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Intesticult organoid growth media

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IntestiCult Organoid Growth Media is a specialized culture medium designed to support the growth and maintenance of intestinal organoids. The media provides the necessary components to facilitate the development and expansion of intestinal epithelial cells in a three-dimensional culture system.

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Lab products found in correlation

Matrigel, by Corning (5 mentions) Gentle cell dissociation reagent (gcdr), by STEMCELL (3 mentions) Hepes, by STEMCELL (3 mentions) Ly294002, by Merck Group (2 mentions) Rapamycin, by Merck Group (2 mentions) A6003, by Merck Group (2 mentions) Intesticult organoid growth media, by Corning (2 mentions) Y 27632, by Merck Group (2 mentions) Recombinant mouse il 13, by R&D Systems (1 mentions) Matrigel, by STEMCELL (1 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions) Tryple express, by Thermo Fisher Scientific (1 mentions) Matrigel, by Thermo Fisher Scientific (1 mentions) Celltiter glo luminescent cell viability assay, by Promega (1 mentions) Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions) Rho kinase inhibitor, by Merck Group (1 mentions) Growth factor reduced matrigel, by Corning (1 mentions) Y 27632, by Axon Medchem (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Mouse recombinant tnf α, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Intesticult (16 citations) IntestiCult media (4 citations) Human IntestiCult organoid growth media (2 citations)

11 protocols using intesticult organoid growth media

1

Intestinal Crypt Isolation and Organoid Culture

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The procedure for Intestinal crypts isolation and in vitro culture was conducted as previously described (Yu et al., 2014 (link); Yu et al., 2018 (link)). Briefly, mouse ileal fragments were harvested, washed in PBS and incubated in PBS containing 5mM EDTA for 40 min. After vigorous vortexing, crypt suspensions were passed through 70μm cell strainer and centrifuged at 200×g for 5 min at 4°C. Then, intestinal crypts were counted, and resuspended in Matrigel (Corning, Catalog No. 354230). After Matrigel polymerization, IntestiCult organoid growth media (StemCell Technologies, Catalog No. 06005) was added to each well After 3-day culture, recombinant mouse IL-13 (R&D systems, Catalog No. 413-ML) was added or not and incubated for 2 more days. Then, intestinal organoids were subjected to fixation by 4% paraformaldehyde followed by immunofluorescence.
Yu S., Balasubramanian I., Laubitz D., Tong K., Bandyopadhyay S., Lin X., Flores J., Singh R., Liu Y., Macazana C., Zhao Y., Béguet-Crespel F., Patil K., Midura-Kiela M.T., Wang D., Yap G.S., Ferraris R.P., Wei Z., Bonder E.M., Häggblom M.M., Zhang L., Douard V., Verzi M.P., Cadwell K., Kiela P.R, & Gao N. (2020). Paneth cell-derived lysozyme defines the composition of mucolytic microbiota and the inflammatory tone of the intestine. Immunity, 53(2), 398-416.e8.
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2

Isolation and Culture of Human Colonoids

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Colonoids were isolated from biopsies taken from healthy subjects undergoing routine colonoscopy as previously described [27] (link). Colonoids were cultured in domes of Matrigel (Corning) and covered by human colonoid media (HCM), prepared by mixing 50% IntestiCult organoid growth media (StemCell Technologies) with 50% L-WRN conditioned media prepared as previously described [28] (link) in advanced DMEM/F12, supplemented with 50 ng/ml EGF (Peprotech). Human derived colonoids (HDCs) were passaged by manually dissociating Matrigel domes using a pipette in cold PBS, pelleting, and incubating in 1:5 TrypLE Express (Thermo Fisher Scientific) before passing through a 26G syringe needle prior to re-seeding dissociated HDCs.
Dougherty M.W., Valdés-Mas R., Wernke K.M., Gharaibeh R.Z., Yang Y., Brant J.O., Riva A., Muehlbauer M., Elinav E., Puschhof J., Herzon S.B, & Jobin C. (2023). The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors. Neoplasia (New York, N.Y.), 43, 100918.
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3

