Post-Mitotic Human Dermal Fibroblasts Efficiently Support the Growth of Human Follicular Keratinocytes.

Abstract

For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.

Spermidine Promotes Human Hair Growth and Is a Novel Modulator of Human Epithelial Stem Cell Functions

“…ORS cultures were kept on a feeder layer of mitomycin-treated human dermal fibroblasts [67] in a 1∶3 mixture of Ham's F12 (Biochrom, Berlin, Germany) and Dulbecco's modified Eagle's medium (Invitrogen-Life Technologies, Inc., Grand Island, NY, USA) supplemented with 0.1 nM cholera toxin, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 2.43 µg/ml adenine, 2 nM triiodothyronine, 10 ng/ml epidermal growth factor, 1 mM ascorbyl-2-phosphate, and antibiotics (all from Sigma-Aldrich Corp.).…”

A two-component pre-seeded dermal–epidermal scaffold

“…These results suggest that the amine-aldehyde reaction created strong imine bonds between the two parts of the composite scaffold. This chemistry has been used to crosslink cells with an aldehyde surface modification to amines on unmodified cells [20], to create glues which bind to tissue amines in situ [21, 22] and in situ crosslinking hydrogels to be used the peritoneum, without any toxic effects [41, 64, 65]. These tissue glues based on an aldehyde-modified dextran which reacts with free amines on the cell surface have approximately two times higher adhesion strength than our system [66].…”

“…Such scaffolds, should be applicable to any deep or shape of the lesion and allow the co-culture of cells by furnishing tailored biological and mechanical properties for each compartment, speeding up the healing process by simultaneous regeneration of dermis and epidermis [14, 20]. We have designed an in situ forming dermal-epidermal scaffold, which is adaptable to differing lesion shapes, and is designed to mimic the bilayer structure of human skin while providing instructive cues for cell adhesion, migration and proliferation.…”

Wound Stimulation by Growth-Arrested Human Keratinocytes and Fibroblasts: HP802-247, a New-Generation Allogeneic Tissue Engineering Product

“…As far as fibroblasts are concerned, postmitotic fibrocytes were recently claimed to stimulate keratinocyte proliferation to a greater extent than their progenitors [16]. In fact, fibroblasts have been used as feeder cells in keratinocyte culture for two decennia [17]. This most probably depends on significantly different patterns of cytokine release [18], which – as shown here – also varies with numbers and ratio of the cocultured cells.…”