Polymer-Coated Magnetic Microspheres Conjugated with Growth Factor Receptor Binding Peptides Enable Cell Sorting.

The separation and sorting of human cells is an important step in the bioprocessing of cell-based therapeutics. Heterogeneous mixtures of cells must be sorted to isolate the desired cell type and purify the final product. This process is often achieved by antibody-based sorting techniques. In this work, we demonstrate that magnetic microspheres may be functionalized with peptides that selectively bind to cells on the basis of their relative concentration of specific surface proteins. Five-micrometer-magnetic microspheres were coated with the synthetic copolymer PVG (poly(poly(ethylene glycol)methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) and functionalized with the vascular endothelial growth factor receptor binding peptide (VRBP), which binds to the vascular endothelial growth factor receptor (VEGFR). These microspheres exhibited low cytotoxicity and bind to cells depending on their relative surface protein expression. Finally, coated, magnetic microspheres were used to separate heterogeneous populations of cells dependent on their VEGFR expression through magnetic-assisted cell sorting (MACS), demonstrating that peptide-based cell sorting mechanisms may be useful in the bioprocessing of human-cell-based products.

Xeno-Free Bioreactor Culture of Human Mesenchymal Stromal Cells on Chemically Defined Microcarriers.

Human mesenchymal stromal cells (hMSC), also called mesenchymal stem cells, are adult cells that have demonstrated their potential in therapeutic applications, highlighted by their ability to differentiate down different lineages, modulate the immune system, and produce biologics. There is a pressing need for scalable culture systems for hMSC due to the large number of cells needed for clinical applications. Most current methods for expanding hMSC fail to provide a reproducible cell product in clinically required cell numbers without the use of serum-containing media or harsh enzymes. In this work, we apply a tailorable, thin, synthetic polymer coating-poly(poly(ethylene glycol) methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) (P(PEGMEMA-r-VDM-r-GMA), PVG)-to the surface of commercially available polystyrene (PS) microcarriers to create chemically defined three-dimensional (3D) surfaces for large-scale cell expansion. These chemically defined microcarriers provide a reproducible surface that does not rely on the adsorption of xenogeneic serum proteins to mediate cell adhesion, enabling their use in xeno-free culture systems. Specifically, this work demonstrates the improved adhesion of hMSC to coated microcarriers over PS microcarriers in xeno-free media and describes their use in a readily scalable, bioreactor-based culture system. Additionally, these surfaces resist the adsorption of media-borne and cell-produced proteins, which result in integrin-mediated cell adhesion throughout the culture period. This feature allows the cells to be efficiently passaged from the microcarrier using a chemical chelating agent (ethylenediaminetetraacetic acid (EDTA)) in the absence of cleavage enzymes, an improvement over other microcarrier products in the field. Bioreactor culture of hMSC on these microcarriers enabled the production of hMSC over 4 days from a scalable, xeno-free environment.