JP2006304791A - Method for forming large amount of cell cluster by using three-dimensional culture device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an artificially constructed tissue and organ applicable in a regenerative medical field, especially a method for forming a cell cluster to form a large amount of cell cluster indispensable for the artificial production of pancreatic islet (having insulin secreting function) for applying to cell transplantation in regenerative medicine and enabling the culture for a long period. <P>SOLUTION: The method for forming a cell cluster to form an artificial pancreatic islet for diabetes treatment is carried out by the cell culture using a clinostat-mounting circulation system comprising a three-dimensional culture device using an n-axis rotation clinostat. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a cell culture method by three-dimensional culture, and more particularly, to a method for forming a large amount of cell clusters of uniform size using a three-dimensional culture apparatus using an n-axis rotating clinostat.

In recent years, expectations for regenerative medicine are increasing in the medical field. Regenerative medicine is a medical method for repairing the function of a diseased or damaged tissue or organ using artificially constructed tissues and organs, and is one of the important technologies that will be responsible for medicine in the future.

Conventionally, as a cell suspension culture method, a method of rotating a culture vessel on which cells are mounted is known (Patent Documents 1 to 3). Moreover, clinostat (three-dimensional culture apparatus) and a method for producing an artificial organ using this apparatus are known (Patent Documents 4 to 5).

JP 2002-45173 A Japanese Patent Laid-Open No. 2003-289851 Japanese Patent Laid-Open No. 2003-304866 Japanese Patent Laid-Open No. 2003-70458 JP 2003-70464 A

Diabetes is one of the diseases that are highly expected to realize regenerative medicine. Among them, type 1 diabetes is a disease that develops because control of blood glucose levels becomes difficult due to the loss of pancreatic β cells having the ability to secrete insulin. At present, the only fundamental treatment includes pancreas transplantation and islet tarnsplantation, which has less invasion at the time of transplantation than pancreas transplantation. However, the absolute shortage of donors has become a problem, and it is currently impossible to obtain the pancreas and pancreatic islets necessary for the treatment of many patients awaiting transplantation from limited donor pancreas .

Therefore, the artificial creation technology of pancreatic islets by regenerative medicine approach aiming at the treatment of diabetes has attracted a great deal of attention as an urgent and important medical technology development issue. That is, there is a demand for the development of a basic technology for producing small higher-order tissue elements called islets in an in vitro culture environment.

The islets form a spherical mass with a size of 0.1 mm to 0.3 mm, and there are about 1 million adults per adult in the pancreas. If this islet can be constructed in large quantities in an in vitro environment, 1
Medical application to patients with type 2 diabetes.

In order to realize regenerative medicine, it is essential to produce a cell cluster having a three-dimensional structure from cells. Furthermore, assuming application in the medical field, when transplanting the produced cell agglomerates to the human body, a large number of cells, both functionally and technically when transplanted, are supplied to more patients. It is necessary to produce agglomerates. In addition, it is desirable that the cell clump size can be adjusted as necessary. If the number and size of cell clumps can be adjusted, cell transplantation at a medical site becomes possible.

Patent Document 5 describes an experiment in which frog cells, that is, amphibian cells, were cultured using a clinostat, but there is no description about the “number” and “size” of the artificial organs to be produced. . In addition, long-term culture is difficult in cell culture using conventional clinostats.

The present invention is applicable to the development of methods relating to the production of artificially constructed tissues and organs that can be applied in the field of regenerative medicine, in particular for the artificial production of islets (having an insulin secretion function) for application to cell transplantation as regenerative medicine. The subject is the development of culture means for forming a large amount of essential cell clumps.

The inventor manufactures a large number of cell clumps for human cells and mouse cells using a clinostat (three-dimensional culture apparatus) equipped with a circulation system, and adjusts the size of the large number of cell clumps. We have found culture means that can do this.

