KR101843952B1 - Methods for isolation adipose-derived stromal vascular fraction from fat tissues - Google Patents

Abstract

The present invention relates to a method for separating adipose-derived stromal vascular fraction (AD-SVF). Since the method of separating the fat tissue-derived substrate blood vessel fraction according to the present invention provides an improved cell yield and cell survival rate compared to the conventional method, even a small amount of fat tissue can be easily transferred to a patient and a fat tissue- Fraction can be obtained. The adipose tissue-derived substrate blood vessel fraction obtained according to the above method can be used to treat various intractable diseases instead of existing cell therapy agents.

Description

Methods for isolation of adipose-derived stromal vascular fraction from fat tissues

The present invention relates to a method for separating adipose-derived stromal vascular fraction (AD-SVF).

According to recent reports, it has been reported that adipose tissue is one of the important sources of the pluripotent stem cells (Non-Patent Documents 1 and 2). Namely, there are undifferentiated adult stem cells in human adipose tissue, and it can be differentiated into various cells such as adipocytes, osteogenic cells, chondroblasts, and the like. Especially, (Non-Patent Document 3). It has also been reported in animal experiments that these cells are capable of promoting different cardiac muscle regeneration and neural cell regeneration. The adipose tissue can extract stem cells in a relatively large amount from patients compared with other tissues, and has the advantage of eliminating the immune related side effects when using autologous adipose tissue, thus being considered as a promising source of stem cells.

Adipose-derived stromal vascular fraction (AD-SVF) has been shown to be involved in preadipocytes, endothelial cells (EC), endothelial progenitor cells (EPC), vascular smooth muscle cells (SMC) It is a heterogeneous cell mixture that includes cells (pericytes), wall cells, macrophages, fibroblasts and adipose stem / stromal cells (ASC). AD-SVF cells have been implicated in the pathogenesis of Crohn's disease, graft-versus-host disease, autoimmune and allergic pathologies such as multiple sclerosis and inflammatory bowel disease, myocardial infarction, Clinical trials for symptoms such as lower limb ischemia, inhaled chronic wounds, radiation injuries, urinary incontinence, and other preliminary disease treatment models have been demonstrated (see Non-patent Document 4). AD-SVF has also been shown to prolong the survival of autologous fat transplantation and thus has a great effect on cosmetic and rehabilitative medicines. A clinical study by Yoshimura et al. (Non-Patent Document 5) disclosed the efficacy of AD-SVF enrichment in fat transplantation for breast molding. Fat transplantation can be applied to post-operative breast reconstruction, cosmetic breast reconstruction, facial skeletal reconstruction, wrinkle correction, and many other soft tissue defects. Animal model studies have shown that the concentration of adipocytes by AD-SVF cells improves transplantation by enhancing the angiogenesis of fat, improving the metabolism of adipocytes, and producing anti-apoptotic factors.

In fact, the heterogeneous composition of AD-SVF, especially high endothelial progenitor cells, is ideal for angiogenic factor cell therapy and vascular regeneration. Several groups of CD34-positive cells have been identified in AD-SVF, either through the release of growth factors such as IGF-1, HGF and VEGF, or directly to stimulate angiogenesis, and SVF cells have been shown to have neuro- Proven.

However, conventional methods of separating AD-SVF have several limitations in terms of clinical application, the most problematic being the number, activity and quality of cells in the obtained AD-SVF. For example, if the number of cells in the AD-SVF is not sufficient for transplantation of the patient, the patient must undergo at least one treatment during treatment, or the patient must be further transplanted with AD-SVF from a non-autologous AD-SVF have.

A. Miranville et al., Circulation 110: 349, 2004 S. Gronthos et al., J. Cell Physiol., 189: 54, 2001 P.A. Zuk et al., Tissue Eng., 7: 211, 2001 Gimble et al. Stem Cell Research & Therapy 2010, 1:19 Yoshimura et al., Aesthetic Plast Surg. 2008 Jan; 32 (1): 48-55

Accordingly, the present inventors intend to provide a method for separating an adipose tissue-derived substrate blood vessel fraction, which has improved cell yield and cell viability compared to the conventional method. The present invention also provides a cell therapy agent comprising an adipose tissue-derived substrate blood vessel fraction obtained according to the method of separating the adipose tissue-derived substrate blood vessel fraction.

