1. Introduction to Intestinal Organoid Culture

The intestinal epithelium is one of the most rapidly renewing tissues in the mammalian body, completely turning over every 4–5 days under homeostatic conditions. This remarkable regenerative capacity is driven by a population of active cycling stem cells located at the base of the crypts, known as crypt base columnar (CBC) cells, which are marked by the expression of Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5). These Lgr5+ stem cells are responsible for generating all the differentiated cell types of the intestinal epithelium, including absorptive enterocytes, mucus-secreting goblet cells, hormone-producing enteroendocrine cells, antimicrobial peptide-producing Paneth cells (in the small intestine), and chemosensory tuft cells [1,2].

Intestinal organoids are three-dimensional, self-organizing structures that are derived from isolated intestinal crypts or single Lgr5+ stem cells and that recapitulate the essential features of the native intestinal epithelium, including the crypt-villus architecture, the stem cell niche, and the full spectrum of epithelial cell types. Since the landmark publication by Sato and Clevers in 2009, which demonstrated that single Lgr5+ intestinal stem cells embedded in Matrigel could form organoid structures when cultured with EGF, Noggin, and R-spondin 1, intestinal organoid culture has become a cornerstone model for studying epithelial homeostasis, host-microbe interactions, infectious diseases, inflammatory bowel disease, and colorectal cancer [1,3].

The adaptation of intestinal organoid culture to human tissues by Sato and colleagues in 2011 revealed that human intestinal crypts require additional culture components compared to mouse organoids, reflecting the increased sensitivity of human epithelial cells to anoikis, oxidative stress, and culture-induced senescence [2]. These additional requirements include nicotinamide, A83-01 (a TGF-β receptor inhibitor), and SB202190 (a p38 MAPK inhibitor), which together stabilize human epithelial cells and enable long-term culture. The establishment of human intestinal organoids from both healthy and diseased tissues has opened unprecedented opportunities for modeling human intestinal biology, including genetic diseases such as cystic fibrosis, inflammatory conditions such as Crohn's disease, and infectious diseases such as Clostridioides difficile and rotavirus infection [2,3].

This article provides a comprehensive, step-by-step protocol for the establishment, maintenance, passaging, and differentiation of intestinal organoids, with specific technical parameters and concentrations validated for reproducible results in both research and translational applications. The protocols described here are applicable to both murine and human intestinal organoids, with notes provided where species-specific modifications are required.

Related resource: Culture medium product

2. Fundamental Principles of Intestinal Organoid Culture

Intestinal organoid culture is based on the principle that isolated intestinal crypts or single Lgr5+ stem cells, when provided with the appropriate combination of niche signals and embedded in a basement membrane matrix, can self-organize into structures that recapitulate the essential features of the native intestinal epithelium. The key signaling pathways that must be recapitulated in the culture medium are:

Related resource: Organoid kits

3. Tissue Procurement and Crypt Isolation Protocols

3.1 Murine Small Intestinal Crypt Isolation

The isolation of murine intestinal crypts is a well-established procedure that can be completed in approximately 2–3 hours from tissue collection to plating. The following protocol is adapted from Sato and Clevers (2013) and has been validated for both small intestinal and colonic crypt isolation [1,6].

Materials Required:

Detailed Procedure:

  1. Euthanize the mouse using an approved method (e.g., CO2 asphyxiation followed by cervical dislocation) and confirm death according to institutional animal care guidelines. Spray the abdomen with 70% ethanol to sanitize the surface.
  2. Make a midline incision through the skin and peritoneum to expose the abdominal cavity. Carefully dissect the entire length of the small intestine (from the pyloric sphincter to the ileocecal junction) or the colon (from the cecum to the rectum). Place the tissue immediately in a 50 mL tube containing ice-cold PBS supplemented with 100 U/mL Penicillin and 100 µg/mL Streptomycin.
  3. Transfer the tissue to a 6 cm petri dish containing fresh ice-cold PBS. Using fine scissors, open the intestine longitudinally along the mesenteric border. Gently wash the luminal surface with ice-cold PBS using a 10 mL pipette to remove fecal contents and mucus. Repeat washing until the PBS is clear.
  4. Transfer the opened tissue to a fresh 50 mL tube containing 30 mL ice-cold PBS. Wash the tissue fragments by gently pipetting up and down with a 10 mL pipette. Allow the fragments to settle by gravity (approximately 30 seconds) and discard the supernatant. Repeat this washing step 10 times until the supernatant is almost clear and free of debris. This step is critical for removing mucus and bacteria that can interfere with crypt isolation.
  5. Transfer the washed tissue fragments to a fresh 50 mL tube containing 25 mL of 10 mM EDTA in PBS. Incubate at 4°C on a rocking platform or rotator for 30 minutes (for small intestine) or 90 minutes (for colon). The EDTA chelates calcium and magnesium ions, disrupting the calcium-dependent adhesion complexes (E-cadherin/catenin complexes) that anchor the crypts to the underlying basement membrane.
  6. After EDTA incubation, carefully remove the supernatant without disturbing the tissue fragments. Add 10 mL of fresh ice-cold PBS to the tube. Using a 10 mL pipette, vigorously pipette the tissue fragments up and down 10–20 times. This mechanical agitation releases the crypts from the tissue. The released crypts will be visible in the suspension as small, translucent, cylindrical or flask-shaped structures under the microscope.
  7. Collect the crypt suspension and pass it through a 70 µm cell strainer into a fresh 50 mL tube. The strainer retains large tissue fragments and villous material while allowing the smaller crypts to pass through. If the strainer becomes clogged, gently agitate the mesh with the pipette tip to dislodge debris.
  8. Centrifuge the filtered crypt suspension at 200 × g for 5 minutes at 4°C. Carefully aspirate the supernatant, leaving approximately 100 µL of residual liquid to avoid disturbing the crypt pellet. The pellet should be visible as a small white or translucent button at the bottom of the tube.
  9. Resuspend the crypt pellet in 10 mL of cold Advanced DMEM/F12 and centrifuge again at 200 × g for 5 minutes at 4°C. This wash step removes residual EDTA and debris. Repeat the wash if the supernatant is still cloudy.
  10. After the final wash, carefully aspirate the supernatant and resuspend the crypt pellet in an appropriate volume of cold Matrigel for plating (see Section 5 below).

3.2 Human Intestinal Crypt Isolation from Biopsy or Surgical Resection

Human intestinal crypt isolation follows the same general principles as murine crypt isolation but requires additional considerations for tissue quality, sterility, and the presence of human pathogens. The protocol is adapted from Sato et al. (2011) and Dekkers et al. (2013) and has been validated for both small intestinal and colonic tissues [2,7].

Materials Required:

Detailed Procedure:

  1. Obtain tissue from the operating room or endoscopy suite. Place the tissue immediately in a sterile transport container containing ice-cold PBS supplemented with 100 µg/mL Primocin (a broad-spectrum antibiotic) and 100 U/mL Penicillin/Streptomycin. Transport on ice and process within 6 hours for optimal viability. If processing must be delayed, store the tissue at 4°C; viability is typically maintained for up to 24 hours under these conditions.
  2. For small tissue samples (endoscopic biopsies, 2–4 mm): Place the biopsy in a 15 mL tube containing 5 mL of 10 mM EDTA in PBS. For larger resection specimens: Using sterile forceps and scissors, dissect the mucosal layer from the underlying submucosa and muscularis. Cut the mucosal tissue into 2–4 mm fragments and place them in a 50 mL tube containing 25 mL of 10 mM EDTA in PBS.
  3. Incubate the tissue in EDTA at 4°C with gentle rocking. For small intestinal tissue, incubate for 30 minutes. For colonic or rectal tissue, extend the incubation to 90–120 minutes, as colonic crypts are more firmly anchored to the basement membrane due to differences in mucosal architecture and mucus composition [2].
  4. After EDTA incubation, transfer the tissue fragments to a fresh tube containing 10 mL of ice-cold PBS. Vigorously shake the tube or pipette the tissue up and down 20–30 times to release the crypts. The crypts will detach from the tissue and float into the suspension.
  5. Collect the crypt suspension and pass it sequentially through a 100 µm strainer (to remove large fragments) and then a 70 µm strainer (to remove single cells and small debris). Collect the filtrate in a 50 mL tube.
  6. Centrifuge the filtered crypt suspension at 300 × g for 5 minutes at 4°C. Carefully aspirate the supernatant and resuspend the pellet in 5 mL of cold Advanced DMEM/F12. Centrifuge again at 300 × g for 5 minutes.
  7. After the final wash, resuspend the crypt pellet in cold Matrigel for plating. Count the crypts using a hemocytometer if precise seeding density is required. Typical plating density is 50–200 crypts per 30 µL Matrigel droplet in a 24-well plate [2,7].
Related resource: Organoid modeling services

