Intesticult media

Manufactured by STEMCELL

IntestiCult media is a cell culture medium developed by STEMCELL Technologies for the maintenance and expansion of intestinal epithelial cells. It is designed to support the growth and proliferation of these cells in vitro.

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Lab products found in correlation

Primocin, by STEMCELL (1 mentions) Primocin, by InvivoGen (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Dasatinib, by Selleck Chemicals (1 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions) Primocin antibiotic, by Thermo Fisher Scientific (1 mentions) Adf 12, by Thermo Fisher Scientific (1 mentions) Lgk974, by Selleck Chemicals (1 mentions) Matrigel, by Corning (1 mentions) Human collagen 4, by Merck Group (1 mentions) Evom2 epithelial voltohmmeter, by World Precision Instruments (1 mentions)

Close spelling variants of this product

Human IntestiCult organoid differentiation media Mouse Intesticult media IntestiCult Organoid Growth Media Human IntestiCult organoid growth media Intesticult

4 protocols using intesticult media

Mouse Small Intestine Organoid Culture

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Mouse small intestine was dissected, gently flushed with ice-cold phosphate-buffered saline (PBS) (pH 7.4)/Primocin (1:1,000) (InvivoGen, catalog ant-pm-1), cut into ~0.5 cm pieces that were placed in 7.5 mL cold PBS/EDTA (3 mM)/Primocin/Y27632 (1:1,000) (ApexBio, catalog A3008-200) containing penicillin-streptomycin (Gibco, catalog 15140-122), and gently shaken for 15 minutes at 4°C. The intestinal tissue was transferred to fresh EDTA/PBS/Primocin/Y27632, shaken for 25 minutes at 4°C, and transferred to PBS. Tissue was then transferred to PBS/Y27632, shaken for 2 minutes, and filtered through a 70 μm mesh, examined under a microscope, and aliquoted at a density of 50 crypts in 15 μL growth factor–reduced Matrigel (Corning, catalog 354230). The suspension was centrifuged at 475g for 5 minutes at 4°C, and the pellet was resuspended in cold growth factor–reduced Matrigel and aliquoted (15 μL/well) in a 48-well plate (Eppendorf, catalog 0030723113). Matrigel was left to polymerize for 30 minutes at 37°C. To each well, we added 200 μL of prewarmed (37°C) Intesticult Media (StemCell Technologies, catalog 06005) containing Primocin. Media were changed every 2 days and organoids were split weekly.

Chandra R., Sokratian A., Chavez K.R., King S., Swain S.M., Snyder J.C., West A.B, & Liddle R.A. (2023). Gut mucosal cells transfer α-synuclein to the vagus nerve. JCI Insight, 8(23), e172192.

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Isolation and culture of intestinal organoids

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Intestinal crypts were isolated from 6‐ to 10‐week‐old C57B/l6 or transgenic mice, following the published protocol from Mahe et al (2013). Briefly, the whole gut was harvested and washed in cold DPBS (Gibco, 14190250). The most proximal 5 cm were cut open, and the villi were removed with a coverslip. The remaining tissue was washed and incubated in 2 mM EDTA for 30 min at 4°C. Two crypt fractions were then mechanically extracted, the first one being filtered through a 70 μM cell strainer before pooling both fractions together. After several low speed washes in ADF‐12 (Gibco, 12634‐010), isolated crypts were resuspended and plated in Matrigel (Corning, 354230). Organoids were cultured in IntestiCult media (Stemcell technologies, 06005) complemented with Primocin antibiotic (Invitrogen, ant‐pm‐05). Organoids were cultured in 24‐well plates for maintaining the cultures and then cultured in 8‐well chambers for drug incubations and immunostaining (Ibidi, 80827). The Porcupine inhibitor LGK974 (Selleck chemicals, S7143) was used at 5 μM for 24 h. For Src inhibition experiments, organoids were treated with 100 nM of Dasatinib (Selleck chemicals, S1021) or 100 nM eCF506 for a period of 4 h. Organoid microscopy was performed with either a Leica SP5 or a Leica SP8 laser‐scanning confocal microscope.

Guillermin O., Angelis N., Sidor C.M., Ridgway R., Baulies A., Kucharska A., Antas P., Rose M.R., Cordero J., Sansom O., Li V.S, & Thompson B.J. (2021). Wnt and Src signals converge on YAP‐TEAD to drive intestinal regeneration. The EMBO Journal, 40(13), e105770.

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Intestinal Organoid Monolayer Culture Protocol

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Monolayer protocols were adapted from previous publications with minor modifications.⁸ Differentiation media was not utilized in the study as we were interested in effects of FS on differentiation. Organoids were initially cultured in Matrigel for 2–3 passages prior to plating on monolayers. To form monolayers, Transwell inserts (24‐well inserts, 0.33 cm² surface area, 0.4 μm pore polyester membrane; Corning) were coated with Human Collagen IV (Millipore Sigma) solution (final concentration of 10 μg/cm²) and incubated overnight at 4°C. Prior to plating, any remaining collagen was removed from wells and washed 2X with Advanced DMEM/F12. Fragments for monolayer plating were obtained using the passaging protocol above, making sure to obtain small fragments. Approximately 50 organoid fragments were obtained per 100 μl Intesticult™ media (Stem Cell Technologies), then added to the membrane and allowed to settle at 37°C; 600 μl of media was also added to the basolateral side. Intesticult™ media was changed every 2 days and monolayer development was tracked using transepithelial electrical resistance (TER) measured by the EVOM2 epithelial voltohmmeter (World Precision Instruments). Monolayers were utilized for experiments when cells reached a minimum 400 Ω. Average baseline TER for all treatments was 575 Ω.

Pearce S.C., Weber G.J., Doherty L.A, & Soares J.W. (2022). Human iPSC colon organoid function is improved by exposure to fecal fermentates. FASEB BioAdvances, 4(7), 468-484.

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Isolation of Murine Colonic Crypts

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To isolate crypts from the murine colon, the large intestine of 1-2 mice was cut, flushed with ice-cold PBS, opened longitudinally and cut into ~5 mm pieces. The tissue pieces were washed in ice-cold PBS for at least 8 times by vigorous shaking. The tissue was incubated in PBS with 5 mM EDTA, shaken vigorously before replacing the solution with fresh PBS and 2.5 mM EDTA. Tissue pieces were incubated on a horizontal shaker at 4°C for 30 minutes, shaken vigorously and the solution was passed through a 70 μm strainer to collect the first fraction. The tissue pieces were incubated again for 10 minutes in PBS with 2.5 mM EDTA and up to 3 additional fractions were collected. The best fractions were pooled and spun down at 300 g for 5 min at 4°C. The pellet was resuspended in PBS with 0.2% BSA or FBS and filtered through a 70 μm strainer again. 400-500 crypts were plated per dome. Large intestinal organoids were grown and maintained in IntestiCult media (Stemcell technologies) with Normocin (1:200) and ROCK inhibitor (1:5000), which was added for the first two days.

Niec R.E., Chu T., Schernthanner M., Gur-Cohen S., Hidalgo L., Pasolli H.A., Luckett K.A., Wang Z., Bhalla S.R., Cambuli F., Kataru R.P., Ganesh K., Mehrara B.J., Pe’er D, & Fuchs E. (2022). Lymphatics act as a signaling hub to regulate intestinal stem cell activity. Cell stem cell, 29(7), 1067-1082.e18.

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