Human embryonic stem cells H9 and mutant cells derived from it were kept in feeder‐free Matrigel plates (Corning) and maintained with PSCeasy medium (Cellapy) daily. Cells were passaged using 0.5 mmol/L calcium and magnesium‐free EDTA solution (HyClone) based on cell status and confluence. Human embryonic stem cells were induced to differentiate into cardiomyocytes by using chemically defined small molecules which can modulate Wnt/β‐catenin pathway.19 , 20 Biochemical differences in glucose and lactate metabolism between cardiomyocytes and non‐cardiomyocytes was exploited for purification of hESC‐CMs.21 , 22 All cells were cultured at 37°C, 5% CO2 humidified incubators.
Psceasy medium
Manufactured by Cellapy
Sourced in China
PSCeasy medium is a cell culture medium specifically designed for the maintenance and expansion of pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). The medium provides the essential nutrients and growth factors required to support the undifferentiated state of PSCs.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
L ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Matrigel, by Corning (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Cellapy (2 mentions)
Six well plate, by Corning (2 mentions)
Iwr 1, by Selleck Chemicals (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Matrigel coated plate, by BD (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Recombinant human albumin, by Merck Group (1 mentions)
Matrigel coated dishes, by Corning (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
11 protocols using psceasy medium
1
Cardiomyocyte Differentiation and Purification from hESCs
2
Cardiomyocyte Differentiation from hiPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hiPSCs were kept in a PSCeasy medium (Cellapy, China) and differentiated when they reached 70–80% confluence. The medium was changed to the basal differentiation medium, consisting of RPMI 1640 medium (Life Technologies, USA), B27 supplement (Life Technologies, USA), and 213-µg/mL l -ascorbic acid 2-phosphate (Sigma, USA). The medium was changed every other day (48 h). From day 0 to day 2, the medium was supplemented with 6-µM CHIR99021 (Selleck Chemicals, USA) and 25-ng/mL Activin A (Cellapy, China). On day 2, the medium was changed to the basal differentiational medium supplemented with 5 µM IWR-1 (Selleck Chemicals, USA). The medium was changed on day 4 and every other day for the basal differentiational medium. Contracting cells were noted from day 9.
3
Expansion and Differentiation of PBMCs and iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs collected by Ficoll-Paque density gradient centrifugation were expanded for 6–10 days in serum-free erythroid culture medium (ECM) as described previously (28 (link)). Human iPSCs were cultured on Matrigel-coated plates (BD Biosciences, San Diego, CA, USA) in PSCeasy medium (Cellapy), which was changed daily. The iPSCs were passaged with EDTA (5 × 10−4 M) when reaching 60% confluence. Human embryonic kidney 239T (HEK293T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 atmosphere at 37°C.
4
Generating iPSCs from PBMCs using Episomal Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nucleofection and generation of iPSCs were performed with peripheral blood mononuclear cells (PBMCs) as previously described (28 (link)). Briefly, pEV SFFV-OCT4-E2A-SOX2 (OS), pEV SFFV-MYC-E2A-KLF4 (MK) and pEV SFFV-BCL-XL (Bcl-XL) episomal vectors were mixed and transferred to the cell pellet (1 × 106 cells). After nucleofection, the cells were transferred to a culture plate pre-seeded with feeder cells. The cells were cultured in reprogramming medium composed of knockout DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% KnockOut™ Serum Replacement (Invitrogen), 1% l -glutamine (Invitrogen), 2 mM nonessential amino acids (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 1% 2-mercaptoethanol (Invitrogen), and 20 ng/ml FGF2 (PeproTech, Cranbury, NJ, USA) for 7 days. The cells were then cultured in PSCeasy medium (Cellapy, Beijing, China) until iPSCs were generated. The iPSCs were characterized by karyotyping, analysis of the expression of embryonic stem cell markers, teratoma formation, and SARS2 sequencing.