4. Culture Media Formulation and Preparation

The formulation of the culture medium is the most critical factor determining the success of intestinal organoid culture. The medium must provide the essential nutrients, growth factors, and signaling molecules that recapitulate the intestinal stem cell niche while maintaining the appropriate balance between proliferation and differentiation. All media components should be prepared from high-quality, tissue culture-grade reagents, and growth factors should be stored at −80°C in small aliquots to prevent repeated freeze-thaw cycles.

4.1 Murine Small Intestinal Organoid Complete Medium

The murine small intestinal organoid complete medium is based on the formulation originally described by Sato et al. (2009) and has been widely adopted with minor modifications [1,4].

Base medium: Advanced DMEM/F12 (Gibco, 12634010)

Supplements to be added fresh to each aliquot of base medium:

Preparation: Add all supplements to Advanced DMEM/F12 base medium, mix thoroughly, and filter-sterilize through a 0.22 µm PES filter. The complete medium can be stored at 4°C for up to 2 weeks. Growth factors (EGF, Noggin, R-spondin 1) should be added fresh to the working medium aliquot immediately before each medium change.

4.2 Human Intestinal Organoid Complete Medium

Human intestinal organoids require additional supplements compared to murine organoids to overcome the increased sensitivity of human epithelial cells to culture stress, anoikis, and senescence. The following formulation is based on the protocol established by Sato et al. (2011) and has been validated for long-term culture of human small intestinal and colonic organoids [2,7].

Base medium: Advanced DMEM/F12 (Gibco, 12634010)

Supplements:

Important Notes for Human Colonic Organoids: The colonic epithelium produces significantly less endogenous Wnt than the small intestinal epithelium. Therefore, human colonic organoids require additional exogenous Wnt3a supplementation (100 ng/mL, recombinant human, PeproTech, 120-03H) to maintain stem cell self-renewal and prevent differentiation. Without supplemental Wnt3a, colonic organoids will gradually lose stem cells and undergo terminal differentiation within 2–3 passages [2,9].

4.3 Defined Commercial Formulations

For researchers who prefer not to prepare complex media from individual components, commercially available defined media formulations offer a convenient and reproducible alternative. IntestiCult™ Organoid Growth Medium (Mouse, STEMCELL Technologies, #06005; Human, #06010) is a ready-to-use, serum-free medium that has been optimized for the establishment and long-term maintenance of intestinal organoids. These formulations contain all necessary growth factors and small molecules in defined concentrations, eliminating the need for conditioned medium preparation and reducing batch-to-batch variability [6,10].

Commercial media are particularly valuable for:

However, commercial media may be more expensive than self-prepared media for large-scale applications, and they offer less flexibility for protocol optimization and modification.

Related resource: Culture medium product

5. Embedding, Plating, and Culture Maintenance

5.1 Matrigel Embedding Protocol

The embedding of crypts in Matrigel is a critical step that determines the efficiency of organoid formation and the quality of the resulting organoids. The following protocol should be performed quickly and with all materials kept cold to prevent premature polymerization of the Matrigel.

