Epidermal and dermal equivalents
a technology of keratinocyte precursor and dermal fibroblast cells, which is applied in the field of epidermal and dermal equivalents, can solve the problems that the dissection of plucked anagen hairs was not required, and achieve the effects of short immobilization, reducing the concentration of allogenic or homologous serum, and reducing the risk of disease transmission
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example 1
Preparation of ORS Cells
[0045] Keratinocyte precursor cells from the outer root sheath (ORS) of the hair follicles are selected and subsequently cultured by use of the following methodology, as disclosed in the present invention.
[0046] Approximately 40 hair follicles were plucked with tweezers from the occipital scalp of individuals, and those in the anagen phase, as detected, for example, by well-developed root sheaths, were then selected under the dissecting microscope (see e.g. Limat & Noser, 87 J. Invest. Dermatol. 485-488 (1986); Limat et al., 92 J. Invest. Dermatol. 758-762 (1989)). The anagen hair was placed directly on the microporous culture insert without performing the previously-utilized micro-dissection to remove the hair bulbs and the infundibular hair shaft.
[0047] Generally, six anagenic hairs were explanted on the microporous membrane of a cell culture insert (Costar) that carried on its undersurface a preformed feeder layer preferably comprised of 20×103 postmito...
example 2
Preparation of Epidermal Equivalents
[0052] ORS cells harvested from primary cultures were seeded at a density of 30×103 cells / cm2 to 100×103 cells / cm2, and preferably 60×103 cells / cm2, on cell culture inserts (Costar) which had been previously inoculated with 10×103 cells / cm2 to 50×103 cells / cm2, and preferably 20×103 cells / cm2, of postmitotic HDF cells on the undersurface of their microporous membrane. Similar to the culture of ORS cells, it is important to keep the HDF feeder cells in close proximity with the ORS cells, while concomitantly keeping them separated by use of the microporous membrane. This culture technique enhances proliferation, differentiation, and thus the homeostasis of the developing tissue.
[0053] Culture medium was identical that that utilized for the preparation of the primary cultures as described supra. After 72 hours, the ORS cells were exposed to air by aspiration of the liquid medium inside the insert (i.e. leaving the underside of the insert in contact...
example 3
Stabilization
[0060] Before delivery, the epidermal equivalents are “coated on-top” by placing a silicone membrane of an appropriate diameter onto the cornified upper aspect of the cultures. To further enhance stability, e.g., in case of thin and / or large epidermal equivalents, as well as to increase adhesion of the silicone membrane, a thin layer of tissue glue, e.g. fibrin glue, may be applied before.
[0061] On-top coating (1) enhances stability and improves handling of the grafts and (2) serves as a protective coat against physical damage as well as the proteolytic milieu and bacteria in the wound.
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