All differentiated tissues used for the organ chip coculture were derived from the same hiPSC line HUMIMIC101 (TissUse GmbH, Berlin, Germany). HUMIMIC101 cells were generated by TissUse GmbH with donor consent for commercial use to study the influence of age and gender on the reprogramming into iPSCs and differentiation into specific cell types [13 (link)]. The cells were maintained on growth factor reduced (GFR) Matrigel coated 6-well plates (Corning, Corning, NY, USA) in StemMACS iPS-Brew XF (Miltenyi Biotec, Bergisch Gladbach, Germany). During the normal maintenance culture, the cells were seeded at densities in the range of 2000–4000 cells/cm² in StemMACS iPS-Brew XF with the addition of 10 µM of ROCK-inhibitor Y-27632 (Cayman Chemicals, Ann Arbor, MI, USA). After 48 h, the medium was removed and replaced by fresh medium without the ROCK-inhibitor. From there on, the media were exchanged every day until the passaging of the cells seven days after seeding when the confluence reached approximately 90%. For passaging, hiPSCs were washed once with PBS without calcium and magnesium and incubated with a sufficient volume of Accutase (Corning, Corning, NY, USA) for five to seven minutes.
Rock inhibitor y27632
Manufactured by Cayman Chemical
Sourced in United States
ROCK inhibitor Y27632 is a small molecule that selectively inhibits the Rho-associated coiled-coil containing protein kinase (ROCK) enzymes. ROCK inhibitors are used in research applications to study the role of the ROCK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Chelex 100, by Merck Group (2 mentions)
Dmem powder, by US Biological (2 mentions)
Matrigel coated 6 well plate, by Corning (1 mentions)
Stemmacs ips brew xf, by Miltenyi Biotec (1 mentions)
Accutase, by Corning (1 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Amg337, by Selleck Chemicals (1 mentions)
Crizotinib, by Selleck Chemicals (1 mentions)
Rab7 d95f2, by Cell Signaling Technology (1 mentions)
Pi3k inhibitor ly294002, by Abcam (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Cayman Chemical (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Mtesr media, by STEMCELL (1 mentions)
Accumax, by Merck Group (1 mentions)
X vivo 10 media, by Lonza (1 mentions)
Vitronectin xf, by STEMCELL (1 mentions)
14 protocols using rock inhibitor y27632
1
Expanding hiPSCs for Organ-on-a-Chip
2
Signaling Pathway Protein Detection
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The primary antibodies used in this study were Myc-Tag (9B11), CDCP1, Met (D1C2), phospho-Met (Tyr1234/1235) (D26), phospho-tyrosine (P-Tyr-1000), phospho-Src (Tyr416), phospho-Akt (Ser473), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Vav2 (C64H2), and Rab7 (D95F2) obtained from Cell Signaling Technology. Anti-GAPDH antibody was purchased from Santa Cruz Biotechnology. Anti-EEA1 and Anti-Rac1 antibodies were purchased from BD Biosciences. Anti-M6PR antibodies were purchased from Thermo Fisher Scientific. Anti-ARHGEF7 antibody was prepared by immunizing rabbits with C-terminal fragments. Dynamin inhibitor Dynasore, PI3K inhibitor LY294002, and SFK inhibitor Dasatinib were purchased from Abcam. ROCK inhibitor Y27632 was purchased from Cayman chemical. RAC1 inhibitor was purchased from Merk. MET inhibitors, AMG337 and Crizotinib, were purchased from Selleck Chemicals.
3
Culturing Intestinal Spheroids with L-WRN Conditioned Medium
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A conditioned medium was produced using a mouse fibroblast cell line that was genetically modified to express and secrete Wnt3a, R-spondin 3 and Noggin (abbreviated L-WRN29 (link); ATCC CRL-3276). Conditioned media was produced as described previously29 (link). The proliferation medium was prepared by diluting the conditioned medium 1:1 with basal medium that contained Advanced DMEM/F12 (Gibco), 20% foetal bovine serum (FBS) (v/v) and 2 mM GlutaMAX Supplement (Gibco). The proliferation medium contained a final concentration of 20% FBS. Spheroids of the small intestine were cultured and expanded in conditioned medium supplemented with 10 μM of ROCK inhibitor (Y-27632; Cayman Chemicals) and 10 μM of TGF-β type I receptor inhibitor (SB-431542; Cayman Chemicals).
