JP2009273444A - Cell-culturing supporter, method for producing the same and cell-culturing method using the supporter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-culturing supporter which can safely proliferate objective cells in a large amount at a low cost, and can be used as a material for biological substitutes for transplantation, artificial organs and the like. <P>SOLUTION: Provided is the cell-culturing supporter comprising a three-dimensional carrier and killed support cells which are immobilized on the three-dimensional carrier and whose inner proteins are chemically modified. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell culture support, a production method thereof, and a cell culture method using the support, and in particular, a material for cell culture, a biomaterial used for cell transplantation treatment, an artificial bone marrow, an artificial organ, etc. The present invention relates to a cell culture support that can be suitably used as a biological substitute.

  In recent years, in the field of regenerative medicine, attempts have been made to reconstruct damaged or lost living tissues and organs and their functions using some biological alternative. In general, tissue substitutes are prepared by three-dimensional culture of cells in vitro on a sponge-like carrier and used for treatment and transplantation. As the cells to be cultured, embryonic stem cells (ES cells) having the ability to differentiate into various cells, stem cells derived from adult tissues (adult stem cells), and the like are mainly used (see Non-Patent Documents 1 to 4). ).

  In addition, hematopoietic stem cell transplantation using bone marrow, peripheral blood and umbilical cord blood is being established as an effective treatment for serious blood diseases such as leukemia and aplastic anemia. However, problems such as insufficient donors, mismatched human lymphocyte antigens (HLA), and a small number of hematopoietic stem cells that can be collected at one time remain. On the other hand, for the purpose of application to hematopoietic stem cell transplantation and gene therapy, research for amplifying hematopoietic cells including hematopoietic stem cells in an in vitro culture system has been actively conducted in recent years (Non-Patent Documents 5 to 7). reference).

  In general, the culture of ES cells and hematopoietic cells described above requires co-culture with supporting cells that support cell growth. For example, ES cells are fibroblasts, and hematopoietic cells are stromal cells. (See Non-Patent Documents 5 and 6).

  However, when these cells are co-cultured with the respective supporting cells, only the supporting cells proliferate and the target cells often do not proliferate so much. Therefore, conventionally, after supporting cells are first cultured on a culture vessel such as a dish, irradiation with radiation or treatment with a DNA synthesis inhibitor to suppress the growth of supporting cells, The proliferation rate of the target cell is improved (see Non-Patent Documents 8 and 9).

Langer R. and Vacanti J. P., Science Vol. 260, p. 920-926, 1993. Aung T. et al., J. Biomed. Mater. Res. Vol. 61, p. 75-82, 2002. Baharvand H. et al., Int. J. Dev. Biol. Vol. 50, p. 645-652, 2006. Lees J. G. et al., Regen. Med. Vol. 2, p. 289-300, 2007. Dexter T. M. et al., J. Cell. Physiol. Vol. 91, p. 335-344, 1977. Ando K. et al., Blood Vol. 107, p. 3371-3377, 2006. Hofmeister C. C. et al., Bone Marrow Transplant. Vol. 39, p. 11-23, 2007. Ohneda O. et al., Blood Vol. 92, p. 908-919, 1998. Kang S. K. et al., Brain Res. Dev. Brain Res. Vol. 145, p. 141-149, 2003.

  The above-mentioned methods using irradiation and DNA synthesis inhibitors increase the cost of culture due to the need for expensive equipment, etc., and the remaining drug may adversely affect the target cells to be co-cultured with feeder cells. There is. Furthermore, for example, when cells are co-cultured with supporting cells immobilized on a carrier for the purpose of transplantation to humans, the carrier includes supporting cells in addition to the cells, so that it is not human from the viewpoint of safety. It is difficult to use different types of feeder cells, and it is necessary to prevent the feeder cells from being mixed around the site to be transplanted. In addition, since it is necessary to cultivate the supporting cells on the carrier in advance, the target cells cannot be cultured immediately when necessary.

  Accordingly, an object of the present invention is to be able to proliferate target cells safely and in large quantities at low cost, and can be used as a material for biological substitutes such as transplantation and artificial organs. It is to provide a culture support.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors fixed the support cells on a three-dimensional support and cultured them, and then chemically treated the support on which the support cells were fixed. The present invention was completed by finding that the target cells can be proliferated even if the protein in the protein is denatured and killed.

  That is, the cell culture support of the present invention comprises a three-dimensional carrier, and a support cell immobilized on the three-dimensional carrier and denatured and killed by intracellular protein. Culture support.

  In a preferred example of the cell culture support of the present invention, the three-dimensional support has a porous structure.

  In another preferred embodiment of the cell culture support of the present invention, the three-dimensional carrier is made of a polyvinyl formal resin.

