Cell culture container, cell culture device, and method for producing cell sheet

Abstract

Provided is a cell culture container having a first cell-adherent region, a second cell-adherent region, and a cell-non-adherent region, wherein the cell-non-adherent region is disposed in a predetermined interval D1 provided between the first cell-adherent region and the second cell-adherent region that are located adjacent to each other. By utilizing the cell culture container, it becomes possible to produce a uniform cell sheet having an arbitrary shape with minimal cell loss, and it also becomes possible to produce a large quantity of cell sheets having stabilized quality all at once.

Description

Cell culture vessel, cell culture device, and method for producing cell sheet

[0001] The present disclosure relates to a cell culture vessel, a cell culture device, and a method for producing a cell sheet.

[0002] Conventional cell culture involves culturing a single cell sheet in a single container by manual culture.

[0003] Patent Document 1 discloses a cell culture plate for culturing cell aggregates, which has a plurality of wells W, and in the bottom 2 of one of the wells W, a plurality of cell adhesion regions 3 of substantially the same size, which are circular, a plurality of cell adhesion regions 4 having linear portions, and a cell non-adhesion region 5 are formed, and the plurality of cell adhesion regions 3 and the plurality of cell adhesion regions 4 are respectively regions where seeded cells form cell aggregates, and the cell non-adhesion region 5 is a region where seeded cells do not substantially form cell aggregates.

[0004] JP 2019-110871 A

[0005] The cell culture plate described in Patent Document 1 forms cell aggregates at multiple cell adhesion sites, but does not produce cell sheets of uniform thickness. Furthermore, in the cell culture plate described in Patent Document 1, in addition to the cell adhesion regions for culturing multiple cell aggregates, a cell adhesion region larger in area than the cell adhesion regions is arranged for the purpose of accommodating surplus cells. However, in order to produce many cell sheets with minimal loss from a valuable cell source that meets various quality assessments, it is desirable that the cell adhesion region accommodating surplus cells be smaller in area than each of the cell adhesion regions for cell sheet culture.

[0006] The object of the present disclosure is to provide a cell culture vessel that can produce uniform cell sheets of any shape with minimal cell loss and that can mass-produce cell sheets of stable quality at once.

[0007] The cell culture vessel of the present disclosure has a first cell adhesive region, a second cell adhesive region, and a cell non-adhesive region, and a predetermined distance D1 is provided between adjacent first and second cell adhesive regions, and the cell non-adhesive region is arranged.

[0008] According to the present disclosure, uniform cell sheets of any shape can be produced with minimal cell loss, making it possible to mass-produce cell sheets of stable quality at once.

[0009] 4A is a top view showing a cell culture vessel of Example 1. FIG. 4B is a top view showing a cell culture vessel of Example 1. FIG. 4C is a top view showing a cell culture vessel of Example 1. FIG. 4D is a top view showing a cell culture vessel of Example 1. FIG. 4E is a top view showing a cell culture vessel of Example 1. FIG. 4F is a top view showing a cell culture vessel of Example 1. FIG. 4G is a top view showing a cell culture vessel of Example 1. FIG. 4H is a top view showing a cell culture vessel of Example 2. FIG. 4I is a top view showing a cell culture vessel of Example 2. FIG. 4I is a top view showing a cell culture vessel of Example 2. FIG. 4I is a top view showing a cell culture vessel of Example 2. FIG. 4I is a schematic configuration diagram showing a closed system automatic culture device of Example 3. FIG. 4I is a partially enlarged top view showing a prototype patterned culture vessel. FIG. 4I is an enlarged image showing the state after a culture test in region (b) of FIG. 4A. FIG. 4I is an enlarged image showing the state after a culture test in region (c) of FIG. 4A.

[0010] The present disclosure relates to a cell culture vessel, a cell culture device, and a method for producing a cell sheet.

[0011] Even if a single cell sheet is produced using a large-area culture vessel, cutting it into pieces of a size suitable for transplantation in a sterile manner is a complicated and difficult process.

[0012] The present inventors investigated a method for culturing multiple cell sheets of a size suitable for transplantation within a single culture vessel. They then fabricated a culture vessel (patterned culture vessel) in which adhesive and non-adhesive regions were formed within the culture vessel by partially surface treating the vessel. As a result of performing a culture test using this patterned culture vessel, they discovered that cells seeded in the non-adhesive regions tended to drift to the adhesive regions, resulting in a problem of higher cell density at the periphery of the adhesive regions than in the center.

