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Accelerating R&D; of Cell-Based Therapies Translational Medicine ECM Mimetic Surfaces Enable the Expansion of Cells with Clinical Therapeutic Potential Marshall Kosovsky, Ph.D. Recent advances in cell biology have created new approaches in cell therapy and regen- erative medicine that are under investigation for the treatment of human diseases. While there is great potential for the develop- ment of new cell-based therapies, research- ers must overcome a number of challenges to realize this potential, including reduc- ing experimental variability and the risk of contamination from pathogens common to animal-sourced material. In order to realize the promise of cell thera- py, it will be necessary to culture specialized cell types (e.g., stem and primary cells) in defined, serum-free environments that include not only the culture medium, but also the growth surface. A number of serum-, xeno-, and animal- free media formulations are commercially available. However, there have been lim- ited commercial alternatives for animal-free, chemically defined growth surfaces that are compatible with these defined media formula- tions. Ultimately, it is recognized that scalable animal component-free cell culture surfaces will be essential for the translation of basic cell research into the clinic. These defined sur- faces must support the production of cells that exhibit sufficient purity, expansion capability, and functional behavior in the target tissue. To address this need, BD Biosciences (www.bdbiosciences.com) has developed nov- el animal-free cultureware surfaces that enable the expansion of cells with therapeutic po- tential, including human mesenchymal stem cells (MSCs), endothelial progenitor cells, and primary keratinocytes. BD PureCoat™ ECM Mimetic Fibronectin Peptide (Fibronectin Mi- metic) and Collagen Type I Peptide (Collagen I Mimetic) surfaces are coated with biologi- cally active synthetic, animal-free peptides that provide a consistent alternative to complex extracellular matrix (ECM) proteins or native ECM-derived peptides. The Fibronectin Mimetic surface is pre- pared using a synthetic peptide that contains the RGD amino acid motif, which supports the attachment of cells that require a fibro- nectin coating, including _5-integrin positive cells. This peptide is rationally designed to mimic cell attachment regions present in na- tive fibronectin. The synthetic peptide is co- valently immobilized on a proprietary surface and presented in a functionally active orien- tation. Our studies have shown that this sur- face is suitable for the growth and expansion of human mesenchymal stem cells (MSCs) in defined, xeno-free culture medium. MSCs are multipotent cells with the abil- A TUTORIAL ity to self-renew and differentiate into adipo- cytes, osteoblasts, and chondrocytes. Also, these cells secrete cytokines and modulate im- mune cell function. MSCs hold great promise for the treatment of a variety of degenerative diseases and immune disorders, and are cur- rently being used for clinical trials. Here we demonstrate the utility of the Fibronectin Mimetic surface for culturing MSCs. Human bone marrow-derived MSCs were cultured for multiple passages in xeno- free medium. MSC attachment, growth, and morphology on the Fibronectin Mimetic surface were comparable to cells grown on a human-origin matrix-coated surface when analyzed by phase contrast microscopy at multiple passages (Figure 1). Cumulative population doublings on the Fibronectin Mimetic and human-origin ma- trix surfaces were comparable, indicating sim- ilar growth properties on both surfaces (data not shown). MSCs cultured under these con- ditions were found to exhibit a characteristic MSC surface marker profile, as established by the International Society for Cell Therapy. The MSC population was shown to be posi- tive for CD90, CD105, and CD73 markers, and negative for CD34, CD11b, CD19, CD45, and HLA-DR (data not shown). Furthermore, MSCs cultured on the Fi- bronectin Mimetic surface maintained their differentiation capacity. After three passages, Figure 1. MSCs exhibit comparable cell growth and morphology when cultured on BD PureCoat ECM Mimetic Fibronectin Peptide (top panels) or human origin matrix (Stemcell Technologies, bottom panels). Images are captured from 1, 3, and 5 passages. B A B Figure 2. MSCs exhibit multilineage differentiation potential when cultured on BD PureCoat ECM Mimetic Fibronectin Peptide. Qualitative analysis of (A) osteogenic differentiation demonstrated by alkaline phosphatase staining and (B) adipogenic differentiation demonstrated by Oil-Red O staining of lipid vacuoles following culture on Fibronectin Mimetic or human origin matrix-coated surfaces for three passages and subsequent induction using standard conditions. Uninduced controls did not exhibit the respective staining. 56 | October 1, 2012 | genengnews.com | Genetic Engineering & Biotechnology News Figure 3. Human neonatal keratinocytes (HKN) exhibit similar (A) morphology and growth and (B) cell migration (wound healing assay) when cultured on BD PureCoat ECM Mimetic Collagen I Peptide, rat tail Collagen I or recombinant Human Collagen I.