Mtesr 1 complete medium

Manufactured by STEMCELL

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MTeSR™1 Complete Medium is a serum-free, xeno-free medium formulated for the feeder-free culture of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). It provides the essential components required for the maintenance of undifferentiated stem cell growth and expansion.

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MTeSR medium (173 citations) Complete mTeSR medium (5 citations) MTeSR Complete (2 citations)

9 protocols using mtesr 1 complete medium

Establishment and Cultivation of CADASIL iPSCs

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The CADASIL iPSC lines were established from skin biopsies of two CADASIL patients carrying NOTCH3 variants Arg153C and Cys224Try, respectively, as reported in our previous study (Kelleher et al., 2019 (link)). The five control iPSC clones were from three healthy individuals with two iPSC clones (02C3 and 02C9) reported by Kelleher et al. (2019) (link), two clones (SW171a and SW174a) reported by Wood et al. (2020) (link), Woods et al. (2020) (link), and OX1-19 line reported by Jarosz-Griffiths et al. (2019) (link). Prior to differentiation, all iPSCs were cultured in Vitronectin Recombinant Human Protein (VTN-N) (ThermoFisher, A14700, Loughborough, UK) pre-coated six-well plates with 2 mL TeSR-E8 medium (STEMCELL Technologies, 05990, Cambridge, UK) at 37^°C with 5% CO₂, except for the OX1-19 iPSCs that were cultured on Matrigel (Corning, 354277, Deeside, UK) pre-coated 6-well plates in mTeSR1 complete medium (STEMCELL Technologies, 85850, Cambridge, UK).
Primary human coronary arterial endothelial cells (HCAECs) (PromoCell, C-12222, Heidelberg, Germany) were cultured in six-well plates (Corning, 3516) with 2 mL Endothelial Cell Growth Medium (MV2, PromoCell, C-22121, Heidelberg, Germany) and maintained at 37^°C incubator with 5% CO₂.

Zhang W., Zhao X., Qi X., Kimber S.J., Hooper N.M, & Wang T. (2023). Induced pluripotent stem cell model revealed impaired neurovascular interaction in genetic small vessel disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy. Frontiers in Cellular Neuroscience, 17, 1195470.

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Establishment of HNmMM Primary Cell Lines

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HNmMM-derived primary cell lines were established from four fresh surgically excised HNmMM tissue samples from the original cohort of 20 patients by culturing them within a Matrigel explant prior to extraction of the cells following abundant growth in 24-well plates (Raylab, Auckland, New Zealand). The extracted cells were cultured in a cell culture medium consisting of DMEM (1X) and GlutaMAX-1 (cat#10569-010, Gibco, Rockford, IL, USA) supplemented with 10% fetal bovine serum (cat# 10091148, Gibco), 5% mTeSR 1 Complete medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin (cat#15140122, Gibco), and 0.2% gentamycin/amphotericin B (cat#R015-10, Gibco). All cultures were maintained in a humidified incubator at 37 °C with 5% CO₂. All primary cell lines used for the experiments were at passages 4–5 (RT-qPCR and WB) or 8–9 (tumorsphere formation).

Yoganandarajah V., Patel J., van Schaijik B., Bockett N., Brasch H.D., Paterson E., Sim D., Davis P.F., Roth I.M., Itinteang T, & Tan S.T. (2020). Identification of Cancer Stem Cell Subpopulations in Head and Neck Metastatic Malignant Melanoma. Cells, 9(2), 324.

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Generation of HD and Healthy iPSC-derived NSCs

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Age and sex-matched iPSCs from an apparently healthy individual (GM23476, female, 20 years old at sampling, RRID:CVCL_T841) and a HD patient (GM23225, female, 20 years old at sampling, RRID:CVCL_F169, CAG repeat length 71) were obtained from MIGMS cell repository through Coriell Institute for Medical Research. iPSCs were cultured in Matrigel-coated plates with mTeSR^™1 complete medium (STEMCELL Technologies). The differentiation of iPSCs to NSCs was performed using the STEMdiff^™ SMADi Neural Induction Kit based on the rosette formation and isolation method according to the manufacture’s instruction (STEMCELL Technologies). After differentiation, NSCs were further cultured and expanded in the complete STEMdiff^™ Neural Progenitor Basal Medium (STEMCELL technologies). NSC expansions were limited to 4 passages. The use of human iPSC lines was approved by Institutional Biosafety Committee of Florida Atlantic University (Approval number B20-21). All iPSCs utilized in this study were used within 10 passages from cryopreserved stocks previously determined to be karyotypically normal. None of the cell lines used in this study (SH-SY5Y, STHdhQ7 and Q111, iPSCs) are listed as commonly misidentified cell lines by the International Cell Line Authentication Committee (ICLAC). No further authentication was performed in the laboratory.

