JP4587370B2 - Cell culture support and cell culture method - Google Patents

Description

  In the present invention, after culturing cells, the cultured cells can be recovered with little damage to the cultured cells, or the cultured cells can be collected while maintaining the binding between the cells, and further, the cultured cells are maintained in a sheet form. The present invention relates to a cell culture recovery method that enables recovery as it is, and a cell culture support used therefor.

  In cell culture, a culture medium and cells are generally placed in a container (dish) such as a microplate, a culture dish, or a culture flask, and the cells are allowed to adhere to the container wall and cultured. At this time, in order to keep the pH constant, it is common to incubate in the presence of an appropriate amount of carbon dioxide, and to exchange the culture solution at regular intervals. After the completion of the culture, the cultured cells are generally collected by trypsin treatment in order to peel the cells from the culture vessel. For example, the petri dish medium in which the cells were cultured was removed with an aspirator, 1 ml of 0.05% trypsin solution kept at 37 ° C. was added, and the trypsin solution was removed with an aspirator. This removes the trypsin inhibitor in the medium. Add 1 ml of trypsin solution again and leave at 37 ° C. for about 3 minutes. Add 5 ml of medium and hop with a pipette to detach and disperse the cells from the container. The medium to be added generally contains a trypsin inhibitor, at which point trypsin is ineffective, and the cells are not affected by trypsin thereafter.

  As a method for recovering cells without trypsin treatment, the temperature of the culture vessel is coated on the bottom of the culture vessel. After culturing, the temperature is changed, and the cells are removed from the vessel using the effect of polymer phase transition and elongation. The method of pulling apart is put into practical use. A typical example is poly (N-isopropylacrylamide), which aggregates in water at 32 ° C. or higher, and utilizes the property that polymer molecules extend at a temperature lower than 32 ° C. That is, at 37 ° C. maintained at the time of culturing, the cells are attached to the container because the polymer is agglomerated, but if the cells are left at room temperature after culturing, the polymer extends between the cells and the container wall so that the cells are removed from the container Peel off mechanically. Using this technique, confluent human nuclear epithelial cells can be detached in a sheet form.

In the current general culture technique, as shown in the background art above, it is a general method to culture in a dish and to float cells by trypsin treatment. First, in the dish culture, in order to prevent the exchange of the culture medium from being easy and to increase the probability of unnecessary contamination, the medium is usually not changed halfway. For this reason, cells are affected by various substances accumulated in the medium during the culture. In order to suppress the pH fluctuation during the culture, the culture is generally performed in a 5% CO ₂ incubator in many cases. Undesirable things accumulate in the medium or growth factors are deficient. Many of these can be solved if the cell line can always be changed to a fresh medium supplemented with an appropriate grade of serum.

  The trypsin treatment after the culture partially degrades the protein on the cell surface. For this reason, it is better to consider that cell surface receptors and transporters are very painful. In fact, once a trypsin-treated cell is subcultured, some cells die. Of course, even if the epithelial cells are used in the form of a sheet, the adhesion factor between the cells is cleaved by trypsin, so that the cells are separated, and the cells change from their specific shape to a spherical shape. The most practical conventional technique for recovering cells in sheet form is a method using a thermosensitive polymer. However, this method also mechanically peels off the cells and gives a considerable stress to the cells. There is no guarantee that factors related to cell adhesion will remain on the cell side when peeled off, and it is thought that a significant portion remains adsorbed on the dish surface. On the side of the cell facing the dish, various things may have fallen off or torn off.

  In order to reduce damage to cells, the present invention proposes a technique for facilitating replacement of the above-described medium and separating the cells from the container wall without damaging the cells.

  In order to solve the above problem and to peel off the cells safely, it is only necessary to dissolve the container side instead of cutting the cell side adhesion factor. Since it is difficult to dissolve the surface of a container made of ordinary plastic, a material that can be easily dissolved by some kind of treatment should be selected. In the present invention, a cellulose membrane is used as this material. Cells are adhered to the cellulose membrane and cultured. By treating with cellulase after culturing, the cellulose membrane is decomposed, dissolved and removed, whereby the cultured cells can be easily recovered.