Materials:

Detailed Protocol:

  1. Pre-warm the 24-well plates at 37°C in a 5% CO2 incubator for at least 30 minutes before plating. This pre-warming step is essential for rapid polymerization of the Matrigel after plating.
  2. Keep the Matrigel on ice at all times. Thaw a vial of Matrigel overnight at 4°C in the refrigerator (do not thaw at room temperature or 37°C). Once thawed, Matrigel can be kept at 4°C for up to 2 weeks. Aliquot into pre-cooled tubes to avoid repeated freeze-thaw cycles.
  3. Count the isolated crypts using a hemocytometer or by estimating the density under an inverted microscope. The optimal plating density is 50–100 crypts per 30 µL Matrigel droplet for 24-well plates. Higher densities can lead to overcrowding and organoid fusion, while lower densities may result in poor organoid formation due to insufficient paracrine signaling between crypts.
  4. Centrifuge the crypt suspension at 200 × g for 5 minutes at 4°C and carefully remove the supernatant, leaving approximately 50 µL of residual liquid above the pellet.
  5. Using a pre-cooled 200 µL pipette tip, add an appropriate volume of cold Matrigel to the crypt pellet. Gently resuspend the crypts in the Matrigel by pipetting up and down 5–10 times, taking care not to introduce bubbles. The mixture should be homogeneous with no visible clumps.
  6. Place 30–50 µL droplets of the crypt-Matrigel mixture in the center of each pre-warmed well of the 24-well plate. The droplet should be compact and dome-shaped, and should not touch the sides of the well. Touching the plastic surface can cause the crypts to adhere to the plastic rather than remaining embedded in the 3D matrix, leading to 2D outgrowth rather than 3D organoid formation.
  7. Immediately transfer the plate to the 37°C, 5% CO2 incubator and incubate for 10–15 minutes to allow the Matrigel to polymerize. Do not disturb the plate during this period.
  8. After polymerization, gently add 500–750 µL of pre-warmed complete intestinal organoid medium to the side of each well, taking care not to dislodge the Matrigel dome. The medium should be added slowly and against the wall of the well to avoid mechanical disruption.
  9. Return the plate to the incubator. The first medium change should be performed after 2–3 days.

5.2 Alternative ECM Systems for Specialized Applications

While Matrigel is the standard ECM for intestinal organoid culture, alternative matrices may be preferred for specific applications:

6. Passaging, Expansion, and Cryopreservation

6.1 Passaging Schedule and Ratios

Murine intestinal organoids typically require passaging every 5–7 days when they reach high density (filling >70% of the Matrigel dome). They can be passaged at a 1:3 to 1:6 ratio, depending on the desired expansion rate and the experimental requirements.

Human intestinal organoids typically grow more slowly and require passaging every 7–10 days. They should be passaged at a 1:3 to 1:4 ratio. Human organoids are more sensitive to over-dissociation, so mechanical passaging (fragmentation by pipetting) is often preferred over enzymatic dissociation to single cells [2,7].

6.2 Mechanical Passaging Protocol

  1. Remove the culture medium from the well and add 1 mL of ice-cold Advanced DMEM/F12.
  2. Using a 1 mL pipette tip, mechanically disrupt the Matrigel dome by scraping the bottom and sides of the well. The Matrigel will break into small pieces, releasing the organoids into the suspension.
  3. Transfer the organoid suspension to a 15 mL conical tube and pipette vigorously with a 1 mL pipette 20–30 times to fragment the organoids into smaller pieces (approximately 10–50 cell fragments). The fragments should be small enough to re-embed efficiently but large enough to retain the stem cell niche and regenerate full organoids. Monitor the fragmentation under a microscope; over-fragmentation to single cells will reduce viability, especially for human organoids.
  4. Centrifuge the fragmented organoids at 200–300 × g for 5 minutes at 4°C.
  5. Carefully aspirate the supernatant and resuspend the pellet in an appropriate volume of cold Matrigel.
  6. Plate in pre-warmed 24-well plates as described in Section 5.1 and add complete medium.