Xu H., Bensalel J., Raju S., Capobianco E., Lu M.L, & Wei J. (2022). Characterization of huntingtin interactomes and their dynamic responses in living cells by proximity proteomics. Journal of neurochemistry, 164(4), 512-528.

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iPS Cell Culture from Blood Monocytes

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The iPS cells derived from peripheral blood monocytes of male AD patients (66540594-1VL), and normal healthy control (66540083-1VL) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture was performed in reference to a reported method [21 (link)]. The iPS cells were cultured in mTesR1 complete medium (STEM CELL Technologies, Vancouver, BC, Canada) diluted with BD Matrigel (BD Biosciences, USA). The culture medium was changed every day. Cells were passaged every 4 days by using 0.5 mM ethylenediaminetetraacetic acid (EDTA) digestion for 3 min. Cells were maintained in a 5% CO₂ incubator at 37 °C.

Kang T., Han Z., Zhu L, & Cao B. (2024). TFR1 knockdown alleviates iron overload and mitochondrial dysfunction during neural differentiation of Alzheimer’s disease-derived induced pluripotent stem cells by interacting with GSK3B. European Journal of Medical Research, 29, 101.

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Establishing Primary Cell Lines from HNmMM Tumors

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Primary cell lines were derived from fresh HNmMM tissue samples from four of the original cohort of 20 patients. To generate primary cell lines, small tissue pieces from these tumor samples were incubated between layers of Matrigel (cat#354234, Corning, Tewksbury, MA, USA) in a 24-well plate with media containing Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax™ (cat#10569010, Gibco, Rockford, IL, USA) supplemented with 2% penicillin-streptomycin (cat#15140122, Gibco) and 0.2% gentamycin-amphotericin (cat#R01510, Gibco). Once sufficient cell growth was achieved to support transfer to a monolayer culture, cells were extracted by dissolving Matrigel with Dispase (cat#354235, Corning) and placed in an adherent culture flask with media containing DMEM with Glutamax™ supplemented with 10% fetal calf serum (cat#10091148, Gibco), 5% mTeSR™1 Complete Medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin, and 0.2% gentamycin-amphotericin in a humidified incubator at 37°C and 5% CO₂. Cells were expanded in culture and harvested between passages 4 and 8.

Siljee S., Pilkington T., Brasch H.D., Bockett N., Patel J., Paterson E., Davis P.F, & Tan S.T. (2020). Cancer Stem Cells in Head and Neck Metastatic Malignant Melanoma Express Components of the Renin-Angiotensin System. Life, 10(11), 268.

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Cardiac Fibroblast and iPSC Culture

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hCFs,
isolated from discarded cardiac surgical
tissue from patients undergoing cardiac surgery through explant outgrowth,
were routinely cultured with low glucose DMEM (Biowest, France) supplemented
with 10% FBS, 5% penicillin/streptomycin, 10 ng/mL fibroblast growth
factor 2 (FGF-2) (Miltenyi Biotec, Germany), and 3 μM glutamine.
Human iPSCs (CBiPSsv-4F-40 line) were cultured on 1:80 Growth Factor
Reduced-Matrigel (Corning, United States)-coated plastic surfaces
in mTeSR1 complete medium (STEMCELL Technologies, Canada) and passaged
every 4–5 days through cell detaching by incubation with 0.5
mM EDTA (Invitrogen, United States).

