scRNA-seq of bovine in vitro meat from bovine ES cells
scRNA-seq of bovine in vitro meat from bovine ES cells
Induction media  From day 0 to day 2: PSM medium The PSM medium is CDMi containing 10 μM CHIR99021 (Sigma, SML1046), 20 ng/ml bFGF, 10 μM SB431542 (Sigma, S4317), and 2 μM DMH1 (Sigma, D8946) as previously described28,33,66.  From day 2 to day 6: Muscle medium_1 The basal medium is DMEM/F12 (Gibco, 11320033) containing 15% Knockout Serum Replacement (Gibco, 10828028), 0.1 mM nonessential amino acids (Gibco, 11140-035), 50 mM 2-mercaptoethanol (Gibco, 31350-010), and 0.5x penicillin/streptomycin (Gibco, 15140-122).  The muscle medium_1 is the basal medium supplemented with 10 ng/ml HGF (R&D, 2207-HG-025/CF), 2 ng/ml IGF (R&D, 791-MG-050), and 20 ng/ml bFGF.  From day 2 to day 6: Muscle/Endothelial Cells (EC) medium_1 The muscle/EC medium_1 is the basal medium supplemented with 10 ng/ml HGF, 2 ng/ml IGF, 20 ng/ml bFGF, 2 µM forskolin (R&D, 2207-HG-025/CF), and 200 ng/ml VEGF165 (R&D, 2207-HG-025/CF).    From day 6 to day 15: Muscle medium_2 The muscle medium_2 is the basal medium supplemented with 10 ng/ml HGF and 2 ng/ml IGF.  From day 6 to day 15: Muscle/EC medium_2 The muscle/EC medium_2 is the basal medium supplemented with 10 ng/ml HGF, 2 ng/ml IGF, and 100 ng/ml VEGF165.  Induction protocols  Bovine muscle induction protocol in 2D: One day before the induction protocol started, bovine ESCs were seeded on a Matrigel-coated dish or well plate in the maintenance medium without IWR1 and with 5 μM Y27632. The maintenance medium was changed to the PSM medium the next day (defined as day 0). The cells were incubated in the PSM medium for 2 days, and the medium was changed every day. On day 2, the PSM medium was switched to the muscle medium_1, and the cells were incubated for 4 days with daily medium changes. On day 6, the muscle medium_1 was switched to the muscle medium_2, and the cells were incubated until day 15 with medium changes every other day.  Bovine muscle and endothelial cell co-induction protocol in 2D: Bovine PSM cells were induced as described above. During days 2-6, the cells were incubated in the muscle/EC medium_1 with daily medium changes. During days 6-15, the cells were incubated in the muscle/EC medium_2 with medium changes every other day.   Bovine muscle induction protocol in 3D: Bovine PSM cells were induced as described above. On day 2, PSM cells were trypsinized into single cells using TrypLE solution at 37 °C for 3 min. The cells were mechanically dissociated by pipetting and transferred into 1 ml of the muscle medium_1 containing 5 µM Y27632. The cell suspension was centrifuged at 258 xg for 3 min and resuspended into 1 ml of the muscle medium_1 containing Y27632. After one more wash with the muscle medium_1 containing Y27632, the supernatant was completely removed. The cell pellet was resuspended into 600-800 µl of the muscle medium_1 containing Y27632. The cell concentration was adjusted to 133.3 cells/µl with a sufficient amount of the pre-warmed muscle medium_1 containing Y27632. Then 150 µl of the cell suspension (= 20,000 cells) was aliquoted into each well of a U-Shaped-Bottom, 96-well-plate (96-well Clear Round Bottom Ultra-Low Attachment Microplate, Corning, 7007) by using a multichannel pipette. The 96-well plate was centrifuged at 258 xg for 2 min for the cells to settle down on the bottom of the plate. On day 3, 100 µl of the medium was carefully removed and replaced with 150 µl of the fresh muscle medium_1 containing 2% Matrigel (growth factor reduced, Corning, 356231). After day 4, the plate was placed on a rotary shaker (Celltron, INFORS HT) with the rotation speed set at 75 r.p.m. The medium was not changed until day 6. On day 6, the aggregates were collected using wide-bore tips and transferred into a 6-well plate containing 3 ml of the muscle medium_2. The aggregates were incubated in the muscle medium_2 until day 15 with medium changes every 3 days.  Bovine muscle and endothelial cell co-induction protocol in 3D: The aggregate of 20,000 PSM cells was made as described above using the muscle/EC medium_1 instead of the muscle medium_1. During days 6-15, the aggregates were incubated in the muscle/EC medium_2 instead of the muscle medium_2. cDNA libraries were constructed with Chromium Next GEM Single Cell 3' Library; Gel Bead Kit v3.1 (10x Genomics) according to manufacturer's protocol. cDNA libraries were constructed with Chromium Next GEM Single Cell 3' Library; Gel Bead Kit v3.1 (10x Genomics) according to manufacturer's protocol.