6.3 Enzymatic Passaging and Single-Cell Dissociation

For applications requiring single cells (e.g., clonal organoid generation, transfection, or flow cytometry), organoids can be dissociated enzymatically:

  1. Collect organoids by mechanical disruption of the Matrigel as described above.
  2. Centrifuge at 200 × g for 5 minutes and resuspend the pellet in TrypLE Express (Gibco, 12604013) or 0.05% Trypsin-EDTA.
  3. Incubate at 37°C for 5–10 minutes, gently pipetting every 2–3 minutes to aid dissociation.
  4. Stop the enzymatic reaction by adding an equal volume of DMEM/F12 containing 10% FBS.
  5. Centrifuge at 300 × g for 5 minutes and resuspend the single cells in cold Matrigel or complete medium.
  6. For single-cell plating, add 10 µM Y-27632 (ROCK inhibitor, STEMCELL Technologies, 72302) to the medium for the first 48 hours to prevent anoikis and improve cell survival [2,13].

6.4 Cryopreservation and Banking

Intestinal organoid lines should be cryopreserved at regular intervals to create backup stocks and to facilitate sharing between laboratories.

  1. Harvest organoids by mechanical disruption of the Matrigel and collect by centrifugation at 200 × g for 5 minutes.
  2. Resuspend the organoid pellet in cryopreservation medium: 80% complete intestinal organoid medium + 10% FBS + 10% DMSO. For improved post-thaw recovery, some protocols recommend using 50% L-WRN conditioned medium as the base for the freezing medium, which provides Wnt, R-spondin, and Noggin during the freeze-thaw process [2].
  3. Transfer the suspension to cryovials (1 mL per vial) and place the vials in a Mr. Frosty freezing container or a controlled-rate freezing device at −80°C for a minimum of 24 hours.
  4. Transfer the cryovials to liquid nitrogen (−196°C) for long-term storage.

Thawing Protocol:

  1. Rapidly warm the cryovial in a 37°C water bath until only a small ice crystal remains.
  2. Transfer the contents to a 15 mL tube containing 10 mL of pre-warmed complete medium.
  3. Centrifuge at 200 × g for 5 minutes.
  4. Resuspend the pellet in cold Matrigel and plate as usual.
  5. Add Y-27632 (10 µM) to the medium for the first 48 hours to improve recovery [2].
Related resource: Organoid reagents

7. Differentiation Protocols for Specialized Cell Types

7.1 General Differentiation Medium

To induce differentiation of intestinal organoids toward mature epithelial lineages (enterocytes, goblet cells, and enteroendocrine cells), the pro-proliferative Wnt signals must be reduced or withdrawn, and differentiation cues must be added. The general differentiation medium is prepared by removing Wnt3a and R-spondin 1 from the complete medium and adding the following components:

Culture organoids in differentiation medium for 5–7 days, with medium changes every 2 days. By day 5–7, the organoids will exhibit increased numbers of goblet cells (visible as clear, mucin-filled vacuoles by brightfield microscopy) and enteroendocrine cells [5,14].

7.2 M Cell Differentiation

Microfold (M) cells are specialized epithelial cells located over Peyer's patches and isolated lymphoid follicles that are responsible for sampling luminal antigens and initiating mucosal immune responses. M cells can be induced in intestinal organoids by adding recombinant RANKL (receptor activator of NF-κB ligand, 100 ng/mL, PeproTech, 315-11) to the complete culture medium for 4–5 days. RANKL signaling through the RANK receptor on enterocytes drives their transdifferentiation into M cells, which can be identified by the expression of M cell-specific markers such as GP2 and SPI-B [15].

7.3 Paneth Cell Enrichment

Paneth cells are specialized secretory cells located at the base of small intestinal crypts that produce antimicrobial peptides (including defensins and lysozyme) and provide niche signals (including Wnt3, EGF, and Notch ligands) that maintain Lgr5+ stem cells. Paneth cell differentiation can be promoted by maintaining high Wnt signaling (Wnt3a + R-spondin 1) and adding DAPT (10 µM) to the medium. Paneth cells are identified by their distinctive granular morphology and lysozyme immunoreactivity [5,14].

