JP2014138557A - Method for producing sheet-like cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a good sheet-like cell culture easily and rapidly.SOLUTION: The above subject is solved by a method for producing a sheet-like cell culture comprising seeding and incubating a cell on a culture substrate having an approximate plane-like surface in which a cell non-adhesive region and a cell adhesive region form a pattern shape.

Description

  The present invention relates to a method for easily producing a sheet-like cell culture.

  Despite recent advances in the treatment of heart disease, a treatment system for severe heart failure has not yet been established. For the treatment of heart failure, medical treatment with β-blockers or ACE inhibitors is performed. For heart failure that has become so severe that these treatments are not successful, replacement treatment such as auxiliary artificial heart and heart transplantation, that is, surgical treatment Is done.

Severe heart failure that is the subject of such surgical treatment includes those caused by advanced valvular disease and severe myocardial ischemia, acute myocardial infarction and its complications, acute myocarditis, ischemic cardiomyopathy (ICM), dilation There are a wide variety of causes such as chronic heart failure due to dilated cardiomyopathy (DCM) and acute aversion.
Depending on these causes and severity, valvuloplasty and replacement, coronary artery bypass surgery, left ventricular plastic surgery, mechanical assisted circulation, etc. are applied.

  Of these, only heart transplantation or replacement treatment with an artificial heart has been regarded as an effective treatment for those who have suffered heart failure due to a severe decrease in left ventricular function caused by ICM or DCM. However, replacement therapy for these patients with severe heart failure has many problems to be solved, such as chronic donor shortages, the need for continuous immunosuppression, and the development of complications. Is hard to say.

On the other hand, recently, development of new regenerative medicine is considered indispensable as a solution for the treatment of severe heart failure.
In severe myocardial infarction and the like, cardiomyocytes become dysfunctional, and fibroblast proliferation and interstitial fibrosis progress, resulting in heart failure. As the heart failure progresses, the cardiomyocytes are damaged and fall into apoptosis, but the cardiomyocytes hardly undergo cell division, so the number of cardiomyocytes decreases and the cardiac function further decreases.
Cell transplantation is considered useful for the recovery of cardiac function in such patients with severe heart failure, and clinical application with autologous skeletal myoblasts has already been started.

  In recent years, as an example, a three-dimensional cell culture that can be applied to the heart including cells derived from parts other than the adult myocardium by using a temperature-responsive culture dish that applies tissue engineering, and its A manufacturing method was provided (Patent Document 1).

  Patent Document 2 discloses a cell culture support that is less damaged, easily peels off, and does not slow down the sheet formation speed by forming a concave-convex structure having a concave portion with a size that prevents cells from entering the cell culture surface. Have been described. Patent Document 3 discloses a cell culture substrate having a cell adhesion region and a cell adhesion inhibitory region. When cells are cultured using the culture substrate, the cells adhere to only the cell adhesion region, thereby patterning the cells. It is described that can be performed.

Special table 2007-528755 gazette JP 2008-11766 A JP 2007-312736 A

  In the method for producing a high-quality and safe sheet-like cell culture, the physical strength of the sheet and the speed of sheet formation are important factors, but the ease of peeling is also an important factor. In particular, in the production of a fragile sheet-shaped cell culture, there are many cases in which the sheet-shaped cell culture is damaged in the peeling process. Accordingly, there is a need for a culture substrate that can easily peel a cultured sheet-like cell culture or a method that allows easy peeling. An object of the present invention is to provide a method for quickly and easily producing a high-quality and safe sheet-like cell culture.

  When the present inventors performed sheet culture in a dish in which serum is plotted at a certain interval on a non-adhesive surface to form an adhesive region, the cells are not affected even if the cell non-adhesive region exists. It was found that a uniform single sheet was formed without patterning, and that the sheet was easily peelable. As a result of further earnest research based on this knowledge, the present invention has been completed.