Furthermore, the present invention is characterized in that, when culturing cells using a clinostat (three-dimensional culture apparatus), the size of the cell clump can be adjusted by adjusting the number of cells mounted on the cell culture vessel. And

In the method of the present invention, the rotation of each axis of the clinostat is preferably 2 rpm or less per minute. By rotating at a low speed of 2 rpm, the cells in the culture solution tend to sink in the direction of gravity, generating a high cell density region at the inner bottom of the cell culture vessel, increasing the contact frequency between the cells, and cell agglomeration. Will be formed in large quantities (over 1000). That is, 2rpm
If it exceeds 1, the diffusibility of the cells in the culture medium increases, the frequency of contact between the cells decreases, and the formation rate of cell clumps decreases, so 2 rpm or less is desirable. Further, it is preferable to circulate the culture solution. In addition, the viscosity and mass of the culture solution are preferably such that the cells sink when the cells are added and allowed to stand. Agglomerates of mammalian cells can be formed by the method of the present invention.

The present invention enables culture of mammalian cells by mounting a culture medium circulation system. Somatic cells established in a culture system can be cultured using a clinostat, and 1000 or more cell clumps can be produced in one culture with the number or size of the cell clumps adjusted. The method of the present invention enables mass production of cell clumps and regulation of cell clump size for mammalian cells.

The cell culture apparatus itself equipped with a clinostat used in the cell culture method is known,
Although some are commercially available, they cannot be used in carrying out the method of the present invention because they do not have a circulation system installed inside the clinostat. The method of the present invention is different from the conventionally known methods. As shown in FIG. 10, the cell culture device disclosed in Patent Document 5 includes a three-dimensional clinostat 11 including a main body 15 including an outer frame 17 and an inner frame 22 supported by a column 16 a and a column 16 b. Yes. The rotary joint 18 rotatably supports the outer frame 17. The culture vessel 12 is connected to the inner frame 22.

As shown in FIG. 4, the culture vessel 12 shown in Document 5 is in the vicinity of the intersection of the rotary shaft 18a and the rotary shaft 20a. The clinostat 11 rotates the culture vessel 12 by n-axis. The culture container 12 rotates together with the inner frame 22. Outer frame 17 and inner frame 2
When 2 and 2 are respectively rotated, the culture vessel 12 rotates n-axis. The supply of the culture solution from the culture solution tank 13 to the culture vessel 12 is performed via the rotary joint 18 and the rotary joint 20 by the pump 14 installed outside. The culture solution supplied to the culture vessel 12 is returned to the culture solution tank 13. The culture solution for culturing the cell mass is used cyclically.

As shown in FIG. 11, the culture vessel shown in Document 5 contains a culture solution and a cell mass to be cultured inside the culture vessel 12. By rotating the culture vessel (12) by n-axis, the direction of gravity acting on the cultured cells is dispersed, and each cell can be propagated and extended in all directions. Culture becomes possible.

FIG. 1 shows a schematic overall configuration of a cell mounting unit in the cell culture method of the present invention. FIG.
As shown in FIG. 2, the cell culture vessel 9 is separated by a medium-interval membrane 3 into a culture layer 1 on which cells are mounted and a medium layer 2 through which a culture solution is circulated. This intermediate spacing membrane 3 has a structure that allows the culture medium to permeate but does not allow cells to pass therethrough (pore density 10 ^{5 to} 6 × 10 ⁶ pore / cm ³ ). Further, a gas permeable membrane 4 is provided outside each layer, and gas is supplied and discharged. The culture medium in the culture medium layer 2 of the cell culture device 9 flows into the culture medium bag 6 storing the culture liquid by the culture medium circulation pump 5 operated by the power supply box 7 and is stored in the culture medium bag 6 at the same time. Culture fluid is culture fluid circulation pump 5
Thus, the circulation system flows into the culture medium layer 2 of the cell culture device 9 through the pump tube 8. The culture medium in the culture medium layer 2 passes through the intermediate interval film 3 and flows into the culture layer 1 to supply nutrients to the cells.

Unlike the method described in Patent Document 5, the circulatory system is mounted inside the rotary shaft and does not go through the n-axis rotator, so that the culture that has been a problem in the existing clinostat with a culture fluid circulating system is used. There is no clogging, contamination, or culture fluid leakage.

In the method of the present invention, nutrients can be constantly and stably supplied by using a culture solution circulation system. That is, long-term culture and three-dimensional culture essential for organ production can be combined.

