KR100637901B1 - Isolation and culture method of mature adipocyte and preadipocyte - Google Patents

Abstract

A method for isolating and culturing mature adipocyte and preadipocyte is provided to offer high recovery rate of the adipocyte and preadipocyte by treating fat tissue with a solution composition for each step to isolate the mature adipocyte and preadipocyte in maximum-activated state, and then culturing each of the isolated adipocyte. The method comprises the steps of: (a) washing fat tissue obtained from human body at least three times with 1X-10X phosphate-buffered solution(PBS) where 0.01-1% of bovine serum albumine(BSA) is added; (b) after adding 0.1-0.3% of collagenase type-II solution to the washed fat tissue, agitating it at a temperature of 36-38 deg.C for 10-60 minutes to perform enzymatic treatment; (c) adding Dulbecco's minimum essential medium solution(DMEM), where 1-20% of fetal bovine serum(FBS) is added, to the fat tissue of the step(b) to stop the enzymatic reaction; (d) after adding a fat cell culture solution to the fat tissue of the step(c), centrifuging it to separate the fat tissue into mature adipocyte and preadipocyte; and (e) after respectively collecting the separated mature adipocyte and preadipocyte, respectively culturing it.

Description

Isolation and culture method of mature adipocyte and preadipocyte}

1 is a process chart showing the manufacturing process of the isolated culture method of mature fat cells and fat precursor cells according to the present invention,

Figure 2 is a photograph showing the mature fat cells separated and cultured from adipose tissue collected from the orbit of the human body by the method of separating and culturing the mature fat cells and fat precursor cells according to the first embodiment of the present invention,

Figure 3 is a photograph showing the fat precursor cells separated and cultured from the adipose tissue collected from the orbit of the human body by the method of separating and culturing mature fat cells and fat precursor cells according to the first embodiment of the present invention,

4 is a photograph showing mature fat cells separated and cultured from adipose tissue adsorbed from the human body by the method of separating and culturing mature fat cells and fat precursor cells according to a second embodiment of the present invention.

5 is a photograph showing the fat precursor cells separated and cultured from adipose tissue adsorbed from the human body by the method of separating and culturing mature fat cells and fat precursor cells according to a second embodiment of the present invention.

The present invention relates to a method for isolating and culturing mature fat cells and fat precursor cells, and in detail, to separate mature fat cells and fat precursor cells in the most activated state by using a solution composition for each step in treating adipose tissue. The present invention relates to a method for isolating and culturing mature fat cells and fat progenitor cells to have a high recovery rate by allowing each of the separated fat cells to be cultured.

According to Chajchir A et al., Adipose tissue plays an important role in maintaining the body shape as well as buffering vital organs. Since most tissues are abundant anywhere in the body, tissues can be easily collected without altering the donor contour. Adipose tissue transplantation has been practiced for several decades. See: 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]

In addition, according to Patrick CW, in the early stages, the adipose tissue was lumped to fill the soft tissue defects, but most of the grafts were necrotic and the complications were frequent. [Reference: Patrick CW Jr, Chauvin PB, Robb GL. Tissue-engineered adipose tissue. 369-382, Houston, Elsevier Science, Ltd, 1998]

Nevertheless, there is a problem that the volume is reduced by more than 40 ~ 60% within one year after transplantation, and the procedure must be repeated several times. This phenomenon is caused by the ischemic state due to insufficient blood flow of transplanted adipose tissue and physical damage during tissue collection. This is caused by cell necrosis. Necrotic adipocytes are removed by the phagocytic cells of the surrounding tissue and fibrosis, which is significantly reduced than the volume initially injected.

In addition, many synthetic and natural composite materials are already commercialized and used as soft tissue reinforcement and filling materials as heterogeneous inserts, and they are mainly used as fillers for subcutaneous tissue defects and depression as a substitute for fatty tissue. While amounts may be useful, they also serve to simply fill in the deficiency rather than being functional reinforcing materials. These substances are not autologous and can cause foreign body reactions, allergies and infections. As a substitute for actually missing tissue, it is most ideal to fill it with the same tissue and have the same functionality.

