Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation

Abstract

Four techniques for dissociation of skin biopsies were compared to identify the method of choice for optimal expansion of isolated keratinocytes. Equivalent biopsies were obtained from 4 healthy human subjects and each divided into four parts. One part was minced and placed in a trypsinizing flask containing 0.05% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA). Released cells were harvested hourly. With the other parts, the epidermis was separated from the dermis after treatment with 0.5 mg/nml thermolysin, 2.5 mg/ml Dispase, or 0.17% trypsin and the epidermal portions were minced and incubated for 1 h in trypsin:EDTA. The cells were cocultivated with irradiated 3T3 fibroblasts to study the keratinocytes proliferative capacity. Freshly isolated cells were immunostained with anti-vimentin antibodies or grown in fibroblast-supportive conditions to detect the presence of human dermal fibroblasts. The mean number of cells dissociated per cm2 biopsy was higher after trypsin:EDTA digestion of a dermis-containing biopsy using a trypsinizing flask (4.0x 10(6) cells/cm2) compared to a biopsy where dermis-epidermis had been separated by thermolysin (2.8x 10(6) cells/cm2), Dispase (2.3x 10(6) cells/cm2) or trypsin (1.1 x 10(6) cells/cm2). Between 0.5% and 4% of the cells dissociated from a dermis-containing biopsy were human fibroblasts. This comprised more than twice the number of fibroblasts obtained by using epidermal/dermal split techniques. The proliferative capacity in primary and secondary culture was higher in cells isolated by trypsin:EDTA incubation in the trypsinizing flask or after epidermal-dermal separation using thermolysin, suggesting that Dispase or trypsin may have a more detrimental effect on the isolated keratinocytes. Our results show that dissociating the cells by trypsin:EDTA incubation in a trypsinizing flask or after epidermal-dermal separation using thermolysin, are preferable methods for isolating keratinocytes from human skin.

Initial Characterization of the Pig Skin Bacteriome and Its Effect on In Vitro Models of Wound Healing

“…Primary Normal Human Dermal Fibroblasts (NHDF) and Normal Human Epidermal Keratinocytes (NHEK) were provided by Dr. Robert Christy lab under an approved institutional review board protocol (# H-11-020) by isolation techniques previously described [29–31]. Briefly, tissue harvested during an abdominoplasty underwent a 2 unit/mL dispase (Gibco) digestion, which was used to isolate epidermal versus dermal layers that were processed separately.…”

ROCK Inhibitor Y-27632 Increases the Cloning Efficiency of Limbal Stem/Progenitor Cells by Improving Their Adherence and ROS-Scavenging Capacity

“…27,28 The colonies were then loaded with 10 mM peroxide-sensitive fluorescence probe 2,7-dichlorodihydrofluorescein diacetateacetyl ester (H2-DCFDA; Molecular Probes, Eugene, OR) for 30 min at 37°C. The images were observed using Nikon confocal microscopy at 488/520 nm.…”

Development of a method for assessing non-targeted radiation damage in an artificial 3D human skin model

“…Although the trypsin incubation adopted for these experiments is longer (30 minutes) than what conventionally used in culturing established cell lines (few minutes), similar protocols have been successfully used. Exposure to trypsin solution up to 1 hr are commonly used and suggested for the dissociation of human biopsies (Hybbinette et al , 1999) resulting in high number of cells with good proliferative potentials. A 35 minute trypsin treatment, in particular, has been demonstrated to be suitable for similar MatTek systems for a variety of biological end-points including micronuclei formation (Curren et al , 2006).…”