The overall goal of this procedure is to isolate and culture primary neural crest stem cells, or N CSCs from human scalp tissues. This is accomplished by first cussing human scalp skin into one centimeter wide strips and incubating in media containing dis bays. The skin is then arranged in the fresh media and the hair follicles are plugged.
The second step of the procedure is to treat the hair follicles with trypsin and single cell suspensions are obtained. The final step of the procedure is to sort CD 2 71 HNK one double positive ncsc using flow cytometry and to plate in culture plates. Ultimately phase contrast microscopy is used to study neuros sphere formation and growth, Where demonstration of this methods is critical as the extracting and isolating health vertical steps are difficult to learn because it is very hard to dissect the skin and health particles under microscope.
To prepare for culturing stem cells coat the bottom of each well of a six Well plate with poly de lycine allow the plates to dry in the hood. Once the wells are dry, rinse with sterile water and aspirate, then allow the wells to dry again. Coat the plates with the 0.17 milligram per milliliter solution of fibronectin.
Then before the fibronectin dries in the plate at NCSC culture medium, prepare and have ready the following media and reagents. 97%D-M-E-M-F 12 2%B 27 supplement 1%and two supplement B-F-G-F-E-G-F IGF one and diss. Collect fresh adult human scalp skin from facelift procedures or fetal scalp tissue and wash it with PBS containing penicillin streptomycin.
Transfer the skin into 50 milliliter tubes and incubated in DMEM with 10 milligrams per milliliter DIS space at 37 degrees Celsius for two to four hours or overnight at four degrees Celsius. Next, transfer the skin into a sterilized Petri dish and remove the hairs from the skin by grasping the hair shaft near the skin surface and pulling firmly and smoothly. The hair follicles will either show an antigen orgen morphology, incubate the isolated follicle fragments in 0.05%trips in EDTA for 15 to 20 minutes at room temperature with occasional shaking.
Then add four milliliters of DMEM with 10%FBS to stop the reaction. Next, pass the follicular epithelium through a 40 micron filter to obtain a single cell suspension containing cells of varying size and shape. Then spin the suspension at 200 times G for five minutes.
Carefully discard the S supernatant and resuspend the pellet in one milliliter of PBS with 2%FBS To isolate N CSCs using facts first, label the cells with antibodies against a PC conjugated CD 2 71 FE C conjugated hnk one or CD 2 71 PE conjugated alpha four integrin by incubating on ice for 40 minutes in the dark centrifuge at 200 times G for five minutes at room temperature and aspirate the supernatant. Re suspend the cells in PBS containing 2%FBS and two micrograms per milliliter. Propidium iodide to gate out the dead cells.
Perform cell sorting by flow cytometry and collect CD 2 71 H and K one double positive or CD 2 71 alpha four integrin double positive cells culture. The cells in ultra-low attachment plates in NCSC medium, changing the medium once per day. Check the cell culture every day under the microscope.
NCSC will start to form floating small aggregates after several days and well-formed spheres in two to five weeks in the culture, depending on the age of the donors. Outgrowth will appear in a few days at the bulge region and well-formed spheres in situ in several weeks when entire hair follicles are cultured in the NCSC medium. To expand NCSC cultures, wash the cells once with PBS prewarm two times tripsin EDTA to 37 degrees Celsius.
Then add it to the cells for seven to 15 minutes for the hail follicle stem cell to detach. To stop the tripsin ADD DMEM with 10%FBS gently pipet up and down to disperse the cells. Then spin down at 200 times G for five minutes.
Re suspend the cells in NCSC medium and culture and fibronectin coated plates at 37 degrees Celsius. NCSC culture. Medium is sufficient to maintain human NCSC in an undifferentiated state.
Without the need for feeder cells, keratinocytes will not proliferate and gradually die in the medium. Some small round cells will proliferate and form small aggregates in suspension. After three to five days, these floating aggregates slowly increase in size generating three dimensional sphere like structures, termed hair spheres.
When cultured encoded plates, the N CSCs attach to the surface and grow faster than sphere forming cells in suspension, both sphere forming and attached stem cells. Express NCSC markers when entire hair follicles are cultured spheres form at the area corresponding to the bulge region. After watching the video, you should have a good understanding of how to isolate and culture.
Primary neuro stem cell from herma scalp tissues.