Mature adipocytes are easy to isolate but do not induce proliferation cultures. Preadipocytes can be easily isolated from adipose tissue by enzymatic treatment to induce proliferation and differentiation.

However, this is also limited to animals only, and in the case of human adipose tissue, it is not yet easy to separate the fat precursor cells and the mature fat cells.

According to Melvin A, there are many reports on the methodology for separating progenitor cells from adipose tissue, but there are limitations in replacing all defective areas using only progenitor cells. [Reference: Melvin A, Shiffman, MD and Sid Mirrafati, MD. Fat transfer techniques: the effect of harvest and transfer methods on adipocyte viability and review of the literature. The American Society for Dermatology Surgery, 27: 819-826, 2001]

The present invention is to solve the above problems, by using the solution composition for each step in the treatment of adipose tissue to separate mature fat cells and fat precursor cells in the most active state, and to separate each fat cells It is an object of the present invention to provide a method for isolating and culturing mature fat cells and fat progenitor cells to have high recovery rate by culturing them.

Features of the present invention for achieving the above object,

Washing adipose tissue by adding 1X ~ 10X Phosphate-Buffered Saline (PBS) solution containing 0.01-1% BSA (Bovine Serum Albumin), which is 5-15 times the weight of adipose tissue, to adipose tissue obtained from the human body Washing step (S100); An enzyme treatment step (S110) of adding 0.1-0.3% collagenase type II solution, which is an enzyme treatment solution, to the washed adipose tissue at a constant volume ratio and then stirring the mixture at a predetermined temperature for a predetermined time; In order to stop the enzyme reaction, the enzyme reaction stopping step of stopping the reaction by adding DMEM (Dulbecco`s Minimum Essential Medium) solution containing 1-20% of FBS (Fetal Bovine Serum), which is an enzyme stopping solution S120); A centrifugation step (S130) of adding adipose cell culture solution to centrifugation at a constant rate for a predetermined time in order to separate the enzyme-activated fat tissue into mature fat cells and fat precursor cells (S130); And a culture step (S140) of collecting and culturing the separated mature fat cells and the fat precursor cells, respectively.

Here, the washing step is repeated at least three times to wash the adipose tissue.

Here also, the water washing solution of 0.01 ~ 1% BSA the 1X ~ 10X PBS was added, the purified water _{900㎖ KH 2 PO 4 0.144 ~ 1.44g} , NaCl 9.000 ~ 90.00g, Na 2 HPO 4 · 7H 2 O Dissolve 0.795 ~ 7.950g to adjust the pH to 7.2 ~ 7.8, make final volume 1000ml with purified water, and then filter, add antibiotic gentamicin (gentamicin) to final concentration of 100㎍ / ㎖ 1X PBS solution was prepared, and 0.05 ~ 5g BSA was dissolved in 400ml 1X PBS solution so that the final volume was 500ml with 1X ~ 10X PBS solution, followed by filtration.

Here, in the enzyme treatment step, the ratio of the volume of the adipose tissue and the volume of the enzyme treatment solution is 1: 4 (v / v), and the mixture is stirred for 10 to 60 minutes in a thermostat at 36 to 38 ° C.

Here, 0.1-0.3% collagenase type II solution, which is the enzyme treatment solution, is prepared by dissolving 0.1-0.3 g collagenase type II in 9 ml of purified water and then adjusting the final volume to 10 ml with purified water and filtering. .

Here, the step of stopping the enzyme reaction stops the reaction by adding the DMEM solution in a volume ratio of 1: 1 (v / v).

Here, the DMEM solution containing 1-20% of FBS, the enzyme stopping solution, was added in 800 ml of purified water, 8-11 g of DMEM powder, 2000-2400 mg of NaHNO ₃ (Sodium Bicarbonate), and HEPES (N-2-Hydroxyethylpiperazine). -N`-2-Ethane Sulfonic Acid) After dissolving 2200 ~ 2400mg, add 10 ~ 200ml of FBS to DMEM solution, mix, adjust pH to 6.8 ~ 7.8, and make final volume 1000ml with purified water. After filtration it is prepared.

