JP5912734B2 - Cell culture equipment - Google Patents

Description

  The present invention relates to a device capable of culturing adherent cells and performing subsequent processing such as washing and peeling, a method for producing a sheet-like cell culture using the device, and a sheet-like cell culture produced by the production method.

  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 therapy such as an artificial heart or a heart transplant, 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).

  In order to produce a cell culture for use in transplantation in this way, various culture vessels are provided. For example, Patent Document 2 discloses a cell culture support substrate having a porous structure part, and discloses that a high-density three-dimensional structure is produced by culturing cells in the pores of the porous structure part. . Patent Document 3 discloses a culture container in which a part or all of a bottom plate having a culture surface and other parts can be separated. With this configuration, a sheet-like culture can be easily placed on a support. It is disclosed that it can be placed.

Special table 2007-528755 gazette JP 2011-172533 A JP 2009-22247 A

  An object of the present invention is to cultivate a large number of cells in a space-saving and simple manner in the cultivation of adherent cells, a method for producing a sheet-shaped cell culture using the device, and the method. An object of the present invention is to provide a sheet-shaped cell culture produced in the above manner.

  In the production of a sheet-like cell culture, it is preferable to use autologous cells from the viewpoint of a low rejection reaction, etc., but when producing a sheet-like cell culture using autologous cells, it takes time to proliferate and differentiate the cells. Therefore, in the conventional method for producing a sheet-shaped cell culture, a sheet is usually formed by adhering to the bottom surface of the culture vessel. We faced a new challenge of being unable to produce dendritic cell cultures. In order to solve such a problem, the present inventor has made a new study that adherent cells can be cultured even if the cell culture surface is formed perpendicular to the horizontal plane under normal culture conditions, while continuing research. As a result of obtaining knowledge and continuing research, the present invention has been completed.

That is, the present invention relates to the following.
[1] A device for an adherent cell comprising one or more carriers having a cell culture surface and a container for storing the carrier and a liquid, the container having an opening on the upper surface, The device, wherein the device has a shape in which at least a part of the cell culture surface is substantially perpendicular to the bottom surface of the container.
[2] The device according to [1], wherein the carrier has a shape that is substantially planar and substantially perpendicular to the bottom surface of the container.
[3] The device according to [1] or [2], wherein one or more carriers are detachably fixed to one connecting means, and the connecting means is detachably fixed to the container.

[4] The device according to [1] to [3], further including a circulation means for circulating the liquid in the container.
[5] The device according to [1] to [4], further comprising movable means capable of changing a distance between two or more carriers.
[6] A method for producing a sheet-shaped cell culture using the device according to [1] to [5],
Culturing the cells forming the sheet on the cell culture surface of the carrier in a container containing the carrier and liquid, and removing the carrier from the container with the sheet adhered to the cell culture surface;
Said method.
[7] The method of [6], wherein the cell is a skeletal myoblast.
[8] A sheet-like cell culture produced using the method of [6] or [7].

  According to the present invention, a large amount of cells can be cultured in a space-saving manner. In addition, since the liquid such as the medium can be exchanged for each container, the procedures such as discarding and refilling of the medium can be performed overwhelmingly and easily compared to the conventional method, and the labor of culture can be greatly reduced. Furthermore, since the culture surface can be easily observed even from outside the culture apparatus, the risk of contamination in follow-up observation can be reduced. In addition, since it can be applied to all adherent cells, a large amount of sheet-like cell culture that takes time to produce can be cultured at the same time. Furthermore, by replacing the medium with a container containing a buffer solution or a peeling liquid composition, operations such as washing and peeling can be easily performed.

FIG. 1 is a photograph showing the state of adherent cells adhered to a slide glass on day 1 and day 4 of culture observed in Example 1. It can be confirmed that the number of adherent cells, which were only slightly observed on the first day, increased on the fourth day. FIG. 2 is a graph showing the number of cells at the start of culture and on the sixth day of culture. As can be seen from the graph, the number of cells doubled on day 6, indicating that adherent cells can be sufficiently cultured by the device of the present invention.

FIG. 3 is a schematic diagram showing an aspect of the movable part of the present invention. In this aspect, the movable part is a carrier arranged in a circle, and the distance between the carriers is widened by expanding the circumference constituting the movable part. FIG. 4 is a schematic diagram showing an aspect of the movable part of the present invention. In this aspect, the movable part is configured to arrange the carriers at equal intervals on a straight line, and the interval between the carriers is widened by the extension of the movable part. FIG. 5 is a schematic diagram showing an aspect of the movable part of the present invention. In this embodiment, the carriers connected to the two movable parts are arranged in a nested manner, and the two movable parts move away from each other so that they are not adjacent to the adjacent carrier, and as a result, the carrier The distance between them changes.

