Hemant P. Joshi

Sun Pharmaceutical Advanced research Centre (SPARC),17/B, Off Mahakali caves road, Mahal Indl. Estate,Andheri (E), Mumbai-400 093,INDIA,email : hemantjoshee@sify.com

To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms.

Keywords: In vitro models, keratinocytes, fibroblasts, skin re-epithelialization, in situ hybridization, cadaver skin

Introduction:

In skin, epithelial cells develop an orderly structured and well-organized epithelium consisting of basal, spinous, granular, and cornified strata. Their development during embryogenesis, regeneration in wound healing, as well as the maintenance of the homoeostasis of the epidermis depends on epithelial interactions with the underlying connective tissue, the dermis. The continuous process involving keratinocyte proliferation and terminal differentiation is mainly regulated by mesenchymal influences¹ . culture systems have been developed to examine cellular growth, signalling processes and cell-cell interactions, combining the two major cell types of skin, keratinocytes and fibroblasts. In simple two-dimensional feeder-layer cocultures combining postmitotic dermal fibroblasts (feeder cells) and epidermal keratinocytes, properly stratified epithelia are not formed. Only in advanced three-dimensional in vitro systems keratinocytes develop well-ordered epithelia, thus offering an opportunity to analyse the cellular mechanisms of tissue formation, such as cell-cell interactions, the regulation of proliferation and differentiation as well as the reepithelialisation process after wounding ². several organ-like culture systems have been established to reconstruct skin in vitro  ^3-6. organotypic cocultures, epidermal keratinocytes can be conveniently grown on an air-exposed matrix of native collagen type I, extracted from rat tail tendon or calf skin. The collagen gels contains viable fibroblasts which reorganize this matrix by producing extracellular matrix components comparable to the wound situation ^{7, 8}. The cultures are nourished by diffusion from below on a permeable support , optionally in serum containing medium or in defined medium ⁹ serving as a functional dermal equivalents.

The characterization of paracrine acting factors produced either by fibroblasts or keratinocytes is difficult to simulate in a homologous skin model, that contains human cells in both compartments. On the other hand, in a heterologous skin equivalent harboring cell types of different species, the source of the respective gene products can be easily and unequivocally distinguished  by assigning to the specific cell types ¹⁰.

 In vitro skin equivalents are generally composed of freshly isolated cells from human skin specimens. However, more preferable tools for large scale examinations are either the molecular analysis of regulatory processes or pharmaco-toxicological studies. Skin equivalents composed of cell lines, which are easier to handle and to standardize. Despite the altered karyotype and unlimited growth potential, HaCaT cells are non tumorigenic and form an orderly structured and differentiated epidermis with essentially all structural and functional features after transplantation onto athymic nude mice^11-13. organotypic cocultures in culture medium supplemented with TGF-α HaCaT cells form well organised and differentiated epithelia in  presence of human or mouse fibroblasts¹⁴.

Cell culture

Human dermal fibroblasts (HDF) can derived from adult skin by trypsinization using a mixture of trypsin and EDTA in a concentration range of 0.25 and .2% respectively ^{8, 9}. HDF can be obtained from an outgrowth of explant cultures, that can be cultured using (Dulbecco’s modified Eagle's medium; Bio Whittaker) supplemented with 10% fetal calf serum (FCS), as growth medium. The medium can be supplemented with 10% FCS. ^16,17.

Normal epidermal keratinocytes (NEK) are derived from adult skin ^{8, 9} and are cultured on X-irradiated fibroblast feeder cells using FAD medium (DMEM:Ham’s F-12 / 3:1) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5% FCS, 5 µg/ml insulin, 1 ng/ml recombinant human EGF, 10^-10 M cholera toxin, 10^-4 M adenine, and 0.4 µg/ml hydrocortisone. ⁸

Organotypic cocultures

Collagen to used for cell culture can be commercially obtained as a lyophilized Collagen type I which can be  resolubilised at the desired concentration in 0.1% acetic acid and stored at 4°C.

(Suppliers e.g. (a) Vitrogen-100, bovine dermal collagen, (type I) (Collagen Corp., Palo Alto, California);

(b) Type I collagen, calf skin (Cerard, Lyon, France);

(c) Type I collagen, rat tail tendons (SIGMA);

(d) Type I collagen, calf skin (IBFB, Leipzig, Germany).

Coating of collagen on to the culture inserts:

The stored frozen samples of collagen,  mixed with Hanks salt 10x containing phenol red. The pH of the medium is adjusted 7.4 using 2 M NaOH/12 ml collagen mixture, on an ice bath  with gentle stirring to avoid entrapment of air bubbles, which inturn leads to premature gelation. The desired density of fibroblast in the gel phase range from 1x10⁵ - 5x10⁵/ml. The same is suspended in FCS and added to the gel solution on ice under cautious stirring. collagen gel mixture is poured into filter inserts (2.5 ml/ filter insert; using frozen pipettes (Fig.1).

Figure 1: Schematic illustration of the organotypic culture system. Keratinocytes grow air exposed on a fibroblast containing collagen gel. The filter inserts are in contact with the culture medium

For gelation, the collagen solution in filter inserts is incubated for 1 h at 37°C in a humidified incubator and thus casted  gel can be confined to a specific  area using glass rings and forceps, providing a confined area for optimal epithelial growth. The gels with the glass rings are placed for 1 h at 37°C in a humified incubator. The excess liquid pressed out of the gel can be gently and carefully aspirated. Eventually, the gels were equilibrated by complete immersion in culture medium for 24 h.