Here, the centrifugation step is centrifuged for 1 to 5 minutes at 1,000 ~ 5,000rpm of the adipose tissue suspended enzyme reaction, and then collecting the cell layer from the upper layer to collect mature fat cells, the collected upper cell layer above A first centrifugation step of pouring the fat cell culture solution about 1 to 5 times, shaking gently to wash off the enzyme solution, and then centrifuging at 1,000 to 5,000 rpm for 1 to 5 minutes to collect the upper cell layer; The upper cell layer is separated and the remaining solution is centrifuged again at 1,000 to 5,000 rpm for 5 to 10 minutes, the cell layer is collected at the lower layer to collect the fat precursor cells, and the adipocyte culture solution is collected at the lower layer layer 1 to 5 Pour about twice and shake gently to wash off the enzyme solution, followed by centrifugation for 5 to 10 minutes at 1,000 ~ 5,000rpm, and then the second centrifugal separation step to collect the lower cell layer.

Here, the adipocyte culture solution was dissolved in DMEM powder 8-11g, NaHNO ₃ 2000-2400mg, HEPES 2200-2400mg in 800ml purified water, the pH of the dissolved solution was adjusted to 6.8-7.8, purified water Prepare the final volume to 1000ml, dissolve 8-11g of F-12 powder and 1000-1200mg of NaHNO _{3 in} 800ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8, and final volume with purified water. Prepare 1000 ml, and mix the solutions prepared above in a volume ratio of 1: 1 (v / v), add 10-200 ml of FBS to make the final volume 1000 ml, and then prepare by filtration.

Here, in the culturing step, the mature adipocytes centrifuged are mixed with a defatted fat cell degreasing solution, put in a culture vessel, and placed in a culture vessel at a constant temperature of 36 to 38 ° C. to supply 5-8% CO ₂ , and cultured. After degreasing for 5 to 20 hours by hanging a vibrator on one side of the container, culturing the mature fat cells while removing the mature fat cells attached to the culture vessel, replacing them with new mature fat cell degreasing solution once degreasing is completed. A first culture step; Centrifuged fat precursor cells are composed of a second culture step of culturing by mixing the adipocyte culture solution.

Here, the mature fat cell degreasing solution was dissolved in DMEM powder 8-11g, NaHNO ₃ 2000-2400mg, HEPES 2200-2400mg in 800ml purified water, the pH of the dissolved solution was adjusted to 6.8-7.8, Prepare final volume to be 1000ml with purified water, dissolve 8-11g of F-12 powder and 1000-1200mg of NaHNO _{3 in} 800ml of purified water, adjust the pH of the dissolved solution to 6.8 ~ 7.8, and finally with purified water. preparation and bulky so that 1000㎖, each prepared solution in the volume ratio 1: mix a 1 (v / v), epinephrine (epinephrine) 1 ~ 100㎍ / ㎖ , dexamethasone (dexamethasone) 10 ^-5 ~ 10 ^{- 7} M, 10-200 ml of FBS is added to make the final final volume of 1000 ml, followed by filtration.

Hereinafter, with reference to the accompanying drawings the manufacturing process of the isolated culture method of mature fat cells and fat precursor cells according to the present invention will be described in detail.

In the following description of the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. Terms to be described later are terms defined in consideration of functions in the present invention, and may be changed according to intentions or customs of users or operators. Therefore, the definition should be made based on the contents throughout the specification.

1 is a process chart showing the manufacturing process of the isolated culture method of mature fat cells and fat precursor cells according to the present invention, Figure 2 is a separate culture method of mature fat cells and fat precursor cells according to a first embodiment of the present invention It is a photograph showing the mature fat cells separated and cultured from the adipose tissue collected from the orbit of the human body, Figure 3 is in the orbit of the human body by the isolation culture method of mature fat cells and fat precursor cells according to the first embodiment of the present invention It is a photograph showing the fat precursor cells separated and cultured from the collected adipose tissue, Figure 4 is separated from the adipose tissue aspirated from the human body by the isolation culture method of mature fat cells and fat precursor cells according to a second embodiment of the present invention Photo shows the cultured mature fat cells, Figure 5 is a phosphorus by the method of separating and culturing mature fat cells and fat precursor cells according to a second embodiment of the present invention From a photograph showing the removed culture from liposuction fat tissue preadipocytes.

