JP2001316285A - Material for regeneration of tissue organ composed of cell and cell growth factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material for the regeneration of a tissue organ in vivo. SOLUTION: The material for the regeneration of a tissue organ in vivo is composed of a cell such as a blast cell, e.g. anaplastic mesenchyme cell, bone marrow cell, peripheral blood stem cell, nerve stem cell, hepatic stem cell (small hepatocyte, oval cell), embryonic stem cell(ES cell), EG cell, muscular satellite cell, osteoblast, odontoblast, chondroblast, epithelial embryonic cell and biliary embryonic cell and a cell growth factor such as basic fibroblast growth factor(bFGF), platelet differentiation growth factor (PDGF), insulin, insulin-like growth factor(IGF), hepatocyte growth factor(HGF), glia-derived neurotrophic factor(GDNF), neurotrophic factor(NF), hormone, cytokine, bone morphogenetic factor(BMP) and transforming growth factor(TGF).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a material for regenerating tissue organs, which is characterized by using a combination of cells and cell growth factors.

[0002]

2. Description of the Related Art Although reconstructive medicine using artificial organs is currently contributing greatly to clinical practice, there are major drawbacks in that the effect is temporary, the invasion is large, and a single function can be assisted. On the other hand, in transplantation medicine, in addition to a shortage of donors, infectious diseases and carcinogenesis due to the side effects of immunosuppressants after transplantation are also problems, and it is difficult to say that these treatments are ideal. Thus, the current two pillars of advanced medicine are reaching their respective limits. In such a situation, new treatment attempts have been made to regenerate the own tissue to restore the missing or devastated tissue or organ. This is regenerative medicine. In other words, by using cells,
It is an attempt to artificially make their own tissues and organs healthy again.

[0003] In mammals including 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 also have a regenerative ability. That is, in a mammal, wound healing for protecting an individual progresses too quickly, so that a site for organ regeneration is lost, and the inherent regeneration ability is lost. For example, bone marrow transplantation is a long-standing therapy and has shown good clinical results. This is considered to be successful because bone marrow tissue is present in the body, a birthplace where blood stem cells in bone marrow cells can proliferate and differentiate. In other words, in order for an individual to survive, humans would not be able to survive unless stem cells are present in tissues and organs. If a place for regeneration can be provided, there is a possibility that self-organization can be regenerated. In recent years, it has been found that stem cells also exist in the nervous system, liver, and the like.

Therefore, various methods of tissue regeneration have been researched and developed based on such a concept. At present, a combination of a scaffold for regeneration, a cell growth factor, and a stem cell is used. The method used is the most realistic.

[0005] In order to promote the adhesion, differentiation and morphogenesis of stem cells and progenitor cells at the regeneration site, a scaffold for regeneration is required. In some cases, the living tissue at the regeneration site can be a scaffold for regeneration, but in other cases, a scaffold matrix is required as a scaffold for regeneration. Bioabsorbable materials are used as this matrix.

However, for example, even if a scaffold matrix is embedded in a regeneration site, if the number of stem cells, progenitor cells, or blast cells required for regeneration is small, or if the concentration of a biological factor that causes the cells to proliferate and differentiate is too low, No matter how good the matrix is, the desired tissue regeneration will not occur. Thus, the first factor to be replenished is a cell growth factor for cell growth or differentiation.

In general, the in vivo life of these factors is short and unstable, and simply dissolving the necessary cell growth factor in water and injecting it into the required site does not provide the expected tissue regeneration effect. Therefore, the concentration of the cell growth factor in the regeneration site must be kept at an effective value for a necessary period. For example, there is a technique in which a cell growth factor is contained in a sustained-release carrier and is continuously released at a regeneration site. The sustained release of cell growth factors enhances the proliferation and differentiation of cells and promotes the regeneration of self-tissues. However, depending on the type of tissue or organ, regeneration is often insufficient with only a cell growth factor.

