University of Wisconsin-Madison,
Keywords: human mesenchymal stromal cells, hMSC, biomanufacturing, synthetic coatings
Summary:Statement of Purpose: Human mesenchymal stem cells, also called mesenchymal stromal cells (hMSC) are adult cells that have attracted interest due to their multipotency and immunomodulatory potential. For use in therapeutic applications, hMSC must be isolated and expanded in vitro before returning to the patient. This expansion process is costly and time consuming, presenting the need for improved cell culture systems. An ideal culture system is one that is scalable and tailorable to multiple cell or media types and may reduce the cost associated with cell culture. Here, we describe a method for applying a chemically defined, tailorable copolymer layer to existing cell culture materials. This copolymer, poly(poly(ethylene glycol) methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) [P(PEGMEMA-r-VDM-r-GMA); PVG], has previously been spin-coated onto flat, symmetrical substrates and shown to inhibit cell adhesion until it is functionalized with integrin-binding peptides (Schmitt, SA. Adv Healthcare Mats. 2015;4:1555-1564). We developed a method to apply PVG copolymer to 2D and 3D cell culture materials without the need for spin coating or other specialized equipment. These coated materials demonstrate the ability to culture hMSC on in both serum-containing and serum-free media. We demonstrate an improvement over the state of the art surfaces used for serum-free hMSC culture with high passaging efficiency and the ability to harvest cells without the use of enzymes. Methods: We anchored PVG to polystyrene (PS) tissue culture materials – including microcarriers, 96-well and 384-well tissue culture plates. First, we exposed the PS surface to a poly-L-lysine (PLL) solution of varying concentration to create a primary amine-presenting layer through adsorption. Next, we applied a 10% (wt) solution of PVG in ethanol to the surface overnight, anchoring the PVG to the surface via the GMA epoxide ring. We characterized the mechanism of PVG attachment using surface analysis techniques including x-ray photoelectron spectroscopy (XPS), polarization modulation-infrared reflection and absorption spectroscopy (PM-IRRAS), water contact angle, and fluorescence imaging. We functionalized the PVG coated materials with an integrin-binding peptide (RGD) and a scrambled peptide to enable cell adhesion and growth. Results: Multiple surface analysis techniques were used to characterize the PVG coating. XPS and PM-IRRAS confirm the presence of the PVG coating on the surface. Contact angle showed a decreased water contact angle of 53° after addition of PVG, which agrees with previously reported values for PEG-based coatings. We applied the PVG coating to PS microcarriers, 96-well and 384-well plates using varying concentrations of PLL. Cells did not adhere to PVG-coated surfaces, and cell adhesion was restored by functionalization with RGD peptide in a PLL concentration-dependent manner. hMSC adhered to and grew on PVG coatings after functionalization with RGD peptide in both serum-containing and serum-free conditions. Conclusions: We have presented a method for creating a functionalizable, chemically defined coating on existing cell culture substrates. This work represents a step toward the creation of biomaterials capable of meeting the needs of hMSC culture for therapeutic applications through low-cost modifications to existing materials that may drive cell growth as well on a highly reproducible surface.