The UofSC School of Medicine Instrumentation Resource Facility (IRF) provided access to imaging and cell sorting equipment and technical assistance. This work was supported in part by grant 1R21CA201853. The MCF and the IRF receive partial support from NIH grant P20GM103499, SC INBRE. The MCF also receives support from NIH grant P20GM109091. Yvon Woappi was supported in part by NIH grants 2R25GM066526-06A1 (PREP) and R25GM076277 (IMSD), and by a Fellowship by the Grace Jordan McFadden Professors Program at UofSC. Geraldine Ezeka and Justin Vercellino were supported by NIH grants 2R25GM066526-10A1 (PREP) at UofSC. Sean M. Bloos was supported by the 2016 Magellan Scholar Award at UofSC.

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 &#34;research not involving human subjects&#34;, 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
<ol>
	<li>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 &#181;m pore size). MCDB 153-LB basal medium can also be used as an alternative wash solution in place of KSFM.</li>
	<li>In a laminar flow hood, wash neonatal foreskin with 5 mL of wash media in a 50 mL conical tube. Repeat twice.</li>
	<li>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.</li>
	<li>Place foreskin epidermis side up in a 6-well plate containing 2 mL of dispase enzyme diluted in wash media (50 U/mL).</li>
	<li>Transfer the plate to an incubator for 4 hours (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>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.</li>
	<li>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 &#176;C with periodic vortexing for 5 s every 5 min.</li>
	<li>After incubation, add 2 mL of soybean trypsin inhibitor and mix by pipetting to neutralize the trypsin.</li>
	<li>Centrifuge cell suspension for 2 min at 450 x g, and then for 8 min at 250 x g.</li>
	<li>Remove floating debris with forceps, being careful not to dislodge the cell pellet. Carefully aspirate supernatant from the cell pellet.</li>
	<li>Resuspend pellet in 12 mL of complete KSFM-scm medium and plate in a 10-cm dish. Incubate dish overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>On the following day, remove medium using an aspirating pipette and replace with 12 mL complete KSFM-scm. Repeat on days 4 and 7.</li>
	<li>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.</li>
</ol>
2. Generating skin epidermosphere cultures in vitro
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Add 3 mL of melted 5% agarose to 12 mL KSFM-scm prewarmed to 42 &#176;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.</li>
	<li>Quickly pipette 200 &#181;L 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 &#176;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 &#176;C up to 24 h or at 4 &#176;C for up to 48 h when sealed with parafilm wrap. Warm plate to 37 &#176;C in a humidified incubator for at least 1 h before use.</li>
	<li>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 &#176;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.</li>
	<li>Resuspend pellet in 1 mL of PBS. Quantify cell viability using trypan blue staining and a hemocytometer or an automated cell counter.</li>
	<li>Aliquot 2 x 104 NHKc in 100 &#181;L of KSFM-scm and seed into individual wells of previously prepared 96-well round-bottom plate. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>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).</li>
</ol>
3. Epidermal spheroid re-plating assay
<ol>
	<li>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.</li>
	<li>Incubate the plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
4. 3D fluorescence cell tracking
<ol>
	<li>In a 6 cm dish, transfect spheroid-forming 2D monolayer NHKc cultures with 1 &#181;g of pMSCV-IRES-EGFP plasmid vector carrying the enhanced green fluorescent protein (eGFP) gene. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity) microscope (Figure 4A). 
	NOTE: It is important that transfection is completed in serum-free antibiotic-free conditions.</li>
	<li>Without removing transfection mix, add 2 mL of prewarmed KSFM-scm to cells. Incubate plate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>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.</li>
	<li>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 &#176;C, 5% CO2, 95% humidity). Visualize and monitor EGFPpos spheroid cell movement under the FITC channel of a fluorescence microscope (Figure 4B-C).</li>
	<li>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 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Observe and analyze propagating SD-NHKcEGFP 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.</li>
	<li>Harvest SD-NHKCEGFP for FACS isolation or assays of choice.</li>
</ol>
5. Characterization of spheroid-derived (SD) sub-populations by FACS
<ol>
	<li>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 &#176;C for 5 min or until all cells completely detach.</li>
	<li>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.</li>
	<li>Resuspend pellets in 1 mL of PBS. Quantify cell viability using trypan blue and an automated cell counter of hemocytometer.</li>