In order to solve the above problems,

The adipose tissue was washed with PBS (phosphate buffer saline) containing anticoagulant,

Collagenase in washed fat tissue; And at least one enzyme selected from the group consisting of dyspazyme and accutase < ( ^R ) >

The fat tissue precipitate treated with the enzyme agent is centrifuged to separate the cell-containing fat layer,

And separating the adipose-derived stromal vascular fraction (AD-SVF) from the resulting cell-containing fat layer

Thereby providing a method for separating adipose tissue-derived substrate blood vessel fraction.

In the present invention, the term " adipose " means a tissue composed of white fat, yellow fat or brown fat.

The term " adipose-derived stromal vascular fraction (AD-SVF) " includes adipose-derived progenitor stem cells and adipose-derived progenitor stem cells, , Adipocytes, endothelial cells, smooth muscle cells, hematopoietic stem cells, other stromal cells, etc.).

The adipose tissue is obtained from a living human or animal, preferably, but not exclusively, derived from the lower abdomen, calf, thigh, forearm, breast or knee adipose tissue.

The resulting adipose tissue is first washed with a physiologically-compatible solution such as PBS (phosphate buffer saline). The present invention is characterized in that the adipose tissue is washed using PBS containing an anticoagulant, as opposed to using only PBS in the known method for separating adipose tissue-derived blood vessel fraction. The use of anticoagulants seems to play a role in inhibiting the coagulation of blood cells.

The washed fat tissue can be separated into fat and cells through tissue dissociation. Enzymes can be used for tissue dissociation. Unlike the known method of separating the adipose tissue-derived substrate blood vessel fraction from collagenase alone, collagenase; And an enzyme preparation for tissue disintegration comprising at least one enzyme selected from dyspazyme and accutase ( ^R ). The use of additional enzymes of Dipazane or Accutase with collagenase was found to show significant differences in cell yield and survival rate of adipose tissue-derived substrate blood vessel fraction.

Preferably, the content of the collagenase in the tissue remover preparation may be 95 to 99.9%. The treatment concentration of the collagenase may be 400 to 800 units / ml. The collagenase may be one or a combination of two or more of collagenases I, II, III, IV and V.

On the other hand, if the content of one or more enzymes is selected from the tissue to de-SPA kinase and Accu other kinase (accutase ^®) of Lyon enzyme preparation may be from 0.1 to 5%. Further, when the tissue to the Lyon enzyme preparation of the di-SPA kinase and Accu other azepin one or more enzymes selected from (accutase ^®) used, these enzymes can each be processed in the following concentrations: D SPA kinase concentration Is 1 to 2.4 Unit / ml, and the concentration of acutase treatment is 400 to 800 Unit / ml.

The enzyme composition and enzyme concentration of the enzyme preparation for tissue disruption are important for improving the cell yield and survival rate of adipose tissue-derived substrate blood vessel fraction.

The tissue detachable enzyme preparation may be treated with the fat tissue washed in the agitator for 0.5 to 6 hours, though it is not limited thereto. When considering the optimization of enzyme activity, it is preferable that the temperature in the stirrer at the time of enzyme treatment is maintained at 25 to 37 占 폚.

The fat tissue precipitate treated with the enzyme agent can be centrifuged to separate the cell-containing fat layer. At this time, it is preferable that the centrifugation condition is 420g to 480g. The lowest layer among the lipid layers separated by centrifugation includes the AD-SVF of the present invention.

In order to remove the remaining adipose tissue from the cell-containing fat layer obtained after the centrifugation and to separate pure AD-SVF, a filtering method and a cell separation method according to density can be used.

In the present invention, the step of obtaining AD-SVF from the cell-containing fat layer obtained by centrifugation can be preferably performed by filtering with a mesh filter.

Preferably, the filtering is performed by a 100 to 150 mm mesh filter.

