Name: isolation, culture, and characterization of primary schwann cells, keratinocytes, and fibroblasts from human foreskin
Uploaded: 2022-03-23T15:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin at JoVE.com

We would like to thank Dr. Fadia Kamal, and Dr. Reyad Elbarbary for allowing us to use lab instruments and technical support. This work was supported by grants from the NIH (K08 AR060164-01A) and DOD (W81XWH-16-1-0725) to J. C. E. in addition to institutional support from the Pennsylvania State University Hershey Medical Center.

Acquisition and use of de-identified human foreskin tissue for research purposes was reviewed and received the determination of &#34;not human research&#34; by the Penn State College of Medicine Institutional Review Board (IRB #17574).
NOTE: By following the protocol below, 2.4 x 106 keratinocytes, 4.4 x 106 fibroblasts and 1.1 x 106 SCs are obtained from a single foreskin. In general, these primary cells can be used for 3 passages, depending on the experimental conditions.
1. Isolation of primary cells and culture conditions
<ol>
	<li>Epidermis and dermis separation procedure
	<ol>
		<li>Acquire neonatal foreskin following standard of care procedures by a physician under approved hospital regulatory protocols. The physician determines the timing of circumcision after birth and the amount of skin removed.</li>
		<li>Place the excised foreskin in a microcentrifuge tube containing 8-10 mL of ice-cold Dulbecco&#39;s Modified Eagle Medium (DMEM) basal medium and immediately transport the foreskin to the cell culture laboratory for cells isolation. Rinse the skin twice with 20-25 mL of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) containing 1x antibiotic/antimycotic. 
		NOTE: For maximum cell viability, the excised foreskin should be placed at 4 &#176;C until processing, and cells should be harvested within 24 h.</li>
		<li>After rinsing in DPBS/antibiotics, soak the foreskin in 25-30 mL of ice-cold DMEM basal medium for 10-15 min in a 10 cm tissue culture dish. Perform all processing steps in a tissue culture hood using sterile technique.</li>
		<li>Using a scalpel, carefully slice the foreskin open and expose the dermis and subcutaneous adipose tissue.</li>
		<li>Remove the subcutaneous adipose tissue using forceps, scalpel blade, and scissors, as needed. Discard the adipose tissue. Cut the cleaned foreskin into three to five smaller pieces.</li>
		<li>Transfer skin pieces to a sterile conical tube containing 16-20 mL of dispase-I/DMEM medium (4 mg/mL in DMEM basal medium).</li>
		<li>Mix the skin pieces in the conical tube by inversion, intermittently, during the first 30 min of incubation with dispase-I. Then place the conical tube at 4 &#176;C for 16-18 h, being mindful of immersing the skin pieces in dispase-I/media. 
		NOTE: Dispase-I solution should be prepared just before addition using cold DMEM basal medium, filtered with a 0.22 &#181;m strainer, and kept at 4 &#176;C.</li>
		<li>After overnight digestion with dispase-I, remove the skin pieces and place them on a dry, sterile, culture dish. Unroll each piece of skin so that the outer epidermal layer is touching the culture dish.</li>
		<li>Using two sets of forceps, carefully separate the epidermis from the dermis. Start on the edge of the tissue and peel the epidermis away from the dermis. 
		NOTE: If the epidermis layer is difficult to separate from the dermis, the dispase-I digestion was not sufficient. Troubleshooting options: 1) return skin pieces to dispase-I/DMEM solution and incubate for an additional 2-3 h at 4 &#176;C; 2) place skin pieces in fresh dispase-I/DMEM and incubate at 4 &#176;C for 2-3 h more.</li>
		<li>Use the separated epidermis and dermis to isolate keratinocytes (epidermis) and fibroblasts and Schwann cells (dermis). 
		NOTE: Follow the steps below for specific cell types isolation and culture conditions.</li>
	</ol>
	</li>
	<li>Keratinocyte isolation from the epidermis
	<ol>
		<li>Add 5 mL of HEPES buffer saline (HBS) to a 10 cm culture dish and add the separated epidermis fragments. Incubate at room temperature (RT) for 10 min.</li>
		<li>After HBS incubation, collect the HBS solution in a 50 mL conical tube. Add 5 mL of trypsin neutralization buffer (TNB) (Table of Materials) in a 50 mL conical tube and set aside.</li>
		<li>Add 3 mL of trypsin solution to the epidermal fragments. Incubate at 37 &#176;C for 10 min.</li>
		<li>Gently agitate the epidermal fragments using forceps until the trypsin solution becomes turbid.</li>
		<li>Add the trypsin solution/keratinocytes to the HBS and TNB containing tube in step 1.2.2.</li>
		<li>For any remaining epidermal fragments that have not dissolved in the initial trypsin digestion, add 3 mL of 0.25% trypsin/Ethylenediaminetetraacetic acid (EDTA). Incubate at 37 &#176;C for 10 min and repeat steps 1.2.4 and 1.2.5.</li>
