COMPOSITION FOR RAPIDLY SEPARATING ADIPOSE TISSUE-DERIVED STROMAL CELLS

Abstract

The present invention provides a composition for isolating adipose-derived stromal cells, which comprises a Type I collagenase of 0.5-8% (v/v); a Trypsin of 0.1-0.6% (v/v); and a metal ion chelating agent of 0.01-0.2% (v/v). The present invention further provides a method for isolating adipose-derived stromal cells, which method comprises obtaining an adipose tissue; treating the adipose tissue with the composition of the present invention; centrifuging the adipose tissue; and isolating the adipose tissue to obtain the adipose-derived stromal cells. The present invention facilitates rapid isolation of mesenchymal stromal cells within a short period of time in operating rooms and can be applied to regenerative medicine in the future.

Claims

1. A composition for isolating adipose-derived stromal cells, which comprises a Type I collagenase of 0.5-8% (v/v); a Trypsin of 0.1-0.6% (v/v); and a metal ion chelating agent of 0.01-0.2% (v/v).

2. The composition according to claim 1, wherein the metal ion chelating agent is selected from ethylenediaminetetraacetic acid (EDTA) or sodium salts thereof, ethylene glycol-bisaminoethyl ether tetraacetic acid (EGTA) or sodium salts thereof, diethylene triamine pentaacetic acid (DTPA) or sodium salts thereof, polyphosphates, organic phosphates, phosphates, polyacrylates, organic phosphates, sodium gluconate, or mixtures thereof.

3. The composition according to claim 2, wherein the metal ion chelating agent is EDTA.

4. A method for isolating adipose-derived stromal cells, which comprises the steps of: (a) obtaining an adipose tissue; (b) adding the composition according to claim 1, homogenizing and allowing for reaction to obtain a digested tissue mixture, wherein the composition comprises a Type I collagenase of 0.5-8% (v/v); a Trypsin of 0.1-0.6% (v/v); and a metal ion chelating agent of 0.01-0.2% (v/v); (c) centrifuging the digested tissue mixture of step (b), removing impurities to obtain a first filtrate containing the adipose-derived stromal cells; (d) adding a hypotonic solution to the first filtrate of step (c) to obtain a second filtrate of hemocyte-free adipose-derived stromal cells; and (e) neutralizing the second filtrate of step (d) and centrifuging.

5. The method according to claim 4, wherein the metal ion chelating agent is selected from ethylenediaminetetraacetic acid (EDTA) or sodium salts thereof, ethylene glycol-bisaminoethyl ether tetraacetic acid (EGTA) or sodium salts thereof, diethylene triamine pentaacetic acid (DTPA) or sodium salts thereof, polyphosphates, organic phosphates, phosphates, polyacrylates, organic phosphates, sodium gluconate, or mixtures thereof.

6. The method according to claim 5, wherein the metal ion chelating agent is EDTA.

7. The method according to claim 4, which is further characterized in that the total reaction time for digesting an adipose tissue is one hour or less to obtain characteristics of the adipose-derived stromal cells.

8. The method according to claim 4, which has an effect of isolating at least one million adipose-derived stromal cells from 5 gram of adipose tissue.

9. The method according to claim 4, wherein the adipose-derived stromal cells are capable of differentiating into adipose cells, hematopoietic cells, vascular endothelial cells, osteoblasts, chondroblasts, nerve cells or epithelial cells.

Description

DETAIL DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows a schematic flow chart of one embodiment of the method for isolating adipose-derived stromal cells of the present invention.

[0028] FIG. 2 shows the results of the number of mesenchymal stromal cells isolated from 5 grams of subcutaneous adipose tissue extracted from the inner thighs of SD rats by different concentrations of enzyme formulations for one hour.

[0029] FIG. 3 shows the results of the number of mesenchymal stromal cells isolated from 5 grams of subcutaneous adipose tissue extracted from the inner thighs of guinea pigs by different concentrations of enzyme formulations for one hour.

[0030] FIG. 4 shows the differentiation capability analysis of the adipose-derived stem cells selected from the selected adipose-derived stromal cells.

[0031] FIG. 5 shows the expression level of biomarkers on the cell surface of the isolated adipose-derived stem cells.