  In another preferred embodiment of the cell culture support of the present invention, the chemical treatment is treatment with an aldehyde.

  In another preferred embodiment of the cell culture support of the present invention, the chemical treatment is treatment with an organic solvent. In addition, as said organic solvent, C1-C3 alcohol or ketone is preferable.

  In another preferred embodiment of the cell culture support of the present invention, the support cell is a stromal cell.

The method for producing the cell culture support of the present invention comprises a step of immobilizing support cells on a three-dimensional carrier,
Culturing the feeder cells on the three-dimensional carrier;
And denaturing the proteins in the feeder cells by chemical treatment to kill the feeder cells.

  The cell culture method of the present invention is characterized in that cells are seeded and cultured on the cell culture support. Examples of the cells include hematopoietic cells, embryonic stem cells, and adult stem cells.

  According to the present invention, target cells can be proliferated safely and in large quantities at low cost, and a cell culture support suitable as a material for biological substitutes such as artificial organs can be easily obtained. There is an advantageous effect.

  Hereinafter, the cell culture support of the present invention will be described in detail. The cell culture support of the present invention is characterized by comprising a three-dimensional support and support cells immobilized on the three-dimensional support and denatured and killed by intracellular protein. In the cell culture support of the present invention, since a three-dimensional carrier is used, target cells can be proliferated in a large amount and efficiently compared to two-dimensional culture such as a dish. Furthermore, since the supporting cells immobilized on the three-dimensional carrier have been killed and sterilized by chemical treatment, for example, cells obtained by growing the target cells on the cell culture support of the present invention are transplanted into the living body. When this is done, living support cells do not get mixed around the transplant site, which is highly safe. In addition, since the supporting cells are dead, the storage cost is low, and the supporting cells can be used immediately when necessary. In addition, since the feeder cells are killed, for example, when the cell culture support of the present invention is transplanted into a human body, heterogeneous feeder cells other than humans can be used, and thus mass production is possible.

  As the three-dimensional carrier constituting the cell culture support of the present invention, the supporting cells are held in the three-dimensional carrier, have biocompatibility, do not flow out to the outside, and support cells adhere smoothly and grow. As long as it is carried out, there is no particular limitation. Here, in order to attach a large number of supporting cells to the three-dimensional support, it is preferable that the surface area per unit area is large. Therefore, it is preferable that the three-dimensional support has a porous structure. A three-dimensional support having a network porous structure is more preferable.

  Here, the three-dimensional porous structure possessed by the three-dimensional carrier means that the substance constituting the three-dimensional carrier forms a network structure in three-dimensional and random directions, resulting in a complicated structure in the three-dimensional carrier. Intricate pores are produced, and these pores are continuous pores, that is, all the pores are connected and continuous, the pore diameter is uniform, the orientation of the pores is not directional, and Those having a high porosity are preferred. Furthermore, the three-dimensional porous structure is different in the pore size of the three-dimensional carrier used depending on the shape and size of the supporting cells to be used, the target cells, the three-dimensional carrier, and the size of the incubator. In view of the efficiency of culturing with the supporting cells carrying the target cells and the cells, the ability to retain the cells, and the growth state of the cells in the three-dimensional carrier, the average pore diameter is 1-1000 μm, preferably 5-600 μm. It is preferable. Similarly, the porosity is preferably 50 to 98%, more preferably 75 to 95%. This makes it possible to more efficiently immobilize a large number of supporting cells and target cells suspended in a predetermined medium on the three-dimensional carrier at high density.

  The shape of the three-dimensional carrier is not particularly limited, and any shape such as a spherical shape, a block shape, a sheet shape, or a honeycomb shape can be used. In particular, it is preferable to use a particulate material from the viewpoint that handling is easy and the ratio of the surface area is large, so that the amount of immobilized supporting cells and target cells can be further increased.

  In addition, when using a particulate three-dimensional support | carrier, it is preferable that a diameter or the magnitude | size of one side is a magnitude | size of 0.1-20 mm from the reason similar to the above. When a sheet-like or honeycomb-like three-dimensional carrier is used, it is preferable that one side has a size of 0.1 to 20 mm.

  As the material of the above three-dimensional carrier, it does not change in water and medium, exhibits chemical resistance to organic substances such as weak acids, alkalis and many organic solvents, is chemically stable, and has a high pressure. Those that can withstand sterilization with steam are preferred. With such a three-dimensional carrier, the three-dimensional carrier can be easily sterilized in a container appropriately selected with high-pressure steam or the like before culturing the feeder cells, and the three-dimensional carrier on which the feeder cells are immobilized. In addition, after culturing the target cells, the three-dimensional carrier is recovered, and the cells are lysed and detached by heat treatment or treatment with a weak acid, alkali, or solvent, Conveniently, the three-dimensional carrier can be reused after washing.