[0013] The above-mentioned uneven cell density may lead to uneven thickness, which may affect the quality of the produced cell sheet.

[0014] Therefore, after extensive research, the inventor discovered a configuration in which a predetermined gap is provided between adjacent first and second cell adhesive regions in a cell culture vessel, and a non-cell adhesive region is arranged.

[0015] Hereinafter, examples of the present disclosure will be described with reference to the drawings.

[0016] FIG. 1A is a top view showing a cell culture vessel (sample 1A) of Example 1. FIG.

[0017] As shown in this figure, the cell culture vessel is a square-shaped petri dish, and its bottom surface is provided with a first cell adhesive region (culture region 1), a second cell adhesive region (dummy region 2), and a non-cell adhesive region 3. The culture region 1 is square-shaped with chamfered vertices. The dummy region 2 is rod-shaped with arc-shaped ends. The culture regions 1 are arranged two-dimensionally (vertically and horizontally) with a predetermined gap between them. The dummy region 2 is arranged so as to surround the multiple culture regions 1 arranged. The dummy region 2 has a predetermined length and is arranged in a row with a predetermined gap between them. The dummy region 2 also has a predetermined width. The width of the dummy region may be equal to or less than the width of the multiple culture regions 1. Furthermore, it is desirable that the area of ​​the dummy region 2 be equal to or less than the area of ​​the culture region 1.

[0018] Samples 1B to 1E of this example will be described below, but only the differences between these samples and the other samples will be described.

[0019] FIG. 1B is a top view showing the cell culture vessel (sample 1B) of this example.

[0020] As shown in the figure, in sample 1B, the culture region 1 has a square shape with no chamfered vertices, and the dummy region 2 is the same as in sample 1A.

[0021] FIG. 1C is a top view showing the cell culture vessel (sample 1C) of this example.

[0022] As shown in this figure, in sample 1C, the culture region 1 is circular, and the dummy region 2 is the same as in sample 1A.

[0023] FIG. 1D is a top view showing the cell culture vessel (sample 1D) of this example.

[0024] As shown in the figure, in sample 1D, the culture region 1 has a circular shape similar to that of sample 1C, and the dummy region 2 has a shape in which a part of the circle is missing.

[0025] FIG. 1E is a top view showing the cell culture vessel (sample 1E) of this example.

[0026] As shown in this figure, sample 1E has a configuration in which dummy regions 2a are added to the gaps in the culture regions 1 similar to sample 1C.

[0027] According to Example 1, it is possible to mass-produce uniform cell sheets of any shape at once. Furthermore, the dummy region surrounding the culture region 1 prevents excessive collection of surplus cells, allowing for the production of cell sheets of stable quality with minimal cell loss.

[0028] FIG. 2A is a top view showing a cell culture vessel (sample 2A) of Example 2. FIG.

[0029] As shown in this figure, the cell culture vessel is a circular petri dish, and its bottom surface is provided with a first cell adhesive region, which is a culture region 1, a second cell adhesive region, which is a dummy region 2, and a non-cell adhesive region 3. The culture region 1 is square-shaped with chamfered vertices. The dummy region 2 is rod-shaped (rectangular) with vertices at both ends not chamfered. The culture regions 1 are arranged two-dimensionally (vertically and horizontally) with a predetermined gap between them. The culture regions 1 are arranged in different numbers vertically and horizontally, resulting in a roughly rectangular shape overall. The dummy region 2 is arranged so as to surround the multiple culture regions 1 that are arranged. The dummy regions 2 have a predetermined length and are arranged in a row with a predetermined gap between them.

[0030] Samples 2B to 2E of this example will be described below, but only the differences between these samples and the other samples will be described.

[0031] FIG. 2B is a top view showing the cell culture vessel (sample 2B) of this example.

[0032] As shown in the figure, in sample 2B, the culture region 1 has a square shape with no chamfered vertices, and the dummy region 2 has a rod shape with arc-shaped ends.

[0033] FIG. 2C is a top view showing the cell culture vessel (sample 2C) of this example.

[0034] As shown in this figure, in sample 2C, the culture region 1 is circular, and the dummy region 2 is the same as in sample 2A.

[0035] FIG. 2D is a top view showing the cell culture vessel (sample 2D) of this example.

[0036] As shown in this figure, in sample 2D, the culture region 1 is circular, similar to sample 2C. The culture region 1 is a dense arrangement of multiple circular regions. The dummy region 2 is arranged in an arc shape along the outer culture region 1. In this figure, the dummy region 2 is arranged so as to correspond to every half circumference of the culture region 1.

