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Hesc qualified matrigel

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HESC-qualified Matrigel is a soluble basement membrane extract of proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is used as a substrate for the in vitro culture of human embryonic stem cells (hESCs) and other cell types.

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Lab products found in correlation

Mtesr1 medium, by STEMCELL (10 mentions) Relesr, by STEMCELL (9 mentions) Mtesr1, by STEMCELL (9 mentions) Glutamax, by Thermo Fisher Scientific (8 mentions) Mtesr plus medium, by STEMCELL (5 mentions) Mtesr1 media, by STEMCELL (4 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Rpmi 1640 media, by Thermo Fisher Scientific (4 mentions) Gsk3 inhibitor chir99021, by Bio-Techne (3 mentions) Wnt inhibitor iwr1, by Bio-Techne (3 mentions) Stemdiff cardiomyocyte differentiation kit, by STEMCELL (3 mentions) Stempro accutase, by Thermo Fisher Scientific (3 mentions) Gentle cell dissociation reagent (gcdr), by STEMCELL (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Growth factor reduced matrigel, by Corning (2 mentions) Atcc hys0103, by American Type Culture Collection (2 mentions) Rh vitronectin, by Thermo Fisher Scientific (2 mentions) Cryostor cs10, by STEMCELL (2 mentions) Rock inhibitor y27632, by American Type Culture Collection (2 mentions)

57 protocols using hesc qualified matrigel

1

Matrigel Coating for hESC Culture

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Matrigel hESC-qualified (Corning, Glendale, AZ, USA) was diluted to a concentration of 0.1 mg/mL in cold Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; GIBCO), which was then applied to TCP dishes. This coating was allowed to polymerize at room temperature during the 2 h incubation. Excess Matrigel-DMEM/F12 solution was aspirated, and the dishes were washed with cold sterile D-PBS followed by seeding of the cells.
Timilsina S., McCandliss K.F., Trivedi E, & Villa-Diaz L.G. (2023). Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions. Bioengineering, 10(9), 999.
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2

Culturing and Characterizing Human Embryonic Stem Cells

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H9WA09 cells were obtained from the European Institute for the Biology of Ageing (ERIBA) at the University of Groningen. H9WA09 cells were cultured on Matrigel hESC-qualified (Corning, 354277) pre-coated 6-well plates in mTeSR1 medium (STEMCELL technologies, 85850). The cells were incubated at 5% CO2 and 37°C. Once the cells grew confluent, the H9WA09 cells were passaged using ReLeSR (STEMCELL technologies, 05872). The pluripotency of the H9WA09 cells was tested regularly by staining for the pluripotency-marker OCT4. In addition, the cells were examined regularly for the presence of mycoplasma.
Goldsteen P.A., Sabogal Guaqueta A.M., Mulder P.P., Bos I.S., Eggens M., Van der Koog L., Soeiro J.T., Halayko A.J., Mathwig K., Kistemaker L.E., Verpoorte E.M., Dolga A.M, & Gosens R. (2022). Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies. Frontiers in Pharmacology, 13, 991072.
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3

Efficient hiPSC Transfection via Lipofectamine

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hiPSCs were cultured in a PGM1 medium (Cellapy, CAT# CA3001500) with 0.5% Plasmocin prophylactic on matrix-coated (Corning Matrigel hESC-Qualified) plate and maintained at 37 °C in humidified air with 5% CO2. Cells were passaged with Cellapy Cell Dissociation Reagent (Stem cell, CAT# 07174) every 3–4 days and plated at a density of 2 × 104 cells/cm2 with a split ratio of around 1:6. During cell generation, 10 µM Y-27632 (STEMCELL Technologies, CAT# 72,302) was added.
Cells were transfected with a complex formed with the Cas9 plasmid (VB190801_1165nbd) and two sgRNA plasmids (VB190801-1166xkk and VB190801-1168fdu), constructed by VectorBuilder, using Lipofectamine Stem transfection reagent (Invitrogen, CAT #STEM00001).
The detailed steps were as follows:
StepsTubeComponentPer well of a 6-well plate
11Opti-MEM™ I Medium100 µl
Lipofectamine™ Stem Reagent4 µl
22Opti-MEM™ I Medium100 µl
DNA2 µg*
3Add diluted DNA to diluted Lipofectamine Stem Reagent
4Incubate for 10 min at room temperature
5Add DNA-lipid complex to cells (200 µl/per well)
6Incubate and monitor the transfected stem cells at 37 °C for 2 days