Seeding of Keratinocytes:

Keratinocytes can seeded inside the glass ring (after removing the medium) at a desired density (1x10⁶⁾ using 1 ml FAD medium/2.5 cm insert. Keratinocytes get attach within 12-24 h and form a nearly confluent layer on top of the collagen gel. 24-30 hours of post seeding, the glass rings were removed, thereby avoiding any mechanical distortion of the epithelial cell sheet. However, commercially the collagen coated culture inserts are also available.

Media replenishment:

Culture medium was changed, and, by lowering the medium level to the lower part of the gels, the cultures were raised to the air-liquid interphase thus restricting nourishment to diffusion from below. This air-lift procedure is defined as the start of the culture time of organotypic cocultures. Organotypic cocultures with primary keratinocytes were grown in FAD-Medium or DMEM with 10% FCS and 50 µm ascorbic acid, those with HaCaT cells were supplemented with 2 ng/ml TGF-α. Medium was changed every 2-3 days. Depending on the purpose of the respective study, alternative media might be used, such as serum free formulations or media with specific supplementations such as inhibitors, neutralizing antibodies, growth factors, etc ^{1, 2, 8, 9}

Characterization

Analysis generally comprises of histological or immunohistochemical studies.  The analysis involves fixing these organotypic cocultures in formaldehyde (3.7%) for at least 24h taking utmost care to avoid any dislodging of cultured epithelium by covering each culture insert with a drop of hand warm agar (2%).  the specimens can be processed for paraffin embedding employing routine histological procedures. Alternatively, for cryosectioning specimens were embedded in Tissue Tek-OTC-compound and subsequently snap frozen in liquid nitrogen vapour.

The most important quality parameter of epidermal tissue reconstitution is its morphologic appearance. In addition , tissue maturation is readily evaluated by in situ-analytical techniques on frozen or fixed tissue sections determinig expression (in situ-hybridization) and distribution of specific epidermal differentiation products (immunohistochemistry) such as involucrin, keratin 1/10, transglutaminase, filaggrin, etc ^{2, 4, 9, 10}. Usually, the formation of a regular epidermal tissue architecture is paralleled by typical, in vivo-like expression patterns of differentiation products and the development of a stratum corneum.

Remarks:

organotypic cocultures, epidermal keratinocytes containing embedded fibroblasts, attach rapidly and forms confluent layers within 1-2 days. Subsequently, these keratinocytes reconstitutes into  an epithelial tissue architecture resembling native epidermis and expressing characteristic epidermal differentiation markers ^{1 – 9, 18 , 19}. In the absence of fibroblasts, only thin epithelia developes leading to rapid loss of proliferation within 2 to 3 days ²⁰. However, the addition of fibroblasts into the collagen matrix - either proliferative or postmitotic cells leads to a sustained keratinocyte growth accompanied by regular differentiation. It could be demonstrated by means of this in vitro-system, that the cocultured fibroblasts produce growth factors which are essential for epidermal morphogenesis ^{8, 20}.

In general, using normal human epidermal keratinocytes  , human dermal fibroblasts and differentiated epithelia features all characteristic epidermal layers including an orthokeratotic stratum corneum after 7-10 days. Besides, variations in epithelial quality can be observed due to the interindividual variability of primary epithelial cells (different donors, body sites, ages) , differences in the isolation procedures as well as culture conditions ²¹.

Density of fibroblast and the genotype to be used depends on the aim of the experiments.

 For example, heterologous skin equivalents consisting of mouse embryonic fibroblasts (MEF) and human keratinocytes are an excellent tool to identify the source of paracrine factors contributing in dermal-epidermal interactions ^{22 - 24}. In both, homologous as well as heterologous organotypic cocultures, forms a typical epidermal tissue within a week, suggesting the potential of responsible factors beyond species barriers ²⁵

Incase of  coculture with mouse fibroblasts, growth of human keratinocytes can be delayed (beyond a week)  resulting in thinner epithelia in comparison with cocultures of human fibroblasts. This is indicative for a reduced efficacy of these cells in supporting the growth of human keratinocytes. Nevertheless, this can be compensated by a prolonged culture time of 10 to 14 days. However, heterologous skin equivalent models have the unique advantage, that genetically modified mouse cells can be exploited such as embryonic fibroblasts from knock-out or transgenic mice. ^{10,16, 25}

To further standardise the in vitro skin equivalent model and to avoid donor-dependent variations, the epidermal cell line HaCaT has been used instead of primary keratinocytes. The HaCaT cell line is a spontaneously transformed keratinocyte line ¹¹ with sustained genetic alterations indicative of transformed but not tumorigenic cells ²⁶. When grown on plastic the HaCaT cells show continuous proliferation, independence of feeder-cells, and a rather typical epithelial morphology with expression of a large panel of keratins ^{12, 13, 26}. When transplanted onto the nude mice, HaCaT cells regenerate and differentiate to well structured epithelia expressing the characteristic epidermal markers¹².

On the other hand, formation of multilayered epithelia by HaCaT cells in organotypic cocultures is delayed and require an increased number of fibroblasts¹³. Which could be attributed to certain limitations of HaCaT cells in the production of IL-1 as well as a low expression of the receptors for KGF and GM-CSF which can be compensated by supplementation of GF-α¹⁴. In organotypic cocultures of HaCaT cells either with human or mouse fibroblasts leads to a well structured and differentiated stratified epithelia, upon supplemented with TGF-α.

In conclusion, their reproducible production and standardized quality renders HaCaT organotypic cocultures an excellent tool for large scale examinations of mechanisms regulating skin re-epithelialization and homoeostasis in a tissue-type context.

Acknowledgements:

Author wish to thank Prof. B.G. Shivananda, Principal of Al-Ameen College of Pharmacy, Bangalore for his kind support and encouragement.

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