Protocol for the long‐term culture of human primary keratinocytes from the normal colorectal mucosa

Abstract

Procedures for in vitro culturing of human primary keratinocytes from normal colon mucosa specimens have not been fully feasible, thus far. The protocol described herein allows primary keratinocytes from small tissue fragments of colorectal mucosa biopsies to grow in vitro. The procedure develops in three steps: (a) the enzymatic digestion of the tissue biopsy; (b) the use of cloning rings to purify primary keratinocyte colonies, (c) a defined keratinocyte medium to grow these cells in long‐term culture. Our cultural method enables normal primary keratinocytes to be obtained by simple and rapid techniques. In our culture condition, primary keratinocytes express specific epithelial markers. Colorectal mucosa keratinocyte colonies require approximately 2 weeks to grow. Compared with previous approaches, our protocol provides a valuable model of study for human primary keratinocytes from normal colorectal (NCR) mucosa both at the cellular and molecular levels. It is well known, that different mutations occurring during the multistep process of carcinogenesis in the NCR mucosa, are strictly associated to the onset/progression of the colorectal carcinoma. On this ground, normal keratinocytes grown with our protocol, may represent an innovative tool to investigate the mechanisms that lead to colorectal carcinoma and other diseases. Our innovative procedure may allow to perform comparative investigations between normal and pathological colorectal cells.

Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients

“…Similar results were reported by a recent study in which, organoid cultures from ulcerative colitis patients were compared to organoid cultures from non-inflamed controls, showing similar self-renewal capacity and comparable molecular features (Dotti et al, 2016). Importantly, the organoid structures that were followed in the present study expressed the cytokeratin-20 (KRT20) protein (Figure 2A), providing proofs of success in achieving human primary keratinocyte cultures as previously demonstrated (Torreggiani et al, 2019), and confirming the epithelial origin of those cells.…”

Smart Nanofibers with Natural Extracts Prevent Senescence Patterning in a Dynamic Cell Culture Model of Human Skin

“…They were isolated and cultured as previously described [31]. Human skin fibroblast 1 (HFF1) and Human skin keratinocytes (HACAT) were purchased from ATCC (Manassas, VA, USA) and cultured in a Dulbecco's modified Eagle's Medium (DMEM) low-glucose medium (Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS Life Technologies), 2 mM l-glutamine (Euroclone, Milano, Italy) and 1% of penicillin/streptomycin (Euroclone) [32].…”

Association Between Simian Virus 40 and Human Tumors

“…As for other DNA tumor viruses (75, 76), several in vitro cellular models have been developed to study SV40 transformation potentials. Tag and tag expression both cause high cell transformation efficiently (3).…”

“…Over the years, with the development of molecular biology techniques (12, 18, 76, 177, 178), SV40 DNA sequences have been investigated and detected in both normal and neoplastic tissues from individuals vaccinated with polio vaccines contaminated with SV40. DNA sequences from this PyV have been detected in pituitary tissues (179) as well as in leukocytes from organ (180) and blood (181) donors.…”