FIG. 6 is a schematic view showing one embodiment of the device of the present invention. In this embodiment, the plurality of carriers are detachably fixed to the connecting means, and the connecting means are detachably fixed to the container (A). Then, by removing the connecting means from the container, it is possible to remove all the carriers from the container at once (B). The removed connecting means can be replaced with a container separately.

  In one aspect of the present invention, the present invention is a device for adherent cells comprising one or more carriers having a cell culture surface and a container containing the carrier and a liquid, the container having an opening on the upper surface. And the carrier has a shape in which at least a part of the cell culture surface thereof is substantially perpendicular to the bottom surface of the container.

In the present invention, the “device for adherent cells” means a device useful in a plurality of operations for handling adherent cells including culture. Examples of such work include, but are not limited to, culture, washing, peeling, and staining.
In the present invention, the “carrier” means a scaffold structure that adheres adherent cells. Adherent cells can adhere to the “cell culture surface” which is the surface of the carrier.

  In the present invention, the “cell culture surface” means a surface on which cells are adhered and cultured in the structure of the carrier, and is typically the surface of the carrier immersed in a liquid. However, the entire surface of the immersed carrier does not have to be a cell culture surface. For example, only the surface suitably formed to adhere cells among the immersed surfaces may be indicated.

  The device of the present invention has a container for containing a liquid having an opening on the upper surface, and one or more carriers having a cell culture surface. Accordingly, the liquid and the carrier can be taken in and out basically from the upper surface opening, and can preferably be covered. The liquid and the carrier are taken in and out from the opening on the upper surface of the container. However, the carrier partially immersed in the liquid in the container may be moved in the horizontal direction. The material of the container may be anything as long as it can contain a liquid, and can be appropriately selected by those skilled in the art. From the viewpoint of easy internal observation, it is preferably a transparent material, can be used once, is durable, non-cytotoxic, and can be sterilized from a plastic material, and adheres cells to the bottom of the container. From the viewpoint of making it difficult to cause it to be made difficult, it is preferably a material with low cell adhesion. Examples of these materials include, but are not limited to, glass, polycarbonate, polystyrene, and polypropylene.

  The carrier used in the present invention is stored so that it can be taken in and out of the container. The device of the present invention may have one or more carriers, each of which can be separated and removed from the device independently. The shape of the carrier of the present invention is such that at least a part of the cell culture surface of the carrier is substantially perpendicular to the bottom surface of the container. In general, the fact that another surface 2 is perpendicular to a certain surface 1 means that an arbitrary normal vector of the surface 2 is orthogonal to an arbitrary normal vector of the surface 1. In the present invention, “substantially vertical” means basically vertical, but is allowed to be inclined to such an extent that it can be regarded as vertical or approximate to vertical. Intended to be. Typically, including, but not limited to, a shape that reduces hydrodynamic resistance when moving through the liquid in the direction of the container opening or in the direction opposite to the opening, for example, horizontal A surface shape, a weight shape, a columnar shape, or the like having a smaller surface area in the direction than the surface area in the vertical direction can be given.

The material of the carrier of the present invention is not particularly limited as long as it has no cytotoxicity. From the viewpoint of improving cell adhesion, hydrophobic polymers, polycarbonates, polystyrenes, polypropylenes and the like are preferable.
As described above, the carrier of the present invention may be subjected to various surface treatments depending on the cell to be handled and the purpose of use of the device. Although not limited thereto, for example, in the case of normal adherent cells, it may be coated with an adhesive protein such as a hydrophobic polymer, fibrin, gelatin, laminin, collagen, fibronectin, etc. from the viewpoint of improving adhesiveness. When manufacturing a sheet-shaped cell culture, it may be coated with a temperature-responsive polymer or the like to control the cell adhesion in order to make it easy to peel the culture while maintaining the sheet shape. In some cases, a surface treatment such as a nano pillar may be performed.

  In the device of the present invention, since the carrier can be easily taken in and out, it is very easy to exchange the liquid in the container. Furthermore, since one or more carriers can function as an attachment for the container, use it as if the attachment was replaced with a container containing a fresh medium or another liquid such as a cleaning solution or a stripping solution. Is possible. Therefore, complicated procedures are not required for discharging and injecting the medium. Moreover, the cell which can use the device of this invention is not specifically limited as long as it is an adherent cell. Therefore, it can be used for a wide range of cells and a wide range of applications. For example, when used in the manufacture of a sheet-like skeletal myoblast culture, a carrier having a flat shape in which a temperature-responsive polymer is coated on the cell culture surface is used. Can be appropriately selected as an attachment.

