Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Podsumowanie

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Streszczenie

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

Wprowadzenie

The goal of this study was to develop a simple and effective protocol to isolate melanocytes from the epidermis of adult skin for biological research and clinical applications. Melanocytes, which are located in the basal epidermis of the skin and in hair follicles, play an important role in pigmentation of the skin and hair by producing melanin¹. The resulting skin pigmentation from epidermal melanocytes acts as an ultraviolet radiation filter that reduces/prevents DNA damage to underlying cells in the skin². The abnormal proliferation of melanocytes in the skin is quite common, such as in the formation of benign nevi (moles) in which melanocytes potentially transform to oncogenic growth followed by cellular senescence³.

Since 1957, the isolation and subsequent culture of human primary melanocytes has been possible⁴, but only since 1982 has there been an efficient method that can reproducibly establish cultures of human melanocytes from the epidermis⁵. The conventional method to isolate primary melanocytes from the epidermis involves a two-step enzymatic digestion. Briefly, the skin is initially digested with dispase to separate the epidermis from the dermis, after which the epidermis is digested with trypsin to produce suspensions containing melanocytes and keratinocytes, which can then be selectively grown in different media. Currently, the initial culture usually takes about 3 to 4 weeks for melanocytes to reach confluency using the conventional method, which is likely due to the low efficiency of their isolation. Therefore, increasing the initial production of melanocytes from adult skin tissues would be quite helpful for both laboratory research and clinical applications.

Many growth factors, most of which have been shown to be secreted by keratinocytes (e.g., α-MSH, ACTH, bFGF, NGF, ET-1, GM-CSF, HGF, LIF and SCF)^5-8, regulate the differentiation and proliferation of mammalian melanocytes. In the absence of feeder layers, the lack of those growth factors leads to the decreased proliferation of melanocytes and their increased apoptosis⁹. The co-culture of keratinocytes as feeder cells and melanocytes could lead to accelerated melanocyte proliferation and reduced apoptosis. However, the co-culture method not only demands more skin tissues to prepare keratinocytes, which is not practical, but also could not work efficiently because the culture conditions required by melanocytes does not favor keratinocyte growth, and vice-versa.

Previous studies have reported that the addition of Y-27632, a ROCK inhibitor, into the growth medium can enhance the yield of human primary epidermal cells from skin tissues^10-14. Therefore, it would be interesting to test whether the isolation of human primary melanocytes would benefit from the presence of Y-27632. Indeed, the addition of Y-27632 into the inoculation TIVA medium¹⁵, which contains TPA, IBMX, Na₃VO₄ and dbcAMP, significantly increased the yield of primary melanocytes isolated from foreskin tissues in the initial cultures¹⁶. Compared to the conventional protocol, this new protocol is significantly more efficient in increasing the yield of melanocytes¹⁶. Therefore, this protocol describes the new method in detail using adult skin tissues based on a previous study¹⁶ in order to promote its application in basic and applied biological research.

Protokół

The use of adult foreskin tissues in this protocol has been approved by the Human Research Ethics Committee (No.2015120401, date: May 12, 2015).

NOTE: Perform all the following procedures in a sterile environment to prevent contaminations of cells and cultures.

1. Preparations

1. Gather fresh adult foreskin tissues from circumcision surgeries in 15 mL tubes containing 10 mL of phosphate buffer saline (PBS) and store in 4 °C.
   NOTE: Separate the tissues as detailed below within 24 h after their excision.
2. Prepare reagents and culture medium.
   1. Prepare 3% P/S (penicillin/streptomycin) as the washing solution. Mix 50 mL of PBS with 1.5 mL of penicillin (100 U/mL) and streptomycin (100 mg/L) (P/S).
   2. Prepare the termination solution (neutralization solution) for neutralization of enzymatic digestion. Supplement 1% P/S, 10% fetal bovine serum (FBS) into DMEM medium.
   3. Prepare TIVA medium to culture melanocytes: Ham’s F12; 10% FBS; 1x P⁄S⁄glutamine; 50 ng/mL 12-O-tetradecanoyl phorbol-13-acetate (TPA); 1 x 10^-4 M 3-isobutyl-1-methyl xanthine (IBMX); 1 μM Na₃VO₄; 1 x 10^-3 M N-6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate (dbcAMP).

2. The conventional method

1. Skin tissue pretreatment
   1. Rinse each adult foreskin tissue once with 10 mL of 75% ethanol (alcohol) for 30 s, and then rinse twice with 10 mL of washing solution (3% P/S), for 5 min each.
      NOTE: Wash foreskin tissues with 10 mL of PBS in the beginning if there is a lot of blood. All washes are prepared in 100 mm cell culture dishes.
   2. Scrape each foreskin tissue with a surgical blade to remove subcutaneous adipose tissues and loose connective tissues in the cover of a 100 mm cell culture dish.
      NOTE: Use a scalpel to scrape the fat layers away until only the thin epidermis and the dense dermis remain.
   3. Transfer each adult foreskin into another 100 mm culture dish with the dermis side down.
      NOTE: Cut each adult foreskin tissue into 3-4 mm wide strips using a scalpel blade to make the dispase work more efficiently.
   4. Add 2.5 mg/mL dispase to each 100 mm culture dish with adult foreskin tissues and incubate for 16 to 20 h at 4 °C.
      NOTE: Each gram of tissue is digested with 10 mL of dispase solution.
2. Separation of the epidermis from the adult foreskin tissue
   1. After digestion for 16 to 20 h, separate the epidermis from the dermis using tweezers. Grab one side of the dermal edge of the tissue with one tweezer and the corresponding position of the epidermal part with another tweezer and gently peel off the epidermis.
   2. Wash the epidermis 3x with washing solution (3% P/S), and then transfer the epidermis into a tube.
   3. Submerge the epidermis by adding 5 mL of 0.05% trypsin into the tube and incubate in a water bath for 30 min at 37 °C.
      NOTE: Shake the tube to ensure the epidermis is completely submerged before incubation.
3. Collection and culture of primary cells
   1. Add an equal volume of termination solution (10% FBS, 1% P/S in DMEM) to neutralize the trypsin and pipette the solution up and down 10-15 times to separate the epidermal cells.
   2. Pass the cell suspension into a new 50 mL tube through a 100 µm mesh filter to remove debris.
   3. Centrifuge at 200 x g for 5 min.
   4. Remove the supernatant and add 10 mL of inoculation TIVA medium to resuspend the cells.
   5. Seed the resuspended cells into a 100 mm culture dish.
   6. Culture in a 37° C incubator with 5% CO₂.
   7. Change the medium every 2 days.
   8. Passage cells when they reach 80% confluence.

