Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

Isolation and culture of DP cells

C57BL/6 mice were obtained from and housed in the laboratory animal center of the Army Medical University, Chongqing, China. All the animal-related procedures were conducted in strict accordance with the approved institutional animal care and maintenance protocols. All experimental protocols were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University. Permission number for producing animals: SCXK-PLA-20120011. Permission number for using animals: SYXK-PLA-20120031.

A 9-day old C57BL/6 mouse was sacrificed according to standard protocol. The vibrissa pads were cut off bilaterally with an iris scissor in a 100-mm plate. Vibrissa pads were rinsed with PBS, and then hair follicles were dissected together with their connective tissue sheath using 27G syringe needles under dissecting microscope. The dissected hair follicles were rinsed with PBS and incubated with 0.25% dispase for 20 min at room temperature.

Dissected hair follicles were transferred into a new 100-mm plate and thoroughly washed with PBS. A horizontal cut directly above dermal papilla was made. After that, dermal papilla was dissected out of dermal sheath using 27G syringe needles under dissecting microscope. Then the dissected DP tissues were transferred into a 10 μg/cm² collagen I coated 24-well plate. DP media were added after 30 min incubation in 37 °C. DP cells presented at about 3 days later. Cells reach confluence after 2 weeks and were passaged onto collagen I coated plates. DP medium should include α-MEM (Gibco, Waltham, MA, USA), 10% FBS (Gibco, USA), 1 × sodium pyruvate (Gibco, USA), 1 × non-essential amino acid (Gibco, USA), 1 × penicillin-streptomycin, 10 ng/ml bFGF (PeproTech, Rocky Hill, NJ, USA). During the optimization process, the classical DP medium was used as control. The control medium is consisted of DMEM (Gibco, USA), 10% FBS (Gibco, USA), 1 × penicillin-streptomycin. Another control medium was the classical DP medium with the addition of 10 ng/ml bFGF (PeproTech, USA).

Establishment of immortalized DP cell line

Retrovirus with SV40 large T antigen which was flanked with FRT sites was prepared as formerly reported (Yang et al., 2012). Primary DP cells were plated in a 60 mm-dish at 50% confluency in the morning. After attachment, polybrene (final concentration 10 μg/mL) and retrovirus (3.0 × 10⁷ TU) were added together. The second day, the supernatant was aspirated out of dish, and new DP medium was refilled. At the same time, hygromycin was added at a final concentration of 200 μg/mL. The culture medium was changed every two days until all the cells in control group died.

Antibiotic-selected DP cells were diluted with DP medium to 1–2 cells per 100 μL, and 100 μL diluted cells were added into every well of 96-well plates. Wells with only one cell were labeled and were monitored every 2 days. Cells in the labeled wells were passaged when the cell number of clones reached 20 or more.

RT-PCR

Total RNA of immortalized DP cells were extracted with Eastep™ super total RNA extraction kit (Promega, Beijing, China) according to manufacturer’s protocol. Complementary DNA was synthesized from RNA using Rever Tre Ace cDNA synthesis Kit (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. Several gene expressions were determined by PCR machine (Bio-Rad, Hercules, CA, USA) with the synthesized cDNA as template. The primers used were shown in Table 1. PCR mastermix (Novoprotein, Shanghai, China) were used when amplifying. The reannealing temperatures (Tm) and product size for the primers were also shown in Table 1.

Immunocytochemistry staining

Cover slides were placed on a 24-well plate, and cells were plated on cover slides. Twenty-four hours later, cover slides were rinsed with PBS and fixed with acetone. Then, the cover slides were rinsed with PBS and incubated with 5% goat serum in PBS at room temperature for 1 h. After that, slides were incubated with a rabbit anti-FGF7 antibody (1:100; Boster, Wuhan, China) or a rabbit anti-α-SMA antibody (1:200; Bioss, Beijing, China) at 4 °C overnight and subsequently with appropriate secondary antibodies (1:500; ZSGB-bio, Beijing, China). The slides were counterstained with DAPI (1:10,000; Beyotime, Shanghai, China) for 10 min. At last, the cover slides were moved to microscope slides, mounted with antifade mounting medium (Beyotime, Shanghai, China), and observed under fluorescent microscope. The immunostaining experiments were repeated three times.

