JP2004331643A - Cell preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell preparation capable of preserving cells secreting physiologically active factors such as a hormone, a protein, useful for patients in a polyvinyl alcohol stably for a long period, and a method for producing the same, and a method for preventing and treating endocrinic and metabolic diseases, hemophilia, bone diseases, cancers, or the like, by administering the cell preparation. <P>SOLUTION: This cell preparation is provided by containing the cells in the polyvinyl alcohol blended with a cell preserving agent. Preferably, it is characterized in that the surfaces of the cells are covered with an extracellular matrix. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell preparation useful as a pharmaceutical composition for humans or animals. More specifically, it is a preparation that stably and long-term retains cells that produce and secrete biologically active factors such as hormones or proteins useful for living bodies, or cells that have the effect of detoxifying harmful substances. The present invention relates to a cell preparation that exerts an effect on prevention / treatment of endocrine / metabolic diseases, hemophilia, bone diseases, cancer, etc. by administration.

  A bioartificial organ contains living cells, living tissues, etc., and provides the patient with metabolic functions necessary for the patient, specifically, biologically active factors such as hormones and proteins that control the metabolic function. A device intended to prevent or treat a patient's disease. Compared to living organ transplantation that has problems such as side effects and donor demand and supply due to immunosuppressive agents, bioartificial organs can protect cells from the defense mechanism of the living body by immune isolation membranes, so no immunosuppressive agents are needed, It is excellent in that not only allogeneic transplantation but also xenotransplantation is possible.

  Bioartificial organs can be used to treat all diseases by changing the types of cells and the like contained therein. For example, a bioartificial islet has insulin-secreting cells, for example, islet cells therein, and is used to supply the insulin hormone secreted from the islet cells to the patient to correct the blood glucose level. The blood coagulation factor-producing bioartificial organ has hepatocytes that produce blood coagulation factors in its interior, and is used for the treatment of hemophilia that has blood coagulation disorders due to the lack of factor VIII or factor XI. Yes. Growth hormone-producing bioartificial organs have growth hormone (hGH) -secreting cells inside and are used for the treatment of pituitary dwarfism caused by insufficient secretion of growth hormone. In addition, by incorporating cells that secrete parathyroid hormone and erythropoietin into the bioartificial organ, other diseases such as hypoparathyroidism and anemia can be treated.

  There are various shapes of bioartificial organs. Examples thereof include a microcapsule type or a macrocapsule type in which cells are encapsulated with a polymer. These are protected from the defense mechanism of the living body by the strong cross-linked structure of the polymer and are secreted from the cell by utilizing the molecular permeability of the polymer. It is characterized by supplying hormones to the living body.

  In recent years, polyvinyl alcohol (hereinafter abbreviated as PVA) has attracted attention as a polymer used in macrocapsule-type artificial organs and the like. PVA has been conventionally used in the food industry and the medical industry, and specifically applied to surgical sutures, containers that come into contact with food, and the like. Moreover, the technique (for example, refer nonpatent literature 1) which uses PVA as an artificial joint cartilage material has been disclosed until now, and clinical safety is also evaluated (for example, refer nonpatent literature 2).

PVA is gelled by chemical or physical treatment and can be formed into any shape. As a chemical treatment, a method of adding glutaraldehyde (crosslinking agent) and hydrochloric acid (catalyst) to an aqueous solution containing PVA (see, for example, Non-Patent Document 3), or an aqueous solution containing PVA is applied to a glass plate or the like, and this is applied. A method of immersing in an aqueous solution of Na ₂ SO ₄ / KOH (see, for example, Non-Patent Document 4) or the like, and a physical treatment such as a method of rapidly cooling an aqueous solution containing PVA at about −80 ° C. to gel is used. . However, when PVA is used as a polymer such as a macrocapsule-type artificial organ, the necessity for these chemical or physical treatments becomes a problem.

  Specifically, there is a method in which PVA is gelled by chemical treatment to produce a bag-like PVA gel film, and cells are placed in the bag to produce a bioartificial organ. However, this method reduces the number of viable cells or biological activity, such as the cells are damaged by the sealing treatment of the bag or the cross-linking agent remaining on the PVA gel film, or the cells in the bag are aggregated to cause necrosis. There is no denying the decrease in the amount of hormone secretion due to the decrease in the level of hormone.

  The physical treatment is preferable in that there is no risk of cell necrosis due to aggregation or the like because it does not use a drug and can be dispersed and included in the PVA gel, but it is rapidly cooled at −80 ° C. In some cases, cells may be destroyed.

It is desirable that the cells contained in the bioartificial organ be in a state in which a biologically active factor that exhibits a necessary metabolic function for the patient can be stably supplied. Therefore, although PVA is a high molecular polymer suitable for macrocapsule-type bioartificial organs due to its characteristics, its application technology has not been established yet. Thus, as in the present invention, a bioartificial organ that utilizes the characteristics of PVA and can stably hold cells in the PVA gel has never been known.
Kobayashi et al. (Kobayashi M., et al.), Preliminary study of polyvinyl alcohol-hydrogel (PVA-H) artificial meniscus, Biomaterials, 2003, Volume 24, p639-647 Kobayashi et al. (Kobayashi M., et al.), Preliminary study of polyvinyl alcohol-hydrogel (PVA-H) artificial meniscus, Biomaterials, 2003, Volume 24, p639-647 CC Demerlis et al., Review of the oral toxicity of PVA, Food and Chemical Toxicology, 2003, Vol. 41, p319-326 Krystyna Burczak et. Al. Long-term in vivo performance and biocompatibility of PVA hydrogel macrocapsules for hybrid-type artificial pancreas , Biomaterials, 1996, Vol. 17, p2351-2356 Tai-Horn Young et. Al. Assessment and modeling of PVA bioartificial pancreas in vivo, Biomaterials, 2002, Vol. 23, p3495 -3501

  An object of the present invention is to provide a cell preparation useful as a pharmaceutical composition for humans or animals. More specifically, cells that secrete biologically active factors such as hormones or proteins useful for patients can be stably and long-term retained in PVA, which is a suitable material as a polymer used in bioartificial organs. It is an object to provide a preparation and a method for producing the cell preparation. It is another object of the present invention to provide a method for preventing / treating endocrine / metabolic diseases, hemophilia, bone diseases or cancer by administering the cell preparation to a patient.