	<li>Aliquot 100 &#181;L 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.</li>
	<li>Add 2 &#181;L of FITC-conjugated anti-integrin&#945;6 and 2 &#181;L 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 &#176;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 integrin&#945;6 and EGFR.</li>
	<li>Perform flow cytometry analysis using flow cytometer of choice containing with appropriate lasers.</li>
	<li>Use the negative and positive controls to establish gates. Sort the subpopulation of integrin&#945;6hi/EGFRlo cells; these are the epidermal stem cell fraction. Integrin&#945;6hi/EGFRhi cells are the proliferative progenitor cell fraction. The integrin&#945;6lo/EGFRhi cells are the committed progenitor cell fraction (Figure 4D). Sorting FACS tube(s) should contain at least 1 mL of ice-cold KSFM-scm.</li>
	<li>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.</li>
	<li>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 &#176;C, 5% CO2, 95% humidity).</li>
	<li>The next day, remove media in plates and add 8 mL pre-warmed KSFM-scm. Incubate at 37 &#176;C, 5% CO2, 95% humidity. Re-feed cells with 12 mL medium every 3 days until 70-80% confluent (Figure 4E).</li>
</ol>
6. Immunofluorescence and staining of epidermospheres for basal stem cell markers
<ol>
	<li>Transfer epidermospheres onto coverslips coated with poly-lysine. Allow SD-NHKc to propagate until 75% confluent.</li>
	<li>Wash cells twice in PBS (4 &#176;C) for 5 min each.</li>
	<li>Fix cells with 4% paraformaldehyde (PFA) for 20 min at room temperature.</li>
	<li>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.</li>
	<li>Incubate samples with antibodies against P63 (1:200) and cytokeratin 14 (1:200) in blocking solution overnight at 4 &#176;C.</li>
	<li>Wash three times with PBS (4 &#176;C) containing Tween 20 (PBST), followed by incubation with FITC-and Alexa 568- conjugated secondary antibodies (1:1000 dilution).</li>
	<li>Stain nuclei with 4&#39;, 6-diamidino-2-phenylindole (DAPI) (1:5000 dilution) before mounting cells.</li>
	<li>Observe cells using a fluorescent-capable microscope with FITC and PE laser lines (Figure 4F).</li>
</ol>
7. Transcriptional analysis of epidermosphere cultures
<ol>
	<li>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.</li>
	<li>Pool 3-5 corresponding epidermospheres into a 1.5 mL microcentrifuge tube. Separately harvest autologous SD-NHKc and 2D monolayer cultures.</li>
	<li>Isolate total RNA from all three groups.</li>
	<li>Perform reverse transcription with 1 &#181;g of total RNA.</li>
	<li>Using cDNA, perform real-time PCR, using GAPDH as an internal control (Table 1).</li>
	<li>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.</li>
	<li>Assess RNA quality using a bioanalyzer to achieve RNA Integrity Numbers (RIN) ranging from at least 9.0 to 9.1.</li>
	<li>Perform microarray experiments by amplifying and biotinylating total RNA samples.</li>
	<li>Reverse transcribe 100 ng of total RNA per sample into ds-cDNA using NNN random primers containing a T7 RNA polymerase promoter sequence.</li>
	<li>Add T7 RNA polymerase to cDNA samples to amplify RNA, then copy RNA to ss-cDNA. Degrade excess RNA by using RNase H.</li>
	<li>Fragment sscDNA molecules and label with biotin.</li>
	<li>Amplify labeled samples for 16 h at 45 &#176;C.</li>
	<li>Hybridize samples using a hybridization oven and a wash and stain kit.</li>
	<li>Wash and stain hybridized arrays.</li>
	<li>Scan arrays using a system and computer workstation.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
8. Assessing SD-NHKc colony-forming efficiency
<ol>
	<li>Aspirate media from plates when cells reach 70-80% confluency. Wash cells three times in PBS (4 &#176;C) for 5 min each.</li>
	<li>Fix cells with 3 mL of 100% methanol (4 &#176;C) for 15 min. Wash three times in PBS for 5 min each.</li>
	<li>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.</li>
	<li>Analyze colony formation the following day and quantify the number of colonies obtained</li>
</ol>
9. Determine SD-NHKc population doublings
<ol>
	<li>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 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Add 2 mL of Soybean Trypsin Inhibitor to plate and transfer cells into a 15 mL tube.</li>
	<li>Centrifuge at 250 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend pellet in 12 mL of KSFM-scm.</li>