Related resource: Organoid modeling services

8. Quality Control, Validation, and Functional Assays

8.1 Morphological Assessment and Growth Monitoring

Daily observation of intestinal organoids by brightfield microscopy is essential for quality control. Healthy intestinal organoids exhibit the following characteristics:

Signs of poor culture quality include:

8.2 Immunofluorescence Staining

Immunofluorescence staining of cryosectioned organoids (10–20 µm sections) is the gold standard for validating the cellular composition of intestinal organoids. The standard fixation and embedding protocol is as follows:

  1. Fix organoids in 4% paraformaldehyde (PFA) in PBS for 30–45 minutes at room temperature.
  2. Wash three times with PBS.
  3. Incubate in 30% sucrose in PBS overnight at 4°C for cryoprotection.
  4. Embed in optimal cutting temperature (OCT) compound and freeze at −80°C.
  5. Cryosection at 10–20 µm using a cryostat.
  6. Permeabilize with 0.3% Triton X-100 in PBS for 15 minutes.
  7. Block with 2% bovine serum albumin (BSA) + 2% normal goat serum in PBS for 1 hour.
  8. Incubate with primary antibodies overnight at 4°C.
  9. Wash and incubate with fluorophore-conjugated secondary antibodies for 1 hour at room temperature.
  10. Counterstain with DAPI and mount with antifade mounting medium.

Key markers for intestinal organoid characterization:

8.3 Forskolin-Induced Swelling (FIS) Assay for CFTR Function

The FIS assay is a functional readout of cystic fibrosis transmembrane conductance regulator (CFTR) activity and is widely used for cystic fibrosis diagnosis, drug screening, and research. The assay is based on the principle that forskolin activates adenylate cyclase, increasing intracellular cAMP levels and activating CFTR-mediated chloride secretion. The influx of chloride and water causes the organoid lumen to swell, which can be quantified by brightfield microscopy [7,16].

Protocol:

  1. Culture intestinal organoids from cystic fibrosis patients or controls in complete medium until they are well-formed (day 5–7 after passage).
  2. Replace the medium with Advanced DMEM/F12 containing 10 µM forskolin (Sigma-Aldrich, F6886).
  3. Incubate at 37°C for 1–4 hours.
  4. Capture brightfield images at baseline (0 hours) and at 1, 2, and 4 hours.
  5. Quantify the change in cross-sectional area using image analysis software (e.g., ImageJ or Fiji). Normal organoids with functional CFTR exhibit 20–50% increase in cross-sectional area within 4 hours. CFTR-deficient organoids show minimal or no swelling (<5% increase).
  6. For drug screening, add CFTR modulators (e.g., VX-770, VX-809) to the medium 24 hours before the FIS assay and compare the swelling response to vehicle-treated controls [7,16].

8.4 Transepithelial Electrical Resistance (TEER)

TEER measures the electrical resistance across the epithelial barrier and is an indicator of tight junction integrity and barrier function. TEER can be measured in intestinal organoids using microelectrodes inserted into the organoid lumen or by plating organoid-derived monolayers on permeable supports. Normal intestinal organoids exhibit TEER values of 200–500 Ω·cm², which is lower than the in vivo intestine but consistent with the immature state of the in vitro epithelium [2,17].

Related resource: Drug screening services

9. Troubleshooting Common Issues in Intestinal Organoid Culture

Issue: Low plating efficiency (<50% of crypts form organoids)

Potential Causes and Solutions:

Issue: Organoid death or poor growth after passage

Potential Causes and Solutions:

Issue: Loss of budding morphology and cystic enlargement

Potential Causes and Solutions:

Issue: Excessive differentiation or premature loss of stem cells

Potential Causes and Solutions:

Related resource: Organoid modeling services

10. Conclusion and Practical Recommendations

Intestinal organoid culture has matured into a robust, reproducible, and widely accessible technology that enables the detailed study of intestinal epithelial homeostasis, host-pathogen interactions, and personalized medicine. The key to successful intestinal organoid culture lies in meticulous attention to tissue processing, growth factor quality, matrix handling, and environmental conditions. By following the detailed protocols and parameters outlined in this guide, researchers can establish and maintain high-quality intestinal organoid cultures for a broad spectrum of applications, from basic developmental biology to clinical drug screening.

