Name: Video: Establishment and Passaging of Small Intestine Organoids
Uploaded: 2023-06-23T12:55:00
Duration: 5 min 32 s
Description: 666 Views. Establishment and Passaging of Small Intestine Organoids

establishment and passaging of small intestine organoids

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

The human small intestinal epithelial architecture and cellular composition are complex, reflecting their physiological functions. The primary role of the small intestine is to absorb nutrients from food passing through its lumen1. To maximize this function, the intestinal surface is organized into finger-like protrusions called villi, which increase the absorptive surface area, and cup-like invaginations called crypts, which house and insulate the stem cells. Within the epithelium, various specialized absorptive and secretory cell types are generated to perform distinct functions1. Because of this complexity, it has been difficult to model tissues like the intestine in high passage-transformed immortalized cell lines. However, the study of stem cells, especially adult stem cells and their differentiation mechanisms, has allowed for the development of 3D intestinal organoid cultures. The use of organoid models has transformed the field, in part due to their recapitulation of some architectural components and cell type heterogeneity found in the intact intestine. Intestinal organoids can be cultured long-term in vitro due to the maintenance of the active stem cell population2.
Intestinal organoids have rapidly become an adaptable model to study stem cell biology, cell physiology, genetic disease, and nutrition3,4, as well as a tool to develop novel drug delivery methods5. In addition, patient-derived organoids are being utilized for disease modeling, drug discovery, and personalized treatment screening, among others6,7,8,9. However, human intestinal organoids still present challenges. Tissue availability, requirements for Institutional Review Board approval, and ethical issues limit the widespread use of human samples. Additionally, human intestinal organoids generated from intestinal crypts require two distinct culture conditions for the maintenance of undifferentiated stem cells or to induce the differentiation of mature cell types10. This contrasts with in vivo, where stem cells and mature differentiated cell types are simultaneously present and continuously generated/maintained1. On the other hand, mouse intestinal organoids, which are grown in a less complex cocktail of growth factors, do not require this switch in media composition and can maintain stem cells and differentiated cells in the same media context2,11. However, key differences in mouse intestine when compared to humans can make mouse organoids a suboptimal model in many instances. Overall, many intestinal organoids from larger mammals, including horses, pigs, sheep, cows, dogs, and cats, have been successfully generated in culture conditions more closely aligned with mouse intestinal organoids than the culture conditions of human intestinal organoids12. The differences in growth factor conditions between mouse and human organoids likely reflect differences in stem cell niche composition and differing requirements for stem cell survival, proliferation, and maintenance. Therefore, there is a need for an easily accessible model organoid system that 1) closely resembles human intestinal cell composition, 2) contains stem cells with growth factor requirements like those of human intestinal organoids, and 3) is capable of continuously maintaining undifferentiated and differentiated compartments. Ideally, the system would be from a commonly used preclinical animal model, such that in vivo and in vitro experiments can be correlated and used in tandem.
Rats are a commonly used preclinical model for intestinal physiology and pharmacology studies due to their very similar intestinal physiology and biochemistry to humans13, particularly with regards to intestinal permeability14. Their relatively larger size compared to mice makes them more amenable to surgical procedures. While large animal models, including pigs, are sometimes used, rats are a more affordable model, require less space for husbandry, and have readily commercially available standard strains15. A drawback of using rat models is that the genetic toolkit for in vivo studies is not well developed compared to mice, and the generation of novel rat lines, including knockouts, knock-ins, and transgenics, is often cost-prohibitive. The development and optimization of a robust rat intestinal organoid model would allow for genetic manipulation, pharmacological treatments, and higher throughput studies in an accessible model that retains key physiological relevance to humans. However, the advantages of one rodent organoid model versus another is highly dependent on the particular process or gene being studied; certain genes found in humans may be pseudogenes in mice but not rats16,17. Additionally, species-specific cell subtypes are increasingly being unveiled by single-cell RNAseq18,19,20. Finally, rat and mouse intestinal disease models often display considerable variations in phenotypes21,22 such that the model that more closely recapitulates the symptoms and disease process seen in humans must be selected for downstream work. The generation of a rat intestinal organoid model provides additional flexibility and choice for researchers in selecting a model system most appropriate for their circumstances. Here, existing protocols23,24 are expanded upon for the generation of rat intestinal organoids and a protocol is outlined for the generation and maintenance of rat intestinal organoids from the duodenum or jejunum. In addition, several downstream applications, including lentiviral infection, whole mount staining, and forskolin swelling assays, are described.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Generation and Manipulation of Rat Intestinal Organoids

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Establishment and Passaging of Small Intestine Organoids  

establishment-and-passaging-of-small-intestine-organoids

Research

JoVE Journal

Biology

666 Views. Establishment and Passaging of Small Intestine Organoids

Video: Establishment and Passaging of Small Intestine Organoids  

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.