Culturing Mouse Colon Stem Cells

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Mouse colon stem cells were cultured using IntestiCult Organoid Growth Media according to the manufacturer’s instructions (06005, STEMCELL Technologies). The whole colon was removed from untreated WT and Nlrc3−/− mice and rinsed with cold PBS. The colon was cut into 2 mm segments and washed 20 times with cold PBS. Colonic segments were incubated in Gentle Cell Dissociation Reagent (07174, STEMCELL Technologies), rotated at 350 g for 15 min at room temperature, followed by resuspension in 0.1% BSA (A6003, Sigma) in PBS. Dissociated colonic crypts were filtered through 70-µm strainers. Dissociated colonic crypts were resuspended in DMEM/F12 medium with 15 mM HEPES (36254, STEMCELL Technologies), counted and resuspended in IntestiCult Organoid Growth Media and Matrigel (356230, Corning) in a 1:1 ratio. Cells were plated in 24-well culture plates (3738, Corning). IntestiCult Organoid Growth Media were added to the cell culture plates to immerse the matrix composed of IntestiCult Organoid Growth Media and Matrigel. For inhibition studies, 50 µM of LY294002 (440202, Millipore) or 10 µg/ml rapamycin (553210, Sigma) was added to the IntestiCult Organoid Growth Media.
Karki R., Man S.M., Malireddi R.K., Kesavardhana S., Zhu Q., Burton A.R., Sharma B.R., Qi X., Pelletier S., Vogel P., Rosenstiel P, & Kanneganti T.D. (2016). NLRC3 is an inhibitory sensor of PI3K–mTOR pathways in cancer. Nature, 540(7634), 583-587.
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4

Organoid Establishment from Mouse Intestine

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Organoids were established from freshly isolated wild‐type, Nedd4 cKO, Nedd4l cKO and DKO small intestine. Tissues were incubated in cold PBS containing 2 mM EDTA for isolating epithelial crypts and culture as previously describe (Sato et al, 2009) except that Matrigel was replaced with Cultrex® BME, Type 2 RGF PathClear (Amsbio 3533‐010‐02). In brief, the organoid basal media contain EGF (Invitrogen PMG8043), Noggin and R‐spondin (ENR) (5%). For the R‐spondin withdrawal experiment, when indicated, R‐spondin was used at 1%. Noggin and R‐spondin‐conditioned media (CM) were generated from HEK293T cells. For all the other organoid experiments, Intesticult™ Organoid Growth media (06005, Stem Cell Technologies) was used. The Rho kinase inhibitor Y‐27632 (Sigma) was added to the culture when trypsinised.
Novellasdemunt L., Kucharska A., Jamieson C., Prange‐Barczynska M., Baulies A., Antas P., van der Vaart J., Gehart H., Maurice M.M, & Li V.S. (2019). NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor. The EMBO Journal, 39(3), e102771.
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5

Organoid Viability Assay Protocol

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Organoids were trypsinized using TrypLE and filtered with a 20 μm cell strainer. 3500 single cells were seeded in BME per 48 well and placed in a 37°C incubator to polymerize for 20 min. 250 μl of Intesticult™ Organoid Growth media (06005, Stem Cell Technologies) (for APCmin organoids) or 250 μl of complete human organoid media (for human patient organoids) with the indicated treatments was added for the indicated times. The media were supplemented with 10 μM Y-27632 for two days after plating. Cell Titer Glo Luminescent Cell viability assay (G7572, Promega) was used to assess viability of the organoids. Experiments were performed at least three times with three triplicates each.
Méndez-Lucas A., Lin W., Driscoll P.C., Legrave N., Novellasdemunt L., Xie C., Charles M., Wilson Z., Jones N.P., Rayport S., Rodríguez-Justo M., Li V., MacRae J.I., Hay N., Chen X, & Yuneva M. (2020). Identifying strategies to target the metabolic flexibility of tumours.. Nature metabolism, 2(4), 335-350.
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6

Organoid Formation and Characterization

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Organoids were dissociated into single cells using TrypLE and further filtered with a 20 μm cell strainer. For each condition, 3500 single cells were seeded in BME per 48 well and placed in a 37°C incubator to polymerize for 20 min. 250 μl of Intesticult™ Organoid Growth media (06005, Stem Cell Technologies) (for APCmin organoids) or 250 μl of complete human organoid media (for human patient organoids) with the indicated conditions. The media were supplemented with 10 μM Y-27632 for two days after plating. Number of spheres formed in each well was counted as plating efficiency. Images were captured at the indicated times with EVOS FL cell imaging system (Life technologies). Experiments were performed at least three times with three triplicates each.
Méndez-Lucas A., Lin W., Driscoll P.C., Legrave N., Novellasdemunt L., Xie C., Charles M., Wilson Z., Jones N.P., Rayport S., Rodríguez-Justo M., Li V., MacRae J.I., Hay N., Chen X, & Yuneva M. (2020). Identifying strategies to target the metabolic flexibility of tumours.. Nature metabolism, 2(4), 335-350.
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7

