Figure 1IMMUNOPHENOTYPING OF CULTURED ADIPOSE-DERIVED STEM CELLS (ASCS) BY FLOW CYTOMETRY, DIAGNOSTIC FOR ASCS
Row A: Scatterplots displaying respective gating to singlets. Row B: Scatterplots and histograms displaying positive immunophenotypes (in the "Cocktail"): CD44 (row B), CD73 (row C), CD 90 (row D), and CD105 (row E).-
Figure 2GROWTH OF ADIPOSE-DERIVED STEM CELLS
Representative bright-field 10x micrograph illustrating plate adherence and colony formation of adipose-derived stem cells.
Figure 3TRILINEAGE DIFFERENTIATION
A. ADIPOCYTE DIFFERENTIATION: Representative bright-field 30x micrograph illustrating lipid droplet staining of adipose-derived stem cells differentiated to adipocytes. B. OSTEOBLAST DIFFERENTIATION: Representative bright-field 15x micrograph illustrating alkaline phosphatase staining of adipose-derived stem cells differentiated to osteoblasts. C. CHONDROBLAST DIFFERENTIATION: Representative indirect micrograph illustrating a sulfated proteoglycan sphere stained with alcian blue & nuclear fast red indicating adipose-derived stem cells differentiated to chrondroblasts. Sphere diameter 2mm.
| 1. Sterile Work-area Mat (1) |
| 2. Biohazard Bag (1) (Biohazard cartridge in Kit) |
| 3. Transfer Hubs (4) (standard) |
| 4. Alcohol Wipe (1) |
| 5. Vial-to-Syringe Adapters (8) |
| 6. 60mL Trituration Syringes (2) (standard and pre-labeled) |
| 7. 35mL Transfer Syringes (4) (patented and pre-labeled) |
| 8. 20mL Injectable Normal Saline (8) (pre-measured and pre-labeled) |
| 9. 35mL Concentrate Syringe (1) (standard and pre-labeled) |
| 10. 10 ML PBS (1) (pre-measured and pre-labeled) |
| 11. Corase® Aliquot (1) (prepared during a pre-operative protocol) |
| 12. 35mL Corase® Syringe (1) (standard and pre-labeled) |
Table 1: Kit contents
Processing Quantity |
Processing Quantitative Standard |
Volume of unconcentrated lipoaspirate |
70 mls |
Volume of saline |
160 mls |
Mass / activity of Corase® (collagenase) |
1 mg / 19-32 Wunsch Units |
Centrifugation, time |
3 minutes |
Centrifugal force (x g) |
340 |
Incubation, time |
20 minutes |
Incubation, temperature |
37⁰C |
Incubation, tilt level |
6 |
Incubation, tilt speed |
30 |
Table 2. Processing Quantitative Standards
Processing Quality |
Processing Qualitative Standard |
Collagenase: CGMP grade |
Confirmed |
Processing time of ≤30 minutes |
30 minutes |
Collective weight of kit contents ≤2 lbs. |
1.63 lbs. |
Transparency of kit components |
Confirmed |
Modular design of kit to allow configuration for a variety of protocols |
Confirmed |
Maintains continuously closed sterile processing environment |
Confirmed |
Table 3. Processing Qualitative Standards
Minimum CQA targets |
Results |
SVF with >70% viability of nucleated cells |
90.7% |
SVF with >10,000 live nucleated cells per ml of concentrated lipoaspirate enzymatically processed |
58,000 live nucleated cells per ml of concentrated lipoaspirate enzymatically processed |
ASC plastic adherence |
ASC plastic adherence: confirmed |
ASC tri-lineage differentiation
|
ASC tri-lineage differentiation
|
ASC Immunophenotype
|
ASC Immunophenotype
all confirmed |
Endotoxin level: <0.01 EU/ml |
Confirmed: <0.004 EU/ml |
Table 4. The minimum Critical Quality Attributes (CQA) targets and results of isolation of Stromal Vascular Fraction and Adipose-Derived Stem Cells from lipoaspirate.