The AD-SVF obtained according to the above method was found to have expression of CD14, CD34, CD36 and / or CD146 of 3% or more. In addition, it was confirmed that the expression level of IL-8, which is known to be an important factor for vascular or tissue regeneration, is about 3,000 times or more as compared with human adipose derived mesenchymal stem cell (AD-MSC).

AD-SVF obtained according to the method of the present invention provides more cell count and superior cell survival rate from adipose tissue under the same conditions as AD-SVF obtained by the conventional method. Thus, even a small amount of adipose tissue can obtain AD-SVF of sufficient quality and quality to be transplanted into a patient.

The present invention also provides a cell therapy agent comprising AD-SVF obtained by the above method as an active ingredient.

The cell therapeutic agent according to the present invention may comprise a pharmaceutically acceptable carrier. The type of pharmaceutically acceptable carrier suitable for parenteral administration (e.g., intravenous, subcutaneous, or topical administration) is well known in the art.

The dosage of the cell therapy agent comprising the adipose tissue-derived substrate blood vessel fraction according to the present invention as an active ingredient can be determined depending on the administration method, age, body weight, sex, pathological condition, food, administration time, administration route, And the like. On the other hand, the dosage of the cell therapy agent containing the adipose tissue-derived substrate blood vessel fraction according to the present invention as the active ingredient is preferably 0.5 to 5 × ^{10 9} .

Since the method of separating the fat tissue-derived substrate blood vessel fraction according to the present invention provides an improved cell yield and cell survival rate compared to the conventional method, even a small amount of fat tissue can be easily transferred to a patient and a fat tissue- Fraction can be obtained. The adipose tissue-derived substrate blood vessel fraction obtained according to the above method can be used to treat various intractable diseases instead of existing cell therapy agents.

FIG. 1 is a graph showing the effect of interleukin-8 (IL) on human adipose-derived mesenchymal stem cells (AD-MSC), human Umbilical Vein Endothelial Cells (HUVEC), adipose tissue- -8). The results of the qRT-PCR are shown.

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.

Example 1: AD-SVF separation

Five fat samples obtained from liposuction were used to isolate AD-SVF according to the method of the present invention.

First, human abdomen or knee-derived fat tissue obtained by liposuction was cut into small pieces and washed with PBS (phosphate buffer saline) solution containing anticoagulant. The washed fat tissue was treated with a lyophilized enzyme preparation (collagenase-I; 99%, Accutase; 1%) for 1 hour at 37 ° C, and the resulting fat tissue precipitate was centrifuged The fat layer was separated. Separated lipid layers separated only the cells in the lowest layer among them. Separated cells were sprayed with PBS five times, filtered with a 100 mm mesh filter, and finally AD-SVF was isolated.

In addition, the same fat sample was separated from the AD-SVF using an automated cell sorting device from Celtdis (herein referred to as " conventional method "). Table 1 shows the cell numbers and cell viability of AD-SVF obtained according to the present invention in comparison with AD-SVF obtained by the conventional method. At this time, cell number and cell viability were measured with a cell automatic measuring device NC-200.

Compared with conventional methods The method of the present invention Cell growth rate (%) 41 ± 25 Cell survival rate (%) 23 ± 17

As can be seen in Table 1, according to the method according to the present invention, the cell number was increased by 41 ± 25% and the cell viability by 23 ± 17% compared with the conventional method.

Example 2: Fluorescence-activated cell sorting (FACS) analysis

AD-SVF obtained according to the method of the invention of Example 1 was suspended in PBS (Cellgro, Manassas, MD) and directly incubated with PE- or FITC-conjugated antibodies for 20 minutes at 4 ° C. Antibodies can be produced by a variety of methods including, but not limited to, anti-CD4, anti-CD14, anti-CD-25, anti-CD-31, anti-CD-34, anti-CD- Respectively. At this time, the corresponding isotype-identical IgG was acted as a negative control. After cell staining, cell composition FACS analysis was performed using a flow cytometer (BD). The results are shown in Table 2.