  In a preferred embodiment of the present invention, the carrier is substantially planar and is accommodated in the container so as to be substantially perpendicular to the bottom surface of the container. In the present invention, “substantially planar” means basically a plane, but is intended to include a curved surface that is regarded as a plane or is flat enough to approximate a plane. Yes. As described above, in the present invention, the carrier means a scaffold structure to which adherent cells adhere, and therefore, when the carrier is substantially planar, a wide surface portion is usually used as a cell culture surface. Therefore, it means that an arbitrary normal vector of the wide surface portion of the carrier is substantially orthogonal to an arbitrary normal vector of the bottom surface of the container.

In such an embodiment, the carrier is preferably planar from the viewpoint of easy handling of the carrier and ease of reflux of the medium. In particular, in the formation of a sheet-like cell culture, there are advantages such as less unevenness in the thickness of the formed sheet.
In another embodiment, one or more carriers are detachably fixed to one connecting means. Therefore, the carrier functions as an attachment to the connecting means, and it is possible to operate a plurality of carriers collectively by operating the connecting means.

  The connecting means is detachably fixed to the container. Accordingly, the communication means can function as an attachment to the container. The connecting means may have any shape as long as the above configuration is satisfied. Although not limited to this, for example, the container may have a lid-like shape in which a plurality of carriers can be inserted, or a cage-like shape that fits inside the container, and the carrier is placed therein. It may be a shape that can be inserted. Therefore, in such an aspect, by detaching the connecting means from the container, it is possible to take them out from the container at a time, even when a plurality of carriers are provided.

  In another aspect of the present invention, the device of the present invention has a circulation means for circulating the liquid in the container. By circulating the liquid in the container, the cells do not stay on the bottom of the container during culture, making it easier for the cells to adhere to the cell culture surface, and a fresh medium for cells that adhere to the cell culture surface. Can be easily supplied, and can be expected to be highly effective in cleaning. The circulating means may have any mechanism as long as the liquid in the container can be circulated. As such a mechanism, for example, stirring means for circulating the liquid in the container by stirring may be used, or a liquid inlet and outlet are provided in the container, the liquid is introduced from the inlet, and the liquid is supplied from the outlet. It is also possible to use a mechanism that circulates by flowing out. For example, the stirrer disposed on the bottom surface of the container may cause a flow in the liquid between the carriers extending vertically from the bottom to the top of the container. The flow generated in this way can travel along the side of the container and return to the bottom of the container.

  In yet another aspect, the device of the present invention comprises two or more carriers and movable means capable of changing the spacing between any of these carriers. By changing the interval between the carriers in the course of culture, the circulation efficiency of the medium on the culture surface where the cells are more confluent can be increased. Such movable means is not limited to this, but, for example, it is shown in FIGS. 3 to 5 in addition to a mechanism for adjusting the interval by moving each carrier horizontally and / or vertically on the rail. Mechanism.

  Since any of the adherent cells can be used in the device of the present invention, a sheet-shaped cell culture can be produced using the device of the present invention. Therefore, in another aspect of the present invention, a method for producing a sheet-shaped cell culture using the device of the present invention is provided. Such methods include culturing the cells forming the sheet on the cell culture surface and removing the carrier from the container with the sheet adhered to the cell culture surface.

  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 that can connect cells at least physically (mechanically), 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 physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected. The sheet-shaped cell culture preferably does not contain a scaffold not derived from a living body.

  A sheet cell culture includes any cell capable of forming a sheet cell culture. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, mesenchymal stem cells, synovial cells, embryonic stem cells, epithelial cells, endothelial cells, etc. Is included. Among these, in the present invention, those forming a monolayer cell culture, such as myoblasts, are preferred.

  The cells can be derived from any organism capable of being treated with cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like. Further, 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.

  In addition to the above steps, various steps useful for forming a sheet-shaped cell culture may be further included. For example, the carrier removed from the container may be subjected to the next step such as cleaning and peeling. In these next steps, the device of the present invention may or may not be used. For example, in the washing step, the container of the present invention is washed by placing a liquid used for washing a sheet-shaped cell culture such as Hanks buffer or phosphate buffer, and mounting the carrier on the container. Alternatively, the liquid may simply be poured over the carrier for cleaning. In addition, the step of peeling may also be carried out by shaking, for example, in a liquid used for peeling a sheet-shaped cell culture such as Hanks buffer or phosphate buffer. The carrier to which the material is attached may be placed on the support as it is, and the sheet-shaped cell culture may be transferred to the support.

  Furthermore, in order to further facilitate the production of the sheet-like cell culture in the device of the present invention, a step of pre-culturing the sheet-forming cells on the cell culture surface before culturing the sheet-formed cell on the device of the present invention may be included. In the preculture, in order to increase the adhesion efficiency of the cells to the cell culture surface, the cell culture surface may be cultured in a state parallel to the horizontal plane. Once the cells have adhered to the cell culture surface, even if the cell culture surface is then cultured vertically, the growth rate does not differ from that when the cells are cultured horizontally. Cell culture and sheet-shaped cell culture can be more efficiently produced by accelerating cell adhesion to the surface, and then performing main culture with the culture surface vertical after adhering to the culture surface to some extent. .

