Medium Flow Rate Regulates Viability and Barrier Function of Engineered Skin Substitutes in Perfusion Culture

Abstract

Perfusion culture of engineered tissues improves mass transfer of nutrients and provides flow-mediated mechanical stimulation to the developing constructs, thereby improving their anatomy and physiology in vitro. In this study, the responses to medium flow rate of engineered skin substitutes (ESS) incubated in perfusion at the air-liquid interface were investigated. ESS fabricated with autologous keratinocytes, fibroblasts, and collagen-glycosaminoglycan (GAG) sponges were incubated for 21 days at the air-liquid interface in a custom-built recirculating bioreactor system at flow rates of 5, 15, and 50 mL/min (n = 8 per condition). ESS were evaluated in vitro using histology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) incorporation, and surface hydration. ESS incubated at 5 and 15 mL/min had histological organization comparable with that of control ESS incubated in static conditions. ESS incubated at 50 mL/min displayed a disorganized epidermal substitute and, at later time points in culture, showed greater degradation of the dermal scaffold. Cell viability measured using MTT assay was significantly higher in ESS incubated at 5 mL/min than in static controls at day 14 (mean +/- standard error of the mean 1.63 +/- 0.11 vs 1.30 +/- 0.14, p < 0.05) and day 21 (1.66 +/- 0.12 vs 1.11+/- 0.15, p < 0.05) of culture. Viability of ESS incubated at 15 mL/min was comparable with that of controls. ESS incubated at 50 mL/min had significantly lower viabilities than controls at all time points. Results of BrdU incorporation data showed that, although ESS incubated at 5 and 15 mL/min were comparable with controls, those incubated at 50 mL/min had fewer proliferating keratinocytes per high-power field than controls (2.77 +/- 0.48 vs 28.1 +/- 0.78, p < 0.05). ESS incubated at 5 mL/min had surface hydration comparable with that of controls, whereas those incubated at 15 mL/min and 50 mL/min had significantly higher surface hydration than static controls at all time points. ESS incubated at a 5 mL/min flow rate and transplanted onto full-thickness wounds on athymic mice demonstrated wound healing comparable with that of controls. From these results, it can be concluded that perfusion culture of ESS at lower flow rates increases cell viability and maintains an epidermal barrier suitable for grafting, whereas higher flow rates lead to deterioration of ESS anatomy and physiology in vitro.

Epidermal differentiation governs engineered skin biomechanics

“…In contrast, the UCMC 161 medium used to culture skin (co-culture of fibroblasts and keratinocytes) was designed to promote epidermal proliferation and differentiation while maintaining fibroblast viability. Prior studies showing BrdU staining of ES culture in this medium indicated that the epidermis was highly proliferative but very few fibroblasts in the dermis were actively proliferating (Kalyanaraman et al, 2008). As the culture medium is formulated to maintain fibroblast viability and promote epidermal proliferation and differentiation, it is not surprising that dermal strength in our model was lower than that of studies that utilize DME-based mediums with FBS supplements where fibroblasts are highly proliferative and deposit large amounts of ECM.…”

The Integration of Nanotechnology and Biology for Cell Engineering: Promises and Challenges

“…Modifying the electrospinning parameters to achieve better porosity and larger pore dimensions has also been attempted. Increased flow-rates were found to increase the pore dimensions of synthetic human elastin nanofibrous scaffolds that were found to be superior to those scaffolds fabricated using low flowrates for skin tissue engineering applications [57]. The difference in the cell response to both scaffolds was attributed to the differences in the pore dimensions.…”

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

“…The re-endothelialization of a porcine jejunal segment, which subsequently could be perfused through the remaining arterial connection, still represents a fundamentally different approach than our experimental setup, arguably rendering the results incomparable to our study: We use a facilitated diffusion approach without a connective vasculature. Kalyanaram et al 35 focused on the effect of flow conditions on bi-layered skin grafts without any angiogenesis occurring in vitro. They revealed that barrier function of a skin graft, cultivated in a perfused bioreactor with a 5 mL/ min flow, increases significantly.…”