HEKns were seeded on the dermis to construct the epidermis and cultured using the EpiLife medium. The next day, the medium was replaced with CnT‐3D‐PR medium (CELLnTEC, Bern, Switzerland) and incubated for 1 day. The formation of epidermal layers was induced by air‐liquid interface (ALI) culture for 10 days. On the third day of ALI culture, the mold was carefully removed from the dermis to ensure full maturation of the epidermis.
Cnt pr 3d medium
Manufactured by CELLnTEC
Sourced in Switzerland
CnT-PR-3D medium is a proprietary cell culture medium formulated by CELLnTEC for the maintenance and proliferation of primary cells. It is designed to support the growth of various cell types, including keratinocytes, fibroblasts, and epithelial cells, without the need for additional supplements.
Automatically generated - may contain errors
Lab products found in correlation
Ker ct, by American Type Culture Collection (3 mentions)
Cnt pr medium, by CELLnTEC (3 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Epilife medium, by Thermo Fisher Scientific (2 mentions)
Rat tail collagen, by BD (2 mentions)
Nunc cell culture inserts, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by CELLnTEC (2 mentions)
Type 1 collagen, by Nitta Gelatin (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Fibrinogen, by Corning (1 mentions)
Human keratinocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
K sfm medium, by Thermo Fisher Scientific (1 mentions)
Thincert, by Greiner (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Cnt 07, by CELLnTEC (1 mentions)
Cnt bm 1, by CELLnTEC (1 mentions)
9 protocols using cnt pr 3d medium
1
Constructing Epidermal Tissue from HEKns
2
Reconstruction of Full-Thickness Human 3D Skin
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A full-thickness human 3-D-skin model was reconstructed through slight modification of our previously described protocols [37 (link),38 (link)]. Briefly, dermal mixture was prepared by mixing Type-I collagen (3 mg/mL, Nitta gelatin, Osaka, Japan), reconstruction buffer (Nitta gelatin), DE concentrated culture solution (Nitta gelatin), fibrinogen (10 mg/mL, Sigma-Aldrich. St. Louis, MO, USA), aprotinin (0.4 TIU/mL, Sigma-Aldrich), and fibroblasts. Then, thrombin (0.625 U/mL, Sigma-Aldrich) was added to initiate polymerization of fibrinogen and the mixture was loaded to each insert of 12 mm Snapwell cell culture inserts (Corning Costar, NY, USA) and incubated for 2 h at 37 °C to allow polymerization. The dermal layer was then cultured in 106 media for 7 days at 37 °C and 5% CO2. Then, HEKns were seeded on dermis and cultured in EpiLife media. The next day, the media was changed with the CnT-3D-PR medium (CELLnTEC, Bern, Switzerland) and incubated for 1 day. The formation of epidermal layers was induced by the air–liquid interface (ALI) culture for 10 days.
3
Human Keratinocyte Air-Liquid Culture
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Insert transwells (Merck, MCHT12H48) were seeded with 105 human foreskin keratinocytes (Ker-CT, ATCC CRL-4048) on the transwells in 300 μL CnT-PR medium (CellnTec) in a 12 well format. After 48 hours, cultures were switched to CnT-PR-3D medium (CELLnTEC, Bern 3014, Switzerland) for 24 hours and then cultured at the air–liquid interface for 17 days. From day 12 to 17 of the air–liquid interphase culture, the Th17 cytokines IL17A (30 ng/mL) and IL22 (30 ng/mL) were added [60 (link)]. Pharmacological treatments were applied from day 14 to 17 and consisted of 100 μM apocynin, 100 μM FK-866, 100 μM olaparib, 10 μM NP and 1 μM ATRA. Culture medium was refreshed every 2 days. At day 17, the tissues were harvested for gene expression analysis.
4
Generation of Human Epidermal Equivalents
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Human N/TERT keratinocyte cell line N/TERT-2G, purchased from J. Rheinwald laboratory (Harvard Medical School, Boston, MA, USA), was cultured in Epilife medium (MEPI500CA, ThermoFisher Scientific, Waltham, MA, USA), complemented with human keratinocyte growth supplement (S0015, ThermoFisher Scientific) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich, Saint-Louis, MO, USA). Human epidermal equivalents (HEEs) were generated as previously described62 (link), with minor adjustments. Briefly, inert Nunc cell culture inserts (141002, ThermoFisher Scientific) were coated with rat tail collagen (100 μg/mL, BD Biosciences, Bedford, USA) at 4°C for 1 hour. 1.5×105 N/TERT-2G keratinocytes (either wildtype, ΔAHR, or ΔTFAP2A keratinocytes) were seeded on the transwells in 150 μL Epilife medium (ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) in a 24 wells format. After 48 h, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 (v/v)) without penicillin/streptomycin for 24 h and then cultured at the air-liquid interface for an additional ten days. Culture medium was refreshed every other day until harvesting at day ten of the air-exposed phase.