*Equimolar amounts of Cas9 plasmid DNA and gRNA plasmid DNA were added

Chen X., Peng M., Cai Y., Zhou C, & Liu L. (2022). Human iPSC-derived neural stem cells with ALDH5A1 mutation as a model of succinic semialdehyde dehydrogenase deficiency. BMC Neuroscience, 23, 77.
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4

Culturing and Maintenance of H9WA09 Stem Cells

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H9WA09 cells were obtained from the European Institute for the Biology of Ageing (ERIBA) at the University of Groningen. H9WA09 cells were cultured on Matrigel hESC-qualified (Corning, 354277) pre-coated 6-well plates (6WP) in mTeSR1 medium (STEMCELL technologies, 85850). The cells were incubated at 5% CO2 and 37C. Once the cells grew confluent, the H9WA09 cells were passaged using ReLeSR (STEMCELL technologies, 05872). The pluripotency of the H9WA09 cells was tested regularly by staining for the pluripotency-marker OCT4. In addition, the cells were examined regularly for the presence of mycoplasma.
Goldsteen P.A., Sabogal-Guáqueta A.M., Bos I.S.T., Kistemaker L., Koog L.v.d., Eggens M., Halayko A.J., Dolga A.M., & Gosens R. (2022). Differentiation of airway cholinergic neurons from human pluripotent stem cells for airway neurobiology studies.
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5

Expansion of WA-09 Stem Cell Line

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The WA-09 embryonic stem cell lineage [15 (link)] was acquired directly from the Wicell Research Institute (WiCell, Madison, WI, USA) and thawed and cultured under current Good Manufacturing Practices (cGMP) conditions at the University of São Paulo (USP) Cell and Molecular Therapy Center (NUCEL) [16 (link)]. Pluripotent cell colonies were plated onto hESC-qualified Matrigel (Corning, NY, USA), maintained in mTeSR-1 medium (Stem Cell Technologies, Vancouver BC, Canada) and manually expanded every 4 to 5 days.
Lojudice F.H., Fernandes R.A., Innocenti F., Franciozi C.E., Cristovam P., Maia M., Sogayar M.C, & Belfort R J.u.n.i.o.r. (2019). In vitro differentiation of cGMP-grade retinal pigmented epithelium from human embryonic stem cells. International Journal of Retina and Vitreous, 5, 45.
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6

Differentiation of hiPSCs into Telencephalic Neural Progenitors

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Previously characterized healthy control hiPSC lines 20b and 18a43 (link) were maintained in mTEsR medium (StemCell Technolgies) on hESC-qualified matrigel (Corning)-coated tissue culture plates and passaged using Dispase (1 mg/ml, Life Technologies). Cell lines were mycoplasma-negative by PCR (LookOut Mycoplasma PCR Detection Kit, Sigma). HiPSCs were differentiated into dorsal telencephalic neural progenitors using a previously published protocol44 (link) without inducing sonic hedgehog signalling (no SHH, see Extended Data Fig. 5). After 18 days, cultures were enzymatically dissociated to single cells using Accutase (StemPro Accutase, Life Technologies) and were replated on Growth Factor Reduced matrigel (1:30, Corning) at 10,000 cells/cm2 in human neurobasal (hNB) medium supplemented with 10 μM ROCK inhibitor (Y27632, Tocris). hNB media was replaced 24 h later and supplemented every 2–3 days thereafter. Dissociation and replating (100,000 cells/cm2) was repeated at day 40. Cultures were silenced on day 81 and then KCl-depolarized (see above) and harvested on day 82. hNB medium: neurobasal medium (no glutamine), with 1× penicillin/streptomycin, 1× GlutaMax, 1× MEM non-essential amino acids, 1× B27-supplement without vitamin A (all Life Technologies), 1× N2-B supplement (StemCell Technologies), 1 μM ascorbic acid (Sigma), 20 ng/ml rhBDNF and 10 ng/ml rhGDNF (Peprotech).
Ataman B., Boulting G.L., Harmin D.A., Yang M.G., Baker-Salisbury M., Yap E.L., Malik A.N., Mei K., Rubin A.A., Spiegel I., Durresi E., Sharma N., Hu L.S., Pletikos M., Griffith E.C., Partlow J.N., Stevens C.R., Adli M., Chahrour M., Sestan N., Walsh C.A., Berezovskii V.K., Livingstone M.S, & Greenberg M.E. (2016). Evolution of Osteocrin as an activity-regulated factor in the primate brain. Nature, 539(7628), 242-247.
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7