		<li>Add 2 mL of fetal bovine serum (FBS) to the trypsin/EDTA solution containing HBS and TNB solution tubes.</li>
		<li>Centrifuge the collection tube containing HBS/TNB/trypsin and keratinocytes at 870 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the cell pellet in 3 mL of keratinocyte complete growth medium (Table of Materials).</li>
		<li>Filter the cell suspension with a sterile 100 &#181;M filter into a sterile 50 mL conical tube to remove debris.</li>
		<li>Quantify the cell number and cell viability. 
		NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
		<li>Seed culture plates as needed for experiments. The recommended seeding density is ~45,000 cells/cm2.</li>
		<li>Place the culture plates in an incubator at 37 &#176;C in 5% CO2 for 2 days to allow keratinocytes to adhere to the dish.</li>
		<li>After 2 days, aspirate any non-adhered cells. Refresh the cell culture media every other day until keratinocytes reach desired confluence for passaging or downstream experiments (50%-80% confluence).</li>
	</ol>
	</li>
	<li>Isolation of Schwann cells (SCs) and fibroblasts from the dermis
	<ol>
		<li>Pre-coat T25 flasks with poly-L-lysine (PLL, 0.01 &#181;g/&#181;L) using 5 mL of 1x DPBS. Place the pre-coated flasks at 37 &#176;C for 3 h. Wash the PLL coating matrix twice using 1x DPBS (5 mL). 
		NOTE: T25 flasks can be prepared ahead of time.</li>
		<li>Rinse the isolated pieces of the dermis with 5 mL of DMEM basal medium.</li>
		<li>Mince the dermis into small pieces with scissors or a scalpel.</li>
		<li>Digest the minced dermis with 5 mL of collagenase (2 mg/mL in DMEM basal medium) at 37 &#176;C for 2.5 h. Gently triturate the minced dermis with a pipette tip every 30 min until the dermis completely dissociates.</li>
		<li>Filter the cell suspension with a 70 &#181;M cell strainer. It is necessary to apply mechanical force using a 1 mL syringe to fully filter the suspension.</li>
		<li>Dilute the filtered cell suspension with 5 mL of complete DMEM medium (DMEM basal medium + 5% FBS + 1x of antibiotic) to stop the enzymatic activity of collagenase.</li>
		<li>Centrifuge the cell suspension at 870 x g for 5 min at RT to pellet the cells.</li>
		<li>From this cell pellet, both fibroblasts and SCs will be obtained. Resuspend the cell pellet in 2 mL of DMEM complete medium to divide the cells (1 mL cell suspension/tube) and centrifuge at 870 x g for 5 min at RT.</li>
		<li>For SCs culture, follow steps 1.3.10-1.3.17 and for fibroblast culture, follow steps 1.3.18-1.3.22.</li>
		<li>From step 1.3.8, aspirate the supernatant. Resuspend the cell pellet in 5 mL of fresh DMEM complete medium.</li>
		<li>Quantify the cell number and viability. 
		NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
		<li>Seed the pre-coated T25 flasks with 5 mL of resuspended cells at a density 4.0 x 103 cells/mL. Incubate the flask at 37 &#176;C in the presence of 5% CO2 overnight (~12-16 h incubation).</li>
		<li>After 16 h, confirm cell adhesion by microscopy (Table of Materials) at 10x magnification.</li>
		<li>Remove the non-adherent cells and wash the flask 3x with 5 mL of 1x DPBS.</li>
		<li>Add 5 mL of 10 &#181;M cytosine arabinoside (antimitotic agent, kills fibroblasts) in DMEM complete medium. Incubate the flask at 37 &#176;C in the presence of 5% CO2&#160;for 24 h.</li>
		<li>Aspirate the cytosine arabinoside containing medium from the flask. Rinse the flask 3x with 5 mL of 1x DPBS.</li>
		<li>Refill the culture flask with 5 mL of SCs complete culture medium (Table of Materials) and incubate at 37 &#176;C in the presence of 5% CO2&#160;for 48 h. Refresh SC complete culture medium every other day until SCs reach 80% confluence.</li>
		<li>Aspirate the supernatant from the dissociated dermal layer (step 1.3.8) and resuspend the cell pellet with 5 mL of fibroblast complete medium (fibroblast basal medium + 5% FBS + 1x antibiotic).</li>
		<li>Quantify cell number and cell viability. 
		&#8203;NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
		<li>Plate the fibroblasts at a cell density of 4.0 x 103 cells/mL in T25 culture flasks (not pre-coated) using 5 mL of fibroblast complete medium. Incubate at 37 &#176;C in the presence of 5% CO2 overnight (~12-16 h incubation).</li>
		<li>After 16-24 h, confirm cell adhesion by microscopy (Table of Materials) at 10x magnification. Aspirate non-adherent cells from the flask and wash the flask twice with 5 mL of 1x DPBS.</li>
		<li>Add 5 mL of fresh fibroblast complete medium and incubate at 37 &#176;C in the presence of 5% CO2 for 48 h. Refresh the culture medium every other day until fibroblasts reach the desired confluence for passaging or downstream assays (50%-80% confluence).</li>
	</ol>
	</li>
</ol>