For new laboratories entering the field, we recommend starting with commercially available defined media (such as IntestiCult™) and established protocols before attempting to optimize custom formulations. As experience is gained, the protocols can be adapted to specific research needs, including the generation of genetically modified organoids, the co-culture with immune cells or microbes, and the integration with organ-on-chip platforms.

GBiowit provides comprehensive intestinal organoid solutions, including IntestiCult-compatible defined media formulations, growth factor cocktails with verified activity, Matrigel and defined matrix alternatives, complete organoid kits for mouse and human intestinal organoid culture, and specialized reagents for quality control and functional validation. Our technical support team can provide guidance on protocol optimization, troubleshooting, and custom media development for specific applications.

Visit: GBiowit homepage (www.gbiowit.com)

References

[1] Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt‑villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262‑265. PubMed DOI
[2] Sato T, Stange DE, Ferrante M, et al. Long‑term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011;141(5):1762‑1772. PubMed DOI
[3] Balaya RDA, Gopinathan A. A Practical Guide to Developing and Troubleshooting Patient‑Derived "Mini‑Gut" Colorectal Organoids for Clinical Research. Bioengineering. 2025;8(5):121. PubMed DOI
[4] Urbischek M, Rannani M, Gopalkrishnan M, et al. Organoid culture media formulated with growth factors of defined cellular activity. Sci Rep. 2019;9:6193. PubMed DOI
[5] Yin X, Farin HF, van Es JH, et al. Niche‑independent high‑purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat Methods. 2014;11(1):106‑112. PubMed DOI
[6] Mahe MM, Aihara E, Schumacher MA, et al. Establishment of Gastrointestinal Epithelial Organoids. Curr Protoc Mouse Biol. 2013;3(4):217‑240. PubMed DOI
[7] Dekkers JF, Wiebrands K, de Jonge HR, et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med. 2013;19(7):939‑945. PubMed DOI
[8] Miyoshi H, Stappenbeck TS. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc. 2013;8(12):2471‑2482. PubMed DOI
[9] Fujii M, Shimokawa M, Date S, et al. A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis. Cell Stem Cell. 2016;18(6):827‑838. PubMed DOI
[10] StemCell Technologies. IntestiCult Organoid Growth Medium (Human) Technical Manual. Visit Website
[11] Bio‑Techne. Cultrex UltiMatrix RGF BME Product Information. Visit Website
[12] Gjorevski N, Sachs N, Manfrin A, et al. Designer matrices for intestinal stem cell and organoid culture. Nature. 2016;539(7630):560‑564. PubMed DOI
[13] Sato T, Clevers H. Growing self‑organizing mini‑guts from a single intestinal stem cell: mechanism and applications. Science. 2013;340(6137):1190‑1194. PubMed DOI
[14] Haber AL, Biton M, Rogel N, et al. A single‑cell survey of the small intestinal epithelium. Nature. 2017;551(7680):333‑339. PubMed DOI
[15] Kawasaki T, Kawai T. RANKL‑induced M cell differentiation in intestinal organoids. Methods Mol Biol. 2019;1960:101‑111. PubMed DOI
[16] Dekkers JF, van der Ent CK, Beekman JM. Novel opportunities for CFTR‑targeting drug development using organoids. Rare Dis. 2013;1:e945047. PubMed DOI
[17] Co JY, Margalef‑Català M, Li X, et al. Controlling Epithelial Polarity: A Human Enteroid Model for Host‑Pathogen Interactions. Cell Rep. 2019;26(9):2509‑2520.e4. PubMed DOI