  As a result of intensive studies to achieve the above object, the present inventors have previously treated PVA with a cell preservative, for example, a Euro-Collins solution, a cell banker, a UW solution or the like, and then mixed with the cells to gel. Thus, it was found that the death rate and damage of the cells in the PVA gel can be reduced, and the cells are stably held in the PVA. More specifically, by dissolving powdered PVA with a liquid cell preservative to prepare a PVA-cell preservative mixture, cells are dispersed in the mixture and rapidly cooled to gel the PVA. The cell viability in PVA and the decrease in the secretory function of biologically active factors are suppressed, and further, a cell preparation capable of stably supplying hormones or proteins to a patient for a long period of time is obtained. I found it. The present inventors have further studied based on these findings and have completed the present invention.

That is, the present invention provides (1) a cell preparation containing cells in polyvinyl alcohol containing a cell preservative,
(2) The cell preparation according to (1), wherein the cell surface is further covered with an extracellular matrix,
(3) The cell preparation according to (1) or (2), further comprising a growth factor,
(4) One or more cells selected from the group consisting of islet cells, pancreatic endocrine cells, hepatocytes, anterior lobe cells, growth hormone secreting cells, bone cells, thyroid hormone secreting cells and parathyroid hormone secreting cells The cell preparation according to any one of (1) to (3), wherein
(5) The cell preparation according to any one of (1) to (4), wherein the cell is treated with a cell preservative,
(6) The cell preparation according to any one of (1) to (5), wherein the cell is a transformant,
(7) The cell preparation according to any one of (1) to (6) above, which has a sheet shape, a plate shape, a rod shape, a tube shape, or a bead shape,

(8) The cell preparation according to any one of (1) to (7) above, wherein the cell preservation solution is a Euro-Collins solution, a cell banker or a UW solution,
(9) The cell preparation according to (1) to (8) above, which is transplanted subcutaneously, intramuscularly or intraperitoneally in a mammal,
(10) A method for preventing and treating an endocrine / metabolic disease, comprising using the cell preparation according to any one of (1) to (9),
(11) The endocrine / metabolic disease prevention and treatment method according to (10), wherein the endocrine / metabolic disease is diabetes or pituitary dwarfism,
(12) A method for preventing and treating hemophilia, comprising using the cell preparation according to any one of (1) to (9),
(13) A method for preventing and treating a bone disease, comprising using the cell preparation according to any one of (1) to (9),
(14) A method for preventing and treating cancer, comprising using the cell preparation according to any one of (1) to (9),
(15) A method for preventing and treating liver failure or inborn errors of metabolism characterized by using the cell preparation according to any one of (1) to (9) above,
(16) A method for producing a cell preparation, comprising mixing a cell preservative with polyvinyl alcohol, mixing the cells with the polyvinyl alcohol, and gelling the obtained cell-containing polyvinyl alcohol,
(17) The cell preparation according to any one of (1) to (9), which is a pharmaceutical for humans or animals,
About.

  According to the present invention, cells that secrete biologically active factors such as hormones or proteins useful for patients can be stably and long-term retained in PVA, which is a material suitable as a polymer used in bioartificial organs. A formulation can be provided. Furthermore, by administering the present invention to a patient, it is possible to prevent and treat endocrine / metabolic diseases, hemophilia, bone diseases or cancer. Since the present invention can have cells in a stable state for a long period of time, the frequency of transplantation can be reduced, and since it can be formed into various shapes, it can be implanted subcutaneously or intramuscularly by injection or the like. It is.

The present invention is characterized in that it contains PVA mixed with a cell preservative and has cells that produce and secrete biologically active factors such as hormones and proteins useful for patients in the PVA.
The PVA used in the present invention may be any one as long as it does not impair the object of the present invention. For example, medical materials such as keratin protective / humectant for eye drops, intestinal adhesion preventive film, and food contact such as food packaging film It is preferable that the material has a purity that can be used as a material. Moreover, even if it is a commercial item, the freeze-dried product of the product obtained by making polyvinyl acetate from vinyl acetate and hydrolyzing this may be sufficient. Moreover, when manufacturing PVA, the manufacturing method and the freeze-drying method may follow a technique known per se. The average polymerization molecular weight of PVA is preferably about 5,000 to 16,600, and the saponification degree is preferably about 85% or more.
These PVAs are finely particulate lyophilized products and desirably have a shape that can be easily suspended in a solution such as physiological saline or phosphate buffer.

  PVA in which a cell preservative is blended means PVA containing a cell preservative in PVA. The cell preservative used in the present invention is not particularly limited, and is a liquid agent usually used for preserving animal cells and the like. For example, a Euro-Collins solution or a cell banker (SERUBANNKA code 630-01601: Wako Pure Chemical Industries, Ltd. Osaka, Japan) or UW solution (Biasol-Myers Squibb Company, USA). These may be commercially available or compositions prepared in the laboratory. When preparing in a laboratory, it is preferable to prepare by sterilizing by adding / mixing each composition component to distilled water or the like and performing filter filtration or the like. For example, the composition of Euro-Collins solution is as shown in Table 1.

The composition of the UW liquid (via span) is as shown in Table 2.

PVA containing a cell preservative in PVA can be prepared, for example, by mixing PVA and a cell preservative. Moreover, it is desirable that the PVA containing the obtained cell preservative is aseptic. The method of mixing the PVA and the cell preservative is not particularly limited. For example, a commercially available particulate PVA is suspended in distilled water or the like, and the PVA suspension is heated and sterilized (autoclave or the like) to be dissolved and sterilized. And a method of adding and mixing a cell preservative aseptically to the obtained sterile PVA aqueous solution. The heat sterilization treatment or aseptic operation may be performed according to a method known per se.
Here, PVA containing a cell preservative prepared by using the above-mentioned mixing means and any other means is referred to as PVA treated with a cell preservative. It goes without saying that PVA is also within the scope of the present invention.

  In addition, the content of PVA in a mixed solution obtained by mixing a PVA suspension aqueous solution and a cell preservative (hereinafter referred to as PVA-cell preservative solution) is usually approximately about the PVA-cell preservative solution. It is 2 to 5% by weight, more preferably about 2.5 to 3.5% by weight, particularly preferably about 2.5 to 3% by weight. This is because PVA is most easily gelled in the concentration range, and a PVA gel having a strength preferable for including the cells of the cell preparation of the present invention can be obtained. The concentration of the cell preservative in the PVA-cell preservative solution is usually about 0.8 to 1 times, more preferably about 0.9 to 1 when the concentration of the cell preservative used for normal cell preservation is 1 time. 1 time, particularly preferably about 0.95 to 1 time. This is because if the concentration is out of the range, the cells may not survive stably. Therefore, in the present invention, a concentrated cell preservation solution having a concentration of about 2 to 10 times the concentration used for normal cell preservation is prepared or purchased, and this and the above PVA suspension aqueous solution are appropriately used (for example, 10 times concentrated cell preservation Liquid: PVA suspension aqueous solution = 1: 9) It is preferable to mix to prepare a PVA-cell preservative solution containing about 1-fold concentration of the cell preservative.

