Nunclon Sphera surface supports formation of embryoid bodies from pluripotent stem cells

Keeping stem cells in an undifferentiated state is important for expansion and maintenance. However studying the process that stem cells follow to become body tissues requires them to differentiate. The formation of spheroids is an important milestone in the differentiation process. Here we introduce a new cell culture surface, Thermo ScientificTM Nunclon Sphera that supports the in vitro formation of stem cell spheroids. The Nunclon Sphera surface coating inhibits cell attachment to the culture dish by blocking the adsorption of extracellular matrix (ECM) proteins that usually mediate cell adhesion, thereby promoting cell-cell aggregation in vitro. The low binding property of Nunclon Sphera surface was demonstrated by low cell attachment of adherent cell lines (e.g. Vero, A549, and U937) as well as by the formation of embryoid bodies (EB) of both human embryonic stem cells (hESCs) and mouse embryonic stem cells (mESCs). EB formation in Nunclon Sphera dishes was compared to either non-treated or cell culture treated dishes. The integrity and consistency of the Nunclon Sphera surface was also showcased through the differentiation of cells derived from the embryoid bodies. These results demonstrate the advantage of the Nunclon Sphera surface for embryoid body formation in the process of pluripotent stem cell differentiation. Introduction Stem cells are often used in research due to their ability to become multiple different cell types. The formation of spheroids in suspension cultures is an intermediate step in producing different cell lineages from pluripotent stem cells and other progenitor cells. The variability seen in experiments using spheroid-forming cultures has been linked to medium composition and volume, cell density, and duration in culture. While these factors all affect the culture, the surface of the culture dish is one of the most critical factors contributing to the success of spheroid formation in vitro. On cell culture surfaces where cell attachment is required, non-covalent interactions between extracellular proteins and the functional groups within the molecular structure of the surface bind the proteins to the culture surface. The cells then use these bound proteins as anchor points to attach to the surface substrate. In cultures where spheroids are desired, this cellular attachment must be prevented in order to promote the cell-cell aggregation required for spheroid formation. Although several different surfaces that offer properties of low adhesion are commercially available, spontaneous cell differentiation resulted from 2 random cell attachment is still a challenge to many researchers. The novel Nunclon Sphera surface by Thermo Fisher Scientific effectively prevents protein adsorption and allows cells to grow in suspension with virtually no cell attachment, providing superior performance over other low binding surfaces. Materials and methods ECM Protein Adsorption Nunclon Sphera 96-well plates and Nunclon Delta (standard cell culture-treated) 96-well plates were coated with 100 μL/well of either 24 μg/mL FITC labeled Bovine Collagen Type I or 20 μg/mL of TAMRA labeled Fibronectin in DPBS. The plates were incubated for 24 hours at 2-8°C or 16 hours at room temperature, respectively. The solution was aspirated and plates were washed 3 times with 200 μL/well PBST (0.05% Tween 20 in PBS). The fluorescence intensity was read at Ex495/ Em525 (Collagen) or Ex543/Em570 (Fibronectin) on a fluorescent plate reader. Stem Cell Line Cultivation Human and mouse ESC lines (Hes-3, B6N #1) were maintained in the appropriate HyClone media containing 20% serum (mESC) or 10% serum replacement (hESC), non-essential amino acids, penicillin/streptomycin, and beta-mercaptoethanol. Cultures were incubated at 37°C and 5% CO2. For passaging, the cells were released from the surface using HyQTase (HyClone). Spheroid Formation Stem cells were seeded into Nunclon Sphera, non-treated, and standard cell culture treated 6-well multidishes at a density of 6.3x104 cells/well (hESC) or 4x104 cells/well (mESC) in the appropriate HyClone media containing Rho-kinase inhibitor. Cells were incubated at 37°C and 5% CO2. For hESCs, fresh media was added after 2 days, and media was replaced with fresh media after 3 days. For mESCs, media change was performed after 2 days. After 7 days in culture, cells were imaged, and the number of EBs and spheroid morphology was assessed. Cell viability was also determined using a NucleoCounter (Chemometec). Differentiation After EB formation, cells were transferred to gelatincoated 12-well Nunclon Delta multidishes, cultured in the appropriate media with Rho-kinase inhibitor, and allowed to grow for 7 days with media changes every 2-3 days (mESC) or for 14 days with media changes every 3-4 days (hESC). Cells were then analyzed by immunocytostaining using markers for the three germ layers: alpha fetoprotein (AFP) for endoderm, beta-tubulin for ectoderm, and smooth muscle actin (alpha-SMA) for mesoderm. Results and discussion The adsorption of ECM proteins to the Nunclon Sphera surface is extremely low, resulting in low attachment of the adherent cells Common ECM proteins are known to mediate cell attachment to culture surfaces. In order for adherent cells to form spheroids in suspension, the culture vessel must encourage the aggregation of cells through cell-cell binding by preventing ECM binding to the plastic surface. In this study, the binding of both Collagen I and Fibronectin on the culture surface was demonstrated by the fluorescence intensity following overnight incubation of solution with the fluorescence labeled ECM proteins (Figure 1). Unlike the standard cell culture-treated surface, the Nunclon Sphera surface has minimal binding interactions with the ECM proteins, consequently discouraging the cells from attaching to the surface.