We used previously established human iPS cell lines reprogrammed from peripheral blood mononuclear cells (PBMCs) isolated from blood samples from SIX6risk allele POAG patients and age‐ and sex‐matched control donors by ectopic expression of KLF4, OCT4, SOX2, and C‐MYC, using a nonintegrating approach.10 Individual colonies of prospective induced pluripotent stem cells (iPSCs) were manually picked and clonally expanded to yield lines of control and patient‐specific iPSCs. Individual colonies were maintained in feeder‐free conditions using mTeSR1 medium (STEMCELL Technologies, Canada, https://www.stemcell.com/ ) in Matrigel‐coated dishes. Cells were passaged at 70%‐80% confluence at a 1:3 to 1:4 split ratio every 5 days using cell dissociation reagent (STEMCELL Technologies). All experiments were carried out using cells from passages 12 to 30. Cells were characterized for expression of pluripotency markers before RGC differentiation.
Cell dissociation reagent
Manufactured by STEMCELL
Sourced in Canada
Cell dissociation reagent is a laboratory product designed to facilitate the separation of cells from their surrounding extracellular matrix or tissue. It is a solution containing enzymes and other components that work to break down the bonds between cells, enabling their isolation for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1 medium, by STEMCELL (1 mentions)
Matrigel, by R&D Systems (1 mentions)
Matrigel, by Corning (1 mentions)
Human epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Human intesticult organoid growth medium, by STEMCELL (1 mentions)
48 well cell culture plate, by Corning (1 mentions)
Y 27632, by STEMCELL (1 mentions)
5 protocols using cell dissociation reagent
1
Induced Pluripotent Stem Cell Lines from POAG Patients
2
Isolation and Culture of Mouse Intestinal Enteroids
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A 20 cm segment of proximal small intestine from both Zip14ΔIEC and Zip14F/F mice were excised and perfused with phosphate-buffered saline (PBS), opened longitudinally, cut into 5 mm lengths and placed in Cell Dissociation Reagent (Stemcell Technologies, Vancouver, BC, Canada). The domes produced, were seeded into Matrigel® (Corning, Corning, NY, USA) and incubated for 20 min at 37 °C, with InstestiCult™ (Stemcell Technologies, Vancouver, Canada) added thereafter. The enteroids were cultured for 10 days. These media were replaced every three days. Enteroids in the Matrigel domes were placed in Organoid Harvesting Solution (Cultrex, R&D Systems, Inc., Minneapolis, MN, USA), washed with PBS, and RNA was isolated using RNeasy® and TRI Reagent® as above.
3
Isolating and Culturing Small Intestinal Organoids
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Small intestinal crypts were isolated from 12-20-week-old mice. Then, cut tissue pieces were incubated in cell dissociation reagent (STEMCELL Technologies, Vancouver, BC, Canada) for 15 min, and isolated crypts, in 50 μl Matrigel, were seeded in each well of a 24-well plate of the following culture medium mixture. The L-WRN cell line (ATCC)-conditioned medium was mixed with an equal volume of basal crypt media (advanced DMEM/F12 (ADF), supplemented with Glutamax (2 mM, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/mL), streptomycin (0.1 mg/mL), N2 supplement (1X, Gibco), B27 supplement (1X, Gibco), human epidermal growth factor (EGF) (50 ng/mL; Peprotech, Rocky Hill, NJ, USA), and NAC (1 mM; Sigma-Aldrich). After ten days, differentiated small intestine organoids were exposed to RIS.
4
Establishing Patient-Derived Colorectal Cancer Organoids
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Biopsies from CRC patients were collected in 5 ml PBS containing penicillin/streptomycin on ice. Following washing and mincing tissues into around 1–2 mm3, samples were digested with 10 ml of cell dissociation reagent (Stem Cell, #07174) on ice on a rocking platform for 30 min. Dissociated cells were passed through 100 μm cell strainer, and then pelleted and suspended in ice-cold PBS. Centrifuge cells for 300 g, 5 min, and then resuspend cells in growth factor reduced (GFR) matrigel (Corning, #356231), and seed cells on 48-well cell culture plate (Corning, #3548). Following solidified in 37 °C and 5% CO2 incubator for 30 min, 300 μl of human IntestiCult™ Organoid Growth Medium (Stem Cell, #06010) which were additionally added 10 μM of Y27632 (Stem Cell, #72304) for the primary culture and were overlaid in the well coated with matrigel. As for the passaging of PDOs, organoids were harvested with ice-cold PBS and pipetted with mechanical force through 1 ml pipette (160 times per well). Dissociated PDOs were pipetted and washed with ice-cold PBS. Resuspend the dissociated cells in GFR matrigel and re-seeded on 48-well flat bottom cell culture plate. PDOs were frozen in FBS containing 10% DMSO for biobanking. The information of the patients from whom the organoids derived is provided in Supplementary Table 1 .
5
Encapsulation of iPSCs in Hydrogel
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