Adult stem-cell epithelial organoid culture
Materials Required
Principle
Adult stem-cell epithelial organoid culture is a three-dimensional culture method in which adult epithelial stem cells or isolated epithelial crypts self-organize into organ-like epithelial structures that retain stem-cell activity and generate differentiated epithelial lineages[1][2][3].
In the intestinal model, Lgr5-positive crypt base columnar cells are adult stem cells that generate all intestinal epithelial lineages, and single Lgr5-positive cells or isolated crypts can form long-term crypt–villus organoids in extracellular matrix culture when supplied with epithelial niche signals[1][2][3].
Organoid growth reflects epithelial stem-cell survival, self-renewal, proliferation, and multilineage differentiation; the main readouts are organoid-forming efficiency, growth, morphology, passaging capacity, marker expression, and lineage composition[2][4][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
Reagents and chemicals
• Use freshly isolated epithelial tissue or sorted adult epithelial stem cells, extracellular matrix such as Matrigel, basal organoid medium, EGF, Noggin, R-spondin, and standard cell-culture supplements; Wnt3A, nicotinamide, A83-01, SB202190, gastrin, CHIR99021, valproic acid, and other small molecules are added only in tissue-specific or species-specific protocols where supported ([2][4][6][7][8]).Antibodies, probes, dyes, or kits
• Use viability dyes or live/dead assays to assess organoid survival, EdU or Ki-67 staining to assess proliferation, lineage-marker antibodies to detect differentiated epithelial cell types, and qPCR or immunostaining for stem-cell and epithelial markers such as Lgr5, Olfm4, E-cadherin, villin, lysozyme, MUC2, and chromogranin A when appropriate for the tissue model ([4][5][9]).Equipment and instruments
• Use sterile tissue-processing tools, a biosafety cabinet, centrifuge, incubator, inverted microscope, fluorescence microscope or confocal microscope, flow cytometer or cell sorter when isolating Lgr5-positive cells, and image-analysis software for organoid counting and size measurement ([4][5][9]).Experimental Procedure
Preparation Steps
• Collect epithelial tissue under sterile conditions, isolate crypts or epithelial fragments using the tissue-specific protocol, enrich or sort stem-cell populations when required, and keep samples cold during preparation before embedding in extracellular matrix[4][5][9].• Prepare extracellular matrix domes or equivalent 3D matrix cultures and overlay them with organoid medium containing the growth factors required for the selected epithelial tissue; mouse small intestinal organoids classically use EGF, Noggin, and R-spondin, while human intestinal and other adult epithelial organoids may require additional pathway modulators[2][4][6][8].
Operation Steps
• Embed isolated crypts or sorted epithelial stem cells in extracellular matrix, allow the matrix to polymerize, add complete organoid medium, culture under standard mammalian cell-culture conditions, and monitor organoid initiation, budding, expansion, and viability by microscopy[2][4][5].• Refresh culture medium regularly according to the selected validated protocol and passage organoids by mechanical or enzymatic dissociation when organoids expand sufficiently; passaged fragments are re-embedded in fresh extracellular matrix with fresh complete medium[4][5][8].
• For Lgr5-positive single-cell organoid culture, sort viable Lgr5-positive epithelial cells, embed them in extracellular matrix, and provide niche factors that support colony formation; Paneth cells or exogenous Wnt signals can improve organoid formation from small-intestinal Lgr5-positive cells[3][5][7].
Data Acquisition and Analysis
• Quantify organoid-forming efficiency by counting organoids relative to the number of seeded crypts or cells, measure organoid size or budding morphology over time, and compare expansion or differentiation across experimental groups using matched passage number and culture conditions[4][5][9].• Validate epithelial identity and lineage composition by morphology, qPCR, immunostaining, or flow cytometry for stem-cell, proliferative, and differentiated lineage markers; organoids should be interpreted as epithelial models that preserve key tissue features but do not fully reproduce all in vivo stromal, immune, vascular, or neural components unless those components are experimentally added[2][4][10].
Troubleshooting
Problem: Few or no organoids form after seeding.
• Possible Cause: Insufficient stem-cell niche signaling or poor starting-cell viability.• Literature-supported Solution: Use validated epithelial niche factors such as EGF, Noggin, and R-spondin for intestinal organoids and verify crypt or Lgr5-positive cell viability before embedding[2][4][5].
Problem: Single Lgr5-positive cells show low colony formation.
• Possible Cause: Loss of niche support after dissociation.• Literature-supported Solution: Co-culture with Paneth cells or provide exogenous Wnt stimulation, because Paneth cells supply essential niche signals and improve organoid formation from Lgr5-positive cells[3].
Problem: Organoids expand but show limited differentiation.
• Possible Cause: Culture conditions favor stem-cell maintenance over lineage differentiation.• Literature-supported Solution: Adjust pathway modulation using validated differentiation conditions; combined Wnt and Notch modulation has been used to direct Lgr5-positive intestinal stem-cell progeny toward enterocytes, goblet cells, or Paneth cells[7].
Problem: Human intestinal organoids lose cellular diversity during expansion.
• Possible Cause: Culture conditions do not adequately preserve both self-renewal and differentiation.• Literature-supported Solution: Use niche-inspired human intestinal organoid conditions validated to maintain self-renewal capacity and cellular diversity[8].
References:
- [1]. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449(7165):1003-1007. [Content Brief]
- [2]. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262-265. [Content Brief]
- [3]. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 2011;469(7330):415-418. [Content Brief]
- [4]. Mahe MM, Aihara E, Schumacher MA, Zavros Y, Montrose MH, Helmrath MA, et al. Establishment of gastrointestinal epithelial organoids. Curr Protoc Mouse Biol. 2013;3(4):217-240. [Content Brief]
- [5]. Liu R, et al. Lgr5+ intestinal stem cell sorting and organoid culture. Anim Model Exp Med. 2019;2(2):132-135. [Content Brief]
- [6]. Sato T, Stange DE, Ferrante M, Vries RG, van Es JH, van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 2011;141(5):1762-1772. [Content Brief]
- [7]. Yin X, et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat Methods. 2014;11(1):106-112. [Content Brief]
- [8]. Fujii M, Matano M, Toshimitsu K, Takano A, Mikami Y, Nishikori S, et al. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell. 2018;23(6):787-793.e6. [Content Brief]
- [9]. Flanagan DJ, et al. Isolation and culture of adult intestinal, gastric, and liver organoids for Cre-recombinase-mediated gene deletion. Methods Mol Biol. 2019;1576:39-64. [Content Brief]
- [10]. Pastuła A, Middelhoff M, Brandtner A, Tobiasch M, Höhl B, Nuber AH, et al. Three-dimensional gastrointestinal organoid culture in combination with nerves or fibroblasts: a method to characterize the gastrointestinal stem cell niche. Stem Cells Int. 2016;2016:3710836. [Content Brief]
LinkedIn
Researchgate
Instagram
X
Facebook
WhatsApp
Reddit
Mail