  According to the present invention, cultured cells can be collected without damaging them. For this reason, it becomes possible to supply the cell as a functional material used for regenerative medicine, in which it is essential to use the cell while maintaining its shape. Furthermore, since a sheet-like cell can be obtained, tissue engineering for creating a three-dimensional cell structure by stacking the cells can be performed. It becomes a reality that functional cell tissues are reconstructed by stacking cell sheets having different functions.

  In the present invention, cells are adhered to a cellulose membrane and cell culture is performed. The cellulose membrane may be coated with an extracellular matrix such as gelatin or laminin in advance. After culturing, the cellulose membrane is decomposed by treatment with cellulase, and the cultured cells are collected. Cellulose membrane is spread on a normal dish and cultured on it. Finally, cellulase is transmitted along the edge of the dish and gently spread throughout the dish, and cellulose is decomposed. In other words, it can be recovered while maintaining the intercellular adhesion state.

  Furthermore, the cellulose membrane is held by placing the cellulose membrane on a substrate having a fine channel structure, and the cellulose membrane is selectively inserted by inserting a cellulase solution between the fine channel structure between the cellulose and the substrate. Can be decomposed and removed. What is important here is that cellulase does not degrade animal cells. This is because animal cells do not have cell walls made of cellulose. In addition to the addition of cellulase, this fine channel structure can exchange the medium during cell culture. As a result, if the cellulose membrane is substantially a cellulose filter and has a molecular weight cut off of about 10,000 to 100,000 daltons, the growth factors in the serum and waste products from the cells can be exchanged or removed.

Example 1
1 (A) to 1 (D) schematically illustrate an example in which cell culture on a cellulose membrane according to Example 1 of the present invention, and the cells after culture are collected in a sheet form, and further, a multilayer cell sheet is configured. FIG.

As shown in FIG. 1A, a cellulose membrane 2 (fractionated molecular weight of 30,000 daltons, 55 mmφ) coated with gelatin is spread on a dish (60 mm) 5 containing 5 ml of a medium 1 containing serum. Pre-incubate for 30 minutes at 5% CO ₂ and 37 ° C. to allow Cellulose Membrane 2 to adapt Medium 1. Add 0.5 ml of myocardial beating cell suspension and culture in a CO ₂ incubator at 37 ° C. During this cultivation, if necessary, the medium 1 is replaced with a fresh one. As the culture progresses, myocardial pulsatile cells spread in a sheet shape over almost the entire cellulose membrane 2. 3 shows myocardial pulsatile cells spreading like a sheet.

When the myocardial pulsating cells are in the form of a sheet, the medium 1 is removed by suction with an aspirator, and immediately, 10 mg / ml cellulase solution 4 (1 ml) is gently added along the edge of the dish 5 using the pipette 6. The dish 5 is gently tilted, and the cellulase solution is spread over the bottom of the dish so as to rinse the sheet-like cells 3. Next, the cellulase solution is removed with an aspirator, and 1 ml of the cellulase solution is added again. The reason why the cellulase solution is treated twice is because it is assumed that a cellulase inhibitor is present in the medium. Place in a CO ₂ incubator at 37 ° C. and incubate until the cellulose membrane 2 is degraded and the cell sheet 3 is released. The decomposition state of the cellulose film 2 can be easily confirmed by microscopic observation.

  FIG. 1B shows a single-layer cell sheet 3 separated from the cellulose membrane 2 in this way.

  FIG. 1 (C) shows a state in which a two-layer cell sheet 10 is formed by stacking an existing cell sheet 3 on a newly generated cell sheet 3 ′ in the same manner as described in FIG. 1 (A). Indicates.

