Background Art The present invention is aimed at volume replacement on necessary tissues in human body using allogenic tissues or solid powder, both of them are used for existing human tissue volume replacement effect or wrinkle removal, wherein culturing cell of necessary tissue and mixing then injecting again to the human body.

Related method and technology of culturing cells in solid particles having a spongy structure as a scaffold then injecting them for human tissue volume replacement effect are already laid by Korea patent application No. 1020020025375,1020020061701, 1020020064189* The laid method is a technology relating to cell culture and injection- transplantation using solid sponge powder, it has a remarkable advantage to use solid sponge powder in view of easiness and efficiency of cell culture.

But it needs a lot of efforts and cost to produce the sponge powder- namely sponge fine particles having fine bubbles-and there is a demerit of limited use in respect that it is impossible to use in material difficult to have fine bubbles.

Nevertheless, existing three dimensional cell culture in the solid is possible in the only sponge structure on the ground that unless it has a sponge structure, the culture fluid cannot infiltrate into the solid of big lump which is not a powder. And it resulted from stereotype that only sponge structure is

positively necessary in case of three dimensional culture, and this stereotype was caused by stressing excessively efficiency of cell culture.

Furthermore the culture fluid used in the process of cell culture includes ingredient such as a bovine serum forbidden to enter the human body, therefore it has to pass through a process of washing and removal before injected to the human body. In the sponge powder structure there is a high possibility that the culture fluid remains behind among the fine holes of sponge.

Since the bovine serum in the culture fluid is a different protein ingredient from that of human, it can cause immune reaction if injected to the human body.

Therefore if at all possible, it is necessary to remove completely.

Washing the culture fluid has to be carried out not to come off the cells by PBS (phosphate buffer saline) harmless to the human body. In the sponge structure it is essential to wash the culture fluid 7 times or more. If the washing frequency increases, no matter how slowly it may be washed, there is a loss of preadipocytes multiplying and adhering to the sponge structure in this process.

Disclosure of the Invention The present invention relates to a method supplementing the demerit of existing cell culture and injection method using sponge structure. And it is an object of the present invention to provide more economical and actually applicable method devised by a fact that as a result of observing electronic microscope, the materials (for example allogenic fascia tissue, allogenic human dermis tissue, gore-tex, PLGA and etc. ) for simple volume replacement have a structure of prominence and depression wherein cells can adhere to.

When the existing injectable materials were injected, they are replaced into fibrous tissues or they have a hard property, so they can't have the same elasticity as that of several human tissues. However by means of adhering necessary cells to the material, there is a most useful merit in view of creating the same elasticity as that of human tissues.

Furthermore in view of volume replacement effect, it can also provide

abundant volume effect than injection without cell adhesion. For instance, if injecting human dermis powder, allogenic fascia powder or chitosan for existing wrinkle removal, in case of human dermis some remains behind and other is replaced into fibrous tissue, there is a demerit of hard feel different from skin elasticity, In case of allogenic fascia, most of it is replaced into fibrous tissue, and initial volume effect is decreased. In this case, if adhering and culturing the preadipocyte then injecting, the preadipocytes exist among the human dermis powder, thus it can maximize the volume replacement effect. And it has the same elasticity as that of human soft tissue while preadipocytes have grown up three dimensionally, on this occasion injected material with cells provides a space wherein sticked cells can grow up three dimensionally in the human body.

As the laid analogous idea, the possibility that it is difficult to embody actually use of powder having a sponge structure is high because the materials able to produce the sponge structure are limited. The present invention reveals that the solid powder not having a sponge structure is also used valuably and efficiently for cell culture as well as an injectable material. And it is an object of the present invention to provide a cell culture method using solid powder, which can be economically and conveniently obtained and utilized in case of excluding efficiency of cell culture.

In order to accomplish the above object of the present invention, there is provided a method of three dimensional cell culture using solid powder which is a previous step required for injection-transplantation of preadipocyte, chondroblast osteoblast, which comprises the steps of mixing a serum extracted from human body or saline solution or culture fluids harmless to the human body ; with subcultured culture such as preadipocyte, chondroblast, osteoblast ; with culture material made up solid powder having no bubbles and a particle size of 20~500gan, culturing them.

