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 diabetes.²

The process of wound healing can be divided into 3 phases: (i) the inflammatory phase, (ii) the proliferative and the (iii) remodeling phase. Upon injury to the skin, a complex signal-cascade starts, leading to the closure of the wound.³ After injury, the wound is bleeding and a blood clot is formed. Fibroblasts move into the blood clot and replace it with new tissue which is subsequently remodeled over years.

The current understanding of the biological processes underlying cutaneous repair is limited. Small animal and pig models have been used to study wound healing. However, these results cannot be directly transferred to humans due to species-specific differences. In addition to these in vivo models, some aspects of wound healing can be studied by simulating a wound situation via scratching of in vitro monolayer cultures based on immortalized cell lines or primary cells.⁴ These scratching models are highly standardized but do not sufficiently reflect the complex in vivo physiology.⁵ Besides two-dimensional models, three-dimensional human skin equivalents have been developed for dermatological research. The dermal part of these models are generated using various scaffolds including decellularized dermis,⁶ collagen hydrogels,^7,8 glycosaminoglycans⁹ or synthetic materials.¹⁰ Employing these skin equivalents, the role of epithelial-mesenchymal interactions¹¹, the re-epithelialization, the cellular crosstalk between fibroblasts and keratinocytes and the influence of different growth factors can be studied. Moreover, these models are useful to gain new knowledge about how fibroblasts migrate into the wounded area and how chemotactic factors influence tissue regeneration.¹²

Not only generation of the wound healing model itself is challenging, but also to establish a highly standardized wound in a model is problematic. Common techniques to create wounds are scratch tests,¹³ burns,¹⁴ tape abrasion,¹⁵ thermal injury,¹⁶ suction blisters,¹⁷ liquid nitrogen,¹⁸ lasers,¹⁹ scalpels,¹⁸ meshers⁶ and biopsy punches.²⁰ Most of these methods have the same pitfalls. Injuries implemented manually are difficult to standardize and to reproduce between multiple tests. Size, shape and depth of the wound vary between studies and thus impair the quality of research data. The use of laser for defined skin wounding can be relatively easily standardized but leads to a situation mimicking burn wounds. Heat applied by laser can cause protein denaturation, platelets aggregation or vessels constriction, which can lead to necrotic tissue.

In an alternative approach, we developed an automated wounding device (aWD), which allows us to generate defined and precise cutaneous wounds under sterile conditions. Wounding parameters, like depth and speed of penetration as well as revolutions of the drill head can be regulated. In this study, we combined the aWD with in house developed full thickness skin equivalents (ftSE) that are comparable to a protocol published by Gangatirkar et al. ⁸ The dermal layer of the skin equivalent is composed of human dermal fibroblasts (hDF), which are embedded in a collagen I hydrogel. On the dermal layer, human epidermal keratinocytes (hEK) are seeded. Within two weeks at the air-liquid interface the hEK build up an epidermis composed of several vital cell layers and a stratum corneum. Besides the generation of this model, this study shows the use of the aWD to create defined and precise wounds in ftSE.

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 ml 3 M HEPES, 2.5 ml chondroitin sulfate (5 mg/ml). Keep gel neutralization solution and collagen solution on ice. Calculate 500 µl of gel per insert/skin equivalent.
   NOTE: As the gel neutralization solution will be mixed with two parts of collagen solution prepare double amount of collagen.
2. Place 24 inserts into a 24 well plate.
3. Resuspend human dermal fibroblasts (hDF) in 10 ml DMEM, centrifuge for 5 min at 270 x g and count the cells. Extract the needed amount of hDF (5 x 10⁴ cells per 500 μl collagen gel) and centrifuge the cells for 5 min at 270 x g.
4. Remove the supernatant and carefully resuspend the hDF in 4 ml cooled gel neutralization solution without producing air bubbles. From this step make sure to work as fast as possible to avoid premature solidifying of the collagen gels.
5. Resuspend the solution with 8 ml of collagen solution.
6. Take the collagen-cell-mixture with a multistep-pipette and fill quickly 500 μl of mixture in each insert. Incubate the gels for 20 min in an incubator (37 °C, 5% CO₂) to jell the gels.
7. Submerge the gels in 2 ml Dulbecco’s Modified Eagle Medium (DMEM) + 10% fetal calf serum (FCS) per well and incubate it for 24 hr in an incubator (37 °C, 5% CO₂).

2. Addition of Human Epidermal Keratinocytes (hEK)

1. Remove medium after 24 hr. Dissolve fibronectin human protein with ultrapure water to a final concentration of 50 µg/ml. Cover each dermal equivalent with 25 μl of fibronectin solution. Incubate the gels for 30 min in an incubator (37 °C, 5% CO₂).
2. In the meantime, resuspend the hEK in 10 ml KGM-2 and set cell concentration to 1 x 10⁶ cells/ml with KGM-2 ready + 5% FCS. Seed 1 x 10⁵ hEK on each gel. Incubate gels for 45 min in the incubator (37 °C, 5% CO₂) to allow cells to adhere.
3. Submerse the gels with KGM-2 ready + 5% FCS and culture them in the incubator (37 °C, 5% CO₂).

