KR20120117386A

KR20120117386A - Adipose-derived stem cell, method for separating the same and method for storing the same

Info

Publication number: KR20120117386A
Application number: KR1020110035117A
Authority: KR
Inventors: 박희섭; 한규범; 한규현
Original assignee: (주)차바이오앤디오스텍
Priority date: 2011-04-15
Filing date: 2011-04-15
Publication date: 2012-10-24

Abstract

The present invention relates to adipose derived stem cells, a method for separating and storage method thereof. More specifically, the present invention comprises the steps of (i) separating the adipose derived stem cells by adding a collagenase solution to the adipose tissue obtained from the human body; And (ii) removing the blood cells by adding a blood cell hemolysis solution to the isolated adipose derived stem cells, a method for separating adipose derived stem cells from adipose tissue, adipose derived stem cells and adipose derived stems obtained therefrom. It is about a cell storage method.

Description

Adipose-derived Stem Cell, Method for Separating the Same and Method for Storing the Same}

Adipose cells, adipose stem cells and adipose tissue are known methods, for example, liposuction and sedimentation from adipose tissue, as disclosed in WO2000 / 53795 and WO2005 / 042730. Fat cells and adipose stem cells can be separated through a process such as enzymatic treatment of collagenase, removal of floating cells such as red blood cells by centrifugation, and the like. In addition, as disclosed in WO2006 / 084284, human adipose tissue in a subcutaneous reservoir is removed by sedimentation blood by liposuction and sedimentation, washing of adipose tissue using Krebs-Ringer buffer, and collagenase treatment. After centrifugation, the isolation of adipose tissue stromal cells through the removal of oil and adipose cell layers, suspended in the medium, attached to a tissue culture dish or flask or cultured to remove suspended cells.

Adipose stem cells and adipose cells used for face shaping and removing traces of wounds are present in the remaining cell mass after separating the adipose tissues using collagenase to improve the subcutaneous fat defects. It is used in plastic surgery etc. for the purpose. These fat stem cells and fat cells are damaged not only about 50% of the fat cells in the process of collecting the fat tissue, but also a problem such as damage to the fat cells under physical stress during the enzyme treatment process. In addition, the problem of reducing the activity and stability of the cells when suspended and filled in sterile saline for injection in order to transplant the isolated adipose stem cells occurs.

Therefore, the method of separating can be used to minimize the separation time by using a high concentration of collagenase, the fat stem cells reduced activity by this separation has a problem that the engraftment rate in the subcutaneous fat after transplantation. Problems such as reduced cell activity and decreased engraftment rate may cause necrosis of isolated adult adipose derived stem cells, and furthermore, may not be beneficial to recipients receiving the cells.

Recently, studies on apoptosis or necrosis have been actively conducted, and materials that can fundamentally prevent or reduce it have been found. In addition, studies of apoptosis or necrosis have revealed that mitochondria in cells play an important role.

Mitochondria are many critical intracellular processes, such as energy metabolism in cells (the mitochondria's basic functions provide energy to the cell), and metabolism of certain substances (e.g. fatty acids). play a key role in processes. Mitochondria are directly involved in the formation and use of free radicals (hereinafter referred to as 'FR') and reactive oxygen species (hereafter referred to as 'ROS') (many processes in living cells Reactive moieties that can affect. Mitochondria have recently been shown to play a key role in the process of programmed cell death.

Many diseases are known to be related to dysfunction of mitochondria. These diseases include the death of one or many cells in all diseases, tissues or organisms associated with increased FR and ROS formation, disorders in apoptosis mechanisms, and disorders in metabolism of fatty acids.

Targeting mitochondria to prevent dysfunction of mitochondria and to inhibit cell necrosis, cells called NecroX (NecroX-1, NecroX-2, NecroX-5 and NecroX-18; LG Life Sciences) Protective agents have been developed.