Enteroid Expansion from Mouse Jejunal and Human Intestinal Crypts

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Mouse jejunal crypts were plated at a density of 100 crypts per well in a 24-well plate in phenol red–free, growth factor–reduced basement membrane Matrigel (#356231; Corning) and fed with enteroid growth media containing 50% Wnt3a, R-spondin 3 and Noggin (L-WRN) conditioned media.45 (link) Seventy-two hours after plating, the number of spherical enteroids growing in each well was counted.
Human intestinal organoids were dissociated as described earlier. The villus fraction was discarded. Crypts were split equally into 6 wells in a 24-well plate in phenol red–free, growth factor–reduced basement membrane Matrigel (#356231; Corning) and fed with human IntestiCult Organoid Growth Media (#06010; STEMCELL Technologies). Ninety-six hours after plating, the number of spherical enteroids growing in each well was counted.
McCauley H.A., Riedman A.M., Enriquez J.R., Zhang X., Watanabe-Chailland M., Sanchez J.G., Kechele D.O., Paul E.F., Riley K., Burger C., Lang R.A, & Wells J.M. (2023). Enteroendocrine Cells Protect the Stem Cell Niche by Regulating Crypt Metabolism in Response to Nutrients. Cellular and Molecular Gastroenterology and Hepatology, 15(6), 1293-1310.
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8

Organoid Culture from APC^min Tumors

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Organoids were stablished from tumours isolated from APCmin mice using a previously described protocol66 , with an additional collagenase and dispase digestion step after the EDTA-chelation step67 where Matrigel was replaced with Cultrex® BME, Type 2 RGF PathClear (Amsbio 3533–010-02). Organoids were cultured in Intesticult™ Organoid Growth media (06005, Stem Cell Technologies). The Rho kinase inhibitor Y-27632 (Sigma) was added to the culture when trypsinised.
Méndez-Lucas A., Lin W., Driscoll P.C., Legrave N., Novellasdemunt L., Xie C., Charles M., Wilson Z., Jones N.P., Rayport S., Rodríguez-Justo M., Li V., MacRae J.I., Hay N., Chen X, & Yuneva M. (2020). Identifying strategies to target the metabolic flexibility of tumours.. Nature metabolism, 2(4), 335-350.
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9

Culturing Mouse Colon Stem Cells

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Mouse colon stem cells were cultured using IntestiCult Organoid Growth Media according to the manufacturer’s instructions (06005, STEMCELL Technologies). The whole colon was removed from untreated WT and Nlrc3−/− mice and rinsed with cold PBS. The colon was cut into 2 mm segments and washed 20 times with cold PBS. Colonic segments were incubated in Gentle Cell Dissociation Reagent (07174, STEMCELL Technologies), rotated at 350 g for 15 min at room temperature, followed by resuspension in 0.1% BSA (A6003, Sigma) in PBS. Dissociated colonic crypts were filtered through 70-µm strainers. Dissociated colonic crypts were resuspended in DMEM/F12 medium with 15 mM HEPES (36254, STEMCELL Technologies), counted and resuspended in IntestiCult Organoid Growth Media and Matrigel (356230, Corning) in a 1:1 ratio. Cells were plated in 24-well culture plates (3738, Corning). IntestiCult Organoid Growth Media were added to the cell culture plates to immerse the matrix composed of IntestiCult Organoid Growth Media and Matrigel. For inhibition studies, 50 µM of LY294002 (440202, Millipore) or 10 µg/ml rapamycin (553210, Sigma) was added to the IntestiCult Organoid Growth Media.
Karki R., Man S.M., Malireddi R.K., Kesavardhana S., Zhu Q., Burton A.R., Sharma B.R., Qi X., Pelletier S., Vogel P., Rosenstiel P, & Kanneganti T.D. (2016). NLRC3 is an inhibitory sensor of PI3K–mTOR pathways in cancer. Nature, 540(7634), 583-587.
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10

Intestinal Organoid Culture Protocol

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Sorted cells were collected at low temperatures, pelleted, and embedded in Growth Factor Reduced Matrigel (354230; Corning), followed by seeding on a prewarmed 96-well plate. Prewarmed Intesticult organoid growth media (06005; Stemcell Technologies) was added after polymerization of the Matrigel. Media was supplemented with CHIR99021 (HY-10182; MedChem Express) and Y-27632 (Axon 1683; Axon MedChem) for the first days. Media was changed every 4 d by removing 50% and adding 50% fresh media.
Chaves-Pérez A., Santos-de-Frutos K., de la Rosa S., Herranz-Montoya I., Perna C, & Djouder N. (2022). Transit-amplifying cells control R-spondins in the mouse crypt to modulate intestinal stem cell proliferation. The Journal of Experimental Medicine, 219(11), e20212405.
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