Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology.

Human intestinal tissue specimens were obtained from endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using qRT-PCR, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions.

MATERIALS AND METHODS Human Tissues Tissues from human subjects were obtained under approval of the Johns Hopkins University School of Medicine Institutional Review Board (protocol NA_00038329). De-identified specimens determined to come from normal intestines were obtained from endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypt isolation, enteroid propagation, differentiation, immunodetection, lentiviral transduction, and mRNA analysis are described in Supplementary Methods .

Reagents

Wnt3a conditioned medium was generated using the L-Wnt3a cell line (ATCC CRL-2647). R-spondin1 conditioned medium was produced using HEK293T cells stably expressing mouse Rspo1-Fc. 7 Noggin conditioned medium was prepared from HEK293T cells stably expressing murine Noggin-Fc. 8 Expansion medium (EM) is made in a base solution of Advanced DMEM/F12, 100 U penicillin/streptomycin, 10 mM HEPES, pH 7.4, 0.2 mM GlutaMAX with 50% v/v Wnt3a conditioned medium, 20% v/v R-spondin1 conditioned medium, 10% v/v murine Noggin conditioned medium, 1x B27 supplement, 1x N2 supplement, 1 mM N -acetylcysteine, 50 ng/mL murine EGF, 1 μg/mL [Leu-15]gastrin, 10 mM nicotinamide, 500 nM A83-01, 10 μM SB202190, and 100 μg/mL Primocin. Differentiation medium (DM) is EM lacking Wnt3a, nicotinamide, and SB202190. A comprehensive list of growth factors/small molecules and their respective sources is included in Supplementary Table 1 .

Enteroid Imaging and Intracellular pH Measurement

Enteroid intracellular pH measurement was adapted from the procedure for murine tissues. 9 , 10 Enteroid fragments were plated in 50% Matrigel (20 μL) on a 35 mm dish with a bottom coverslip (P35G-1.5-10-C, MatTek), and then overlaid with 1.5 mL Expansion Medium (EM) for 3–5 d, followed by Differentiation Medium (DM) for 5 days. Enteroids were loaded with SNARF-4F-AM (5 μM) in 50 mM NH 4 Cl-prepulse buffer (3 mL) for 30 min at 37 °C, 5% CO 2 . Probenecid (1 mM) was present in buffers to inhibit SNARF-4F export. 35-mm dishes were mounted in a preheated chamber (37 °C, DH-35, Warne r Instruments) and optical sections were collected at 10 μm intervals using a 25X/1.05 NA water immersion objective (XLPLN, Olympus) on a multiphoton laser scanning microscope (Fluoview FV1000-MPE, Olympus) at 780 nm excitation, 580/640 nm emission. For each condition, fluid was aspirated and replaced with 3 mL fresh buffer maintained at 37 °C. Additional images were collected after 15 min incubation in Na + -free tetramethylammonium chloride (TMA) buffer and at various intervals in Na + buffer, both containing HOE-694 (50 μM). In some experiments, HCO 3 − buffer was also used (Na + buffer but with 25 mM NaHCO 3 , 98 mM NaCl). Intracellular pH was calibrated at the end of each experiment by exposure to 20 μM nigericin for 10 min in K + clamp buffers to set the pH at 6.25, 6.70, and 7.50. Image analysis and pH calculations are outlined in Supplementary Methods . Time-lapse confocal imaging used to capture enteroid cross-sections over time was based on the method previously described 11 and detailed in Supplementary Methods .

Statistics

Data are presented as the mean of at least 3 independent experiments ± SEM. Significance was determined using Student’s t-tests or ANOVA analysis where appropriate. Values of P