(393e) Development of a Bioreactor System for Tissue Engineered Skin Substitutes with Integrated Convective Flow Networks

Of all tissue engineered (TE), TE skin substitutes have achieved great success in the treatment of wound healing or reconstructing injured. However, lack of vascularization that allows nutrients and oxygen delivery and waste removal between host blood vessels and TE skin substitutes after transplantation has been suggested as one of the limitation for current TE skin substitutes. The goal of this work is to design a perfusion bioreactor for TE skin substitutes and develop an in vitro TE skin substitutes with a built-in microchannels in it to eliminate this mass transfer limitation. A bioreactor, which consists of cell culture chamber and two polyvinylidene fluoride (PVDF) ports for submerged culture condition and two for air/liquid interface culture condition, has been designed for TE skin substitutes specifically with a built-in microchannels (Figure A and B). In the closed system, human keratinocytes (HKs) were differentiated on the collagen-glycosaminoglycan (CG) membrane with built-in microchannels. Medium was perfused into chamber using syringe pump. HKs were cultured in submerged condition for three days to reach confluence and then exposed to air/liquid interface for 14 days by perfusing medium through closed flow network with perfusion rate of 100 μl/hr. TE skin substitutes were cultured in six-well tissue culture plate in static condition as a control. The efficacy of microchannels was evaluated from epithelial organization from histology results. Viability of HKs was assessed by performing 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and proliferation was also be evaluated from bromo-deoxyuridine (BrdU) results. Figure 1. (A) Schematic diagram of the bioreactor design. The culture system consists of (1) a culture chamber, (2) PDMS flat membrane, (3) CG membrane with built-in microchannels, (4) microchannels for internal flow, and (5) gas permeable FEP membrane. For submerged culture, medium are delivered into chamber with (6) syringe and drained to (9) collection bottle through (7) inlet and (8) outlet respectively. For air/liquid interface culture, medium are pumped into chamber with (10, 11) syringe pump and drained to (14) collection bottle through (12) inlet and (12) outlet respectively. (B) Bioreactor setup with syringe pump. (C) Microfluidics in composite CG networks inside bioreactor.