	<li>Seed cells at low density 1-2 x 104 NHKc into individual 10 cm dishes and incubate overnight (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Feed plates with 8 mL KSMF-scm the following day. Feed every 4 days until at least 25% confluent, then feed every 2 days.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Patel, G. K., Wilson, C. H., Harding, K. G., Finlay, A. Y., Bowden, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerous+keratinocyte+subtypes+involved+in+wound+re-epithelialization.">Numerous keratinocyte subtypes involved in wound re-epithelialization.</a> Journal of Investigative Dermatology. 126 (2), 497-502 (2006).</li><li>Dekoninck, S., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics,+migration+and+plasticity+during+wound+healing.">Stem cell dynamics, migration and plasticity during wound healing.</a> Nature Cell Biology. 21 (1), 18-24 (2019).</li><li>Byrd, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+within+Stratified+Epithelial+Stem+Cell+Populations+Maintains+the+Oral+Mucosa+in+Response+to+Physiological+Stress.">Heterogeneity within Stratified Epithelial Stem Cell Populations Maintains the Oral Mucosa in Response to Physiological Stress.</a> Cell Stem Cell. , (2019).</li><li>Rothbauer, M., Rosser, J. M., Zirath, H., Ertl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomorrow+today:+organ-on-a-chip+advances+towards+clinically+relevant+pharmaceutical+and+medical+in+vitro+models.">Tomorrow today: organ-on-a-chip advances towards clinically relevant pharmaceutical and medical in vitro models.</a> Current Opinion in Biotechnology. 55, 81-86 (2019).</li><li>Kim, S. J., Kim, E. M., Yamamoto, M., Park, H., Shin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Multi-Cellular+Spheroids+for+Tissue+Engineering+and+Regenerative+Medicine.">Engineering Multi-Cellular Spheroids for Tissue Engineering and Regenerative Medicine.</a> Advanced Healthcare Materials. , 2000608 (2020).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-bearing+human+skin+generated+entirely+from+pluripotent+stem+cells.">Hair-bearing human skin generated entirely from pluripotent stem cells.</a> Nature. 582 (7812), 399-404 (2020).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-stage+bilayer+tissue-engineered+skin+substitute+formed+by+adult+skin+progenitor+cells+produces+an+improved+skin+structure+in+vivo.">Early-stage bilayer tissue-engineered skin substitute formed by adult skin progenitor cells produces an improved skin structure in vivo.</a> Stem Cell Research &amp; Therapy. 11 (1), 407 (2020).</li><li>Borena, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphere-forming+capacity+as+an+enrichment+strategy+for+epithelial-like+stem+cells+from+equine+skin.">Sphere-forming capacity as an enrichment strategy for epithelial-like stem cells from equine skin.</a> Cellular Physiology and Biochemistry. 34 (4), 1291-1303 (2014).</li><li>Vollmers, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-+and+three-dimensional+culture+of+keratinocyte+stem+and+precursor+cells+derived+from+primary+murine+epidermal+cultures.">Two- and three-dimensional culture of keratinocyte stem and precursor cells derived from primary murine epidermal cultures.</a> Stem Cell Reviews and Reports. 8 (2), 402-413 (2012).</li><li>Kang, B. M., Kwack, M. H., Kim, M. K., Kim, J. C., Sung, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphere+formation+increases+the+ability+of+cultured+human+dermal+papilla+cells+to+induce+hair+follicles+from+mouse+epidermal+cells+in+a+reconstitution+assay.">Sphere formation increases the ability of cultured human dermal papilla cells to induce hair follicles from mouse epidermal cells in a reconstitution assay.</a> Journal of Investigative Dermatology. 132 (1), 237-239 (2012).</li><li>Woappi, Y., Hosseinipour, M., Creek, K. E., Pirisi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Properties+of+Normal+Human+Keratinocytes+Determine+Transformation+Responses+to+Human+Papillomavirus+16+DNA.">Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to Human Papillomavirus 16 DNA.</a> Journal of Virology. 92 (11), (2018).</li><li>Woappi, Y., Altomare, D., Creek, K., Pirisi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+3D+spheroid+cultures+of+human+neonatal+keratinocytes+have+enhanced+regenerative+properties.">Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties.</a> Stem Cell Research. , (2020).</li><li>Toma, J. G., McKenzie, I. A., Bagli, D., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+skin-derived+precursors+from+human+skin.">Isolation and characterization of multipotent skin-derived precursors from human skin.</a> Stem Cells. 23 (6), 727-737 (2005).</li><li>Li, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+Epigenetic+Mark+5-hmC+in+Psoriasis:+Implications+for+Epidermal+Stem+Cell+Dysregulation.">Loss of the Epigenetic Mark 5-hmC in Psoriasis: Implications for Epidermal Stem Cell Dysregulation.</a> Journal of Investigative Dermatology. , (2020).</li><li>Kuo, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+spheroid+culture+targeting+versatile+tissue+bioassays+using+a+PDMS-based+hanging+drop+array.">Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.</a> Scientific Reports. , (2017).</li><li>Woappi, Y., Ezeka, G., Vercellino, J., Bloos, S. M., Creek, K. E., Pirisi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSE94244+-+Expression+data+from+normal+human+spheroid-forming+keratinocytes+in+monolayer+mass+culture+and+from+corresponding+cornified-like+spheroid+ring+structures.">GSE94244 - Expression data from normal human spheroid-forming keratinocytes in monolayer mass culture and from corresponding cornified-like spheroid ring structures.</a> Gene Expression Omnibus (GEO). , (2020).</li></ol>

