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

Abstract

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.

Effect of Pig-Adipose-Derived Stem Cells’ Conditioned Media on Skin Wound-Healing Characteristics In Vitro

“…One explanation that may account for this observation involves the specific requirements for an optimal cell culture of pKERs in terms of culture media composition and coating substrates. A recent study by Ponce et al, in which several culture media and adhesion substrates were examined, clearly demonstrates that media rich in hormones and specific growth factors together with the collagen-based matrix used to cover growth surface are necessary for the effective cultivation of keratinocytes isolated from the skin of newborn piglets [ 47 ]. In light of these findings, we cannot exclude that in our study, although CMs were rich in VEGF, bFGF, and MCP-1, they did not contain the full package of factors necessary for supporting pKERs’ migration.…”

“…Skin samples were sliced using a dermatome (Zimmer ® Electric Dermatome; Zimmer Biomet Surgical, Inc., Dover, OH, USA). Skin layers were incubated in 1.6 U/mL dispase (Gibco, Life Technologies Corporation, Grand Island, NY, USA) overnight at 4 °C to separate the epidermis from dermis [ 47 ]. The next day, the separated epidermis was digested for 3 min in 0.05% trypsin-EDTA solution (Sigma-Aldrich Co., St. Louis, MO, USA) and filtered through 70-μm strainers (Corning Incorporated, NY, USA) for keratinocyte isolation.…”

Engineering a multicompartment in vitro model for dorsal root ganglia phenotypic assessment

“…45,57 Despite this knowledge, the majority of in vitro DRG platforms utilize 2D culture, as represented in Table 1. [23][24][25]45 Commonly, the methods of 2D DRG culture include direct plating onto a rigid surface or onto a coating of varying substrate stiffness. 39,[62][63][64][65][66] However, in 3D culture, cells are embedded within a matrix where the mechanical properties are tunable by modifying the composition.…”

“…Specifically, previous work has shown that the stiffness of the culture environment influences axonal growth, 59,60 branching, 61 and neuronal excitability of DRGs 45,57 . Despite this knowledge, the majority of in vitro DRG platforms utilize 2D culture, as represented in Table 1 23–25,45 . Commonly, the methods of 2D DRG culture include direct plating onto a rigid surface or onto a coating of varying substrate stiffness 39,62–66 .…”

“…However, a literature review of current dorsal root ganglia (DRG) culture models yields no culture systems that incorporate these three features-to our knowledge-, highlighting a gap in the field (Table 1). [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] Thus, the goal of this work was to develop a physiologically relevant phenotypic screening DRG platform of neuronal excitability that maintains key anatomical features to recapitulate the in vivo environment (Figure 1).…”