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.

Customized hydrogel substrates for serum-free expansion of functional hMSCs.

We describe a screening approach to identify customized substrates for serum-free human mesenchymal stromal cell (hMSC) culture. In particular, we combine a biomaterials screening approach with design of experiments (DOE) and multivariate analysis (MVA) to understand the effects of substrate stiffness, substrate adhesivity, and media composition on hMSC behavior in vitro. This approach enabled identification of poly(ethylene glycol)-based and integrin binding hydrogel substrate compositions that supported functional hMSC expansion in multiple serum-containing and serum-free media, as well as the expansion of MSCs from multiple, distinct sources. The identified substrates were compatible with standard thaw, seed, and harvest protocols. Finally, we used MVA on the screening data to reveal the importance of serum and substrate stiffness on hMSC expansion, highlighting the need for customized cell culture substrates in optimal hMSC biomanufacturing processes.

Synthetic, Chemically Defined Polymer-Coated Microcarriers for the Expansion of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC), also called marrow stromal cells, are adult cells that have attracted interest for their potential uses in therapeutic applications. There is a pressing need for scalable culture systems due to the large number of cells needed for clinical treatments. Here, a tailorable thin 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 and glass microcarriers to create chemically defined surfaces for large-scale cell expansion is applied. These chemically defined microcarriers create a reproducible surface that does not rely on the adsorption of xenogenic serum proteins to mediate cell adhesion. Specifically, this coating method anchors PVG copolymer through ring opening nucleophilic attack by amine residues on poly-l-lysine that is pre-adsorbed to the surface of microcarriers. Importantly, this anchoring reaction preserves the monomer VDM reactivity for subsequent functionalization with an integrin-specific Arg-Gly-Asp peptide to enable cell adhesion and expansion via a one-step reaction in aqueous media. MSCs cultured on PVG-coated microcarriers achieve sixfold expansion-similar to the expansion achieved on PS microcarriers-and retain their ability to differentiate after harvesting.

Surface functionalization and dynamics of polymeric cell culture substrates.

The promise of growing tissues to replace or improve the function of failing ones, a practice often referred to as regenerative medicine, has been driven in recent years by the development of stem cells and cell lines. Stem cells are typically cultured outside the body to increase cell number or differentiate the cells into mature cell types. In order to maximize the regenerative potential of these cells, there is a need to understand cell-material interactions that direct cell behavior and cell-material dynamics. Most synthetic surfaces used for growth and differentiation of cells in the lab are impractical and cost prohibitive in clinical labs. This review focuses on the modification of low cost polymer substrates that are already widely used for cell culture so that they may be used to control and understand cell-material interactions. In addition, we discuss the ability of cells to exert dynamic control over the microenvironment leading to a more complex, less controlled surface.

Peptide Conjugation to a Polymer Coating via Native Chemical Ligation of Azlactones for Cell Culture.

Conjugation of biomolecules for stable presentation is an essential step toward reliable chemically defined platforms for cell culture studies. In this work, we describe the formation of a stable and site-specific amide bond via the coupling of a cysteine terminated peptide at low concentration to an azlactone containing copolymer coating. A copolymer of polyethylene glycol methyl ether methacrylate-ran-vinyl azlactone-ran-glycidyl methacrylate P(PEGMEMA-r-VDM-r-GMA) was used to form a thin coating (20-30 nm) on silicon and polycarbonate substrates. The formation and stability of coating-peptide bonds for peptides containing free thiols and amines were quantified by X-ray photoelectron spectroscopy (XPS) after exposure to cell culture conditions. Peptides containing a thiol as the only nucleophile coupled via a thioester bond; however, the bond was labile under cell culture conditions and almost all the bound peptides were displaced from the surface over a period of 2 days. Coupling with N-terminal primary amine peptides resulted in the formation of an amide bond with low efficiency (<20%). In contrast, peptides containing an N-terminal cysteine, which contain both nucleophiles (free thiol and amine) in close proximity, bound with 67% efficiency under neutral pH, and were stable under the same conditions for 2 weeks. Control studies confirm that the stable amide formation was a result of an intramolecular rearrangement through a N-acyl intermediate that resembles native chemical ligation. Through a combination of XPS and cell culture studies, we show that the cysteine terminated peptides undergo a native chemical ligation process at low peptide concentration in aqueous media, short reaction time, and at room temperature resulting in the stable presentation of peptides beyond 2 weeks for cell culture studies.