  Specifically, as the material of the above three-dimensional carrier, a polyvinyl formal resin having a three-dimensional network continuous porous structure, a polyurethane foam, or a known polymer material (especially polylactic acid, Glycolic acid, polycaprolactone, and copolymers thereof) foamed or made porous, stainless steel sintered metal support, porous glass and ceramics, and natural materials such as chitosan, cellulose, dextran, etc. Examples thereof include those derived from a high molecular weight material having a porous structure. Among these, from the viewpoint of cell retention ability, physicochemical properties of the carrier, or cost, the polyvinyl formal resin has a formalization degree of 60 to 90% and an average pore diameter of about 20 to 600 μm. Are preferred.

  In addition, the above three-dimensional carrier can be used by coating with a substance that promotes adherence of supporting cells, for example, a substance such as collagen, polylysine, fibronectin, laminin, histone, gelatin, or by treatment such as crosslinking. It can also be used by changing the surface characteristics such as the charged state of the surface.

  The feeder cell used in the present invention is a cell for proliferating a target cell itself that is grown using the cell culture support of the present invention. Examples of such support cells include stromal cells. Here, a stromal cell is also called a matrix cell or a stromal cell, and is a general term for cells that support and support cells and tissues that are responsible for their functions in organs. Some have the function of exerting some action on the partner cell by interaction. Such stromal cells include various types of cells such as fibroblasts, epithelial cells, reticulum cells, adipocytes, vascular cells, nerve cells, macrophages and the like. In the present invention, in addition to stromal cells isolated from a living body, stromal cell lines established by subculturing the cells or, in some cases, performing genetic recombination, can be used. Such cell lines are also intended to be included in the term “stromal cells” as used herein.

  The feeder cells used in the present invention can be selected according to the target cells to be proliferated. For example, when the target cells are ES cells, fibroblasts can be used as the feeder cells. When the cells are hematopoietic cells, bone cells or chondrocytes, stromal cells such as those derived from bone marrow, placenta and umbilical cord blood can be used. In addition, when the target cell is an undifferentiated cell, it is preferable that the supporting cell suppresses differentiation of the target cell. Further, the feeder cells may be different from the target cells.

  Immobilization of the supporting cells on the three-dimensional carrier is performed by injecting the seeding cells into the three-dimensional carrier in a predetermined container, seeding the cells, and culturing the cells in a predetermined medium for a predetermined period. A layer of feeder cells is formed inside or on the surface, or a medium containing feeder cells and a three-dimensional carrier are placed in a predetermined container, and the container is centrifuged and cultured for a predetermined period. be able to. From the viewpoint of increasing the number of cells that can be immobilized in the three-dimensional carrier and performing stronger immobilization, it is preferably immobilized by centrifugation. Centrifugation is preferably performed by applying a centrifugal force of 100 G or more, more preferably 200 G or more. Thereby, the animal cells suspended in the predetermined medium can be more efficiently immobilized. The upper limit of the centrifugal force is not particularly limited, but is preferably 500G. This eliminates the risk of damaging animal cells to be immobilized during centrifugation. The centrifugation time is preferably 1 to 10 minutes. And the centrifugation process performed continuously in the time in this range is made into 1 term, Preferably it is 1-10 terms.

Further, in order to increase the immobilization density of the supporting cells to be immobilized on the three-dimensional carrier and increase the amount of immobilizing the supporting cells per three-dimensional carrier, the number of seeding cells of the supporting cells in the medium is one. It is preferable that it is × 10 ⁶ pieces / cm ³ or more, and further preferably 5 × 10 ⁶ pieces / cm ³ or more. In the case of centrifugation, when the number of seeded cells suspended in the medium before centrifugation is increased as described above, if a centrifugal force of up to 400 G is applied, In addition, the animal cells can be efficiently immobilized without damaging the animal cells.

  As a container used for immobilizing feeder cells on a three-dimensional carrier, the container should be resistant to organic solvents, and in the case of using a centrifugal treatment, it must have mechanical strength that can withstand this. is necessary. Specifically, it is preferable to use an engineering plastic container such as polycarbonate. And before putting the culture medium containing a supporting cell in the said container, it sterilizes by high pressure steam at 121-150 degreeC for 20 minutes as needed.

  Moreover, the culture medium used for culture | cultivation of a support cell can be suitably selected according to the kind of cell, and can also add serum according to the objective. In addition, when the cell culture support of the present invention is used for cord blood transplantation or the like, plasma separated from blood or blood itself can be used. The period for culturing the feeder cells is not particularly limited as long as the feeder cell layer is sufficiently formed on the three-dimensional carrier, and may be, for example, 2 to 7 days.

