Human blastoids induction and culture was performed as described previously (Kagawa et al., 2021 (link)) with little modification. For the induction of blastoids, naive H9 hPSCs cultured with PXGL medium on MMC-MEF feeders were harvested with Accutase (Biozym). Cells were resuspended in PXGL medium, supplemented with 10 μM Y-27632 (MedChemExpress) and seeded onto gelatin-coated plates and incubated at 37°C for 70 min to deplete feeders. Unattached cells were then collected, centrifuged to pellet, and resuspended in N2B27 medium containing 10 μM Y-27632, after which 30,000 cells were seeded onto a well of a 96 well plate containing 200 μm microwell array. Note that microwell arrays comprising microwells were imprinted into 96-well plates as previously described (Rivron et al., 2012 (link); Vrij et al., 2016 (link)). After 24 h, the aggregation medium was replaced with N2B27 medium supplemented with PD0325901 (1 μM), A 83-01 (1 μM, MedChemExpress, HY-10432), 1-Oleoyl lysophosphatidic acid sodium salt (LPA) (500 nM, Tocris, 3854), human LIF (10 ng/mL), and Y-27632 (Tocris, 10 μM). The medium was refreshed every 24 h. 48 h after blastoid induction the medium was replaced with N2B27 supplemented with LPA (500 nM) and Y-27632 (10 μM). Blastoids were used for the downstream analysis 96 h after induction.
Accutase
Manufactured by Biozym
Accutase is a cell detachment solution used for the dissociation of adherent cells. It is a proteolytic and collagenolytic enzyme mixture that effectively removes cells from cell culture surfaces without affecting cell viability or functionality.
Automatically generated - may contain errors
2 protocols using accutase
1
Human Blastoids Induction and Culture
2
Blastoid Induction from Naive hPSCs
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Naive hPSCs cultured on MEFs in PXGL medium were pre-treated with PRC2 inhibitor (1 μM UNC1999) for 4 d before blastoid induction. Blastoids were induced19 as follows. Naive hPSCs cultured in untreated or pre-treated conditions were harvested using accutase (Biozym). The cells were resuspended in PXGL medium supplemented with 10 µM Y-27632 (MedChemExpress), seeded onto gelatin-coated plates and incubated at 37 °C for 70 min to deplete the MEFs. The unattached cells were collected, pelleted through centrifugation and resuspended in N2B27 medium containing 10 µM Y-27632 with or without PRC2 inhibitor (aggregation medium), after which 30,000 cells were seeded onto an array of 200-µm microwells inserted into a well of a 96-well plate. Note that microwell arrays comprising microwells were imprinted into 96-well plates77 (link),78 (link). After 24 h, the aggregation medium was replaced with N2B27 medium supplemented with 1 μM PD0325901, 1 μM A83-01 (MedChemExpress, HY-10432), 500 nM 1-oleoyl lysophosphatidic acid sodium salt (Tocris, 3854), 10 ng ml−1 hLIF and 10 µM Y-27632, with DMSO for control or 1 μM UNC1999 for pre-treated samples. The medium was refreshed every 24 h.
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