Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Summary

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Abstract

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

Introduction

The skin is the largest organ of the body. It creates a barrier between the external environment and the internal organs. Moreover, the skin protects the body from fluid loss, environmental influences, injuries and infections and helps to regulate body temperature¹. Due to its exposed location, the skin is often affected by mechanical, thermal or chemical trauma. Though the skin is generally capable of self-repair, multiple local factors such as infection, oxygenation, and venous sufficiency can lead to impaired wound healing. Wound healing can also be interfered by systemic factors as obesity, alcoholism, smoking, medication, nutrition and diseases such as....

Protocol

NOTE: The protocol is designed for the production of 24 full thickness skin equivalents. Human dermal fibroblasts and epidermal keratinocytes were isolated from skin biopsies according to a previously published protocol.^21,22 Informed consent was obtained beforehand and the study was approved by the institutional ethics committee on human research of the Julius-Maximilians-University Würzburg (vote 182/10).

1. Production of Dermal Component

1. Dissolve collagen with 0.1% acetic acid to a final concentration of 6 mg/ml. For the gel neutralization solution, mix 232.5 ml 2x DMEM, 7.5 ml fetal calf serum, 7.5&....

Results

The isolated hDF and hEK differ both in morphology and in expression of typical markers. The hDF showed typical spindle-shape morphology, whereas morphology of hEK can be described by a cobblestone morphology. The cells were characterized by immunohistochemical staining (Figure 1) before using them for ftSE. The hDF are positive for vimentin (Figure 1A), a marker for fibroblasts. Primary hEK highly express early differentiation protein cytokeratin-14 (Figure 1B) but near.......

Discussion

In vitro cells are usually expanded in two-dimensional cell cultures, in which cells adhere to plastic surfaces. However, these culture conditions do not reflect the physiological three-dimensional conditions in which cells grow in vivo. Under three-dimensional conditions, cells can form natural cell-cell and cell-matrix attachments and migrate in three-dimensions. Especially in the cutaneous wound healing the resemblance of the in vivo situation is pivotal to generate meaningful data, as cell .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trypsin EDTA (1:250) 0.5% in DPBS	PAA	L11-003	0.05%
Dulbecco’s Phosphate Buffered Saline	Sigma	D8537
Collagen (6 mg/ml in 0.1% acetic acid)	Produced in house
Fibronectin Human Protein, Plasma (50 µg/ml)	Life Technologies	33016-015
Inserts	Nunc	140627
6 well plate	Nunc	140685
24 well plate	Nunc	142485
Microscope slides	R. Langenbrinck	03-0070
Fibroblasts culturing (500 ml):
DMEM, high glucose	Life Technologies	11965-092	89% (445 ml)
Fetal bovine serum	Bio & Sell	FCS.ADD.0500	10% (50 ml)
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122	1% (5 ml)
Keratinozyten culture medium (500 ml):
Keratinocyte Growth Medium 2	Promocell	C-20111	89% (445 ml)
Keratinocyte Growth Medium 2 SupplementPack	Promocell	C-39011
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122	1% (5 ml)
Gel neutralization solution (250 ml):
Dulbecco’s Modified Eagle Medium, high Glucose Powder with L-Glutamine	PAA	G0001,3010	93% (232.5 ml)
Chondroitin sulfate sodium salt from shark cartilage	Sigma	C4384-1g	1% (2.5 ml)
Fetal bovine serum	Bio & Sell	FCS.ADD.0500	3% (7.5 ml)
HEPES	Sigma	H3375-1kg	3% (7.5 ml)
Skin model submers medium (500 ml):
Keratinocyte Growth Medium 2	Promocell	C-20111
Keratinocyte Growth Medium 2 SupplementPack	Promocell	C-39011
Fetal calf serum	Bio & Sell	FCS.ADD.0500	5%-2% (25 ml-10 ml)
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122	1% (5 ml)
Skin model air-liquid interface medium (500 ml):
Keratinocyte Growth Medium 2
Keratinocyte Growth Medium 2 Supplement Pack	Promocell	C-39011	adding only supplements: insulin, hydrocortisone, epinephrine, transferrin, CaCl2
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122	1% (5 ml)
CaCl2 (300 mM)	Sigma	C7902-500g	0.62% (3.1 ml)
Histology:
IHC-Kit DCS SuperVision 2 HRP	DCS	PD000KIT
Vimentin antibody	Abcam	ab92547
CK14 antibody	Sigma	HPA023040-100µl
CK10 antibody	Dako	M7002
Filaggrin antibody	Abcam	ab81468
H&E staining:
Mayer´s Haemalaun	AppliChem	A0884,2500
Xylol	Sigma Aldrich	296325-4X2L
Ethanol	Sigma Aldrich	32205-4X2.5L
HCl	Sigma Aldrich	H1758-500ML
Eosin	Sigma Aldrich	E4009-5G
2-Propanolol	Sigma Aldrich	I9516-500ML
Mounting Medium	Sigma Aldrich	M1289-10ML