[0008] Although mastectomy has been performed as a curative operation for breast cancer, there is no clinically satisfactory method for breast reconstruction after surgery. Therefore, in recent years, a mastectomy in which a resection site is made as small as possible and a part of the breast is preserved has been performed. However, reconstruction is difficult for patients with small breasts or patients who cannot perform conservative surgery. In such cases, filling of the defective part with subcutaneous fat or subcutaneous fat with muscle has hitherto been performed. However, the transplanted adipose tissue is absorbed and replaced with fibrous tissue with time, even though the blood flow is controlled by blood vessels, and the intended purpose cannot be achieved. on the other hand,
Although artificial breasts made of silicone have been used, the presence of such materials often causes problems such as difficulty in seeing during X-ray photography, calcification and encapsulation during long-term implantation.

[0009] In addition to breast reconstruction, for example, in the field of plastic surgery, subcutaneous fat is used for filling in a facial dent. However, absorption in vivo and subsequent fibrosis of tissue are clinical. Above, it is a problem.

[0010] It has been reported in many animal experiments that the implanted adipose tissue is simply absorbed over time by simply implanting the collected adipose tissue into a living body (for example, Eppley, BL, etc.). Plast. Reconstr. Surg.,
90 volumes, p1022, 1992, etc.). Even if allogeneic, allogeneic, an inflammatory response to the transplanted tissues occurs, and the mature fat cells in those tissues are destined to die in vivo and will become mature fat cells in the future. This indicates that the generation of adipose tissue cannot be expected without the presence of differentiable progenitor cells.

[0011] Differentiation into fat cells has been investigated using fat precursor cells or fat precursor cell lines derived from animal or human adipose tissue. In addition, advances in genetic technology have made it possible to purchase various cell growth factors and to examine their effects on adipocyte differentiation. However,
All of these studies have been performed in vitro (eg, Hausman, GJ et al., J. Lipid Res., 21, p657, 198).
0, Hauner, H. et al., Klin. Wochenschr, 65, p803, 198
7, Butterwith, SC, etc., J. Endocrinology, 137, p3
69, 1993, Gregoire, FM, etc., Physiological Review,
78, p783, 1998, etc.), no in vivo studies.

It has been reported that subcutaneous adipose tissue was formed 5 weeks after subcutaneous administration of a mouse preadipocyte cell line to nude mice (Green, H. et al., J. Am.
CellPhysiol., Volume 101, p169, 1979). However,
The preadipocytes used are cell lines, and there is no description of the use of autologous progenitor cells or their combined use with cell growth factors.

The extracellular matrix (Matrigel) produced by mouse EHS cancer cells contains a large amount of basement membrane components, which can be implanted subcutaneously into mice or together with a cell line of fat precursor cells. It has been reported that tissue formation is observed and that regeneration is enhanced by the addition of cell growth factors (Kawagu
Chic, N. et al., Proc. Natl. Acad. Sci. USA., 95, p106
2, 1998), but also does not describe the use of preadipocytes isolated from animals.

After seeding fat precursor cells isolated from human adipose tissue on a non-degradable and absorbable polymer Teflon (registered trademark) mesh scaffold, differentiation into adipocytes in vitro has been examined. . (Krai, JG, etc., Plast. R
econstr. Surg., 104, p1732, 1999)
In this study, the complex of those cells and scaffolds
No implantation in vivo has been performed, and no mention is made of adipose tissue regeneration in vivo.

[0015] It has been reported that rat autologous adipose tissue was regenerated by disseminating rat autologous fat precursor cells into a mesh made of bioabsorbable polylactic acid and implanting the cells under the skin of the rat (CW Patrick). . et. al. Tiss
ue Engineering, 5 (2), 1999), however, did not examine the effects of adding cell growth factors in this study, nor did it discuss any of its potential.

[0016]

[Problems to be Solved by the Invention] In vivo regeneration of tissue organs
There is no doubt that the cells that make up that tissue or organ are needed to do it ivo. However,
It is extremely difficult to regenerate a tissue or organ at that site simply by injecting it into the site where the cell is to be regenerated. The reason is that the added cells can hardly be expected to proliferate 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, progenitor, or blast cells can increase their numbers in the presence of appropriate growth factors,
It has been proven to differentiate. But in vitro
Cell environment is completely different between in vivo and in vivo.
It is often difficult to reproduce and predict the results in vivo in vivo.