[0037] FIG. 2E is a top view showing the cell culture vessel (sample 2E) of this example.

[0038] As shown in the figure, in sample 2E, the culture region 1 is the same as in sample 2D, and the dummy region 2 has a circular shape with a part missing.

[0039] Although Examples 1 and 2 show examples in which the culture region 1 is divided into multiple regions, the cell culture vessel of the present disclosure is not limited to the above examples and also includes cases in which there is only one culture region 1. Whether there are multiple culture regions 1 or only one culture region 1, it is desirable that the dummy region 2 be arranged so as to surround the entire culture region 1.

[0040] The method for producing a cell sheet according to the present disclosure may include a step of automatically performing the process using a cell culture device, which includes a cell culture vessel installation unit capable of connecting any number of the cell culture vessels according to the present disclosure in a sterile and closed manner.

[0041] FIG. 3 is a schematic diagram showing the closed-system automatic culture device of this embodiment.

[0042] The closed-system automatic culture device 100 (cell culture device) includes a cell culture vessel installation unit for installing cell culture vessels, a culture medium supply pipe, and a culture medium discharge pipe. The culture medium supply pipe, the culture medium discharge pipe, and the cell culture vessels are disposable. The cell culture vessel installation unit is a portion to which the cell culture vessels can be connected in a sterile manner.

[0043] The closed-system automatic culture apparatus 100 also has a first bottle 101 for storing a first liquid. The first bottle 101 is a container for storing a cell culture medium, which is the first liquid, and has an air pressure adjustment pipe 103 that is open to the outside air via a filter 102. The air pressure adjustment pipe 103 has an end in the gas phase portion of the first bottle 101.

[0044] The first patterning culture vessel 105 is a cell culture vessel attached to a cell culture vessel installation section, into which a first liquid is introduced to culture cells. The first patterning culture vessel 105 is also attached with an air pressure adjustment pipe 109 that is open to the outside air via a filter 108. The air pressure adjustment pipe 109 is connected to the gas phase within the first patterning culture vessel 105.

[0045] The closed-system automatic culture device 100 has a first liquid transfer pipe 104 for transferring the first liquid in the first bottle 101, and a second liquid transfer pipe 107 for transferring the first liquid in the first liquid transfer pipe 104 to a first patterning culture vessel 105. The second liquid transfer pipe 107 has a first liquid transfer pump 106, which adjusts the amount of liquid transferred in the second liquid transfer pipe 107. The first liquid transfer pipe 104 has a first valve 110. CO 2 is introduced into the first liquid transfer pipe 104 via a filter 112. ２ A pipe for supplying air containing 5% of acetic acid is connected to the second liquid supply pipe 107. A second valve 111 is provided in this pipe. By alternately opening and closing the first valve 110 and the second valve 111, it is possible to switch between supplying liquid and supplying air. Here, the second liquid supply pipe 107 functions as a culture medium supply pipe.

[0046] The closed-system automatic culture device 100 also has a second bottle 113 for discharging the first liquid in the first patterning culture vessel 105. The second bottle 113 has an air pressure adjustment tube 115 that is open to the outside air via a filter 114. The air pressure adjustment tube 115 has an end in the gas phase portion within the second bottle 113.

[0047] The closed-system automatic culture device 100 further includes a third liquid feed pipe 116 for discharging the first liquid in the first patterning culture vessel 105, and a fourth liquid feed pipe 118 connected to the third liquid feed pipe 116 and discharging the first liquid in the first patterning culture vessel 105 into the second bottle 113. The third liquid feed pipe 116 includes a second liquid feed pump 117 that adjusts the amount of liquid fed in the third liquid feed pipe 116. Here, the third liquid feed pipe 116 functions as a culture solution discharge pipe.

[0048] A second patterning culture vessel 120, which is a cell culture vessel similar to the first patterning culture vessel 105, can also be connected in parallel to the cell culture vessel installation section. In this case, a branch can be provided to the second liquid supply pipe 107, a fifth valve 119 can be installed in the second liquid supply pipe 107, and a sixth valve 121 can be installed in the branch pipe connected to the second patterning culture vessel 120. Furthermore, to discharge the first liquid from the second patterning culture vessel 120, a pipe can be provided that joins with the third liquid supply pipe 116, a seventh valve 122 can be installed in the third liquid supply pipe 116, and an eighth valve 123 can be installed in the pipe connected to the second patterning culture vessel 120. To adjust the pressure inside the second patterning culture vessel 120, an air pressure adjustment pipe 124 can be provided for connection to the filter 108.

