WO2003013588A1

WO2003013588A1 - Kidney regeneration material comprising cells and cell growth factor

Info

Publication number: WO2003013588A1
Application number: PCT/JP2002/007995
Authority: WO
Inventors: Yasuhiko Tabata; Osamu Ogawa
Original assignee: Kaken Pharmaceutical Co., Ltd.
Priority date: 2001-08-08
Filing date: 2002-08-06
Publication date: 2003-02-20
Also published as: JPWO2003013588A1; JP4358621B2

Abstract

A material for the regeneration of kidney in vivo which comprises undifferentiated mesenchymal stem cells and a cell growth factor; and a method for kidney regeneration.

Description

Specification

Materials for regeneration of kidneys composed of cells and cell growth factors

The present invention relates to a material for regenerating kidney in vivo, characterized by using a combination of undifferentiated mesenchymal stem cells and a cell growth factor. Background art

Reconstructive medicine using artificial organs is currently making significant contributions to the clinic, but has the major drawback of being temporary, invasive, and having a single function that can be supported. On the other hand, transplantation medicine has problems such as infections and carcinogenesis due to side effects of immunosuppressive drugs after transplantation, in addition to the shortage of donors. In this way, the current two pillars of advanced medicine have reached their limits. Under such circumstances, a new treatment attempt is being made to restore a defective or devastated tissue or organ by regenerating its own tissue. This is regenerative medicine. It is an attempt to artificially reestablish one's own tissues and organs by using the.

In mammals containing humans, it has been considered that the ability to regenerate highly differentiated organs has disappeared. However, in recent years, it has been considered that these animals may have regenerative ability. In other words, wound healing to protect individuals progresses too quickly in mammals, depriving them of organ regeneration and losing sight of the inherent regeneration ability. For example, bone marrow transplantation is a long-standing treatment and has good clinical results. This is considered a success because the bone marrow tissue exists in the body, a natural place where blood stem cells in the bone marrow cells can proliferate and differentiate. In other words, even for human beings, in order for an individual to survive, it must be able to survive without the presence of stem cells in tissues and organs, which is sufficient for stem cells that would be widely present in the body. If it is possible to provide a place for renewal, it is possible to regenerate the self-organization. In recent years, it has been found that stem cells also exist in the nervous system and liver. Therefore, various methods of tissue regeneration have been researched and developed based on this idea, but at present, a combination of the scaffold for regeneration, cell growth factor, and stem cells is used. The method to do is the most realistic.

In order to promote adhesion, differentiation, and morphogenesis of stem cells and progenitor cells at the regeneration site, a scaffold for regeneration is required. In some cases, the biological tissue at the regeneration site can serve as a scaffold for regeneration, but there are also cases where a scaffold Matritta is required as a scaffold for regeneration. Bioabsorbable materials are used as this matrix.

However, for example, even if the scaffold matrix is embedded in the regeneration site, if the number of stem cells, progenitor cells, or blasts necessary for regeneration is small, or the concentration of biological factors that cause the cells to grow and differentiate is too low, No matter how good Matritta is, the desired regeneration will not occur. Therefore, what is first considered as a supplement is a cell growth factor for cell growth or differentiation.

In general, these factors have a short in vivo life span and are unstable, and the expected tissue regeneration effect cannot be obtained simply by dissolving the necessary cell growth factor in water and injecting it into the required site. Therefore, the concentration of cell growth factor at the regeneration site must be kept at an effective value for the required period. For example, it is a technology that does not contain cell growth factors in a sustained-release carrier and releases them continuously in the field of regeneration. Sustained release of cell growth factors enhances cell proliferation and differentiation, and promotes self-organization regeneration. However, depending on the type of tissue or organ, regeneration with cell growth factors alone is often insufficient.

The kidney is a blood filtration organ that excretes the end products of body metabolism in the form of urine and regulates the concentration of hydrogen, sodium, potassium, phosphorus and other ions in the extracellular fluid. Depending on the balance between excretion and reabsorption mechanism, it has functions such as regulation of water, electrolyte, fluid osmotic pressure, and acid-base balance. When kidney function fails, blood pressure increases, nitrogen metabolites such as urea and creatine accumulate in the blood, and blood levels of biowaste increase to harmful levels. In order to eliminate such pathology, it is necessary to replace the function of the failing kidney.