[0017]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems relating to the proliferation and differentiation of cells in vivo, and as a result, by using cells in combination with cell growth factors, The present inventors have found that regeneration of tissue organs in vivo is extremely advantageous, and have completed the present invention. Therefore, the present invention provides a method for in vivo tissue organs by using a combination of cells and cell growth factors.
Supply material for regeneration in o.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The technical configuration of the present invention will be described below. Cells used in the present invention are not particularly limited, for example, undifferentiated mesenchymal cells, bone marrow cells, peripheral blood stem cells, neural stem cells, hepatic stem cells (small hepatocytes, oval cells), embryonic stem cells (ES cells ), EG cells, muscle satellite cells, osteoblasts, rhododendron buds, chondroblasts, epithelial germ cells, cholangioblasts and other blast cells. These cells can be prepared by collecting the cells from animals or humans, or by increasing the number of the collected cells in a culture system, or by performing a treatment for differentiation in a specific direction.

Preparation of cells used in the present invention, for example, preadipocytes, mixing with cell growth factors, and regeneration of adipose tissue using the tissue engineering materials can be performed by the following methods.

The adipose tissue mass collected from human subcutaneous fat is made as small as possible with scissors. A small piece of this adipose tissue mass was placed in a 2 mg / ml collagenase solution at 37 ° C. for 5 minutes.
Shaking treatment was performed for 0 minutes. After this treatment, the cells were collected by filtration and centrifuged through a mesh.

The preadipocytes obtained are cultured in an incubator at 37 ° C. and 5%, and the cells are proliferated. The cells are dispersed in a culture solution, and an appropriate concentration of a cell growth factor is mixed therein. The obtained complex is implanted, for example, under the back of a nude mouse. After 5 weeks, regeneration of adipose tissue is seen at the site of implantation.

As long as the tissue mass to be collected is a tissue containing adipose tissue, any tissue can be used in the present invention irrespective of the type, age, and site of the animal to be collected. Generally, immune rejection is a biological response to cellular components. As a method for avoiding the immune rejection, for example, there is a method using preadipocytes collected from an own adipose tissue. According to this, it is considered that the problem of the immune reaction can be cleared. These techniques are used for other progenitor cells, blast cells,
The same applies to stem cells and the like, and the present invention can also regenerate its own fat tissue by utilizing its own cell components.

The cell growth factor used in the present invention is
Increase in number of preadipocytes, but not limited to
What has the effect | action which makes it do is preferable. For example, basic fibers
Blast growth factor (bFGF), platelet differentiation growth factor (P
DGF), insulin, insulin-like growth factor (IG
F), hepatocyte growth factor (HGF), glial-induced neurotrophic
Factors (GDNF), neurotrophic factors (NF), hormones,
Cytokine, bone morphogenetic factor (BMP), transform
Mining growth factor (TGF) and the like. these
Among them, bFGF is particularly desirable in the present invention. That dark
The degree is 10 cells ^Five-10⁸0.0001-10μ per piece
g, preferably 0.001 to 1 μg.

These cell growth factors used in the present invention are preferably mixed with cells in a state of being contained in a suitable sustained release carrier. When used in a state of being contained in a suitable sustained release carrier, the sustained release period is preferably 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, a copolymer of lactic acid and glycolic acid, poly-ε-caprolactone, a copolymer of ε-caprolactone and lactic acid or glycolic acid,
Polycitric acid, polymalic acid, poly-α-cyanoacrylate, poly-β-hydroxybutyric acid, polytrimethylene oxalate, polytetramethylene oxalate, polyorthoester, polyorthocarbonate, polyethylenecarbonate, polypropylenecarbonate, poly-
γ-benzyl-L-glutamate, poly-γ-methyl-
Synthetic polymers such as L-glutamate and poly-L-alanine; polysaccharides such as starch, alginic acid, hyaluronic acid, chitin, pectic acid and derivatives thereof; gelatin; collagen (collagen type and extraction method may be any) , Albumin, fibrin and the like. A carrier for sustained release of a cell growth factor can be prepared from these materials, but a carrier in the form of particles is preferred for the purpose of uniform mixing with a basement membrane component. The diameter of the particles is 10 to 500 μm, preferably 20 to 100 μm.