2. Verification of epidermal keratinocytes, dermal SCs and fibroblasts protein marker by immunofluorescence
<ol>
	<li>Day 1- Coat chamber glass slides and detach cells
	<ol>
		<li>Pre-coat 4-well chamber glass slides with collagen-I for keratinocytes, or PLL for SCs or, only 1x DPBS for fibroblasts (0.5 mL each well). Ensure to coat the entire surface. Incubate chamber slides at 37 &#176;C for 2 h. During incubation, prepare the cells.</li>
		<li>After cultured cells reach 50%-80% confluence, wash the flasks with 1x DPBS 3x (1 mL for each well).</li>
		<li>Detach the adhered cells with 5 mL of 0.25% trypsin-EDTA (TE) solution by incubating at 37 &#176;C in the presence of 5% CO2 for 2 min, until cells start to become round. Visualize cell detachment with microscopy (Table of Materials) at 10x magnification.</li>
		<li>After incubation, transfer TE solution containing cells to a conical tube with 5 mL fetal bovine serum (FBS).</li>
		<li>Add 5 mL of trypsin neutralization buffer (TNB) to the culture flask and then transfer the contents to the above-mentioned centrifuge tube (cells with TE and FBS). Visualize cell detachment with microscopy (Table of Materials) at 10x magnification. 
		NOTE: TNB or FBS or both can be used to neutralize trypsin activity.</li>
		<li>Centrifuge the conical tube containing cells/TE/TNB at 870 x g for 5 min. Transfer the supernatant to another tube and resuspend the cell pellet in the corresponding complete culture medium. Measure the cell viability as mentioned in step 1.2.11.</li>
		<li>Rinse the pre-coated chamber glass slides (step 2.1.1) with 1x DPBS (1 mL for each well) 3x.</li>
		<li>Semi-dry the slides inside the tissue culture hood and seed keratinocytes, SCs, or fibroblasts at an appropriate density (30,000 cells/0.5 mL/chamber) in an appropriate complete cell culture medium.</li>
		<li>Incubate the chamber slides at 37 &#176;C overnight to allow cells to adhere.</li>
	</ol>
	</li>
	<li>Day 2- Blocking the chamber glass slide with Bovine Serum Albumin (BSA) and primary antibodies incubation
	<ol>
		<li>Aspirate the cell media from the chamber slides and wash 3x with 1 mL of 1x DPBS.</li>
		<li>Fix the adhered cells with 4% paraformaldehyde in 1x DPBS (1 mL for each well) and incubate for 15 min at RT inside the tissue culture hood.</li>
		<li>Aspirate 4% paraformaldehyde from the chamber slides and wash the chamber slides 3x with 1x DPBS (1 mL for each well).</li>
		<li>Permeabilize the cell membranes with 1% TritonX-100 in 1x DPBS (1 mL for each well) and incubate for 15 min at RT.</li>
		<li>Aspirate the blocking solution from the chamber slides, wash 3x with 1x DPBS (1 mL for each well) for 30 s and then aspirate 1x DPBS from the well.</li>
		<li>Block the chamber slides with 5% BSA (0.5 mL for each well) and incubate for 45 min at RT.</li>
		<li>Aspirate the blocking solution from the chamber slides, wash 3x with 1x DPBS (1 mL for each well) for 30 s each and then aspirate 1x DPBS from the well.</li>
		<li>Dilute the primary antibodies with 5% BSA (0.2 mL for each well) (Primary antibodies dilutions: Keratinocytes: mouse cytokeratin 14 antibody (1:100) and, cytokeratin 10 antibody (1:100); Schwann cells: mouse S100 antibody (1:200), and rabbit nerve growth factor receptor (p75-NTR) antibody (1:500); Fibroblasts: rabbit vimentin antibody (1:200), and mouse alpha-smooth muscle actin (&#945;-SMA) antibody (1:200)).</li>
		<li>Add the primary antibodies to the corresponding cells on the chamber glass slide and incubate for about 15-16 h (overnight) at 4 &#176;C in a humidified chamber.</li>
		<li>Aspirate the primary antibodies from the chamber slides, wash 3 times with 1x DPBS (1 mL for each well) for 30 s each and then aspirate 1x DPBS from the well.</li>
		<li>Dilute the secondary antibodies (Goat anti-rabbit IgG Secondary Antibody-Alexa Fluor 488 (1:500) and Goat anti-Mouse IgG Secondary Antibody-Alexa Fluor 594 (1:500)) with 0.5% BSA. Add appropriate secondary antibody to each well (0.2 mL for each well). Incubate for 45 min at RT.</li>
		<li>Aspirate the secondary antibody from chamber slides, wash 3x with 1x DPBS (1 mL for each well) for 30 s each and then aspirate 1x DBPS from the well.</li>
		<li>Remove the gasket on the 4-well chamber slide using gasket remover. Try not to leave any adhesive on the slide as this interferes with coverslip application. Add a drop of mounting medium with 4&#39;,6-diamidino-2-phenylindole (DAPI). Alternatively, counter-stain the wells with DAPI and use a non-label-containing mounting medium.</li>
		<li>Carefully place the coverslip on the glass slide by avoiding air bubbles and seal the end of the coverslip with glue. Observe immunofluorescence staining patterns by microscopy (Table of Materials) at 10x and/or 20x magnification.</li>
	</ol>
	</li>
</ol>

