International Journal of Stem Cells : eISSN 2005-5447

Table. 1.

Table. 1.

Troubleshooting table

Problem Possible reason Solution
Poor attachment Old Y27632 Use fresh Y27632 and do not withdraw within 24 h.
Cells do not recover or grow slowly Unhealthy cells at cryopreservation Thaw a healthy one
Insufficient habituation of hPSCs on Laminin Gradually reduce the concentration of Laminin 521 at an interval of 2 passages until reaching the target concentration of 3 μg/ml
Poor endoderm/mesoderm commitment Variability between lines Redefine the most efficient concentration of BMP4 (10∼20 ng/ml) and mTeSR1 (5∼10%) in EMM1/2
Poor quality of hPSC Recheck the pluripotent markers and karyotype of hPSC
High passage cultures Initiate the differentiation within 10 passages after cell thawing
Inappropriate starting confluence Strictly control the plating density and starting confluence
Insufficient pre-stimulation by pre-EMM Prolong pre-stimulation time (4∼10 h)
Old mTeSR1 mTeSR1 in EMM1/2 should be freshly prepared
Poor B27 batch Screen B27 batches for their capacity to support efficient differentiation
Low proportion of AFP SOX cells by day 15 Suboptimal performance of previous steps Check and optimize the efficiency of differentiation during day 0∼4 as described above
Variability between lines Optimization may be required for specific lines. Advanced DMEM/F12 (1% B27) can replace RPMI (2% B27) for selected lines
Reduced activity of cytokines Use cytokines that have undergone <3 freeze–thaw cycles
Poor B27 batch Change the B27 batch as described above
Poor efficiency of hepatobiliary differentiation Suboptimal performance of previous steps Check and optimize the efficiency of previous differentiation steps
Variability between lines Redefine the optimal concentration of cholesterol-MIX (5∼10%) in HBMM.
Poor functional performance Variability between lines Reduce the concentration of cholesterol-MIX as described above. Besides, Hepatozyme-SFM (Gibco) may replace HCM in HBMM for selected lines
Delayed differentiation Insufficient habituation of hPSCs on rhLaminin Gradually reduce the concentration of Laminin 521 at an interval of 2 passages until reaching the target concentration of 3 μg/ml.
Low ratio of rhLaminin 521 Ensure the ratio of Laminin 521/111 is not lower than 1:3 since Laminin 111 is not ideal for cell expansion before day 9. Alternatively, redefine the ratio as 1:1 for selected lines.
Exhausted cytokines/other nutrients Ensure the medium refreshment for every 24 h.
Unexpected cell death Inappropriate composition of culture medium Prepare new medium and check the concentration of cytokines. Besides, test the reagents for lot-to-lot variability
Incorrect parameter-setting of cell incubator Check and calibrate CO2 and humidity levels in the incubator
Contamination of the culture Try the differentiation again by sterile operation
Low concentration of Laminin The working concentration of Laminin 521/111 should not be lower than 3 μg/ml
International Journal of Stem Cells 2021;14:119-26 https://doi.org/10.15283/ijsc20152
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