Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

The protocol for the collection and handling of skin specimens and isolation of human keratinocytes has been reviewed by the University of South Carolina (UofSC) IRB and classified as "research not involving human subjects", as the foreskin specimens were surgical discards produced during routine surgical procedures (circumcision of neonate boys) and were completely devoid of identifying information. The protocol was also reviewed and approved by the UofSC Biosafety Committee on a regular basis, and all laboratory personnel underwent laboratory biosafety training. All procedures were conducted in concordance to the safety and ethics standards of UofSC.

1. Isolation and culture of human keratinocytes from neonatal foreskin tissue

1. Prepare wash medium by adding 0.1 M HEPES buffer to 500 mL of KSFM medium and adjust it to a pH of 7.2. Sterilize medium in a 500 mL vacuum filter (0.22 µm pore size). MCDB 153-LB basal medium can also be used as an alternative wash solution in place of KSFM.
2. In a laminar flow hood, wash neonatal foreskin with 5 mL of wash media in a 50 mL conical tube. Repeat twice.
3. Transfer washed foreskin to a sterile Petri dish. Using a scalpel and forceps, scrape off adipose and loose connective tissues from the dermal layer. Rewash foreskin with 5 mL of wash media in a 50 mL conical tube.
4. Place foreskin epidermis side up in a 6-well plate containing 2 mL of dispase enzyme diluted in wash media (50 U/mL).
5. Transfer the plate to an incubator for 4 hours (37 °C, 5% CO₂, 95% humidity).
6. Remove foreskin from the incubator and, under sterile conditions, transfer it to a Petri dish. Using fine-tip forceps, separate the epidermis from dermis layer.
7. Place epidermis into a 15 mL conical tube containing 2 mL of 0.25% Trypsin-EDTA. Using a 5 mL serological pipet, mechanically crush the floating epidermis. Incubate for 15 min at 37 °C with periodic vortexing for 5 s every 5 min.
8. After incubation, add 2 mL of soybean trypsin inhibitor and mix by pipetting to neutralize the trypsin.
9. Centrifuge cell suspension for 2 min at 450 x g, and then for 8 min at 250 x g.
10. Remove floating debris with forceps, being careful not to dislodge the cell pellet. Carefully aspirate supernatant from the cell pellet.
11. Resuspend pellet in 12 mL of complete KSFM-scm medium and plate in a 10-cm dish. Incubate dish overnight (37 °C, 5% CO₂, 95% humidity).
12. On the following day, remove medium using an aspirating pipette and replace with 12 mL complete KSFM-scm. Repeat on days 4 and 7.
13. To maintain optimal growth of normal human keratinocyte (NHKc) cultures, passage cells 1:5 on day 10 to prevent cells from achieving greater than 80% confluency.