[0049] It is also possible to further connect a cell culture vessel similar to the second patterning culture vessel 120 in parallel.

[0050] Next, a prototype patterning culture vessel was fabricated and an actual culture test was carried out, which will be described.

[0051] A patterned culture vessel (cell culture vessel) was fabricated by patterning a culture region, a dummy region, and a cell non-adhesive region using a 500 cm2 Square TC-treated Culture Dish (Corning (registered trademark), 431110) (hereinafter referred to as the "test culture vessel"). A photosensitive polymer (BIOSURFINE (registered trademark)-MRH, manufactured by Toyo Gosei Co., Ltd.) was used for patterning.

[0052] First, a photosensitive polymer was applied to the culture surface of a test culture vessel. Then, a photomask was placed over the photosensitive polymer, and the uncured photosensitive polymer was removed by development to create a patterned culture vessel.

[0053] FIG. 4A is a partially enlarged top view showing an example of a prototype patterning culture vessel.

[0054] On the bottom surface of the patterned culture vessel shown in this figure, the culture region 1 has a square shape with no chamfered vertices. The distance between it and the adjacent culture region 1 on the left is 10 mm. The distance between it and the culture region 1 below in the figure is 8 mm. The distance between it and the adjacent rod-shaped dummy region 2 on the right is 2 mm. The width of the dummy region 2 is 1 mm. The distance between the dummy region 2 and the inner surface of the side wall of the cell culture vessel is 15 mm. The area other than the culture region 1 and dummy region 2 is a non-cell adhesive region 3. Regions (b) and (c) in the figure indicate the observation areas after the culture test.

[0055] In this cell culture vessel, 1.2 × 10 NIH / 3T3 cells were cultured. ５ cells / cm ２ and incubated at 37°C, 5% CO for 3 days. ２ A cell sheet was prepared by culturing in an incubator, and the edge of culture area 1 (adhesive area) was observed.

[0056] FIG. 4B is an enlarged image showing the state of region (b) in FIG. 4A after the culture test.

[0057] As shown in Figure 4B, the culture area in region (b) is the edge of a culture area without an adjacent dummy region. Cells that had remained in the liquid above the non-adhesive region drifted to the nearby culture area, attached to the periphery of the culture area, and proliferated. As a result, the seeding density was locally high at the periphery of the culture area (approximately 0.2 to 0.3 mm wide), which resulted in problems with the uniformity of the cell sheet.

[0058] FIG. 4C is an enlarged image showing the state of region (c) in FIG. 4A after the culture test.

[0059] As shown in Figure 4C, the culture area in region (c) is the edge of a culture area with a nearby dummy area (on the right side of the figure). Cells that had been suspended in the liquid above the non-adhesive area were trapped in the dummy area. As a result, the cell density in the culture area was consistent at both the periphery and the center. Therefore, it was found that a uniform cell sheet could be easily produced by providing a nearby dummy area.

[0060] From the above results, it is considered that the narrower the distance between the culture regions, the better, as long as the distance is maintained so that the attached cells do not overlap.

[0061] The method for forming a cell adhesive surface is not limited to the method using the photosensitive polymer described above, but may also be a method using plasma treatment, corona discharge treatment, or the like.

[0062] Finally, preferred embodiments of the present disclosure will be summarized.

[0063] The second cell adhesive region has a smaller area than the first cell adhesive region.

[0064] The first cell adhesive region has a configuration in which it is divided into multiple regions, and a predetermined distance D2 is provided between adjacent first cell adhesive regions, and a non-cell adhesive region is disposed between the first cell adhesive regions.

[0065] The second cell adhesive region is disposed so as to entirely surround the first cell adhesive region.

[0066] The width of the second cell adhesive region is equal to or less than the width of the first cell adhesive region.

[0067] The first cell adhesive region is square-shaped, and the second cell adhesive regions are arranged so as to correspond to each side of the first cell adhesive region.

[0068] The first cell adhesive region is circular, and the second cell adhesive regions are arranged to correspond to each half of the circumference of the first cell adhesive region.

[0069] The distance D1 between adjacent first and second cell adhesive regions is approximately equal to the distance D2 between adjacent first cell adhesive regions.

[0070] The plurality of first cell adhesive regions includes regions of two or more different shapes.

[0071] The predetermined distance D1 is equal to or greater than 100 μm and less than 10 mm.

