The limitation of self-repair and proliferation capacity of chondrocytes in cartilage reconstruction lead to alternative
search of cell source that can improve the auricular regeneration. Human adipose-derived stem cells (HADSC) are an
alternative cell source that have unique characteristics to self-renew and differentiate into various tissues making it
suitable for cell therapy and tissue engineering. This study aimed to examine the chondrogenic differentiation potential of
(HADSC) in monolayer culture by the presence of different transforming growth factor beta’s, TFG-β1, -β2 and -β3. HADSC
at passage 3 (1.5 × 105 cell/mL) were cultured in chondrogenic medium containing 5 ng/mL of different transforming
growth factor beta’s, TFG-β1, -β2 and -β3 for 7, 14 and 21 days. Data analysis was evaluated based on the growth
rate of cells, cells morphological changed, production of collagen type II and glycosaminoglycan sulphate (sGAG). The
quantitative RT-PCR was carried out to determine the chondrogenic, fibrogenic and hypertrophic gene expression levels.
Differentiation of HADSC into chondrocytes using TFG-β indicates the occurrence of the chondrogenesis process. The best
chondrogenic differentiation was observed in HADSC induced by TFG-β3 through the chondrocytes-like cells morphology
with cells aggregation and high production of proteoglycan matrices compared to other TGF-βs groups. Additionally,
the expression of chondrocytes-specific genes such as Type II collagen, Aggrecan core protein, Elastin and Sox 9 was
high. In conclusion, this study has showed that TGF-β3 is the potential growth factor in producing chondrogenic cells
for auricular cartilage tissue engineering.
Skin plays an important role in defense against infection and other harmful biological agents. Due to its fragile structure, skin can be easily damaged by heat, chemicals, traumatic injuries and diseases. An autologous bilayered human skin equivalent, MyDerm™, was engineered to provide a living skin substitute to treat critical skin loss. However, one of the disadvantages of living skin substitute is its short shelf-life, hence limiting its distribution worldwide. The aim of this study was to evaluate the shelf-life of MyDerm™ through assessment of cell morphology, cell viability, population doubling time and functional gene expression levels before transplantation. Skin samples were digested with 0.6% Collagenase Type I followed by epithelial cells dissociation with TrypLE Select. Dermal fibroblasts and keratinocytes were culture-expanded to obtain sufficient cells for MyDerm™ construction. MyDerm™ was constructed with plasma-fibrin as temporary biomaterial and evaluated at 0, 24, 48 and 72 hours after storage at 4°C for its shelf-life determination. The morphology of skin cells derived from MyDerm™ remained unchanged across storage times. Cells harvested from MyDerm™ after storage appeared in good viability (90.5%±2.7% to 94.9%±1.6%) and had short population doubling time (58.4±8.7 to 76.9±19 hours). The modest drop in cell viability and increased in population doubling time at longer storage duration did not demonstrate a significant difference. Gene expression for CK10, CK14 and COL III were also comparable between different storage times. In conclusion, MyDerm™ can be stored in basal medium at 4°C for at least 72 hours before transplantation without compromising its functionality.
Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.