Formula (I)

NecroX-1 (C ₂₄ H ₂₆ N ₃ O ₃ S), NecroX-2 (C ₂₅ H ₃₂ N ₄ O ₄ S ₂ , [[5- (1,1-Dioxo-thiomorpholin-4-ylmethyl) -2 -phenyl-1H-indol-7-yl]-(1-methanesulfonyl-piperidin-4-yl) -amine]), NecroX-5 (C ₂₅ H ₃₁ N ₃ O ₃ S, [[5- (1,1 -Dioxo-thiomorpholin-4-ylmethyl) -2-phenyl-1H-indol-7-yl]-(tetrahydro-pyran-4-ylmethyl) -amine]), and NecroX-18 (C ₂₉ H ₃₆ N ₄ O ₄ S) is a novel substance that has an excellent effect of inhibiting Necrosis and is a compound that does not have a clear mechanism of action as a mitochondrial-specific potent cell necrosis inhibitor. Unlike the conventional substances reported in the literature, NecroX is a substance having an effect of inhibiting cell death due to toxin or stress, an effect of increasing cell viability, and an antioxidant and anti-inflammatory effect.

NecroX is a cell-permeable cell necrosis inhibitor that inhibits cell necrosis such as antioxidant activity and is a substance that prevents cell death by oxidative stress. In vitro experiments have been shown to have cytoprotective effects on cold shock, hypoxia, oxidative stress, and CCl ₄ induced rodent models and chronic liver fibrosis.

The basic object of the present invention is the step of (i) separating the adipose derived stem cells by adding a collagenase solution to the adipose tissue obtained from the human body; And (ii) adding blood cell hemolysis solution to the isolated adipose derived stem cells to remove blood cells, thereby providing a method for separating adipose derived stem cells from adipose tissue.

Another object of the present invention is to provide adipose derived stem cells obtained by the above method, which exhibit positive immunological properties for CD49d, CD73, and CD105 and negative immunological properties for CD106. will be.

Still another object of the present invention is to provide a fat-derived stem cell storage method of adding fat-derived stem cells to sterile saline containing NecroX.

The basic object of the present invention described above is (i) adding a collagenase solution to the adipose tissue obtained from the human body to separate the adipose derived stem cells; And (ii) it can be achieved by providing a method for separating adipose derived stem cells from adipose tissue comprising the step of removing blood cells by adding a hemolysis solution to the isolated adipose derived stem cells.

The collagenase is preferably I type. In addition, the collagenase solution may contain, but is not limited to, DMEM medium and Hepes solution.

Moreover, the collagenase solution may comprise NecroX. The NecroX improves the survival rate, proliferation rate, activity and engraftment rate of isolated adipose derived stem cells. In addition, the concentration of NecroX is preferably 0.1 μM to 10 μM.

Step (i) of the method for separating adipose derived stem cells from adipose tissue of the present invention can be performed by centrifugation, but is not limited thereto.

The blood cell hemolysis solution may include, but is not limited to, water, ammonium chloride, potassium bicarbonate, and EDTA.

The method for separating the adipose derived stem cells from the adipose tissue of the present invention may further comprise the step of (iii) washing the adipose derived stem cells obtained in step (ii). The washing may be performed using sterile saline, but is not limited thereto.

Another object of the present invention described above is adipose derived stem cells obtained by the method of isolating adipose derived stem cells from the adipose tissue of the present invention, and show positive immunological properties to CD49d, CD73 and CD105, It can be achieved by providing adipose derived stem cells exhibiting immunological properties negative for.

Adipose-derived stem cells obtainable according to the method of the present invention are attached to plastic and grow, and have a spindle form.

Another object of the present invention described above can be achieved by providing a fat-derived stem cell storage method, by adding adipose derived stem cells to sterile saline containing NecroX.

The concentration of NecroX is preferably 0.1 μM to 10 μM. The NecroX improves the survival rate, proliferation rate, activity and engraftment rate of isolated adipose derived stem cells.

Adipose derived stem cells obtained according to the adipose derived stem cell separation method of the present invention have excellent survival rate, proliferation rate, activity and engraftment rate.

In addition, when the adipose derived stem cells are stored in sterile saline containing NecroX, the survival rate, proliferation rate, activity and activity of the adipose derived stem cells are improved.