The control of sustained release can be performed by controlling the degradability of the carrier for sustained release. The degradability can be adjusted, for example, by changing the degree of crosslinking at the time of preparing the carrier. In order to set the sustained release period to 1 to 3 weeks, for example, the concentration of the cross-linking agent or the reaction time at the time of preparing the carrier is adjusted, the water content is set to 98 to 94%, and the sustained release is decomposed and absorbed in 1 to 3 weeks. A carrier may be prepared.

The conditions for preparing the tissue engineering material comprising cells and cell growth factors are not particularly limited. For example, the two may be simply mixed, or may be a buffer, saline, or injection solution. It may be mixed with a solvent or a liquid such as a collagen solution. The number of cells to be used is preferably 100,000 to 5,000,000. Furthermore, a mixture of cells and a cell growth factor can be injected into a scaffold such as a sponge, a mesh, or a non-woven fabric made of a bioabsorbable material, or can be used in a state of being mixed with such a scaffold.

It is essential that the scaffold material used at this time is bioabsorbable, and if it is non-absorbable, it is not preferable because it hinders tissue regeneration physically. It is necessary to select and use an appropriate scaffolding material so that it does not hinder tissue regeneration. When the scaffold is a synthetic polymer, its absorbency can be controlled by its molecular weight and chemical composition, and when it is a natural polymer, its absorbency can be controlled by the degree of crosslinking. Known methods can be used for these methods.

The scaffold used for injecting and mixing the cells and cell growth factors used in the present invention must have a property of being decomposed and absorbed in vivo, for example, The material used for the carrier for sustained release of cell growth factor can be used. The same material may be used for the scaffold material and the sustained-release carrier, or different materials may be used. The form is a disk, film,
Examples include, but are not limited to, rods, particles, and pastes. A mixture of the cell of the present invention and a cell growth factor or a mixed complex thereof with a scaffold material is
It can be injected into the body by incising the skin and implanting or injecting by injection.

[0030]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

Example 1 Under the consent of a breast cancer patient, adipose tissue at a normal site was collected at the time of surgery. This tissue was washed with a 0.05 M phosphate buffered saline solution (PBS (pH 7.4)) to remove attached blood and the like. Adipose tissue mass is removed with surgical scissors for approximately 5
After cutting into dice with a square cube, a collagenase solution (serum-free DMEM medium containing 2 mg / ml collagenase 20 mg / ml bovine serum albumin) was added, and the mixture was infiltrated with a shaker (37 ° C.) with a water bath for about 50 minutes. Tissues were collagenase treated. Processing solution with pore size 250 μm
To remove tissue fragments that had not been digested with collagenase. MEM of equal volume to filtrate
After adding 199 medium (10% fetal calf serum FCS) to stop the collagenase activity, the cell suspension was centrifuged at 1,000 rpm for 5 minutes to obtain fat precursor cells as a sediment. Discard the supernatant and add a new MEM1
99 medium (10% FCS) was added and the cells were dispersed well by pipetting, and then seeded in a 75 cm ² culture flask (cell density of 1 × 10 ⁵ cells / ml, 15 ml).

Twenty-four hours after seeding the isolated preadipocytes into the flask, erythrocytes not adhered to the bottom of the flask and unadhered cells are removed together with the medium.
10% FCSMEM19 containing 1 μg / ml bFGF
Nine media was added to the flask and the culture continued. The state of the cells immediately after the addition of the bFGF-containing medium is shown in FIG. When the culture is continued for about 10 days, the cells become confluent (FIG. 1 (B)). When 10 mM dexamethasone was added to the cells, after 2 weeks of culture, the cells differentiated into mature adipocytes so as to accumulate lipid droplets (FIG. 1 (C)). This indicates that the isolated cells are preadipocytes.

