Proven Protocols for Intestinal Organoid Culture
Download this resource to access optimized protocols for getting started with first-generation IntestiCult鈩 and work your way up to advanced downstream assays.
Intestinal organoids have come a long way since their development in 2009. Considering their application in a variety of fields, from developmental biology to drug discovery, personalized medicine, and more, intestinal organoids are a powerful tool for any researcher.
This e-book provides an introduction for researchers who are new to intestinal organoids or are considering integrating them into future research, as well as more advanced protocols to bring your research to the next level.
In this e-book, you will find:
- Strategies for successful isolation and subsequent culture of first-generation human-derived intestinal organoids.
- Proven protocols for a wide variety of well-established assays, from immunocytochemical staining, toxicity testing, and genetic manipulations, to protocols on creating and using intestinal organoid-derived monolayers.
Continue reading for a preview from the e-book.
Page 4, Introduction
From the Intestinal Epithelium to Intestinal Organoids
The intestinal epithelium incorporates several distinct cell populations, including rapidly dividing intestinal stem cells (ISCs) that facilitate the typical four-to-five-day turnover cycle of the adult intestinal epithelium.4 This property of rapid regeneration makes the intestine a uniquely convenient model system for epithelial cell biology and adult stem cell biology studies both inside and outside the specific context of intestinal function. The intestinal niche has been well-characterized and consists of spatial gradients of high wingless-related integration site (Wnt) and epithelial growth factor (EGF), and low bone morphogenetic protein (BMP). The discovery of LGR5 expression, a protein in the Wnt signaling pathway, in ISCs, which can give rise to all cell types in the adult intestine, heavily informed the development of the culture conditions for intestinal organoids.
Figure 1. Spatial Arrangements of Key Epithelial Cell Types in the Intestinal Epithelium and Intestinal Organoids
(A) The adult intestinal epithelium is primarily composed of six cell types that are arranged in a crypt-villus structure.6 At the base of the intestinal crypt, the intestinal stem cells are found intercalated with Paneth cells and are credited with much of the signaling required to maintain the stem cell niche.6 Transit-amplifying cells are partially differentiated cells that migrate upward via a physical crypt exclusion mechanism as the ISCs below them divide. As these cells move upward out of the crypt, they move along signaling gradients that trigger them to differentiate, giving rise to the mature cell types that populate the villus domain. Mature cells include enterocytes, which make up the majority of the villus epithelium and carry out nutrient absorption; goblet cells, which secrete mucus to protect the epithelial lining and help move intestinal contents through the lumen; and enteroendocrine cells, which respond to chemical actuators in the luminal contents by secreting hormones into the body to maintain nutrient metabolism. (B) Intestinal organoids derived from TSCs mature and develop distinct crypt and villus domains with similar cellular distribution to that of the intestinal epithelium.
Page 22, Experimental Techniques Applied to Intestinal Organoid Cultures
Toxicity Testing for Drug Development Using Human Intestinal Organoids and IntestiCult鈩
Gastrointestinal toxicity is often a limiting factor in the development of therapeutic drugs, and traditional toxicity testing methods using immortalized cell lines or animal models may not show the true clinical effects.8 Due to their increased complexity, intestinal organoids are a physiologically relevant in vitro cell model that can overcome the limitations of traditional methods for investigating intestinal epithelial cell biology and modeling disease.9
The following protocol provides a step-by-step guide for carrying out toxicity testing using intestinal organoids, providing researchers with the tools to effectively assess new therapeutic targets. Researchers working with specific applications may need to optimize this protocol further.
Materials
- IntestiCult鈩 Organoid Growth Medium (OGM) (Human) (Catalog #06010)
- 25% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)
- DMEM/F-12 with 15 mM HEPES (Catalog #36254)
- Corning庐 Matrigel庐 Matrix, Growth Factor Reduced (GFR), Phenol Red-Free (e.g. Corning Catalog #356231)
- Costar庐 24-Well Flat-Bottom Plate, Tissue Culture-Treated (Catalog #38017)
- Costar庐 96 Flat-Bottom Plate, Tissue Culture-Treated (e.g. Sigma CLS Catalog #3595)
- Falcon庐 70 渭m Cell Strainer (e.g. Corning Catalog #352350)
- Y-27632 (Catalog #72302)
- STEMvision鈩 (Catalog #22000)
- CellTiter-Glo庐 3D Cell Viability Assay (Promega Catalog #G9683)
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- Gentle Cell Dissociation Reagent (Catalog #07174)
- Pipettor (e.g. Corning庐 Lambda鈩 Plus Pipettor, Catalog #38060)
- Pipette tips (e.g. Corning庐 Filtered Pipette Tips, Catalog #38034)
- Serological pipettes (e.g. Falcon庐 Serological Pipettes, 10 mL, Catalog #38004)
- Imaging device capable of reading luminescence in multiwell plates (e.g. Promega GloMax庐 Microplate Reader)
Protocol
A. Expansion of Organoids
- Expand organoids in a 24-well plate according to standard protocols. Estimate 1 x 50 渭L dome in a
24-well plate to seed 8 x 10 渭L domes of a 96-well plate. Note: This is an estimate only and should be adjusted based on organoid size and density.
- Grow organoids for 7 to 10 days, until they have reached full size but have not begun to differentiate and shed dead cells.
- Follow standard protocols for passaging intestinal organoids as described on page 8.
- Centrifuge the sample at 200 x g for 5 minutes.
- Remove as much supernatant as possible and store on ice.
- Resuspend the organoid pellet in an appropriate amount of Matrigel庐 (10 渭L per well to be seeded) and mix gently.
- Remove a pre-warmed 96-well plate from the 37掳C incubator.
- Remove 10 渭L of the organoid-Matrigel庐 solution from the tube and slowly, while keeping the pipette completely upright, add the 10 渭L droplet to the center of the target well.
- Repeat for the remainder of the samples.
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