[0032] FIG. 6 shows the effect of different concentrations of enzyme formulations on the survival of mesenchymal stromal cells.

[0033] FIG. 7 is a comparison when the content of the composition of the present invention is replaced with different species of enzymes.

DESCRIPTION OF THE CODINGS IN THE DRAWINGS

[0034] A 0.5% Type I collagenase+0.05% Trypsin+0.02% EDTA [0035] B 0.5% Type I collagenase+0.1% Trypsin+0.02% EDTA [0036] C 0.5% Type I collagenase+0.2% Trypsin+0.02% EDTA [0037] D 1% Type I collagenase+0.1% Trypsin+0.02% EDTA [0038] E 2% Type I collagenase+0.1% Trypsin+0.02% EDTA [0039] F 2.5% Type I collagenase+0.1% Trypsin+0.02% EDTA [0040] G 3% Type I collagenase+0.1% Trypsin+0.02% EDTA [0041] H 4% Type I collagenase+0.1% Trypsin+0.02% EDTA [0042] E 2% Type IV collagenase+0.1% Trypsin+0.02% EDTA [0043] F 2.5% Type IV collagenase+0.1% Trypsin+0.02% EDTA [0044] G 3% Type IV collagenase+0.1% Trypsin+0.02% EDTA [0045] I 3.5% Type IV collagenase+0.1% Trypsin+0.02% EDTA [0046] H 4% Type IV collagenase+0.1% Trypsin+0.02% EDTA

EXAMPLES

[0047] The present invention may be implemented in many different forms and should not be construed as limited to the examples set forth herein. The examples below are non-limiting and are merely representative of various aspects and features of the present invention.

[0048] The One-way Analysis of Variance (ANOVA) was used to test for statistical differences and the Scheffe's test was used for multiple comparisons. Significant difference is defined as a significant probability value (p-value) <0.05.

Example 1

Rapid Isolation of Mesenchymal Stromal Cells

[0049] The schematic flow chart as shown in FIG. 1 shows the rapid isolation of mesenchymal stromal cells from adipose tissues of Sprague-Dawley rats (SD rats) and Genia pigs to which the enzyme formulations of the present invention were applied.

[0050] 5 grams of adipose tissue were removed from the inner thighs of SD rats and guinea pigs, an equal amount (about 5 mL) of enzyme formulation buffer solution of the present invention (0.5-3% (v/v) of type I collagenase, 0.1-0.3% (v/v) of trypsin and 0.02% (v/v) of EDTA dissolved in a phosphate buffer solution (PBS)) was added; placed in a sterile tissue homogenizer (GentleMACs, Miltenyi Biotec, Bergisch Gladbach, Germany) to homogenize the tissues into single cells for 1 minute and then soaked in a 37 C. water bath to react for 10 minutes, then placed in a sterile tissue homogenizer again for 1 minute and soaked in a 37 C. water bath for 10 minutes. Subsequently, after it was centrifuged at (300g) for 10 minutes, the upper layer containing lipid, connective tissues, or non-homogenized tissues were removed, washed with PBS (5 mL) and then centrifuged (300g) for 10 minutes, an equal amount of hypotonic solution (5 mL) was added, stood at room temperature for 5 minutes to remove blood cells and then 10mL of PBS buffer solution was added to neutralize the reaction. The solution was removed after it was centrifuged at 3,000 rpm for 10 minutes, the cells in the lower layer were re-suspended in 1 mL of PBS and then sampled to count the number of cells. The total reaction time was less than one hour.

Example 2

Mesenchymal Stromal Cells Yield Analysis

[0051] 20 L was removed from the adipose-derived stromal cells sample isolated in Example 1, 20 L of Trypan blue was added, mixed thoroughly for staining, dripped into a cell counting dish to count viable cells (non-stained cells).

[0052] FIG. 2 shows the number of mesenchymal stromal cells isolated by different concentrations of enzyme formulations for one hour from 5 grams of subcutaneous adipose tissues removed from the inner thighs of SD rats. Results showed that when the enzyme formulation containing 3% (v/v) of Type I collagenase, 0.1% (v/v) of Trypsin and 0.02% (v/v) of EDTA (encoded as G) was used for one hour, the number of extracted cells was the highest, approximately 9><10.sup.6 cells/mL could be isolated.

