Gentle cell dissociation buffer

Human iPS cells were differentiated into CMs using the established matrix sandwich method^{35 (link)} with modifications. Briefly, 6 days before initiating differentiation, hiPS cell colonies were passaged on human embryonic stem cell–qualified Matrigel-coated plates (0.05 mg/mL; BD Biosciences) using Gentle Cell Dissociation Buffer (Stemcell Technologies) and cultured as a monolayer in mTeSR1 with 1× Y-27632 ROCK inhibitor (Stemcell Technologies) in a normal oxygen atmosphere. When cells reached 80% confluence, cold mTeSR1 with Growth Factor Reduced Matrigel (0.033 mg/mL) was added to create an overlay of Matrigel. Differentiation was initiated 24 hours later (day 0) by culturing the cells in RPMI-1640 medium (Life Technologies) supplemented with B27 (without insulin; Life Technologies), 100 ng/mL activin A (Miltenyi Biotec), and 10 ng/mL FGF2 (Miltenyi Biotec) for 24 hours. On the next day, the medium was replaced by RPMI-1640 medium supplemented with B27 without insulin, 10 ng/mL BMP4 (Miltenyi Biotec), and 5 ng/mL FGF2 for 4 days. By day 5, cells were cultured in RPMI-1640 medium supplemented with B27 complete (Life Technologies) and 1% NEAA and changed every 2 to 3 days.

Mesenchymal cells were generated as previously described26 (link). FH-iPSC±LDLR were passaged onto Matrigel-coated plates using Gentle Cell Dissociation Buffer (Stem Cell Technologies) and introduced to 100% mTeSR1 in a graded manner. One day after passaging, transfected cultures received 1 μg/ml Hygromycin B to maintain selective pressure. Once confluent and in 100% mTeSR1, the media was changed to EGM2-MV (Lonza) for 20 days for differentiation into FH-MC. Once fully differentiated, FH-MC were passaged and used in LDL internalization assays.

Colonic organoids were derived as previously described from Muc2^+/+ and Muc2^-/- littermates (23 (link), 24 (link)). Briefly, the colons were excised, and crypts digested using a gentle cell dissociation buffer (StemCell Technologies) and approximately 100 crypts were embedded into GFR-matrigel (BD) domes. Organoids were fed every two days with advanced DMEM supplemented with L-WRN (ATCC CRL-3276) as well as N2, B27, GlutaMax, SB202190, Nicotinamide, N-acetylcysteine, A83-01, and mEGF. Colonoids were passaged once every 7-10 days with TrypeLE. For experiments, colonoids were grown for 7 days in complete media and then fed with L-WRN-conditioned media containing only N2, B27, Nicotinamide, and Glutamax for 3 days. Five µM DAPT was administered to the experimental media 24 hours prior to harvesting for experiments using colonoids skewed to a goblet cell phenotype.