FIG. 2 shows the rotation trajectories of the X-axis, Y-axis, and Z-axis of the clinostat in the cell culture method of the present invention. The culture for creating a cell conglomerate has a horizontal axis (X axis) as an outer axis.
Orbit control operation with 15 rotations per minute, vertical axis (Y axis) as inner axis and 17 rotations per 10 minutes, rotating so that gravity dispersion of each axis of X, Y, Z is 1: 1: 1 Let As a result, the center of rotation becomes a simulated microgravity environment due to the dispersion of gravity, and the cells seem to float in the culture medium.

FIG. 3 shows the relationship between the center of rotation of the clinostat and the arrangement of the cell incubator in the cell culture method of the present invention. The clinostat is a device that suspends cells by dispersing gravity in the X, Y, and Z axis directions (simulated microgravity environment) to perform three-dimensional culture. The place where gravity is most dispersed is the rotation axis X This is the rotation center C where the rotation axes Y intersect. Therefore, by mounting the cell culture device 9 near the rotation center C, the gravity on the cultured cells is dispersed.

As a medium for loading cells, a generally used culture solution can be selected and used. This culture has a lower viscosity and mass than cells. Therefore, when the clinostat is rotated at a low speed of 2 rpm or less per minute and the gravity is dispersed, the action of the centrifugal force is reduced, and when the three-dimensional culture is performed with the clinostat, the cells sink in the direction of the earth's gravity. Tend to.

In order for cells to form a cell clump, in addition to the connection between mother and daughter cells in which individual cells have proliferated, it is necessary to bond with other cells in a floating state. The cell-to-cell connection is performed by the adhesion of the cells to each other. However, it is necessary to meet between cells as a pre-stage for connection. In addition, even when cells are adhered to each other, it is necessary to efficiently produce a spherical agglomeration having three dimensions, not a two-dimensional sheet-like adhesion formed when stationary culture is performed. is there.

Therefore, in the present invention, a cell culture device is mounted near the center of rotation, and the cells are cultured while dispersing the gravity. However, by using a commonly used cell culture solution at a low speed of 2 rpm, Animal cells having a specific gravity higher than that of the culture solution sink in the direction of gravity, and the cells are unevenly distributed at the bottom in the cell culture vessel. Furthermore, since the incubator always rotates n-axis at 2 rpm or less, the cells are cultured while rolling the portion corresponding to the bottom of the rotating inner wall of the incubator. From these things, the contact frequency of cells increases more than the cell contact frequency of the state currently disperse | distributed in a culture solution, and comes to form a large quantity (1000 or more) cell clumps.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
1. Three-dimensional culture (test method) Three-dimensional culture of cells was performed using a clinostat equipped with a culture medium circulation system. The culture period was 48 hours (short-term culture) and 144 hours (6 days, long-term culture). In the long-term culture, the culture medium (medium) circulation system installed in the present clinostat was used.
The system is set up to circulate approximately 1 μl of culture fluid per minute (approximately 1.44 ml per day). The operating conditions of the clinostat were set so that the average gravity level was 1 × 10 ⁻² G or less and the gravity dispersion values were 0.333 for all of the X axis, Y axis, and Z axis. The operation mode is set to orbit control, the rotation time of one cycle is 10 minutes, the rotation speed is 17 rotations / cycle on the inner shaft, and on the outer shaft
15 rotations / cycle. By this orbit control setting, the average gravity (X axis, Y axis and Z axis during operation)
G) values are -0.000G, -0.001G and -0.000G, respectively.

2. Culture conditions The gas phase during the culture was 95% Air (approximately 20% O ₂ ) and 5% CO ₂ , and the culture temperature was 37 ° C. 10% Fetal Bovine Serum (FBS: GIBCO, California, USA) and 1% Penicillin-Strepto
Dalbecco's Modified Eagle Medium (DMEM, P-4) with mycin (GIBCO, NY, USA)
417: Sigma-Aldrich co, St Louis, Mo, USA).

3. Test cells 3.1 Short-term culture (48 hours)
A 48-hour three-dimensional culture was performed using two types of cells: Hep G2, a human hepatoblastoma-derived cell line derived from an endoderm organ similar to the islet component cells, and MLE 10, a mouse liver epithelium-derived cell line. It was. The cell density is 2 x 10 ⁵ cells / ml, 2 x 10 ⁵ cells / ml and 2 x 10 ⁶
Cells / ml.

