Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells.
BACKGROUND
Before the potential of adipose tissue-derived stem cells can fully be exploited for a broad scope of tissue-engineering and cell-based therapeutical applications, an effective and reproducible method for isolation is needed.


OBJECTIVE
To comparatively analyze five highly defined protease formulations, Blendzyme 1-4, liberase H1 and a crude collagenase mixture in the course of digestion that consisted of three 1-h intervals.


METHODS
The resulting digests of human adipose tissue aspirates were evaluated for the yield of nucleated cells, viability and frequency of specific lineages, in particular CD90, CD34 and CD45, by flow cytometry. The functionality of the cells was assessed as to the colony-forming capacity in limiting dilution assays.


RESULTS
Based on all evaluation criteria, Blendzymes 1 and 2 and liberase H1 demonstrated a superior performance and highest consistency. Blendzyme 3 clearly underperformed compared with all other enzymes, and the performance of the rest of enzymes appeared erratic. As for the length of digestion, a 2-h interval appeared optimal when weighing both the yield and functionality of the cells in the stromal vascular fractions obtained from different adipose tissue samples.


CONCLUSIONS
Our results demonstrate that the highly purified proteases provide a valuable alternative to crude collagenase preparations, especially in scenarios where a high definition and reproducibility of the digestion process is of importance.
Pilgaard
Linda
L
Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg, Denmark.
Lund
Pia
P
Rasmussen
Jeppe G
JG
Fink
Trine
T
Zachar
Vladimir
V
D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D018952:Antigens, CD34 / Q000096:biosynthesis; D017364:Collagenases / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008875:Middle Aged; D008954:Models, Biological; D044968:Regenerative Medicine / Q000379:methods; D013234:Stem Cells / Q000166:cytology*; D018800:Thy-1 Antigens / Q000096:biosynthesis; D023822:Tissue Engineering / Q000379:methods*
D018952:Antigens, CD34; D018800:Thy-1 Antigens; D017493:Leukocyte Common Antigens; D017364:Collagenases