That is, the present invention relates to the following.
[1] A method for producing a sheet-shaped cell culture, in which cells are seeded on a culture substrate having a substantially planar surface in which a cell non-adhesive region and a cell adhesive region form a pattern shape. Incubating said method.
[2] The method according to [1], further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.
[3] The interval in at least a part between one cell adhesion region and another cell adhesion region adjacent to the cell adhesion region is larger than the major axis of the seeded cells [1] or [2] the method of.
[4] The method of [1] to [3], wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.
[5] The method of [1] to [4], wherein the cell is a skeletal myoblast.
[6] The method according to [1] to [5], wherein the pattern shape is a sea-island structure in which either one of the cell non-adhesion region or the cell adhesion region is an island and the other is the sea.
[7] The method of [6], wherein the cell adhesion region is an island and the cell non-adhesion region is the sea.
[8] The method according to [1] to [7], wherein the ratio of the cell adhesive region is 4% to 65% of the entire surface.

  According to the present invention, since a sheet-like cell culture having sufficient physical strength that can withstand a washing operation such as immersion stirring and a transplanting operation such as removal, holding, and transfer is obtained, a cell sheet obtained by a conventional production method This makes it possible to perform the manufacturing / transplanting operation more easily and reliably. In addition, since the sheet-like cell culture can be obtained with a high cell recovery rate by the production method of the present invention, the cells to be used can be effectively used, which contributes to cost reduction and the like. In addition, the sheet-like cell culture obtained by the production method of the present invention has a high production of factors having an effect on the effectiveness such as angiogenesis, while the production of inflammatory cytokines is low, so that it is therapeutically effective. Sexuality and safety to recipients are extremely high and are clinically very useful. For this reason, a great contribution can be expected in human medicine and veterinary medicine. Furthermore, according to the production method of the present invention, the time required to form a sheet is short and can be easily peeled off, thereby reducing the risk of sheet breakage in the production process and producing a large amount of high-quality sheet-like cell cultures. It is suitable for manufacturing.

FIG. 1 is a photograph of a sheet-like cell culture obtained when an adhesive region is formed on a non-adhesive region and sheet culture is performed for 6 hours. It can be seen that a uniform sheet-shaped cell culture without wrinkles, kinks and breakage is obtained. FIG. 2 is a photograph of a sheet-like cell culture obtained when a non-adhesive region is formed on an adhesive region and sheet culture is performed for 6 hours. It can be seen that a uniform sheet-shaped cell culture without wrinkles, kinks and breakage is obtained.

The present invention relates to a sheet-like cell culture comprising seeding and incubating cells on a culture substrate having a substantially planar surface in which a cell non-adhesive region and a cell adhesive region form a pattern shape. It relates to a manufacturing method.
In the present invention, the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are made. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it is a substance capable of mechanically connecting cells to each other, and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, in particular, derived from the cells constituting the cell culture. The cells are at least mechanically linked, but may be further functionally, eg, chemically or electrically linked.

  The sheet-shaped cell culture of the present invention preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF) Although manufactured membranes are known, the cell culture of the present invention can maintain its physical integrity even without such a scaffold. In addition, the cell culture of the present invention preferably consists only of substances derived from the cells constituting the cell culture and does not contain any other substances.

In the present invention, “cell adhesion” means that adherent cells can adhere or are easily adhered. Thus, conversely, “cell non-adhesive” means that adherent cells are not adherent or difficult to adhere. In the present invention, the “cell adhesive region” means a portion that is cell adhesive on the culture surface of the culture substrate, and the “cell non-adhesive region” means a non-cell on the culture surface of the culture substrate. It means the part that is adhesive. Accordingly, in the present invention, the term “cell adhesive surface” is used synonymously with “cell adhesive region”, and similarly the term “cell nonadhesive surface” is used synonymously with “cell nonadhesive region”. .
It is known that adherent cells are difficult to adhere if the surface or surface to be contacted has a higher hydrophobicity or hydrophilicity than a certain level. Therefore, in one embodiment of the present invention, the non-cell-adhesive surface has a certain degree of hydrophobicity or hydrophilicity. The degree of hydrophobicity or hydrophilicity of the surface can be expressed by, for example, a water contact angle. In the present invention, the water contact angle of the non-cell-adhesive surface is preferably 70 ° or more or 50 ° or less, particularly 75. It is more than or less than 40 degrees.