Hemodialysis is a procedure that cleans and filters blood that is in renal failure. This measure reduces the concentration of hazardous waste. However, this treatment It takes 3-4 times a week, 2-4 hours once a week, and there are problems of side effects and complications.

Kidney transplantation is a procedure in which a healthy kidney from a donor is implanted in an individual suffering from renal failure. However, lifelong administration of immunosuppressive drugs is required, and the resulting side effects such as infectivity and canceration are problematic. Another problem is that there is not a sufficient amount of donors for all individuals requiring transplantation.

Instead of transplanting the kidney itself, transplantation of kidney cells has also been reported (Pediatrics, 1996, 98S, 615). According to it, C 5 7 black mouse kidney cells were cultured, encapsulated in a polycarbonate tube, and transplanted subcutaneously in an athymic mouse. In the observation up to 8 weeks later, angiogenesis, glomeruli and urine 'tubules' A similar structure was observed. Alkaline phosphatase and fibronectin were found in the tubule-like structure, and uric acid was detected from the secreted yellow liquid.

According to another report, New Zealand White Rabbit kidney cells were fractionated into distal tubules, glomeruli, and proximal tubules, and each was cultured on a polylactic acid-polyglycolic acid sheet. When implanted subcutaneously, nephron formation was observed.

(J. Urol, 1995, 153 (suppl.), 4).

WO 9 8/0 9 5 8 2 discloses an artificial kidney to which the above technique is applied. However, this artificial kidney is a hybrid type artificial kidney, that is, a kidney that has been removed, cultured, and then reconstructed into an organ. Such differentiated cells have a limited life span, and it is difficult to maintain their function as kidney cells, so there are problems with the durability and reliability of the artificial kidney. Also, when using non-self cells, such as from different animals, there is always a problem of immune rejection.

There is no doubt that in order to regenerate a tissue organ in vivo, the cells constituting the tissue or organ are necessary. However, it is extremely difficult to regenerate tissues and organs at the site by simply injecting the cells into the site where the cells are to be regenerated. The reason is that added cells can hardly be expected to grow and differentiate at the site. In order to actively promote cell growth and differentiation in vivo, the presence of cell growth factors is essential. Already, many in vitro experiments have shown that undifferentiated mesenchymal stem cells, progenitor cells, or blasts can be It has been proven that the number will increase and differentiate in the country. However, the cellular environment is quite different between in vitro and in vivo, and in vitro results are often difficult to reproduce and predict in vivo.

As a result of diligent studies to solve the above-described problems related to cell proliferation and differentiation in vivo, the present inventors have found that regeneration of kidney in in vivo by using a combination of undifferentiated mesenchymal stem cells and cell growth factors. Was found to be extremely advantageous, and the present invention was completed. Disclosure of the invention

Therefore, the present invention provides a material for regenerating the kidney in vivo by using a combination of undifferentiated mesenchymal stem cells and cell growth factors. The present invention also relates to a method for regenerating a kidney, comprising using the above material. Brief Description of Drawings

Figure 1

A group of collagen sponges containing undifferentiated mesenchymal stem cells and bFGF-impregnated gelatin particles. A: X 1 0 0, B: X 4 0 0, arrows indicate regenerated glomeruli (embedding

3 after 6 weeks).

Figure 2

Collagen sponge embedded group containing undifferentiated mesenchymal stem cells. A: X 1 0 0, B: X

4 0 0 (3 weeks after placement).

Fig 3

b Collagen sponge embedded group containing FGF impregnated gelatin particles. A: X 1 00, B: X 4 0 0, (4 weeks after implantation). BEST MODE FOR CARRYING OUT THE INVENTION

The technical configuration of the present invention will be described below.

The cells used in the present invention are undifferentiated mesenchymal stem cells. These cells can be collected from animals or humans, or the number of collected cells can be increased in culture. Can.

The undifferentiated mesenchymal stem cells used in the present invention, their mixing with cell growth factors, and regeneration of kidney tissue using these tissue engineering materials can be performed by the following methods. Although some modification points can be added to cell isolation methods, they do not contribute significantly to the differentiation properties of cells. The method for collecting undifferentiated mesenchymal stem cells can be performed by a conventional method. For example, bone marrow fluid is collected from bone marrow such as femur and osteoid, and the cells are dispersed by pipetting or the like, suspended in an appropriate medium or saline such as _α -MEM, and the bone marrow cell suspension is obtained. Prepare. The cells are cultured in an incubator under conditions of 37 ° C and 5% carbon dioxide, for example, for 7 to 10 days, and then the adherent cells are detached using, for example, 0.05% trypsin and collected. To do.

