Biopatterning of Keratinocytes in Aqueous Two-Phase Systems as a Potential Tool for Skin Tissue Engineering
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Biopatterning of Keratinocytes in Aqueous Two-Phase Systems as a Potential Tool for Skin Tissue Engineering Rishima Agarwal1, Kristin Robin Ko1, Paul F. Gratzer1 and John P. Frampton1 1 School of Biomedical Engineering, Dalhousie University, Halifax, NS, B3H 4R2, Canada ABSTRACT Extrusion-based bioprinting (EBP) is limited by loss of pattern fidelity when printing on wet substrates. This can be overcome using aqueous two-phase systems (ATPSs) as novel ink formulations for EBP. In this study, optimal concentrations of ATPS “inks” were determined and used to pattern human epidermal keratinocyte (HEK001) colonies on a wet substrate for promoting epidermal growth. Four equilibrated and non-equilibrated ATPS formulations were tested for stable ATPS formation and uniform cell patterning. We identified an optimal formulation that produced stable droplets on a standard tissue culture plate coated with PEG. This process was also tested on an acellular dermal matrix (DermGEN™) to evaluate biopattern fidelity on a tissue matrix. Cell proliferation and formation of adherens junctions between cells were analyzed by immunocytochemistry. Non-equilibrated 5.0% PEG and 5.0% DEX solutions formed tighter colonies than equilibrated solutions containing identical total polymer concentrations. Cells patterned in colonies displayed higher cell viability and increased formation of E-cadherin junctions compared to non-patterned cells. Finally, when the cells were patterned on DermGEN™, discrete cell colonies were observed. This suggests that ATPS EBP holds promise for biopatterning epidermal keratinocyte cells to improve skin tissue engineering. INTRODUCTION Bioprinting allows precise positioning of a fluid containing cells to analyze cell-cell interactions, cell migration, cell proliferation, and cell differentiation [1]. Commonly used bioprinting approaches include laser-assisted bioprinting (LaBP), inkjet-based bioprinting (IBP), and extrusion-based bioprinting (EBP). While LaBP and IBP are very efficient, they can harm cells and can only print bioinks over a limited viscosity range [2]. Moreover, LaBP and IBP require highly specialized equipment and expertise. EBP overcomes many of these limitations. This technique uses pneumatic or mechanical fluid displacement to extrude a fluid through a nozzle [3]. EBP systems typically require less complex instrumentation and are compatible with a broad range of viscoelastic bioinks. Due to the versatility and affordability of this technique, extrusion-based printers have been used to print a variety of cells [4] and tissue constructs [5]. For instance, Kim et al. used a pneumatic driven extrusion-based system to print a collagen and alginate scaffold on a cryogenic stage. This scaffold was then co-cultured with keratinocytes and fibroblasts to fabricate a functional dermal skin substitute [6]. Despite these benefits, the major limitation of EBP is that the printed pattern fidelity is sometimes lost when the bioink is deposited in cell culture medium or on a wet substrate. Thus, there is a need for
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