The cell preparation of the present invention further includes dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of protecting cells, antibiotics, etc. for the purpose of preventing contamination by bacteria, and for maintaining cell activity. Vitamins such as nicotinamide may be included. Furthermore, the present invention may be supplemented with other additive components that are normally allowed to be added to pharmaceutical preparations, such as sustained release imparting agents, tonicity agents, pH adjusting agents and the like. The method for adding these other additive components to the cell preparation is not particularly limited, but when the PVA-cell preservative solution is prepared, it is preferably mixed aseptically into the solution. These contents are preferably in a range that does not inhibit the growth / survival of cells and / or secretion of biologically active factors included in the present invention and that do not impair the object of the present invention.
Since PVA treated with a cell preservative is aseptically produced as described above, the cell preparation of the present invention has a low probability of contamination by various bacteria and is stored at room temperature (about 15 to 35 ° C.) for a long time. Is possible.

  Examples of the cells used in the present invention include islet cells, pancreatic endocrine cells, hepatocytes, anterior lobe cells, growth hormone secreting cells, bone cells, thyroid hormone secreting cells, or parathyroid hormone secreting cells. These cells are preferably cells that are derived from mammals such as humans, pigs, rats, or mice, and that produce and secrete biologically active factors such as hormones or proteins useful for patients. The choice of cell type is preferably determined by the type of disease in the patient being administered. For example, pancreatic islet cells or pancreatic endocrine cells that produce insulin for diabetic patients, hepatocytes that produce vascular coagulation factors, etc. for patients with hemophilia, etc. for patients with pituitary dwarfism, etc. It is preferable to use anterior lobe cells that produce growth hormone, growth hormone-secreting cells, and the like, and bone cells that produce bone morphogenetic protein (BMP) for bone disease patients such as fractures. For cancer patients and the like, transformed cells that can produce a large amount of a tumor growth inhibitor by genetic recombination can also be used. In addition, for patients with diseases in which toxic metabolites accumulate in the body due to liver failure, renal failure or inborn errors of metabolism, cells that have the ability to selectively metabolize and detoxify toxic metabolites may be used. it can. When a patient needs a plurality of biologically active factors such as having a plurality of diseases, two or more of the above cells may be combined. By containing these cells, the present invention can supply biologically active factors useful for the above patients and the like. Therefore, by using the cell preparation of the present invention, it is possible to prevent or treat the onset of various diseases that can be treated by supplying biologically active factors or detoxifying harmful metabolites. .

The cells used in the present invention may be any of cells established for laboratories or cells separated from living tissue, but are preferably differentiated non-dividing cells. This is because differentiated non-dividing cells can produce and secrete the target hormones and proteins more than undifferentiated dividing cells.
The method for separating cells from living tissue is not particularly limited, and may be in accordance with a known technique. For example, a method of treating a tissue extracted by an appropriate means with Dispase or EDTA and then treating with trypsin to separate it into a single cell can be mentioned.

For example, when the cells used in the present invention are islet cells, the separation of the cells from the living tissue can be performed by a known collagenase treatment separation method such as J. Adam Van Der Vliet et al., Transplantation, 45 (2), p493-495 (1988) etc.
When the cells used in the present invention are pancreatic endocrine cells, for example, the method described in JP-A-2001-231548 may be followed.
As described above, it is desirable that the pathogen such as pathogenic virus is removed from the cells separated from the living tissue.

  Cells already established for laboratories or single cells obtained from living tissues are cultured until they become confluent in an appropriate medium, and subculture is repeated 2-3 times before being used as the cells of the present invention. It is preferred that For example, when the cells used in the present invention are islet cells or pancreatic endocrine cells (hereinafter abbreviated as pancreatic endocrine cells and the like), these cells are treated with, for example, 10% fetal calf serum (hereinafter abbreviated as FBS) and nicotinamide. Subculture is preferably performed in supplemented CMRL-1066 medium (Sigma, St. Louis, MO, USA). The subcultured cells are separated again as single cells according to known means such as trypsin treatment and collagenase treatment, and can be used as the cells of the present invention.

  The cell used in the present invention may be a transformed cell into which a gene encoding a biologically active factor peptide such as a hormone or protein necessary for a patient to be administered is introduced. By using the transformed cells, the desired biologically active factor peptide can be produced efficiently and in large quantities. The base sequence of a gene encoding a biologically active factor peptide has already been made public and can be easily obtained from depository agencies such as the American Type Culture Collection (ATCC) and general commercial sources. An oligonucleotide probe is prepared from a known gene or a known base sequence, and a gene synthesized using a known means such as a PCR amplification method or a DNA synthesis method using the probe is exemplified. For example, the cell preparation of the present invention can be used as an agent for preventing and treating cancer by using a transformed cell having a gene encoding a tumor growth inhibitory protein.

The host cell into which the gene encoding the biologically active factor peptide is introduced is not particularly limited, and a known host cell that is usually used in the field of gene recombination technology may be used. For example, as host cells, in addition to the above-mentioned pancreatic islet cells, pancreatic endocrine cells, hepatocytes, anterior lobe cells, growth hormone secreting cells, bone cells, monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter referred to as CHO cells). Abbreviation), dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cell), mouse cell BALB / 3T3, mouse L cell, mouse AtT-20, mouse C127 cell, mouse myeloma cell, rat GH3, Examples include human cells HeLa and human FL cells.

  The method for introducing the gene into the cells used in the present invention is not particularly limited, and may be in accordance with known means. For example, a known method such as introduction into a cell after inclusion in a recombinant expression vector such as a plasmid or virus or an artificial vector such as a liposome or microcapsule can be mentioned. When a recombinant expression vector is used, a competent cell method [J. Mol. Biol., 53, p154 (1970)], a DEAE dextran method [Science, 215, p166 (1982)], an in vitro packaging method [Proc Natl. Acad. Sci., USA, 72, p581 (1975)], viral vector method [Cell, 37, p1053 (1984)], microinjection method [Exp. Cell. Res., 153, p347 (1984)] Electroporation method [Cytotechnology, 3, p133 (1990)], calcium phosphate method [Science, 221, p551 (1983)], lipofection method [Proc. Natl. Acad. Sci., USA, 84, p7413 (1987)] And the protoplast method [Japanese Unexamined Patent Publication No. 63-2483942, Gene, 17, p107, (1982), Molecular & General Genetics, 168, p111 (1979)].