Referring to Figure 1, the method for separating and culturing mature fat cells and fat precursor cells according to the present invention, first, 0.01-1% BSA (Bovine Serum), which is a 5-15 times the amount of adipose tissue weight in adipose tissue obtained from the human body. Albumin) is added to the 1X ~ 10X Phosphate-Buffered Saline (PBS) solution is added to wash the adipose tissue, and repeatedly washed at least three times the adipose tissue (S100).

In addition, 0.1-0.3% collagenase type II solution, which is an enzyme treatment solution, was mixed with the adipose tissue in a ratio of 1: 4 (v / v) in the ratio of the volume of the adipose tissue and the volume of the enzyme treatment solution. Stir for 10 to 60 minutes in a thermostat of 36 ~ 38 ℃ (S110).

Then, to stop the enzymatic reaction, a Dulbecco's Minimum Essential Medium (DMEM) solution containing 1-20% of FBS (Fetal Bovine Serum), an enzyme stopping solution, was added at a volume ratio of 1: 1 (v / v). The reaction was stopped by the addition (S120).

After centrifugation of enzyme-sustained adipose tissue at 1,000 to 5,000 rpm for 1 to 5 minutes (S131), the cell layer is collected from the upper layer to collect mature fat cells (S132), and the adipocyte culture is performed on the collected upper cell layer. Pour the solution about 1 to 5 times and shake gently to wash away the enzyme solution (S141), and then centrifuge for 1 to 5 minutes at 1,000 to 5,000 rpm and then collect the upper cell layer. (S142)

On the other hand, the upper cell layer is separated and the remaining solution is centrifuged again at 1,000 to 5,000 rpm for 5 to 10 minutes (S133), the cell layer is collected at the lower layer to collect the fat precursor cells (S143), and the collected lower layer is on the cell layer. Pour 1 ~ 5 times the adipocyte culture solution and gently shake to wash away the enzyme solution (S143), and then centrifuged at 1,000 ~ 5,000rpm for 5-10 minutes and then collect the lower layer (S144).

The mature fat cells centrifuged above were mixed with the mature fat cell degreasing solution, put in a culture vessel, and put the culture vessel in a thermostat at 36-38 ° C. that supplies 5-8% CO ₂ , and vibrator on one side of the culture vessel. Defatting for 5 to 20 hours by walking (S151), and when degreasing is completed, the mature fat cells are cultured again while exchanging with new mature fat cell degreasing solution except for the mature fat cells attached to the culture vessel (S152). .

In addition, the centrifuged adipocytes are mixed with the adipocyte culture solution and cultured (S153).

In addition, 1X ~ 10X PBS solution to which 0.01-1% BSA, which is a water washing solution, was added in 900 ml of purified water, 0.144-1.44g of KH ₂ PO ₄ , NaCl 9.000-90.00g, Na ₂ HPO ₄ · 7H ₂ O 0.795 ~ 7.950g Dissolved to adjust the pH to 7.2-7.8, and the final volume to 1000ml with purified water, and then filtered, and the filtered solution was added to the final concentration of antibiotic gentamicin (gentamicin) to a final concentration of 100㎍ / ㎖ 1X PBS solution To prepare, prepare a final volume of 500ml by dissolving 0.05 ~ 5g BSA in 400ml 1X PBS solution to 1X ~ 10X PBS solution and then prepared by filtration.

Here, 0.1-0.3% collagenase type II solution, which is the enzyme treatment solution, is prepared by dissolving 0.1-0.3 g collagenase type II in 9 ml of purified water and then adjusting the final volume to 10 ml with purified water and filtering. .