Example 2 Olive oil (375 ml) was added to a 1000 ml round bottom flask, and a Teflon stirring propeller was attached to a fixed stirring motor (Shinto Kagaku, three one motor).
Fixed together with the flask. Olive oil at 30 ° C, 42
While stirring at 0 rpm, an aqueous solution (10 wt%, 10 wt%) of alkali-treated gelatin (manufactured by Nitta Gelatin Co., Ltd.) having an isoelectric point of 4.9.
ml) was added dropwise to prepare a W / O emulsion. 10
After stirring for minutes, the flask was cooled to 10-20 ° C and stirred for an additional 30 minutes. 100 ml of acetone was added to the emulsion, and the mixture was further stirred for 1 hour.
(00 rpm, 4 ° C, 5 minutes) to recover the gelatin particles. Uncrosslinked gelatin particles were obtained by centrifugally washing the particles with acetone and 2-propanol. The obtained uncrosslinked gelatin particles (500 mg) were mixed with 0.0
0.1wt% Tw containing glutaraldehyde at 1wt% concentration
The resulting mixture was suspended in an aqueous solution (100 ml) of een 80 and gently stirred at 4 ° C. for 15 hours to carry out a crosslinking reaction of gelatin. After that, the particles were dissolved in an aqueous solution of 0.1 wt% Tween 80
After washing twice with 2-propanol and distilled water, each was freeze-dried. Air drying from 2-propanol or PB
In S, the diameter of the particles during equilibrium swelling at 37 ° C. was measured with a microscope for each 100 particles, and the water content was calculated as the ratio of the volume of water contained in the particles to the volume of the particles in the swollen state. , Its water content is about 95vol
%Met. The average particle size of the particles at the time of swelling is 4
It was 0 μm.

After labeling with ¹²⁵ I, the water content
% Of the particles administered subcutaneously to the mice, the radioactivity at the administration site decreased over time. Its radioactivity is 14
Zero days later. Next, bFGF was labeled with ¹²⁵ I and impregnated into gelatin particles, and then in vivo as described above.
o After administration, the time-dependent change in radioactivity was examined, and the time-dependence of the remaining radioactivity was almost the same as in the case of particles. In this way, bFG
F was sustained-released in vivo. BFGF from this particle
The sustained release period was 14 days.

Example 3 The confluent human preadipocytes prepared in Example 1 were thoroughly rinsed with PBS, added with a 0.25% trypsin solution, and incubated for 5 minutes to detach the cells. Fresh medium was added, centrifuged, and the number of cells was counted using a hemocytometer. Example 2
0 μg and 10 μg of bFGF to lyophilized gelatin particles (2 mg) prepared in (1) (provided by Kaken Pharmaceutical Co., Ltd.)
Then, 10 μl of an aqueous solution containing was dropped and allowed to stand at 25 ° C. for 1 hour to impregnate the particles with bFGF. afterwards,
100 μl of PBS was added to disperse the bFGF-impregnated gelatin particles. After well mixing 100 μl of the suspension containing 1 × 10 ⁵ cells and the particle suspension, a type I collagen sponge (Gunze Co., Ltd.) cut into about 1 cm square and 3 mm thick was prepared.
(Provided by the Company) was carefully injected with a syringe. The sponge was left in a 37 ° C. incubator for 1 hour to allow the cells to settle in the sponge. As a control, a collagen sponge into which only cells were injected was prepared. The collagen sponge obtained in this manner was implanted into a subcutaneous back of a nude mouse at a site free of adipose tissue. After about 5 weeks, the skin was peeled off, the newly formed adipose tissue was taken out, a tissue section was prepared, and histological evaluation was performed by HE staining, Sudan III staining and the like.

After 5 weeks from the implantation, the skin on the back was peeled off and the implantation site was observed. As a result, only a group in which a collagen sponge containing 10 μg of bFGF-impregnated gelatin particles and cells was implanted was visually observed to be fat. Tissue regeneration was observed (FIG. 2 (A)). Such a phenomenon was not observed in empty gelatin particles and cells (FIG. 2 (B)), and those in which only cells were placed in a sponge (FIG. 2 (C)).

Example 4 In the same manner as in Example 3, the doses were different (0.01,
0.1 and 10 μg) bFGF-impregnated gelatin particles or collagen sponges into which empty particles and cells were injected were implanted subcutaneously in the back of mice, and adipose tissue regeneration after 5 weeks was evaluated from tissue sections. Tissue sections of the regenerated tissue were stained with Sudan III, which stains neutral fat (FIGS. 3 and 4). FIG.
Is (1) a collagen sponge in which only cells are injected,
(2) a collagen sponge into which cells and empty gelatin particles are injected, (3) a collagen sponge into which cells and gelatin particles impregnated with 0.01 μg of bFGF are injected,
(4) Collagen sponge injected with cells and gelatin particles impregnated with 0.1 μg of bFGF, FIG. 4 shows (1)
Collagen sponge infused with cells and gelatin particles impregnated with 1 μg bFGF, (2) cells and 10 μg bFGF
5 is a micrograph of a tissue section when a collagen sponge into which gelatin particles impregnated with FGF are injected is embedded. In FIG. 4, (3) is an enlarged photograph of (1), and (4) is an enlarged photograph of (2).