Paz-Artigas L., González-Lana S., Polo N., Vicente P., Montero-Calle P., Martínez M.A., Rábago G., Serra M., Prósper F., Mazo M.M., González A., Ochoa I, & Ciriza J. (2024). Generation of Self-Induced Myocardial Ischemia in Large-Sized Cardiac Spheroids without Alteration of Environmental Conditions Recreates Fibrotic Remodeling and Tissue Stiffening Revealed by Constriction Assays. ACS Biomaterials Science & Engineering, 10(2), 987-997.

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Establishment of AVM Primary Cell Lines

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Primary cell lines were derived from three available fresh AVM tissue samples from the original cohort of 13 patients. Fragments of tissue were initially encased in Matrigel (cat# 354234, Corning, Tewkesbury, MA, USA) in 24-well plates with an explant culture media comprised of Dulbecco' Modified Eagle Medium (DMEM) and Glutamax™ (cat# 10569010, Gibco, Rockford, IL, USA) supplemented with 2% penicillin-streptomycin (cat# 15140122, Gibco) and 0.2% gentamicin-amphotericin B (cat# R01510, Gibco). Once sufficient cell growth was achieved to support transfer to a monolayer culture, cells were extracted by dissolving the Matrigel with Dispase (cat#354235, Corning) and transferred to an adherent culture flask with media containing DMEM with Glutamax™ supplemented with 10% fetal bovine serum (cat#10091148, Gibco), 5% mTeSR™1 Complete Medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin, and 0.2% gentamycin-amphotericin in a humidified incubator at 37°C and 5% CO₂. Cells for the AVM-derived primary cell lines were expanded in culture and harvested between passages four and nine for use in Western blotting, RT-qPCR, and enzymatic activity assay analyses.

Hansen L., Brasch H.D., Paterson E., Patel J., Bockett N., Davis P.F, & Tan S.T. (2021). Expression of Cathepsins B, D, and G in Extracranial Arterio-Venous Malformation. Frontiers in Surgery, 8, 676871.

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Primary Cell Lines from mHNcSCC Tissue

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Primary cell lines were generated from four available freshly excised mHNcSCC tissue samples of the original cohort of 20 patients. Samples were cut into small pieces and incubated between layers of Matrigel (cat#354234, Corning Life Sciences, Tewksbury, USA) in 24-well plates with a culture media containing Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax™ (cat#15140122, Gibco, Rockford, IL, USA) supplemented with 2% penicillin-streptomycin (cat#15140122, Gibco) and 0.2% gentamycin/amphotericin B (cat#R01510, Gibco). Once sufficient cell growth was achieved to support transfer to a monolayer culture, cells were extracted by dissolving the Matrigel with Dispase (cat#354235, Corning Life Sciences) and transferred to an adherent culture flask with media consisting of DMEM (1X) (Gibco) with Glutamax™ supplemented with 10% fetal bovine serum (cat#10091148, Gibco), 5% mTeSR™1 Complete Medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin and 0.2% gentamycin-amphotericin (Gibco) in a humidified incubator at 37°C and 5% CO₂. Cells were expanded in culture and harvested between passages 4 and 8.

Humphries F., Chang-McDonald B., Patel J., Bockett N., Paterson E., Davis P.F, & Tan S.T. (2021). Cathepsins B, D, and G Are Expressed in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma. Frontiers in Oncology, 11, 690460.

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Induced Pluripotent Stem Cell Protocol

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Human iPSCs were obtained as reported previously (Cui et al., 2020 (link)), which were reprogrammed by an inactivated Sendai virus from healthy female peripheral blood mononuclear cells, and cultured in mTesR1 complete medium (STEM CELL Technologies, BC, Canada) on diluted Matrigel (1:100, Corning, NY, United States). The cells were subcultured every 4 days by incubation with 0.5 mM EDTA (Thermo Fisher Scientific, United States) at 37°C for 3 min. Human beta cells were cultured in high glucose DMEM supplemented with 10% serum replacement. At 80% confluence, the cells were trypsinized with 0.25% trypsin and 0.01% EDTA (Gibco).

Bai C., Ren Q., Liu H., Li X., Guan W, & Gao Y. (2021). miR-212/132-Enriched Extracellular Vesicles Promote Differentiation of Induced Pluripotent Stem Cells Into Pancreatic Beta Cells. Frontiers in Cell and Developmental Biology, 9, 673231.

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