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

37C

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

  • Adipogenic
  • Chondrogenic
  • Osteogenic

ASC tri-lineage differentiation

  • Adipogenic: confirmed
  • Chondrogenic: confirmed
  • Osteogenic: confirmed

ASC Immunophenotype

  • +: CD90, CD73,CD105,CD44
  • -: CD11b,CD19,CD34,CD45,HLA-DR

ASC Immunophenotype

  • +: CD90, CD73: confirmed
  • +CD105: confirmed
  • +CD44: confirmed
  • -: CD11b,CD19,CD34,CD45,HLA-DR:

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.