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Proliferation and Growth Factor Exression of Human Keratinocytes on Microcarrier Beads
Author(s) -
Williams R.,
Rees R.S.,
Lindblad W.J.
Publication year - 2004
Publication title -
wound repair and regeneration
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.847
H-Index - 109
eISSN - 1524-475X
pISSN - 1067-1927
DOI - 10.1111/j.1067-1927.2004.0abstractbk.x
Subject(s) - microcarrier , trypsinization , keratinocyte , cell growth , growth factor , chemistry , staining , microbiology and biotechnology , platelet derived growth factor receptor , cell counting , cell culture , flow cytometry , cell , andrology , biology , cell cycle , trypsin , in vitro , biochemistry , medicine , genetics , receptor , enzyme
Human keratinocytes have been extensively used for the treatment of large body surface area burn injuries. We have studied the potential for treating non‐thermal, chronic wounds with keratinocytes grown on microcarrier beads placed into porous retrievable bags. The current studies were undertaken to examine the growth characteristics of these cells on Cytoline‐1™ microcarrier beads, and how this growth correlated with the expression of growth factors. Human keratinocytes were expanded in Keratinocyte Basal Medium‐2 with standard supplements (Clonetics, Inc.). The cells were trypsinized and added to Cytoline‐1™(Pharmacia Biotech) beads at 13.3 × 10 6 cells per mL pre‐equilibrated beads. The beads were seeded into roller bottles and cultured in a humidified 37° C incubator for up to two weeks. Cell numbers were monitored by direct cell counting on the beads following hematoxylin staining; and by protein and DNA content. Aliquots of media were also assayed for levels of PDGF and VEGF by immunoassay (Quantikine R , R&D Systems). Keratinocytes showed a lag in growth with a sustained decrement in cells per bead from 456 to 346 cells/bead by day 7. This was followed by a robust growth response to 551 cells/bead by day 11. These values correlated with total cellular protein (994, 738, and 1164 ug); and DNA content. The secretion of PDGF and VEGF by human keratinocyte under these conditions, did not directly correlate to the cell number but rather indirectly to the increase in cell number after the 7 days of stagnant growth. These results suggest that the vulnerary effect observed with human keratincytes on microcarrier beads is dependent on acculturation of the cells to the microbead environment, after which they begin to express elevated levels of growth factors important for healing.