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All other authors declare that they have no competing financial interests.

This protocol describes a method to isolate three distinct cell populations from a single piece of the foreskin, namely keratinocytes, fibroblasts, and Schwann cells. There are a few isolation protocols available to isolate keratinocytes and fibroblasts2,3,5,6, but none describe SC isolation. Apart from the key structural cells in skin, keratinocytes, and fibroblasts, skin is also highly innerv...

Primary cells derived from living tissue and cultured under in vitro conditions closely resemble the physiological state1, making them an ideal model for investigating physiological and pathophysiological processes. The skin contains multiple cell types, including keratinocytes, fibroblasts, sebocytes, melanocytes, and Schwann cells (SCs), which can be isolated and cultured for in vitro experiments. Methods to isolate and culture keratinocytes, fibroblasts, and SCs, from a single piece of skin, have not been described. The goal of this protocol is twofold: 1) to establish a reliable and reproducible method for the isolation of and culturing of dermal SCs and 2) to use an efficient, robust method for the isolation of keratinocytes, fibroblasts, and SCs from a single human foreskin.
At present, there are established protocols to isolate skin keratinocytes2,3,4 and fibroblasts5,6. These studies describe the isolation of either keratinocytes, fibroblasts, or both from the skin, but no protocol addresses how to establish cultures of primary SCs from human skin. Recent studies suggest that neuronal SCs modulate keratinocyte and fibroblast cellular processes and regulate normal skin physiological functions7. SCs are thus critical to skin homeostasis and contribute substantially to the regulate physiology that influences the behavior of neighboring skin cell types present8. Therefore, a protocol that allows for the isolation of each of these cell types is ideal for in vitro experiments involving cell-cell communication or cross-talk between cell types.
This protocol describes the establishment of individual cell cultures of primary cells from a single piece of skin. This protocol is particularly useful when the amount of tissue available is limited. Moreover, isolation of all three cell types from a single donor allows for robust comparisons between cell types or co-culture experiments while mitigating the influence of genetics during the desired experiment.

<table><tbody><tr><td>0.22 &amp;micro;M sterile filters (Millex-GP Syringe Filter Unit,polyethersulfone)</td><td>MilliporeSigma</td><td>SLGPR33RS</td><td></td></tr><tr><td>70 &amp;micro;M cell strainers</td><td>CELLTREAT</td><td>229483</td><td></td></tr><tr><td>100 &amp;micro;M cell strainers</td><td>CELLTREAT</td><td>229485</td><td></td></tr><tr><td>1 mL disposable syringes</td><td>BD Luer-Lok</td><td>BD-309659</td><td></td></tr><tr><td>5 mL disposable syringes (Syringe sterile, single use)</td><td>BD Luer-Lok</td><td>BD309646</td><td></td></tr><tr><td>10 mL disposable syringes</td><td>BD Luer-Lok</td><td>BD305462</td><td></td></tr><tr><td>1% TritonX-100</td><td>Sigma</td><td>X100-1L</td><td>Prepared at the time of use</td></tr><tr><td>4% paraformaldehyde solution</td><td>ThermoFisher Scientific</td><td>J19943.K2</td><td>Ready to use and store at 4 &amp;deg;C</td></tr><tr><td>5% BSA</td><td>Sigma</td><td>A7906-100G</td><td>Prepared at the time of use</td></tr><tr><td>70% ethanol</td><td>Pharmco</td><td>111000200</td><td></td></tr><tr><td>Antibiotic</td><td>ScienCell Research</td><td>503</td><td></td></tr><tr><td>Chemometec Vial1-Cassette</td><td>Fisher