  In addition, a vector for introducing the gene into a host cell is not particularly limited, and any expression vector that can express a desired gene in the introduced cell and effectively produce a biologically active factor peptide can be used. That's fine. For example, bacteriophage such as λ phage, adenovirus, adeno-associated virus (AAV), retrovirus, pox virus, herpes virus, herpes simplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV), vaccinia In addition to animal viruses such as viruses, polioviruses, synbisviruses, and SV40, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like can be mentioned.

The cells used in the present invention are preferably included in PVA by the following method.
Cells are added and mixed in the PVA-cell preservative solution prepared above. The cells to be added / mixed are preferably treated with a cell preservative in advance. Specifically, it is preferable to suspend cells in a cell preservative in advance, collect the cells by centrifugation, and then mix with the PVA-cell preservative solution. Since the number of cells contained in the cell preparation of the present invention varies depending on the type of disease and the degree of disease of the patient to whom the cell preparation is administered, it cannot be said unconditionally and is determined by the judgment of a doctor. Preferably, for example, in a PVA-cell preservative solution, it is preferably about 1 × 10 ^{7 to} 5 × 10 ⁷ cells / ml. By setting the number of cells within this range, the cells are evenly dispersed in the PVA gel, and the cells can survive stably for a long period of time without inhibiting the supply of oxygen and nutrients to the cells due to cell aggregation and the like. Can do.

  In addition, since the cells used in the present invention are stably maintained in a state where the biologically active factor can be optimally supplied, the cell preparation of the present invention further includes mucopolysaccharides such as hyaluronic acid, chondroitin sulfate, and dermatanic acid. , An extracellular matrix containing one or more selected from elastin, collagen, fibrin and the like, and / or hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), human basic fibroblast growth factor (bFGF) Further, it may contain a growth factor such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), or growth hormone (GH). These growth factors may be contained alone or in combination of two or more. Examples of the method of incorporating an extracellular matrix and / or growth factor into the present invention include, for example, a cell culture medium containing an extracellular matrix and growth factor before mixing the PVA-cell preservative solution and cells. Examples of the method include immersing cells in a cell to form a membrane such as an extracellular matrix on the cell surface, or preliminarily containing an extracellular matrix and / or growth factor in a PVA-cell preservative solution. The content of the extracellular matrix and / or growth factor in the cell preparation of the present invention is not particularly limited, but is within a range that does not inhibit the retention of cells used in the present invention and the secretion function of biologically active factors, It is desirable to be within a range that can add an effect of extending the survival period of the cell and the secretion period of the biologically active factor.

After mixing the PVA-cell preservative solution and the cells, the obtained cell mixture (hereinafter referred to as cell-containing PVA-cell preservative solution) is cooled, and the PVA is gelled to produce the cell preparation of the present invention. Can be produced. The cooling is preferably performed by standing in an ultra-low temperature chamber of about -20 to -80 ° C for about half a day to 3 days, or by storing in an ultra-low temperature chamber of about -80 ° C for about 18 to 30 hours.
In the present invention, since a cell preservative is preliminarily blended with PVA, cell death and damage can be suppressed even when quenched at the above temperature. Moreover, the cells can be dispersed and contained in the PVA gel, and aggregation of the cells can be prevented.
By the gelation of PVA, the present invention can be formed into various shapes such as a sheet shape, a plate shape, a disk shape, a rod shape, a tube shape or a bead shape. For example, the cell-containing PVA-cell preservative solution is smeared on a glass plate, and the whole glass plate is cooled to prepare a cell preparation in the form of a thin film sheet. A cell preparation having a desired thickness can be prepared by appropriately changing the amount of the cell-containing PVA-cell preservative solution smear per unit area of the glass plate, but it is usually about 100 to 300 μl / mm ² . .

  The cell preparation of the present invention may be used in combination with a reinforcing material in order to reinforce or / and simplify operability. For example, when a cell preparation is formed into a thin film sheet, the cell-containing PVA-cell preservative solution is fixed to a resin mesh sheet or the like for gelation in order to reinforce and simplify operability. Good. Specifically, in FIG. 1, for example, a PET (polyethylene terephthalate) resin mesh sheet (4) is placed on a glass plate (5), and a cell-containing PVA-cell preservative solution is placed on the mesh sheet (4). Apply. Another mesh sheet (6) is covered from above, and another glass plate (7) is further covered thereon, and the cell-containing PVA-cell preservative solution and the mesh sheets (4) and (6) are applied to the glass plate (5 ) And (7). It can be formed into a sheet-like gel by cooling at about -20 to -80 ° C. The mesh sheet is preferably preliminarily immersed in a PVA-cell preservative solution.

  The reinforcing material such as a mesh sheet is preferably a material that is safe for a living body, that is, a material that is non-degradable and excellent in biocompatibility in a living body, such as PET. This is because when the mesh sheet in the cell preparation of the present invention is decomposed in the living body, the cell preparation adheres to the living tissue, and it is difficult not only to exert the effects of the present invention for a long time, but also to the living body. This is because there are cases where it can be. Of course, it is needless to say that a reinforcing material that is not harmful to the living body even if decomposed in the living body can be used in the present invention.

  The cell preparation in the frozen state is preferably administered to the living body after activating the cells in the frozen state. Activation of the cells in the frozen cell preparation can be performed by thawing the cells and re-culturing in an appropriate medium. Specifically, a frozen cell preparation is quickly immersed in a cell culture medium at about 37 ° C., such as CMRL-1966, and then thawed, and then further cultured in a new cell culture medium for about 24 hours, for example. . When the cell preparation contains DMSO, the thawed cell preparation is washed with a new cell preservation solution, such as UW solution, and then immersed in the UW solution, for example, at about 4 ° C. for about 24 hours. By doing so, it is preferable to remove DMSO in the gel. Needless to say, a cell preparation obtained by thawing and removing DMSO as described above is also a cell preparation of the present invention.