  In the present invention, the supporting cells immobilized on the three-dimensional carrier must be denatured by killing intracellular proteins by chemical treatment. Here, “denaturing and killing proteins in cells by chemical treatment” means that proteins such as enzymes in cells are denatured by chemical treatment to make the structure and form of the cells as alive as possible. This means that the cells are killed while at the same time preventing changes such as cell autolysis, and is synonymous with cell fixation for the preparation of specimens of biological tissues and the like in histology.

  In the present invention, the chemical treatment for denaturing and killing the proteins in the cells of the feeder cells includes those used in the fixation of tissues and cells in so-called histology. Fixatives commonly used for fixing tissues and cells such as organic solvents such as aldehydes such as tar aldehyde and paraformaldehyde, or alcohols having 1 to 3 carbon atoms such as methanol and ethanol, or ketones having 1 to 3 carbon atoms such as acetone. The processing by.

  The treatment with a fixative such as an aldehyde or an organic solvent can be carried out, for example, by adding a fixative having an appropriate concentration to a container containing a three-dimensional carrier on which the above support cells are fixed. In the case of treatment with aldehyde, an aqueous solution in which aldehyde is dissolved in a suitably selected buffer is generally used. The concentration of the aldehyde in the aqueous aldehyde solution varies depending on the type of aldehyde, but may be a concentration that is usually used for fixation for preparing a tissue specimen. In the case of formaldehyde, 10 to 20% (volume / volume) ) In the case of glutaraldehyde, taking into account its toxicity, it may be 0.2 to 10% (volume / volume). The treatment time is not particularly limited as long as the protein is crosslinked in the case of an aldehyde and the cell is sufficiently dehydrated in the case of an organic solvent. When treatment with an aqueous solution of aldehyde is performed, it is desirable to wash the three-dimensional carrier having supporting cells with an aqueous solution such as a buffer solution. However, in the case of treatment with an organic solvent, the organic solvent is volatilized, and thus the washing treatment is performed. Is not particularly necessary.

  The washing treatment of the three-dimensional carrier containing the supporting cells after the treatment with aldehyde can be performed by rinsing with a washing solution such as an appropriately selected buffer solution or by immersing in the washing solution. The time to do can be selected according to the kind of aldehyde used and the concentration of aldehyde in the aldehyde solution. For example, in the case of a carrier that forms support cells and is treated with 10% formalin buffer as an aldehyde, the carrier is rinsed three or more times with a washing solution, and the carrier is washed by immersing in the washing solution for 24 hours or more for safety. it can.

  The cell culture support of the present invention is a cell that particularly requires co-culture with support cells, specifically, stem cells such as ES cells, induced pluripotent stem (iPS) cells, adult stem cells, hematopoietic stem cells, hematopoiesis Suitable for culturing hematopoietic cells other than stem cells, bone cells, chondrocytes, muscle cells, nerve cells, fibroblasts, vascular cells, liver-derived cells, pancreas-derived cells and kidney-derived cells. Culturing of such cells using the cell culture support of the present invention is carried out, for example, by seeding and culturing target cells on the cell culture support in a predetermined container containing an appropriately selected medium. The target cells can be proliferated in large quantities. The medium can be appropriately selected according to the target cells to be cultured, and serum can be added according to the purpose. In addition, when the cell culture support of the present invention is used for transplantation of hematopoietic stem cells, plasma separated from blood or blood itself can be used.

  In addition, target cells can be cultured on the cell culture support of the present invention, and the whole cell culture support containing the target cells can be transplanted in vivo. Furthermore, biological substitutes such as artificial organs and artificial bone marrow can be produced using a cell culture support containing such cells of interest. Since the support cells contained in the cell culture support of the present invention can be different from the target cells, and the support cells are killed and sterilized by the above-described chemical treatment, the cell culture support of the present invention Is highly safe and can be transplanted into a living body as it is, and can also be used as a material for a biological substitute such as an artificial organ.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

  In this example, a stromal cell layer subjected to various chemical treatments using a hematopoietic cell as a cell to be amplified using the cell culture support of the present invention as an example and a stromal cell as a supporting cell is a hematopoietic cell. The effect on the amplification was investigated.

<Experiment method>
(Cells used for culture)
In this example, fetal liver cells collected from embryonic day 14 C57BL / 6 Cr Slc mice (Japan SLC, Shizuoka) were used as hematopoietic cells (Miyoshi H. et al., ASAIO J. Vol. 46). , p. 397-402, 2000). After the mouse was dislocated and euthanized by cervical vertebrae, the abdomen was opened, the uterus was removed, the placenta was separated, and the fetus in the amniotic membrane was removed. The liver was isolated from these fetuses under a stereomicroscope, and the cells were dispersed by pipetting using a syringe equipped with a 22G injection needle in the HAVA medium described below. The cell suspension was passed through a cell strainer (Falcon 352350; Becton Dickinson Labware, NJ, USA) with a pore size of 70 μm to remove impurities and cell clumps. The obtained cell number and cell viability were measured by the trypan blue exclusion method.