  The culture substrate used in the method of the present invention has a cell non-adhesive region and a cell adhesive region on its surface. The cell non-adhesive region and the cell adhesive region form a pattern shape. In the present invention, the “pattern shape” means a state in which both regions are dispersed and present over the entire culture surface with a certain degree of variation, and examples include a repeated pattern, but it is necessary to form a repeated pattern. There is no. Examples of the pattern shape include, but are not limited to, a sea island shape, a lattice shape, a stripe shape, a concentric circle shape, a radial shape, a checkered shape, or a combination of one or two or more thereof.

You may form the pattern shape of a cell non-adhesion area | region and a cell adhesion area | region using the arbitrary methods known in the said technical field. Examples of methods for forming a pattern shape include, but are not limited to, a method of applying a cell adhesive component to a cell non-adhesive culture surface, and a cell non-adhesive component to a cell adhesive culture surface. Examples thereof include a method of applying, and a method of performing cell adhesion treatment on a non-cell-adhesive culture surface. The cell adhesion treatment is not limited to this, and examples thereof include plasma treatment (charge treatment).
“Cell-adhesive component” means any component to which cells can adhere. Conversely, the “cell non-adhesive component” means any component to which cells cannot adhere or are difficult to adhere. Examples of cell adhesion components include, but are not limited to, serum / extracellular matrix components such as fibronectin, vitronectin, ramenin, collagen, proteoglycan, cell adhesion peptides such as RGD and CS-1, polylysine, polyethyleneimine And polymer compounds such as polyallylamine. Examples of the cell non-adhesive component include hydrogels such as polyhydroxyethyl methacrylate (polyHEMA) and 2-methacryloyloxyethylphosphorischoline (MPC).

  In one embodiment of the present invention, the culture surface of the culture substrate may have an uneven shape, but preferably the culture surface is substantially planar. In the present invention, “substantially planar” means a state where there is no difference in level that hinders cell-cell adhesion and uniform distribution of cells, and is not limited thereto, but has, for example, irregularities of 10 μm or more. There is nothing.

  The incubation in the method of the present invention is an incubation for forming a sheet-like cell culture. Therefore, any conditions can be used as long as they can form cell-cell adhesion, but usually the same conditions as general cell culture conditions may be used. Any person who has ordinary knowledge in the art can select optimal conditions according to the type of cells to be seeded.

  In the method of the present invention, cells form an intercellular adhesion without patterning even on a non-adhesive region, and a sheet-like cell culture can be produced. This fact was thought to be due to the fact that cells cannot normally adhere to non-cell-adherent areas, so that it was thought that cell-cell adhesion could not be formed sufficiently. In view of the fact that the technology for forming the sex region and the cell adhesion region on the culture surface has been known (see Patent Document 3), it is surprising.

  In the method of the present invention, since the formed sheet-shaped cell culture and the culture substrate are bound only by the cell adhesive region on the culture surface, the sheet-shaped cell culture formed by the conventional method Compared with, the adhesive force with the culture surface is weak. Therefore, when the cells are cultured for a long time, the cell-cell binding force of the sheet-shaped cell culture exceeds the binding force between the sheet-shaped cell culture and the culture surface, and peeling occurs naturally. Therefore, in one embodiment of the present invention, incubation may be performed until separation occurs spontaneously. However, the peeled sheet-shaped cell culture is very fragile, and if the incubation is continued as it is, the cell-like cell culture may cause wrinkles or wrinkles due to the contraction of the sheet-shaped cell culture, or it will stick in a folded state. Therefore, it is preferable to actively perform the peeling before the peeling naturally occurs.