Scientific</td><td>NC1420193</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>17018-029</td><td></td></tr><tr><td>Coverslip</td><td>Fisherbrand</td><td>12544D 22*50-1.5</td><td></td></tr><tr><td>Dispase I</td><td>Sigma-Aldrich</td><td>D46693</td><td></td></tr><tr><td>DMEM basal medium</td><td>ScienCell Research</td><td>9221</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline free from Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>Corning</td><td>&amp;nbsp;21-031-CV)</td><td></td></tr><tr><td>Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>ThermoFisher Scientific</td><td>339651</td><td></td></tr><tr><td>Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>ThermoFisher Scientific</td><td>339653</td><td></td></tr><tr><td>1.5 mL micro-centrifuge tubes</td><td>Fisherbrand</td><td>02-681-5</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>ThermoFisher Scientific</td><td>10082147</td><td></td></tr><tr><td>Fibroblast complete medium</td><td>ScienCell Research</td><td>2331</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td><td>Invitrogen</td><td>A11032</td><td>Dilution (1:500)</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A11008</td><td>Dilution (1:500)</td></tr><tr><td>Hanks&#039; Buffered Saline Solution (HBSS buffer)</td><td>Lonza</td><td>CC-5022</td><td></td></tr><tr><td>Human foreskin</td><td></td><td></td><td>De-identified human foreskin tissue for research purposes (Institutional Review Board- IRB #17574).</td></tr><tr><td>KGM-GOLD keratinocyte medium (KGM gold and supplements)</td><td>Lonza</td><td>00192151 and 00192152</td><td></td></tr><tr><td>Mouse alpha-smooth muscle actin antibody</td><td>ThermoFisher Scientific</td><td>14-9760-82</td><td>Dilution (1:200)</td></tr><tr><td>Mouse Cytokeratin14 antibody</td><td>Abcam</td><td>ab7800</td><td>Dilution (1:100)</td></tr><tr><td>Mouse S100 antibody</td><td>ThermoFisher Scientific</td><td>MA5-12969</td><td>Dilution (1:200)</td></tr><tr><td>Multi chambered (4 well glass slide)</td><td>Tab-Tek</td><td>154526</td><td></td></tr><tr><td>NucleoCounter -Via1-Cassette</td><td>Chemometec</td><td>941-0012</td><td></td></tr><tr><td>Poly-L-Lysisne (PLL)</td><td>ScienCell Research</td><td>32503</td><td></td></tr><tr><td>ProLong Gold Anti-fade Mountant with DAPI</td><td>Invitrogen</td><td>P36935</td><td></td></tr><tr><td>Rabbit K10 antibody</td><td>Sigma-Aldrich</td><td>SAB4501656</td><td>Dilution (1:100)</td></tr><tr><td>Rabbit p75-NTR antibody</td><td>Millipore</td><td>AB1554</td><td>Dilution (1:500)</td></tr><tr><td>Rabbit vimentin</td><td>ProteinTech</td><td>10366-1-AP</td><td>Dilution (1:200)</td></tr><tr><td>Schwann cell culture medium</td><td>ScienCell Research</td><td>1701</td><td></td></tr><tr><td>Precision tweezers DUMONT straight with extra fine tips Dumostar, 5</td><td>ROTH</td><td>LH75.1</td><td>Sterilize with 70% alcohol before use</td></tr><tr><td>IRIS Scissors, sharp/sharp. Length 4&amp;ndash;3/8&quot;(111mm)</td><td>Codman</td><td>54-6500</td><td>Sterilize with 70% alcohol before use</td></tr><tr><td>Sterilized surgical - sharp blade (Duro Edge Economy Single Edge Blades)</td><td>Razor blade company</td><td>94-0120</td><td>Sterilize with 70% alcohol before use</td></tr><tr><td>T25 culture flask</td><td>Corning</td><td>353109</td><td></td></tr><tr><td>Trypsin neutralization buffer (TNS)</td><td>Lonza</td><td>CC-5002</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>Inverted microscope</td><td>ZEISS</td><td>Axio Observer 7- Axiocam 506 mono &amp;ndash; Apotome.2 microscope</td><td>For immunofluorescence of chamber slide containing stained cells</td></tr><tr><td>Inverted microscope</td><td>ZEISS</td><td>Primovert</td><td>For visulaizing/observing cell attachment or detachment</td></tr></tbody></table>

