Biomedical Engineering Reference
In-Depth Information
Skin biopsy
Isolate &
culture cells
Keratinocytes
Melanocytes
Endothelial cells
Fibroblasts
Biopolymer
substrate
Cultured skin substitute
12.1
Schematic of the general process for tissue engineering of skin. A
small biopsy of split-thickness skin is separated using enzymes and/or
mechanical disaggregation to isolate epidermal keratinocytes and
dermal fibroblasts. Epidermal melanocytes and dermal microvascular
endothelial cells may also be isolated and cultured. Skin cells are
grown into large populations and inoculated onto collagen-GAG
substrates. Incubation of the skin substitutes at the air-liquid interface
promotes epidermal differentiation and morphogenesis of the cells into
an analog of human skin that may be transplanted as a graft to ex-
cised, full-thickness wounds.
12.3.1
Collection, isolation and expansion of cells from a
patient
To fabricate autologous engineered skin substitutes, a split-thickness skin biopsy
(~250-300 µm, 0.010-0.012 in thick) from a patient is collected. From this initial
biopsy, keratinocytes, fibroblasts, melanocytes and microvascular endothelial
cells can be isolated by serial disaggregation using enzymatic and mechanical
techniques.
40
Each cell type is placed into culture in a selective growth medium
(
Fig. 12.2
). For current chemical use of CSS, primary cultures of keratinocytes and
fibroblasts are grown to sub-confluence to generate stocks of cells that are
cryopreserved by controlled rate freezing and subsequently stored in liquid or gas
phase nitrogen at -196 ºC. Cells are recovered by rapid thawing and are then
inoculated into the intermediate cultures in conventional flasks at a calculated
