October 1, 2012 (Vol. 32, No. 17)
ECM Mimetic Surfaces Enable the Expansion of Cells with Clinical Therapeutic Potential
Recent advances in cell biology have created new approaches in cell therapy and regenerative medicine that are under investigation for the treatment of human diseases. While there is great potential for the development of new cell-based therapies, researchers must overcome a number of challenges to realize this potential, including reducing experimental variability and the risk of contamination from pathogens common to animal-sourced material.
In order to realize the promise of cell therapy, 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 limited commercial alternatives for animal-free, chemically defined growth surfaces that are compatible with these defined media formulations. 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 surfaces 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 has developed novel animal-free cultureware surfaces that enable the expansion of cells with therapeutic potential, including human mesenchymal stem cells (MSCs), endothelial progenitor cells, and primary keratinocytes. BD PureCoat™ ECM Mimetic Fibronectin Peptide (Fibronectin Mimetic) and Collagen Type I Peptide (Collagen I Mimetic) surfaces are coated with biologically 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 prepared using a synthetic peptide that contains the RGD amino acid motif, which supports the attachment of cells that require a fibronectin coating, including α5-integrin positive cells. This peptide is rationally designed to mimic cell attachment regions present in native fibronectin. The synthetic peptide is covalently immobilized on a proprietary surface and presented in a functionally active orientation. Our studies have shown that this surface 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 ability to self-renew and differentiate into adipocytes, osteoblasts, and chondrocytes. Also, these cells secrete cytokines and modulate immune cell function. MSCs hold great promise for the treatment of a variety of degenerative diseases and immune disorders, and are currently 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 matrix surfaces were comparable, indicating similar growth properties on both surfaces (data not shown). MSCs cultured under these conditions 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 positive for CD90, CD105, and CD73 markers, and negative for CD34, CD11b, CD19, CD45, and HLA-DR (data not shown).
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.
Furthermore, MSCs cultured on the Fibronectin Mimetic surface maintained their differentiation capacity. After three passages, MSCs were shown to exhibit osteogenic differentiation following induction, fixation, and staining for alkaline phosphatase activity (Figure 2A). Mineralization by osteocytes was quantified using fluorescence-based staining of hydroxyapatite, and revealed that cells grown on the Fibronectin Mimetic surface exhibit comparable mineralization to cells grown on the human origin matrix (data not shown). MSCs cultured on the Fibronectin surface were also differentiated to adipogenic lineage following induction, fixation, and staining for lipid vacuoles (Figure 2B). Taken together, these results demonstrate that human MSCs cultured on the Fibronectin Mimetic surface retain their differentiation potential.
The BD PureCoat ECM Mimetic Collagen Type I Peptide surface is prepared using a peptide that contains the GFOGER amino acid motif, which supports the attachment of cells that require a collagen type I coating, including α2-integrin positive cells. This peptide is rationally designed to mimic cell attachment regions present in native collagen type I. The synthetic peptide is covalently immobilized on a proprietary surface and presented in a functionally active orientation. This surface is an animal-free alternative to native collagen I or other human-derived ECM surfaces that have been used for keratinocyte growth and expansion.
Keratinocyte accessibility, proliferation potential, and ease of culture has enabled the use of these cells for cell therapy and regenerative medicine. Ex vivo expansion of keratinocytes requires either coating a culture vessel with human or animal-derived ECM proteins or culturing cells in growth medium containing bovine serum or animal-derived components. However, the use of animal-origin components can raise concerns about introducing human-derived or animal-derived pathogens into the culture system and can result in batch-to-batch variability.
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.
Our studies demonstrate that human primary keratinocytes can be cultured under serum-free and animal-free conditions on the Collagen I Mimetic surface for multiple passages (Figure 3A) while exhibiting morphology and growth comparable to cells cultured on native rat tail collagen I or recombinant human collagen I.
Figure 3A. Human neonatal keratinocytes (HKN) exhibit similar morphology and growth.
In addition, keratinocytes retain their functionality after several passages on the Collagen I Mimetic surface as demonstrated by a wound healing assay (Figure 3B). Cells migrated to seal the scratch in the cell monolayer equally well on all three collagen-based surfaces, resulting in wound healing after 4 hours in culture. Furthermore, the Fibronectin Mimetic and Collagen I Mimetic surfaces have been shown to support the growth and functionality of human endothelial colony forming cells (endothelial progenitor cells) under low serum conditions (data not shown).
In conclusion, our studies demonstrate that BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide supports the xeno-free expansion of human MSCs and maintains their capacity for differentiation. Moreover, BD PureCoat ECM Mimetic Cultureware Collagen I Peptide enables the attachment, growth, and functionality of human primary keratinocytes in a completely animal-free culture environment. The fully defined and synthetic nature of these novel surfaces greatly reduces the risk of contamination from animal-derived pathogens.
In addition, these surfaces are manufactured in a cGMP-compliant facility, are pre-coated to ensure batch-to-batch consistency, and are available in a variety of scalable formats that are room temperature stable for up to 18 months. BD PureCoat ECM Mimetic Cultureware meets an important need in clinical research by providing animal-free surfaces suitable for the expansion of a variety of cell types.
Figure 3B. HKN exhibit similar cell migration (wound healing assay) when cultured on BD PureCoat ECM Mimetic Collagen I Peptide, rat tail Collagen I or recombinant Human Collagen I
Marshall Kosovsky, Ph.D. ([email protected]), is technical support manager at BD Biosciences–Discovery Labware. For further information, please contact Dr. Kosovsky or BD Biosciences–Discovery Labware Technical Support at 877-232-8995 (select prompt 3, then 1).
