Development of Defined Media for the Serum-Free Expansion of Primary Keratinocytes and Human Embryonic Stem Cells

Abstract

Primary keratinocyte (Kc) cells and human embryonic stem (hES) cells are routinely propagated on a mouse fibroblast feeder layer in media containing fetal bovine serum or other nondefined factors. One disadvantage of using these nondefined factors is that they may inadvertently contaminate the culture system with infectious agents; thus, there remains a need to develop safe culture conditions free from poorly defined and/or animal products. Our laboratory has discovered that growth factors (GFs) and vitronectin (VN) can bind to each other resulting in synergistic short-term functional effects in several cell types. The aim of the current study was to determine whether primary Kc and hES cells can be established and serially propagated serum-free using medium containing VN, insulin-like growth factor-I, and insulin-like growth factor binding protein-3 (VN:GF). Here we demonstrate that primary Kc cells can be isolated, established, serially propagated, and re-form an epidermal layer using the VN:GF combination. Additionally, cell proliferation studies indicate that the Kcs proliferate using the VN:GF combination at a rate comparable to cells grown using serum. Similarly, we verified that this VN:GF combination could be employed for the serial propagation of hES cells. Importantly, both the Kc and hES cells retain their undifferentiated phenotype when cultured using the VN:GF combinations as a serum-free medium for up to 4 passages for Kc and at least 10 passages for hES cells as indicated by the expression of a range of cell surface markers. This study demonstrates that the novel, fully defined VN:GF medium is a viable alternative to media containing serum and highlights the potential of this technology for generating therapeutically viable cells and tissues.

Interleukin 32 (IL-32) Contains a Typical α-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1

“…Culturing cells often require attachment to a surface, which is facilitated by serum adhesive proteins. In FCS, serum adhesive proteins such as fibronectin and vitronectin are present (64,65) that contain RGD motifs. Presumably, these serum adhesive proteins inhibit the IL-32/integrin interactions through their RGD motifs, resulting in reduced cytokine production.…”

Epidermal cells delivered for cutaneous wound healing

“…Adding serum to the medium provides optimal growth rate for epidermal cells but clinical practice would prefer the culturing media without xenologic serum (126)(127)(128). Using collagen type IV coating with medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF), keratinocytes, isolated from 1 cm 2 of skin, were reported to be obtained to cover 400 cm 2 of wound surface in 2 weeks without the need of a fibroblast feeder layer and xenologic serum and these cells are still able to create a fully differentiated epidermis (129).…”

“…However, cells cultured by this technique have proved invaluable for many cell biology studies, including the development of complex 3D tissue models and for grafting in wound therapy such as in severe burn patients (130). Moreover, these products still require, more often than not, the inclusion of undefined human and/or animal products, such as purified human serum albumin or bovine pituitary extract, for the long-term survival of the cells (128).…”

Isolation and culture of human primary keratinocytes—a methods review

“…Supplementation with insulin (within the range of the concentration from 0.1 to 50 μg/mL) increases keratinocyte proliferation rate, and its reduction (along with EGF and transferrin) leads to substantial decrease in keratinocyte growth . Recent findings show that insulin‐like growth factor 1 (IGF‐1) can induce keratinocyte growth even better than insulin alone, and medium supplemented with IGF‐1, insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) and vitronectin (major component of serum) stimulates keratinocyte proliferation to the rate observed in cultivation in serum‐rich media …”