For stromal cells, DAS (dorsal aorta derived-stromal) 104-8, a cell line derived from the dorsal aortic vascular endothelial cells of embryonic day 11, was used (Ohneda O. et al., Blood Vol. 92, p. 908-919, 1998). This DAS 104-8 cell line was subcultured using a 75 cm ² flask (Falcon 353135). When the cell density reached about 2.0 × 10 ⁵ cells / cm ² , the cells were detached using 0.05% trypsin-EDTA (Gibco BRL, NY, USA) and used for passage or experiment.

(Medium and three-dimensional culture carrier)
In all the culture experiments, HAVA medium using DMEM-H (high glucose Dulbecco's modified Eagle's medium; Gibco BRL) as a basic medium was used. This medium contains 10% FBS (fetal bovine serum characterized; Sigma, MO, USA), MEM nonessential amino acid (0.1 mmol / L; Gibco BRL), L-glutamine (2 mmol / L; Gibco) as additives to DMEM-H. BRL), 2-mercaptoethanol (1 × 10 ⁻⁴ mol / L; Wako Pure Chemical Industries, Osaka) and antibiotics (0.5% penicillin / streptomycin; Gibco BRL).

  In this example, polyvinyl formal (PVF) resin (Aion, Osaka), which is a porous resin, was used as a carrier for three-dimensional cell culture (Ehashi T. et al., J. Biomed. Mater. Res. A Vol. 77, p. 90-96, 2006; Ehashi T. et al., J. Biomed. Mater. Res. A Vol. 82, p. 73-79, 2007). After chopping PVF resin with an average pore size of 130μm into 2x2x2mm cubes, sterilizing them in a centrifuge bottle and autoclaving, infiltrate with 0.06% collagen (Cellgen I-PC; Koken, Tokyo) for more than half a day. Collagen coat. The obtained PVF resin carrier was washed stepwise with PBS (−) (phosphate buffered saline; Nissui Pharmaceutical, Tokyo), Hank's solution (Sigma), and HAVA medium, and cells were seeded.

(Seeding of cells into a three-dimensional culture carrier)
In three-dimensional culture, seeding of cells on a culture carrier was performed using a centrifugal cell immobilization (CCI) method utilizing centrifugation (Yang TH et al., J. Biomed. Mater. Res. Vol. 55, p. 379-386, 2001). First, 100 collagen-coated carriers were placed in a centrifuge bottle. After adding 4.0 ml of a cell suspension containing a predetermined amount of cells to this bottle, the cells were attached to the carrier by centrifugation at 300 × g for 1 minute. . Cells that did not adhere to the carrier and settled to the bottom of the bottle were floated again by gently stirring the bottle and centrifuged again. By repeating these operations 6 times, the cells were seeded on a carrier.

(Culture experiment)
In this example, three-dimensional culture using PVF resin as a carrier and monolayer culture using a dish, which is a normal culture method, were performed. In each culture, stromal cells were first seeded and cultured to form a stromal layer, which was then chemically treated and stored. The stromal cells were replated with mouse fetal liver cells and cultured. The general flow of the culture experiment is shown in FIG.

In the three-dimensional culture, 1 × 10 ⁷ cells / cm ³ of stromal cells were seeded on a carrier and cultured for 2 days or 7 days to form a stromal layer. In this culture, 30 carriers each were transferred to a 60-mm Petri dish, and 7.5 ml of HAVA medium was added to perform static culture. Next, an experiment was conducted in which the PVF resin in which the stromal layer was formed was chemically treated together with the carrier, and after a certain period of time, these were washed and re-seeded with fetal liver cells.

In the culture of fetal liver cells, 1 × 10 ⁸ cells / cm ³ of mouse fetal liver cells were seeded on a carrier having a stromal layer and cultured for another 2 weeks. At this time, 10 carriers each were transferred to a 35-mm Petri dish and statically cultured in 2.5 ml of medium.

In monolayer culture, collagen-coated 35-mm Petri dishes were seeded with 5 × 10 ⁵ cells / dish stromal cells, cultured for 2 days to form a stromal layer, and then subjected to chemical treatment. The dish was replated with 5 × 10 ⁶ cells / dish of mouse fetal liver cells and cultured for 2 weeks. The medium was changed every other day for both monolayer culture and three-dimensional culture.