1 is a photograph taken at 100 and 400 times the human adipose tissue-derived stem cells according to the present invention.
2 is a photograph at 100-fold magnification of immunostaining results using CD49d, a specific positive marker of human adipose tissue-derived stem cells, and CD106, a specific negative marker.
Figure 3 is a view showing the engraftment rate improvement effect of the human adipose tissue-derived stem cells according to the present invention, Figure 3a is a micrograph of the adipocytes grafted on the plate, Figure 3b is a graph quantifying the engraftment of adipocytes.
Figure 4 is a graph of the proliferation rate is superior to fetal calf serum-containing DMEM medium in DMEM medium containing cell necrosis inhibitors in the culture of human adipose tissue-derived stem cells according to the present invention.
5 shows surface antigen analysis (FACS Analysis) results using CD73 and CD105, which are specific positive markers of stem cells derived from human adipose tissue according to the present invention.
Figure 6 is a graph showing the effect of improving the survival rate in sterile physiological saline containing apoptosis inhibitors when filling the human adipose tissue-derived stem cells according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following embodiments. However, the following description of the embodiments is only intended to specifically describe the specific embodiments of the present invention, it is not intended to limit or limit the scope of the present invention to the contents described therein.

Example One. Necrox Plus -5 Collagenase Stem Cell Isolation from Adipose Tissue

A collagenase solution was prepared by mixing 200 mg of collagenase (type I), 500 ml of DMEM medium, and 12.4 ml of Hepes solution, followed by filtration through a sterile filter. Human adipose tissue was taken from the human abdomen. Human adipose tissue was divided into a control group containing a collagenase solution and an experimental group containing a collagenase solution (NecroX-5 final concentration 0.5 μM) to which NecroX-5, a cell necrosis inhibitor, was added. 50 ml of human adipose tissue was transferred to a 150Φ culture dish using a 25 ml pipette, and then 50 ml of the collagenase solution was added to the 150Φ culture dish in the control group and NecroX-5 added to the experimental group (NecroX-5). , 0.5 μM) 50 ml were added respectively. Thereafter, the enzyme was enzymatically treated at 37 ° C. in a 5% CO ₂ incubator for 2 hours. The reaction solution was pipetted for 5 minutes using a 25 ml pipette and 100 μm mesh-filters were assembled into a 50 ml tube using tweezers to filter the fat and collagenase mixture. The 100 μm mesh-filter was removed and centrifuged at 250 g for 6 minutes. Free fat, other adipose tissue and collagenase solution in the upper layer were removed and only the precipitated cell mass was obtained. Ammonium chloride (150 mM), potassium bicarbonate (1 mM) and EDTA (0.1 mM) were mixed with 1 L of purified water and filtered through a sterile filter to prepare a hemocytic hemolysis solution. Cells were suspended by adding 5 ml of hemolysis solution to each cell mass obtained above. Each suspended cell was collected into a new 50 ml tube and centrifuged for 6 min at 250 g. The supernatant solution was removed and only precipitated cell masses were obtained. 20 ml of sterile physiological saline was added to the cell mass thus obtained, the cells were suspended and centrifuged at 250 g for 6 minutes. After the first wash, the solution of the upper layer was removed and only precipitated cell masses were obtained. 20 ml of sterile physiological saline was added to the cell mass, the cells were suspended and centrifuged at 250 g for 6 minutes. After the second wash, the solution of the upper layer was removed and only precipitated cell masses were obtained. 20 ml of sterile physiological saline was added to the cell mass thus obtained, the cells were suspended, 10 μl was collected, the cell number was measured, and centrifuged at 250 g for 6 minutes. After the third wash, the solution of the upper layer was removed and only precipitated cell masses were obtained. According to the measured cell number, sterile saline solution was added so that the cell number was 1 × 10 ⁶ / ml or more.

1 is a flask culture result of isolated adipose tissue-derived stem cells, it was confirmed that the morphological characteristics of the cells appear on the fourth day of culture compared to the first day of culture.

Example 2. Morphological Characteristics and Immunostaining Analysis of Adipose Tissue-Derived Stem Cells

Adipose tissue-derived stem cells obtained in Example 1 were washed three times with PBS and fixed for 30 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, it was permeabilized with PBS containing 0.1% Triton-X100 for 10 minutes. After washing three times with PBS, it was reacted with 10% NGS for 1 hour, and reacted overnight with PBS containing the primary antibody. Washed three times with PBS and reacted for 1 hour in the dark with a secondary antibody. After washing three times with PBS, it was mounted.

As a result, as shown in Figure 2, the stem cells according to the present invention showed a positive and negative response to CD49d and CD106, which can be said to be a cell marker of adipose tissue-derived stem cells, respectively.