5
Airway Epithelial Cell Culture Protocol
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Insert transwells (Merck, Rahway, NJ, USA, MCHT12H48) were seeded with 200,000 human foreskin keratinocytes (Ker-CT, ATCC CRL-4048) on the transwells in 300 μL CnT-PR medium (CellnTec) in a 12-well format. After 48 h, the cultures were switched to CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) for 24 h and then cultured in the air–liquid interface for 17 days. From day 12 to 17 of the air–liquid interphase culture, Th2 cytokines IL13 (10 ng/mL) and IL4 (10 ng/mL) (PeproTech, London, UK) were added [32 (link)]. Pharmacological treatments were applied from day 14 to 17 and consisted of 100 μM Olaparib, 100 μM FK-866 or their combination. The culture medium was refreshed every 2 days. On day 17, tissues were harvested for protein and gene expression analysis.
6
Generating N/TERT Keratinocyte Air-Liquid Culture
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N/TERT-HEEs were generated as previously described32 ,95 (link) with few modifications (Supplemental Fig. 3 ). Briefly, inert transwells (ThinCerts, Greiner Bio-One GmbH) were coated with rat tail collagen (100 µg/mL, BD Biosciences, Bedford, USA) at 4 °C for 1 hour. 105 N/TERT1 or N/TERT2G keratinocytes were seeded on the transwells in 100 µL K-SFM medium (Gibco) in a 24 wells format. After 48 hours, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 (v/v)) for 24 hours and then cultured at the air-liquid interface for 10 days. Culture medium was refreshed every other day. Optimisation of the culture protocol is shown in Supplemental Fig. 4A–C .
7
Engineered Human Epidermal Equivalents
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To generate HEEs, primary human KCs were trypsinized 72 hours after infection and seeded at a density of 3.4 × 105 on inserts with a pore size of 0.4 μm (Merck Millipore, Burlington, MA) in CnT-basal KC medium (CnT-BM.1) supplemented with human KC growth supplements (CnT-07) (CELLnTEC Advanced Cell Systems) (Blunder et al., 2017 (link)). After 48 hours, the medium was switched to a mixture of CnT-PR-3D medium (CELLnTEC Advanced Cell Systems) and DMEM (Lonza, Basel, Switzerland) medium (60:40 [v/v]) according to the procedure by Smits et al. (2020) (link). After 16 hours, HEEs were lifted to the air‒liquid interface and cultivated at a humidity of 55–60% at 37 °C and 5% carbon dioxide for an additional 8 days until harvesting. All cell cultures were grown in the absence of antibiotics and antifungals.
8
Generation of Epidermal Equivalents for CRISPR Editing
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Epidermal equivalents were generated as previously described (Smits et al., 2017) , with minor adjustments. Briefly, inert Nunc cell culture inserts (141002, Thermo Fisher Scientific) were coated with rat tail collagen (100 mg/ml, BD Biosciences, Bedford, MA) at 4 C for 1 hour. A total of 1.5 Â 10 5 N/TERT-2G KCs were seeded on the transwells in 150 ml Epilife medium (Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) in a 24wells format. After 48 hours, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 [v/v]) without penicillin/streptomycin for 24 hours and then cultured at the airÀliquid interface for an additional 10 days. The culture medium was refreshed every other day until harvesting on day 10 of the air-exposed phase.
sgRNA design, single-strand donor oligonucleotide, and synthetic Cas9
Synthetic sgRNAs to knockout FLG gene and purified Edit-R Cas9 nuclease protein (nuclear localization signal, number CAS11200) were obtained from Synthego (Menlo Park, CA) and IDT Technologies (Coralville, IA), respectively. Custom synthetic Alt-R sgRNAs and single-strand donor oligonucleotide to correct FLG expression were ordered from IDT Technologies. See Supplementary Table S4 for details on the sgRNAs and single-strand donor oligonucleotide used.
sgRNA design, single-strand donor oligonucleotide, and synthetic Cas9
Synthetic sgRNAs to knockout FLG gene and purified Edit-R Cas9 nuclease protein (nuclear localization signal, number CAS11200) were obtained from Synthego (Menlo Park, CA) and IDT Technologies (Coralville, IA), respectively. Custom synthetic Alt-R sgRNAs and single-strand donor oligonucleotide to correct FLG expression were ordered from IDT Technologies. See Supplementary Table S4 for details on the sgRNAs and single-strand donor oligonucleotide used.
9
Keratinocyte Differentiation and Th17 Modulation
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Insert transwells (Sigma-Aldrich MCHT12H48) were seeded with human foreskin keratinocytes (Ker-CT, ATCC CRL-4048) on the transwells in 300 μL CnT-PR medium (CellnTec) in a 12-well format. After 48 h, cultures were switched to CnTPR-3D medium (CELLnTEC) for 24 h and then cultured at the air-liquid interface for 17 days [30 (link)]. From day 12 to 17 of the air-liquid interphase culture, the Th17 cytokines IL17A (30 ng/mL) and IL22 (30 ng/mL) were added. Pharmacological treatments consisting of WZB117 at 100 μM, DPP4 at 100 μM, and all-trans retinoic acid (ATRA) at 1 μM (Table S2 ), were applied from day 14 to 17. The culture medium was refreshed every two days. On day 17, the tissues were harvested for gene expression analysis.
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