KR20100123995A - Composition for transplant comprising adipose tissue and stromal vascular fraction - Google Patents

Composition for transplant comprising adipose tissue and stromal vascular fraction Download PDF

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KR20100123995A
KR20100123995A KR1020090043012A KR20090043012A KR20100123995A KR 20100123995 A KR20100123995 A KR 20100123995A KR 1020090043012 A KR1020090043012 A KR 1020090043012A KR 20090043012 A KR20090043012 A KR 20090043012A KR 20100123995 A KR20100123995 A KR 20100123995A
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adipose tissue
vascular fraction
cryopreservation
composition
interstitial
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KR1020090043012A
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Korean (ko)
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방상일
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방상일
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Abstract

The present invention relates to a graft composition containing adipose tissue and interstitial vascular fraction, specifically to a graft composition containing adipose tissue and interstitial vascular fraction, or cryopreserved adipose tissue and cryopreserved treated interstitial vascular fraction It is about.

Description

Composition for transplant comprising adipose tissue and stromal vascular fraction

The present invention relates to a graft composition containing adipose tissue and interstitial vascular fraction, specifically to a graft composition containing adipose tissue and interstitial vascular fraction, or cryopreserved adipose tissue and cryopreserved treated interstitial vascular fraction It is about. The implantable composition of the present invention is prepared by mixing the adipose tissue and the interstitial vascular fraction extracted therefrom, thereby inducing capillaries into the injected composition through proper interaction with surrounding tissues, and supplying them through the induced capillaries. The nutrients that cause the proliferation and differentiation of the cells, the implantable composition of the present invention is able to maintain a constant volume and regeneration even with a single injection. In addition, the cryopreservation of the adipose tissue and interstitial vascular fraction is more effective, and in particular, the cryopreservation of the adipose tissue and interstitial vascular fraction is excellent in commercial use value.

In general, adipose tissue is known to be divided into a stromal vascular fraction (SVF) composed of many microvascular endothelial cells, endothelial cells, fibroblasts, muscle cells, and fat progenitor cells in addition to fat cells (B. Cousin et. al., BBRC, 301: 1016, 2003; A. Miranville et al., Circulation, 110: 349, 2004; S. Gronthos et al., J. Cell Physiol., 189: 54, 2001; MJ Seo et al. , BBRC, 328: 258, 2005).

The fat tissue plays an important role in maintaining the body shape along with the buffering function of the vital organs of the human body, and since the tissue is abundantly present anywhere in the human body, most tissues can be easily collected without modifying the contour of the donor, and thus fat has been produced for decades. Tissue transplantation has been performed clinically (Chajchir A, Benzaquen I. Fat-grafting injection for soft-tissue augumentation.Plast Reconstr Surg, 84: 921-934, 1989; Ersek RA.Transplatation of purified autogenous fat: a 3-years follow -up is disappointing.Plast Reconstr Surg, 87: 21-227, 1991).

The adipose graft is a procedure for re-injecting adipose tissue extracted from one part of the body to another part of the body (Coleman, 1995) to see the effects of cosmetic surgery, such as removing wrinkles on various body surfaces and melting of specific body parts. (Bircoll et al., 1987), there is no ethical problem, it is possible to extract large quantities, it can be collected under local anesthesia, it can cause minimal pain for the patient, accumulated accumulated know-how and Can be safely performed (Patricia et al., 2001). In addition, adipose tissue has the advantage of being relatively easy to obtain in comparison with other tissues that may be a source of adult stem cells such as bone marrow, skin, muscle, liver and brain (Commons etal., 2001).

In relation to the adipose tissue transplantation, initially, the soft tissue defects were filled by the adipose tissue itself, but most of the transplanted tissues were necrotic and complications were frequent. After that, the small adipose tissue was divided into pieces and transplanted into the surrounding blood flow. (Catrick CW Jr, Chauvin PB, Robb GL. Tissue-engineered adipose tissue. 369-382, Houston, Elsevier Science, Ltd, 1998). However, as described above, when only the adipose tissue is implanted, the tissue is injected into the body in a lump, and thus, new capillaries are not formed well in the dense adipose tissue injected into the body, resulting in a decrease in engraftment rate of the transplanted adipose tissue. It was. As a result, the induction of proliferation and differentiation of cells existing in the newly injected adipose tissue is reduced, resulting in a decrease in the volume of the injected composition and a problem of injecting the adipose tissue again to fill the reduced volume.

Since then, various compositions for transplantation using adipose tissue have begun to be developed. Today, adipose stem cells, fibroblasts, and adipose cells are separated from adipose tissue and used in combination or mixed with biomaterials. Most of the way. However, when the cells are simply mixed and used, there is a problem in that sufficient volume cannot be filled with only the cells to be filled with the volume. In addition, when the cells are mixed with other biomaterials, a considerable amount of biomaterials must be used, resulting in economic problems. Above all, there was a problem that it takes a considerable time and cost to separate the cells.

