JP2010263868A - Cell culture carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture carrier which can flexibly deal with various conditions such as the kinds and degree of growth of cells, and objectives, in cell culture, includes a simple constitution, and can culture a uniform-shaped three-dimensional cell mass easily in a large amount. <P>SOLUTION: This cell culture carrier has a structure of two stages of an upper member 1 and a lower member 2, wherein the lower member 2 has a plate-like body comprised of a porous material and arranging a plurality of recessed parts 21 for housing the cells regularly on its upper surface, and the upper member 1 is a plate-like body comprising a minute material and installed on the upper surface of the lower member 2 as demountable and having penetrating holes 11 at positions corresponding to the recessed parts 21, and capable of housing the cells cultured in the recessed parts 21 through the penetrating holes 11. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell culture carrier for forming a three-dimensional cell mass such as embryonic stem cells (ES cells) in vitro.

In recent years, animal experiments and cell culture experiments have been performed for the purpose of developing medical techniques and pharmaceuticals.
Cell culture experiments can be significantly reduced in cost, labor, and experiment period compared to animal experiments, and are reproducible without the problems of strains and individual differences as in animal experiments. Experimental results can be obtained. Furthermore, it has an advantage that the intended action can be directly examined by culturing not only animal-derived but also human-derived cells.
In such cell culture experiments, tissue cells of animals including humans are three-dimensionally cultured, and these cultured cells can be applied to artificial organs, biosensors, bioreactors, etc. Attempts have been made.

  Conventionally, plastic petri dishes, glass petri dishes, glass plates, well plates, and the like have been generally used as cell culture carriers used in such cell culture experiments.

  In recent years, using a substrate with a patterned surface so that the adhesion to cells is different, culturing the cells on this surface, allowing the cells to adhere only to the processed surface with cell adhesion, Techniques for arranging cultured cells have also been reported (see, for example, Patent Documents 1 and 2).

  Furthermore, seed cells are cultured in a uniform cell size in a cell culture cell that is regularly arranged on the surface of a chip substrate made of an inorganic material such as glass, a metal material such as stainless steel, a synthetic resin, or rubber. Thus, a cell culture chip capable of supporting each cell mass in a regularly arranged state is disclosed (for example, see Patent Document 3).

JP-A-2-245181 Japanese Patent Laid-Open No. 3-7576 JP-A-2005-27598

  However, the cell culture methods described in Patent Documents 1 and 2 described above are difficult to control the cell morphology when cell masses are formed on the surface of a substrate having portions with different adhesion properties. It was difficult to obtain a uniform spherical cell mass due to large variations in size and carrying position.

  Therefore, when such a cell mass is used as a biosensor in a biological function check, a new drug screening, etc., a sufficient biological reaction cannot be obtained, the responsiveness and reliability as a sensor are lacking, and the application range is limited. Therefore, there was a problem of lack of versatility.

Moreover, even when the cell culture chip as described in Patent Document 3 is used, it is difficult to set a minute depth, and if the cell culture cell is too shallow, the cell culture chip was cultured. The cell moves to the next cell. On the other hand, if the depth of the cell culture cell is too deep, there is a problem that it is difficult to collect the cultured cells.
That is, since cells vary in size depending on the type and degree of growth, when cells are three-dimensionally cultured with a conventional cell culture carrier having a concave portion as described above, each cell depends on each cell. It was necessary to appropriately adjust the depth of the recess for accommodating the.

  Accordingly, a carrier for culturing a large amount of a three-dimensional cell cluster having a uniform shape is required to have a structure that can flexibly cope with various situations such as cell type, growth rate, and purpose. It was.

  The present invention has been made to solve the above technical problem, and can flexibly cope with various situations such as cell type, growth degree, and purpose in cell culture, and has a simple configuration. It is an object of the present invention to provide a cell culture carrier that can easily cultivate a uniform three-dimensional cell mass in large quantities.

The cell culture carrier according to the present invention has a two-stage structure of a lower member and an upper member, and the lower member is made of a porous body, and has a plate shape in which a plurality of recesses for accommodating and culturing cells are arranged on the upper surface. The upper member is a dense body, is a plate-like body that is detachably provided on the upper surface of the lower member, and has a plurality of through holes at positions corresponding to the recesses. The cell to be cultured can be accommodated in the recess.
According to the cell culture carrier having such a structure, a uniform three-dimensional cell mass can be easily cultured in large quantities, and the upper member can be removed as necessary. It can be easily collected, and can flexibly cope with various situations such as cell type, growth degree, and purpose in cell culture.