2. Generating skin epidermosphere cultures in vitro

1. Prepare a 5% agarose mixture by adding 2.5 g of agarose to 50 mL of 1x phosphate buffered saline (PBS), in a 50 mL glass bottle. Autoclave bottle under liquid cycle. Allow to cool down to room temperature.
2. To prepare plates, place the cooled glass bottle containing agarose solution in a 1 L plastic beaker filled with 200 mL deionized water (dH₂O). Melt agarose mixture in a research-grade microwave for up to 2 minutes, mixing the agar every 60 s by gently tilting the bottle side to side.
   CAUTION: Melting agar in the microwave will cause the flask container to become extremely hot resulting in pressure buildup. Release pressure buildup every 30 s. Wear appropriate protective equipment to prevent injury.
3. Add 3 mL of melted 5% agarose to 12 mL KSFM-scm prewarmed to 42 °C for a final concentration of 1% agarose (wt/vol).
   Optional: pre-warm the serological pipettes and pipette tips to prevent premature polymerization of the soft agar.
4. Quickly pipette 200 µL of the 1% agar mix into individual wells of a 96-well round-bottom plate using a multichannel pipettor (Figure 1A). Leave plate in sterile environment at 25 °C for 4 h to allow agarose to fully polymerize.
   NOTE: Experimentation can be stopped here and continued the next day. Polymerized agarose plates can be maintained at 37 °C up to 24 h or at 4 °C for up to 48 h when sealed with parafilm wrap. Warm plate to 37 °C in a humidified incubator for at least 1 h before use.
5. Passage spheroid-forming NHKc by aspirating media and washing cells in 2 mL of PBS. Aspirate PBS and add 2 mL of 0.25% Trypsin-EDTA to washed cells. Incubate for 5 min at 37 °C or until all cells completely detach. Add 2 mL of Soybean Trypsin Inhibitor to plate and wash off cells from the plate into a 15 mL tube. Centrifuge at 250 x g for 5 min.
6. Resuspend pellet in 1 mL of PBS. Quantify cell viability using trypan blue staining and a hemocytometer or an automated cell counter.
7. Aliquot 2 x 10⁴ NHKc in 100 µL of KSFM-scm and seed into individual wells of previously prepared 96-well round-bottom plate. Incubate plate overnight (37 °C, 5% CO₂, 95% humidity).
8. Using an inverted phase contrast microscope, analyze seeded well for epidermosphere formation.
   NOTE: Human epidermospheres from normal human keratinocytes can remain viable in 3D suspension culture for up to 96 h, although with considerable decrease in cell viability (Figure 2).

3. Epidermal spheroid re-plating assay

1. 24-48 h after 3D epidermosphere formation, add 4 mL of prewarmed KSFM-scm to a 6 cm dish. Use a wide bore 1 mL pipette tip to transfer a single spheroid to the plate, ensuring to not break it apart. Alternatively, widen a 1 mL pipette tip using a sterile razor blade to cut the tip.
2. Incubate the plate overnight (37 °C, 5% CO₂, 95% humidity).
3. Analyze seeded spheroid for attachment to the bottom of the plate and observe for propagating cells using an inverted phase contrast microscope (Figure 3). Feed cells every 96 h by gently removing 2 mL of the media inside the plate and slowly adding 2 mL fresh KSFM-scm media to the plate.
4. Passage spheroid-derived (SD) NHKc once they reach 70-80% confluency and continue with assay of choice. Monitor SD-NHKc frequently to prevent cells from reaching full confluency in culture, as this results in premature differentiation and cell growth arrest (Figure 3E-F).
5. The epidermal spheroid replating assay models keratinocyte-mediated wound repair by capturing each of the key sequential phases of epidermal regenerative plasticity: a) homeostatic maintenance, b) differentiation halt/reversal c) stress lineage mobility, and d) tissue restoration (Figure 1B; Table 2). This assay can also be used to produce cell populations for organotypic raft cultures or to model HPV-mediated neoplasia as demonstrated in our previous work ^11,12.

4. 3D fluorescence cell tracking

1. In a 6 cm dish, transfect spheroid-forming 2D monolayer NHKc cultures with 1 µg of pMSCV-IRES-EGFP plasmid vector carrying the enhanced green fluorescent protein (eGFP) gene. Incubate plate overnight (37 °C, 5% CO₂, 95% humidity) microscope (Figure 4A).
   NOTE: It is important that transfection is completed in serum-free antibiotic-free conditions.
2. Without removing transfection mix, add 2 mL of prewarmed KSFM-scm to cells. Incubate plate overnight (37 °C, 5% CO₂, 95% humidity).
3. Aspirate all transfection media from plate and feed cells with 3 mL of prewarmed KSFM-scm. Assess for presence of EGFP-expressing cells under the FITC channel of a fluorescent microscope.
4. Passage and seed 2 x 10⁴ EGFP-transfected cells into individual wells of a previously prepared 96-well round-bottom plate. Incubate plate overnight (37 °C, 5% CO₂, 95% humidity). Visualize and monitor EGFP^pos spheroid cell movement under the FITC channel of a fluorescence microscope (Figure 4B-C).
5. 24 h after plating, add 3 mL of prewarmed KSFM-scm to a 6 cm dish. Use a wide bore 1 mL pipette tip to transfer a single spheroid to the plate, ensuring to not break it apart. Incubate plate overnight (37 °C, 5% CO₂, 95% humidity).
6. Observe and analyze propagating SD-NHKc^EGFP using a fluorescence microscope. Feed cells every 96 h by gently removing 2 mL the media inside the plate and slowly adding 2 mL fresh KSFM-scm media to the plate.
7. Harvest SD-NHKC^EGFP for FACS isolation or assays of choice.