In addition, since the techniques related to the conventional fat tissue transplantation are mostly mixed with fresh adipose tissue inhaled on the same day, fat stem cells, fibroblasts, fat cells, etc. separated from the same, or with biomaterials, There was a problem that can not be used for a long time. Therefore, in the case of transplantation with the above techniques, since the adipose tissue must be separated at each implantation procedure, it caused considerable inconvenience to the patient.

Accordingly, the present inventors studied the effective and convenient transplant composition using adipose tissue, and when the implantable composition in which the adipose tissue and the interstitial vascular fraction were properly mixed was implanted, the volume of the transplanted site was maintained for a long time and the engraftment rate was increased. The present invention was completed by confirming high. In addition, in the case of an implantable composition containing cryopreserved adipose tissue and cryopreserved treated interstitial vascular fraction, the present invention was completed by confirming that the volume of the transplanted site is maintained as it is and the engraftment rate is high.

Accordingly, an object of the present invention is to provide an implantable composition containing adipose tissue and stromal vascular fraction.

It is another object of the present invention to provide a composition for transplantation containing cryopreserved adipose tissue and cryopreserved treated stromal vascular fraction.

In addition, the present invention (a) washing the isolated adipose tissue; (b) administering the washed adipose tissue to a cryopreservation solution containing 1-20 wt% dimethyl sulfoxide (DMSO) and 80-99 wt% human serum albumin or fetal bovine serum for 2-20 minutes. It is an object of the present invention to provide a cryopreservation method of adipose tissue for transplantation, comprising the step and (c) cryopreservation of the adipose tissue administered to the cryopreservation solution at -70 to -196 ° C.

In another aspect, the present invention (a) washing the extracted stromal vascular fraction (stromal vascular fraction); (b) administering the washed interstitial vascular fraction to a cryopreservation solution containing 1-20% by weight of dimethyl sulfoxide (DMSO) and 80-99% by weight of human serum albumin or fetal bovine serum; And (c) storing the interstitial vascular fraction administered to the cryopreservative at a rate of 1-5 ° C./min and storing it at −56˜80 ° C. for 24 to 168 hours, and (d) the stored epilepsy. An object of the present invention is to provide a cryopreservation method of an interstitial vascular fraction for transplantation, which comprises cryopreserving a blood vessel fraction at -169 to -196 ° C.

The present invention also comprises the steps of: (a) thawing the cryopreserved transplanted adipose tissue; (b) thawing the cryopreserved graft interstitial vascular fraction; and (c) mixing the adipose tissue thawed in (a) and the stromal vascular fraction thawed in (b). An object of the present invention is to provide a manufacturing method.

In order to solve the above problems, the present invention provides a composition for transplantation containing adipose tissue and stromal vascular fraction.

The present invention also provides a composition for transplantation containing cryopreserved adipose tissue and cryopreserved treated stromal vascular fraction.

In addition, the present invention (a) washing the isolated adipose tissue; (b) administering the washed adipose tissue to a cryopreservation solution containing 1-20 wt% dimethyl sulfoxide (DMSO) and 80-99 wt% human serum albumin or fetal bovine serum for 2-20 minutes. It provides a cryopreservation method of adipose tissue for transplantation comprising the step and (c) cryopreservation of the adipose tissue administered to the cryopreservation solution at -70 ~ -196 ℃.

In addition, the present invention (a) washing the extracted stromal vascular fraction (stromal vascular fraction); (b) administering the washed interstitial vascular fraction to a cryopreservation solution containing 1-20% by weight of dimethyl sulfoxide (DMSO) and 80-99% by weight of human serum albumin or fetal bovine serum; And (c) storing the interstitial vascular fraction administered to the cryopreservative at a rate of 1-5 ° C./min and storing it at −56˜80 ° C. for 24 to 168 hours, and (d) the stored epilepsy. Provided is a cryopreservation method of an interstitial vascular fraction for transplantation comprising cryopreservation of a blood vessel fraction at -169 to -196 ° C.

The present invention also comprises the steps of: (a) thawing the cryopreserved transplanted adipose tissue; (b) thawing the cryopreserved graft interstitial vascular fraction; and (c) mixing the adipose tissue thawed in (a) and the stromal vascular fraction thawed in (b). It provides a method of manufacturing.

Hereinafter, the present invention will be described in more detail.

In the present invention, "fat tissue" is one of the connective tissue is a connective tissue consisting of fat cells and fibers. Adipose cells are usually ball-shaped, larger than normal cells by 50 ~ 100um. Each adipocyte is wrapped in fiber, and capillaries are distributed densely between cells. These adipose tissues can be found in the subcutaneous, retina, and mesentery, especially in the kidneys, heart surface, around the joints, and in the long bones.