3. The new method

NOTE: The procedure for tissue preparation and digestion in the new method is the same as described above for the conventional method, the only difference being that the isolated fresh cells are resuspended in 10 mL of TIVA medium containing 10 µM Y-27632.

1. Two days after seeding, replace the media with normal TIVA medium without Y-27632. After that, the culture conditions are the same between the new and the conventional methods.

4. Cell passaging

1. Remove the TIVA medium and rinse each culture dish twice with PBS.
2. Add 2 mL of 0.05% trypsin per 100 mm cell culture dish.
   NOTE: Shake the dish to ensure adequate contact between digestive enzymes and the bottom of the dish.
3. Digest in a 37 °C incubator for 2 min.
4. Use a microscope to check the cells, and make sure that most cells have dissociated from the cell culture dish.
   NOTE: Gently tap the dish to release the cells to round up and float in the solution. Prolong the digestion time if most cells did not suspend after tapping, but no more than 5 more min.
5. Transfer the melanocytes into a 15 mL tube after neutralizing the trypsin activity by adding 2 mL of termination solution. Centrifuge at 200 x g for 5 min.
6. Resuspend each cell pellet with 10 mL of TIVA medium after slowly removing the supernatant, and then count the number of melanocytes.
7. Plate about 1 x 10⁶ melanocytes in 10 mL of TIVA medium per 100 mm cell culture dish.
8. Renew the cell culture medium every 48 h.

Wyniki

Figure 1 shows a schematic diagram comparing the conventional and the new methods. The procedure for tissue preparation and digestion with the new method is the same as the procedure for the conventional method, the only difference being that the isolated cells are resuspended in 10 mL of TIVA medium in the presence of 10 µM Y-27632. Two days after seeding, replace the media with normal TIVA medium without Y-27632. The conventional method of isolating primary melanocytes from skin tissu...

Dyskusje

The protocol described here was based on a recent publication¹⁶. Some attention should be paid to the following critical steps to achieve the best results with the new method. First, successful separation of the epidermis and the dermis is crucial. Cut the adult foreskin tissues into 3-4 mm wide strips using a scalpel blade to make the dispase work more thoroughly and easily to separate the epidermis from the dermis. Second, when separating those two layers, contamination of the dermis should be a...

Materiały

Name	Company	Catalog Number	Comments
Alexa fluor-594 donkey anti-mouse IgG	Thermo Fisher Scientific	A21203	For Immunofluorescence
Alexa fluor-594 donkey anti-rabbit IgG	Thermo Fisher Scientific	R37119	For Immunofluorescence
Cell Culture Dish	Eppendorf	30702115	For cell culture
Cell Strainer	Corning incorporated	431792	Cell filtration
15 mL Centrifuge Tube	KIRGEN	171003	For cell centrifuge
50 mL Centrifuge Tube	KIRGEN	171003	For cell centrifuge
CO2 Incubator	Thermo Scientific	51026333	For cell incubating
Constant Temperature Shaker	Shanghai Boxun	150036	For water bath
DAPI	Abcam	ab104139	For Immunofluorescence
Dispase	Gibco	17105-041	For melanocyte isolation
DMEM	Thermo Scientific	C11995500	Component of neutralization medium
Fetal Bovine Serum	Biological Industries	04-001-1AC5	Component of neutralization medium
Fluorescence microscope	Olympus	5E44316	For Immunofluorescence
Forskolin	MCE	HY-15371	Induce pigmentation
Glutamine	Thermo Fisher Scientific	25030081	melanocyte culture medium
Ham's F12	Thermo Scientific	11330032	melanocyte culture medium
Inverted microscope	Olympus	5C42258	For cell microscopic observation
3-isobutyl-1-methyl xanthine (IBMX)	Sigma	17018	melanocyte culture medium
mouse anti-human MITF	Abcam	ab12039	For Immunofluorescence
Na3VO4	Sigma	S6508	melanocytes culture medium
N6,2'-O-dibutyryladeno-sine 3',5'-cyclic monophosphate (dbcAMP)	Sigma	D0627	melanocyte culture medium
12-O-tetradecanoyl phorbol-13-acetate (TPA）	Sigma	79346	melanocyte culture medium
Penicillin Streptomycin	Thermo Scientific	15140-122	Antibiotics
rabbit anti-human Ki-67	Abcam	ab15580	For Immunofluorescence
Phosphate buffered solution	Solarbio Life Science	P1020-500	Washing solution
Sorvall ST 16R Centrifuge	Thermo Scientific	75004380	Cell centrifugation
TC20TM automated cell counter	Bio-Rad	1450102	Automatic cell counting
0.05% Trypsin	Life Technologies	25300-062	For melanocyte dissociation
Y-27632	Sigma	Y0503	For melanocyte isolation