Adherence and viability of primary human keratinocytes and primary human dermal fibroblasts on acrylonitrile-based copolymers with different concentrations of positively charged functional groups

AbstractThe long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

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Receptors for ?2-Macroglobulin Are Present on Human Dermal Fibroblasts, but Not on Human Keratinocytes

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1994.tb44348.x ◽

1994 ◽

Vol 737 (1 Biology of 2-) ◽

pp. 490-492

Author(s):

STEVEN R. FELDMAN ◽

TORU SEO ◽

RICHARD W. ST. CLAIR

Keyword(s):

Human Keratinocytes ◽

Dermal Fibroblasts ◽

Human Dermal Fibroblasts

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Biodegradable citrate-based polyesters with S-nitrosothiol functional groups for nitric oxide release

Journal of Materials Chemistry B ◽

10.1039/c5tb01625h ◽

2015 ◽

Vol 3 (48) ◽

pp. 9233-9241 ◽

Cited By ~ 14

Author(s):

J. P. Yapor ◽

A. Lutzke ◽

A. Pegalajar-Jurado ◽

B. H. Neufeld ◽

V. B. Damodaran ◽

...

Keyword(s):

Nitric Oxide ◽

Functional Groups ◽

Dermal Fibroblasts ◽

Synthesis And Characterization ◽

Human Dermal Fibroblasts ◽

Nitric Oxide Release

Herein, we report the synthesis and characterization of biodegradable citrate-based polyesters that were functionalized for nitric oxide release. The material extracts did not exhibit cytotoxicity as evaluated with human dermal fibroblasts.

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In VivoApplicability ofNeosartorya fischeriAntifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01777-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01777-18 ◽

Cited By ~ 8

Author(s):

Renátó Kovács ◽

Jeanett Holzknecht ◽

Zoltán Hargitai ◽

Csaba Papp ◽

Attila Farkas ◽

...

Keyword(s):

Human Keratinocytes ◽

Dermal Fibroblasts ◽

Vulvovaginal Candidiasis ◽

Antifungal Protein ◽

Primary Human Keratinocytes ◽

Fungal Cell ◽

Efficient Treatment ◽

Content Type

ABSTRACTAs a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-formingCandidaspecies, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of severalCandidaspp.in vitro; however, no information is available about theirin vivoantifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis,Neosartorya fischeriantifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistantCandida albicansstrain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilmin vitro. We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MICin vitro. In vivomurine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistantC. albicanscells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

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In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

10.1101/381178 ◽

2018 ◽

Author(s):

Chee-Wai Wong ◽

Beverley F. Kinnear ◽

Radoslaw M. Sobota ◽

Rajkumar Ramalingam ◽

Catherine F. LeGrand ◽

...

Keyword(s):

Cell Fate ◽

Dermal Fibroblast ◽

Human Keratinocytes ◽

Human Dermal Fibroblast ◽

Dermal Fibroblasts ◽

Primary Human Keratinocytes ◽

Matrix Deposition ◽

Forming Efficiency

SummaryThe long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the dermal fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.

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