In the present invention, "stromal vascular fraction" refers to a fraction composed of microvascular endothelial cells, endothelial cells, fibroblasts, muscle cells, and fat progenitor cells excluding fat cells from adipose tissue.

The adipose tissue may be separated in vitro according to methods known in the art, but is not limited thereto, and may be separated from the subcutaneous, retinal, mesenteric, particularly kidney, heart surface, periarticular, and iliac bone. . Preferably, the adipose tissue can be obtained from the subcutaneous adipose tissue by a liposuction method (see <Production Example 1-1>).

In addition, the interstitial vascular fraction can be prepared by removing fat cells from the isolated adipose tissue according to methods known in the art. Preferably, the obtained adipose tissue can be obtained by washing the blood and impurities, followed by treatment with collagenase (see <Preparation Example 1-2>). (Young Joon Jun: Recent Development Trend and Prospects of Adipose-derived Stem Cells on Nerve Regeneration, Tissue Engineering and Regenerative Medicine , Vol. 5, No. 1, pp 51-56, 2008)

Implantable composition of the present invention is characterized by using a mixture of adipose tissue and interstitial vascular fraction obtained as described above.

In the case of using only the adipose tissue, the tissue is injected into the body in the form of agglomerates, so that new capillary blood formation is not well formed into the adipose tissue injected into the body, so that the proliferation and differentiation of cells existing in the adipose tissue are reduced. The volume of is reduced (see <Example 1>).

In addition, when using only the interstitial vascular fraction, there was a problem in that it is not suitable for soft tissue regeneration that requires a sense of volume because it cannot fill a sufficient volume with cells only at the volume to be filled (see <Example 1>).

In the case of the graft composition in which the adipose tissue and the interstitial vascular fraction of the present invention are mixed, it is understood that the volume of the transplanted site is maintained well over time compared to the case of using only the adipose tissue and only the interstitial vascular fraction. Could. This is because the implantable composition of the present invention disperses the cells by separating the cells densely packed into adipose tissue into some interstitial vascular fraction and artificially mixing them. In addition, because the cells dispersed as described above to induce capillaries into the injected composition through proper interaction with the surrounding tissue, nutrients supplied through the induced capillaries cause the proliferation and differentiation of the cells. Therefore, the implantable composition of the present invention allows continuous volume maintenance and regeneration even with a single injection (see <Example 1>).

In particular, by using the interstitial vascular fraction as it is without using cells extracted from adipose tissue, it is possible to maintain engraftment and efficacy maintenance after infusion by the interaction of various cells in the stromal vascular fraction.

In addition, the implantable composition of the present invention is not limited to this, but preferably, the interstitial vascular fraction may be contained by mixing 1 × 10 5 to 1 × 10 7 cells per cc of adipose tissue. Most preferably, the interstitial vascular fraction may be contained in a mixture of 1 × 10 6 cells per cc of adipose tissue. This is because the volume of the implanted site can be maintained as it is by inducing an appropriate interaction between the adipose tissue and the interstitial vascular fraction (see <Example 1>).

Meanwhile, the implantable composition of the present invention is characterized by containing cryopreserved adipose tissue and cryopreserved treated interstitial vascular fraction.

The cryopreservation liquid used for cryopreservation may include, but is not limited to, 1-20% by weight of dimethyl sulfoxide (DMSO) and 80-99% by weight of human serum albumin or fetal bovine serum. . Most preferably the cryopreservation solution may comprise 10% by weight of dimethylsulfoxide and 90% by weight of human serum albumin. In the case of DMSO, the cryopreservation effect is hardly exerted below the numerical range, and the cell may be killed by the toxicity of DMSO above the numerical range. In addition, the use of the human serum albumin is because when using a culture solution containing a water-soluble component may contain a large amount of water-soluble components, which may adversely affect the freezing of tissues or cells. However, if the cryopreservation liquid having the composition as described above, it may further include one or more nutrients selected from the group consisting of autologous serum and bovine serum albumin.

In one embodiment of the present invention, in order to determine the composition of the cryopreservation solution, cryopreservation solutions having various compositions were prepared and administered to the interstitial vascular fraction, and then stained with a standard cell count solution (trypan blue) to test cell viability, Cryopreservation solution containing 10% DMSO (dimethyl sulfoxide) and 90% human serum albumin or fetal bovine serum was found to be the most effective (see <Example 2-1>).

In particular, after thawing the interstitial vascular fraction 180 days after cryopreservation as described above, the cell viability contained in the stromal vascular fraction was compared with the cell viability of the stromal vascular fraction directly isolated from adipose tissue (<Example 2-2>).

In addition, when thawing cryopreserved adipose tissue and cryopreserved interstitial vascular fractions to be mixed and transplanted, the volume of the transplantation site can be better maintained than when cryopreservation is not carried out. In particular, cryopreserved adipose tissue and cryopreserved interstitial vascular fractions were thawed and mixed, and then maintained well without decreasing in volume even after about 3 months of transplantation (see <Example 2-4>). In addition, in the case of containing cryopreserved adipose tissue and cryopreserved interstitial vascular fractions, cell proliferation and differentiation are better induced in the body than in cryopreservation (see <Example 2-5>).

