Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice

Abstract

Protocols for preparing and culturing primary keratinocytes from newborn and adult mouse epidermis have evolved over the past 35 years. This protocol is now routinely applied to mice of various genetic backgrounds for in vitro studies of signaling pathways in differentiation and cell transformation, and for assessing the in vivo phenotype of altered keratinocytes in grafts of cells on immunodeficient mice. Crucial in the development and application of the procedure was the observation that keratinocytes proliferate in media of low calcium concentration, but rapidly commit to differentiation at calcium concentrations >0.07 mM after the initial attachment period. Preparing primary keratinocytes from ten newborn mice requires 2-3 h of hands-on time. Related procedures are also provided: preparing immature hair follicle buds, developing dermal hair follicles and fibroblasts from newborn mice, preparing primary keratinocytes from adult mice and grafting cell mixtures on athymic nude mice.

Fig. 1

Steps in the procedure for obtaining epidermis and dermis from newborn mice: a. removal of lower forelimbs; b. removal of lower hind limbs; c. severing tail close to the body; d. cutting the skin along dorsal midline starting at the tail end of the body; e. ending the dorsal midline cut at the tip of the nose; f. loosening the skin on both sides of the dorsal midline cut; g. pulling skin over the hind leg stumps; h. gathering skin on the ventral side and pulling it toward the head; i. pulling the skin over the forelimb stumps and over the head; j. flattening skin, dermis side down, on the culture surface of culture dish; k. uncurling folded down edges of skin as necessary after transferring it, dermis side down, to the surface of the trypsin solution contained in culture dish; l. dermis being lifted above the adhering epidermis of skin, that had been floating overnight on trypsin and was transferred, epidermis side down, to the inner surface of a dry lid of a sterile culture dish and gently spread; m. gathering of epidermis to enclose loosely adhering cells prior to transfer to a container with HiCa medium; also shown is the dermis lifted in the previous panel and deposited on the lid surface.

Fig. 2

Phase contrast images of freshly prepared and cultured cell fractions from epidermis and dermis of newborn BALB/c mice: a, c, e: freshly prepared preparations captured through 10× objective; b, d, f: fractions cultured for 3 days, captured through 4× objective. a, b: total epidermal cell fraction plated at 0.5 mouse equivalent per 60 mm dish; white arrow near top edge of panel (a) points to an immature hair follicle bud; c, d: epidermal cell fraction after removing immature hair follicle buds by centrifugation at 20g, plated at 1 mouse equivalent per 60 mm dish; e, f: dermal cell fraction (fibroblasts) after removing dermal hair follicles by low speed centrifugation and final filtration through 20μ Nytex cloth, plated at 2.9 million cells per 60 mm dish. Epidermal fractions were plated in 0.2 mM Ca²⁺ medium overnight; attached cells were washed with PBS and switched LoCa medium; medium was changed on day 3 before image capture. Fibroblast fraction was plated in HiCa medium overnight followed by a medium change on day 1 and 3. Bars indicates 20 μm for top row panels and 50 μm for bottom row panels

Fig. 3

Phase contrast images of freshly prepared and cultured hair follicle fractions from epidermis and dermis of newborn BALB/c mice: a, c, e: freshly prepared hair follicle fractions captured through 10× objective; b, d, f: hair follicle fractions cultured for 3 days, captured through 4× objective. a, b: hair follicle bud preparation from epidermis suitable for grafting; white arrows in panel a point to contaminating suprabasal cell aggregates and to granular cells; plated at 0.75 mouse equivalents per 60 mm dish; c, d: more highly purified hair follicle bud preparations from epidermis devoid of extraneous cells and cell aggregates, suitable for collagen matrix co-culture experiments; plated at 5 mouse equivalents per 60 mm dish. Extremely high cell density on day 3 indicated underestimation of yield based on volume of final pellet. e, f: dermal hair follicles plated at 0.75 mouse equivalent per 60 mm dish. All hair follicle fractions were plated in 0.2 mM Ca²⁺ medium overnight, followed by washing attached cells with PBS and feeding with LoCa medium; medium was changed on day 3 before image capture. Bars indicates 20 μm for top row panels and 50 μm for bottom row panels.

Fig. 4

Steps in the procedure for grafting cells mixtures on the backs of athymic nude mice to determine their in vivo phenotype: a. decontamination of the graft area of sedated mice with Betadine (to be followed by wiping area with 70% ethanol); b. cutting an approximately 1 cm diameter piece of full thickness skin with curved scissors after lifting skin with forceps; c. appearance of graft area after removing the skin piece; d. lifting the skin and moistening the area under the skin around the graft area with PBS using forceps; e. sliding part of the PBS-moistened dome flange under the skin on one side of the graft area; f. appearance of partially inserted dome prior to pulling the skin on the other side over the remaining exposed flange; g. appearance of fully inserted and snugly seated dome; h. appearance of loosely fitting dome fastened to the skin with wound clips (clip on other side not visible); i. application of cell suspension to the graft area.