100 μm cell strainer

To detect the CSC population, A2780, ES-2, and R182 cells were cultured in 2D and 3D for 7 days and were harvested by pipetting. They were washed with Hanks’ balanced salt solution (HBSS, Gibco/Thermo Fisher Scientific) containing 0.1% BSA and 0.1% sodium azide and filtered through a 100-μm cell strainer (SPL Life Sciences). Phenotypic analysis of cell surface marker expression was performed by flow cytometry. Briefly, cells were washed twice with HBSS and resuspended in the cell-staining buffer. Cells were immunostained for cell surface markers by incubating them for 30 min with phycoerythrin (PE)-Cy7-labeled anti-CD44 (1:10, Invitrogen, Life Technologies, Carlsbad, CA, USA), PE-labeled anti-CD117 (1:10, BioLegend, San Diego, CA, USA), and allophycocyanin-labeled anti-CD133 (1:10, BioLegend) monoclonal antibodies. For control, 2D-cultured cells were used. Fluorescence-activated cell sorting analysis was performed using a FACS Canto-II flow cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometry data were analyzed using FlowJo 10.3.0 (Tree Star, Ashland, OR, USA).

Six-week-old pigs raised on a Kangwon National University experimental farm (Republic of Korea) were used in this study. Five pigs were randomly selected for fecal sample collection. Feces from 5 pigs were mixed, 15 g of which was equally distributed into three 50 ml tubes for reproducibility. Before incubation, to avoid the effects of fecal residues, fecal samples were mixed thoroughly with 50 ml 1× phosphate-buffered saline (PBS) and filtered with a 100-μm cell strainer (SPL Life Sciences, Republic of Korea). To make media, freeze-dried beef (round), pork (sirloin) and chicken (breast) were purchased from a local online shop, and skim milk was obtained from MB Cell (Republic of Korea). The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Kangwon National University (KIACUC, KW-190429-1).

The crypts of porcine were harvested from the jejunum tissue of 5-week-old pigs (n = 3, body weight: 7.80 ± 0.96 kg). All piglets were healthy without diarrhea symptoms. A total of 3–4 cm of tissue was harvested and opened longitudinally. To remove luminal content, mucus, and part of villus, tissue was scraped using slide glass and washed with phosphate-buffered saline (PBS) 3–4 times. Next, 0.5 × 0.5 cm² jejunum segments were cut and incubated for 30 min on ice at horizontal shaking of 100 rpm using 30 mM ethylene-diamine-tetra acetic acid (EDTA) and 1 mM DL-dithiothreitol (DTT). After incubation step, tissue fragments were transferred to cold crypt washing buffer (54.9 mM D-sorbitol and 43.4 mM sucrose in PBS) and vigorously shaken for 2 min to release intestinal crypts. Then, to remove villus and debris, supernatant was transferred to new 50 mL tube by using 100 μm cell strainer (SPL Life Sciences, Pocheon, Korea) and was centrifuged at 200× g for 2 min (two times repeat for removing debris and single cells). The crypt pellet was suspended with advanced Dulbecco’s modified Eagle medium/F12 (advanced DMEM/F12) (Thermo Fisher Scientific, Waltham, MA, USA) and counted for culturing organoids.