As described above, cryopreservation of adipose tissue and interstitial vascular fractions is then thawed and mixed, and even after transplantation, the biological activity is retained even when transplanted. It is well maintained as it is, and induces the proliferation and differentiation of cells in the body.

In addition, the graft composition of the present invention is not limited thereto, and preferably, the interstitial vascular fraction treated with cryopreservation may be mixed into 1 × 10 5 to 1 × 10 7 cells per cc of cryopreserved adipose tissue. Most preferably, the cryopreserved interstitial vascular fraction may be mixed and contained in 1 × 10 6 cells per cc of cryopreserved adipose tissue. This is because the volume of the transplanted site can be maintained by inducing proper interaction of cryopreserved adipose tissue with cryopreserved interstitial vascular fraction.

Meanwhile, the implantable composition of the present invention may additionally include biomaterials and nutrients commonly used in the implantable composition.

The biomaterial is not limited thereto, but preferably, fibrin gel, hyaluronic acid, collagen, chitosan, alginic acid, gelatin, and dextran It may be one or more selected from the group consisting of. In particular, the biomaterial is not limited thereto, and may preferably include 1 to 25% by weight based on the total weight of the graft composition.

In addition, the nutrient may be one or more selected from the group consisting of autoplatelets, essential fatty acids, human serum albumin, bovine serum albumin, and autologous serum, but not limited thereto. The essential fatty acid may be, but is not limited to, at least one selected from the group consisting of linoleic acid, linoleinic acid, and arachidonic acid.

Meanwhile, the cryopreservation method of the adipose tissue for transplantation of the present invention includes the steps of: (a) washing the isolated adipose tissue; (b) administering the washed adipose tissue to a cryopreservation solution containing 1-20 wt% dimethyl sulfoxide (DMSO) and 80-99 wt% human serum albumin or fetal bovine serum for 2-20 minutes. And (c) cryopreservation of the adipose tissue for transplantation, which comprises cryopreserving the adipose tissue administered to the cryopreservation solution at -70 to -196 ° C.

In the step (a), the separated adipose tissue may be washed several times with a solution containing physiological saline, but not limited thereto, and effectively removes impurities such as blood remaining in the separated adipose tissue through the washing process. In vivo transplantation can increase engraftment rate.

In the step (b), the washed adipose tissue 2 to 20 in cryopreservation solution containing 1 to 20% by weight of dimethyl sulfoxide (DMSO) and 80 to 99% by weight of human serum albumin or fetal bovine serum It can be administered for a minute. In the case of administration to the cryopreservation solution, the volume of the transplant site can be maintained in the future. The reason for administration to the cryopreservation solution during this time is that administration over time is meaningless and uneconomical, and administration below that time is not effective. Most preferably, the cryopreservation liquid may be administered for 2 minutes.

In the step (c), the adipose tissue can be stored for a long time by freezing the adipose tissue administered to the cryopreservation solution at -70 ~ -196 ℃. When the temperature range is exceeded, crystallization of the cells is caused, and when the temperature is lower than the temperature range, long-term storage is difficult, so the temperature range is preferable. Most preferably it can be cryopreserved in -196 ° C liquid nitrogen.

Meanwhile, the cryopreservation method of the transplanted interstitial vascular fraction of the present invention comprises the steps of: (a) washing the extracted stromal vascular fraction; (b) administering the washed interstitial vascular fraction to a cryopreservation solution containing 1-20% by weight of dimethyl sulfoxide (DMSO) and 80-99% by weight of human serum albumin or fetal bovine serum; And (c) storing the interstitial vascular fraction administered to the cryopreservative at a rate of 1-5 ° C./min and storing it at -56 ~ -80 ° C. for 24 to 168 hours, and (d) The interstitial vascular fraction may comprise the step of cryopreservation at -169 ~ -196 ℃.

In the step (a), the separated interstitial vascular fraction may be washed several times with a solution containing physiological saline, but not limited thereto, and effectively removes impurities such as blood remaining in the separated interstitial vascular fraction through the washing process. In the future transplantation can increase the engraftment rate.

In the step (b), the washed interstitial vascular fraction is 2 ~ in cryopreservation solution containing 1 to 20% by weight of dimethyl sulfoxide (DMSO) and 80 to 99% by weight of human serum albumin or fetal calf serum. Administration for 20 minutes may prevent crystallization of the cells following rapid freezing in step (d). In addition, when the cryopreservation solution is administered, the volume of the transplant site may be maintained in the future. The reason why the cryopreservation solution is administered during this time is that administration over time is meaningless and uneconomical, and administration below that time is not effective. Most preferably, the interstitial vascular fraction may be administered to the cryopreservation solution for 2 minutes.