5
Differentiation of hESCs into Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H1 human embryonic stem cells (WiCell Research Institute, USA) were cultured in PSCeasy medium (Cellapy, China) in Matrigel-coated dishes (Corning, USA) and passaged with 0.5 mM EDTA at 70–80% confluence. At ~ 90% confluence, H1 cells were moved into CDM3 medium with different supplements to induce directional differentiation. CDM3 medium consists of RPMI 1640 medium (Thermo Fisher, USA), recombinant human albumin (Sigma-Aldrich, USA) and L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA). During days 0–2, the medium was supplemented with 6 μM CHIR99021 (Sigma-Aldrich, USA). On day 2, 50 ng/mL bFGF (Novoprotein, USA) was added. After that, 50 ng/mL VEGF (Novoprotein, USA), 25 ng/mL BMP4 (PeproTech, USA) and 25 ng/mL bFGF (Novoprotein, USA) were used for the next 3 days. The medium was changed daily. On day 6, CD34+ cells were enriched using anti-CD34 antibody-conjugated magnetic beads (Miltenyi, USA) according to the manufacturer’s instructions. The sorted cells were defined as human embryonic stem cell-derived endothelial cells (hESC-ECs).
6
Cardiomyocyte Differentiation and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Matrigel (Corning Life Science, Tewksbury, MA, USA) coated 6-well plates were seeded with hiPSCs at a density of 1×105 cells / well and cultured in PSCeasy medium (Cellapy, Beijing, China) at 37°C and 5% CO2. hiPSCs were differentiated into cardiomyocytes using the small molecule-based method as previously described [51 (link)]. Myocardial purification is performed by the lactate metabolism-selection method at day 10 post myocardial differentiation. And the cells were reseeded on new Matrigel-coated culture plates. Here, the first day of myocardial differentiation was defined as Day 1.
7
Culturing Human Pluripotent Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human PSCs including hESCs-H9 (H9, provided by WiCell Institute Inc., Madison, WI, USA) and urine-derived iPSCs (UiPSCs, provided by Cellapy: CA1002008, Beijing, China) were cultured in PSCeasy medium (Cellapy, China) on six-well plates (Corning, US) coated with a 1:500 dilution of hESC-matrigel (5 μg/cm2, Corning). Medium was changed every day. PSCs were passaged every 3–4 days at 70–80% confluence with 0.5 mM EDTA (Cellapy). All cells were maintained at 37 °C, 5% CO2 in incubator (Thermo Fisher Scientific, USA).
8
Directed Differentiation of hESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hESC line was purchased from Cellapy (Beijing, China) was routinely maintained in the presence of PSCeasy medium (Cellapy, China) on six-well plates (Corning, USA) coated with 5% Matrigel (Corning, USA). Medium was changed every day and passaged every 2–3 days with EDTA (Cellapy, China). The cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The hESCs were differentiated when they reached 70–80% confluence. Medium was changed to the basal differentiation medium. For day 0 to day 2, medium was changed to the basal medium C01 (Cellapy, China). For day 2 to day 4, medium was changed to the basal differentiational medium C02 (Cellapy, China). On day 4, medium was changed to the basal differentiational medium C03 (Cellapy, China) and changed medium every other day. Contracting cells were noted from day 9.
9
Culturing Primary Murine Fibroblasts and Human iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary murine embryonic fibroblasts (MEFs) with p53 knockout were obtained from 13.5-day CD-1 IGS mouse embryos. HEK293T and MEF cells were cultured in standard DMEM containing 10% FBS (HyClone, Logan) and passaged routinely with trypsin-EDTA solution. Human iPSCs were maintained in a feeder-free culture system. Briefly, the wells of plates were precoated with Matrigel (BD Biosciences), and then we seeded the iPSCs and cultured them in PSCeasy medium (Cellapy).
10
Cell Line Maintenance and Authentication
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HEK-293T human embryonic kidney cell line and Vero E6 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The HEK-293F-ACE2 cell line was purchased from Novoprotein Scientific Inc. (Shanghai, China). The human induced pluripotent stem cells (hiPSC) were maintained in our lab, which has been registered at hPSCreg (https://hpscreg.eu/ , hPSCreg Name: IBTCMi002-A). HEK-293T, HEK-293F-ACE2 and Vero E6 cell lines were maintained in Dulbecco's modified essential medium (DMEM) with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco, Carlsbad, CA, USA). The HEK-293F-ACE2 cells were also supplemented with 25 µg/ml Geneticin (G418, Gibco) for stable expression of ACE2. The hiPSC cells were kept in PSCeasy medium (Cellapy, Beijing, China). All cells were grown in a humidified incubator at 37°C with 5% CO2. HEK-293T and Vero E6 cells between 18-30 passages, HEK-293F-ACE2 at a passage number between 12-25, and the hiPSC between 35-42 passages were used. The hiPSC-derived AECII at a passage number lower than 5 were applied.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!