The obtained undifferentiated mesenchymal stem cells can be cultured in an incubator under conditions of 37 ° C and 5% carbon dioxide gas, and the cells can be proliferated and subcultured. Disperse these cells in the culture medium and mix them with appropriate concentrations of cell growth factors. The resulting complex is implanted, for example, in a SD rat kidney defect. After 36 weeks, regeneration of renal tissue is seen at the site of implantation.

As long as the cells to be collected are undifferentiated mesenchymal stem cells, any tissue can be used in the present invention regardless of the kind of animal to be collected, age, and site thereof. In general, immune rejection is a biological response to cellular components. As a method for avoiding this immune rejection, for example, there is a method using undifferentiated mesenchymal stem cells collected from its own tissue. According to this, it is considered that the problem of immune reaction can be solved. In other words, in the present invention, the self-renal tissue can be regenerated by utilizing the self-cell component.

The cell growth factor used in the present invention is not particularly limited, but preferably has a function of increasing the number of undifferentiated mesenchymal stem cells. For example, basic fibroblast growth factor (b FGF), platelet-derived growth factor (PDGF), insulin, insulin-like growth factor (I GF), hepatocyte growth factor (HGF), glia-induced neurotrophic factor (GDNF) ), Neurotrophic factor (NF), hormone, site force-in, bone morphogenetic factor (BMP), transforming growth factor (TGF), and the like. Among these, in the present invention, bFGF is particularly desirable. Its concentration is the number of cells Per 10 ⁵ to 10 ⁸ pieces, 0.0 0:! To 1 0 / g, preferably 0.0 1 to 1.

These cell growth factors used in the present invention are preferably mixed with cells in a state of being contained in an appropriate sustained release carrier. When used in an appropriate sustained release carrier, the sustained release period should be in the range of about 1 to 3 weeks.

The carrier for sustained release of cell growth factor is preferably one having the property of being decomposed and absorbed in vivo. For example, polylactic acid, polyglycolic acid, copolymer of lactic acid and dalcholic acid, poly-f-force prolatatone, f-copolymer of force prolatatone and lactic acid or glycolic acid, polyquenic acid, polymalic acid, poly 1α-cyanoacrylate, poly 1j3-hydroxybutyrate, polytrimethylene oxalate, poly tramethylene oxalate, polyorthoester, polyorthocarbonate, polyethylene carbonate, polypropylene carbonate, poly 1 γ-benzen ^^ー Synthetic polymers such as L-glutamate, poly-γ-methyl-L-glutamate, poly-l-alanine, polysaccharides such as starch, alginic acid, hyaluronic acid, chitin, pectinic acid and their derivatives, gelatin, collagen ( Collagen type and its Deho may be either), albumin, and the like proteins, such as fibrin. Carriers for sustained release of cell growth factors can be prepared from these materials, but the forms include, but are not limited to, discs, films, rods, particles, and pastes. . For example, in the case of aiming at uniform mixing with the basement membrane component, a particulate carrier is preferable. The diameter of the particles is from 10 to 500 μπι, preferably from 20 to 100.

The sustained release can be adjusted by adjusting the degradability of the sustained release carrier. Degradability can be adjusted, for example, by changing the degree of crosslinking during carrier production. In order to set the sustained release period to 1 to 3 weeks, for example, by adjusting the concentration of the crosslinking agent or the reaction time at the time of preparing the carrier, the moisture content is 98 to 94%, and it is decomposed and absorbed in 1 to 3 weeks A sustained release carrier may be prepared.

The conditions for preparing the tissue engineering material composed of cells and cell growth factors are not particularly limited. For example, they may be simply mixed with each other, or buffer, physiological saline, solvent for injection, Or mixed with a liquid such as a collagen solution Also good. The number of cells to be used is preferably from 100,000 to 500,000. Furthermore, a mixture of cells and cell growth factors can be injected into a scaffold material such as a sponge, mesh, or non-woven fabric molding made of a bioabsorbable material, or used in a state of being mixed with the scaffold material. .