[0053] FIG. 3 shows the number of mesenchymal stromal cells isolated by using different concentrations of enzyme formulations for one hour from 5 grams of subcutaneous adipose tissues removed from the inner thighs of guinea pigs. Results showed that when the enzyme formulation containing 3-4% (v/v) of type I collagenase, 0.1% (v/v) of Trypsin and 0.02% (v/v) of EDTA (encoded as G, H) was used for one hour, the number of extracted cells was the highest, approximately 810.sup.6 to 8.510.sup.6 cells/mL could be isolated.

Example 3

Differentiation Capability of Adipose-Derived Stem Cells After Isolation

[0054] According to the definition of mesenchymal stem cell published by the International Association of Mesenchymal Stem Cells in 2006, a mesenchymal stem cell has three characteristics: [0055] 1. the cell must be attached to a cell culture dish; [0056] 2. surface antigens are required to express CD105, CD73, or CD90 but not CD45, CD34, CD14, or CD11b, CD79a, or CD19, or HLA-DR; [0057] 3. after being induced, the mesenchymal stem cell is required to be capable of differentiating into adipocytes, osteoblasts and chondrocytes.

[0058] When the adipose-derived stromal cells isolated in Example 1 were cultivated in a selective medium (Kerationcytr-SFM; Product Number: 10724-001; GIBCO, New York, USA) (mesenchymal stem cells were selectively retained, non-mesenchymal stem cells underwent apoptosis), adipose-derived mesenchymal stem cells could be isolated after one week of cultivation. As shown in FIG. 4 (a), the isolated adipose-derived mesenchymal stem cells were adherent cells. When the isolated adipose-derived stromal cells were analyzed by using a flow cytometry, the expression levels of CD271, CD73 and CD90 on the cell surface were significantly higher than that of the control group, and the expression level of CD34 was lower than that of the control group, as shown in FIG. 5. Therefore, the isolated adipose-derived stromal cells belong to mesenchymal stem cells. The following further examined whether the mesenchymal stem cells were capable of being induced to differentiate into adipocytes, osteoblasts and chondrocytes.

[0059] After being subcultured, the isolated adipose-derived mesenchymal stem cells were cultured for two weeks in different differentiation-inducing culture media (Adipo-medium: a medium inducing differentiation into adipocytes; Osteo-medium: a medium inducing differentiation into osteoblasts; and Chondro-medium: a medium inducing differentiation into chondrocytes; wherein Adipo-medium: DMEM medium (Dulbecco's Modified Eagle Medium), 10% of fetal bovine serum (FBS), 1% of penicillin/streptomycin, 500 M of 3-isobutyl-1-methylxanthine IBMX, 1 M of dexamethasone, 1 M of indomethasin, 10 Mg/mL of insulin; Chondro-medium: DMEM medium (Dulbecco's Modified Eagle Medium), 10% of fetal bovine serum (FBS), 1% of penicillin/streptomycin, 50 nM of L-ascorbic acid-2-phosphate (L-Ascobate-2-phosphate), 6.25 g/mL of insulin, 10 ng/mL of TGF-; and Osteo-medium: DMEM medium (Dulbecco's Modified Eagle Medium), 10% of fetal bovine serum (FBS), 1% of penicillin Streptomyces, 50 M of L-ascorbyl-2-phosphate , 0.1 M of dexamethasone, 10 mM of -glycerophosphate (-glycerophosphate).

[0060] The differentiation capability of mesenchymal stem cells into chondrocytes was evaluated by using a glycosaminoglycan (abbreviated as GAG) assay, the GAG test was carried out with Alcian blue staining. After two weeks of cultivation in Chondro-medium, the old medium was removed and fixed with 10% of formalin or 4% of paraformaldehyde for 10 minutes. It is washed twice with distilled water (3 mL), and then shaken with 3% of acetic acid (3 mL) for 5 minutes. After all liquids were removed, Nelson Blue (1%) was added into the dish, shaken for 15 minutes, after the stain was removed and then washed with distilled water (3 mL) for 2-3 times, photos were taken.

[0061] As shown in FIG. 4(b), the extracellular matrix GAG was stained by Nelson blue, the stained GAG was blue and the cells aggregated, which were characteristics of chondrocytes.

