Normal human dermal fibroblasts (nhdf)
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NHDF is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed to perform a specific core function, but a detailed description while maintaining an unbiased and factual approach is not available at this time.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (3 mentions)
Normal human dermal fibroblasts (nhdf), by PromoCell (2 mentions)
Primary human hepatocytes, by Thermo Fisher Scientific (2 mentions)
Egm 2 media, by Lonza (2 mentions)
Penicillin streptomycin penstrep, by Avantor (2 mentions)
Normal human dermal fibroblasts (nhdf), by Lonza (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Trypan blue exclusion method, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Htb 14, by LGC (1 mentions)
Gentamicin glutamine solution, by Thermo Fisher Scientific (1 mentions)
10 protocols using normal human dermal fibroblasts (nhdf)
1
Fibroblast Culture and HCMV Strains
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Normal Human Dermal Fibroblasts (NHDF; Gibco) and IRF3 -/- cells were maintained in Dulbecco’s modified high glucose media (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Invitrogen). IRF3 -/- cells were provided by Victor DeFillipis. A low passage HCMV strain TB40E-GFP [45 (link)], which expresses GFP from an SV40 promoter was used for arrayed ISG expression library screening, western blot analysis, Real-Time PCR, and flow cytometry analysis. Laboratory adapted HCMV strain AD169 was used for immunofluorescence experiments.
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2
Culturing NHDF and RAW 264.7 Cells
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The Normal human dermal fibroblasts (NHDF, Promocell) and the murine macrophage cell line Raw 264.7 were grown respectively in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) and Roswell Park Memorial Institute (RPMI1640, Gibco BRL) medium, respectively. Media were supplemented with 10% heat-inactivated FBS (Gibco BRL), penicillin (100 units.mL -1 ), streptomycin (100 μg.mL -1 , from Gibco BRL) and fungizone (0.25 μg.mL -1 , Gibco BRL), at 37 °C in 95% humidity and a 5% CO 2 atmosphere. At confluence, cells were removed from cultured flasks by treatment with 0.1% trypsin and 0.02% EDTA (Gibco BRL). Cells were rinsed and resuspended in the supplemented DMEM or RPMI1640 medium.
Of note, NHDF cells were used at passage 6-7 for the experiments.
Of note, NHDF cells were used at passage 6-7 for the experiments.
3
Culturing and Seeding Normal Human Dermal Fibroblasts on Hydrogels
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The primary cells, normal human dermal fibroblasts (NHDF) (Promocell, Germany), were cultured in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic, at 37°C, with a controlled atmosphere of 5% CO2 and 95% relative humidity. Monolayer of NHDF in their growth phase (∼90% confluence) was detached using trypsin/1 mM ethylenediaminetetraacetic (EDTA) (Life Tech., Germany) in PBS, centrifuged and resuspended in complete cell culture medium. Cells were counted using trypan blue exclusion method (Sigma-Aldrich, Germany) before seeding on hydrogels.
The prepared circular films of alginate and ADA-GEL hydrogels were placed in the wells of 24-well plates (VWR Int., Germany) and washed with DMEM. For analysis of cell adhesion, 85000 cells/film were seeded and incubated in a humidified atmosphere of 95% relative humidity and 5% CO2, at 37°C. Culture medium was changed on the next day after seeding, and then every two days.
The prepared circular films of alginate and ADA-GEL hydrogels were placed in the wells of 24-well plates (VWR Int., Germany) and washed with DMEM. For analysis of cell adhesion, 85000 cells/film were seeded and incubated in a humidified atmosphere of 95% relative humidity and 5% CO2, at 37°C. Culture medium was changed on the next day after seeding, and then every two days.
4
Comparative Analysis of Cell Cultures
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Following cells were used: (1) Normal and cancerous standard permanent cell lines represented by primary normal human dermal fibroblasts (NHDF, PromoCell, Heidelberg, Germany) isolated from the dermis of juvenile foreskin or adult skin and highly radioresistant U-87 glioblastoma cells (ATCC HTB-14, LGC Standards, United Kingdom), respectively. (2) Tumor and tumor-adjacent primary cell cultures isolated from patients with spinocellular head and neck tumors (histologically verified tumor and tumoradjacent tissues). While the permanent cell lines represented biologically homogeneous and technically relatively easy samples, tumor cell primary cultures were involved as highly heterogeneous and challenging samples.
The protocol for patients' primary culture isolation was described in [75] (link). The primary culture was cultivated in RPMI-1640 medium with the Pen/Strep antibiotic solution (PAA Laboratories GmbH, Austria) and 10 % fetal bovine serum, FBS (Biochrom, USA), at 37 °C and 5.0 % CO2 in a humidified atmosphere up to 50 % confluence. NHDF and U-87 were grown in Dulbecco's modified essential medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS) and a 1% gentamicin-glutamine solution (all reagents from Sigma-Aldrich).
The protocol for patients' primary culture isolation was described in [75] (link). The primary culture was cultivated in RPMI-1640 medium with the Pen/Strep antibiotic solution (PAA Laboratories GmbH, Austria) and 10 % fetal bovine serum, FBS (Biochrom, USA), at 37 °C and 5.0 % CO2 in a humidified atmosphere up to 50 % confluence. NHDF and U-87 were grown in Dulbecco's modified essential medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS) and a 1% gentamicin-glutamine solution (all reagents from Sigma-Aldrich).
5
Cryopreserved Human Hepatocytes and Cell Lines
6
Culturing Neonatal Human Dermal Fibroblasts
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The neonatal human dermal fibroblast cell line (NHDF; C0045C) was purchased from Thermo Fischer Scientific, Inc. The cells were grown in a humidified incubator at 37°C with a 5% CO2 atmosphere in Medium 106 (Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 0.5% (v/v) penicillin-streptomycin and used up to passage seven. When they were 80% confluent, the cells were sub-cultured by trypsinization. The culture medium was replenished every 48 h.
