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 laborator...

<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 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Affymetrix platform</td>
			<td>Affymetrix</td>
			<td></td>
			<td>For microarray experiments</td>
		</tr>
		<tr>
			<td>Affymetrix&#8217;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&#945;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 
			basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 &#181;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 
			basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 &#181;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&#8482; 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 ...

This protocol presents a novel method for the cultivation and maintenance of epidermal sphere cultures, and the strategy to closely investigate them using the spheroid replating assay. Begin by preparing wash medium as described in the text manuscript. In a laminar flow hood, wash neonatal foreskin twice with five milliliters of wash media in a five milliliter conical tube, then transfer the washed foreskin to a sterile Petri dish.Using a scalpel and forceps, scrape off adipose tissue and loose connective tissues from the dermal layer, then rewash the foreskin with wash media. Place the foreskin epidermis side up in a six-well plate containing two milliliters of Dispase enzyme diluted in wash media. Transfer the plate to an incubator for four hours.After incubation, transfer the foreskin to a Petri dish and use fine-tip forceps to separate the epidermis from the dermis layer. Place the epidermis into a 15 milliliter conical tube containing two milliliters of 0.25%Trypsin-EDTA. Crush the floating epidermis using a five milliliter serological pipette and incubate it for 15 minutes at 37 degrees Celsius with periodic vortexing for five seconds every five minutes.After incubation, add two milliliters of soybean trypsin inhibitor and mix it by pipetting to neutralize the trypsin, then centrifuge the cell suspension. Resuspend the pellet in 12 milliliters of complete KSFM-SCM medium and plate the cells in a 10 centimeter dish to incubate overnight at 37 degrees Celsius and 5%carbon dioxide. On the next day, remove the medium using an aspirating pipette and replace it with 12 milliliters of complete KSFM-SCM.Repeat this on days four and seven. Prepare 5%agarose mixture by adding 2.5 grams of agarose to 50 milliliters of PBS in a 100 milliliter glass bottle. Autoclave the bottle onto the liquid cycle and allow it to cool to room temperature.To prepare plates, place the cooled glass containing agarose solution in a one liter beaker filled with 200 milliliters of deionized water. Melt the agarose mixture in a research-grade microwave for up to two minutes, mixing the agarose every 60 seconds by gently tilting the bottle side to side. Add three milliliters of melted 5%agarose to 12 milliliters of prewarmed KSFM-SCM at 42 degrees Celsius for a final concentration of 1%agarose.Add 200 microliters of the 1%agar mix to each well of a 96-well plate using a multi-channel pipetter. Then leave the plate in a sterile environment at 25 degrees Celsius for four hours. Passage spheroid-forming normal human keratinocytes by aspirating media and washing cells in two milliliters of PBS.Aspirate PBS and add two milliliters of 0.25%Trypsin-EDTA to the washed cells. Then incubate for five minutes at 37 degrees Celsius. After the incubation, add two milliliters of soybean trypsin inhibitor to the plate and wash the cells from the plate into a 15 milliliter tube.Centrifuge at 250 times G for five minutes. Resuspend the cell pellet in one milliliter of PBS and quantify the cells using Trypan Blue staining. Aliquot 20, 000 normal human keratinocytes in 100 microliters of KSFM-SCM and seed the cells in each well of the previously prepared 96-well plate.Then incubate the plate overnight. Using an inverted phase contrast microscope, analyze the seeded wells for epidermis sphere formation. 24 to 48 hours after 3D epidermis sphere formation, add four milliliters of prewarmed KSFM-SCM at 37 degrees Celsius to a six centimeter dish.Using a wide bore one milliliter pipette tip, transfer a single spheroid to the plate and incubate the plate overnight. Analyze the seeded spheroid for attaching or propagating cells using an inverted phase contrast microscope. Feed cells every 96 hours by removing the media and adding two milliliters of fresh KSFM-SCM media to the plate.Passage 70 to 80%confluent spheroid-derived normal human keratinocytes and 2D monolayer cultures as previously demonstrated. Then quantify cell viability using Trypan Blue and an automated cell counter. Aliquot 100 microliters containing 100, 000 to 4 million normal human keratinocytes into 1.5 milliliter microcentrifuge tubes.Place the tubes on ice in a dark environment by turning off the bright lights within the laminar hood. Add two microliters of FITC-conjugated anti-integrin alpha six and two microliters of PE-conjugated anti-EGFR to the tubes to achieve a one to 50 dilution. Prepare a tube with no antibodies added to serve as the unstained control.Incubate the tubes on ice in the dark for 30 minutes. Perform flow cytometry analysis with appropriate lasers. Use the negative and positive controls to establish gates.Sort the subpopulation of epidermal stem cell fraction, the proliferative progenitor cell fraction, and committed progenitor cell fraction. Test for proliferative capacity of sorted cell subpopulations by transferring the content of each respective sorting tube into a 15 milliliter conical tube containing 10 times the volume of sorted cells in PBS. Centrifuge the tubes at 250 times G for five minutes.Remove the supernatant and resuspend the pellet in four milliliters of KSFM-SCM. Transfer the resuspended cells to a six centimeter plate and incubate overnight at 37 degrees Celsius in a 5%carbon dioxide incubator with 95%humidity. On the next day, remove media from the plates and add four milliliters of prewarmed KSFM-SCM at 37 degrees Celsius every three days until 70 to 80%confluency is reached.Autonomous epidermal spheroid-forming ability of normal human keratinocytes in 3D culture was assessed using the skin epidermis sphere assay. Non-spherical cell aggregation was not considered as adequate epidermis sphere formation. Dense sphere-shaped aggregation was considered a hallmark of spontaneous spheroid formation.It was necessary to use more than two times 10 to the four cells to ensure proper spontaneous aggregation. Planting of epidermis spheres in 2D culture resulted in the proliferation of a small-sized viable normal human keratinocytes. It's important to maintain these cultures below 100%confluency as this can considerably impair their growth and stem cell state in culture.The process of epidermal spheroid formation was functionally tracked at the single cell level by transfected cells with a fluorescent reporter. Spheroid-derived normal human keratinocytes subpopulation are integrin alpha six high and EGF are low cells. The cells generally make up about 25%of cultures.Characterization of this stem-like keratinocyte subpopulation was achieved by immunostaining analysis of basal cytokeratin 14 and tumor protein 63. The spheroid replating assay is an effective strategy for epidermal stem cell enrichment from neonatal human skin. This system can be employed as a high-throughput cell-based model to investigate cutaneous diseases such as wound healing and cancer.

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Developmental Biology

3.6K visualizações.  University of South Carolina School of Medicine. Este protocolo apresenta um novo método para o cultivo e manutenção de culturas epidérmicas da esfera, e a estratégia de investigá-las de perto usando o ensaio de replação esferoide. Comece preparando o meio de lavagem como descrito no manuscrito do texto. Em uma capa de fluxo laminar, lave o prepúcio neonatal duas vezes com cinco mililitros de mídia de lavagem em um tubo cônico de cinco mililitros, em seguida, transfira o prepúcio lavado para uma placa de Petri estéril.Us...