isolation, culture, and characterization of primary schwann cells, keratinocytes, and fibroblasts from human foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

Normal neonatal foreskin was used for the isolation of primary epidermal keratinocytes and dermal SCs and fibroblasts. The isolated primary cells were cultured in respective cell culture media containing growth factors. After seeding of SCs and fibroblast in culture flasks, most of the cells adhered to the bottom of the flask within 2 h. In the case of keratinocytes, most keratinocytes adhered by 24 h. Isolated epidermal primary keratinocytes reached 85% confluence by day 7 and exhibited characteristic cell morphology (c...

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Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

isolation-culture-characterization-primary-schwann-cells

Research

JoVE Journal

Neuroscience

6.0K Views.  The Pennsylvania State University College of Medicine. The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin. 

Video: Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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. ...

Developmental Biology

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 ...

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.

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Biology

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Medicine

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...

JoVE Encyclopedia of Experiments

Cancer Research

Skin Cancer

Assays and techniques

Isolation of Schwann Cells from Mouse Spinal Roots

Source: He, Q., et al., &#38; Ding, F. <a href="https://app.jove.com/v/60952">Purification of fibroblasts and schwann cells from sensory and motor nerves in vitro.</a> J. Vis. Exp. (2020)
This video outlines the isolation and culturing of Schwann cells from mouse spinal roots. Isolated cells are then cultured in specific media. Schwann cells are isolated via differential digestion and adherence. They are subsequently transferred to extracellular matrix-coated coverslips for attachment and proliferation.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...