Naïve State Induction of hESCs

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Primed hESCs were routinely cultured with TeSR-E8 (StemCell Technologies) on hESC Qualified Matrigel (Corning). hESCs were reverted to naive state using a one-step induction adapted from published protocols (Guo et al., 2017 (link)). Further details are provided in Supplemental Experimental Procedures.
For consistency, all stem cells used in RNA-seq were cultured in 5% O2.
Cinkornpumin J.K., Kwon S.Y., Guo Y., Hossain I., Sirois J., Russett C.S., Tseng H.W., Okae H., Arima T., Duchaine T.F., Liu W, & Pastor W.A. (2020). Naive Human Embryonic Stem Cells Can Give Rise to Cells with a Trophoblast-like Transcriptome and Methylome. Stem Cell Reports, 15(1), 198-213.
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8

Culturing hiPSC and hESC lines

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The established hiPSC lines and WA16 hESCs were cultured on hESC-qualified Matrigel (Corning) coated six-well-plates in E8 medium and passaged with Versene in E8 medium supplemented with RevitaCell™ (Thermo Fisher Scientific). The hiPSC lines were incubated at 37°C in the presence of 5% CO2 and 5% O2.
Astro V., Fiacco E., Cardona-Londoño K.J., De Toma I., Alzahrani H.S., Alama J., Kokandi A., Hamoda T.A., Felemban M, & Adamo A. (2023). A transcriptomic signature of X chromosome overdosage in Saudi Klinefelter syndrome induced pluripotent stem cells. Endocrine Connections, 12(5), e220515.
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9

Differentiation of FMRP-WT and FMRP-KO hiPSCs into Cortical Neurons

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FMRP-WT and FMRP-KO hiPSC lines were cultured in Nutristem-XF (Biological Industries) supplemented with 0.1% Penicillin-Streptomycin (Thermo Fisher Scientific) onto hESC-qualified Matrigel (CORNING) functionalized plates. The culture medium was refreshed every day and cells were passaged every 4-5 days using 1 mg/mL Dispase II (Thermo Fisher Scientific).
hiPSCs were differentiated into cortical neurons according to a previously published protocol [37] with minor modifications. Briefly, hiPSCs were treated with Accutase (Thermo Fisher Scientific
Brighi C., Salaris F., Soloperto A., Cordella F., Ghirga S., Turris V.d., Rosito M., Porceddu P.F., Reggiani A., Rosa A., & Angelantonio S.D. (2020). Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs.
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10

Feeder-free Expansion of Human iPSCs

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Hepatic fibroblast derived iPS line ATCC-HYS0103 Human Induced Pluripotent Stem (IPS) Cells were purchased directly from the vendor. hESC-Qualified Matrigel (Corning 354277) Matrigel was used coat culture dishes for feeder-free expansion of induced pluripotent stem cells and were routinely cultured and maintained in mTeSR™1 (STEMCELL Technologies, 85850) in a humidified incubator at 37 °C with 5% CO2. Cells were passaged once a week using selective dissociation reagent ReLeSR™ (STEMCELL Technologies 05872) and seeded using the cell aggregate counting method described in 'Plating Human ES and iPS Cells Using the Cell Aggregate Count Method' (Appendix 1) from the STEMCELL Technologies-'Maintenance of Human Pluripotent Stem Cells in mTeSR™1' technical handbook to assess the size of aggregates and seed them in low, medium or high densities, as described. All cryopreservation of cells was performed in CryoStor® CS10 (STEMCELL Technologies 07930) freezing media.
For the glass controls in the experiments -sterile rh-Vitronectin (Gibco, Life Technologies, A14700) was used to coat glass cover slips. In case of all experiments, cells were dissociated into a single cell suspension using StemPro™ Accutase™ (Gibco A1110501) cell dissociation reagent to facilitate cell counting. In case of single cell dissociation, cells were always seeded with 10uM Rock inhibitor Y27632 (ATCC® ACS-3030™).
Srivastava P., Romanazzo S., Ireland J., Nemec S., Molley T.G., Jayathilaka P.B., Pandžić E., Yeola A., Chandrakanthan V., Pimanda J.E., & Kilian K. (2021). Defined microenvironments trigger in vitro gastrulation in human pluripotent stem cells.
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