Since the administration form of the cell preparation obtained by the above method to the living body differs depending on the type of disease, disease site, and degree of disease of the patient to be administered, it cannot be generally stated, and is determined by the judgment of a doctor, but preferable administration The form is described below.
Subjects to which the cell preparation of the present invention is administered include mammals other than humans such as humans, dogs, cats, monkeys, rabbits and mice.
As described above, the present invention can be administered after being shaped into a shape suitable for an administration site in a living body. In addition, the present invention may be transplanted to the administration site in direct contact with the diseased tissue, but can also be implanted subcutaneously or intramuscularly that can be administered with a relatively slight invasiveness. For example, the tubular cell preparation can be shredded and transplanted subcutaneously using a transplant needle or the like, or the cell preparation can be filled into a syringe or the like and injected intramuscularly or subcutaneously. Moreover, it is easy to recover the cell preparation from the site. When transplanting the cell preparation subcutaneously or intramuscularly, if the blood vessel distribution density at the transplant site is sparse and it is considered difficult to supply oxygen and nutrients for cell growth and survival, a known appropriate It can also be transplanted in combination with various angiogenesis-inducing agents. The angiogenesis inducer may be previously contained in the cell preparation, or may be administered in a form different from the cell preparation.

  The amount of a biologically active factor such as a hormone or protein supplied to the patient from the cell preparation of the present invention takes into consideration the secretory ability of the biologically active factor of the cell used and the transition of the cell viability during the transplantation period. And it can set suitably by a doctor's judgment. For example, when the patient's disease is diabetes, the insulin secretion ability of cells to be used, such as pancreatic endocrine cells, is measured beforehand in vitro, and the insulin secretion ability, the number of cells, the administration period, and the cell survival rate The amount of insulin supply of the cell preparation of the present invention can be determined from the transition of the above. Since the cell preparation of the present invention can stably hold contained cells for a long period of time, the rate of decrease in the ability to secrete biologically active factors over time is low. Therefore, if this invention is used, the biologically active factor required for a patient can be stably supplied to a patient for a long period of time, and the frequency of transplantation of a cell preparation can also be reduced.

  The secretory ability of the biologically active factor of the cell used in the cell preparation of the present invention may be in accordance with a known quantitative method suitable for measuring each biologically active factor. For example, the method for quantifying insulin when the cells are pancreatic endocrine cells and the like will be described in detail in the following examples.

  Hereinafter, the present invention will be specifically described with reference to examples and the like. Needless to say, the present invention is not limited to these examples. Note that “%” represents “% by mass”.

(Production Example 1) Production of sheet-like cell preparation containing islet cells (1) Preparation of islet cells From Wister rats (Shimazu animal Co. Ltd. Kyoto, Japan) weighing 8 to 10 weeks and weighing 300 to 350 g, The islet cells were isolated by the method described above.
First, type I collagenase (Sigma, St. Louis, MO, USA, 350 U / mg) and type XI collagenase (Sigma, St. Louis, MO, USA, 2,200 U / mg) were respectively contained in the pancreatic duct of the isolated rat pancreas. 10 ml of Hank's solution containing 800 U / ml and 1500 U / ml concentrations were injected and subjected to enzyme treatment at 37 ° C. for 15-20 minutes. A cooling Hanks solution was added to stop the enzyme reaction. The pancreatic tissue was dispersed using a pipette, the tissue pellet was collected by centrifugation, and then the tissue pellet was washed twice with Hanks' solution. The centrifugation was performed at 1,000 rpm for 1 minute. The tissue suspension was filtered using an 800 μm size mesh sheet. The filtrate was collected in a 50 ml conical tube and centrifuged (1500 rpm, 3 minutes) to obtain a tissue pellet. The pellet was suspended in Hank's solution containing 27% dextran (Sigma, St. Louis, MO, USA, MW 70.000) and subjected to discontinuous density gradient centrifugation. At this time, the lowermost layer was composed of two layers of 27% dextran (density 1.094 g / ml) and the upper layer of 23% dextran (density 1.081 g / ml) and 11% (density 1.041 g / ml) dextran. The discontinuous density gradient centrifugation was performed at 400 rpm for 4 minutes and further at 1,700 rpm for 10 minutes. After centrifugation, the islet cell-containing fraction was collected from the interface of the uppermost two layers using a Pasteur pipette. The obtained islet cells were washed twice with CMRL-1066 medium (1,000 rpm, 3 minutes).

(2) Preparation of PVA-Euro-collins solution 3 g of PVA (Gunsei PVA-180H, Lot37435) was suspended in 75 ml of distilled water, and then heat-dissolved and sterilized by autoclave (121 ° C, 15 minutes) twice. did. The obtained sterile PVA aqueous solution was aseptically mixed and stirred with 10 ml of 10-fold concentrated Euro-collins solution (hereinafter abbreviated as EC solution), DMSO 5 ml, FBS 10 ml and 0.122 g nicotinamide, and a sterile 3% PVA-EC solution 100 ml was obtained. The above 10-fold concentrated EC solution was prepared by adding and mixing the components shown in Table 1 to 100 ml of distilled water, followed by 0.22 μm sterilization by filtration. After production, it was stored at room temperature.

(3) Mixing of cells The islet cells obtained in (1) were further mixed with a carbon dioxide incubator (5% CO ₂ , 37 ° C.) using a CMRL-1066 medium containing 10% FBS and 1.22 g / L nicotinamide. ) For 24 hours. A culture solution containing 1,000 islet cells was placed in a 1.5 ml centrifuge tube and centrifuged at 800 rpm for 1 minute. To the obtained cell pellet, 0.1 ml of a cell banker (SERUBANNKA code 630-01601: Wako Pure Chemical Industries, Ltd. Osaka, Japan) was added, and the cells were suspended and allowed to stand for 5 minutes. The cells were collected by centrifugation again. The collected cells were suspended in 100 μl of the sterile 3% PVA-EC solution prepared in (2) to obtain an islet cell-containing PVA-EC solution.

(4) Formation of sheet Two 10 mm × 10 mm square PET mesh sheets (GUNSE: TNO60SS) were previously immersed in a sterile 3% PVA aqueous solution for 5 minutes, and one of these (4) was placed on a glass plate (5 ) Installed on top. The pancreatic islet cell-containing PVA-EC solution obtained in (3) above is uniformly applied on the mesh sheet (4), and another mesh sheet (6) is covered from above, mesh sheet (4) -islet A three-layered sheet of cell-containing PVA-EC solution-mesh sheet (6) was obtained. Further, another glass plate (7) is covered from above, and pressure is applied to adjust it to a three-layer sheet having a thickness of about 1 mm, and this is sandwiched between two glass plates at −80 ° C., 24 The PVA was gelled by storage for a period of time (FIG. 1).