Alkaline phosphatase staining

Cover slides were placed on a 24-well plate, and cells were plated on cover slides. Twenty-four hours later, cover slides were rinsed with PBS and fixed with in situ fixation solution (Beyotime, Shanghai, China) for 10 min. Then the cover slides were rinsed with PBS five times. Fresh made NBT/BCIP staining buffer (Beyotime, Shanghai, China) or BM purple (Roche, Indianapolis, IN, USA) were added into the wells. The plate was covered with aluminium foil in the dark. Color change was monitored every 15 min to avoid non-specific staining. After the colour change appeared, the staining solution was aspirated out and the cells were washed twice with 1 × PBS. At last, the cover slides were dehydrated, cleared, moved to microscope slides, mounted with permount (ZSGB-bio, Beijing, China), and observed under microscope. The AP staining experiments were performed twice.

Detection of immortalization

Primary DP cells and iDP6 cells were cultured. The iDP6 cells were treated with AdGFP (adenovirus with the ability to express GFP protein), AdFlip (adenovirus with the ability to express flip recombinase, which can interact with FRT thus remove the expression of SV40) or PBS. Forty-eight hours later, cells were collected and total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China). Then, total proteins were loaded to 1% SDS-PAGE gel (Beyotime, China) and transmitted to PVDF membrane (Bio-Rad, Hercules, CA, USA). The PVDF membrane were incubated with anti-SV40 (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-GAPDH (1:500; ZSGB-bio, Beijing, China) antibodies. HRP labelled secondary antibodies were used, and the results were observed under ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA). The experiment on reversing immortalization was performed twice.

DP cells can be long-term cultured with the optimized strategy

We optimized the culture strategy for DP cells from three dimensions, plate coating, dissecting method, and culture media (Fig. 1). The optimized dissecting method worked well in obtaining primary DP cells. DP cells grew better on plate coated with collagen I than on uncoated plate. The morphology of DP cells did not have any significant difference between classical DP culture medium (DMEM with 10% FBS) and classical DP culture medium with the addition of bFGF (data not shown). Compared with classical DP culture medium, primary DP cells grew better in the optimized culture medium (Figs. 2A–2D). The morphology of passaged DP cells was much more resemble in primary DP cells in the optimized culture medium. The cultured DP cells still had the characteristics of agglutinative growth in the optimized culture medium, but not in the control medium (Figs. 2E–2H).

DP cells are heterogeneous

Primary DP cells were immortalized by SV40 system. DP cells before antibiotic-selection were named with 0#. After antibiotic-selection, DP cell strains were selected by infinite dilution method. Not every single cell grew to clone at last. Cell strains were named with the time sequence when they grew to clone beginning with just one single cell. Totally 19 cell strains survived at last, named with iDP1 to iDP19 (1#–19#). The morphologic characteristics of the selected cell strains were different from each other (Fig. 3). Some cells still look like fibroblast, whereas some cells changed into epithelial-like cells (Fig. 3G). iDP6 still had the characteristic of agglutinative growth, while others lost this characteristic. Specially, iDP10 grew clonally, which implied that the cell line was more primitive. For these cell strains, the expression patterns of the markers for DP cells were also determined by RT-PCR, including FGF7, BMP6, Sox2, Tbx18, Sostdc, α-SMA and noggin (Fig. 4). All these data indicate that the cell strains were totally different from each other. Since each cell line grew from one single DP cell, the DP cells from one DP tissue were heterogeneous.

iDP6 keeps the molecular characteristics of primary DP cells

Taken morphology and mRNA expression characteristics together, iDP6 is the one that most similar to the primary DP cells. To determine whether iDP6 can be used in DP research, the activity of alkaline phosphatase was determined by AP staining, and the expression of FGF7 and α-SMA were determined by immunocytochemistry as well. At protein level, just like in situ, some iDP6 cells still had high AP activity (Figs. 5A–5F). FGF7 was expressed in the cytoplasm of all iDP6 cells (Figs. 5G–5L). α-SMA was expressed in both the cytoplasm and the nucleus of all iDP6 cells (Figs. 5M–5R). Although the AP activity in iDP6 was lower than primary DP cells, the expression patterns of FGF7 and α-SMA was similar to primary DP cells.

The immortal process of DP cells is reversible

At first, the expression of SV40 were determined to make sure that iDP6 were immortalized. Western blot showed that iDP6 cells expressed SV40, while primary DP cells did not express SV40 (Fig. 6A). Then, AdFlip was used to remove the expression of SV40 in iDP6 cells. AdGFP and PBS were used as control. Results showed that compared with control groups, the expression of SV40 decreased at 48 h after being treated with AdFlip (Fig. 6B). These results demonstrated that iDP6 was successfully immortalized and the immortal process was reversible.