The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

Parenteau, Nancy L.; Bilbo, Patrick; Nolte, Cynthia J. M.; Mason, Valerie S.; Rosenberg, Mireille

doi:10.1007/bf02521744

Cited by 141 publications

(85 citation statements)

References 23 publications

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“…Organotypic raft cultures were constructed by seeding 5 ϫ 10 5 of the transduced keratinocytes onto a collagen matrix containing 75,000 human fibroblasts (16,42) and maintaining the rafts at the air-liquid interface for 3 days until grafting was performed. Grafting was performed on 4-to 5-week-old NIH male Swiss nu/nu mice (Taconic Farms).…”

Section: Methodsmentioning

confidence: 99%

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

Kühn

Terunuma

Pfützner

et al. 2002

J Virol

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For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. Achieving persistent and high-level gene expression in a significant percentage of target cells is important for establishing successful clinical applications of gene therapy. For gene therapy of the skin, a renewable tissue undergoing constant turnover, and for achieving long-term expression in a significant percentage of keratinocytes, gene targeting to keratinocyte stem cells (KSC) or long-lasting keratinocyte progenitor cells will be required (36). In ex vivo skin gene therapy, keratinocytes are removed from the donor and transduced with retroviral vectors in vitro without selectively targeting KSC. The genetically modified keratinocytes can then be grafted back onto the donor. Even though progress in skin gene therapy has enabled gene expression from retroviral vectors to be detected for sustained periods in grafted keratinocytes (9,24,32,56), transgene expression in vivo is frequently decreased over time, either in the duration of expression or in the percentage of keratinocytes expressing the gene (12,14,18,35,50). The factors contributing to this decreased expression may be similar to those for other tissues (14,27,41,51) and may include in...

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Section: Methodsmentioning

confidence: 99%

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

Kühn

Terunuma

Pfützner

et al. 2002

J Virol

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“…epidermal keratinocytes (9)(10)(11). Most likely this is due to the permanent production of a set of biologically active growth factors that can act in physiological concentrations at their appropriate locations.…”

Section: Introductionmentioning

confidence: 99%

Modified Plastic Compression of Collagen Hydrogels Provides an Ideal Matrix for Clinically Applicable Skin Substitutes

Braziulis

Diezi

Biedermann

et al. 2012

Tissue Engineering Part C: Methods

108

126

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Tissue engineering of clinically applicable dermo-epidermal skin substitutes is crucially dependent on the three-dimensional extracellular matrix, supporting the biological function of epidermal and dermal cells. This matrix essentially determines the mechanical stability of these substitutes to allow for safe and convenient surgical handling. Collagen type I hydrogels yield excellent biological functionality but their mechanical weakness and their tendency to contract and degrade does not allow producing clinically applicable transplants of larger sizes. We show here that plastically compressed collagen type I hydrogels can be produced in clinically relevant sizes (7 x 7 cm), and can be safely and conveniently handled by the surgeon. Most importantly, these dermo-epidermal skin substitutes mature into a near normal skin that can successfully reconstitute full thickness skin defects in an animal model. This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. We show here that plastically compressed collagen type I hydrogels can be produced in clinically relevant sizes (7 x 7 cm), and can be safely and conveniently handled by the surgeon. Most importantly, these dermo-epidermal skin substitutes mature into a near normal skin that can successfully reconstitute full thickness skin defects in an animal model.

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“…22,29 Furthermore, limited mass diffusion, 30 an issue especially associated with high cell seeding densities, and periodic shifts in culture environments due to media exchanges 31 can hinder optimal growth of cultured tissues. Adding the ''airlift'' culture as a specific requirement for the cultivation of differentiated skin grafts 32 raises another important issue related to an investigator's manipulation: Usually, airlift is achieved by using permeable membrane cell culture inserts and lowering the culture medium amount manually in a way that it marginally nurtures the skin graft from its basal side, leaving the apical side free. 33 Depending on the total medium volume, adjusting and controlling media levels by hand can be a rather fickle and laborious obligation.…”

Section: Discussionmentioning

confidence: 99%

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

Helmedag

Weinandy

Marquardt

et al. 2015

Tissue Engineering Part A

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Organotypic full-thickness skin grafts (OTSG) are already an important technology for treating various skin conditions and are well established for skin research and development. These obvious benefits are often impaired by the need of laborious production, their noncomplete autologous composition, and, most importantly, their lack of included vasculature. Therefore, our study focused on combining a prevascularized dermal layer with an epidermis to cultivate full-thickness skin grafts incorporating capillary-like networks. It has been shown that prevascularization accelerates ingrowth of tissue-engineered grafts, and it is a prerequisite to circumvent diffusion limits due to graft thickness. To obtain such a graft, we chose a dermal layer incorporating human umbilical vein endothelial cells (HuVEC) amid human dermal fibroblasts within a fibrin-based scaffold, seeded apically with human foreskin keratinocytes (hfKC). Our research investigated the used concept's feasibility, as well as the effect of hfKC addition on the development of a well-connected capillary-like network after approximately 21 days. In addition, we evaluated the utilization of a custom-made constant flow bioreactor for simplified cultivation of these grafts, therefore possibly easing graft production and presumably increasing their cost effectiveness. Skin grafts were assessed by conventional two-dimensional histology. In addition, software-assisted three-dimensional evaluation of the capillary-like structure networks was performed by twophoton laser scanning microscopy (TPLSM) and subsequent image processing was done with ImagePro Ò Analyzer 7.0 software, thereby evaluating its platform technology power in the field of prevascularized skin grafts. All samples showed a capillary-like structure network, but we could report a significant reduction of its total length after 14 days of tri-culture with 5 · 10 5 /cm 2 seeded hfKC, possibly indicating nutritional deficiencies for this particular high cell density experimental setup. Lower concentrations of hfKC did not affect the formation of the capillary-like structures significantly. The developed bioreactor simplified cultivation of prevascularized OTSG. However, a flow-dependent reduction of capillary-like structures in 1 and 5 mL/min flow conditions occurred. We conclude that our technique for creating prevascularized OTSG is feasible. In addition, TPLSM is well suited for analyzing the prevascularization process. We hypothesize that the handling benefits of our bioreactor can be preserved by using considerably lower flow rates while not impairing the forming of capillary-like structure networks.

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The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

Cited by 141 publications

References 23 publications

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

Modified Plastic Compression of Collagen Hydrogels Provides an Ideal Matrix for Clinically Applicable Skin Substitutes

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

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