Hi, I'm Maya Sibu Bloom from the Institute of Human Genetics and the Northeast England Stem Cell Institute at Newcastle University. And this is from the Medical College of Wisconsin. Today we're gonna show you our procedure for preparing Al Explan from Visco Follicles of mice.
B Explan are the starting tissue for preparing epidermal neuro crest stem cell cultures. This procedure involves dissecting the wi whisker pad and micro dissecting the hair follicles, and then isolating the BULs and placing them into a culture plate. So let's get started.
This procedure begins with the dissection of the bulge from adult mouse whisker follicles. Typically, we use mice two to six months old, however, younger mice tend to yield more stem cells. Animals are ized and entirely submerged in a one-to-one mixture of Betadine and hydrogen peroxide for about three minutes for disinfection, subsequently squirt entire mouse, especially the facial region, with 75%ethanol and carry the mouse to the dissecting microscope.
In the laminar flow hood, dissect WHI pads carefully, avoiding cutting into hair bulbs and put in hangs balance salt solution at room temperature. To dissect whi follicles, hold the skin next to the follicle with forceps and then cut around the follicle with straight scissors. Cut deeply.
To avoid injuring the hair bulbs, lift the whisker follicle out of the whisker pad and put it into a new plate. With fresh HBSS pull the follicles in the same plate. Pin a hair follicle with sharpened ts and needles onto a silk guard coated glass Petri dish that contains HBSS.
Flush loose adipose tissue and dermal tissue with squirts of buffer until whisker follicle is clean. If necessary, cut nerve to whisker follicle and remove adherent tissue by scraping and repeated flushes of HBSS. Cut visco follicle longitudinally with a microblade.
Avoid cutting too deeply as this will injure the bulge. Remove any blood with repeated squits of HBSS until gone. Appearance of blood is a good indicator that the cut was deep enough.
Next, make a transverse cut above the level of the coness sinus and subsequently a second transverse cut at the level within the ring sinus very close to the skin within the isolated piece of follicle. You will now see the bulge inside the capsule. See, there it is.
Grab an end of the capsule with the forceps and remove the bulge. You will now see the empty capsule and the isolated bulge pool. Isolated bulges in a separate culture plate in HBSS at room temperature.
Make sure no other tissue is contaminating the pooled bulges. At this point, bulge plants can be cultured prior to culture In bul plants. 35 millimeter culture plates are coated with collagen by placing drops of 50 microliter collagen and 10 microliter sterile.
6%saline next to each other. Mix well with a double bent poster pipette and push towards the edge of the culture plate incubate overnight in a clean environment but do not let dry. We use a desiccate with a plate of water at the bottom for this purpose.
The next day, the day of culture. Rinse the plates with saline, pre incubate, collagen coated, and rinsed culture plates for approximately three hours. With culture medium in the incubator culture medium consists of 85%alpha modified MEM, 10%fetal bovine serum, and 5%day 11 chick embryo extract.
After pre incubation, remove culture medium from plates and add several BULs with as little medium as possible. Remove excess culture medium. Incubate for one hour in cell incubator in a humidified atmosphere with 10%oxygen and 5%CO2.
After one hour, slowly add 1.5 milliliter of culture. Medium CHE plants should adhere to the col collagen substratum replace 50%of the cultural medium daily. Okay, now let's go.
And now a look at the cultures. Within three to four days, highly migratory cells will emigrate from the ball checks. Plants note their stellate morphology and predominant absence of cell cell contacts.
Over the next few days, more cells will emigrate and emigrated cells will proliferate rapidly important rare cells with flattened morphology, which becomes sometimes apparent several days later than epidermal neuro crest. Stem cells are sometimes seen in these cultures. These cells are not epidermal neuro crest stem cells, but are instead less motile, putative, epidermal stem cells or progenitors cultures consisting of these cells should be discarded.
We have just shown you how to prepare bul explan cultures which contain epidermal neuro neurore stem cells. Because neurore cells are very migratory, they will leave the balch explan within three to four days. After explanation, the bulge is then removed and the pure population of neurore cells remains in the culture plate.
When doing this procedure, it's important to remember to carefully disinfect the mouse in order to avoid contamination. One also has to cut deeply in the risk of pad in order not to injure risk of follicles. With regard to the cultures, it is important to differentiate between the migratory distillate, neuro crest cells, and the non migratory cobblestone shaped keratinocytes.
So that's it. Thanks for watching and good luck with your experiments.