In addition, DMEM solution containing 1 ~ 20% FBS, enzyme stopping solution, 8 ~ 11g DMEM powder, 2000 ~ 2400mg NaHNO ₃ (Sodium Bicarbonate), HEPES (N-2-Hydroxyethylpiperazine-N`) -2-Ethane Sulfonic Acid) After dissolving 2200 ~ 2400mg, add 10 ~ 200ml of FBS to DMEM solution, mix, adjust pH to 6.8 ~ 7.8, filter to final volume of 1000ml with purified water Manufacture. Here, 9.6 g of DMEM powder, 2200 mg of NaHNO ₃ and 2383 mg of HEPES are preferably added.

In addition, in the adipocyte culture solution, dissolve 8-11 g of DMEM powder, 2000-2400 mg of NaHNO ₃ , and 2200-2400 mg of HEPES in 800 ml of purified water, and adjust the pH of the dissolved solution to 6.8-7.8. Was prepared to make 1000 ml, and dissolved 8-12 g of F-12 powder and 1000-1200 mg of NaHNO _{3 in} 800 ml of purified water, and then adjusted the pH of the dissolved solution to 6.8-7.8, and the final volume was 1000 ml with purified water. Prepare to be prepared, and the solutions prepared above are mixed in a volume ratio of 1: 1 (v / v), and 10 to 200 ml of FBS is added so that the final volume is 1000 ml, followed by filtration. Here, 9.6 g of DMEM powder, 2200 mg of NaHNO ₃ and 2383 mg of HEPES are preferably added, and 9.6 g of F-12 powder, 1176 mg of NaHNO ₃ , and 100 ml of FBS are added.

On the other hand, in mature fat cell degreasing solution, dissolve DMEM powder 8-11g, NaHNO ₃ 2000-2400mg, HEPES 2200-2400mg in 800ml purified water, adjust the pH of the dissolved solution to 6.8-7.8 Prepare the volume to 1000ml, dissolve 8-11g of F-12 powder and 1000-1200mg of NaHNO _{3 in} 800ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8, and final volume 1000 with purified water. Prepare to ㎖, mix the solution prepared in each of the volume ratio 1: 1 (v / v), 1 ~ 100 ㎍ / ㎖ epinephrine (epinephrine), dexamethasone (dexamethasone) 10 ^-5 ~ 10 ^-7 M, 10 to 200 ml of FBS is added to make the final volume of 1000 ml, followed by filtration. Here, 9.6 g of DMEM powder, 2200 mg of NaHNO ₃ and 2383 mg of HEPES are preferably added, and 9.6 g of F-12 powder and 1176 mg of NaHNO ₃ are added.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these embodiments.

Example 1 Isolation and Culture of Adipocytes from Human Orbital Adipose Tissue

The method of separating and culturing fat cells from human orbital, ie, eye adipose tissue collected on the day is as follows.

First, in order to weigh the eye adipose tissue that has been moved to the refrigerated state at -20 to 4 ° C., the weight of the container containing no 50 ml centrifuge tube is weighed, Measure the total weight and subtract it from the weight of the tissue.

After weighing the tissue, add a flushing solution 5 to 15 times the tissue weight to close the 50 ml centrifuge tube lid and gently shake to wash the tissue. The washing process is repeated three times in total. Pour 1-10 mL of flushing solution into the washed tissue, cut it into small pieces on a petri-dish and place it back into a 50 mL centrifuge tube.

Enzyme solution is added to the washed and cut adipose tissue. At this time, the volume ratio of the tissue to the collagenase is 1: 4 (v / v). A 50 ml centrifuge tube containing enzymatic adipose tissue is stirred in a 37 ° C. thermostat with agitation time of 10-20 minutes.

In order to stop the enzyme reaction after the end of the enzyme treatment, stop the reaction by adding the enzyme reaction stop solution, that is, the medium in a volume ratio of 1: 1 (v / v).