  In FIG. 1C, after the cell sheet 3 'is formed and before the cellulase solution 4 is added, it is important to continue the culture by stacking the existing cell sheet 3. Cultivation after stacking the existing cell sheet 3 is preferably performed under the same conditions as described above. After culturing, as described in FIG. 1 (A), when the cellulase solution 4 is added and cellulase treatment is performed in the same manner, a two-layer cell sheet 12 can be obtained as shown in FIG. 1 (D). it can.

  By repeating this operation, cell layers can be stacked up to about four layers. Furthermore, an 8-layer cell sheet can also be created by stacking 4 layers of cells. That is, in the same manner as described above, first, a four-layer sheet is prepared and then cellulase is used to obtain a four-layer cell sheet. Next, in the same manner as described above, a four-layer sheet is prepared, and then the previously formed four-layer cell sheet is stacked thereon to continue the culture. After the culture is continued, the cellulase solution 4 is added in the same manner as described above, followed by cellulase treatment to obtain an 8-layer cell sheet.

(Example 2)
FIG. 2 (A) is a plan view of a cell culture support that is devised as a support for holding a cellulose sheet and has a structure that prevents cellulase from contacting the entire cell surface. (B) is sectional drawing seen in the arrow direction in the AA position of FIG. 2 (A). (C) is sectional drawing seen in the arrow direction in the BB position of FIG. 2 (A).

  The substrate 100 is a container having a diameter of 60 mm and has two bottom surfaces 101 and 102 on the inner surface. The higher bottom surface 101 is approximately 10 mm from the top surface of the container, and the lower bottom surface 102 is approximately 12 mm from the top surface of the container. The lower bottom surface 102 is provided with a beam 105 having a width of 1 mm. The height of the beam 105 is such that the top of the beam 105 is at the same level as or slightly lower than the bottom surface 101. The distance between the beams 105 is approximately 1 mm. The bottom surface 102 is provided with a recess 103 for injecting or sucking a culture solution or the like. Further, when the liquid is injected from the depression 103, the diffusion plate 106 is installed between the depression 103 and the beam 105 so that the liquid is evenly distributed between the beams 105. The height of the diffusion plate is approximately 3/4 of the height of the beam so that the liquid leaks and spreads. Without the diffusion plate, when a solution such as a culture medium is exchanged, the liquid flows only in the shortest flow path, and there is a problem that the liquid does not easily flow between the beams in the peripheral portion.

  FIG. 3A is a diagram illustrating a situation in which the cell sheet described in the first embodiment is created using the substrate 100 described in FIG. 2, and is a diagram viewed in the arrow direction at the AA position. FIG. FIG. 3B is also a cross-sectional view corresponding to the view seen in the arrow direction at the BB position. A cellulose film 104 is placed on the upper surface of the surface constituted by the beam 105 and the bottom surface 101. A lid 110 is placed on the upper surface of the substrate 100, and tubes 111-1 and 111-2 extending to the bottom surface of the recess 103 are attached to the lid 110. When the lid 110 is attached, the tip of the tube descends to the vicinity of the space 103.

The cell culture procedure using the board | substrate 100 of Example 2 is shown. First, a medium that fills the upper side of the beam 105 of the substrate 100 is added. That is, the medium is added to such an extent that the surface of the higher bottom surface 101 gets wet with the medium. Next, the cellulose film 104 that has been familiarized with the culture medium is sunk and placed on the structure of the beam 105 and the higher bottom surface 101. The lid 110 is attached, and the medium is supplied from the tube 111-1 using a peristaltic pump and discharged from the 111-2. The medium is preliminarily kept at 37 ° C. Pre-incubate for 30 minutes in a CO ₂ incubator (5% CO ₂ , 37 ° C.).

Remove from the CO ₂ incubator, open the lid, and seed myocardial pulsatile cells as in Example 1. The lid 110 is put on and the culture is continued while changing the culture medium. When the muscle pulsation cells spread over almost the entire cellulose membrane, a 10 mg / ml cellulase solution is continuously supplied from the tube 111-1 instead of the supplied medium to fill the upper side of the beam 105. Since the cellulose film 104 and the upper part of the beam 105 are not completely in close contact with each other, the cellulase solution also flows around the valley portion between the beam 105 and the beam 105. Subsequently, when incubated at 37 ° C., the cellulose membrane 104 is decomposed and the cultured cells are peeled off in a sheet form.

