferably 200% or less, more preferably 50% or less, and still more preferably about 100% of the number of cells at the time of seeding. When the cells are skeletal myoblasts, for example, the method described in JP2011-110368 can be used.

As the medium used in the method for producing a sheet-shaped cell culture of the present invention, a medium suitable for the cells to be cultured can be appropriately selected and used. For example, MEM, DMEM, F12, IMEM, RPMI-1640 etc. can be mentioned as a culture medium which can be generally used. You may purchase and use these culture media. Moreover, these culture media may be used independently or may be used in combination of 2 or more types.
Furthermore, you may use for a culture medium, adding an appropriate additive as needed. Examples of the additive include amino acids (for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-trionine, L-tryptophan, L-tyrosine, L-valine, L-alanyl-L-glutamine, etc.), vitamins (eg, folic acid, riboflavin, thiamine, etc.), D-glucose, pyrubin It may contain sodium acid. Moreover, you may add buffer (liquid), such as PBS, HEPES, and HANK'S, to a culture medium suitably. Furthermore, a cell growth factor or the like may be added as appropriate according to the characteristics of the cells to be cultured.

Cell culture for forming a sheet-like structure of cells can be performed under conditions ordinarily performed in the technical field. For example, the culture temperature can be 30 to 40 ° C., preferably 36 to 38 ° C., and the CO ₂ concentration can be 0 to 10%, preferably 4 to 6%. However, this condition is not limited. Depending on the characteristics of the cells to be cultured, the culture temperature and CO ₂ concentration can be appropriately selected. The culture time is not particularly limited as long as it is a time necessary for forming a desired sheet-shaped cell culture. For example, the culture time is about 10 hours to 30 hours, about 12 hours to 26 hours. It can be illustrated.

After the cell culture, a sheet-like cell culture having a desired shape, structure, etc. is formed, and then the sheet-like cell culture is peeled off from the culture surface of the cell culture substrate, and the sheet-like cell culture is recovered. I do. The peeling of the sheet-shaped cell culture from the culture surface can be performed by a method that does not damage the sheet-shaped structure. In the present invention, since the culture surface is coated with vitronectin, the sheet-like cell culture can be easily detached. For example, the sheet-like cell culture is directly picked with tweezers and detached from the culture surface. can do. Furthermore, if necessary, a process of mechanically separating the cells from the culture surface by pipetting may be included. Alternatively, as long as the sheet-like structure is not damaged, an enzyme treatment such as trypsin or collagenase may be performed, and an appropriate method can be selected according to the properties of the cells. Alternatively, a substrate having affinity for cells (eg, PVDF membrane, nitrocellulose membrane, fibrin gel, collagen gel, etc.) is placed on the upper surface of the sheet-shaped cell culture, and the cells are detached by copying the cells to the membrane. It can also be recovered.
When a cell culture substrate whose surface is coated with a temperature-responsive material (for example, an acrylamide derivative such as poly-N-isopropylacrylamide) is used, the temperature of the container is lowered to, for example, about 0 to 30 ° C. Later, the cells may be detached and collected. Alternatively, a cell culture substrate whose surface is made of a material made of a non-temperature-responsive material can be used.

  The present invention also includes a sheet-shaped cell culture produced by the method of the present invention. Since the sheet-shaped cell culture is cultured and manufactured on a culture surface coated with vitronectin, vitronectin is attached to the culture-side surface of the sheet-shaped cell culture. Such a sheet-shaped cell culture requires less mechanical load on the cells upon detachment, so that viability and cytokine-producing ability are higher than those produced on a culture surface that is not coated with vitronectin. Therefore, it can be expected to have an excellent therapeutic effect.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

  Human skeletal myoblasts were grown in MCDB131 medium (Life Technology Japan) containing 20% (v / v) FBS, recovered, and then frozen using a cryopreservation solution containing 10% DMSO (Pure Chemical). Cryopreserved. The cryopreserved cells were thawed in a water bath set at about 37 ° C. and washed with a buffer containing 0.5% human serum albumin and used in the following examples.