(5) Thawing of sheet-like islet cell preparation and cell activation The frozen sheet-like islet cell preparation obtained in (4) above was quickly thawed in a CMRL-1066 medium at 37 ° C. The thawed sheet-like islet cell preparation was immersed in 5 ml of UW solution (Biasol-Myers Squibb Company, USA) for 5 minutes. This operation was repeated three times to remove DMSO contained in the sheet. The sheet was further immersed in 5 ml of UW liquid at 4 ° C. for 24 hours. The soaked preparation was washed twice with CMRL-1066 medium, and further cultured in CMRL-1066 medium for 24 hours (37 ° C., 5% CO ₂ ) to produce a cell-like activated islet cell preparation (manufactured) Example 1) was obtained. The obtained Production Example 1 was used in the following test examples. In addition, a 10% concentrated EC solution 10 ml, DMSO 5 ml, FBS 10 ml and 0.122 g nicotinamide were added in the above (2) to prepare a sterile 3% PVA aqueous solution. It was.

(Test Example 1) Stability confirmation of cells in preparation The production example 1, comparative example 1 or free islet cells (200 cells) were cultured in CMRL-1066 medium (5% CO ₂ , 37 ° C). The number of viable cells and the number of free islet cells in Production Example 1 or Comparative Example 1 after 1 day of culture were counted under a microscope, respectively, and the recovery rate (%) of viable cells was determined. The recovery rate was expressed with the number of cells before culture as 100.
The results are shown in FIG. The recovery rate of the living cells of Production Example 1 was about 82%, which was very high compared to the recovery rate of Comparative Example 1 (about 35%), which was higher than that of free cells.

Test Example 2 Confirmation of Insulin Content in Formulation The above Production Example 1, Comparative Example 1 or free islet cells (200 cells) were cultured in CMRL-1066 medium (5% CO ₂ , 37 ° C.) and cultured. Each insulin content after one day was examined. One day after the cultivation, Production Example 1, Comparative Example 1 or free islet cells were collected in hydrochloric acid-EtOH, ground using a spatula or the like, and then vortexed to stir and suspend the gel and cells in the preparation in hydrochloric acid-EtOH. It became cloudy (−20 ° C., 24 hours). Insulin concentration in each cell suspension was measured. Insulin was measured using a commercially available insulin measurement ELISA kit (Levis Insulin Kit (Shiba Goat)).
The results are shown in FIG. The insulin content (about 205 ng / ml) in Production Example 1 was significantly higher than that of Comparative Example 1 (about 20 ng / ml), which was almost the same as that of free cells.

(Test Example 3)
Confirmation of insulin secretion ability and cell state of the preparation The above Production Example 1, Comparative Example 1 or free islet cells (200 cells) were cultured in CMRL-1066 medium (5% CO ₂ , 37 ° C.), and after 1 day of culture, Each was recovered after 7 days and 14 days. After the collection, a glucose reaction test was performed for each. The glucose response test is as follows. First, the recovered production example 1, comparative example 1 or free islet cells were cultured in CMRL-1066 medium containing 3.3 mM glucose and 0.1% bovine serum albumin (BSA) for 1 hour (5% CO ₂ , 37 ° C.) and then washed with glucose-free CMRL-1066 medium. This operation was repeated again and further cultured in CMRL-1066 medium containing 3.3 mM glucose for 1 hour, and then the insulin concentration in each supernatant was measured. Subsequently, it culture | cultivated for 1 hour with the CMRL-1066 culture medium containing 16.7 mM glucose, and measured the insulin concentration in each supernatant liquid.

The results are shown in FIG. In FIG. 4, (a) shows the amount of insulin secretion (concentration) after 1 day of culture, (b) after 7 days of culture, and (c) after 14 days of culture. In Comparative Example 1, an increase in the amount of insulin secretion depending on the increase in the glucose concentration was not observed. In contrast, in Production Example 1, the amount of insulin secretion (concentration) increased depending on the increase in glucose concentration after 1 day, 7 days, and 14 days after culture. Moreover, in the free cells, the increase in insulin secretion depending on the glucose concentration was no longer observed after 7 days, whereas in Production Example 1, the increase was observed even after 14 days.
Moreover, as a result of observing the cells contained in Production Example 1 or Comparative Example 1 under a microscope, the cells in Comparative Example 1 had already undergone cell destruction from the first day. In contrast, the cells in Production Example 1 did not show significant cell destruction, and remained relatively stable after 14 days (FIG. 5). In FIG. 5, (a) shows a cell observation image after 1 day of culture, (b) after 7 days of culture, and (c) after 14 days of culture.

  From the above results, it was found that islet cells survived stably in Production Example 1, maintained their activity stably for a long period of time, and secreted insulin in response to an increase in glucose concentration. Therefore, by blending the EC solution in advance with PVA, cell death and cell damage due to rapid cooling during PVA gelation are suppressed, and as a result, the survival rate of islet cells contained in PVA is high, In addition, it was proved that a cell preparation capable of maintaining insulin production ability and responsiveness to an increase in glucose concentration can be produced.

(Production Example 2) Production of sheet-like cell preparation containing islet cells (1) Preparation of islet cells Wister rats were anesthetized by absorption of diethyl ether and intraperitoneal administration of Nembutal 1 mg / kg. After laparotomy, the common bile duct was identified and clamped with a forceps near the Vater papilla. A small incision is placed at the junction of the gallbladder duct, a cannulation tube is inserted into the common bile duct, ligated and fixed, and a type XI collagenase (Sigma, St. Louis, MO, USA) equivalent to 1200 to 1500 u / ml is injected to remove the pancreas Swelled. The whole pancreas was removed so as not to damage the pancreas, placed in a flask, placed in a thermostatic bath at 37 ° C., and digested warmly in about 18 minutes. After digestion, the flask was shaken several times to disrupt the pancreas and washed 3 times with Hank's solution supplemented with 0.1% bovine serum albumin. After the cells were suspended in the Hanks solution, they were passed through a mesh filter having a diameter of 850 μm, and the filtered contents were collected.

Islet isolation was performed with a specific gravity gradient of dextran (Dextran 70). That is, the pancreatic cell component from which the supernatant was discarded was stirred in a dextran + Hank's solution having a specific gravity of 1.094 until uniform, and then a dextran solution having a specific gravity of 1.094 was gently introduced. A dextran solution having a specific gravity of 1.081 and 1.041 was gradually and gently put on the cell layer to form 4 layers. Centrifugation was carried out at 400 rpm for 4 minutes and further at 1700 rpm for 13 minutes. The islets separated between the first layer and the second layer were collected with a hand pick, washed, and then purified with a hand pick. After washing 4 times, the cells were cultured overnight in an incubator under an environment of 37 ° C. and 5% CO ₂ 95% air. As the medium, CMRL 1066 + 10% inactivated fetal bovine serum + antibiotic-antimycotic solution (Gibco, BRL) + nicotinamide was used.

