For preparation of cortical neural stem cells [54 (link)], cells were plated into Nunc 90 mm petri dishes, previously coated with Cell Start (Gibco), at a seeding density of 500 x105 cells/ml. Neural stem cells were cultured in StemPro NSC SFM composed of: Knockout D-MEM / F12; Glutamax (2mM); bFGF (20ng/ml); EGF (20ng/ml); StemPro Neural Supplement (2%); all from (Gibco). Under these growth conditions at 7 DIV cells were proliferative and were Nestin and Ki67 positive.
Cellstart
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
CELLstart is a cell culture substrate that promotes cell attachment and growth. It is designed for use in a variety of cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (7 mentions)
12 well plate, by BD (5 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (3 mentions)
Tryple, by Thermo Fisher Scientific (3 mentions)
Tryple select, by Thermo Fisher Scientific (3 mentions)
Aof collagenase a, by Worthington (3 mentions)
Fibrogro medium, by Merck Group (3 mentions)
Sb431542, by Merck Group (3 mentions)
Matrigel, by Corning (3 mentions)
Cnt pr 3d differentiation medium, by CELLnTEC (3 mentions)
Cnt prime medium, by CELLnTEC (3 mentions)
Epilife kg2, by Kurabo (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Stemprosfm cts, by Thermo Fisher Scientific (3 mentions)
Knockout dmem f 12, by Thermo Fisher Scientific (2 mentions)
Stempro neural supplement, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
52 protocols using cellstart
1
Culturing Cortical Neural Stem Cells
2
Generation and Maintenance of Human iPSCs
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The human iPSCs lines SFCi55 (parental, control) and SFCi55-iKLF1.2 were generated in house20 (link). The SFCi55 iPSC line was originally generated using fibroblasts obtained from blood group O Rhesus negative individuals by R Biomedical under REC 1/AL/0020 ethical approval and programmed to iPSCs using Yamanaka factors on episomal vectors. Both lines were confirmed to be pluripotent and have normal karyotype20 (link). All IPSC lines were routinely tested for mycoplasma and maintained in StemPro hESC SFM media (Gibco) with 20 ng/ml bFGF (R&D) (Maintenance media). Wells were pre-coated with CELLstart (Gibco) for 1 h before. Cells were passaged using the StemPro EZPassage tool (Thermo Fisher Scientific). Media change was performed every day and cells passaged at a ratio of 1:4 every 3–4 days, when cells reached 70% confluency.
3
Cell Culture Protocols for Sarcoma Research
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ES cell lines TC32, TC71, A4573 and STA-ET-7.2, were grown in RPMI with 10% FBS and 1% HEPES. SKES and RDES cells were grown in McCoy's 5A medium with 15% FBS. hMSC were obtained from Lonza and maintained in StemPro MSC SFM supplement CTS and GlutaMax for complete media (Gibco by Life technologies). hMSC cells were grown in the flasks coated with CELLstart (Gibco by Life technologies). hMSC cells were subcultured when cell confluency reaches 60–80%, cells were in mid-logarithmic phase of growth and cell viability is at least 90%. All cell lines were grown at 37°C in 5% CO2 and ES cells were passaged every 2–4 days. Cell line integrity was confirmed by fingerprinting. Cell lines were tested mycoplasma negative in domo.
4
Isolation and Culture of Neural Stem Cells
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Rip was purchased from Developmental Studies Hybridoma bank (DSHB, USA) and CD133 was purchased from Abcam (USA). GFAP and Dulbecco’s phosphate-buffered saline (DPBS) with and without Ca2+ and Mg2+ were purchased from Sigma-Aldrich (MO, USA). Cell start, Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM/F-12 medium, StemPro neural supplement, neurobasal medium, TrypLE, B-27, glutamine, and Glutamax were purchased from Gibco (Carlsbad, CA, USA). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from Alomone Lab (Israel). Platelet-derived growth factor-AA (PDGF-AA, Millipore, USA), tri-iodothyronine (T3, Sigma), poly-D-lysine (PDL, Sigma), matrigel matrix (Corning Inc) were also used.
5
hNSC Culture and Maintenance Protocol
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hNSCs (H9 hESC-Derived, Gibco, Waltham, MA, USA, USA) were seeded on T75 flasks (Corning, New York, NY, USA) coated with defined substrate (diluted 1:100, CELLstart, Gibco, Waltham, MA, USA) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) F-12 (Gibco, USA) with 2% Neural Supplement (Gibco, USA), 2 mM glutamine (Gibco, USA), 20 ng/mL fibroblast growth factor-basic (bFGF, Gibco, USA), and 20 ng/mL epidermal growth factor (EGF, Gibco, USA). After about a week, the hNSCs were washed with Dulbecco’s Phosphate Buffered Saline (D-PBS) without Ca2+ and Mg2+ (WAKO, Tokyo, Japan), dissociated into single cells using dissociation reagent (Accutase, Gibco, USA), and passaged at a 1:4 ratio. All cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C, and the culture medium was fully replaced every 2–3 days. hNSCs at passage number ≤7 were used in extracellular [3H]choline uptake experiments and at passage number ≤10 were used in all other experiments.
