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The efficacy of human autologous adipose mesenchymal stem cells isolated with cGMP collagenase to obtain osteogenic-bone like tissue

Daela Xhema, Najima Aouassar, Rose-Marie Goebbels, Denis Dufrane

Endocrine Cell Therapy, University Clinical Hospital Saint-Luc/UCL, Brussels, Belgium

Background: Multipotent adipose-derived mesenchymal stem cells (AMSCs) can be isolated from animal and human subcutaneous tissue for osteogenic differentiation and bone tissue engineering. However, the collagenase digestion of adipose tissue remains a crucial step in view to obtain a maximal yield of stem cells in Good Manufacturing Practice (cGMP) for cell banking. This study investigated the impact of collagenase (crude or GMP collagenase) on human AMSCs in term of isolation, expansion and osteogenic-differentiation.

Methods: Human adipose tissues were digested with a standard static digestion method using GMP- (provided by Serva, n=12) or crude-collagenase (Sigma, n=7) and were compared to pre-clinical pig AMSCs isolation with crude collagenase (n=8) at the concentration of 1.5 mg/ml. Time course of Proliferation Phase (PP, Passage 0^th to Passage 4^th) and Osteogenic-Differentiation Phase (DP), AMSCs cellular yield at passage 4^th and the osteogenic phenotype expression (Red Alizarin, Osteocalcin,Von Kossa staining) were assessed for each experimental groups.

Results: No significant difference of adipose tissue weight (prior digestion) was observed between groups (p=0.784). Human AMSCs (isolated with GMP collagenase) demonstrated a significant shorter PP than pig AMSCs (28±10 vs. 60±25 days, p=0.003, respectively). No significant difference of proliferation phase was found for human AMSCs isolated with both GMP and crude collagenase.

A significant lower yield of AMSCs was found at Passage 4^th (after PP) from human adipose tissues isolated with crude collagenase in comparison to GMP collagenase (2.3±0.6 x10⁶ vs. 9.8±3.5 x10⁶ cells, respectively, p=0.025). No significant difference of time (required for DP) was observed between experimental groups (mean of 30 days, p=0.131). All differentiated AMSCs expressed osteogenic phenotype with mineralization process at the end of the differentiation phase.

Conclusions: cGMP clinical grade collagenase can provide a safety procedure to obtain rapidly a high yield of human AMSCs differentiated in osteogenic-like tissue for bone reconstruction.