5. Characterization of spheroid-derived (SD) sub-populations by FACS

1. Passage SD-NHKc and corresponding autologous 2D monolayer cultures by aspirating old media and washing cells in 2 mL of PBS. Remove PBS and add 2 mL of 0.25% Trypsin-EDTA to washed cells. Incubate at 37 °C for 5 min or until all cells completely detach.
2. Add 2 mL of Soybean Trypsin Inhibitor to plate and transfer cells into a 15 mL tube. Centrifuge at 250 x g for 5 min.
3. Resuspend pellets in 1 mL of PBS. Quantify cell viability using trypan blue and an automated cell counter of hemocytometer.
4. Aliquot 100 µL containing 0.1-4 x 10⁶ NHKc cells to 1.5 mL microcentrifuge tubes. Place tube on ice in a dark environment, created by turning off bright lights within the laminar hood.
5. Add 2 µL of FITC-conjugated anti-integrinα6 and 2 µL of PE-conjugated anti-EGFR to tubes to achieve a 1:50 dilutions. Prepare a tube with no antibodies added to serve as the unstained control. Incubate tubes on ice in dark or at 4 °C for 30 min. The use of beads or a skin SCC line can serve as positive control, as skin SCC cells express elevated levels of integrinα6 and EGFR.
6. Perform flow cytometry analysis using flow cytometer of choice containing with appropriate lasers.
7. Use the negative and positive controls to establish gates. Sort the subpopulation of integrinα6^hi/EGFR^lo cells; these are the epidermal stem cell fraction. Integrinα6^hi/EGFR^hi cells are the proliferative progenitor cell fraction. The integrinα6^lo/EGFR^hi cells are the committed progenitor cell fraction (Figure 4D). Sorting FACS tube(s) should contain at least 1 mL of ice-cold KSFM-scm.
8. Test for proliferative capacity of sorted cell subpopulations by transferring the content of each respective sorting tube into a 15 mL conical tube containing 10x the volume of sorted cells in PBS. NOTE: It is important to remove all cells attached to the rim by pipetting the wall of the sorting FACS tube(s) several times, as keratinocytes can often adhere there.
9. Centrifuge 15 mL tubes at 250 x g for 5 min. Remove supernatant and resuspend pellet in 12 mL of KSFM-scm. Transfer the resuspended cells into a 6 cm plate and incubate overnight (37 °C, 5% CO₂, 95% humidity).
10. The next day, remove media in plates and add 8 mL pre-warmed KSFM-scm. Incubate at 37 °C, 5% CO₂, 95% humidity. Re-feed cells with 12 mL medium every 3 days until 70-80% confluent (Figure 4E).

6. Immunofluorescence and staining of epidermospheres for basal stem cell markers

1. Transfer epidermospheres onto coverslips coated with poly-lysine. Allow SD-NHKc to propagate until 75% confluent.
2. Wash cells twice in PBS (4 °C) for 5 min each.
3. Fix cells with 4% paraformaldehyde (PFA) for 20 min at room temperature.
4. Permeabilize cells with 0.5% Triton X-100 in 1% glycine. Block using 0.5% BSA and 5% goat serum for 30 min at room temperature.
5. Incubate samples with antibodies against P63 (1:200) and cytokeratin 14 (1:200) in blocking solution overnight at 4 °C.
6. Wash three times with PBS (4 °C) containing Tween 20 (PBST), followed by incubation with FITC-and Alexa 568- conjugated secondary antibodies (1:1000 dilution).
7. Stain nuclei with 4', 6-diamidino-2-phenylindole (DAPI) (1:5000 dilution) before mounting cells.
8. Observe cells using a fluorescent-capable microscope with FITC and PE laser lines (Figure 4F).