As a result, formation of adipose tissue was observed when the bFGF dose of the bFGF-impregnated particles was 1 and 10 μg. The globular cavities characteristic of fat cells and their corresponding spots were stained orange by Sudan. No fat cells were found in the other experimental groups.

Example 5 10 μl of an aqueous solution containing 0.05 μg of TGF-β1 (purchased from Sigma Chemical) was added dropwise to 2 mg of the freeze-dried gelatin particles prepared in Example 2, and the TGF was allowed to stand at 25 ° C. for 1 hour. −β1 was impregnated in the particles.
Under anesthesia, a 2 mm diameter hole was made in the femur with a dental drill under anesthesia, and 1 ml of PBS was injected. Thereafter, the PBS was collected and washed by centrifugation with fresh PBS to prepare rabbit bone marrow cells. 100 μl of a suspension containing 1 × 10 ⁶ rabbit bone marrow cells and TGF-β1-impregnated gelatin particles were mixed well. The head of a Japanese white rabbit (male, 2-2.5 kg) that had been under general anesthesia with Nembutal was shaved with a hair clipper, disinfected with isodine, and subjected to local anesthesia with xylocaine. A midline incision was made on the parietal skin, and the periosteum was peeled and incised. While the exposed crown of the skull was cooled with a saline solution, a bone-drilled hole having a diameter of 6 mm (the dura was preserved) was formed one by one in a sagittal suture with a dental drill. A mixture of gelatin particles impregnated with TGF-β1 and bone marrow cells was inserted into the bone defect, the periosteum was covered with 4-0 nylon thread, and
Each of them was sutured with −0 silk thread. As a control, a mixture of empty gelatin particles not impregnated with TGF-β1 and 1 × 10 ⁶ or 1 × 10 ⁷ rabbit bone marrow cells only with PBS, gelatin particles impregnated with 0.05 μg of TGF-β1 was used as a control. Only was used. Six weeks after the operation, he was anesthetized with Nembutal, and the bone including the defect was cut out.
The removed bone sample was subjected to soft X-ray photography. Next, fixation and decalcification with formalin were performed, and the tissue section was stained with hematoxylin-eosin and then observed with an optical microscope. FIG.
It is a tissue section photograph of a bone defect part. FIG. 5A shows PBS.
Treatment group, (B) a mixture treated group of gelatin particles and bone marrow cells without TGF-β1 (10 ⁶ cells / bone defect),
(C) is a group treated with a mixture of gelatin particles not containing TGF-β1 and bone marrow cells (10 ⁷ cells / bone defect).
GF-β1-containing gelatin particles (0.05 μg TGF-
.beta.1 / bone defects) treated groups, indicating the mixture treated group (E) and the TGF-.beta.1-containing gelatin particles (0.05 [mu] g TGF-.beta.1 / bone defect) and bone marrow cells (10 ⁶ cells / bone defect).

FIG. 5 shows that only the bone defect in the group treated with the mixture of gelatin particles impregnated with TGF-β1 and bone marrow cells was
It became clear that the bone defect was repaired by the regenerated bone tissue. In other control experiment groups,
No such bone regeneration was observed, and soft tissue invasion was observed in the bone defect. These results indicate that the mixture of bone marrow cells and cell growth factors is essential for tissue regeneration.

The same effect was obtained by performing the same treatment using a collagen sponge as a scaffold material.

[0042]

According to the present invention, tissue organs can be in vivo by using a combination of cells and cell growth factors.
And a material to be regenerated can be obtained. In addition, in the present invention, the problem of immune rejection can be solved by using cells from a self-tissue as cells to be used, and self-organs can be regenerated, and its utility in medical treatment can be said to be very large.