In order to separate the cells contained in the 50ml centrifuge tube into mature adipocytes and adipocytes, centrifuge for 1 to 5 minutes at 1,000 to 5,000 rpm. At this time, the cell layer collected on the upper layer, that is, the mature adipocytes are separately placed in a 15 ml centrifuge tube. Then, the fat cell culture solution is poured 1-5 times in the collected cell layer, gently shaken to wash off the enzyme solution, and centrifuged at 1,000-5,000 rpm for 1-5 minutes to collect the upper layer.

Centrifuge the solution in which the enzymatic reaction was stopped in the 50 ml centrifuge tube from which the upper layer was separated once again at 1,000 to 5,000 rpm for 5 to 10 minutes. After the centrifugation, remove the solution of the collected cell layer in the lower layer of the 50 ml centrifuge tube and pour 1-5 times the adipose tissue culture solution into the lower cell layer and gently stir to wash the remaining enzyme solution. Collect the lower layer by centrifugation for 5-10 minutes at ˜5,000 rpm.

Take only a part of the mature fat cells separated from the upper layer, measure the cell number, add the mature fat cell degreasing solution, mix them evenly with the cells, and transfer to the adipocyte culture vessel.

Then, defatting the vibrator on one side of the adipocyte culture vessel for 12 hours in a 37 ° C thermostat supplying 5 to 8% CO ₂ , and degreasing after completion of degreasing, new mature fat cells except the attached cells. Replace with degreasing solution, and incubate by replacing the mature fat cell degreasing solution every three days.

In addition, measure the cell number by taking only a part of the fat precursor cells separated from the lower layer, and then evenly mix the fat cell culture solution, that is, 10 ml of medium, select the vent cap flask according to the total cell number, Transfer to the flask and incubate.

Mature fat cells and fat precursor cells cultured as described above are as shown in Figs.

Example 2 Isolation and Culture of Adipocytes from Adipose-Aspirated Adipose Tissue

The method of separating and culturing fat cells of adipose tissue (liposuction) from a person collected on the day is as follows.

First, in order to weigh the liposuctioned adipose tissue that has been moved to the chilled state at -20 to 4 ° C., the weight of the container containing no tissue of the 50 ml centrifuge tube is measured, and the container in the state where the tissue is contained. Measure the total weight of and subtract that value as the weight of the tissue.

After weighing the tissue, add a flushing solution 5 to 15 times the tissue weight, and then close the 50 ml centrifuge tube lid and shake gently to wash the tissue. The washing process is repeated three times in total.

The enzyme treatment solution is added to the washed adipose tissue. At this time, the volume ratio of the tissue to the collagenase is 1: 4 (v / v) so that the final concentration of the enzyme treatment solution is 0.23%. A 50 ml centrifuge tube containing enzymatic adipose tissue is stirred in a 37 ° C. thermostat, with agitation time of 15-60 minutes.

In order to stop the enzyme reaction after the end of the enzyme treatment, stop the reaction by adding the enzyme reaction stop solution, that is, the medium in a volume ratio of 1: 1 (v / v).

In order to separate the cells contained in the 50ml centrifuge tube into mature adipocytes and adipocytes, centrifuge for 1 to 5 minutes at 1,000 to 5,000 rpm. At this time, the cell layer collected on the upper layer, that is, the mature adipocytes are separately placed in a 15 ml centrifuge tube. Then, the fat cell culture solution is poured into the collected cell layer about 1 to 5 times, gently shaken to wash off the enzyme solution, and the upper layer is collected by centrifugation for 1 to 5 minutes at 1,000 to 5,000 rpm.

Centrifuge the solution in which the enzymatic reaction was stopped in the 50 ml centrifuge tube from which the upper layer was separated once again at 1,000 to 5,000 rpm for 5 to 10 minutes. After the centrifugation, remove the solution of the collected cell layer in the lower layer of the 50 ml centrifuge tube and pour 1-5 times the adipose tissue culture solution into the lower cell layer and gently stir to wash the remaining enzyme solution. Collect the lower layer by centrifugation for 5-10 minutes at ˜5,000 rpm.

Take only a part of the mature fat cells separated from the upper layer, measure the cell number, add the mature fat cell degreasing solution, mix them evenly with the cells, and transfer to the adipocyte culture vessel.

