a-simplified-efficient-method-to-isolate-primary-human-keratinocytes

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Shandong University

Here, we report a model for studying wound repair based on skin regenerated from human tissue culture-expanded cells. The reconstituted skin (hRSK) responds to injury similar to that of intact human skin, and its constituent cells contribute to the healing process. As we have demonstrated that hRSK composed of GFP-labelled cells also heals "normally," we believe this model will be useful in analysing the wound repair process using genetically modified human cells.

An advanced mouse model for human skin wound healing.

We tested whether the a simple injection known as the patch assay could reconstitute mature hair follicles by culture-expanded human cells and explored whether the assay could reflect the trichogenicity of cultured cells.

The patch assay reconstitutes mature hair follicles by culture-expanded human cells.

Primary skin epidermal cells isolation and in vitro expansion culture have been widely used for laboratory research and clinical applications. The conventional methods involving sequential enzymatic digestion of adult tissues have given low cell recovery rate and reduced cell viability. We report here an advanced method for human primary epidermal progenitor cells isolation from skin tissues including the Rho kinase inhibitor Y-27632. Compared with traditional protocols, the current protocol is simple, easy, and faster; moreover, it gives a greater yield of integrin-expressing epithelial stem cells. In addition, our new methodology does not require a separation of epidermis from dermis because the medium selectively blocks focal adhesion and growth of dermal cells. Importantly, the cells isolated from this method can maintain their regeneration potential and quickly reconstitute a mature human skin in vivo after grafting onto nude mice. In brief, we describe here a simple (one step) and serum-free method for isolating primary epidermal stem cells from adult tissues. The isolated cells may be widely used for both laboratory studies and clinical application, especially in the field of tissue engineering and regeneration.

Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.

Stem cells play a crucial role in maintaining and repairing tissues during homeostasis and following injury. The efficient procurement of high quantity and quality of skin stem cells is important for both laboratory studies and clinical applications. Here, we describe a one-step isolation procedure to efficiently obtain both epidermal and dermal cell population from human skin specimen, based on the different influence of the Rho kinase inhibitor Y27632 on the growth of epidermal and dermal cells during the initial culture. Compared with the conventional methods, our protocol shows that it is simpler and less time consuming and can efficiently obtain the high quality of skin stem cells and can maintain the stem cell features after culture expansion.

One-Step Simple Isolation Method to Obtain Both Epidermal and Dermal Stem Cells from Human Skin Specimen.

The purpose of this study was to develop a new, easily executed hair follicle regeneration system and assay, which could be further developed for clinical or cosmetic applications. Dissociated epidermal and dermal progenitor cells, isolated either from neonatal C57BL/6 mice or human foetal scalp tissues, were suspended in (10 μL) F12 medium and pipetted into a 1 or 2 mm-diameter punch biopsy wounds on the back skin of immunodeficient mice. At 3 weeks after transplantation, although pigmented mouse hairs could efficiently form at the injection sites with delivery of mouse cells, none hair formed on the host mouse skin at 3 months after delivery of human cells. Under the same conditions, human follicles could be regenerated when the human skin cells were delivered onto a 2 mm size punch created on a reconstituted human skin (hRSK), which previously generated on the back of an immunodeficient mouse, but the efficiency of hair formation was low. We demonstrated that both mouse and human regenerated follicles showed normal histology and differentiation markers; moreover, the cell chasing experiment confirmed that the regenerated hair follicles were formed from transplanted cells. Compared to other current hair reconstituted assays, the Punch Assay is relatively simple and generates normal hair follicles within a smaller wound. We suggest that the punch assay is a better in vivo assay of cell trichogenicity.

Xue Leng

Shandong Provincial Key Laboratory of Oral Tissue Regeneration and Laboratory for Tissue Engineering and Regeneration,
School of Stomatology,
Shandong Provincial Key Laboratory of Oral Tissue Regeneration and Laboratory for Tissue Engineering and Regeneration, School of Stomatology

Shandong University

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue