Wild-type keratinocytes were obtained from CELLnTEC (Bern, Switzerland) or isolated from healthy donors. JEB keratinocytes (JEB654) were isolated from a female patient (age at time of sample taking was 43) carrying a homozygous frameshift mutation (c.3899_3900delCT) within exon 52 of COL17A1. Cell isolation was performed with written consent of the patient. Patient keratinocytes were subsequently immortalized by transduction of human papilloma virus proteins E6 and E7.34 (link) Both wild-type (WT) and JEB keratinocytes were cultured in CnT-Prime Epithelial Proliferation Medium (CELLnTEC, Bern, Switzerland) at 37°C and 5% CO2 in a humidified incubator. 3 × 105 cells were used for the electroporation procedure. Cells were carefully trypsinized and washed two times with PBS. Following filtering to remove cell clumps, a single-cell suspension was combined with pre-complexed RNPs and electroporated with the Neon transfection system (Thermo Fisher Scientific, Waltham, MA, USA). Treated cells were then seeded on collagen I (Sigma-Aldrich, St. Louis, MO, USA) pre-coated 6-well plates. After 1 day of incubation, primocin (InvivoGen, Toulouse, France) was added, and cells were cultured until they reached confluence. In the case of primary keratinocytes, 2 × 105 feeder cells were added 1 day after electroporation. Ethics approval number for patient-derived cells: 415-E/2118/34-2021.
Cnt prime epithelial proliferation medium
Manufactured by CELLnTEC
Sourced in Switzerland
CnT-Prime Epithelial Proliferation Medium is a cell culture medium designed to support the growth and proliferation of epithelial cells. It provides the necessary nutrients and growth factors to maintain the viability and expansion of epithelial cell lines in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Primocin, by InvivoGen (3 mentions)
Cnt fibroblast medium, by CELLnTEC (3 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Collagen 1, by Merck Group (1 mentions)
Mscbm, by Lonza (1 mentions)
Human bmsc, by Lonza (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Hblak cells, by CELLnTEC (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
9 protocols using cnt prime epithelial proliferation medium
1
CRISPR-Mediated Gene Editing in Keratinocytes
2
CRISPR-Mediated Genome Editing in Keratinocytes
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JEB keratinocytes were obtained from a skin biopsy of a patient bearing a homozygous frameshift mutation (c.3899_3900delCT) within exon 52 of COL17A1 upon informed consent of the patient. Wild-type keratinocytes, which served as a positive control, were isolated from healthy donors upon informed consent. Healthy and patient primary keratinocytes were cultured in CnT-Prime Epithelial Proliferation Medium (CELLnTEC, Bern, Switzerland) at 37°C and 5% CO2 in a humidified incubator. One day before electroporation, cells were moved to 32°C and antibiotic-free medium. 3 × 105 cells were electroporated in suspension with RNPs (3 μg HiFiCas9/Cas9n + 750 ng sgRNA) and ssODN (250 ng) using the Neon transfection system (Thermo Fisher Scientific, Waltham, MA, United States) at 1,400 V, 20 ms, 2 pulses. Transfected cells were maintained at 32°C for an additional 24 h before moving to 37°C.
3
Isolation and Cultivation of Keratinocytes and Fibroblasts
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JEB keratinocytes (JEB-282-Kc) were isolated from a patient biopsy and carry a homozygous frameshift mutation (c.3899-3900delCT/c.3899-3900delCT) within exon 52 of COL17A1 [7 (link),8 (link)]. Wild-type keratinocytes (hKc-1090 and WT-200-Kc) and wild-type fibroblasts (HC-941-Fibs) were isolated from healthy donors upon informed consent. All cells were subsequently immortalized through transduction of the human papillomavirus (HPV) proteins E6 and E7 and grown at 37 °C and 5% CO2 in a humidified incubator [15 (link)]. WT Kc as well as JEB Kc were cultured in CnT-Prime Epithelial Proliferation Medium (CELLnTEC, Bern, Switzerland) with primocin (InvivoGen, Toulouse, France). Wild-type fibroblasts were cultivated in CnT Fibroblast Medium (CELLnTEC, Bern, Switzerland) with primocin (InvivoGen, Toulouse, France).
4
Isolation and Characterization of Corneal Epithelial Cells and Mesenchymal Stem Cells
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Human corneal epithelial cells (HCECs) were obtained from the corneas of cadaveric donors, and were isolated and characterized as previously described [25 (link)]. Cells were maintained in CnT-Prime Epithelial Proliferation Medium (CellnTEC, Bern, Switzerland), 10% XerumFreeTM XF212 supplement medium (TNC Bio BV, Eindhoven, The Netherlands) and 1% penicillin-streptomycin (10 mg/mL, Life Technologies Corporation, Grand Island, NY, USA). Human BMSCs, purchased from Lonza (Basel, Switzerland), were cultured in mesenchymal stem cell basal medium (MSCBM, Lonza, Basel, Switzerland) as previously reported [26 (link)].
5
Isolation and Culture of Murine Epidermal Keratinocytes
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To isolate the MEKs, neonatal mice were sacrificed. Dispase II was used to separate the epidermis. The next day, accutase (A11105-01, Gibco) was used to digest epidermis. The MEKs were cultured with CnT-Prime epithelial proliferation medium (#CnT-PR, CELLnTEC, Bern Switzerland).
6
Isolation and Immortalization of RDEB Cells
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RDEB keratinocytes and fibroblasts were isolated from a 3 mm skin biopsy of patients. HC-Kcs and fibroblasts were isolated from a healthy donor. Cells were E6/E7 immortalized and cultured under standardized conditions (37 °C and 5% CO2 in a humidified incubator) in CnT-Prime Epithelial Proliferation Medium (CELLnTEC, Bern, Switzerland) or in CnT Fibroblast Medium (CELLnTEC, Bern, Switzerland), respectively.
7
Bladder and Kidney Cell Culture Protocols
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Commercially available primary human bladder urothelial cells (HBLAK cells, CELLnTEC Advanced Cell Systems, Bern, Switzerland), derived from the bladder of an 80-y-old male, were cultured in a CnT-Prime Epithelial Proliferation Medium (CELLnTec Advanced Cell Systems) media (62 (link), 63 (link)). Commercially available primary human kidney medullary epithelial cells obtained from a 3-y-old Hispanic female (Lifeline Cell Technology, Frederick, MD) were cultured in Renalife Media (Lifeline Cell Technology, as previously published (31 (link)). Once reaching 90 to 95% confluency, cells were treated with the indicated drugs. At the indicated time points, RNA was isolated from cells for qRT-PCR or cell lysates were generated for Western blot. Conditioned media were collected for ELISA or antibacterial assays.
8
Culturing Primary Human Epidermal Keratinocytes
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Human primary epidermal KCs (HPEK) isolated from the juvenile foreskin of multiple donors (>three individuals) were purchased cryopreserved at an early passage from CELLnTEC (Bern, Switzerland). Cells were cultured according to the manufacturer’s protocol in CnT-Prime Epithelial Proliferation medium (CnT-PR) fully supplemented, phenol red-free medium with low calcium formulation (0.07 mM) and a combination of Growth factors and Progenitor Cell Targeted factors maximizing retention of proliferative progenitor cells and minimizing cell differentiation (CELLnTec, Bern, Switzerland). HPEK were seeded after thawing or passaging at a density of 4000 cells/cm2 and were incubated at 37 °C in a humidified atmosphere under 5% CO2. The medium was replaced every two days. Cells were passaged at ~80% confluency (day 6) after trypsinization with 0.05% trypsin–EDTA solution (Gibco, Paisley, UK). Cells between the second and fourth passages were used for experiments.
9
Isolation and Culture of RDEB Cells
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RDEB keratinocytes were isolated from the hair follicles of a patient carrying a homozygous frameshift mutation (c.6081delC/c.6081delC) within exon 73 of COL7A1. RDEB fibroblasts were isolated from hair follicles carrying a mutation in exon 115 (c.8441-15del20/c.8505-8506dupCG) and from a phimosis carrying a mutation in exon 105 (c.7795G>T/c.7795G>T) (Table 1 ). Wild-type fibroblasts (WT Fib) and human keratinocytes (hKc) were isolated from a healthy donor upon informed consent. All cells were subsequently immortalised through transduction of the human papillomavirus proteins E6 and E7 and grown at 37 °C and 5% CO2 in a humidified incubator. Healthy keratinocytes, as well as patient-derived keratinocytes, were cultured in CnT-Prime Epithelial Proliferation Medium (CELLnTEC, Bern, Switzerland) with Primocin (InvivoGen, Toulouse, France) or in Gibco serum-free keratinocyte medium (SFM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 U/mL penicillin/streptomycin (Biochrom, Berlin, Germany). Wild-type fibroblasts and RDEB fibroblasts were cultivated in CnT Fibroblast Medium (CELLnTEC, Bern, Switzerland) with Primocin (InvivoGen, Toulouse, France).
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