The authors do not have financial relationships to disclose.

The use of 3D spheroid culture systems has had broad utility in assessing cell stemness. These systems have been demonstrated to enhance enrichment of tissue stem cells13, yet their utility for the study of human epidermal stem cells has been limitedly explored. Here, we describe a strategy for enriching human keratinocyte stem cells using 3D culture techniques. In this system, NHKc are cultivated as self-assembling multicellular spheroid suspensions, comprised of several keratinocyte subtypes sus...

The mammalian stratified epithelium is the most complex epithelial architecture in all living systems and is most often subject to damage and injury. As a protective tissue, stratified epithelium has evolved to generate a complex and effective tissue damage response. Upon injury, these cells must activate lineage plasticity programs, which enable them to migrate to the injured site and carry out repair1,2,3. This multifaceted response occurs in several sequential steps which remain poorly understood.
A major obstacle in studying the intricate process of epithelial regeneration lies in the dearth of high throughput model systems that can capture dynamic cellular activities at defined stages of cell regeneration. While in vivo mouse models offer relevant insight into wound healing and most closely recapitulate the human regenerative process, their development require laborious efforts and significant cost, limiting their throughput capacity. There exists, therefore, a critical need for establishing systems that enable functional investigation of human epithelial tissue regeneration at high throughput scale.
In recent years, several attempts have been made to meet the scalability challenge. This is seen through great expansion of innovative in vitro&#160;and ex vivo cell-based models that closely mimic the in vivo regenerative context. This include advances in organ-on-chip4, spheroid5, organoid6, and organotypic cultures7. These 3D cell-based systems each offer unique advantages and present distinct experimental limitations. To date, spheroid culture remains the most cost-effective and widely used 3D cell culture model. And while several reports have indicated that spheroid cultures can be used to study skin stem cell characteristics, these studies have largely been conducted with animal tissue8,9, or with dermal fibroblasts10, with virtually no reports thoroughly characterizing the regenerative properties of human epidermal spheroid cultures. In this protocol we detail the functional development, culture, and maintenance of epidermal spheroid cultures from normal human keratinocytes (NHKc). We equally describe the utility of this system to model the sequential phases of epidermal regeneration and keratinocyte stem cell plasticity in vitro.

