Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes |
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Authors: | Hongjun Wang Clemens A van Blitterswijk Marion Bertrand-De Haas Arnold H Schuurman Evert N Lamme |
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Institution: | (1) Institute of Biomedical Technology, Twente University, 7500 AE Enschede, The Netherlands;(2) IsoTis N.V., 3723MB Bilthoven, The Netherlands;(3) Department of Plastic, Reconstructive, and Hand Surgery, University Medical Center, Utrecht, The Netherlands;(4) Department of Dermatology, University Medical Center, St. Radbound Hospital, Rene Descartesdreef 1, 6525GL Nijmegen, The Netherlands |
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Abstract: | Summary The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure
to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability,
and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and
importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small
tissue fragments (2–3 mm3) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields
were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity
had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long
exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter
digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase
U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700
U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells
were seeded at 1×104 cells/cm2. These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability
but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable
time frame of 3 wk. |
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Keywords: | tissue digestion crude Clostridium histolyticum collagenase cell isolation fibroblasts |
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