The said solid powder is as an injectable material, although it is a fine particle having no bubbles, the culture fluid is possible to infiltrate since spaces between the particles acted as bubbles. The present invention has view on the

fact that cell adhesion is possible, if seeing the enlarged picture with high magnifications although there are no bubbles in the said solid powder but it has protuberances where cells can adhere to. <BR> <BR> <P>There are Alloderm (USA Life Cell Inc. ), Sheba (Korea Hansbiomed Inc.),<BR> Fascian (Fascia Biosystem Inc. ) which are produced into powder without a sponge structure by treating human corium tissue, chitosan powder having no fine bubbles, gore-tex powder (PTFE), PLGA as an applicable solid powder which is an injectable material, sufficiently stabilized, proved safety, obtained easily and conveniently.

These materials can adhere and culture the cells, they have a size of 20~500gan enough to transplant with a injector.

In case of the laid sponge structure, it is advantageous to limit the size of particle from 20 to 250, can in order to wash completely the culture fluid since cell culture is performed between the bubbles inside particles and the culture fluid infiltrates deep into the sponge structure. But in case of solid powder having no bubbles, its size can be enlarged from 20 to 500,.

Moreover in case of applying solid powder, thick syringe needle has to be used because the space between particles has to be utilized unlike sponge structure. However it is advantageous to have big particle size that it can reduce possibility of cell loss, possibility of changing relative location is little since adhesion surface of singular particle is large.

Also, carrying out this, it has advantages that it is possible to decrease efforts to assort the particle size in manufacturing process, to reduce the cost, to simplify the process and to improve working efficiency.

If using solid powder having no bubbles, efficiency of cell culture may decrease a little but it has some advantages as followings. It can lower the production cost of culture material, shorten the manufacturing process, expand application range, select the materials free, and simultaneously the solid powder having no bubbles can be acted as a cell culture material injected into the human body by injection-transplantation like use of solid sponge powder after

culture.

Except existing method of using sponge powder, the method of present invention is the first trial as injectable cell culture and injection-transplantable.

It may have rather suffer loss in efficiency of cell culture than sponge powder.

But in accordance with kind of the materials, there may be not such difference, and it can diversify the kind of material and produce conveniently.

The said solid powders shown in FIG. 1 through FIG. 4 have conditions of scaffold available to culture, exactly, they includes cubic structure where cell can adhere to, space where cell can grow up, free infiltration of culture fluid, hydrophile property and etc.

Among the said solid powders under the condition that cell can adhere to if they have a surface in contact with the cell, cell adhesion is possible even if they don't have a dissepiment or complex cubic structure. If also seeing them shown in high magnification enlarged photo, it is possible for them to attach the cell as a concavo-convex structure.

Spaces among the fine particles acted as the spaces where the culture fluid is infiltrated by the capillary as well as simultaneously where the cell can grow up, their functions are similar to those of bubbles.

In the process needed for injection, if particles'own relative position is changed, a demerit may be raised that the spaces are transformed then where the cells are swept away in comparison with the sponge structure having spaces where each particle never change its own relative position. But there are many particles that never change their relative position among numerous particles, so it is nothing more than probability rise. And it doesn't matter even if cell adhesion came off there is a possibility of re-adhesion after injection- transplantation.

The said method has features that it provides very useful method needed in culture material and injection-transplantation of cultured precursor cell as well as it takes advantage of human dermis powder or allogenic fascia powder for this method, which never attempted yet.

Moreover, the said method means a successive development in material for injection-transplantation of human cell in respect that it utilizes initially PLGA powder wherein autologous was cultured easy to manufacture and obtain at present, chitosan powder apt to obtain easily and etc as an injectable material for the human soft tissue volume replacement.

When manufacturing gore-tex into powder, it is feasible to utilize as an injection material and on this occasion it can be also used for the purpose of this invention.

Existing three dimensional cell culture in solid is possible in only sponge structure on account of big lump of solid not a powder state, the culture fluid cannot infiltrate unless it has sponge structure. But Alloderm, Sheba, Facian, gore-tex, chitosan powder having no fine bubbles or PLGA is possible to culture cell three dimensionally. Although they are not a sponge structure, the culture fluid can infiltrate free.