In the step (c), the interstitial vascular fraction administered to the cryopreservation solution is lowered at a rate of 1 ~ 5 ℃ / min and stored for 24 to 168 hours at -56 ~ -80 ℃ to prevent the crystallization of the interstitial vascular fraction The biological activity can be maintained as it is. In one embodiment of the present invention using an isopropanol cooling box to lower the temperature at a rate of 1 ~ 2 ℃ / min and stored at -70 ℃ for 1 day. In particular, in the case of adipose tissue, the tissue itself acts as a matrix, but rapid freezing is possible. However, in the case of interstitial vascular fraction consisting of various cells, rapid freezing causes crystallization of cells. desirable.

In the step (d), the interstitial vascular fraction administered to the cryopreservation liquid can be frozen for a long time by freezing at -169 ~ -196 ℃. When the temperature range is exceeded, crystallization of the cells is caused, and when the temperature is lower than the temperature range, long-term storage is difficult, so the temperature range is preferable. Most preferably it can be cryopreserved in -196 ° C liquid nitrogen.

Implantable compositions of the present invention can be prepared using cryopreserved adipose tissue and cryopreserved interstitial vascular fraction as described above. Specifically, the method for producing a graft composition of the present invention comprises the steps of: (a) thawing cryopreserved adipose tissue cryopreserved by the above method; (b) thawing the interstitial vascular fraction for cryopreservation in said method; and (c) mixing the adipose tissue thawed in (a) and the stromal vascular fraction thawed in (b). have.

The thawing of (a) and (b) may use a thawing method known to those skilled in the art, and in (c), but not limited thereto, preferably, cryopreserved adipose tissue and cryopreserved interstitial vascular fractions. It can be mixed into 1x10 5 ~ 1x10 7 cells per 1cc of adipose tissue. Most preferably, the cryopreserved interstitial vascular fraction may be mixed at 1 × 10 6 cells per cc of cryopreserved fatty tissue.

Implantable compositions of the present invention can be useful in a variety of fields, such as skin care, molding and treatment of lost soft tissues, urinary enlargement, specifically for breast augmentation or wrinkle removal through the formation of breast tissue, facial wrinkles of the face and It can be applied to various fields such as removing wrinkles and maintaining elasticity and expanding urinary tract.

The implantable composition of the present invention is prepared by mixing the adipose tissue and the interstitial vascular fraction extracted therefrom, thereby inducing capillaries into the injected composition through proper interaction with surrounding tissues, and supplying them through the induced capillaries. The nutrients that cause the proliferation and differentiation of the cells, the implantable composition of the present invention is able to maintain a constant volume and regeneration even with a single injection. In addition, the cryopreservation of the adipose tissue and interstitial vascular fraction is more effective, and in particular, the cryopreservation of the adipose tissue and interstitial vascular fraction is excellent in commercial use value.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Production Example  1>

Preparation of Adipose Tissue and Interstitial Vascular Fractions

<1-1> Production of Adipose Tissue

The adipose tissue inhaled on the day was washed three times with physiological saline containing 1% human serum albumin, and then centrifuged at 1,200 rpm for 5 minutes. In each centrifugation, impurities including blood of the lower layer were removed, and free-oil layer of the upper layer was removed to obtain adipose tissue.

<1-2> Preparation of Interstitial Vascular Fraction

After transporting the adipose tissue obtained in <Example 1-1> in the refrigerated state of -4 ℃, washed three times with physiological saline containing 1% human serum albumin containing physiological saline. After washing with water, the adipose tissue was centrifuged at 1200 rpm for 5 minutes to remove blood and impurities from the lower layer. After the removal was treated with 0.03% collagenase (collagenase) and stirred for 15 minutes at 37 ℃. After the stirring, DMEM / F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, 1: 1, v / v, invitrogen) medium containing 10% FBS was added to stop the enzyme reaction. After the addition, the supernatant was removed after centrifugation at 1200 rpm for 5 minutes, and washed with the addition of 1% DMEM / F-12 (1: 1, v / v) medium containing human serum albumin. After centrifugation at 1200rpm for 5 minutes, impurities and debris were removed by a filter on a 100um mesh, and again, only pellets were taken after centrifugation at 1,200rpm for 5 minutes to prepare an interstitial blood vessel fraction.

< Example  1>

Effect of Adipose Tissue and Interstitial Vascular Fraction

The group in which the adipose tissue of <Production Example 1-1> was transplanted was used as a control group 1, and the group in which the interstitial vascular fraction of <Production Example 1-2> was transplanted was a control group 2, and the adipose tissue and the interstitial blood vessel fraction were adipose tissue. 1 x 10 3 cells per 1cc of tissue was transplanted into experimental group 1, adipose tissue and interstitial vascular fractions were mixed into 1x10 5 cells per 1cc of adipose tissue and transplanted into experimental group 2, and adipose tissue and interstitial blood vessels were divided into fat Experimental group 3 was used as a mixed group of 1x10 6 cells per 1cc of tissue and experimental group 3 was mixed with 1x10 7 cells per 1cc of adipose tissue.

More specifically, BALB c / Nu 8 weeks old, 5 female mice were used in each group, one month after injection to each control and experimental group nude mouse scapula to have a total volume of 1cc to obtain a sample at the expense of nude mice The mass size of the sample was measured. The results are shown in FIG.

As shown in FIG. 1, the control group 1 was found to have significantly reduced volume than the initial injection amount, and the control group 2 could not find the composition itself injected into the mouse. However, in the experimental group injected with a mixture of adipose tissue and interstitial vascular fraction, it was found that the volume of the transplanted sample was well maintained as compared with the control groups 1 and 2. In particular, there was a difference in the transplanted volume according to the mixing ratio of the adipose tissue and the interstitial vascular fraction, and the most suitable ratio was 1x10 5 to 1x10 7 cells per 1cc of adipose tissue to maintain the transplant volume. It was found to be effective. Adipose and interstitial vascular fractions were found to be the most desirable when mixed with 1x10 6 cells per 1cc of adipose tissue.

< Example  2>

Effect of Cryopreservation of Adipose Tissue and Interstitial Vascular Fraction

<2-1> Determination of Cryopreservative Composition

As shown in Table 1 below, cryopreservation liquids were prepared with various compositions, and the interstitial vascular fractions prepared in <Preparation Example 1> were cryopreserved. The cell viability of the interstitial vascular fraction was examined to determine the optimal cryopreservative composition.

Composition of cryopreservation solution (unit: wt%) division Dimethyl sulfoxide (DMSO) Fetal bovine serum Human serum albumin DMEM / F-12 culture Experimental group 1 10 10 80 Experiment group 2 10 10 80 Experiment group 3 10 90 Experimental Group 4 10 90

Specifically, the interstitial vascular fractions prepared in <Preparation Example 1> were added to the cryopreservation solution, respectively, and placed in an isopropanol cooling box for 1 day at -70 ° C, and then transferred to -196 ° C liquid nitrogen. After storage, 0, 1, 3, 6, 9, 12 months after thawing the interstitial vascular fraction and stained with a standard cell count solution (trypan blue) known in the art to check the cell viability and it is shown in Figure 2 Indicated.

As shown in FIG. 2, it was found that dimethyl sulfoxide (DMSO) and human serum albumin or fetal bovine serum were mixed at a weight ratio of 1: 9.

<2-2> Effect of Cryopreservation of Interstitial Vascular Fraction

Morphological observations of the cells contained in the interstitial vascular fractions cryopreserved with the cryopreservation solution of Experimental Group 4 of <Example 2-1> were performed.

Specifically, after thawing the interstitial vascular fraction 180 days after cryopreservation, cells of 4 days of culture and 7 days of cultivation were not cryopreserved cells or cells of 7 days of culture. The results were compared with the morphology of cells after 13 days, and the results are shown in FIGS. 3 (freezing) and 4 (freezing). The interstitial vascular fraction was cultured by adding the interstitial vascular fraction to DMEM / F-12 (1: 1. V / v) medium in a 5% CO 2 incubator at 37 ° C.

As described in FIGS. 3 and 4, the cell viability (93%) after thawing cryopreserved interstitial vascular fraction 180 days was similar to the cell viability of the interstitial vascular fraction immediately separated from adipose tissue (95%). In addition, the interstitial vascular fraction of 180 days after cryopreservation retained the fibroblastic-morphology, a morphological characteristic of typical mesenchymal stem cells, even after 7 days of culture.

<2-3> Preparation of Implant Composition Containing Cryopreserved Adipose Tissue and Interstitial Vascular Fraction

The adipose tissue obtained in <Preparation Example 1> was placed in the cryopreservation solution of Experimental Group 4 of <Example 2-1> for 2 minutes and then cryopreserved in -196 ° C liquid nitrogen.

In addition, the interstitial vascular fraction obtained in <Production Example 1> was put in the cryopreservation solution of Experimental Group 4 of <Example 2-1>, and put it in an isopropanol cooling device (1 ° C / min temperature drop). After storage at 1 ° C for 1 day, it was transferred to -196 ° C liquid nitrogen and cryopreserved. The cryopreserved adipose tissue and interstitial vascular fractions were thawed and washed twice with a wash solution containing 1% human serum albumin to remove the cryopreservation solution. Thereafter, the thawed adipose tissue and the interstitial vascular fraction were mixed with 1 X 10 5 cells per 1cc of adipose tissue (Experimental Group 1), or the adipose tissue and the interstitial vascular fraction were mixed with 1 X 10 6 cells per 1cc of Adipose tissue (Experimental Group 2). The implantable composition of the present invention was prepared by further mixing the tissue-compatible biomaterial fibrin gel with other linoleic acid and autologous platelets at 1: 0.5: 0.5 (v / v / v).