(Chemical treatment of stromal cells)
Chemical treatment of stromal cell lines was performed using 10% neutral buffered formalin solution, 0.2% glutaraldehyde-containing phosphate buffer (pH 7.3), or acetone, which is commonly used as a cell fixative. . Rinse the stroma-layered carrier with PBS, remove the medium in the carrier, transfer the carrier to a 50 ml centrifuge tube, add 1 ml of fixative to 10 carriers, and perform chemical treatment at 4 ° C for 1-7 days. Went. The carrier after the chemical treatment was washed with PBS and then used for culture experiments or stored at 4 ° C. in a centrifuge tube to which PBS was added. In the chemical treatment with acetone, chemical treatment was performed at −20 ° C. using a glass centrifuge tube. The chemical treatment was carried out for 3 to 7 days, degassed before reseeding the fetal liver cells, and acetone was removed from the carrier before use in culture experiments.

(Measurement of cell number)
In this example, the sustained change in the number of cells in the culture of mouse fetal liver cells was measured using the DNA method or MTT method described below.

(1) DNA measurement method The number of cells immobilized on a carrier was measured by a method of measuring the amount of DNA using a fluorescent dye Hoechst 33342 (Lydon MJ et al., J. Cell. Physiol. Vol. 102, p. 175-181, 1980; Sriram M. et al., Biochemistry Vol. 31, p. 11823-11834, 1992). Hoechst 33342 is a fluorescent dye that specifically binds to the AT sequence of DNA, and the amount of DNA in all cells can be determined by measuring the fluorescence intensity.

  First, 10 carriers each having a cell attached thereto were put into a test tube, 2 ml of 0.05 U / ml pronase solution (Sigma) was added to the test tube, and allowed to stand at 37 ° C. overnight. Next, this test tube was subjected to an ultrasonic crusher to destroy the cells. 200 μl of the obtained pronase solution was added to 1.8 ml of 1 mM Hoechst 33342 solution (Dojindo Laboratories), and a fluorescent dye was bound to DNA. The amount of DNA was determined by measuring the fluorescence intensity of this solution with a spectrofluorometer (excitation light 352 nm, incident light 461 nm; FP-6300 JASCO Corporation, Tokyo). The calibration curve was prepared using a known amount of herring sperm DNA (Sigma) and a known amount of cells.

(2) MTT method
The number of living cells was measured using the MTT (3- [4,5-dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium bromide) method (Mossman T., J. Immunol. Methods Vol. 65, p. 55-63, 1983; Yamaji H. and Fukuda H., Appl. Microbiol. Biotechnol. Vol. 37, p. 244-251, 1992). This method is a method of measuring living cells by measuring the amount of MTT formazan that is insoluble in water, utilizing the fact that MTT is converted to MTT formazan by an enzyme contained in mitochondria in the cell.

The MTT solution used for the measurement was prepared by dissolving MTT (Dojin Research Institute, Kumamoto) in PBS (−) at a concentration of 0.5 g / L. In the case of monolayer culture, 2 ml of MTT solution was added to the dish from which the medium was removed. In 3D culture, 10 carriers with attached cells were transferred to a 15 ml centrifuge tube and 2 ml of MTT solution was added. After these dishes and centrifuge tubes were incubated at 37 ° C. for 3 hours, when using the dishes, adherent cells were detached by pipetting and transferred to a 15 ml centrifuge tube. The resulting centrifuge tube was centrifuged at 1500 rpm for 3 minutes to precipitate the produced MTT formazan. Remove the supernatant MTT solution from the centrifuge tube, add 2 ml of isopropyl alcohol to dissolve MTT formazan, perform further centrifugation to remove unwanted substances, and use the spectrophotometer to measure the absorbance of the resulting supernatant. Measured. At this time, the absorbance at 560 nm (measurement wavelength) and 700 nm (control wavelength) was measured to determine the difference between the two (OD _560-700 ). The number of immobilized cells cultured was calculated from a calibration curve prepared using a known amount of cells in advance.

(Analysis of blood cells in cultured cells)
In order to identify the types of hematopoietic cells in the cultured cells, analysis using flow cytometry (FACS) was performed. First, 1.0 ml of a 0.25% trypsin-EDTA / 0.2% collagenase solution (Gibco BRL) was dropped onto 10 carriers to which cells were attached, and then 2 ml of PBS containing 2% FBS was added. These cell suspensions were passed through a cell strainer (Falcon 352340) having a pore diameter of 70 μm to remove cell clumps and impurities, and further washed with PBS containing FBS. After that, add a staining medium (PBS with 2% FBS and 0.1% sodium azide (Wako Pure Chemical Industries, Ltd.)) to a cell density of 2-5 × 10 ⁵ cells / ml, and add it to a 1.5 ml sampling tube. 250 μl was dispensed. These tubes were centrifuged at 1500 ° C. for 3 minutes at 4 ° C. to remove the supernatant, and 50 μl of the staining medium was added again.