According to the present invention by means of using solid powder having a size of 20~500, sn and no bubbles for the cell culture, it can lower the manufacturing price of culture material, shorten the manufacturing process, expand its application range, simultaneously the said solid powder can function as an injectable cell culture material similar to the case of using solid sponge powder.

Except the method using existing sponge powder, method of the present invention is the first trial as injectable cell culture and injection-transplantable material. Though it suffers some loss rather than sponge powder in efficiency of cell culture, it can diversify kind of materials and simplify the manufacture and it is safer to the human body.

Brief Description of the Drawings FIG. 1 and FIG. 2 enlarged photo of Sheba having a concavo-convex structure FIG. 3 and FIG. 4 enlarged photo of chitosan having a concavo-convex

structure Figure 5 enlarged photo by highest magnification Best Mode for-Carrying Out the Invention Example 1 (surgery for replacing volume of facial cheek) First, about 3cc of fat tissue was extracted from patient's hypodermis by liposuction. It was treated with collagenase then preadipocytes were separated apart. After separation, they passed through a subculture 4-6 times. The cells increased by subculture were treated with trypsin, and the each cell was separated, then they were floated on sufficient amount of culture fluid. Herein human dermis powder was floated together and mixed. When mixing equally, excessive culture fluid was removed up to appropriate amount. Thereafter the mixture cultured in the culture medium for 3-10 hours and adhering the preadipocytes to the human dermis powder. The culture fluid was washed with PBS 2-3 times, then mixed with a serum extracted from the human body. The last mixture injected by an injector for human volume replacement.

Example 2 (rhinoplasty for cosmetic purpose: nasal augmentation and nasal tip correction-About lOg cartilage tissue is needed.) First, about 200mg of cartilage tissue was extracted from patient's ear. It was treated with collagenase then chondroblasts were separated. After separation, they passed through a subculture 4-6 times. The cells increased by subculture were treated with trypsin, and the each cell was separated apart.

Then they were floated on sufficient amount of culture fluid. Herein allogenic fascia powder was floated together and mixed. When mixing equally, excessive culture fluid was removed up to appropriate amount. Thereafter the mixture cultured in the culture medium for 3-10 hours and adhering the preadipocytes to the allogenic fascia powder. The culture fluid was washed with PBS 2-3

times, then mixed with a serum extracted from the human body. The last mixture injected by an injector for human volume replacement.

According to the said examples, serum extracted from human body is available as a mixture fluid before injection, and preadipocyte, chondroblast or osteoblast is available usefully as a subculture object, and allogenic human dermis powder, chitosan powder, PLGA powder, allogenic fascia powder or gore-tex powder is available usefully as a culture material.

Furthermore, for existing three dimensional culture it needs to inject into the inside of sponge by injector in order to infiltrate cells into a lump of sponge including the culture fluid. This method is some demerits that it is hard to adjust uniform distribution of cells as well as loss of cells may exist due to injection pressure and friction at the time of injection. But these demerits can be overcome using powder and osmotic action by pressure.

Cultured cells of the said examples are put into the injector with culture material together and injected into the replacement site of human volume by means of an injector, therefore injection-transplantation is performed.

In case of the said solid powder, there is a problem which relates to washing of the culture fluid in sponge structure, that is, it is necessary to wash slowly with PBS (phosphate buffer saline) harmless to the human body in order not to come off the cells. In sponge structure it is essential to pass through the washing process 7 times or more, and if the frequency of washing is increased no matter how slowly it may be washed there is a loss of preadipocytes manipulating and adhering.

However in case of solid powder, there is little chance that the culture fluid remains behind in solid powder itself. Even though washing only 2 times with phosphate buffer saline, it is possible to lower the culture fluid content up to secure level before injecting to the human body.

Therefore, when injecting solid powder having no bubble structure to the human body, there are some advantages that danger of immune reaction by

hetero protein is little, the washing process can be simplified, simultaneously possibility cells are swept away is little. For these reasons decline in cell culture efficiency doesn't matter since there isn't such a big difference in comparison with the case of using sponge structural powder.