Further, the cell culture carrier according to the present invention has a two-stage structure of a lower member and an upper member, and the lower member is made of a porous body, and has a plurality of recesses for accommodating and culturing cells on the upper surface. The upper member is detachably provided on the upper surface of the lower member, the lower surface portion of the lower surface in contact with the lower member is made of a porous body, and the upper surface portion of the upper surface facing the lower surface is a dense body And a plate-like body having a plurality of through holes at positions corresponding to the recesses, and the cells to be cultured in the recesses can be accommodated through the through holes.
According to the culture carrier having such a structure, a three-dimensional cell cluster having a uniform shape can be easily cultured in large quantities, and the upper member can be removed as necessary. In cell culture, it can flexibly respond to various situations such as cell type, growth rate, purpose, etc., and the lower surface portion of the lower surface contacting the lower member of the upper member is a porous body. Since it is comprised, it has the effect that oxygen becomes easy to be supplied to the cell accommodated in the recessed part of the lower member from the said lower surface part.

The lower member and the upper member are preferably made of any one of ceramics selected from hydroxyapatite, β-tricalcium phosphate, zirconia, alumina, and titania.
These ceramics are excellent in biocompatibility and biocompatibility, and can be suitably used as a cell culture carrier.

The porous body preferably has pores having a pore diameter of 5 μm or less and a porosity of 10% or more and 70% or less.
If it is the above porous bodies, the adhesiveness of a cell is moderate, the circulation of a culture medium is accelerated | stimulated, it is suitable for cell culture, and the intensity | strength as a cell culture support | carrier can be maintained.

Moreover, it is preferable that the width | variety of the said recessed part and the said through-hole is 10 micrometers or more and 1000 micrometers or less.
If the widths of the recesses and the through holes are within the above ranges, cells can be accommodated in the recesses through the through holes at a density sufficient for culture, so that an appropriate cell mass can be formed.

If the cell culture carrier according to the present invention is used, a three-dimensional cell cluster having a uniform shape can be easily cultured in large quantities.
In addition, since the cell culture carrier according to the present invention can flexibly respond to the type and purpose of the target cell and has a simple configuration, the type of ceramic to be configured and the differentiation induction method are appropriately selected. By doing so, it becomes possible to induce differentiation of ES cells more efficiently than before.

It is a perspective view which shows one aspect | mode of the cell culture carrier which concerns on this invention. It is a top view of the cell culture support | carrier of FIG. It is AA sectional drawing of FIG. It is AA sectional drawing of FIG. 2 corresponding to the other one aspect | mode of the cell culture carrier which concerns on this invention.

Hereinafter, the present invention will be described in more detail with reference to the drawings.
1 and 2 show an embodiment of the cell culture carrier according to the present invention.
This cell culture carrier has a two-stage structure in which the upper member 1 and the lower member 2 are detachable. The lower member 2 is a plate-like body that is made of a porous body and has a plurality of recesses 21 that accommodate and culture cells on the upper surface. On the other hand, the upper member 1 is provided on the upper surface of the lower member 2 and is a plate-like body made of a dense body and having a plurality of through holes 11 at positions corresponding to the recesses 21.
Further, this cell culture carrier has a configuration capable of accommodating cells (seed cells) to be cultured in the recesses 21 through the through holes 11 when the upper member 1 is disposed on the upper surface of the lower member 2.
By configuring the cell culture carrier in such a form, a three-dimensional cell mass having a uniform shape can be cultured in each recess, and the upper member can be removed as necessary. The cells can be easily collected by the above method, and in the cell culture, it is possible to flexibly cope with various situations such as the kind of cells, the degree of growth, and the purpose.

It is preferable that the plurality of recesses (micropatterns) 21 of the lower member 2 are regularly arranged at regular intervals with a size matching the diameter of the desired cultured cell mass.
In consideration of the uniformity of cells to be cultured, the preparation of cell culture carriers, the ease of cell culture operations, and the like, the above micropattern configuration is preferable.
The shape of the micropattern is not particularly limited, but it is preferable that the horizontal cross-sectional shape is circular considering the same points as described above.
The number of recesses 21 depends on the shape and size of the cell culture carrier, but in the case of a circular plate carrier having a diameter of 13 mm, it is possible to culture several thousand cell clusters with one carrier. .