7. Transcriptional analysis of epidermosphere cultures

1. Set up triplicate plates of low passage NHKc cultures to isolate RNA can be from monolayer, spheroid, and spheroid-derived cultures from the same autologous cell line.
2. Pool 3-5 corresponding epidermospheres into a 1.5 mL microcentrifuge tube. Separately harvest autologous SD-NHKc and 2D monolayer cultures.
3. Isolate total RNA from all three groups.
4. Perform reverse transcription with 1 µg of total RNA.
5. Using cDNA, perform real-time PCR, using GAPDH as an internal control (Table 1).
6. Validate amplicon product size by agarose gel electrophoresis (2% v/v).
   NOTE: For transcriptomic-wide profiling of epidermosphere cultures using whole-genome microarray analysis, isolate total RNA from mass cultures of a single NHKc donor and corresponding SD-NHKc from the same donor, in six replicates each.
7. Assess RNA quality using a bioanalyzer to achieve RNA Integrity Numbers (RIN) ranging from at least 9.0 to 9.1.
8. Perform microarray experiments by amplifying and biotinylating total RNA samples.
9. Reverse transcribe 100 ng of total RNA per sample into ds-cDNA using NNN random primers containing a T7 RNA polymerase promoter sequence.
10. Add T7 RNA polymerase to cDNA samples to amplify RNA, then copy RNA to ss-cDNA. Degrade excess RNA by using RNase H.
11. Fragment sscDNA molecules and label with biotin.
12. Amplify labeled samples for 16 h at 45 °C.
13. Hybridize samples using a hybridization oven and a wash and stain kit.
14. Wash and stain hybridized arrays.
15. Scan arrays using a system and computer workstation.
16. Following completion of array scans, import probe cell intensity (CEL) files into expression console software and process at the gene-level using library file and Robust Multichip Analysis (RMA) algorithm to generate CHP files.
17. After confirming data quality within Expression Console, import CHP files containing log2 expression signals for each probe into a transcriptome analysis software to analyze cell type specific transcriptional responses using one-way between-subject analysis of variance.

8. Assessing SD-NHKc colony-forming efficiency

1. Aspirate media from plates when cells reach 70-80% confluency. Wash cells three times in PBS (4 °C) for 5 min each.
2. Fix cells with 3 mL of 100% methanol (4 °C) for 15 min. Wash three times in PBS for 5 min each.
3. Stain cells in 3 mL of 10% Giemsa for 30 min. Wash three times in PBS for 5 min each. Allow cells to air dry overnight.
4. Analyze colony formation the following day and quantify the number of colonies obtained

9. Determine SD-NHKc population doublings

1. Passage SD-NHKc and corresponding autologous 2D monolayer cultures by aspirating old media and washing cells in 2mL PBS. Remove PBS and add 2 mL 0.25% Trypsin-EDTA to washed cells. Incubate for 5 min or until all cells completely detach (37 °C, 5% CO₂, 95% humidity).
2. Add 2 mL of Soybean Trypsin Inhibitor to plate and transfer cells into a 15 mL tube.
3. Centrifuge at 250 x g for 5 min.
4. Remove the supernatant and resuspend pellet in 12 mL of KSFM-scm.
5. Seed cells at low density 1-2 x 10⁴ NHKc into individual 10 cm dishes and incubate overnight (37 °C, 5% CO₂, 95% humidity).
6. Feed plates with 8 mL KSMF-scm the following day. Feed every 4 days until at least 25% confluent, then feed every 2 days.
7. Serially passage cells 1:5 in 60 cm dishes until cell proliferative capacity is exhausted. Quantify cell viability by trypan blue staining. Determine population doublings at each passage using the formula: log(N/N₀) / log2, where N represents the total cell number obtained at each passage and N₀ represents the number of cells plated at the beginning of the experiment.