generation of rat intestinal 2d monolayers from 3d organoids

大鼠肠隐窝分离和肠道类器官生成

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.

作者聚焦：大鼠肠道类器官的产生和操作  

大鼠肠道类器官的产生和操作

In this study we established a rat intestinal organoid model that is robust in long-term cell culture. We also demonstrate the first successful genetic manipulations using both lentiviral infection and transient transfection. This model is an excellent tool for manipulating intestinal biology in the rat where the genetic tools available in vivo are limited.Rat intestinal organoids have previously been difficult to culture long-term. By generating a robust, genetically-manipulatable rat organoid model, we are providing researchers additional flexibility and choice to select an organoid model best suited for their particular circumstances. Mouse intestinal organoids are used to study stem cell behavior, cell fate and physiology, but they can be a suboptimal model.Since there are key differences between mouse and human biology. On the other hand, human intestinal organoids can be difficult to obtain, and have complex cell culture requirements. The rat organoid model we describe here retains key physiological relevance to human biology, while being considerably easier to access and care for than human intestinal organoids.Developing and optimizing the rat intestinal organoid model allows for genetic manipulation, pharmacological treatments, and higher throughput studies of intestinal biology and accessible model with key physiological relevance to humans. Additionally, these rat organoids will allow for the study of species-specific physiology, cell fates, and phenotypic variations and disease models in the intestine.

Watch this Scientific Journal Video about Generation of Rat Intestinal 2D Monolayers from 3D Organoids at JoVE.com

Generation of Rat Intestinal 2D Monolayers from 3D Organoids  

从3D类器官生成大鼠肠道2D单层

要用 EME 包被板，请在冷的 Advanced DMEM+ 中稀释 EME 1 至 20 对于用胶原蛋白包被，在 Advanced DMEM+ 中将每毫升 5 毫克的胶原蛋白 I 稀释至 100 微克/毫升。用 200 微升稀释的 EME 或胶原蛋白涂覆板以完全覆盖孔表面，并在 37 摄氏度下孵育 1 至 2 小时。要生成单层，请从含有类器官的 35 毫米板中吸出培养基。加入一毫升PBS，用P1000吸头破坏孔中的EME，上下移液约20次以松开所有EME。将内容物转移到 15 毫升锥形管中。向平板中加入一毫升PBS以收集其他类器官并将它们转移到同一个锥形管中。将样品以350G离心两分钟，并除去上清液，包括任何EME残留物。然后，向类器官沉淀中加入一毫升胰蛋白酶溶液，并在 37 摄氏度下孵育两分钟。使用 P1000 吸头上下移液 10 次，并加入 2 毫升 Advanced DMEM+ 以中和胰蛋白酶。以 350 G 离心 5 分钟并吸出 supernant，然后将沉淀重悬于 4.8 毫升 rIOM2D 中。从孔中去除高级DMEM +中多余的EME或胶原蛋白。然后，在每个预包被孔中加入 200 微升 rIOM2D 类器官和 10 微摩尔 Y27632。4 至 16 小时后，收集培养基并以 1， 000 G 离心 1 分钟。将上清液转移到新的15毫升锥形管中，弃去沉淀。用 300 微升 PBS 洗涤每个孔，然后向每个孔中加入 200 微升离心的 rIOM2D。每两到三天更换一次 rIOM2D，暂停使用镀在 EME 上的 Y27632.2D 单层，当 EME 加回电池顶部时，这些单层很容易重整为小球体。相比之下，I型胶原基质不足以改造3D结构。

generation-of-rat-intestinal-2d-monolayers-from-3d-organoids

Research

JoVE Journal

Biology

Generation of Rat Intestinal 2D Monolayers from 3D Organoids

419 次观看.  Yale School of Medicine. 要用 EME 包被板，请在冷的 Advanced DMEM+ 中稀释 EME 1 至 20 对于用胶原蛋白包被，在 Advanced DMEM+ 中将每毫升 5 毫克的胶原蛋白 I 稀释至 100 微克/毫升。用 200 微升稀释的 EME 或胶原蛋白涂覆板以完全覆盖孔表面，并在 37 摄氏度下孵育 1 至 2 小时。要生成单层，请从含有类器官的 35 毫米板中吸出培养基。加入一毫升PBS，用P1000吸头破坏孔中的EME，上下移液约20次以松开所有EME...

视频: Generation of Rat Intestinal 2D Monolayers from 3D Organoids  

Generation and Manipulation of Rat Intestinal 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.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