Moreover, it is preferable that the width | variety of the through-hole 11 and the recessed part 21 is 10 micrometers or more and 1000 micrometers or less.
When the widths of the recesses 21 and the through holes 11 are less than 10 μm, the number of cells that can be accommodated in each recess 21 is reduced, and cell culture at a sufficient density becomes difficult.
On the other hand, when the width exceeds 1000 μm, the number of cultured cells is increased, and the supply of the culture solution to the cells in the recess 21 and the replacement of the waste products discharged from the cells become insufficient.

On the other hand, the upper member 1 has a through hole 11 at a position corresponding to the micro pattern of the lower member 2.
The shape of the through-hole 11 is a lattice shape in FIGS. 1 and 2 for the sake of convenience in consideration of easy alignment with the micropattern 21 of the lower member 2 and ease of manufacture. It is not particularly limited, and may be circular or angular.
However, the width of the through hole 11 is, as described above, from the viewpoint of accommodating cells (seed cells) to be cultured in the recess 21 through the through hole 11 and from the viewpoint of culturing a uniform three-dimensional cell mass. The width of the recess 21 is preferably equal to or greater than the width.
The cells in each recess 21 are preferably separated by the upper member 1 so that the cells do not contact the upper member 1.

The upper member 1 and the lower member 2 may include an alignment portion (not shown) that is detachable at a location where the through hole 11 and the concave portion 21 are not provided (for example, the upper member 1 is a convex portion and the lower member 2 is a concave portion). preferable.
By providing such an alignment portion, it is possible to easily align the upper member 1 and the lower member 2.

The cell culture carrier is preferably made of ceramics for both the upper member 1 and the lower member 2.
With ceramics, the porosity can be easily adjusted, and a cell culture carrier comprising a porous body or a dense body with a desired porosity can be easily produced.
Among ceramics, any one of hydroxyapatite, β-tricalcium phosphate, zirconia, alumina, and titania is preferable.
These ceramics are all excellent in biocompatibility and biocompatibility, and are suitable as cell scaffolds. Among these, it is preferable to select appropriately according to cell adhesion, proliferation efficiency, and the like.

The porous body constituting the lower member 2 has pores having a pore diameter of 5 μm or less and preferably has a porosity of 10% or more and 70% or less from the viewpoint of loosely adhering the cell mass.
By making the lower member 2 a porous body having the above-described micropores, cell adhesion becomes appropriate, circulation of the medium is promoted, suitable for cell culture, and strength as a cell culture carrier. Can be maintained.
On the other hand, when the pore diameter exceeds 5 μm, cells enter the pores and are difficult to collect.

Moreover, when the porosity of the porous body constituting the lower member 2 exceeds 70%, it is difficult to obtain sufficient strength as a carrier.
On the other hand, when the porosity is less than 10%, the substance supply to the cells is insufficient.
The cell adhesion to the cell culture carrier can be adjusted by changing the porosity within a range of 10% to 70%.

  On the other hand, the upper member 1 is formed of a dense body because it is preferable that the cells do not adhere when the cells are seeded and collected. Specifically, the porosity is preferably less than 10%.

FIG. 3 is a cross-sectional view of the cell culture carrier of FIG. The depth a of the concave portion 21 of the lower member 2 is appropriately adjusted according to the cell size and the height b of the upper member 1, but is preferably 10 μm or more and 1000 μm or less.
When the depth a is less than 10 μm, it is difficult to culture a three-dimensional cell mass in the recess.
On the other hand, when the depth a exceeds 1000 μm, it is difficult to collect the cultured cells, and the cells are likely to remain in the recesses.

The recess 21 is preferably U-shaped or semicircular in cross section.
When the recess 21 has a corner, it becomes easy for cells to adhere to the corner, making it difficult to obtain a uniform three-dimensional spherical mass, and when the cultured cells are collected by pipetting or the like, the cells remain. It becomes easy to do.

Further, the height b of the upper member 1 is appropriately adjusted according to the cell size and the depth a of the lower member 2, but is preferably in the range of 10 μm or more and 1000 μm or less.
When the height b is less than 10 μm, it is difficult to separate cells for each through hole 11.
On the other hand, when the depth b exceeds 1000 μm, a medium exchange operation or the like becomes difficult.