(2) Preparation of sheet-like islet cell preparation Using the culture solution of islet cells obtained above, in the same manner as in (2), (3), (4) and (5) of Production Example 1 in Example 1 A sheet-like islet cell preparation with activated cells (Production Example 2) was obtained.

(Test Example 4) Insulin secretion ability of the preparation As in Test Example 3, low-concentration glucose (3.3 mM) was added to the above-obtained islet cell preparation (Production Example 2, one day after preparation) or free islet cells. ) And high concentration glucose (16.7 mM) were added for 1 hour at a time, and the amount of insulin secreted was measured by ELISA.
The results are shown in FIG. As can be seen from FIG. 6, the islet cell preparation according to the present invention secreted insulin in response to glucose stimulation and was not inferior to that of free islet cells at the same time.

(Test Example 5) Transplant Test (1) Preparation of Diabetes Model Mice Streptozotocin 200 mg / kg dissolved in citrate buffer (pH 4.5) was intraperitoneally administered to male C57BL / 6 mice aged 8 to 10 weeks. One week after administration, blood glucose was measured 3 times, and a mouse whose blood glucose was 350 mg / dl or more twice or more was judged to be diabetic and used in the experiment. In addition, the mouse | mouth which does not perform streptozotocin administration was made into the normal mouse group.

(2) Transplantation The transplantation of the islet cell preparation was performed as follows. Specifically, a diabetic model mouse (one week or more after the creation of diabetes) was opened by midline incision under ether anesthesia, and the islet cell preparation (Production Example 2) obtained in Production Example 2 was placed in the abdominal cavity. The abdominal wall was closed in two layers. This mouse was used as a transplanted mouse group. In addition, the mouse | mouth which transplanted the preparation (prepared similarly to the comparative example 1) which does not contain a pancreatic islet cell was made into the diabetic mouse group.

(3) Examination of survival time The transplantation time was 0 day, and the subsequent survival time was observed.
The results are shown in FIG. As is clear from FIG. 7, in the diabetic mouse group, the majority died within 4 weeks after transplantation, and there was no mouse that survived until 8 weeks during the follow-up period. On the other hand, in the group of transplanted mice transplanted with the islet cell preparation, all surviving were 8 weeks or more except for 2 mice that remained hyperglycemic and died (survival rate up to 8 weeks was 0% vs. 81.8%, p <0 .001).

(4) Measurement of blood glucose and body weight Both blood glucose and body weight were measured before transplantation, on the 1st day, 3rd day, 5th day, and 7th day after transplantation, and thereafter once a week. The blood glucose change in each group is shown in FIG. In the transplanted group, post-transplantation blood glucose decreased rapidly to less than 300 mg / dl (13/16), and more than half were normalized but gradually increased (maintained below 300 mg / dl). Number of days is 3-7 weeks). However, the level of blood glucose elevation was mild, and blood glucose was significantly improved compared to the diabetic mice group (p <0.0001), and the life prognosis until 8 weeks after transplantation was also clearly improved. Of the 9 animals observed up to 8 weeks, 6 were subjected to excision of the islet cell preparation, and 5 of them were found to have increased blood glucose again.

  The change in the average body weight of each group is shown in FIG. In the transplanted mice group, after transplantation, the body weight was considered to be due to poor postprandial diet, but a repeated measurement analysis of variance until 3 weeks after transplantation showed no significant difference from the diabetic mice group. However, it started to increase at 3 weeks after transplantation, and a significant increase was observed after 3 weeks (p <0.05) compared to week 1 when the body weight decreased most during the course. Furthermore, at 3 weeks and 4 weeks, the mean body weight of the group of diabetic mice was significantly exceeded.

(5) Measurement of renal function BUN (blood urea nitrogen) and creatinine were measured once a week before and after transplantation. At 8 weeks after transplantation, the islet cell preparation was excised and then measured up to 1 week.
The change in BUN is shown in FIG. Mean ± SE of normal blood glucose mouse BUN measured before transplantation of the islet cell preparation was 25.53 mg / dl ± 1.804 (20-38.9). BUN increased in 41.3% of diabetic mice at 1 week after administration of streptozotocin (24/58, diabetic mice: 13/35 vs. transplanted mice: 11/23, BUN> 35 mg). / Dl). Of the 14 diabetic mouse groups used in the experiment, 13 showed high BUN values during the course of the experiment.

On the other hand, in the study of 16 transplanted mice, all 9 mice whose BUN increased before surgery were normalized in 1 week after transplantation. As a mean value, the BUN value that decreased one week after transplantation passed almost unchanged from the normal value, and a significant difference from the diabetic mouse group was observed (p = 0.002).
Furthermore, among the 6 animals from which the pancreatic islet cell preparation was removed 8 weeks after transplantation, the BUN value of 1 week after the removal was measured in 4 animals, but all of them were higher than before the removal.

  The change in creatinine is shown in FIG. The normal value of creatinine was 0.351 mg / dl ± 0.032 (0.16-0.5). Nearly half of the mice in the diabetic group showed a high value of 0.5 mg / dl one week after administration of streptozotocin, and all mice showed abnormal creatinine values four weeks after administration. In addition, after the rise of creatinine, none of the mice died within a few weeks, and no mice were able to survive until 10 weeks after streptozotocin administration. In the transplanted mice group, 6 animals showed an increase in creatinine after transplantation, but all were mild and viable during the follow-up period. The average value of creatinine exceeded 0.5 mg / dl in the transplanted mice group only for 5 weeks after transplantation, but remained at the 0.4 mg / dl level in any other period, and was significantly lower than that in the diabetic mice group. (P = 0.0478). In addition, after the removal of the islet cell preparation, all mice were elevated again as in BUN.

(6) Urinalysis 24 hours urine was collected and urine volume, 24 hours urinary glucose excretion, 24 hours urine albumin excretion and urine ketone body were measured. The transplanted mouse group, the diabetic mouse group, and the normoglycemic mouse group were reared in a metabolic cage for 24 hours under free drinking and eating conditions, and the 24-hour urine was collected. Urine sugar was measured by Fuji DRI-CHEM system, and the product of urine volume multiplied by urine volume was used as urinary glucose excretion. The 24-hour urinary albumin excretion was calculated by measuring the urinary albumin concentration by ELISA (Albuwell M (Exocill)) and multiplying by the 24-hour urine volume. The urine ketone body was evaluated by using urotape (Eiken) and quantifying the color tone.

  The change in 24-hour urine volume is shown in FIG. The transplanted mouse group was not significantly different from the diabetic mouse group, but showed a slightly lower value.