3.2 Long-term culture (144 hours)
144 hours (6 days) of three-dimensional culture was performed using MLE 10 cells.

4. Evaluation method Evaluation items include the presence or absence of spheroid formation, spheroid count measurement, spheroid size measurement, calculation of the number of cells constituting the spheroid, calculation of the survival rate of the cells constituting the spheroid, culture medium The glucose concentration and albumin concentration in the medium were measured.

4.1 Measurement of the number of spheroids was calculated using an image obtained by collecting the spheroids collected from the cell culture vessel for clinostat in the center of the petri dish and photographing with a stereomicroscope.

4.2 Measurement of spheroid size was determined by determining the average of the major axis and minor axis of 100 randomly selected spheroids as the diameter.

4.3 Measurement of the number of cells constituting the spheroid is confirmed by first confirming the size of one spheroid and recognizing each individual, and then adding 10% Hoechst 33258 (WAKO Osaka, Japan)
Spheroids were exposed to Buffered Saline (PBS: Sigma-Aldrich co, St Louis, MO, USA) for 2 minutes to stain nuclei. Next, spheroids were placed on the slide glass, sandwiched so as to be slightly crushed with a cover glass, and sealed with nail polish. Thereafter, the number of nuclei was calculated from the spheroid images taken with a fluorescence microscope, and this value was used as the number of spheroid-constituting cells.

4.4 Calculation of the viability of the cells that make up the spheroid is 0.4% T, which selectively stains dead cells.
Using rypan Blue Stain (GIBCO, California, USA), the spheroid size and the number of constituent cells were calculated.

4.5 Glucose concentration in the culture solution is the enzyme method reagent glucose CII-Test Wako (Mutarotase GOD method: Wako, Osaka, Jap) combining mutarotase and glucose oxidase.
an). In addition, measurement of albumin in culture broth
ELISA Quantitation Kit (BETHYL, TX, USA) was used.

(1) The two cell lines (Hep G2 and MLE 10) both formed a large number of cell clumps when mounted on a clinostat with 1 × 10 ⁵ cells / ml, 2 × 10 ⁵ cells / ml and 2 × 10 ⁶ cells / ml. . The white suspended matter seen in the cell culture container shown in FIG. 4 (a) is a large amount of cell aggregate floating in the culture solution (cell loading density 2 × 10 ⁶ cells / ml). (B) shows an enlarged photograph of a cell cluster.

(2) Hep G2 is loaded into the cell culture vessel at 1x10 ⁵ cells / ml, 2x10 ⁵ cells / ml and 2x10 ⁶ cells / ml, respectively, and the rotation center of the cell culture device is from the rotation center of the clinostat. 2.5
The samples were mounted so that they were separated from each other by 10 cm, and three-dimensional culture was performed for 48 hours with 15 rotations of the X axis and 17 rotations of the Y axis in 10 minutes. The rotation speed was 1.5 rpm for the X axis and 1.7 rpm for the Y axis. As a result, it was shown that the number and size of the cell clumps can be adjusted by the loaded cell density because the number of cell clumps and the average size were different due to the difference in the loaded cell density.

The Hep G2 cell cluster is shown in FIG. The trypan blue-stained photograph shows that cells stained blue are dead cells and white cells are live cells. Therefore, it can be seen that many of the cells constituting the side of the cell cluster are living cells. A Hoechst-stained photograph shows the number of cells constituting a cell cluster. From the paraffin section and HE staining, it can be seen that the inner cell of the cell cluster is a living cell. The distribution of the Hep G2 cell clump size is shown in FIG. From FIG. 6, it can be seen that the higher the cell loading density, the smaller the cell clump size. In addition, 2 x 10 ⁵ cells / ml and 2 x 10 ⁶ cel
The cell loading density of ls / ml shows that the cell clump size is unimodally distributed.