Example 3. Morphological Characteristics of Adipose Tissue-Derived Stem Cells Engraftment rate compare

Adipose tissue-derived stem cells isolated through the isolation method as in Example 1 were centrifuged at 250 g for 6 minutes. The supernatant solution was removed and only precipitated cell masses were obtained. 1 ml of DMEM medium was added to the cell mass to suspend the cells, 10 μl was taken to measure the number of cells, and 1 × 10 ⁶ cells were stored in 10% FBS according to the measured number of cells. It was suspended in 15 ml of medium and inoculated in a T-75 flask. After inoculation, the cells were incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. The cultured T-75 flask was taken out of the incubator and the state of the cells was observed under a microscope. After removing the medium of each T-75 flask and washed with PBS. After washing, 10 ml of trypsin-EDTA solution (0.5%) was added to each T-75 flask, and the cells were detached by reacting for 5 minutes in a 37 ° C., 5% CO ₂ incubator. After the reaction was completed, trypsin-EDTA solution was neutralized by adding DMEM medium containing 10% FBS to each T-75 flask. The neutralized solution was transferred to a 50 ml tube and centrifuged at 250 g for 6 minutes. The supernatant solution was removed and only precipitated cell masses were obtained. 1 ml of DMEM medium was added to the cell mass to float the cells, and 10 μl was collected to measure the cell number.

As a result, as shown in Figure 3, in the comparison of the number of attached cells and the number of cells attached after 24 hours of stem cells according to the present invention in the experimental group separated using NecroX-5 it was superior in the survival rate and adhesion rate than the control group You can check it.

Example 4. NecroX-5 Of Stem Cells Derived from Adipose Tissue Growth rate Research

Adipose tissue-derived stem cells isolated through the isolation method as in Example 1 were centrifuged at 250 g for 6 minutes. The upper solution was removed and only the precipitated cell masses of each experimental and control group were obtained. 1 ml of DMEM medium was added to the cell mass to float the cells, and 10 μl was collected to measure the cell number. Depending on the number of cells measured, 1 × 10 ⁵ viable cell numbers were suspended in 15 ml of DMEM medium contained in 10% FBS and inoculated into T-75 flasks. After inoculation, the cells were incubated for 48 hours in a 37 ° C., 5% CO ₂ incubator. The cultured T-75 flask was taken out of the incubator and the state of the cells was observed under a microscope. After removing the medium of each T-75 flask and washed with PBS. After washing, 10 ml of trypsin-EDTA solution (0.5%) was added to each T-75 flask, and the cells were detached by reacting for 5 minutes in a 37 ° C., 5% CO ₂ incubator. After the reaction was completed, trypsin-EDTA solution was neutralized by adding DMEM medium containing 10% FBS to each T-75 flask. The neutralized solution was transferred to a 50 ml tube and centrifuged at 250 g for 6 minutes. The supernatant solution was removed and only precipitated cell masses were obtained. To the cell mass thus obtained, 1 ml of DMEM medium was added to suspend the cells, and 10 μl was collected to measure the cell number. Thereafter, cells obtained from the experimental group and the control group were suspended in 15 ml of DMEM medium contained in 10% FBS and inoculated into T-75 flasks. After inoculation, the cells were incubated for 96 hours in a 37 ° C., 5% CO ₂ incubator. The medium of each T-75 flask taken out of the incubator was removed and washed with PBS. After washing, 10 ml of trypsin-EDTA solution (0.5%) was added to each T-75 flask, and the cells were detached by reacting for 5 minutes in a 37 ° C., 5% CO ₂ incubator. After the reaction was completed, trypsin-EDTA solution was neutralized by adding DMEM medium containing 10% FBS to each T-75 flask. The neutralized solution was transferred to a 50 ml tube and centrifuged at 250 g for 6 minutes. The supernatant solution was removed and only precipitated cell masses were obtained. To the cell mass thus obtained, 1 ml of DMEM medium was added to suspend the cells, and 10 μl was collected to measure the cell number.

As a result, as shown in Figure 4, the comparison of the number of cells proliferated after 48 hours and 144 hours of the stem cells according to the present invention was superior to the control group in the experimental group separated using NecroX-5.

