Cell culture and human crypt isolation
This protocol is extracted from research article:
Type III interferon signaling restricts enterovirus 71 infection of goblet cells
Sci Adv, Mar 6, 2019; DOI: 10.1126/sciadv.aau4255

Human fetal intestinal crypts were isolated from the entire small intestine and cultured as described previously (16). Human fetal tissue (<24 weeks of gestation) that resulted from elective terminations were obtained from the University of Pittsburgh Biospecimen Core through an honest broker system after approval from the University of Pittsburgh Institutional Review Board and in accordance with the University of Pittsburgh anatomical tissue procurement guidelines. All tissue was genetically normal. Approximately 100 isolated crypts were plated into each well of a 24-well T-clear (0.4-μm pore size) Transwell insert and were grown in crypt culture medium composed of Advanced DMEM/F12 (Invitrogen) with 20% HyClone ES (embryonic stem) Cell Screened Fetal Bovine Serum (Thermo Fisher Scientific), 1% penicillin/streptomycin (Invitrogen), 1% l-glutamine, gentamycin, 0.2% amphotericin B, 1% N-acetylcysteine (100 mM; Sigma-Aldrich), 1% N-2 supplement (100×; Invitrogen), 2% B27 supplement (50×; Invitrogen), Gibco Hepes (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, 0.05 mM; Invitrogen), ROCK Inhibitor Y-27632 (1 mM, 100×; Sigma) and supplemented with the following growth factors: WNT3a (100 ng/ml; Thermo Fisher Scientific), R-spondin (500 ng/ml; R&D Systems), Noggin (100 ng/ml; PeproTech), and EGF (50 ng/ml; Thermo Fisher Scientific) (37, 38) for the remainder of the respective experiments, with medium changes occurring every 48 hours. Unless otherwise stated, monolayers of HIE were used in studies at 6 days after plating and when TER values were >600 ohm.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.