The general goal of the cells based protocol is to isolate and culture human melanocytes cells, improving the shortcomings of conventional methods. From the new method we can get larger amounts of primary melanocytes cells in a relatively short time, based on materials and equipments. Human tissues used in this protocol are fresh adult for skin tissues discarded from circumcision surgery at the hospital and handled according to the guidelines of the Institution's Human Research Ethics Committee number 2015120401, date May 12 2015.
This is a display of all required reagents. Prepare sterile instruments and materials in advance and set the tissue into a dish, wash the tissue with 75%ethanol for 30 seconds and with washes solution ties five minutes each cuts the tissue into convenient pieces and scrap the tissue with surgical blade to remove the fat layer. Flatten the tissues with the dermis side down add 2.5 milligram per milliliter the space solution to the skin tissues.
Cover and store the pieces at four degrees centigrade overnight. On the second day, peel off the epidermis from the dermis and immediately transfer the epidermis into another three dishes with washing solution in turn submerges 0.05%trypsin in the tube incubated for 30 minutes in a water bath at 37 degrees centigrade. Add an equal volume of the neutralization solution.
Pipettes the solution up and down for 10 to 15 times. Pass the solution through a 100 micrometer mash filter. centrifuge at 200 times g for five minutes remove the supernatant re-suspend the cell pallet in 10 milliliter of tiva medium.
Add 10 micrometer of Y-27632. Mark the information this is the field of view under the microscope. It shows cells floating in the medium incubate in a 37 degrees centigrade incubator.
This view shows the view on day two the cells have started to attach the bottom of the dish. And the view on day nine shows obvious deposits of melanocytes exist. Remove the supernatant wash the cells with PBS add two millimeter of 0.05%trypsin incubate the cells for two minutes add two milliliter of the neutralization solution.
centrifuge at 200 times g for five minutes. Remove the supernatant slowly re-suspend the cell pallet with 10 millimeter of tiva medium. Change the medium every two days.
These pictures show the views of passage one schematic diagram so the new method and the conventional method are presented in this figure. The procedure for tissue preparation and the digestion of new method is the same as the conventional method. The only difference is that the isolated fat cells inoculated in 10 minute in her tiva medium with the presence of 10 micrometer Y-27632.
After two days replace the media with tiva medium without supplements at Y-27632. This panel shows representative images of melanocytes prepared by the conventional method top row and by the new method bottom row at day three, day six and day eight after the initial inoculation we call the number of melanocytes at day three, day six and eight, we found that the new method increased the melanocytes yield by about five times. After eight days counter we did dozen and count the number of melanocytes in each single dish.
The results showed that the number of melanocytes isolated by new method is much more than the conventional method. Ki67 standing confirm that melanocytes isolated by new method can normally proliferate more importantly we obtained normal function of melanocytes with the new method. We checked the purity of melanocytes after initial passage from the isolation.
The passage one melanocytes for stand for MITF expression which expressed only in melanocytes. And we found nearly 100%cells were positive for MITF indicating pure melanocytes obtained after first passage. To test melanocytes isolated from the new methods can function normally.
We treated melanocytes with force cooling for over 90 in vitro and observe that force cooling can induce the pigmentation of melanocytes. Taking these data together suggests that the melanocytes isolated from the new method can normally produce pigmentation. Primary melanocytes, as stated above skin and extended in culture have been readily used for laboratory research and the clinical applications.
Having reported that ID Y-27632 into the initial culture medium before two days can dramatically increases the out of melanocytes. In summary, we successfully established a new simple and highly efficient method to isolate pure primary melanocytes from adult tissues.