Then, defatting the vibrator on one side of the adipocyte culture vessel for 12 hours in a 37 ° C thermostat supplying 5 to 8% CO ₂ , and degreasing after completion of degreasing, new mature fat cells except the attached cells. Replace with degreasing solution, and incubate by replacing the mature fat cell degreasing solution every three days.

In addition, measure the cell number by taking only a part of the fat precursor cells separated from the lower layer, and then evenly mix the fat cell culture solution, that is, 10 ml of medium, select the vent cap flask according to the total cell number, Transfer to the flask and incubate.

Mature fat cells and fat precursor cells cultured as above are shown in FIGS. 4 and 5.

According to the isolated culture method of the mature fat cells and fat precursor cells of the present invention configured as described above, by selecting a solution for each process and separating and culturing the mature fat cells and fat precursor cells from adipose tissue as fast as the adipose tissue is treated In addition to recovering the activated adipocytes in time, new adipocyte transplantation may be possible by easily isolating and culturing mature adipocytes, which had been difficult to isolate and culture.

As those skilled in the art would realize, the described embodiments may be modified in various ways, all without departing from the spirit or scope of the present invention. It is to be understood, however, that the present invention is not limited to the specific forms referred to in the description, but rather includes all modifications, equivalents, and substitutions within the spirit and scope of the invention as defined by the appended claims. Should be.

Claims (11)