  By producing a sheet-shaped cell culture using the device of the present invention, it becomes possible to easily and in large quantities produce a sheet-shaped cell culture that takes time and labor for production. This is especially true when producing sheet-like cell cultures for transplantation, so that they are preferably produced using autologous cells in order to prevent rejection, and large quantities of custom-made sheet-like cell cultures are prepared. This is advantageous. The cell culture device of the present invention has recently been shown to be useful in regenerative treatment of various diseases, and autologous cells can be used, but it takes a long time to differentiate and proliferate. It can be particularly preferably used for the production of a culture.

  As described above, the sheet-shaped cell culture produced using the device of the present invention can select a suitable shape and size according to the application by selecting the shape of the carrier or the cell culture surface in various ways. Can do. Therefore, a sheet-like cell culture produced using the device of the present invention is also included in the present invention.

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

Example 1: An MCDB131 medium mixed with 20% FBS and 1 × 10 ⁵ skeletal myoblasts were placed in a vertical culture experimental container, and the slide glass was placed vertically in a holder, and the holder was placed in the container. Fixed to. The container was cultured in an incubator while stirring with a magnetic stirrer, and the state of cells adhered to the slide glass on the first and fourth days of culture was observed under a phase contrast microscope.

  On the 6th day of culture, 20 mL of trypsin-like proteolytic enzyme per cell was added as a cell dissociating agent to dissociate the adhered cells. Cells in the culture solution containing the collected cells were counted visually under a microscope, and the total cell number was calculated.

The results are shown in FIG. 1 and FIG. FIG. 1 shows the state of cells adhered to a slide glass on the first day (upper) and the fourth day (lower) of culture. On day 1, it was confirmed that the cells were slightly adhered, and on day 4, an increase in the number of adherent cells was confirmed. Therefore, it was confirmed that skeletal myoblasts can be cultured even when the culture surface is vertical.
Furthermore, in order to confirm this, the number of cells on day 6 of culture was compared with the number of cells at the start of culture. The results are shown in FIG. On the sixth day of culture, it was confirmed that the number of cells had doubled. From this, it was confirmed that adhesion culture of skeletal myoblasts with the culture surface vertical was possible.

  According to the present invention, it is possible to perform medium replacement, washing, and detachment, which are troublesome in the conventional adherent cell culture device, very easily, and culture can be performed in a small space. It is very useful for producing a cell culture such as a cell sheet culture which takes a long time to produce and is necessary in large quantities. Moreover, it can be used not only for culturing but also for other processes accompanying adherent cell culture such as washing and peeling. Furthermore, since the material and shape of the carrier can be arbitrarily selected according to the purpose of use and work, the shape of the sheet-shaped cell culture, which has conventionally depended on the shape of the bottom surface of the container, can be arbitrarily changed, and the transplant site It is also possible to select an optimum shape according to the condition, and it is very useful in the medical field.

1 movable means 2 carrier 3 container 4 connecting means

Claims (10)

  A device for an adherent cell comprising two or more carriers having a cell culture surface and a container for storing the carrier and a liquid, further comprising movable means capable of changing an interval between the carriers in the course of culturing, The device has an opening on the top surface, and the carrier has a shape in which at least a part of the cell culture surface is substantially perpendicular to the bottom surface of the container.   The device of claim 1, wherein the carrier is substantially planar and has a shape that is substantially perpendicular to the bottom surface of the container.   The device according to claim 1 or 2, wherein two or more carriers are detachably fixed to one connecting means, and the connecting means is detachably fixed to the container.   Furthermore, the device as described in any one of Claims 1-3 containing the circulation means to circulate the liquid in a container.   The device according to any one of claims 1 to 4, wherein the movable means arranges the carrier in a circular shape, and a distance between the carriers is widened by expanding a circumference constituting the movable means.   The device according to any one of claims 1 to 4, wherein the movable means arranges carriers at equal intervals on a straight line, and the distance between the carriers is widened by extending the movable means.   The carriers connected to the two movable means are respectively arranged in a nested manner, and when the two movable means move away from each other, they are not adjacent to the adjacent carrier, and the distance between the carriers changes. The device according to claim 1. A method for producing a sheet-shaped cell culture using the device according to any one of claims 1 to 7,
Culturing the cells forming the sheet on the cell culture surface of the carrier in a container containing the carrier and liquid, and removing the carrier from the container with the sheet adhered to the cell culture surface;
Said method.   The method according to claim 8, wherein the cells are cultured by performing pre-culture in a state where the cell culture surface is parallel to a horizontal plane, and then performing main culture with the cell culture surface vertical.   The method according to claim 8 or 9, wherein the cell is a skeletal myoblast.