The overall goal of this procedure is to isolate and culture adult epithelial stem cells from human skin. This is accomplished by first cutting fresh human skin from facelift or other surgery into one centimeter wide strips and incubating in media containing disc space. The skin is then rinsed in fresh media and the hair follicles are plucked.
Next follicles are separated into telogen and antigen follicles by examining them under a dissecting microscope, the final step of the procedure is trypsin follicles into single cell suspension and plating in culture dishes. Ultimately, results can be obtained that show clon genic cell growth through phase contrast. Microscopy Visual demonstration of this method is critical as a cutting plucking and identification steps are difficult to learn because few people are familiar with the microscopic assisted dissection of skin and hair follicles.
To begin extraction of epithelial stem cells from human skin cut fresh human scalp skin into one centimeter wide strips cut parallel to hair growth, so as not to cut across follicles. Add the skin to a conical tube with DMEM containing 10%FBS and four milligrams per milliliter. Disc base.
Incubate the sample for two to four hours at 37 degrees Celsius. After the incubation, rinse skin with fresh DMEM containing 10%FBS and place in a sterile petri dish. Collect the follicles by gently pulling each hair firmly and smoothly from the base with forceps.
Place isolated hair follicles in fresh media. Next, use a dissecting microscope to select follicles at telogen stage. Based on their morphology, cut out the bulge region and transfer follicles into a 15 milliliter sterilized tube.
Additionally, antigen follicles can be used. Cut the upper one third of follicles to obtain the hair follicle bulge region of stem cells, or cut the bulb bridge into isolate matrix transit amplifying cells and add to the collection tube. Then add four milliliters of a one-to-one solution of 0.05%tripsin EDTA and 0.53 millimolar tetra sodium EDTA for 15 to 20 minutes.
At room temperature, shake the tube periodically to mix detached cells can be visualized under a dissecting scope. To monitor progress of digestion. To stop the digestion, add four milliliters of DMEM plus FPS and centrifuge for five minutes.
At 800 RPM, discard the supernat carefully saving approximately 0.2 to 0.5 milliliters to avoid losing cells and resuspend in one milliliter of keratinocyte medium without EGF. Before plating the epithelial stem cells for culture A six will plate of mitomycin C treated three T three J two feeder cells should be prepared according to the written protocol. Then see the isolated hair follicle stem cells incubate the cells overnight in a humidified incubator at 37 degrees Celsius, 5%carbon dioxide the following day.
The media has changed to keratinocyte medium with epidermal growth factor and return to the incubator. Feed the cells every two days with keratinocyte medium plus EGF and grow them for 14 to 20 days. To passage the stem cells, the feeder cells must be removed.
First, wash the cells once with PBS and add enough room temperature, 0.53 millimolar tetra sodium EDTA to cover the cells. Incubate the cells at room temperature for five minutes. After the incubation, gently shake the dish and the three T three J two feeder cells will detach, aspirate off the feeder cells.
Rinse the plate of stem cells with PBS once and add enough prewarm 37 degrees Celsius, two x trips in EDTA to each well to cover. The cells incubate at 37 degrees Celsius for seven to 15 minutes to allow the hair follicle stem cells to fully detach. After the cells have detached, add approximately 500 microliters of keratinocyte medium without EGF to stop the trypsin and gently pipette up and down to disperse the cells.
Collect the cells in a conical tube and centrifuge at 200 G for five minutes. Aspirate the supernat and resuspend the cells in keratinocyte medium without EGF. Finally, count and reflate the cells onto a mitomycin C treated three T three J two cell layer.
To immortalize the stem cells plate 350, 000 stem cells per well of a six well plate onto a layer of mitomycin C treated three T three J two feeder cells. Culture the cells for two days on the second day treat PA three 17 LX SN 16 E six E seven cells, which encode HPV 16, E six, and E seven with mitomycin C for two hours as described in the written protocol. After the incubation, add the treated cells to the primary epithelial stem cells, leaving one well of stem cells untreated as a selection control co-culture for six days in keratinocyte medium with EGF, changing the media every two days after six days, remove the feeder and PA three 17 cells by treating with 0.53 millimolar tetra sodium e dta.
As before then add 200, 000 mitomycin C treated three T three J two NHP cells per well. The three T three J two NHP cells are feeder cells that are neomycin, hygromycin and pur mycin resistant, which is required for the selection procedure. For selection culture, the cells under 0.2 milligrams per milliliter of medicine or G four 18 for an additional six days.
Renew fresh G four 18 containing KERATINOCYTE medium plus EGF every two days. Surviving epithelial stem cells will be immortalized stem cells in this brightfield image. The epithelial stem cells from skin form tight epithelial colonies designated by the E when cultured in keratinocyte medium with a feeder layer of cells designated by the F shown here are hair follicle implants that give rise to epithelial outgrowths.
The telogen bulge region designated by the T is attached to the dish and surrounded by the epithelial cell colony along with feeder cells. Stem cell colonies in culture maintain expression of epithelial markers such as cytokeratin 15 shown here in green and maintain pluripotency when differentiated along the hair follicle lineage. Skin epithelial stem cells express K six hf, a hair follicle marker.
Additionally, the cells can be cultured on de epidermis, dermis, or other matrices at the air liquid interface, and will form a stratified epidermis with a cornified layer, granular layer and spinous layer. And finally, oil red positive globules can be seen from stem cells induced along the sebaceous lineage. Following this procedure, stem cell assays for self-renew proliferation, cell migration, adhesion and differentiation can be performed to answer additional questions about genes involved in maintaining the stem cell phenotype.