On the other hand, in order to examine the change in volume according to the cryopreservation, as a control group, the adipose tissue prepared in <Production Example 1> was mixed with the adipose stem cells (Control group 1), the adipose tissue and epilepsy prepared in <Production Example 1> A group in which blood vessel fractions were mixed (control 2) was used. The control group was the same as the implantable composition of the present invention, except for the condition that was not cryopreserved.

<2-4> Transplantation Volume Changes by Cryopreservation of Adipose and Interstitial Vascular Fractions

BALB c / Nu 8-week-old, female mice were used in 5 mice in each group. Specifically, the injection composition prepared in Example 2-3 was injected into the nude mouse scapula of each control group 1 and 2 and the experimental group so that the total volume was 1 ml. After the transplantation, the samples were obtained by sacrificing nude mice every day after sampling, taking into account the mass size of the implanted samples every 0, 4, 8, 12, 22, 30, 60, 90, and 180 days. The measurement results are shown in FIG. 5.

As shown in FIG. 5, the controls 1 and 2 showed similar volume changes as time passed, but when about 22 days had elapsed, the volume rapidly decreased. Therefore, in the case of a transplant composition corresponding to the control group, it was found that after about 22 days, the composition should be transplanted again. On the other hand, the implantable composition of the present invention was confirmed that the volume is maintained without shrinking even after about three months.

In addition, the shape and size of the sample when 30 days have passed since the transplant composition of the present invention is described in FIG.

As shown in FIG. 6, when the composition for transplantation of the present invention was used, it was found that volume and shape were well maintained as compared with the control group 1.

Therefore, it was found that the graft composition containing the cryopreserved adipose tissue and interstitial vascular fraction of the present invention can be usefully used in various fields such as skin care, plastic surgery, treatment of lost soft tissues, urinary enlargement, and the like.

<2-5> Changes of Adipocyte Differentiation in Transplanted Sites by Cryopreservation of Adipose Tissue and Interstitial Vascular Fractions

Samples were taken when 30 days had passed since the transplantation composition of the present invention was transplanted, and the change of adipocyte differentiation was observed in comparison with the control 1 of <Example 2-3>. For the observation, oil-red O tissue staining was performed or the expression level of PPAR-γ gene, which is an indicator of adipocyte differentiation, was measured by PCR.

First, oil-red O tissue staining was performed as follows.

(a) After fixing the adipose tissue sample to the calcium (formal calcium) solution for 15 seconds, reacted with the sucrose (gum-sucrose) solution for 20 minutes to relieve fat.

(b) Make frozen sections with a thickness of about 10um. (** Formal calcium is used to fix the sections so that the sections fall well.

(c) React with 100% propylene glycol solution for 5 minutes.

(d) Into the Oil red O solution, and reacted for 60 minutes at 7 ℃ (R.T 15 minutes).

(e) 3 minutes in 85% propylene glycol, deionized water 20 times.

(f) React in dilute harri's hematoxylin solution for 2 minutes.

(g) Tap. After 2 minutes of washing with water, it is washed again with deionized water for 2 minutes.

(h) After encapsulation with glycerin jelly, a water-soluble encapsulant, observed under a microscope.

The oil-red O tissue staining result through the above process is shown in FIG. 7.

As shown in FIG. 7, it can be seen that the proliferation and differentiation of cells are well induced in the implantable composition of the present invention as compared to the control group.

In addition, the degree of PPAR-γ gene expression was performed according to a known method. Specifically, the RNA extracted from each sample was used as a template to measure the expression level of PPAR-γ gene using an intron RT-PCR kit (AJAY CHAWLA, EMILY J. SCHWARZ et al: Departments of Medicine and Genetics, University of Pennsylvania School of Peroxisome Proliferator-Activated Receptor (WAR) y: Adipose-Predominant Expression And Induction Early In Adipocyte Differentiation, Endocrinology, Vol. 135, No. 2, 798-800, 2009).

As shown in FIG. 8, since the expression of the peroxisome proliferator activated receptor-γ gene is well induced in the graft composition of the present invention as compared to the control group, it can be seen that the proliferation and differentiation of cells occurs in the body. there was.

1 is a photograph showing the effect of mixing and mixing ratio of adipose tissue and interstitial vascular fraction.

Figure 2 is the result of measuring the cell viability of the interstitial vascular fraction according to the various cryopreservation composition.

Figure 3 is the result of measuring the cell viability of cryopreserved interstitial vascular fraction.

Figure 4 is the result of measuring the cell viability of the interstitial vascular fraction not cryopreserved.

Figure 5 is the result of measuring the volume change of the transplanted site according to cryopreservation of adipose tissue and interstitial vascular fraction.

