Keratinocytes cell isolation

Living, bi-layered skin substitute containing type 1 bovine collagen Extracted and purified from bovine tendons and viable allogeneic human fibroblast and keratinocytes cells isolated from human infant foreskin... 

Cultures of isolated keratinocytes have facilitated the study of epithelial HA metabolism. Basal keratinocytes synthesize copious quantities of HA. When Ca++ of the culture medium is increased, from 0.05 to 1.20 mM, these cells begin to differentiate, HA synthesis levels drop,148 and there is an onset of hyaluronidase activity.149 This increase in calcium that appears to simulate in culture the natural in situ differentiation of basal keratinocytes parallels the increasing calcium gradient observed in the epidermis. There may be intracellular stores of calcium that are released as keratinocytes mature. 

Isolated keratinocytes subjected to cyclic strain exhibit a significant increase in cell proliferation, DNA synthesis, and protein synthesis compared to stationary or constantly loaded cells, which appear to involve changes in cyclic AMP. Takei et al. (1997) reported a strain-induced reduction in the levels of cyclic adenosine monophosphate, protein kinase A (PKA), and prostaglandin E2 (PGE2) as compared to stationary controls. Takei et al. (1997) also studied the effects of cyclic strain on protein kinase C (PKC) activation and translocation in cultured keratinocytes. 

FcyRIIa (CD32a) is expressed in small-vessel endothelium in the papillary (superficial and periadnexal) dermis [27], Binding of IgG or immune complexes to isolated human dermal microvessel endothelial cells initiates rapid receptor cross-linking, intracellular Ca fluxes, and endosomal internalization of the IgG-FCyRIIa complexes [27], FcRn is expressed principally in basal and suprabasal human keratinocytes in normal epidermis [176] and is also expressed in mouse skin [62], In healthy adult human volunteers, dermal interstitial fluid endogenous IgG levels were demonstrated to be 30% of plasma IgG levels [59]. 

Various tissue constructs have been reassembled from isolated constituents, including resident cell types whose numbers have been amplified or modified in culture. A three-dimensional co-culture system for human skin keratinocytes layered upon a synthetic mesh infiltrated with dermal fibroblasts, when floated to allow contact of the uppermost keratinocytes with air, exhibits stratification and cornification remarkably similar to in vivo squamous epithelia. This reconstructed epithelial model has been recommended as an in vitro replacement for dermal corrosivity testing. It has been anticipated that this and a similar noncomified model will have application in dermal and ocular irritation testing, but thus far validation studies have yielded mixed results. Reconstructed tissues can also provide context for basic toxicological research on aberrant cellular interactions with cellular and acellular constituents, as illustrated by invasion of cancerous epithelial cells into underlying dermis of a skin equivalent model. 

Choi et al. 86) were able to isolate a glycoprotein fraction (5.5 kDa) that is involved in the wound healing effect of A. vera via cell proliferation and migration. This glycoprotein fraction was able to accelerate the wound heahng on a monolayer of human keratinocytes and also to enhance wound healing in hairless mice with significant cell proliferation. 

FIGURE 6.3 SM induces phosphorylation of p53. Normal human epidermal keratinocytes were exposed to 12.5, 25, 50, 100, or 200 p.M SM for 5, 15, 30, 60,120, or 240 min. Whole cell extracts were isolated, proteins fractionated hy SDS-PAGE, and immunoblotted (IB) using antibodies that specifically recognize phosphoiyl-ated p53 (top panels). Total p53 was also analyzed. (Reprinted from Minsavage, G.D., Dillman IB, J.F., J. Pharmacol. Exp. Ther., 321, 202, 2007. With permission.)... 

Site-of-contact tissues might be preferable for such compounds (Burlinson 1989 Furihata et al. 1984 Furihata and Matsushima 1987 Mori et al. 1999 Sawyer et al. 1988), if sufficiently validated. The main technical limitation of this assay with respect to other tissues is the need to isolate the cells after in vivo treatment and to get them to incorporate tritiated thymidine in vitro. Because UDS measurement does not require cell division, it can potentially be applied to many different tissues, provided that the cells can be isolated and maintained in primary culture for the few hours required for tritiated thymidine incorporation. The literature contains reports of UDS-based studies of stomach, colon, kidney, pancreas, tracheal epithelium, nasal epithelium, epidermis, keratinocytes, and spermatocytes (Burlinson 1989 Furihata et al. 1984 Furihata and Matsushima 1987 Sawyer et al. 1988 Loury et al. 1987 Mori et al. 1999 Latt et al. 1981 Helleday 2003). 

Sanguinarine, isolated from the root of Sanguinaria canadensis, possesses both anti-inflammatory and antioxidant properties. In addition, this alkaloid has displayed anti-proliferative and apoptotic effects against human epidermoid carcinoma cells and normal human epidermal keratinocytes. While treatment with sanguinarine has been shown to decrease the viability of both kinds of cells, this loss of viability occurred at lower doses of the compound and was much more pronounced in the carcinoma cells than in the normal keratinocytes [108]. 

Since the initial development of methods for in vitro cnlture of human keratinocytes (Rheinwald and Green, 1975), two-dimensional monolayer cultures of submerged keratinocytes have been widely utilized for skin-related research. Primary and immortalized fibroblast and melanocyte cultures are also now widely available for in vitro research and toxicology testing applications (Hsu and Herlyn, 1996 Costin and Hearing, 2007). Skin-resident immune cells (e.g., Langerhans cells) are difficult to isolate and maintain in primary culture. However, monocyte-derived dendritic cells and dendritic cells derived from cord blood precursors are well established, as are a variety of immortalized dendritic-like cell lines (Ayehunie et al., 2009 Reuter et al., 2011). 

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