6
Isolation and Culture of Dermal Fibroblasts
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Pieces of skin biopsy (1 mm2) were incubated with 1 mg/ml AOF Collagenase A (LS00415; Worthington Biochemical) for 1 h. Released cells were washed twice and plated on dishes, coated with CellStart™ (A1014201; Gibco), in Fibrogro™ medium (SCM037; EMD Millipore). Once confluent, the cells were passaged using TrypLE™ Select (12563-011; Invitrogen), and their purity was determined by the proportion of cells expressing fibroblast cell markers. The cells were stable in culture for at least five passages.
7
Reprogramming Cynomolgus Monkey Fibroblasts
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Example 6
The reprogramming experiments using cynomolgus monkey fibroblasts were conducted in 6-well tissue culture plates coated with CELLstart (Gibco) substrate in accordance with the manufacturer's directions. Target cells were plated in Allele Biotech's serum-free media plus antibiotics. Media was replaced daily during and after reprogramming with 2-Thio-modified mRNA/transfection reagent mix delivered together with the fresh media for 12 consecutive days with or without the use of B18R (
8
Seeding and Culturing hNSCs
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The hNSCs were seeded on 48- or 24-multiwell plates coated with defined substrate (diluted 1:100, CELLstart, Gibco, USA). Hemicholinium-3 (HC-3) was added 48 h after hNSCs plating, and the final culture medium in each well was 0.5 or 1.0 mL. To maintain the cell conditions, all culture media and HC-3 were changed every day. The cell numbers were measured with a cell viability assay kit (ATPLite, PerkinElmer, USA), and their luminescence was measured using a microplate reader (FilterMax F5, Molecular Devices, San Jose, CA, USA).
9
Cell Culture Protocols for Dermal, Stem, and Epidermal Cells
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Example 5
Human dermal microvascular endothelial cells (EC), (HDMEC, PromoCell GmbH, Germany) isolated from dermis from adult donor were grown in culture flasks coated with gelatin (Sigma Aldrich) in complete endothelial cell media MV, containing 5% fetal bovine serum (PromoCell GmbH, Germany).
Human mesenchymal stem cells (hMSC, Gibco) from bone marrow were grown in culture flasks coated with CELLstart (Gibco) in complete StemPro MSC serum free medium CTS (Gibco) containing 25 ng/μl fibroblast growth factor 13 (Gibco) and 2 mM Glutamax (Gibco).
Normal human epidermal keratinocytes from adult skin (NHEK-ad) were purchased from Lonza. Subculture, proliferation and migration experiments were done in KGM-Gold (Lonza), containing bovine pituitary extract, whereas adhesion experiments were performed in KGM-CD (chemically defined), supplemented with CaCl2) to give 1.2 mM Ca2+.
Keratinocyte and mesenchymal stem cell cultures, as well as experiments, were performed under serum-free conditions to avoid possible interactions between the matrices and serum proteins that potentially could give rise to increased cell adherence.
Medium was changed every 2-3 days. Cells were harvested with TrypLE (Life Technologies) when reaching a confluency of 80% for subculture or experiments. All experiments were performed at 37° C. with 5% CO2 and 95% humidity.
10
Cultivation of Human Dermal, Stem, and Epidermal Cells
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Example 5
Human dermal microvascular endothelial cells (EC), (HDMEC, PromoCell GmbH, Germany) isolated from dermis from adult donor were grown in culture flasks coated with gelatin (Sigma Aldrich) in complete endothelial cell media MV, containing 5% fetal bovine serum (PromoCell GmbH, Germany).
Human mesenchymal stem cells (hMSC, Gibco) from bone marrow were grown in culture flasks coated with CELLstart (Gibco) in complete StemPro MSC serum free medium CTS (Gibco) containing 25 ng/μl fibroblast growth factor β (Gibco) and 2 mM Glutamax (Gibco).
Normal human epidermal keratinocytes from adult skin (NHEK-ad) were purchased from Lonza. Subculture, proliferation and migration experiments were done in KGM-Gold (Lonza), containing bovine pituitary extract, whereas adhesion experiments were performed in KGM-CD (chemically defined), supplemented with CaCl2 to give 1.2 mM Ca2+.
Keratinocyte and mesenchymal stem cell cultures, as well as experiments, were performed under serum-free conditions to avoid possible interactions between the matrices and serum proteins that potentially could give rise to increased cell adherence.
Medium was changed every 2-3 days. Cells were harvested with TrypLE (Life Technologies) when reaching a confluency of 80% for subculture or experiments. All experiments were performed at 37° C. with 5% CO2 and 95% humidity.
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