(A)-(D) schematically illustrates an example in which cell culture on a cellulose membrane according to Example 1 of the present invention, and the cells after culture are collected in a sheet form, and further, a multilayer cell sheet is configured. FIG. (A) is a plan view of a cell culture support having a structure that prevents cellulase from contacting the entire cell surface by devising a support that holds the cellulose sheet. (B) is sectional drawing seen in the arrow direction in the AA position of FIG. 2 (A). (C) is sectional drawing seen in the arrow direction in the BB position of (A). (A) is a figure explaining the condition which produces the cell sheet demonstrated in Example 1 using the board | substrate 100 demonstrated in FIG. 2, and respond | corresponds to the figure seen in the arrow direction in the AA position. It is sectional drawing. (B) is also a cross-sectional view corresponding to the view seen in the direction of the arrow at the BB position.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Medium with serum, 2 ... Cellulose membrane, 3, 3 '... Myocardial pulsatile cell spreading in sheet form, 4 ... Cellulase solution, 5 ... Dish, 100 ... Substrate, 101, 102 ... Bottom, 103 ... Depression, 105 ... Beam, 106 ... Diffusion plate, 110 ... Lid, 111-1, 111-2 ... Tube.

Claims (7)

A cell culture support comprising a container having a two-stage bottom surface on the inner surface and a cellulose membrane, allowing the cultured cells to be collected while being kept in a sheet shape,
A plurality of beams having upper portions opened to the lower bottom surface of the two-stage bottom surfaces are formed , and the height of the beams is the same as or slightly lower than the height of the higher bottom surface,
The cellulose membrane is placed to hold the cellulose membrane on the top and higher bottom of the beam ;
A cell culture support , wherein a space between the lower bottom surface and the beam and an upper side of the beam are filled with a culture solution . A lid used to overlap the cell culture support, and the lid is provided with a tube for supplying or sucking a culture solution supplied between the beams or sucked from between the beams. and which, according to claim 1 cell culture support according. A lid used to overlap the cell culture support, and the lid is provided with a tube for supplying or sucking a culture solution supplied between the beams or sucked from between the beams; In addition, a depression is formed on the lower bottom surface corresponding to the position where the tube is provided, and a diffusion plate adjacent to the depression for helping the culture medium to diffuse is formed on the lower bottom surface. It is provided, according to claim 1 cell culture support according. A method for culturing cells using the cell culture support according to any one of claims 1 to 3,
A cell culture method comprising culturing cells on the cellulose membrane, and injecting cellulase into a flow path formed between the plurality of beams after cell culture to decompose the cellulose membrane. A method for culturing cells using the cell culture support according to any one of claims 1 to 3,
In order to culture cells on the cellulose membrane, a culture solution is circulated through a channel formed between the plurality of beams, and after completion of the culture, cellulase is injected into the channel and cellulase is injected into the cellulose membrane. A cell culturing method characterized by decomposing a cellulose membrane by acting to recover cells or sheet-like cells. A method for culturing cells using the cell culture support according to any one of claims 1 to 3,
In order to culture cells on the cellulose membrane, a culture solution is circulated through a channel formed between the plurality of beams, and after completion of the culture, cellulase is injected into the channel and cellulase is injected into the cellulose membrane. The cellulose membrane is decomposed by acting to collect the sheet-like cells, and the cellulase is cultured on the cellulose membrane after culturing the previously collected sheet-like cells on the newly-formed sheet-like cells. A cell culturing method characterized by decomposing a cellulose membrane by acting and recovering two-layered sheet-like cells. The cell culture method according to claim 6, wherein the two-layered sheet-like cells are further superimposed on a new sheet-like cell.

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