  Changes in urinary glucose excretion are shown in FIG. The amount of diabetic excretion was also lower in the transplanted mouse group than in the diabetic mouse group, and a significant difference was observed until 3 weeks after transplantation (p <0.0452).

  Moreover, the change of the amount of urinary ketone bodies is shown in FIG. The amount of urine ketone body increased before transplantation in both diabetic mice and transplanted mice. In the transplanted mouse group, normalization occurred one week after the transplantation, and after that, it was significantly lower than that in the diabetic mouse group (p = 0.0294).

  Furthermore, the change in the amount of urinary albumin is shown in FIG. The amount of urinary albumin was lower in the transplanted mouse group than in the diabetic mouse group, but no significant difference was observed.

(7) 1,5-Anhydro-D-glucitol (1,5-AG) measurement In order to examine the degree of diabetes, 1,5-AG measurement was performed. 1,5-AG is a polyol having a structure very similar to that of glucose, and is mainly ingested and absorbed from food, and is widely accumulated in the body. And it is reabsorbed in the kidney. It has a competitive effect with glucose, and AG reabsorption of urine sugar is inhibited during diabetes, leading to an increase in urinary AG excretion and a decrease in blood AG. Therefore, since 1,5-AG responds extremely sensitively to glucose kinetics, it can be a marker that reflects the state of real-time diabetes. In the transplanted mouse group, diabetic mouse group, and normoglycemic mouse group, 25 μl of blood was collected from the tail vein before transplantation and once weekly after transplantation, and measured with a 1,5-AG kit for animals (Nippon Kayaku).

  The results are shown in FIG. As is apparent from this figure, the 1,5-AG concentration was clearly lower in the diabetic mouse group than in the normoglycemic mouse group, which well reflected the state of diabetes. The average value of 1,5-AG in the transplanted mice group increased in one week compared with before surgery. In addition, a significant increase was observed compared with the diabetic mouse group during the period (p = 0.0203).

(8) Discussion As is clear from the above transplantation experiment, the islet cell preparation of the present invention improves the diabetic state by transplanting it, and is effective in the prevention of death due to diabetes and diabetic renal injury. I understand. Moreover, transplantation of the islet cell preparation of the present invention is effective not only for the cure of diabetes but also for the prevention of complications.

It is a schematic diagram which shows the manufacturing method of a sheet-like islet cell formulation. It is a figure which shows the result of having investigated the collection | recovery rate of the living cell 1 day after culture | cultivation of the manufacture example 1, the comparative example 1, or a free islet cell. It is a figure which shows the result of having investigated the insulin content after 1 day of culture | cultivation in manufacture example 1, comparative example 1, or a free islet cell. It is a figure which shows the result of having investigated the insulin secretory ability of manufacture example 1, the comparative example 1, or a free islet cell over time. It is a figure which shows the microscope picture which observed the pancreatic islet cell contained in the manufacture example 1 or the comparative example 1 over time. It is a figure which shows the result of having investigated the insulin secretion ability of the manufacture example 2 or the free islet cell. It is a figure which shows the survival time of a transplant mouse group and a diabetic mouse group. It is a figure which shows the change of the blood glucose of a transplant mouse group, a diabetic mouse group, and a normoglycemic mouse group. It is a figure which shows the change of the body weight of a transplant mouse group, a diabetic mouse group, and a euglycemic mouse group. It is a figure which shows the change of BUN (blood urea nitrogen) of a transplant mouse group, a diabetic mouse group, and a euglycemic mouse group. It is a figure which shows the change of creatinine of a transplant mouse group, a diabetic mouse group, and a normoglycemic mouse group. It is a figure which shows the change of 24-hour urine volume of a transplant mouse group, a diabetic mouse group, and a euglycemic mouse group. It is a figure which shows the change of the 24-hour urinary glucose excretion amount of a transplant mouse group, a diabetic mouse group, and a euglycemic mouse group. It is a figure which shows the change of the amount of urine ketone bodies of a transplant mouse group, a diabetic mouse group, and a euglycemic mouse group. It is a figure which shows the change of the 24-hour urinary albumin excretion amount of a transplant mouse group, a diabetic mouse group, and a euglycemic mouse group. It is a figure which shows the change of the 1, 5- AG amount of a transplant mouse group, a diabetic mouse group, and a normoglycemic mouse group.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Islet cell 2 PVA + EC solution 3 PVA + EC gel 4, 6 Mesh sheet 5, 7 Glass plate 8 Sheet-like islet cell containing preparation

Claims (17)

  A cell preparation containing cells in polyvinyl alcohol containing a cell preservative.   The cell preparation according to claim 1, wherein the cell surface is further covered with an extracellular matrix.   The cell preparation according to claim 1 or 2, further comprising a growth factor.   The cell is one or more cells selected from the group consisting of islet cells, pancreatic endocrine cells, hepatocytes, anterior lobe cells, growth hormone secreting cells, bone cells, thyroid hormone secreting cells and parathyroid hormone secreting cells. The cell preparation according to any one of claims 1 to 3, wherein the cell preparation is provided.   The cell preparation according to any one of claims 1 to 4, wherein the cell is treated with a cell preservative.   The cell preparation according to any one of claims 1 to 5, wherein the cell is a transformant.   The cell preparation according to any one of claims 1 to 6, which has a sheet shape, a plate shape, a rod shape, a tube shape, or a bead shape.   The cell preparation according to any one of claims 1 to 7, wherein the cell preservation solution is a Euro-Collins solution, a cell banker or a UW solution.   The cell preparation according to claim 1, which is transplanted subcutaneously, intramuscularly or intraperitoneally in a mammal.   A method for preventing and treating endocrine / metabolic diseases, comprising using the cell preparation according to any one of claims 1 to 9.   The method for preventing and treating an endocrine / metabolic disease according to claim 10, wherein the endocrine / metabolic disease is diabetes or pituitary dwarfism.   A method for preventing and treating hemophilia, comprising using the cell preparation according to any one of claims 1 to 9.   A method for preventing and treating a bone disease, comprising using the cell preparation according to any one of claims 1 to 9.   A method for preventing and treating cancer, which comprises using the cell preparation according to any one of claims 1 to 9.   A method for preventing and treating liver failure or inborn errors of metabolism characterized by using the cell preparation according to any one of claims 1 to 9.   A method for producing a cell preparation, comprising mixing a cell preservative with polyvinyl alcohol, mixing the cells with the polyvinyl alcohol, and gelling the obtained cell-containing polyvinyl alcohol. The cell preparation according to any one of claims 1 to 9, which is a pharmaceutical for humans or animals.