Example 1
Various cell adhesion components were coated on the culture surface of the cell culture substrate, and the formation and recovery status of the sheet-shaped cell culture were compared.
Collagen solution (3%, 0.3% and 0.03%) (Nitta Gelatin), laminin solution (10 μg / mL, 1 μg / mL and 150 ng / mL) (Nippon Becton Dickinson), fibronectin solution (20 μg / mL, 12 μg / mL) (mL, 6 μg / mL and 1 μg / mL) (Nippon Becton Dickinson), vitronectin solution (60 μg / mL to 1.5 μg / mL) (AGC Techno Glass) 500 μL each in a temperature-responsive culture dish (24 well, well bottom area) 1.9 cm ² ) (Cellseed) and incubated for 12 to 72 hours (set values: 37 ° C., 5% CO ₂ ). After removing each culture medium from the temperature-responsive culture dish, 2.0 × 10 ⁶ skeletal myoblasts are suspended in 500 μL of each solution or basal medium (DMEM / F12: Life Technology Japan) and temperature-responsive culture is performed. The whole amount was added to the dish and cultured in a CO ₂ incubator (setting value: 37 ° C., 5% CO ₂ ) for 16 to 26 hours. After culture, remove the supernatant from the temperature-responsive culture dish, add 1 mL of Hanks' Balanced Salt Solutions (HBSS: Life Technology Japan), and then pipette the cell layer formed on the temperature-responsive culture dish. It peeled.
As a result, a sheet-like cell culture was obtained only from 1.5 μg / mL to 60 μg / mL of vitronectin solution. Under other conditions, a cell layer was not formed on the surface of the culture substrate, or the cell layer was used as a culture medium. The cell layer formed in the step of peeling from the material was damaged, and the desired sheet-like culture could not be obtained (see Table 1).

Example 2
Next, the formation and recovery of sheet-like cell cultures were examined when cell adhesion components other than vitronectin were used together with vitronectin.
Add 500 μL of the mixture of fibronectin and albumin and the mixture of vitronectin and albumin shown in Table 2 to a temperature-responsive culture dish (24 well) and incubate for 12 to 72 hours (setting value: 37 ° C., 5% CO ₂ ). did. After removing each culture solution from the temperature-responsive culture dish, suspend 2.0 × 10 ⁶ skeletal myoblasts in 500 μL of each solution and add the total amount to the temperature-responsive culture dish for 16 to 26 hours. _The cells were cultured in an incubator (set values: 37 ° C., 5% CO ₂ ). After culturing, the supernatant was removed from the temperature-responsive culture dish, 1 mL of Hanks' Balanced Salt Solutions (HBSS) was added, and then the cell layer formed on the temperature-responsive culture dish was detached.
As a result, the fibronectin solution failed to achieve the step of forming a cell layer on the surface of the culture substrate due to the cell adhesion inhibitory effect of albumin, whereas vitronectin did not show the cell adhesion inhibitory effect, and the sheet under any conditions The cell culture was peeled off and obtained.

Example 3
500 μL of the culture solution described in Table 3 (FN: fibronectin, VIN: vitronectin, ALB: albumin) is added to each temperature-responsive culture dish (24 well) and incubated for 12 to 72 hours (setting value: 37 ° C., 5% CO ₂ ). After each culture medium was removed from the temperature-responsive culture dish, 500 μL of each culture liquid in which 2.0 × 10 ⁶ skeletal myoblasts were suspended was added to the temperature-responsive culture dish, and the CO ₂ was added for 16 to 26 hours. The cells were cultured in an incubator (setting values: 37 ° C., 5% CO ₂ ). After culturing, the supernatant was removed from the temperature-responsive culture dish, 1 mL of Hanks' Balanced Salt Solutions (HBSS) was added, and then the cell layer formed on the temperature-responsive culture dish was detached.
As a result, the cell sheet was peelable when 300 ng / mL vitronectin was contained in the presence of fibronectin and albumin and 600 ng / mL in the presence of fibronectin.

  From the above results, the cells were cultured with a solution containing at least 300 ng / mL, preferably 600 ng / mL, and more preferably 1.5 μg / mL vitronectin, or a culture medium coated with a vitronectin solution corresponding to the above concentration. By culturing cells on the culture surface on the material, even if other components that enhance cell adhesion, such as fibronectin, are contained, the sheet cell culture is surely detached from the culture surface and prepared. It was possible.

  Since the present invention provides a method for reliably and stably producing a desired sheet-shaped cell culture, the present invention is highly applicable in the field of regenerative medicine and contributes to the development of this field.

Claims (5)

A method for producing a sheet-shaped cell culture comprising the following steps (a) to (c):
(A) coating the culture substrate with vitronectin;
(B) culturing cells on a culture substrate coated with vitronectin to form a sheet-like cell culture of the cells;
(C) Step of peeling the sheet-shaped cell culture from the culture substrate   The method according to claim 1, wherein the cell culture substrate is coated with a cell adhesion component and / or a cell adhesion inhibitory component other than vitronectin.   The method according to claim 1 or 2, wherein the surface of the cell substrate is made of a temperature-responsive material or a non-temperature-responsive material.   The method according to any one of claims 1 to 3, wherein the cell is a skeletal myoblast.   A sheet-like cell culture produced by the method according to claim 1.