Murine recombinant epidermal growth factor

Human intestinal organoids were generated following previously established protocols (Spence et al, 2011 (link)) using multiple pluripotent stem cell (PSC) lines (Table EV2). Briefly, PSCs were differentiated through the definitive endoderm stage via the addition of Activin A (Cell Guidance Systems) between day‐0 (D0) and D2, BMP4 (R&D Systems) at D1 and CHIR99021 (Stemgent)/FGF4 (R&D Systems) from D3 to D6. The addition of CHIR99021/FGF4 prompted the formation of midgut tube spheroids from the definitive endoderm monolayer. At D7 midgut tube spheroids were collected and suspended in a Matrigel dome (Corning). Spheroids were then grown in gut media: DMEM/F12 (Gibco) supplemented with N‐2 (Gibco), B‐27 (Gibco), HEPES solution (Millipore‐Sigma), recombinant murine epidermal growth factor (Peprotech), L‐Glutamine (Gibco), Penicillin/Streptomycin (Gibco). At D21, sample density was reduced to ≈1–3 per 50 µl Matrigel bubble. Samples were grown for an additional 14 days in gut media, resulting in terminal HIO cultures (Spence et al, 2011 (link)). Individual HIOs were considered as biological replicates for all experiments. All experimentation using human tissues described herein was approved by an IRB at CCHMC (IRB#2014‐0427). Informed consent for tissue collection, storage, and use of the samples was obtained from the donors at CCHMC.

According to Hassanpour et al.’s protocol[11 (link)], POCs were prepared from 4-week-old female Kunming mice and then encapsulated in the bioink. Briefly, each mouse was intraperitoneally injected with 10 IU Pregnant Mare Serum Gonadotropin (PMSG, Solarbio) followed by 16 U Chorionic Gonadotrophin for Injection (Harbin Sanma Animal Pharmaceutical Co. LTD) after 48 h, and then, the ovaries were isolated after 6 h. The ovaries were incubated in α-MEM medium (Gibco) with 1% penicillin-streptomycin (PS, Millipore) at 4°C. Afterward, the ovaries were, manually, dissected with fine needles into smaller fragments and incubated in a digestion solution consisting of Dispase II (Cat. No. D4693-1G, Sigma) and Collagenase I (Cat. No. 1904MG100, BioFROXX) at 37°C for 30 min. Then, the enzymatic process was terminated by equal volumes of α-MEM medium containing 10% FBS (BI). The suspension was filtered through a 100 μm cell strainer (Life Sciences, USA) and washed twice with the following culture medium: α-MEM medium with 10% FBS, 3 ng/ml Recombinant Murine Epidermal Growth Factor (Cat. no. 315-09, Pepro Tech), 100 mIU/ml Follicle Stimulating Hormone for Injection (Harbin Sanma Animal Pharmaceutical Co. LTD), and 1.5 U/ml chorionic gonadotrophin for injection and 1% PS. Cells were resuspended in 200 ml culture medium after centrifugation and then mixed well with the bioink for printing.