[Brief description of the drawings]

FIG. 1 is an optical micrograph of human preadipocytes isolated and cultured from an adipose tissue mass. (A) Cells immediately after isolation (B) Cells grown by adding bFGF (C) Cells differentiated into mature adipocytes by adding dexamethasone

FIG. 2 is an optical micrograph of an implanted site 5 weeks after a collagen sponge into which human preadipocytes and bFGF-impregnated gelatin particles were injected was implanted subcutaneously in the back of a nude mouse. (A) Collagen sponge injected with cells and gelatin particles impregnated with 10 μg of bFGF (B) Collagen sponge injected with cells and empty gelatin particles (C) Collagen sponge injected only with cells

FIG. 3 shows a tissue section (Zudan III staining for neutral fat) of an implanted site 5 weeks after a collagen sponge into which human preadipocytes and bFGF-impregnated gelatin particles were injected was implanted subcutaneously in the back of a nude mouse. It is a microscope picture. (1) Collagen sponge injected with cells only (2) Collagen sponge injected with cells and empty gelatin particles (3) Collagen sponge injected with cells and gelatin particles impregnated with 0.01 μg bFGF (4) Cells Collagen sponge infused with gelatin particles impregnated with 0.1 μg bFGF

FIG. 4 shows a tissue section (Sudan III staining for neutral fat) of an implant site 5 weeks after a collagen sponge into which human preadipocytes and bFGF-impregnated gelatin particles were injected was implanted subcutaneously in the back of a nude mouse. It is a microscope picture. (1) Collagen sponge injected with cells and gelatin particles impregnated with 1 μg bFGF (2) Collagen sponge injected with cells and gelatin particles impregnated with 10 μg bFGF (3) Enlarged photograph of (1) (4) (2) Enlarged photo

FIG. 5 is a micrograph of a tissue section of a defect after a mixture of gelatin particles impregnated with TGF-β1 and bone marrow cells was implanted in a defect in a rabbit skull. (A) PBS-treated group (B) Mixture-treated group of gelatin particles without TGF-β1 and bone marrow cells (10 ⁶ cells / bone defect) (C) Gelatin particles without TGF-β1 and bone marrow cells (10 ⁷ (D) TGF-β1-containing gelatin particles (0.05 μg)
(E) TGF-β1-containing gelatin particles (0.05 μg)
TGF-.beta.1 / bone defect) and the mixture treated group of bone marrow cells (10 ⁶ cells / bone defect)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C07K 14/50 A61K 37/24 C12N 5/06 C12N 5/00 EFterm (reference) 4B065 AA90X BB19 BB23 BB34 CA44 4C076 AA76 CC30 EE42 FF32 4C081 AB11 CD29 CD34 4C084 AA01 AA02 BA01 BA44 DA01 DB52 DB54 DB55 DB58 DB62 MA01 MA05 NA14 ZB222 ZC032 4H045 AA10 BA10 CA40 DA22 EA28 EA34 FA71

Claims (10)

[Claims] 1. A material for in vivo regeneration of a tissue organ comprising cells and cell growth factors. 2. The material according to claim 1, wherein the cells are undifferentiated mesenchymal cells, progenitor cells, or blast cells. 3. The material according to claim 1, wherein the cell growth factor is sustained release. 4. The material according to claim 3, wherein the cell growth factor is sustained-released by a sustained-release carrier. 5. The method of claim 1, further comprising a scaffold material.
A material according to any one of the preceding claims. 6. The material according to claim 2, wherein the cells are pre-adipocytes, the cell growth factor is bFGF, and the tissue organ is adipose tissue. 7. The material according to claim 4, wherein the cells are preadipocytes, the cell growth factor is bFGF, the sustained-release carrier is cross-linked gelatin, and the tissue organ is adipose tissue. 8. The method according to claim 5, wherein the cells are pre-adipocytes, the cell growth factor is bFGF, the sustained-release carrier is cross-linked gelatin, the scaffold is collagen, and the tissue organ is adipose tissue. Material. 9. The material according to claim 4, wherein the cells are bone marrow cells, the cell growth factor is TGF-β1, the sustained release carrier is cross-linked gelatin, and the tissue organ is bone tissue. 10. The cell according to claim 5, wherein the cell is a bone marrow cell, the cell growth factor is TGF-β1, the sustained-release carrier is cross-linked gelatin, the scaffold is collagen, and the tissue organ is bone tissue. The described material.