4 is a cross-sectional view taken along line AA of FIG. 2 corresponding to another embodiment of the cell culture carrier according to the present invention.
That is, as shown in FIG. 4, the upper member 1 is detachably provided on the upper surface of the lower member 2, and the lower surface portion 1 a of the lower surface contacting the lower member 2 is made of a porous body, and the upper surface portion of the upper surface facing the lower surface. 1b may be composed of a dense body.
According to the culture carrier having such a configuration, in addition to the above-described effects, the lower surface portion 1a of the lower surface in contact with the lower member 2 of the upper member 1 is formed of a porous body. This is preferable because oxygen is easily supplied to the cells accommodated in the recesses 21 of the cell.
The porous body constituting the lower surface portion 1a also has pores having a pore diameter of 5 μm or less and a porosity of 10% or more and 70% or less from the same viewpoint as the lower member 2 described above.

The type of cells that can be cultured using the culture carrier according to the present invention is not particularly limited, and examples thereof include cells having adhesive properties such as ES cells, fibroblasts, stem cells, etc. ES cells are preferred for obtaining the original cell mass.
When the culture carrier according to the present invention is used, it is not necessary to form a feeder cell (supporting cell) layer on the culture carrier, and it is possible to directly seed and culture cells on the culture carrier, It is preferable to do so. Further, the shape and size of the formed culture mass can be controlled by the shape and size of the culture carrier.

Moreover, the culture medium used for cell culture is not specifically limited, According to the cell type to culture, it can select suitably. For example, MEM, α-MEM, DMEM, Eagle medium, etc. are preferably used.
These media are preferably used after adding various substances according to the cell type. Examples include FBS (fetal bovine serum), KSR (KnockOut ™ Serum Replacement), LIF (leukemia inhibitory factor), non-essential amino acids, pyruvate, insulin, dexamethasone, antibiotics, and the like.

  In order to induce differentiation of a three-dimensional cell mass cultured on a carrier into a target cell, for example, in the case of induction into osteoblasts as bone, FGF (fibroblast growth factor), IGF- I, IGF-II (insulin-like growth factor), PDGF (platelet-derived growth factor), TGF-B (transforming growth factor), BMP-Z (bone-inducing protein), HGH (human growth hormone), human-derived growth factor It is preferable to appropriately add a growth factor or the like corresponding to each target cell, such as adding a cell growth factor such as a concentrate in the medium.

In addition, after culturing a three-dimensional cell mass of ES cells on a culture carrier, it is necessary to form an embryoid body (EB) in order to induce differentiation into a target cell.
Also in the case of EB formation, if the culture carrier according to the present invention is used, EB can be formed as it is on the carrier from the cultured cell mass, or alternatively, the cell mass can be transferred to a petri dish and subjected to suspension culture. A large amount of EB can be easily formed.

Furthermore, when differentiation of EB obtained as described above is induced, differentiation can be induced as it is on the carrier, or alternatively, EB can be transferred to a petri dish and adhered to the petri dish to induce differentiation.
When induction of differentiation on the culture carrier according to the present invention, if the stationary culture is performed with the upper member set on the lower member, medium exchange or drug addition to the differentiation-inducing medium can be reliably and easily performed. . Moreover, when differentiation induction is performed with EB adhered to the recess of the lower member of the culture carrier, the upper member is attached to the lower member, and after replacing the differentiation induction medium, the upper member is removed and allowed to stand. Culture may be performed.
On the other hand, when transferring EB to a petri dish, the formed cell mass was transferred to the petri dish by pipetting several times with the EB medium, and suspension culture was performed in the petri dish. By simply pipetting, the EB is peeled off from the carrier, and adhesion culture in a petri dish can be performed.

EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not restrict | limited by the following Example.
[Example 1]
A cell culture support made of zirconia ceramics having a diameter of 13 mm and a height of 2 mm comprising a two-stage structure of an upper member and a lower member was prepared.
The upper member is a dense body having a lattice porosity of less than 10% with a height of 300 μm, a lattice width of 20 μm, and a lattice spacing of 200 μm, and the lower member is formed with a micropattern (concave portion) having a diameter of 200 μm and a depth of 100 μm. A porous body having porosity of 0.1 to 0.5 μm and a porosity of 50% was obtained.
In a carrier in a state where the upper member is set, 5 × 10 ⁴ ES cells are seeded, and in a DMEM medium containing KSR, pyruvate, non-essential amino acids, LIF, streptomycin, penicillin in a 5% CO ₂ incubator, The cells were cultured at 37 ° C. for 3 days. The medium was changed every day.