Figure 6 is a result of measuring the volume change of the implanted site according to cryopreservation of adipose tissue and interstitial vascular fraction.

Figure 7 is a result of confirming the fat cell differentiation change of the transplanted site according to cryopreservation of adipose tissue and stromal vascular fraction by oil-red O tissue staining.

8 is a result of confirming the change in the differentiation of fat cells of the transplanted site according to cryopreservation of adipose tissue and interstitial vascular fraction by measuring the expression level of PPAR-γ gene.

Claims (16)

Implantable composition comprising adipose tissue and stromal vascular fraction. The implantable composition of claim 1, wherein the interstitial vascular fraction contains 1x10 5 to 1x10 7 cells per cc of adipose tissue. The method of claim 1, wherein the fibrin gel, hyaluronic acid, collagen, collagen, chitosan, alginic acid, gelatin, and dextran are selected from the group consisting of: Implantable composition further comprising at least one biomaterial. The implantable composition of claim 3, wherein the biomaterial comprises 1 to 25 wt% based on the total weight of the implantable composition. The implantable composition of claim 1, further comprising one or more nutrients selected from the group consisting of autologous platelets, essential fatty acids, human serum albumin, bovine serum albumin, and autologous serum. The composition of claim 5, wherein the essential fatty acid is at least one selected from the group consisting of linoleic acid, linoleinic acid, and arachidonic acid. An implantable composition comprising cryopreserved adipose tissue and cryopreserved stromal vascular fraction. The method of claim 7, wherein the graft composition containing a stromal vascular fraction in fat tissue ~1x10 7 1x10 5 cells per 1cc. The method of claim 7, wherein the fibrin gel is selected from the group consisting of fibrin gel, hyaluronic acid, collagen, chitosan, alginic acid, gelatin and dextran. Implantable composition further comprising at least one biomaterial. 10. The implantable composition of claim 9, comprising 1 to 25% by weight of the biomaterial based on the total weight of the implantable composition. 8. The implantable composition of claim 7, further comprising one or more nutrients selected from the group consisting of autoplatelets, essential fatty acids, human serum albumin, bovine serum albumin, and autologous serum. The composition of claim 11, wherein the essential fatty acid is at least one selected from the group consisting of linoleic acid, linoleic acid, and arachidonic acid. The cryopreservation of claim 7 wherein the adipose tissue and interstitial vascular fractions are cryopreserved with cryopreservation solution containing 1-20% by weight of dimethyl sulfoxide (DMSO) and 80-99% by weight of human serum albumin or fetal bovine serum. Implantable composition that is treated. (a) washing the isolated adipose tissue; (b) administering the washed adipose tissue to a cryopreservation solution containing 1-20 wt% dimethyl sulfoxide (DMSO) and 80-99 wt% human serum albumin or fetal bovine serum for 2-20 minutes. Steps and (c) cryopreservation method of adipose tissue for transplantation comprising the step of cryopreservation of the adipose tissue administered to the cryopreservation solution at -70 ~ -196 ℃. (a) washing the extracted stromal vascular fraction; (b) administering the washed interstitial vascular fraction to a cryopreservation solution containing 1-20% by weight of dimethyl sulfoxide (DMSO) and 80-99% by weight of human serum albumin or fetal bovine serum. Steps and (c) lowering the temperature of the interstitial vascular fraction administered to the cryopreservation solution at a rate of 1-5 ° C./min and storing it at −56˜80 ° C. for 24 to 168 hours; and (d) cryopreservation method of interstitial vascular fraction for transplantation comprising cryopreserving the stored interstitial vascular fraction at -169 ~ -196 ℃. (a) thawing cryopreserved adipose tissue for cryopreservation by the method of claim 14; (b) thawing the interstitial vascular fraction for cryopreservation by the method of claim 15; and (C) a method for producing a composition for implantation comprising the step of mixing the thawed tissue in (a) and the interstitial vascular fraction thawed in (b).
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014051173A1 (en) * 2012-09-27 2014-04-03 (주)세포바이오 Composition comprising plant-derived recombinant human serum albumin, lipids, and plant protein hydrolysates as active ingredients for cryopreservation of stem cells or primary cells
KR20160119609A (en) * 2015-04-06 2016-10-14 박준한 Method for obtaining stromal vascular fraction from adipose tissue

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014051173A1 (en) * 2012-09-27 2014-04-03 (주)세포바이오 Composition comprising plant-derived recombinant human serum albumin, lipids, and plant protein hydrolysates as active ingredients for cryopreservation of stem cells or primary cells
US10104881B2 (en) 2012-09-27 2018-10-23 Cefo Co., Ltd. Composition comprising plant-derived recombinant human serum albumin, lipids, and plant protein hydrolysates as active ingredients for cryopreservation of stem cells or primary cells
KR20160119609A (en) * 2015-04-06 2016-10-14 박준한 Method for obtaining stromal vascular fraction from adipose tissue

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