  As described above, since the incubation of the present invention is an incubation for forming a sheet, cell proliferation does not need to occur. Therefore, the incubation may be performed for a time sufficient for the formation of cell-cell adhesion. The incubation time is preferably 1 to 24 hours, more preferably 2 to 12 hours. If it is shorter than 1 hour, cell-cell adhesion is not sufficiently formed, so that the sheet formation becomes insufficient. If it is longer than 24 hours, the peeled sheet-shaped cell culture may wrinkle or wrinkle, or the shape of the sheet may collapse. End up.

  In a preferred embodiment of the present invention, the distance between one cell adhesion region and another cell adhesion region adjacent to the cell adhesion region is larger than the major axis of the cells to be seeded in at least one place. That is, in order to form a sheet on the entire culture substrate surface without breakage, it is necessary to cover the cell non-adherent region without any gap, and for that purpose, at least one cell non-adherent region requires two or more cells. It becomes. More preferably, at least one cell is present in at least one cell non-adherent region. The presence of at least one cell in the cell non-adherent region means that there is at least one cell that is not in contact with the cell adhesive region among the cells in contact with the cell non-adherent region. According to the method of the present invention, even if cells that are not in contact with the cell adhesion region exist, it is possible to form a single sheet that is not damaged on the entire surface of the culture substrate.

  In a preferred embodiment of the invention, the seeded cells do not substantially grow during the incubation. “Substantially does not proliferate” means that the cell does not proliferate beyond the range of measurement error, and whether or not the cell has proliferated is, for example, the number of cells at the time of seeding and the number of cells after formation of the cell culture. And can be evaluated by comparing. In the present invention, the number of cells after forming the sheet-shaped cell culture is typically 300% or less of the number of cells at the time of seeding, preferably 200% or less, more preferably 150% or less, more preferably 125% or less, Particularly preferably, it is 100% or less.

  Whether or not the cells substantially proliferate is determined by various conditions, for example, the number of seeded cells (seeded cell density), a medium, incubation conditions, and the like. In the present invention, it is necessary for the seeded cells to form a sheet-like cell culture, and it is necessary to control proliferation without reducing the biological activity of the cells. It is preferable to control. Thus, in a preferred embodiment of the present invention, the cells are seeded at a density that can form a sheet cell culture without substantial growth.

“The density at which a sheet-like cell culture can be formed without substantially growing” means that a sheet-like cell culture can be formed when cultured in a non-proliferating culture medium that does not contain growth factors. Refers to cell density. For example, in the case of skeletal myoblasts, in a method using a culture solution containing a growth factor, cells having a density of about 6,500 cells / cm ² were seeded on a plate in order to form a sheet-like cell culture. However, even if cells having such a density are cultured in a culture solution not containing a growth factor, a sheet-like cell culture cannot be formed. Accordingly, the seeding density of the sheet-forming cells in the present invention is higher than that in a method using a culture solution containing a growth factor, that is, in a culture in which cell proliferation is at least a part of the purpose. Specifically, for example, for skeletal myoblasts, such density is typically 300,000 cells / cm ² or more. The upper limit of the cell density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not shift to differentiation, but for skeletal myoblasts, for example, it is less than 3.4 × 10 ⁶ cells / cm ^2. . A person skilled in the art can appropriately determine the cell density suitable for the present invention by experiments. During the culture period, the cells may or may not proliferate, but even if they proliferate, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts start to differentiate when they become confluent, but in the present invention, skeletal myoblasts are seeded at a density that forms a sheet-like cell culture but does not shift to differentiation.