[0072] The predetermined distance D2 is equal to or greater than 100 μm and less than 10 mm.

[0073] The predetermined intervals D1 and D2 are more preferably 100 μm or more and 2 mm or less, and even more preferably 100 μm or more and 1 mm or less.

[0074] The method for producing a cell sheet includes a step of introducing a culture medium into a cell culture vessel to form a cell sheet, the cell culture vessel having a first cell adhesive region, a second cell adhesive region, and a cell non-adhesive region, and a predetermined distance D1 is provided between adjacent first and second cell adhesive regions, and the cell non-adhesive regions are arranged.

[0075] The effects of the present disclosure will be summarized below.

[0076] According to the present disclosure, the size, shape, and number of adhesive regions can be freely set.

[0077] Furthermore, it becomes possible to mass-produce cell sheets of desired shapes using large culture vessels, and the quality of the cell sheets can be stabilized.

[0078] Furthermore, multiple cell sheets with different shapes can be simultaneously produced using culture vessels of the same shape.

[0079] Furthermore, before collecting the cell sheet, the cells in the dummy region can be removed using tweezers, a scraper, or the like.

[0080] 1: culture area, 2: dummy area, 3: cell non-adhesive area, 100: closed system automatic culture device, 101: first bottle, 102: filter, 103: air pressure adjustment tube, 104: first liquid supply tube, 105: first patterning culture vessel, 106: first liquid supply pump, 107: second liquid supply tube, 108: filter, 109: air pressure adjustment tube, 110: first valve, 111: second valve, 112: filter, 113: second bottle, 114: filter, 115: air pressure adjustment tube, 116: third liquid supply tube, 117: second liquid supply pump, 118: fourth liquid supply tube, 119: fifth valve, 120: second patterning culture vessel, 121: sixth valve, 122: seventh valve, 123: eighth valve, 124: air pressure adjustment tube.

Claims

1. A cell culture vessel having a first cell adhesive region, a second cell adhesive region, and a cell non-adhesive region, wherein a predetermined distance D1 is provided between adjacent first cell adhesive regions and second cell adhesive regions, and the cell non-adhesive regions are arranged.

2. The cell culture vessel according to claim 1, wherein the second cell adhesive region has an area smaller than that of the first cell adhesive region.

3. The cell culture vessel according to claim 1, wherein the first cell adhesive region is divided into multiple regions, a predetermined distance D2 is provided between adjacent first cell adhesive regions, and the non-cell adhesive regions are arranged.

4. The cell culture vessel according to claim 1, wherein the second cell adhesive region is disposed so as to entirely surround the first cell adhesive region.

5. The cell culture vessel according to claim 1, wherein the width of the second cell adhesive region is equal to or less than the width of the first cell adhesive region.

6. The cell culture vessel according to claim 1, wherein the first cell adhesive region is square-shaped, and the second cell adhesive region is arranged to correspond to each side of the first cell adhesive region.

7. The cell culture vessel according to claim 1, wherein the first cell adhesive region is circular, and the second cell adhesive regions are arranged to correspond to each half of the circumference of the first cell adhesive region.

8. A cell culture vessel as described in claim 3, wherein the distance D1 between adjacent first and second cell adhesive regions and the distance D2 between adjacent first cell adhesive regions are approximately equal.

9. The cell culture vessel according to claim 3, wherein the plurality of first cell adhesive regions include regions of two or more different shapes.

10. The cell culture vessel according to claim 1, wherein the predetermined distance D1 is equal to or greater than 100 μm and less than 10 mm.

11. The cell culture vessel according to claim 3, wherein the predetermined distance D2 is equal to or greater than 100 μm and less than 10 mm.

12. A cell culture device comprising: a cell culture vessel installation section; a culture medium supply piping; and a culture medium discharge piping, wherein the cell culture vessel installation section is capable of connecting any number of cell culture vessels, wherein the cell culture vessels have a first cell adhesive region, a second cell adhesive region, and a cell non-adhesive region, and wherein a predetermined distance D1 is provided between adjacent first cell adhesive regions and second cell adhesive regions, and the cell non-adhesive regions are arranged.

13. A method for producing a cell sheet, comprising the step of introducing a culture medium into a cell culture vessel to form a cell sheet, the cell culture vessel having a first cell adhesive region, a second cell adhesive region, and a cell non-adhesive region, the non-cell adhesive regions being arranged with a predetermined distance D1 provided between adjacent first cell adhesive regions and second cell adhesive regions.

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