  For analysis by flow cytometry, anti-TER119 antibody (PE label), which is an indicator of erythroblasts, anti-CD45R / B220 antibody (FITC label), which is an indicator of B cells, and anti-c-, which is an indicator of hematopoietic progenitor cells Kit (CD117) antibody (PE label) and anti-CD34 antibody (FITC label) which is an indicator of hematopoietic stem / progenitor cells (all PharMingen International, CA, USA) were used. 1.0 μl of each of these antibodies was added to a sampling tube and incubated on ice for 30 minutes. A tube to which no antibody was added was used as a control. Then, add 500 μl of staining medium, centrifuge at 1500 ° C. for 3 minutes at 4 ° C. to remove the supernatant, and then add propidium iodide (PI; 0.5 mg / ml, Sigma), a staining dye for dead cells, to the staining medium. 500 μl of the solution added at 2 μl / ml was added. The obtained cell suspension was passed through a cell strainer having a pore size of 35 μm to remove the cell mass, and then transferred to a 5 ml round tube (Falcon 352235). Stained cells were counted using a FACSCalibur (Becton Dickinson Labware) equipped with a 488 nm argon laser and a 599 nm dye laser. In each sample, the results obtained from 10,000 cells were analyzed using Cell Quest Software (Becton Dickinson Labware).

  The number of cells of each lineage in cultured cells is the number of living cells measured by MTT measurement method or DNA measurement method, and TER119 positive cells, B220 positive cells, c-Kit positive cells, or CD34 obtained from FACS results. It calculated | required from the ratio of the positive cell. All measured values were expressed as mean ± standard deviation.

<Example 1>
(Effects of formalin chemically treated stromal cells on hematopoietic cells)
First, a stromal layer is formed on a three-dimensional carrier, stromal cells on the carrier are chemically treated with formalin, and the obtained carrier is rinsed three times or more with a washing solution, and immersed in the washing solution for safety for 24 hours or more. . This stroma layer was replated with mouse fetal liver cells and cultured. FIG. 2 shows the change over time in the cell amplification rate after reseeding. The total number of cells replated decreased continually (FIG. 2a). Regarding the change over time of hematopoietic cells, TER 119 positive cells decreased rapidly (FIG. 2b), and B220 positive cells proliferated (FIG. 2c). On the other hand, the cell density of c-Kit positive cells (FIG. 2d) and CD34 positive cells (FIG. 2e) decreased.

  From the above results, when hematopoietic cells were cultured using a formalin chemically treated stroma layer, B220 positive cells were amplified, but undifferentiated hematopoietic cells (c-Kit positive cells and CD34 positive cells) were not. It was not amplified.

<Example 2>
(Effect of glutaraldehyde chemically treated stromal cells on hematopoietic cells)
FIG. 3 shows the change in the number of cells after re-seeding when the mouse fetal liver cells were replated on the stroma layer chemically treated with glutaraldehyde and cultured. Regarding the cell density of live cells (FIG. 3a), the number of viable cells increased throughout the culture period in three-dimensional culture (black circle), and a constant number of cells was maintained in monolayer culture (white circle). In the long-lasting change of hematopoietic cells (FIGS. 3b to 3e), first, the number of TER119 positive cells slightly increased in three-dimensional culture, and the number of cells was maintained even in monolayer culture (FIG. 3b). On the other hand, B220 positive cells proliferated in both three-dimensional culture and monolayer culture. In addition, c-Kit positive cells had a high amplification rate on the 14th day of culture (FIG. 3d). CD34 positive cells were also amplified in the three-dimensional culture after the seventh day of culture (FIG. 3e).

  Based on the above results, in the culture of hematopoietic cells using glutaraldehyde chemically treated stroma layers, undifferentiated cells such as c-Kit positive cells and CD34 positive cells were amplified, and the effect was remarkable in three-dimensional culture. It was. Furthermore, it was shown that TER119 positive cells can be amplified slightly.

<Example 3>
(Effect of acetone chemically treated stromal cells on hematopoietic cells)
FIG. 4 shows time-dependent changes in the cell growth rate after reseeding when mouse fetal liver cells were reseeded on the acetone chemically treated stroma layer. The number of viable cells increased better than when a three-dimensional cryopreserved stromal cell layer was used (FIG. 4a). Regarding chronological changes in hematopoietic cells after reseeding, TER119-positive cells increased slightly until the seventh day of culture (FIG. 4b), whereas B220-positive cells were amplified after the seventh day of culture. (Figure 4c). Further, c-Kit positive cells and CD34 positive cells were also amplified throughout the culture period (FIG. 4c).