4
Maintenance of Human mc-iPS Cells
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Human mc-iPS cells21 were obtained from System Biosciences (SC301A-1) and were maintained on Matrigel (BD Bioscience) coated plates in mTeSR media (Stem Cell Technologies) with 50 units/ml penicillin-streptomycin (Thermo Fisher Scientific) and with daily medium changes22 (link). For passaging, the cells were washed with PBS and treated with Accutase (Thermo Fisher Scientific) at 37 °C°C for 5 min. The cells were re-suspended in mTeSR media, centrifuged at 80 g for 5 min and cell pellets re-plated in mTeSR media supplemented with 10 μM ROCK Inhibitor Y-27632 (Cayman Chemical).
5
Expansion and Passaging of hiPSC-Derived RPE Cells
6
Cytoskeleton Modulation in Cellular Injury
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Inhibitors were added in fresh growth medium 30 min prior to injury. DMSO (Fisher Scientific #BP231) was added at 0.2% (v/v) as a vehicle control. Blebbistatin (Cayman Chemical #24171) was used at 100 μM. Myosin light chain kinase inhibitory peptide 18 (P18, Cayman Chemical #19181) was used at 10 μM. ROCK inhibitor Y27632 (Cayman Chemical #10005583) was used at 10 μM. Nocodazole (Cell Signaling #2190s) was used at 100 nM. Dynasore (Sigma #D7693) was used at 60 μM. Latrunculin A (Sigma #428026) was used at 1 μM.
7
Focal Adhesion Protein Analysis
8
Maintenance of human induced pluripotent stem cells
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We used 3 previously published hiPSC cell lines in this study; PLZ (Darabi et al., 2012 (link)), MYL2-mEGFP (Roberts et al., 2019 (link)), and DF 19-9-11T (Wheelwright et al., 2018 (link)). Cells were maintained in mTeSR™-1 medium (STEMCELL Technologies) on growth factor-reduced (GFR) Matrigel (∼8 μg/cm2, Corning) at 5% CO2 and 37°C under normoxia conditions. Cells were singularized with Accutase (Innovative Cell Technologies, Inc.) for 3–5 min and passaged every 4 days at a density of 1.25 × 104/cm2 with the addition of 10 μM ROCK inhibitor (Y-27632, Cayman Chemical) for the first 24 h after each passage. Cell lines were routinely tested for mycoplasma contamination every 3 months (Applied Biological Materials, Inc.) following the manufacturer’s recommendation.
9
HaCaT Keratinocyte Differentiation Induction
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The HaCaT line of immortalized keratinocytes was provided by Yu-Ying He (University of Chicago) and was originally developed by the laboratory of Norbert Fusenig (Schoop et al., 1999 (link)). HaCaTs were maintained in low calcium DMEM prepared from calcium-free DMEM powder (#09800; US Biological), spiked with 40 μM CaCl2, penicillin-streptomycin, L-glutamine, and 10% fetal bovine serum depleted of calcium by Chelex-100 (Sigma-Aldrich) treatment. For calcium-induced differentiation experiments, calcium concentration was raised to 2.8 mM for the indicated time span. ROCK-inhibitor (Y-27632; Cayman Chemical) was used at 10 μM.
10
Hematopoietic Differentiation of hPSCs
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hPSCs were maintained and passaged on Matrigel in mTeSR1 media (WiCell). Hematopoietic differentiation was performed on collagen IV (ColIV)-coated plates in chemically defined serum-free medium as described.14 (link) The iSOX18 H1 line from hPSCs (H1 hESC line from WiCell) was maintained and passaged on Matrigel in mTeSR1 media (WiCell). The cell lines were differentiated on a collagen IV (ColIV)-coated plate.14 (link) To initiate differentiation, cells were plated at 5,000 cells/cm2 onto 6 well plates with E8 media and 10 μM Rock inhibitor (Y-27632, Cayman Chemicals). This media was changed the following day to IF9S media with 50 ng/mL FGF2 (PeproTech), 50 ng/mL BMP4 (PeproTech), 15 ng/mL Activin A (PeproTech), and 2 mM LiCl (Sigma), and cells were cultured in hypoxia (5% CO2, 5% O2). On day 2, the media was changed to IF9S media with 50 ng/mL FGF2, 50 ng/mL VEGF (PeproTech), and 2.5 μM TGF-β inhibitor (SB-431542, Cayman), and cells were cultured in hypoxia (5% CO2, 5% O2). On days 4 and 6, the media was changed to IF9S media with 50 ng/mL FGF2, 50 ng/mL VEGF, 50 ng/mL TPO (PeproTech), 50 ng/mL IL-6 (PeproTech), 20 ng/mL SCF (PeproTech), and 10 ng/mL IL-3 (PeproTech), and cells were cultured in normoxia (20% CO2, 5% O2).
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