The scaffold material used in this case is considered to be essential to be bioabsorbable. In the case of non-absorbability, it is preferable because it physically interferes with tissue regeneration. It is necessary to select and use an appropriate scaffold so that the scaffold material does not interfere with tissue regeneration. If the scaffold material is a synthetic polymer, its absorbency can be controlled by its molecular weight and chemical composition, and if it is a natural polymer, its degree of cross-linking can be controlled. About these methods, a well-known method can be used.

As a scaffold material used for injection and mixing of cells and cell growth factors used in the present invention, it is essential to have a property of being decomposed and absorbed in the living body. Materials used as a carrier for sustained release of cell growth factors can be used. The same material may be used for the scaffold material and the sustained-release carrier, or different materials may be used. Examples of the form include, but are not limited to, a disk shape, a film shape, a rod shape, a particle shape, and a paste shape.

The mixture of the cell of the present invention and a cell growth factor or a mixture of the scaffold can be injected into the body by incision through the skin and implantation or injection. Example

EXAMPLES Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples. Example 1 Scaffold preparation

After heat treatment at 140 ° C. for 2 hours, after chemical crosslinking in a 0.2 wt% aqueous solution of dartal aldehyde, unreacted aldehyde groups were blocked in the aqueous glycine solution. Thereafter, the collagen sponge obtained by washing with water and freeze-drying was punched into a cylindrical shape having a diameter of about 5 mm and a thickness of 3 mm. In order to maintain the shape of the collagen sponge with a sterilized pore size 200 μηι polypropylene mesh The mesh was further fixed with 7.0 nylon thread. This was embedded in a cylindrical defect created in the kidney. The reason for using the mesh is to isolate the implanted collagen sponge site from the kidney tissue site in the tissue, and to clarify whether the regenerated tissue is newly born in the defect, or whether the tissue is derived from the kidney tissue. . Example 2 Isolation and purification of undifferentiated mesenchymal stem cells

The femur and tibia of a 6-week-old female SD rat (Shimizu Experimental Animal Co., Ltd.) were removed, and the bone marrow was collected by a clean operation. The initial medium (<< MEM, GIBCO BRL) 1 0.2 wt%, bicarbonate Sodium (Nacalai Tesque) 26.2 mM, penicillin (500 U / 1), 1 streptomycin (500 combination (SIGMA), pup sera cultured in 15%. After 7-10 days, adherent cells were treated with 0.05% trypsin. Collected with ZED TA solution and passaged using the same medium Example 3 Preparation of gelatin particles

375 ml of olive oil was added to a 1000 ml round bottom flask, and a Teflon stirring propeller was attached to the fixed stirring motor (manufactured by Shinto Kagaku Co., Ltd., Three-One Motor) and fixed together with the flask. An aqueous solution (10 wt%, 10 ml) of alkali-treated gelatin (made by Nitta Gelatin Co., Ltd.) with an isoelectric point of 4.9 was dripped while stirring olive oil at 30 ° C and 420 rpm to prepare a WZO type emulsion. . After stirring for 10 minutes, the flask was cooled to 10-20 ° C and further stirred for 30 minutes. To the emulsion, 100 ml of acetone was added, and the mixture was further stirred for 1 hour, and then the gelatin particles were collected by centrifugation (5000 rpm, 4 ° C, 5 minutes). Uncrosslinked gelatin particles were obtained by centrifugally washing the particles with acetone and 2-propanol. The resulting non-crosslinked gelatin particles (500 mg) containing glutaraldehyde de of 0. 01wt% concentration, were suspended in an aqueous solution of ^{_{0. lwt 0/0 Tween 80 (}} 100ml), 4 ° C, 15 hours gentle stirring As a result, the gelatin cross-linking reaction was carried out. Thereafter, the particles were washed twice with an aqueous solution of 0.1 wt% Tween 80, 2-propanol and distilled water, and then lyophilized. 100 particles each when dried in air from 2-propanol or in equilibrium swelling at 37 ° C in PBS The water content was calculated as a ratio of the volume of water contained in the particles to the volume of the swollen particles, and the water content was about 95 vol%. The average particle size of the particles during swelling was 40 μπι.