The seeding density of cells in the production method of the present invention is 3.0 × 10 ^{5 to} 3.4 × 10 ⁶ cells / cm ² in one embodiment, and 3.5 × 10 ^{5 to} 3.4 × 10 ^{6 in} another embodiment. Pieces / cm ² , in yet another aspect, 1.0 × 10 ^{6 to} 3.4 × 10 ⁶ pieces / cm ² , and in yet another aspect, 3.0 × 10 ^{5 to} 1.7 × 10 ⁶ pieces / cm ^2. In another embodiment, it may be 3.5 × 10 ^{5 to} 1.7 × 10 ⁶ pieces / cm ² , and in another embodiment, 1.0 × 10 ^{6 to} 1.7 × 10 ⁶ pieces / cm ² . As long as an upper limit is less than 3.4 * 10 < ⁶ > piece / cm < ² >, the said range may include both an upper limit and a lower limit, or any one thereof. Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is, for example, 3.0 × 10 ⁵ cells / cm ² or more and less than 3.4 × 10 ⁶ cells / cm ² (including the lower limit and not including the upper limit). ), 3.5 × 10 ⁵ pieces / cm ² or more and less than 3.4 × 10 ⁶ pieces / cm ² (including the lower limit, not including the upper limit), 1.0 × 10 ⁶ pieces / cm ² or more and 3.4 × 10 less than ⁶ / cm ² (including the lower and the upper limit is not included), 1.0 × 10 ⁶ cells / cm ² ultra 3.4 × 10 than ⁶ / cm ² (lower limit also contains no upper limit), 1 It may be more than 0.0 × 10 ⁶ pieces / cm ^{2 and} 1.7 × 10 ⁶ pieces / cm ² or less (not including the lower limit but including the upper limit). A person skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments according to the teaching of the present specification.

  As described above, the incubation of the present invention is an incubation for forming a sheet-like cell culture, and thus the cell is a cell capable of forming a sheet-like cell culture. In addition, the incubation for forming the sheet-shaped cell culture may be simply referred to as “sheet culture”. Specific examples of the cells include, but are not limited to, skeletal myoblasts, skin cells, corneal epithelial cells, periodontal ligament cells, cardiomyocytes, hepatocytes, pancreatic cells, oral mucosal epithelial cells, and the like. Preferably, skeletal myoblasts are used.

  In a preferred embodiment of the present invention, the pattern shape is a sea-island structure. In the present specification, the “sea-island structure” refers to a structure in which one region that is relatively continuous (referred to as “the sea”) is discontinuously mixed with the other region (referred to as “island”). . In this specification, a shape having a “sea-island structure” may be expressed as a “sea-island shape”. The sea side region does not necessarily need to be completely continuous as long as it is relatively continuous. However, from the viewpoint of ease of separation of the formed sheet-shaped cell culture, the island side region is a part of the culture surface. It is not preferable to be unevenly distributed in the region of the part.

  In the sea-island structure of the present invention, the cell non-adhesive region may be the sea or the cell adhesive region may be the sea. Any method known in the art can be used as a method for forming the sea-island structure, and the method is not limited to this. For example, a portion of the serum is plotted by spotting on a non-cell-adhesive substrate. And a method of partially coating a non-cell-adhesive polymer in a spot shape on a cell-adhesive substrate. However, in view of ease of formation, ease of peeling after formation of the sheet-shaped cell culture, and the like, the cell adhesion region is preferably an island.

  If the cell adhesion region existing on the culture surface of the present invention is too wide, the adhesion with the sheet-shaped cell culture is increased, which is convenient for the formation of the sheet, but the peelability of the sheet-shaped cell culture is reduced. . On the other hand, if it is too narrow, the adhesiveness with the sheet-shaped cell culture is lowered, so that it becomes difficult to form the sheet-shaped cell culture, but if formed, peeling becomes easy. Accordingly, the cell adhesion region of the present invention is preferably 4% to 65% of the entire surface, more preferably 8% to 52%.

