Promoting the Selection and Maintenance of Fetal Liver Stem/ Progenitor Cell Colonies by Layer-by-Layer Polypeptide Tethered Supported Lipid Bilayer I-Chi Lee, † Yung-Chiang Liu, † Hsuan-Ang Tsai, ‡,§ Chia-Ning Shen, ‡ and Ying-Chih Chang* ,‡ † Graduate Institute of Biochemical and Biomedical Engineering, Chang-Gung University, Tao-yuan 333, Taiwan, R.O.C. ‡ Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, R.O.C. * S Supporting Information ABSTRACT: In this study, we designed and constructed a series of layer-by-layer polypeptide adsorbed supported lipid bilayer (SLB) films as a novel and label-free platform for the isolation and maintenance of rare populated stem cells. In particular, four alternative layers of anionic poly-L-glutamic acid and cationic poly-L-lysine were sequentially deposited on an anionic SLB. We found that the fetal liver stem/progenitor cells from the primary culture were selected and formed colonies on all layer-by-layer polypeptide adsorbed SLB surfaces, regardless of the number of alternative layers and the net charges on those layers. Interestingly, these isolated stem/progenitor cells formed colonies which were maintained for an 8 day observation period. Quartz crystal microbalance with dissipation measurements showed that all SLB-polypeptide films were protein resistant with serum levels significantly lower than those on the polypeptide multilayer films without an underlying SLB. We suggest the fluidic SLB promotes selective binding while minimizing the cell-surface interaction due to its nonfouling nature, thus limiting stem cell colonies from spreading. KEYWORDS: label free, supported lipid bilayer (SLB), layer-by-layer polypeptide films, fetal liver stem/progenitor cells, rare cell isolation, stem cell maintenance ■ INTRODUCTION There is a strong incentive to purify, enrich, maintain, and facilitate production of a large quantity of stem cells for a wide variety of biological and medical applications. It is known that the microenvironment interactions, such as chemical stimulant induced by soluble signaling molecules, electric fields, and mechanical force mediated through integrin-mediated cell- matrix and cell-cell interactions, 1-4 are critical for the stem cell fate. Previously, selective culturing, flow cytometric sorting, and magnetic-activated cell sorting 5-8 have been applied as the first step to isolate the stem cells from primary cells. These affinity- based methods require well-defined biomarkers to isolate scarce stem cells from the entire population and are unusable when a target cell lacks effective biomarkers. Therefore, it is considered that variation of the physical microenvironment could regulate stem cell behaviors and may offer opportunities for stem cell selection that does not require biomarkers. Surface properties, such as surface charges, hydrophilicity, protein surface modification, and surface roughness, have been evaluated for their effects on cell adhesion and stem cell behavior via integrin mediated interactions. 9,10 A layer-by-layer technique for making polyelectrolyte multilayer (PEM) films 11 offered a simple method to prepare highly tunable thin films and versatile tools to form a series of surfaces with adjustable properties. It has been shown that, by varying the electrolytes or process conditions, 12-16 the properties of the films, including thickness, rigidity, chemical composition, hydrophilicity, and surface charges, could be altered at will. 17-19 Among the PEM films that have been used, recent studies have used PEM films composed of the synthetic polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA) to explore the effects of film properties on cells. 20,21 We have used PLL/PLGA PEM films to successfully identify the optimal conditions for selecting and maintaining fetal mouse liver stem/progenitor cells (FLSPCs) colonies. 22,23 Specifically, it was found that the FLSPCs form colonies and are maintained on those thick films, while adhesive cells such as fibroblast and hepatocytes tend to be adhesive and spread on thinner, rigid PEM films. For a prolonged period of culture up to 8 days, the FLSPCs colonies maintain small colonies on the thick films, while the majority of FLSPCs differentiated and spread on the thin films. With further investigation on the surface properties of these films, it was discovered that thick films that promote the FLSPCs colony formation and maintenance are more hydrated, elastic, and protein resistant than those thin films, while chemical Received: June 19, 2014 Accepted: September 22, 2014 Published: September 22, 2014 Research Article www.acsami.org © 2014 American Chemical Society 20654 dx.doi.org/10.1021/am503928u | ACS Appl. Mater. Interfaces 2014, 6, 20654-20663