After labeling with 1 ²⁵ I, Toko filtrate treated with this water content of 95% of the particles in mice subcutaneously, radioactivity at the site of administration was decreased with time. Its radioactivity reached zero after 14 days. Next, b FGF was labeled with ¹²⁵ I, impregnated into gelatin particles, then administered in vivo in the same manner as described above, and the temporal change of radioactivity was examined. It was almost the same as the case of particles. Thus, b FGF was gradually released by in vivo as the particles were degraded. The sustained release period of bFGF from these particles was 14 days. Example 4 Release of basic fibroblast growth factor (b FGF)

Add 10 μΐ of an aqueous solution containing 0 g and 100 g of bFGF (provided by Kaken Pharmaceutical Co., Ltd.) to the freeze-dried gelatin particles (2 mg) prepared in Example 3 and leave them at 25 ° C for 1 hour. FGF was impregnated into the particles. after that,

100 1 PBS was added and b FGF impregnated gelatin particles were dispersed. Example 5 Encapsulation of cells and sustained release factor

The cell suspension 50 containing the cells prepared in Example 2 (2 × 10 ⁴ ) was carefully injected into the collagen scaffold prepared in Example 1 using a syringe equipped with a 22 gauge needle. The scaffold was left for three hours in a C_〇 ₂ incubator 37 ° C, cells were fixed in a collagen sponge. Thereafter, the sustained release bFGF-impregnated gelatin particle suspension 50 ^ 1 prepared in Example 3 was added. As a control mouth, scaffolds were prepared in which only cells or b FGF-impregnated gelatin particles were injected. Example 6 Transplantation of scaffold

SD rats were anesthetized with 15 mg / kg phenobarbital per body weight and dissected from the left 11 1 ribs until reaching the abdominal cavity. Exposure while protecting left kidney with gauze I let you. After clamping the renal artery to prevent bleeding, a defect (defect: 5 mm in diameter, 5 mm in depth) was formed in the lateral cortex of the left renal midrenal fistula. A scaffold containing a sustained release body was embedded. The outer skin of the kidney was sewn with 7.0 nylon to prevent detachment. After replacing the kidney, the peritoneal muscle layer and the outer skin were sutured. 3 Six weeks later, the mice were sacrificed with jetyl ether, and the left kidney was collected. When making an incision in the left kidney, care was taken to cut at the center of the major axis of the polypropylene-coated scaffold. This tissue section was prepared for histological evaluation. Staining was performed with normal hematoxylin eosin staining.

When the specimens of the obtained implantation sites were observed, regeneration of kidney tissue was observed only in the group in which collagen sponges containing gelatin particles impregnated with FGF and undifferentiated mesenchymal stem cells were implanted (Fig. 1 A, 1 B). As is clear from the photograph, regeneration of the glomerular tissue was confirmed inside the propylene mesh in this experimental group. Angiogenesis as well as tubules were also seen. In control, only undifferentiated mesenchymal stem cells were placed in a sponge (Figs. 2A and 2B), and b FGF-impregnated gelatin particles only in a sponge (Figs. 3A and 3B). As a result, formation of tubules was confirmed, but glomerular regeneration was not observed. Although not shown here, in the case of embedding with sponge only, the invasion of fibrous tissue was lighter than that of other groups, and no angiogenesis was induced at all. This result indicates that mixing of undifferentiated mesenchymal stem cells and cell growth factors is essential for the regeneration of renal tissue. Industrial applicability

According to the present invention, a material capable of regenerating the kidney in vivo can be obtained by using a combination of undifferentiated mesenchymal stem cells and cell growth factors. Further, in the present invention, since cells from the self tissue can be used, the problem of immune rejection is solved and it can be said that its utility for medical treatment is very large.

Claims

The scope of the claims

1. Materials for kidney regeneration in vivo, including undifferentiated mesenchymal stem cells and cell growth factors.

2. The material according to claim 1, wherein the cell growth factor is sustained-release.

3. The material according to claim 2, wherein the cell growth factor is sustained-released by a sustained-release carrier.

4. The material according to any one of claims 1 to 3, further comprising a scaffold material.

5. The material according to any one of claims 1 to 4, wherein the cell growth factor is bFGF.

6. The material according to claim 3, wherein the cell growth factor is bFGF and the sustained release carrier is cross-linked gelatin.

7. The material according to claim 4, wherein the cell growth factor is bFGF, the sustained release carrier is cross-linked gelatin, and the scaffold is collagen.

8. A method for regenerating a kidney, comprising using the material according to any one of claims 1 to 7.