  The present invention also relates to a sheet-like cell culture produced by the production method. The sheet-shaped cell culture of the present invention has sufficient physical strength for washing operations such as immersion stirring and transplantation operations such as removal, holding and transfer. Having sufficient physical strength means that even if the above operation is performed, the sheet-like structure of the cell culture is not impaired. For example, the obtained sheet-like cell culture is actually subjected to the above operation. It can be confirmed by conducting a macroscopic or microscopic investigation that the sheet-like structure is maintained. Here, the washing operation by immersing and stirring typically means adding a sufficient amount of a buffer solution in which the culture is immersed in a culture vessel containing a sheet-like cell culture, and stirring and agitating the whole culture vessel. Say. Therefore, having sufficient physical strength can also be confirmed by applying physical stress similar to the washing operation by soaking and stirring to the sheet-like cell culture. In addition, a sheet-like cell culture produced by the above production method usually has sufficient physical strength.

  Based on the following examples, the present invention will be described in more detail with reference to specific embodiments, but the present invention is not limited to these specific examples.

Example 1
A non-adherent surface of a Petri dish (BD, code: 35-1008) for floating cells having a cell non-adhesive surface, with 20% FBS-containing MCDB131 medium having a diameter of about 1 mm at intervals of 4 mm. Plotted and incubated for 40 hours to form an adhesive surface. The ratio of adhesive surface to total culture surface was about 10%. Cells suspended in MCDB131 medium containing 20% (v / v) FBS were seeded in a dish at 9.3 × 10 ⁶ cells / plate. After culturing the cells at 37 ° C. and 5% CO ₂ for 6 hours, the cell culture formed into a sheet by pipetting was detached from the plate.
As shown in FIG. 1, those cultured for 6 hours can be easily detached by pipetting, so that a high quality sheet-shaped cell culture with few wrinkles was obtained. In addition, when cultured for 15 hours under the same conditions, the sheet-like cell culture was formed and spontaneously detached without pipetting, but it was wrinkled or wrinkled.

Example 2
A non-adhesive polymer (poly-HEMA) is plotted on the adhesive surface of a multiwell plate for cell culture (BD, code: 353046) at an interval of 4 mm so as to have a diameter of about 3 mm, and dried at room temperature for about 1 hour. I let you. The ratio of the adhesive surface to the entire culture surface was 60% or less. Cells suspended in 20% (v / v) FBS-containing MCDB131 medium were seeded in a dish at 9.3 × 10 ⁶ cells / plate. After culturing the cells at 37 ° C. and 5% CO ₂ for 6 hours, the cell culture formed into a sheet by pipetting was detached from the plate.
As shown in FIG. 2, those cultured for 6 hours can be easily detached by pipetting, so that a high quality sheet-shaped cell culture with few wrinkles was obtained.

  According to the present invention, it is possible to easily and quickly produce a high-quality sheet-shaped cell culture having sufficient physical strength that can withstand a washing operation such as immersion stirring and a transplanting operation such as removal, holding, and transfer. It becomes. In addition, when the method of the present invention is used, the peeling process that is most likely to cause damage to the sheet-shaped cell culture in the normal manufacturing process can be performed very easily or does not need to be performed. Cell culture can be produced.

Claims (8)

  A method for producing a sheet-shaped cell culture, in which cells are seeded and incubated on a culture substrate having a substantially planar surface in which a non-cell-adhesive region and a cell-adhesive region form a pattern shape Said method.   The method according to claim 1, further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.   The method according to claim 1 or 2, wherein an interval in at least a part between one cell adhesion region and another cell adhesion region adjacent to the cell adhesion region is larger than a major axis of cells to be seeded.   The method according to any one of claims 1 to 3, wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.   The method according to any one of claims 1 to 4, wherein the cell is a skeletal myoblast.   The method according to any one of claims 1 to 5, wherein the pattern shape is a sea-island structure in which either one of the cell non-adhesion region or the cell adhesion region is an island and the other is the sea.   The method according to claim 6, wherein the cell adhesion region is an island and the cell non-adhesion region is the sea.   The method according to any one of claims 1 to 7, wherein a proportion of the cell adhesion region is 4% to 65% of the entire surface.