  From the above results, it was possible to amplify cells other than TER119 positive cells in the culture of hematopoietic cells using an acetone chemically treated stroma layer.

<Example 4>
(Comparison of chemical treatment methods)
Table 1 summarizes the amplification rate of hematopoietic cells when a chemically treated stromal cell layer is used. First, TER119-positive cells were amplified when acetone chemical treatment or glutaraldehyde chemical treatment stromal cells were used, but the amplification rate was slight. B220-positive cells were amplified only up to day 7 when formalin chemical treatment was used, but were amplified up to day 14 of culture when cells treated with glutaraldehyde or acetone were used. it was high. As for undifferentiated hematopoietic cells, c-Kit positive cells were amplified 2 to 5 times by using glutaraldehyde or acetone chemically treated stromal cells. On the other hand, the amplification rate of CD34 positive cells under these conditions was about twice, which was lower than the amplification rate of c-Kit positive cells.

  From the above results, it was revealed that the hematopoietic cells to be amplified differ depending on the chemical treatment.

FA；三次元ホルマリン化学処理DAS 104-8細胞、GA(3-D)；三次元グルタールアルデヒド化学処理DAS 104-8細胞、GA(2-D)；グルタールアルデヒド化学処理DAS 104-8細胞（単層）、アセトン；三次元アセトン化学処理DAS 104-8細胞

FA; 3D formalin chemically treated DAS 104-8 cells, GA (3-D); 3D glutaraldehyde chemically treated DAS 104-8 cells, GA (2-D); glutaraldehyde chemically treated DAS 104-8 cells (Monolayer), acetone; three-dimensional acetone chemical treatment DAS 104-8 cells

  From these results, it was found that hematopoietic cells can be proliferated by three-dimensional culture using a chemically treated stromal cell layer. Such a culture method is a system for amplifying hematopoietic cells in vitro. It was thought that it could become an effective means in building.

FIG. 1 is a diagram showing a flowchart of a culture experiment. FIG. 2 is a graph showing the time-dependent change (n = 3) in cell growth rate after reseeding in three-dimensional culture of hematopoietic cells using a three-dimensional formalin chemically treated stroma layer (black diamond; formalin chemical treatment ( FA), a) total cell density, b) TER119 positive cells, c) B220 positive cells, d) c-Kit positive cells, e) CD34 positive cells). FIG. 3 is a graph showing the change over time in cell growth rate after re-seeding in the culture of hematopoietic cells using a glutaraldehyde chemically treated stroma layer (black circle; glutaraldehyde chemical treatment (three-dimensional culture), ( GA (3-D), n = 5), white circle; glutaraldehyde chemical treatment (monolayer culture), (GA (2-D), n = 3), a) total cell density, b) TER119 positive cell, c) B220 positive cells, d) c-Kit positive cells, e) CD34 positive cells). FIG. 4 is a graph showing the time-dependent change (n = 3) in cell growth rate after reseeding in three-dimensional culture of hematopoietic cells using a three-dimensional acetone chemical treatment stroma layer (black diamond; acetone chemical treatment, a) total cell density, b) TER119 positive cells, c) B220 positive cells, d) c-Kit positive cells, e) CD34 positive cells).

Claims (11)

  A cell culture support comprising: a three-dimensional carrier; and a support cell immobilized on the three-dimensional carrier and denatured and killed by intracellular protein.   The cell culture support according to claim 1, wherein the three-dimensional support has a porous structure.   The cell culture support according to claim 1 or 2, wherein the three-dimensional carrier is made of polyvinyl formal resin.   The cell culture support according to any one of claims 1 to 3, wherein the chemical treatment is treatment with an aldehyde.   The cell culture support according to any one of claims 1 to 3, wherein the chemical treatment is treatment with an organic solvent.   The cell culture support according to claim 5, wherein the organic solvent is an alcohol or ketone having 1 to 3 carbon atoms.   The cell culture support according to any one of claims 1 to 6, wherein the support cells are stromal cells. Immobilizing supporting cells on a three-dimensional carrier;
Culturing the feeder cells on the three-dimensional carrier;
The method for producing a cell culture support according to any one of claims 1 to 7, further comprising a step of denaturing the protein in the feeder cells by chemical treatment to kill the feeder cells.   A cell culture method comprising seeding and culturing cells on the cell culture support according to any one of claims 1 to 7.   The cell culture method according to claim 9, wherein the cell is a hematopoietic cell.   The cell culture method according to claim 9, wherein the cell is an embryonic stem cell or an adult stem cell.

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