7
Culturing HaCaT and NHDF Cells
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The human keratinocyte cell line, HaCaT, was purchased from CLS (#300493, Cell Lines Service, Eppelheim, Germany). Normal human dermal fibroblasts, neonatal (NHDF), were purchased from Thermo Fisher Scientific (#C-004-5C, Waltham, MA, USA). HaCaT cells and NHDF were cultured in Dulbecco’s modified Eagle’s medium (DMEM; #12-604F, Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (#10082-147, Thermo Fisher Scientific), 100 U/mL potassium penicillin and 100 mg/mL streptomycin sulfate (#17-602E, Lonza) at 37 °C in a humidified 5% CO2 incubator. The rate of cell viability was quantified using the Cell Counting Kit-8 (CCK-8) reagent (#CK04-11, DOJINDO, Tokyo, Japan) according to the manufacturer’s instructions.
8
Cryopreserved Human Hepatocytes and Cell Lines
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Cryopreserved primary human hepatocytes were purchased from Thermo Fisher Scientific, lot hu1880 (34-year-old, white, female donor) was used in Figure 1 – Figure 3 , lot hu8375 (19-year-old, white, female donor) was used for Figure 4 . Primary human umbilical endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF) were purchased from Lonza and used at passage < 8. HUVEC were maintained in dishes in EGM-2 media (Lonza), and NHDF were maintained in DMEM (Thermofisher Scientific) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (penstrep, VWR).
9
Comparative Analysis of Neonatal and Adult Fibroblasts
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Neonatal (nHDF) (Cat # C-004-5C) and adult human dermal fibroblasts (aHDF) (Cat # C-013-5C) were purchased from ThermoFisher Scientific (Waltham, MA,USA) Cells were grown in DMEM supplemented with 10% foetal bovine serum (FBS) (Gibco-Thermo Fisher Scientific) according to standard procedures (Quaglino et al., 2000) .
Initially, cell lines were sub-cultured weekly, whereas slow growing cultures were subcultured biweekly. The number of population doublings (PD) was calculated using the formula (van der Loo et al., 1998) :
PD= ln (number of cells harvested) -ln (number of cells seeded) / ln2
The calculated population doubling increase was then added to the previous population doubling value, to yield the cumulative population doubling level (CPD). The end of the replicative lifespan was defined by failure of cell population to double after 20 days in culture despite 3 changes/week of the culture medium.
In this study, we have analysed cell lines at low CPD (i.e. CPD values between 4-6 in both nHDF and aHDF) and at high CPD (i.e. CPD values between 61-64 and 45-49 for nHDF and aHDF, respectively).
Cells were always grown in parallel and regularly observed under the inverted light microscope.
Initially, cell lines were sub-cultured weekly, whereas slow growing cultures were subcultured biweekly. The number of population doublings (PD) was calculated using the formula (van der Loo et al., 1998) :
PD= ln (number of cells harvested) -ln (number of cells seeded) / ln2
The calculated population doubling increase was then added to the previous population doubling value, to yield the cumulative population doubling level (CPD). The end of the replicative lifespan was defined by failure of cell population to double after 20 days in culture despite 3 changes/week of the culture medium.
In this study, we have analysed cell lines at low CPD (i.e. CPD values between 4-6 in both nHDF and aHDF) and at high CPD (i.e. CPD values between 61-64 and 45-49 for nHDF and aHDF, respectively).
Cells were always grown in parallel and regularly observed under the inverted light microscope.
10
Endothelial-Fibroblast Co-Culture Protocol
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All chemicals and cell culture reagents were purchased from Sigma-Aldrich Ltd. (Dorset, UK), unless otherwise stated.
Cell culture. Primary human umbilical vein endothelial cells (HUVEC) and primary normal human dermal fibroblasts (NHDF) were obtained from Clonetics (Lonza, Slough, UK). HUVEC were maintained in endothelial basal medium (EBM-2) supplemented with EGM-2 SingleQuots (i.e., EGM-2 medium) whilst NHDF were maintained in fibroblast basal medium (FBM) supplemented with FGM-2 SingleQuots; all from Clonetics; Lonza (i.e., FGM-2 medium).
The co-culture system was set up using HUVEC (between passages 1-8) and NHDF (between passages 1-12) following the protocol established by Bishop et al (15) and adapted by Barron et al (14) . HUVEC and NHDF were mixed and seeded in 24-well plates (Thermo Fisher Scientific Nunc, Loughborough, UK) in EGM-2 medium. Co-cultured cells were incubated for up to 14 days at 37˚C in a 5% Co 2 in air humidified incubator.
Cell culture. Primary human umbilical vein endothelial cells (HUVEC) and primary normal human dermal fibroblasts (NHDF) were obtained from Clonetics (Lonza, Slough, UK). HUVEC were maintained in endothelial basal medium (EBM-2) supplemented with EGM-2 SingleQuots (i.e., EGM-2 medium) whilst NHDF were maintained in fibroblast basal medium (FBM) supplemented with FGM-2 SingleQuots; all from Clonetics; Lonza (i.e., FGM-2 medium).
The co-culture system was set up using HUVEC (between passages 1-8) and NHDF (between passages 1-12) following the protocol established by Bishop et al (15) and adapted by Barron et al (14) . HUVEC and NHDF were mixed and seeded in 24-well plates (Thermo Fisher Scientific Nunc, Loughborough, UK) in EGM-2 medium. Co-cultured cells were incubated for up to 14 days at 37˚C in a 5% Co 2 in air humidified incubator.
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