3. Culture of Full Thickness Skin Equivalents (ftSE)

1. Culture ftSE for 6-7 days in descending FCS-concentration (5%, 2%, and 0% FCS). Change the medium every 2-3 days with the next lower FCS concentration. For this, completely remove the medium and add 1.6 ml of fresh medium.
2. On day 7, remove medium completely.
   NOTE: Do not touch the ftSE surface with the tip of pipette while doing so.
3. Place every insert into 1 well of a 6 well plate with sterile forceps. Add 1.5 ml airlift medium per well, ensure not to wet the ftSE surface. Change medium every 2-3 days for additional 14 days.
   NOTE: The filling level of the medium is up to the meniscus of the ftSE.

4. Histological Analysis

1. Fixing and paraffin-embedding of the ftSE
   1. Remove culture medium, transfer inserts in fresh 24 well plates and fix tissues by adding 1.6 ml of 4% paraformaldehyde to each well and incubate the tissues for 2 hr at RT. Caution! Paraformaldehyd is toxic, manipulate under fume hood. Transfer the ftSE to a tissue-embedding cassette. Remove remaining fixative by washing with water and dehydrate the tissue using ascending ethanol concentrations.
   2. Divide the skin equivalent by a vertical cut in the middle. Place the two pieces in a paraffin-filled metal base mold with cut surfaces downwards. Add the tissue cassette on top of the mold as a backing.
   3. Cut 3-5 µm slices and float them on a 40 °C water bath for straitening, mount slides onto suitable microscope slides. Dry slices thoroughly.
2. Hematoxylin & Eosin (H&E) and immunohistochemical (IHC) staining
   1. Place slides into rack and incubate for 1 hr at 60 °C. Make sure that paraffin is melted. Stain rehydrated slices with H&E as a standardized overview staining.
   2. For H&E staining prepare 4x glass staining cuvettes with xylol, 3x with ethanol 96%, 2x with ethanol 70%, 1x with ethanol 50%, 4x deionized water, 1x hematoxylin, 1x HCl-ethanol (13.7 ml 1 M HCl ad 200 ml 50% ethanol), 1x tab water, 1x eosin (1 g eosin in 100 ml deionized water) and 2x 2-propanol.
   3. Place slides 10 min in xylol I and 10 min in xylol II to deparaffinize and rehydrate slides. Dip slides 3x in ethanol 96% I, 3x in ethanol 96% II, 3x in ethanol 70% and 3x in ethanol 50%. Rotate slides in deionized water. To differentiate hematoxylin dye, dip 2x in HCl-ethanol. Rinse in deionized water. For blueing, place slides 5 min in tab water. For eosin staining, place slides 1 min in eosin and wash afterwards in deionized water. For dehydration, dip slides 2x in ethanol 70%, 2x in ethanol 96% and place them for 5 min in 2-propanol I, for 5 min in 2-propanol II, 5 min in xylol I and 5 min in xylol II. After H&E staining, cell nuclei are stained in blue, cytoplasm and extracellular matrix are stained in red.
   4. For antigen retrieval, remove paraffin with xylene and rehydrate slices for staining. Place slices in steam cooker and cook deparaffinized and rehydrated slices for 20 min in pre-heated 10 mM citrate buffer (pH 6).
   5. Place slides in washing buffer (PBS buffer + 0.5% Polysorbat 20) and circle slices with a liquid repellent slide marker pen or diamond stylus.
   6. Place slides in a moisture chamber. Block endogenous peroxidase, by adding 100 µl 3% hydrogen peroxide solution onto each slice. Caution! Very hazardous in case of skin and eye contact. Handle with personal protective equipment. Wash slides in washing buffer.
   7. Apply primary antibody and incubate for 1 hr at RT. Wash slides three times with washing buffer. From here on, samples should be kept in the dark.
   8. Use biotin-streptavidin detection system for detection of specific antigen-antibody binding.
   9. Counterstain nuclei with Hematoxylin for 1 min. Dehydrate and mount slides.
   10. Image samples using an inverse microscope.

5. Injury by Use of the Automated Wounding Device (aWD)

1. Prepare the aWD by disinfecting the working area with 70% ethanol and ultraviolet light. Make sure the drilling head of the desired size (e.g., 1 mm) is attached.
2. Place skin equivalents into designated areas of the autoclaved sample carrier plate using sterile forceps. Place the sample carrier plate into the socket below the drilling head.
3. Use the control software to set wounding parameters as follows: spin velocity: 15,000 rpm; penetration: 1.5 mm; propulsion: 100 Hz. These parameters need to be defined for each sample type individually. Transfer wounding parameters to the aWD.
4. Start wounding procedure. Remove sample carrier plate from the socket. Transfer wounded ftSE back into inserts used for culture using sterile forceps.
5. Proceed with culture of wounded ftSE in the incubator (37 °C, 5% CO₂) for investigation of regeneration. Alternatively, use the models for histological analysis at any time.

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