Example 5. Immunological Characteristics of Adipose Tissue-Derived Stem Cells

Adipose tissue-derived stem cells obtained in Example 1 were collected and centrifuged at 250 g for 6 minutes. After discarding the solution of the upper layer and put the mixture of 2% FBS and PBS washed, and centrifuged for 6 minutes at 250g. After discarding the supernatant, the cells were suspended in PBS, and 1 × 10 ⁵ cells were dispensed as many as the number of samples. Antibodies were placed in each well and incubated for 40 minutes on ice. After incubation, centrifugation was performed at 250 g for 6 minutes. After removing the solution of the upper layer was washed with PBS and centrifuged for 6 minutes at 250g. Once again, the solution of the upper layer was removed, washed with PBS and centrifuged for 6 minutes at 250g. The solution of the upper layer was removed and analyzed using a flow cytometer.

In addition, the adipose tissue-derived stem cells obtained up to two passages through the same culture method as in Example 4 were analyzed using a flow cytometer through the same method as described above.

As a result, as shown in Figure 5, the stem cells according to the present invention for CD73 which can be said to be a cell marker of adipose tissue-derived stem cells 98.21% immediately after separation, 94.16% after passage 1, 99.52% after passage 2 positive CD105 showed a positive response of 96.52% immediately after separation, 99.65% after the first passage, and 100.00% after the second passage.

Example 6. Sterile saline NecroX-5 Added Under filling conditions Comparison of Survival Rate of Adipose Tissue-Derived Stem Cells

Adipose tissue-derived stem cells isolated through the isolation method as in Example 1 were used in the control group and the experimental group. Control group was 1 × 10 ⁶ / ml were more than the addition of a sterile physiological saline such that, the experimental group, depending on the number of measurement cells 1 × 10 ⁶ / ml or more to be sterilized by the addition of 0.5 μM NecroX-5 physiological saline based on the number of measurement cells Was added. Immediately after the preparation of the control group and the experimental group, samples were taken and centrifuged at 250 g for 6 minutes. The supernatant solution was removed and only precipitated cell masses were obtained. 1 ml of DMEM medium was added to the cell mass to float the cells, and 10 μl was collected to measure the viable and total cell numbers, and cell viability was measured immediately after preparation. After that, the experimental group and the control group were stored at room temperature (15-25 ° C.), and samples were collected after 48 hours. Specimens from control and experimental groups stored for 48 hours were collected and centrifuged at 250 g for 6 minutes. The supernatant solution was removed and only precipitated cell masses were obtained. To the cell mass thus obtained, 1 ml of DMEM medium was added to suspend the cells, and 10 μl was collected, and the number of living cells and the total cell number were measured, and the cell viability of the sample was measured after 48 hours of storage.

As a result, as shown in Figure 6, the cell survival rate of the experimental group added NecroX-5 after 48 hours at room temperature when stored in sterile saline of the stem cells according to the present invention cells of the control group not added NecroX-5 Three times higher survival rates were measured.

Claims

(i) separating the adipose derived stem cells by adding a collagenase solution to the adipose tissue obtained from the human body; And
(ii) removing the blood cells by adding blood cell hemolysis solution to the isolated adipose derived stem cells, the method for separating adipose derived stem cells from adipose tissue.

The method of claim 1, wherein the collagenase solution comprises NecroX.

The method of claim 2, wherein the concentration of NecroX is 0.1 μM to 10 μM.

The method of claim 1, wherein the step (i) separates the adipose derived stem cells from adipose tissue, characterized in that it is carried out by centrifugation.

The method of claim 1, further comprising the step of (iii) washing the adipose derived stem cells obtained in the step (ii).

An adipose derived stem cell according to any one of claims 1 to 5, wherein the adipose derived stem cell shows positive immunological characteristics with respect to CD49d, CD73 and CD105, and shows negative immunological characteristics with respect to CD106.

7. The fat-derived stem cell according to claim 6, wherein the fat-derived stem cell grows by attaching to plastic and has a spindle form.

A fat-derived stem cell storage method comprising adding adipose derived stem cells to sterile saline containing NecroX.

9. The method of claim 8, wherein the concentration of NecroX-5 is 0.1 μM to 10 μM of the fat-derived stem cell storage method.