<table><tbody><tr><td>Affymetrix platform</td><td>Affymetrix</td><td></td><td>For microarray experiments</td></tr><tr><td>Affymetrix&amp;rsquo;s HuGene-2_0-st library file</td><td>Affymetrix</td><td></td><td>Process</td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent</td><td></td><td>For microarray experiments</td></tr><tr><td>All Prep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td>Used for RNA isolation</td></tr><tr><td>Analysis Console Software version 3.0.0.466</td><td></td><td></td><td>analyze cell type specific transcriptional responses using one-way between-subject analysis of variance</td></tr><tr><td>BD FACSAria II flow cytometer</td><td>Beckman</td><td></td><td>For flow cytometry</td></tr><tr><td>Console Software version 3.0.0.466/Expression console Software</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>For confirming data quality</td></tr><tr><td>Cytokeratin 14</td><td>Santa Cruz Biotechnology</td><td>sc-53253</td><td>1:200 dilution</td></tr><tr><td>Dispase</td><td>Sigma-Aldrich</td><td>D4818</td><td>For cell media</td></tr><tr><td>FITC-conjugated anti-integrin&amp;alpha;6</td><td>Abcam</td><td>ab30496</td><td>For FACS analysis</td></tr><tr><td>GeneChip Command Console 4.0 software</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>For confirming data quality</td></tr><tr><td>GeneChip Fluidics Stations 450 (Affymetrix/Thermo Fisher Scientific)</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>For washing and staining of hybridized arrays</td></tr><tr><td>GeneChip HuGene 2.0 ST Arrays</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>For hybridization and amplifycation of total RNA</td></tr><tr><td>GeneChip Hybridization Oven 640</td><td>Thermo Fisher Scientific</td><td></td><td>For hybridization and amplifycation of total RNA | Amplify labeled samples</td></tr><tr><td>GeneChip Hybridization Wash, and Stain Kit (Affymetrix/Thermo Fisher Scientific).</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>For washing and staining of hybridized arrays</td></tr><tr><td>GeneChip Scanner 3000 7G system</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>Scanning hybridized arrays</td></tr><tr><td>GeneChip WT PLUS Reagent Kit</td><td>Affymetrix/Thermo Fisher Scientific</td><td></td><td>For amplifycation of biotinylating total RNA</td></tr><tr><td>Human Basic Fibroblast Growth Factor (hFGF basic/FGF2)</td><td>Cell Signaling Technology</td><td>8910</td><td>For cell media</td></tr><tr><td>Human Epidermal Growth Factor (hEGF)</td><td>Cell Signaling Technology</td><td>8916</td><td>For cell media</td></tr><tr><td>Human Insulin</td><td>Millipore Sigma</td><td>9011-M</td><td>For cell media</td></tr><tr><td>iQ SYBR Green Supermix (Bio-Rad)</td><td>Bio-Rad</td><td>1708880</td><td>Used for RT-qPCR</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>1708890</td><td>Used for RT-qPCR</td></tr><tr><td>KSFM</td><td>ThermoFisher Scientific</td><td>17005041</td><td>Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml&lt;br/&gt; basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 &amp;micro;g/ml insulin</td></tr><tr><td>KSFM-scm</td><td>ThermoFisher Scientific</td><td>17005042</td><td>Supplemented with 1% Penicillin/Streptomycin, 20 ng/ml EGF, 10 ng/ml&lt;br/&gt; basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 &amp;micro;g/ml insulin</td></tr><tr><td>MCDB 153-LB basal medium</td><td>Sigma-Aldrich</td><td>M7403</td><td>MCDB 153-LB basal media w/ HEPES buffer</td></tr><tr><td>NEST Scientific 1-Well Cell Culture Chamber Slide, BLACK Walls on Glass Slide, 6/PK, 12/CS</td><td>Stellar Scientific</td><td>NST230111</td><td>For immunostaining</td></tr><tr><td>P63</td><td>Thermo Scientific</td><td>703809</td><td>1:200 dilution</td></tr><tr><td>PE-conjugated anti-EGFR ( San Jose, CA; catalog number )</td><td>BD Pharmingen</td><td>555997</td><td>For FACS analysis</td></tr><tr><td>pMSCV-IRES-EGFP plasmid vector</td><td>Addgene</td><td>20672</td><td>For transfection</td></tr><tr><td>Promega TransFast kit</td><td>Promega</td><td>E2431</td><td>For transfection</td></tr><tr><td>Qiagen RNeasy Plus Micro Kit</td><td>Qiagen</td><td></td><td>For microarray experiments</td></tr><tr><td>Thermo Scientific&amp;trade; Sterile Single Use Vacuum Filter Units</td><td>Thermo Scientific</td><td>09-740-63D</td><td>For cell media</td></tr><tr><td>Zeiss Axionvert 135 fluorescence microscope</td><td>Zeiss</td><td></td><td>Use with Axiovision Rel. 4.5 software</td></tr></tbody></table>

establishing a high throughput epidermal spheroid culture system to model keratinocyte stem cell plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

During the skin epidermosphere assay, NHKc cultures are seeded in agarose-coated wells of a 96-well plate (Figure 1A). Spheroid-forming cells should self-aggregate within 48h. Autonomous spheroid formation can be assessed as early as 24 h using a standard inverted phase-contrast microscope. skin epidermosphere formation and re-plating assay model various phases of epidermal tissue regeneration (Figure 1B). Figure 2 shows high resolu...

Watch this Scientific Journal Video about Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity at JoVE.com

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

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

establishing-high-throughput-epidermal-spheroid-culture-system-to

Research

JoVE Journal

Developmental Biology

3.7K Views.  University of South Carolina School of Medicine. Dit protocol presenteert een nieuwe methode voor de teelt en het onderhoud van epidermale bolculturen, en de strategie om ze nauwkeurig te onderzoeken met behulp van de sferoïde replating assay. Begin met het voorbereiden van wasmedium zoals beschreven in het teksthandschrift. Was in een laminaire flowkap de neonatale voorhuid tweemaal met vijf milliliter wasmedia in een conische buis van vijf milliliter en breng de gewassen voorhuid vervolgens over in een steriele petrischaal.Schraap...

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Cancer Research

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Bioengineering

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Biology

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro.&#160;Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

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

Samenvatting

Meer video's verkennen