科研

生物学

Establishment and Passaging of Small Intestine Organoids

To begin, place the euthanized rat onto the dissection surface with the ventral side up. Pinch the skin layer with sterile forceps and make cuts at the surface level, avoiding damage to internal organs. Using large sharp dissection scissors, make a longitudinal surface level cut in the center of the abdomen.Then stemming from that cut, make two shorter horizontal cuts one on each side. Using forceps, peel the skin away to expose the abdominal cavity. Cut through the peritoneal membrane to fully expose the internal organs in the abdominal cavity with ready access to the intestine.Using scissors and forceps, locate the stomach and identify the duodenum, approximately two to three centimeters distal to it which appears as a yellowish segment. The proximal jejunum is located approximately four to five centimeters distal to the ligament of Treitz. A landmark between the duodenum and jejunum.Place the isolated intestinal fragment into a 10 centimeter Petri dish. Flush the desired intestinal segment of mesentery with 10 milliliters of ice cold PBS until cleared of luminal contents. On a paper towel, cut the intestinal segment into two centimeter long pieces.Then open each intestinal piece longitudinally to expose the epithelium. Scrape the exposed luminal surface using a glass microscope slide to remove villi. Place the intestinal pieces into the EDTA solution on ice and rotate it four degrees Celsius for 30 minutes on a tube revolver set to 10 RPM.At the dissecting microscope, pour the contents of the tube into a 10 centimeter Petri dish. Add in an additional five milliliters of ice cold PBS. Using fine forceps, hold an intestinal segment and shake vigorously to observe the epithelium release into the PBS.Initially, the PBS will contain mostly villi. Shake the intestinal fragments continuously, periodically discard the PBS containing villi, and add 10 milliliters of fresh PBS to the intestinal fragments. Continue to shake the fragments and repeat the PBS washing step until villi are no longer released into the PBS.But instead, the PBS primarily contains crypts. Discard the remaining intestinal fragments and enrich the remaining PBS in the Petri dish for intestinal crypts. In a tissue culture hood, collect the PBS containing crypts and filter through a 70 micron cells drainer.Centrifuge the filtrate at 250 x g for five minutes. Then remove their supernatant and resuspend the pellet in five millimeters of advanced DMEM plus. Centrifuge again at 250 x g for five minutes.And remove the supernatant, leaving 50 microliters of media with the pellet. Resuspend the pellet in the remaining media and add it to the aliquot of the extracellular matrix extract or EME on ice. Gently pipet up and down to suspend the crypts evenly throughout the EME avoiding making bubbles.Place 50 microliters of the EME domes into a 35 millimeter tissue culture dish. An incubated 37 degrees Celsius with 5%carbon dioxide for 20 minutes. After incubation, add a two milliliters of rat intestinal organoid media, or RIOM, containing 10 micromolar each of Y27632 and CHIR99021.Next, to passage rat intestinal organoids, aspirate the medium from a plate containing organoids and add one milliliter of dissociation reagent to release the organoids from the EME domes. Transfer the dissociation reagent with fragmented organoids to a 15 milliliter conical tube. Wash the culture plate with two milliliters of advanced DMEM plus and add it to the 15 milliliter conical tube containing the fragmented organoids.Using a glass pasture pipette, gently pipette up and down 15 to 20 times to fragment the organoids. Centrifuge the organoids at 350 x g for two minutes and add 50 microliters of solution from the bottom of the tube into the EME. After mixing the solution, plate 50 microliters of the EME domes into a 35 millimeter tissue culture dish and incubate at 37 degrees Celsius with 5%carbon dioxide for 20 minutes.After incubation, add two milliliters of RIOM containing 10 micromolar each of Y27632 and CHIR99021. Representative images of villi fragments and crypts are shown. Crypts are smaller in size compared to villi.Plated crypts expand over the next few days and begin to bud and differentiate by days four to seven. After two days of thawing, a healthy organoid culture showed the presence of both spheroids and budded organoids. The same organoid line after passaging showed the presence of single crypt-like domains.

小肠类器官的建立和传代

To coat the plate with EME, dilute EME 1 to 20 in cold Advanced DMEM+For coating with collagen, dilute five milligrams per milliliter of collagen I in Advanced DMEM+to 100 micrograms per milliliter. Coat with the plate with 200 microliters of diluted EME or collagen to cover the well surface entirely and incubate for one to two hours at 37 degrees Celsius. To generate monolayers, aspirate the media from a 35 millimeter plate containing organoids.Add one milliliter of PBS and disrupt the EME in the wells with a P1000 tip, pipetting up and down approximately 20 times to loosen all the EME. Transfer the contents to a 15 milliliter conical tube. Add one milliliter of PBS to the plate to collect additional organoids and transfer them to the same conical tube.Centrifuge the sample at 350 G for two minutes and remove the supernatant, including any EME residue. Then, add one milliliter of trypsin solution to the organoid pellet and incubate at 37 degrees Celsius for two minutes. Pipette up and down 10 times using a P1000 tip and add two milliliters of Advanced DMEM+to neutralize trypsin.Centrifuge at 350 G for five minutes and aspirate the supernant before resuspending the pellet in 4.8 milliliters of rIOM2D. Remove excess EME or collagen in Advanced DMEM+from the wells. Then, add 200 microliters of organoids in rIOM2D and 10 micromolar Y27632 to each pre-coated well.After 4 to 16 hours, collect the media and centrifuge at 1, 000 G for one minute. Transfer the supernatant into a new 15 milliliter conical tube and discard the pellet. Wash each well with 300 microliters of PBS before adding 200 microliters of the centrifuged rIOM2D to each well.Change the rIOM2D every two to three days, suspending the use of Y27632.2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells. In contrast, a collagen I substrate was insufficient to reform 3D structures.