Retroviral gene transfer into porcine keratinocytes following improved methods of cultivation

Bevan, Steve; Woodward, B.; Ng, Roy L.H; Green, C.; Martin, Robert H.

doi:10.1016/s0305-4179(97)00018-1

Cited by 12 publications

(16 citation statements)

References 24 publications

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“…To isolate porcine keratinocytes from piglets several methods as described for humans [1][2][3][4][5][6], mice [2,[13][14][15][16][17][18][19], and adult pigs [13,18,[25][26][27][28] were performed without satisfactory results. These methods resulted in low cell yields with rather high percentages of senescent cells.…”

Section: Discussionmentioning

confidence: 99%

“…• C for both tissue dissociation and cell disaggregation [13]. Therefore, taking the information from those published protocols, we combined different isolation procedures, in order to obtain the appropriate conditions for the isolation of porcine keratinocytes.…”

Section: Isolation and Morphologymentioning

confidence: 99%

“…Bevan et al used OptiMEM ® 1 medium (Gibco) supplemented with 0.5 mM calcium chloride, 2 mM L-glutamine, 1.25 g/l amphotericin and 1% fetal bovine serum. This method also necessitates the use 3T3 feeder cells [13].…”

Section: Medium-dependent Proliferation and Viability Of Keratinocytesmentioning

confidence: 99%

“…Subsequent cell dissociation was performed by fine mincing of epidermal pieces and shaking in Trypsin solution. Debris was removed by filtering through a fine mesh sieve [13].…”

Section: Tissue Disaggregation and Cell Dissociationmentioning

confidence: 99%

“…Performing this tissue disaggregation as described in the literature [13,18] with either Trypsin/EDTA, higher Dispase concentrations or at higher temperatures (37…”

Section: Tissue Disaggregation and Cell Dissociationmentioning

confidence: 99%

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Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

Ponce

Heintz

Schäfer

et al. 2017

JCB

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Abstract. Keratinocytes are the main cell population in the epidermis, where they coexist with a variety of other cell types. Their successful isolation and cultivation have afforded opportunities to study epidermal functions. Human keratinocytes have been studied most extensively, but their source is limited by the skin supply. In a previous work, we developed an in vitro co-culture model of porcine keratinocytes with porcine sensory neurites to investigate functional interaction. However, a detailed description of the isolation of porcine keratinocytes and their culture conditions has not been given in detail. Here, we present the isolation procedure and a characterization of keratinocytes derived from new-born piglets, using simple assays based on conventional and fluorescence microscopy. Media, coating substrates and plating densities were tested with respect to cell viability, proliferation and morphology. Growing keratinocytes in EpiLife keratinocyte growth medium (EKGM) on human collagen type I substrate was best to support proliferation. The minimum plated density was 500 viable cells/cm 2 for primary and 1000 viable cells/cm 2 for subcultured cells. Population doubling (PD) and generation time (tg) depended on the plating densities. Keratinocytes seeded at a density of 5000 viable cells/cm 2 had a PD of 4.36 ± 0.60 per passage and tg of 1.69 ± 0.24 days. Our results show that the optimal isolation and culture conditions for keratinocytes from piglets differ from those for keratinocytes from adult pigs and humans. Thus, the information obtained from the characterization allowed the performance and optimization of a co-culture and contributes to further investigations in epidermal homeostasis and cutaneous sensation.

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Section: Discussionmentioning

confidence: 99%

Section: Isolation and Morphologymentioning

confidence: 99%

Section: Medium-dependent Proliferation and Viability Of Keratinocytesmentioning

confidence: 99%

“…Subsequent cell dissociation was performed by fine mincing of epidermal pieces and shaking in Trypsin solution. Debris was removed by filtering through a fine mesh sieve [13].…”

Section: Tissue Disaggregation and Cell Dissociationmentioning

confidence: 99%

“…Performing this tissue disaggregation as described in the literature [13,18] with either Trypsin/EDTA, higher Dispase concentrations or at higher temperatures (37…”

Section: Tissue Disaggregation and Cell Dissociationmentioning

confidence: 99%

See 3 more Smart Citations

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

Ponce

Heintz

Schäfer

et al. 2017

JCB

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show abstract

A large preclinical animal model to assess ex vivo skin gene therapy applications

et al. 2006

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Because of its easy accessibility, the skin is a very attractive target for gene therapy purposes. To study potential clinical applications in a preclinical setting, appropriate animal models are needed. Pig skin is very similar to human skin, and a variety of human diseases that are potentially amenable to gene therapy applications also occur in pigs. Only a few studies have analyzed the engraftment of transduced keratinocytes (KC) in pigs, however, with limited success. We describe a porcine model in which pig KC were transduced ex vivo with a retroviral vector encoding a marker gene and subsequently grafted onto the autologous host, utilizing a relatively simple grafting technique. Enhanced transduction efficiency was achieved by an optimized transduction protocol including centrifugation of the retroviral vector at a temperature of 32 degrees C. Transduced KC were then seeded onto acellular dermis, forming a stratified epidermis. Grafting was performed by creating full thickness wounds and placing the skin graft onto the muscle fascia, covered by a protective skin flap for several days. Successful engraftment of transduced KC was demonstrated by immunohistochemistry of biopsies taken at different time points, showing transgene expression in 40-50% of grafted KC. After 4 weeks, KC expressing a foreign marker gene was lost, suggesting a transgene-specific immune response in the immunocompetent pigs and highlighting the potential problems for clinical gene therapy studies when transferring new genetic material into a patient. The model presented here may be used to examine applications of skin gene therapy, where retroviral vectors encoding endogenous pig genes will be expressed in the skin.

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Demonstration of epidermal transfer from a polymer membrane using genetically marked porcine keratinocytes

Grant

Woodward

et al. 2001

Burns

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Retroviral gene transfer into porcine keratinocytes following improved methods of cultivation

Cited by 12 publications

References 24 publications

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

A large preclinical animal model to assess ex vivo skin gene therapy applications

Demonstration of epidermal transfer from a polymer membrane using genetically marked porcine keratinocytes

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