Reconstructing human skin equivalents on fibrin-based dermal matrix

In vitro human skin equivalents are physiologically complex three-dimensional (3D) models of human skin. These human skin equivalents (also referred to as organotypic skin cultures) are increasingly gaining attention for their importance in basic research, industrial (toxicity studies, drug screening, drug permeation studies and development of treatment strategies) and clinical applications [1]. Due to 3D organization of the skin cells (keratinocytes in various differentiation stages and/or fibroblasts), these organotypic skin cultures offer a superior platform to study skin physiology, wound healing and various skin pathologies. Further, the need for these complex human skin models is amplified due to EU regulations that encourage replacement, reduction and refinement of animal models (EU Directive 2010/63/EU) and enforces ban on testing cosmetic products in animals (EU Cosmetic Directive 76/768/EEC, REACH regulation 1907/2006). In addition to these applications, these human skin equivalents are also used to study skin development in health and disease through various models that recapitulate wound healing, infection and various skin disorders such as psoriasis [2] and [3], vitiligo [4], squamous cell carcinoma [5] and [6], malignant melanoma [7] and [8], aging [9] and [10], photo-aging [11] and [12], and wounds [13] and [14]. Recently, with the advancements in biomaterial and microfluidics technology, the culture of human skin equivalents is moving a step ahead to develop microfluidic skin-on-chip [15] and multiorgan-on-chip [16]models. Furthermore, certain academic institutions and companies are currently developing 3D printed skin tissue for clinical and industrial applications [17] and [18].

The fabrication of these human skin equivalents is generally a multi-step process that consists of (1) construction of dermal equivalents by culturing fibroblasts within biological or synthetic matrices, followed by (2) seeding keratinocytes on top of these dermal equivalents, and (3) culturing the fibroblast–keratinocyte 3D co-culture at air–liquid interface [19]. This unique culture process at air–liquid interface mimics the physiology of skin by providing nutrition from below (dermis) and exposure of the keratinocyte surface to air. This process results in keratinocyte differentiation, stratification and development of the cornified envelope that contributes to the barrier properties of the skin. Various dermal equivalents used include decellularized dermis [20] and [21], collagen [22], fibrin[23], glycosaminoglycans [24] and synthetic polymers [24] and [25]. Currently available models of human skin equivalents are limited by major obstacles in obtaining sufficient number of skin cells from donor skin biopsies, limited propagation in culture, contraction of the dermal matrix and inferior barrier properties compared to native human skin[19] and [26].

We have recently developed a highly reproducible platform for generation of in vitrohuman skin equivalents using immortalized keratinocytes and primary fibroblasts cultured over a fibrin-based dermal equivalent and serum-free conditions [1]. The fibrin-based dermal matrix is fabricated utilizing the self-assembling properties of fibrinogen under appropriate concentration of thrombin and calcium. Further, the physical properties of these fibrin-based matrices were improved by conjugating the fibrinogen with polyethylene glycol. The in vitro human skin equivalents developed over these fibrin-based matrices were superior to conventional collagen-based matrices in terms of absence of shrinkage of the matrix and superior epidermis as evidenced by the formation of a well-defined granular layer and orthokeratinized (without residual nuclear remnants) corneal layer [1]. Furthermore, fibrin-based in vitro human skin equivalents mimic the normal human skin in terms of well-defined stratified layers, expression of major differentiation markers, and similar lipid compositions in the corneal layer.

The scanning electron microscopic image presented on this issue's cover displays the topographic features of a cross-section of the human skin equivalent reconstructed in vitro using immortalized keratinocytes and primary fibroblasts cultured on a fibrin-based dermal equivalent under serum-free conditions for 4 weeks. The fiber-like structures in the foreground are the fibers of the fibrin-based dermal matrix, while the background shows the epidermis with a uniform corneal layer over stratified layers of keratinocytes. The artifactual dehiscence of epidermis from the underlying dermis due to processing also enables the visualization of the basement membrane from beneath.

In conclusion, using immortalized keratinocytes, a non-contracting fibrin-based matrix and serum-free culture conditions, we have developed a reproducible human skin equivalent with superior epidermal reconstruction. Further, toward developing a high-throughput platform to assess skin toxicity and permeation, we are currently developing a novel microfluidic ‘Skin-on-Chip’ device. These human skin equivalents would provide immense opportunities to explore its potential in basic research, safety, toxicology and skin permeation studies, and industrial applications.

Further reading:

[1] G. Sriram, et al.
Eur. J. Cell Biol. (2015)

[2] F.X. Bernard, et al.
J. Allergy (Cairo), 2012 (2012), p. 718725

[3] J. Jean, et al.
J. Dermatol. Sci., 53 (1) (2009), p. 19

[4] M. Cario-Andre, et al.
Pigment Cell Res., 20 (5) (2007), p. 385

[5] S. Commandeur, et al.
Cancer Sci., 103 (12) (2012), p. 2120

[6] S. Depner, et al.
Int. J. Cancer, 135 (3) (2014), p. 551

[7] L. Li, et al.
J. Vis. Exp. (54) (2011)

[8] L. Li, et al.
Cancer Res., 70 (11) (2010), p. 4509

[9] H. Pageon
Pathologie-biologie, 58 (3) (2010), p. 226

[10] H. Pageon, et al.
Eur. J. Dermatol. EJD, 17 (1) (2007), p. 12

[11] F. Bernerd, D. Asselineau
J. Am. Acad. Dermatol., 58 (5 Suppl. 2) (2008), p. S155

[12] F. Bernerd, J. Indian, et al.
Dermatol. Venereol. Leprol., 78 (Suppl. 1) (2012), p. S15

[13] M.E. Smithmyer, et al.
Biomater. Sci., 2 (5) (2014), p. 634

[14] J.W. van Kilsdonk, et al.
Wound Repair Regen., 21 (6) (2013), p. 890

[15] B. Atac, et al.
Lab Chip, 13 (18) (2013), p. 3555

[16] I. Wagner, et al.
Lab Chip, 13 (18) (2013), p. 3538

[17] I.T. Ozbolat, Y. Yu
IEEE Trans. Biomed. Eng., 60 (3) (2013), p. 691

[18] K.W. Binder
In Situ Bioprinting of the Skin
Wake Forest Univ., Winston-Salem, NC, USA (2011)

[19] M. Ponec
Adv. Drug. Deliv. Rev., 54 (Suppl. 1) (2002), p. S19

[20] A. El Ghalbzouri, et al.
Cell Tissue Res., 310 (2) (2002), p. 189

[21] R. Lamb, C.A. Ambler
PLOS ONE, 8 (1) (2013), p. e52494

[22] P. Gangatirkar, et al.
Nat. Protoc., 2 (1) (2007), p. 178

[23] K. Boehnke, et al.
Eur. J. Cell Biol., 86 (11–12) (2007), p. 731

[24] M. Varkey, et al.
Tissue Eng. A, 20 (3–4) (2014), p. 540

[25] M.T. Cerqueira, et al.
Mater. Today, 18 (8) (2015), pp. 468–469

[26] Y. Poumay, A. Coquette
Arch. Dermatol. Res., 298 (8) (2007), p. 361

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DOI: 10.1016/j.mattod.2016.01.015