Washing adipose tissue by adding 1X ~ 10X Phosphate-Buffered Saline (PBS) solution containing 0.01-1% BSA (Bovine Serum Albumin), which is 5-15 times the weight of adipose tissue, to adipose tissue obtained from the human body Washing step; An enzyme treatment step of adding 0.1-0.3% collagenase type II solution, which is an enzyme treatment solution, to the washed adipose tissue at a constant volume ratio and then stirring the mixture at a predetermined temperature for a predetermined time; To stop the reaction by stopping the reaction by adding DMEM (Dulbecco's Minimum Essential Medium) solution containing 1 ~ 20% FBS (Fetal Bovine Serum), which is the enzyme stopping solution, to stop the enzyme reaction. ; A centrifugation step of adding a fat cell culture solution to centrifugation at a constant rate for a predetermined time to separate the enzyme-activated fat tissue into mature fat cells and fat precursor cells; And Isolation and culturing method of mature fat cells and fat precursor cells, characterized in that the culture step of collecting and culturing the separated mature fat cells and fat precursor cells, respectively. The method of claim 1, The washing step, Repeatedly culturing the fat cells and mature fat cells, characterized in that to wash the fat tissue at least three or more times. The method of claim 1, The 1X-10X PBS solution to which 0.01-1% BSA which is the said water washing solution is added, After dissolving 0.144 ~ 1.44g of KH ₂ PO ₄ , 9.000 ~ 90.00g of NaCl, 0.795 ~ 7.950g of Na ₂ HPO ₄ · 7H ₂ O in 900ml of purified water, adjust the pH to 7.2 ~ 8.8 After filtering, and adding the antibiotic gentamicin (gentamicin) to a final concentration of 100 ㎍ / ㎖ to prepare a 1X PBS solution, dissolved 0.05 ~ 5g BSA in 400ml 1X PBS solution to 1X ~ Separation and culturing method of mature fat cells and fat precursor cells, characterized in that the final volume to 500ml with 10X PBS solution and then prepared by filtration. The method of claim 1, The enzyme treatment step, The ratio of the volume of adipose tissue and the volume of the enzyme treatment solution is 1: 4 (v / v), Separation culture method of mature fat cells and fat precursor cells, characterized in that the stirring for 10 to 60 minutes in a thermostat of 36 ~ 38 ℃. The method of claim 1, 0.1-0.3% collagenase type II solution, which is the enzyme treatment solution, Dissolving and culturing the mature fat cells and fat precursor cells, characterized in that the final volume is dissolved in 10 ml of purified water and purified to 9 ~ 0.3g collagenase type II in 10 ml of purified water. The method of claim 1, The enzyme reaction stop step, Separation culture method of mature fat cells and fat precursor cells, characterized in that the reaction is stopped by adding the DMEM solution in a volume ratio of 1: 1 (v / v). The method of claim 1, The DMEM solution to which the 1 to 20% FBS is added, the enzyme stop solution, Dissolve 8-11 g of DMEM powder, 2000-2400 mg of NaHNO ₃ (Sodium Bicarbonate) and 2200-2400 mg of HEPES (N-2-Hydroxyethylpiperazine-N`-2-Ethane Sulfonic Acid) in 800 ml of purified water. After the addition of 10 ~ 200ml FBS, the pH is adjusted to 6.8 ~ 7.8, and the final volume is 1000ml with purified water, and then filtered and prepared by culturing mature fat cells and fat precursor cells, characterized in that the production. The method of claim 1, The centrifugation step, After centrifuging the enzyme-sustained adipose tissue at 1,000 to 5,000 rpm for 1 to 5 minutes, the cell layer was collected from the upper layer to collect mature adipocytes, and the adipocyte culture solution was collected 1 to 5 times into the collected upper cell layer. Pour about and gently shake the enzyme solution to wash away, and then centrifuged again for 1 to 5 minutes at 1,000 ~ 5,000rpm and then the first centrifugation step of collecting the upper cell layer; The upper cell layer is separated and the remaining solution is centrifuged again at 1,000 to 5,000 rpm for 5 to 10 minutes, the cell layer is collected at the lower layer to collect the fat precursor cells, and the adipocyte culture solution is collected at the lower layer layer 1 to 5 Pour about twice and gently shake the enzyme solution to wash away, and then centrifugation for 5 to 10 minutes at 1,000 to 5,000 rpm, followed by a second centrifugal separation step to collect the lower cell layer. Separation Culture Method. The method according to claim 1 or 8, The adipocyte culture solution, After dissolving 8-11 g of DMEM powder, 2000-2400 mg of NaHNO ₃ and 2200-2400 mg of HEPES in 800 ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8 and prepare the final volume to 1000 ml with purified water. , Dissolve 8-11 g of F-12 powder and 1000-1200 mg of NaHNO _{3 in} 800 ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8, and prepare the final volume to 1000 ml with purified water. Mix each prepared solution in a volume ratio of 1: 1 (v / v), add 10-200 ml of FBS to make the final volume 1000 ml, and then filter and prepare the mature fat cells and the fat precursor cells. Separation Culture Method. The method according to claim 1 or 8, The culturing step, Centrifuged mature fat cells were mixed with the mature fat cell degreasing solution, put in a culture vessel, put the culture vessel in a thermostat at 36-38 ° C. that supplies 5-8% CO ₂ , and put a vibrator on one side of the culture vessel. A first culture step of defatting for ˜20 hours, and culturing the mature fat cells while removing the mature fat cells attached to the culture vessel while degreasing is completed, exchanging them with new mature fat cell degreasing solution; Separation culture method of mature fat cells and fat precursor cells, characterized in that consisting of a second culture step of culturing by mixing the centrifuged fat precursor cells in the adipocyte culture solution. The method of claim 10, The mature fat cell degreasing solution, After dissolving 8-11 g of DMEM powder, 2000-2400 mg of NaHNO ₃ and 2200-2400 mg of HEPES in 800 ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8 and prepare the final volume to 1000 ml with purified water. , Dissolve 8-11 g of F-12 powder and 1000-1200 mg of NaHNO _{3 in} 800 ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8, and prepare the final volume to 1000 ml with purified water. Each of the prepared solution in the volume ratio 1: 1 (v / v) with mixing, epinephrine (epinephrine) with 1 ~ 100㎍ / ㎖, dexamethasone (dexamethasone) for 10 ^-5 ~ 10 ^-7 M, the FBS 10 ~ 200㎖ Separation and culturing method of mature fat cells and fat precursor cells, characterized in that the final final volume is added to 1000ml by addition and then filtered.

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