As a result, it was confirmed that a three-dimensional cell mass of ES cells was formed in the recess of the lower member of the carrier without the ES cells moving or spreading to the adjacent recess.
Further, when this three-dimensional cell mass was stained with alkaline phosphatase (ALP), it became blue-purple, confirming that ES cells were proliferating while maintaining an undifferentiated state.

  Thereafter, using EB medium, IMDM medium containing FBS, pyruvic acid, non-essential amino acids, β-mercaptoethanol, penicillin, and streptomycin, the following two methods (a) and (b) were used to form EB. went.

First, in the method (a), static culture was performed with the upper member set, after changing the medium to the EB medium, and holding the cell mass on the carrier.
After changing to the EB medium, the differentiation state on days 3, 5, and 7 was evaluated by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction).

As a result, expression of mesoderm differentiation markers was not observed on the 5th day, but expression of all three germ layers of endoderm, mesoderm, and ectoderm was confirmed on the 7th day.
Therefore, it was confirmed that EB can be formed with the cell mass adhered on the carrier.

  On the other hand, in the method (b), after forming the cell mass, the upper member was removed, and the formed cell mass was transferred to a petri dish by pipetting several times with an EB medium, and suspension culture was performed in the petri dish. At this time, since the upper member is a dense body, there is no adhesion of cells when it is removed, and the adhesion of the cell mass to the lower member of the porous body is loose. The move was also easy.

And when the differentiation state was evaluated similarly to the said method (a), the expression of the differentiation marker of all three germ layers was confirmed on the 5th day.
Therefore, it was recognized that EB can be formed also by the method (b).

[Comparative Example 1]
Made of one-stage zirconia ceramics with a diameter of 13 mm and a height of 2 mm made of a porous body with a porosity of 50% having pores with a pore diameter of 0.1 to 1.0 μm in which micropatterns (recesses) having a diameter of 200 μm and a depth of 100 μm are formed. Using the cell culture carrier, ES cells were cultured in the same manner as in Example 1 except for that.

  As a result, ES cells moved or spread to the adjacent recesses and adhered to portions other than the recesses, so that a large amount of a three-dimensional cell cluster having a uniform shape could not be obtained. It was also difficult to collect cultured cells by pipetting.

[Comparative Example 2]
One stage zirconia ceramics with a diameter of 13 mm and a height of 2 mm made of a porous body with a porosity of 50% having pores with a diameter of 0.1 to 1.0 μm in which micropatterns (recesses) having a diameter of 200 μm and a depth of 400 μm are formed. Using the cell culture carrier, ES cells were cultured in the same manner as in Example 1 except for that.

  As a result, the ES cells did not move or spread to the adjacent recesses, and did not adhere to parts other than the recesses, but formation of a three-dimensional cell cluster with a uniform shape was observed on the third day of culture. The collection of cell mass by pipetting was difficult due to the deep recess.

DESCRIPTION OF SYMBOLS 1 Upper member 2 Lower member 11 Through-hole 21 Recessed part (micro pattern)

Claims (5)

It consists of a two-stage structure with a lower member and an upper member,
The lower member is a plate-like body made of a porous body, containing a plurality of recesses for accommodating and culturing cells on the upper surface,
The upper member is a plate-like body made of a dense body, detachably provided on the upper surface of the lower member, and having a plurality of through holes at positions corresponding to the recesses,
A cell culture carrier characterized in that it can accommodate cells to be cultured in the recess through the through-hole. It consists of a two-stage structure with a lower member and an upper member,
The lower member is a plate-like body made of a porous body, containing a plurality of recesses for accommodating and culturing cells on the upper surface,
The upper member is detachably provided on the upper surface of the lower member, the lower surface portion of the lower surface in contact with the lower member is made of a porous body, the upper surface portion of the upper surface facing the lower surface is made of a dense body, and It is a plate-like body having a plurality of through holes at corresponding positions,
A cell culture carrier characterized in that it can accommodate cells to be cultured in the recess through the through-hole.   The cell culture carrier according to claim 1 or 2, wherein the lower member and the upper member are made of any one of ceramics selected from hydroxyapatite, β-tricalcium phosphate, zirconia, alumina, and titania.   The cell culture carrier according to any one of claims 1 to 3, wherein the porous body has pores having a pore diameter of 5 µm or less and has a porosity of 10% to 70%.   The cell culture carrier according to any one of claims 1 to 4, wherein a width of the recess and the through hole is 10 µm or more and 1000 µm or less.

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