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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

Acquisition and use of de-identified human foreskin tissue for research purposes was reviewed and received the determination of "not human research" 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 experimenta.......

Discussion

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 fibroblasts^2,3,5,6, but none describe SC isolation. Apart from the key structural cells in skin, keratinocytes, and fibroblasts, skin is also highly innerv.......

Materials

Name	Company	Catalog Number	Comments
0.22 µM sterile filters (Millex-GP Syringe Filter Unit,polyethersulfone)	MilliporeSigma	SLGPR33RS
70 µM cell strainers	CELLTREAT	229483
100 µM cell strainers	CELLTREAT	229485
1 mL disposable syringes	BD Luer-Lok	BD-309659
5 mL disposable syringes (Syringe sterile, single use)	BD Luer-Lok	BD309646
10 mL disposable syringes	BD Luer-Lok	BD305462
1% TritonX-100	Sigma	X100-1L	Prepared at the time of use
4% paraformaldehyde solution	ThermoFisher Scientific	J19943.K2	Ready to use and store at 4 °C
5% BSA	Sigma	A7906-100G	Prepared at the time of use
70% ethanol	Pharmco	111000200
Antibiotic	ScienCell Research	503
Chemometec Vial1-Cassette	Fisher Scientific	NC1420193
Collagenase	Gibco	17018-029
Coverslip	Fisherbrand	12544D 22*50-1.5
Dispase I	Sigma-Aldrich	D46693
DMEM basal medium	ScienCell Research	9221
Dulbecco's phosphate-buffered saline free from Ca2+ and Mg2+ (DPBS)	Corning	21-031-CV)
Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes	ThermoFisher Scientific	339651
Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes	ThermoFisher Scientific	339653
1.5 mL micro-centrifuge tubes	Fisherbrand	02-681-5
Fetal bovine serum (FBS)	ThermoFisher Scientific	10082147
Fibroblast complete medium	ScienCell Research	2331
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594	Invitrogen	A11032	Dilution (1:500)
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Invitrogen	A11008	Dilution (1:500)
Hanks' Buffered Saline Solution (HBSS buffer)	Lonza	CC-5022
Human foreskin			De-identified human foreskin tissue for research purposes (Institutional Review Board- IRB #17574).
KGM-GOLD keratinocyte medium (KGM gold and supplements)	Lonza	00192151 and 00192152
Mouse alpha-smooth muscle actin antibody	ThermoFisher Scientific	14-9760-82	Dilution (1:200)
Mouse Cytokeratin14 antibody	Abcam	ab7800	Dilution (1:100)
Mouse S100 antibody	ThermoFisher Scientific	MA5-12969	Dilution (1:200)
Multi chambered (4 well glass slide)	Tab-Tek	154526
NucleoCounter -Via1-Cassette	Chemometec	941-0012
Poly-L-Lysisne (PLL)	ScienCell Research	32503
ProLong Gold Anti-fade Mountant with DAPI	Invitrogen	P36935
Rabbit K10 antibody	Sigma-Aldrich	SAB4501656	Dilution (1:100)
Rabbit p75-NTR antibody	Millipore	AB1554	Dilution (1:500)
Rabbit vimentin	ProteinTech	10366-1-AP	Dilution (1:200)
Schwann cell culture medium	ScienCell Research	1701
Precision tweezers DUMONT straight with extra fine tips Dumostar, 5	ROTH	LH75.1	Sterilize with 70% alcohol before use
IRIS Scissors, sharp/sharp. Length 4–3/8"(111mm)	Codman	54-6500	Sterilize with 70% alcohol before use
Sterilized surgical - sharp blade (Duro Edge Economy Single Edge Blades)	Razor blade company	94-0120	Sterilize with 70% alcohol before use
T25 culture flask	Corning	353109
Trypsin neutralization buffer (TNS)	Lonza	CC-5002
Trypsin/EDTA	Lonza	CC-5012
Inverted microscope	ZEISS	Axio Observer 7- Axiocam 506 mono – Apotome.2 microscope	For immunofluorescence of chamber slide containing stained cells
Inverted microscope	ZEISS	Primovert	For visulaizing/observing cell attachment or detachment