[0062] The ability of the mesenchymal stem cells to differentiate into adipocytes was evaluated by staining oil droplets in the adipocytes with Oil Red O. Two weeks after the Adipo-medium incubation, the old medium was removed and fixed in 10% formalin or 4% paraformaldehyde for 10 minutes. Distilled water (3 mL) was used to wash for 2 to 3 times. After all the liquids were removed, Oil Red O (0.5%) was added into the dish, shaken for 10 minutes, distilled water (3 mL) was used to wash 2-3 times to remove stains. Photos were taken for record after distilled water was added.

[0063] As shown in FIG. 4(c), the oil droplets in the stained cells were in the form of red dots, which is a characteristic of the adipocytes.

[0064] Alizarin-red staining was used to examine the effect of isolated adipose-derived mesenchymal stem cells on osteogenesis. After two weeks of incubation in Osteo-medium, the old medium was removed and fixed in 4% formalin for 15 minutes. Distilled water (3 mL) was used to wash for two times. After all the liquids were removed, 2% bismuth red solution was added into the dish, reacted at room temperature for 20 minutes, photos were taken after distilled water was used to wash for 2-3 time to remove stains.

[0065] As shown in FIG. 4(d), the calcium deposition generated by the stained cells was in the form of red dots, which is a characteristic of osteoblasts.

Example 4

Effect of Different Concentrations of Enzyme Formulations on the Survival of Mesenchymal Stromal Cells

[0066] When the adipose-derived stromal cells isolated in Example 1 were cultivated in a selective medium (Kerationcytr-SFM; Product Number: 10724-001; GIBCO) (mesenchymal stem cells were selectively retained, non-mesenchymal stem cells underwent apoptosis), adipose-derived mesenchymal stem cells could be isolated after one week of cultivation. The following three preferred enzyme formulations which yielded more adipose-derived stromal cells in Example 1 were used to carry out citytoxicity tests: [0067] 1. enzyme formulation of 2.5% (v/v) Type I collagenase, 0.1% (v/v) Trypsin, and 0.02% (v/v) EDTA; [0068] 2. enzyme formulation of 2. 3% (v/v) Type I collagenase, 0.1% (v/v) Trypsin and 0.02% (v/v) EDTA; and [0069] 3. enzyme formulation of 4% (v/v) Type I collagenase, 0.1% (v/v) Trypsin and 0.02% (v/v) EDTA.
10.sup.5 adipose-derived mesenchymal stem cells were placed in these enzyme formulations and incubated for 0.5, 1 and 2 hours. After 0.5, 1 and 2 hours of cultivation, cells were removed and subjected to Trypan-blue staining for calculating cell survival rate. As shown in FIG. 6, the formulations did not show significant cytotoxicity.

Example 5

Comparison of Substitution of Different Enzymes

[0070] The enzymes in the enzyme formulations were replaced with similar but different species of enzyme while the concentrations remained the same: 2% (v/v) of Type 4 collagenase, 0.1% (v/v) of Trypsin, 0.02% (v/v) of EDTA; 2.5% of Type 4 collagenase, 0.1% of Trypsin, 0.02% of EDTA; 3% of Type 4 collagenase, 0.1% of Trypsin, 0.02% EDTA; 3.5% Type 4 of collagenase, 0.1% of Trypsin, 0.02% of EDTA; 4% of Type 4 collagenase, 0.1% of Trypsin, 0.02% of EDTA, and the adipose-derived stromal cells isolation method disclosed in Example 1 was used in this example. The results were shown in FIG. 7, the number of cells yielded by the formulation of the this example was unable to match the number of cells yielded by the formulation of 3% (v/v) Type 1 collagenase, 0.1% (v/v) Trypsin, and 0.02% (v/v) EDTA. Therefore, enzyme formulation of 3% (v/v) Type I collagenase, 0.1% (v/v) Trypsin, and 0.02% (v/v) EDTA was irreplaceable for rapid isolation of adipose-derived stromal cells.

[0071] The above content sets forth many specific details in order to fully understand the present invention, however, the present invention may be implemented in many forms different from what is described; one skilled in the art may modify and vary the examples without departing from the spirit and scope of the present invention, therefore, the examples should not be construed as the limitation of the claims.