Antibody CD4 CD14 CD25 CD31 CD34 CD36 CD90 CD105 CD146 % 0.3 ± 0.1 4.5 ± 2.3 0.4 ± 0.2 0.9 ± 0.3 5.2 ± 3.7 3.1 ± 2.5 17.3 ± 5.1 1.5 ± 0.9 5.6 ± 3.2

FACS analysis showed that AD-SVF cells expressed less than 1% of blood cell markers (CD4), 17.3% of CD90, markers of mesenchymal stem cells, and 5.2% of CD34, a marker of hematopoietic stem cells . Therefore, since the content of mesenchymal stem cells and hematopoietic stem cells is more than 5%, it is useful to be used as a cell therapy agent.

Example 3: Quantitative RT-PCR (qRT-PCR) analysis

In order to quantitatively confirm the expression of IL-8 (IL-8) in AD-SVF obtained according to the method of the present invention in Example 1, qRT-PCR analysis was performed, and the result was expressed in human adipose derived mesenchymal stem cells (AD-MSC ) And IL-8 (IL-8) expression of human Umbilical Vein Endothelial Cells (HUVEC).

(AD-MSC) and Human Umbilical Vein Endothelial Cells (HUVEC), respectively, using the RNA-stat protocol (Iso-Tex Diagnostics, Friendswood, TX) RNA (total RNA) was isolated. RNA extracted in equal volumes from each cell was then reverse transcribed using Taqman reverse transcription reagent (Applied Biosystems, Foster City, CA) for cDNA synthesis. For real-time reverse-transcription-polymerase chain reaction (RT-PCR), human-specific primers and probes were used. The amount of RNA was quantitated using the ABI PRISM 7000 Sequence Detection System. After relative normalization to the housekeeping control gene GAPDH and correlation to the calibrator, the relative expression level of the target gene in the experimental sample was determined using the formula Rel Exp = 2 ^{- ΔCT} (difference in multiple) where ΔCt = (Ct of the target gene) - (endogenous control gene, Ct of GAPDH). The number of PCR cycles was measured using Lightcycler 3.5 software (Roche Molecular Biochemicals, Indianapolis, Ind.).

(IL-8) in the AD-SVF group compared to human adipose-derived mesenchymal stem cells (AD-MSC) and human umbilical vein endothelial cells (HUVEC) (3.6 ± 0.2; HUVEC, 0.6 ± 0.03; AD-MSC, 2141 ± 96; AD-SVF) (Fig. Therefore, high expression of IL-8, a vascular or tissue regenerating agent, is useful for the treatment of ischemic diseases.

Claims (11)

The adipose tissue was washed with PBS (phosphate buffer saline) containing anticoagulant,
Collagenase in washed fat tissue; And at least one enzyme selected from the group consisting of dyspazyme and accutase < ( ^R ) >
The fat tissue precipitate treated with the enzyme agent is centrifuged to separate the cell-containing fat layer,
Comprising separating adipose-derived stromal vascular fraction (AD-SVF) from a cell-containing fat layer,
The AD-SVF expresses CD14, CD34, CD36 and CD146 in an amount of 3% or more, the expression level of IL-8 is 3,000 times or more as compared with human adipose derived mesenchymal stem cells,
The treatment concentrations of the collagenase and the accutase were 400 to 800 units / ml, respectively, and the treatment concentration was 1 to 2.4 units / ml,
A method for separating adipose tissue-derived substrate blood vessel fraction. The method according to claim 1,
Wherein said adipose tissue is derived from lower abdomen, calf, thigh, forearm, breast or knee adipose tissue. The method according to claim 1,
Wherein the tissue remover enzyme preparation is treated with the fat tissue washed in the stirrer for 0.5 to 6 hours. The method according to claim 1,
Wherein the content of the collagenase in the enzyme-degrading enzyme preparation is 95 to 99.9%. The method according to claim 1,
The tissue to Lyon enzyme preparation of the di-SPA kinase and another kinase Accu content of one or more enzymes selected from the group consisting of (accutase ^®) is from 0.1 to 5% of the adipose tissue-derived stromal vascular fraction of the separation process. The method according to claim 1,
Wherein the step of separating AD-SVF from the cell-containing lipid layer is performed by filtering with a mesh filter. The method according to claim 6,
Wherein the filtering is performed by a 100-150 mm mesh filter. delete delete delete delete

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