(3) Mount MLE 10 in a cell culture device at 1 × 10 ⁵ cells / ml, 2 × 10 ⁵ cells / ml and 2 × 10 ⁶ cells / ml respectively, and the center of the cell culture device is 2.5 from the center of rotation of the clinostat. cm
It was mounted so that it was detached, and three-dimensional culture was performed for 48 hours with the X axis rotating 15 times and the Y axis rotating 17 minutes in 10 minutes. As a result, there was a difference in the number of cell clumps due to the difference in the loaded cell density, indicating that the number of cell clumps can be adjusted by the loaded cell density.

The MLE 10 cell cluster is shown in FIG. The trypan blue-stained photograph shows that cells stained blue are dead cells and white cells are live cells. Therefore, it can be seen that many of the cells constituting the side of the cell cluster are living cells. A Hoechst-stained photograph shows the number of cells constituting a cell cluster. From the frozen section and HE staining, it can be seen that the inner cell of the cell cluster is a living cell. The distribution of MLE 10 cell clump size is shown in FIG. FIG. 8 shows that the cell clump size is unimodally distributed at a cell loading density of 1 × 10 ⁵ cells / ml.

(4) Mount Hep G2 on a cell culture vessel with a cell count of 2 × 10 ⁵ cells / mll and attach the cell culture device so that the center of the cell culture device is 2.5 cm away from the center of rotation of the clinostat. 144-hour three-dimensional culture was performed with 15 rotations and 17 Y-axis rotations. A culture medium circulation system was used during the culture period. As a result, it was confirmed that the cell survival was high when the culture solution circulation system was used, and it was shown that a large amount of cell aggregates can be cultured for a long period of time.

A Hep G2 cell cluster that has been cultured for a long time is shown in FIG. The trypan blue-stained photograph shows that cells stained blue-black are dead cells and white cells are live cells. Therefore, it can be seen that when the circulation system is used, the viability of the cells constituting the cell cluster is better than when the circulation system is not used. From the frozen section and HE staining, it can be seen that when the circulation system is used, the viability of the internal cells constituting the cell cluster is better than when the circulation system is not used.
The above results are summarized in Table 1.

According to the present invention, it is possible to efficiently produce cells to be transplanted to a patient in a medical field,
It can be used as a transplanted cell in place of a donor cell that has been absolutely deficient until now. In addition, by artificially mass-producing it, it becomes possible to provide many patients with transplant cells that have been insufficient until now at a low price.

It is a general | schematic whole block diagram of the cell mounting unit in the cell culture method of this invention. It is a figure which shows the rotation track | orbit of the X-axis of the clinostat in the cell culture method of this invention, a Y-axis, and a Z-axis. It is a schematic diagram which shows the relationship between the rotation center part of a clinostat and the arrangement | positioning of a cell culture device in the cell culture method of this invention. 2 is a drawing-substitute optical photograph (A) and a drawing-substitution micrograph (B) of the Hep G2 spheroid cultured in Example 1. FIG. FIG. 2 is a drawing-substituting micrograph of Hep G2 spheroid cultured in Example 1 (A: trypan blue staining, B: Hoechst staining, C: paraffin section, HE staining). It is a distribution map of Hep G2 spheroid size. FIG. 2 is a drawing-substituting micrograph of MLE 10 spheroids cultured in Example 1 (A: trypan blue staining, B: Hoechst staining, C: frozen section, HE staining). It is a distribution map of MLE 10 spheroid size. FIG. 2 is a drawing-substituting micrograph (upper; trypan blue staining, lower; frozen section, HE staining) showing a comparison of spheroids when the culture medium circulation system is used (A) and when not used (B). It is a perspective view which shows the structure of one form of a clinostat. It is a perspective view which shows the structure of one form of the culture container used for the clinostat shown in FIG.

Claims (4)

A method of forming a cell agglomeration comprising culturing cells using a three-dimensional culture apparatus using an n-axis rotating clinostat and forming an artificial islet for treating diabetes by using a clinostat-mounted circulation system. The method of forming a cell agglomeration according to claim 1, wherein the culture solution is circulated by mounting the culture solution circulation system inside the clinostat. The cell clump formation method according to claim 1, wherein the rotation of each axis of the clinostat is 2 rpm or less per minute. The method of forming a cell clump according to any one of claims 1 to 3, wherein the cell is a mammalian cell.

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