Name: recapitulating suckling-to-weaning transition in vitro using fetal intestinal organoids
Uploaded: 2019-11-15T11:00:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids at JoVE.com

This study was conducted in accordance with institutional guidelines for the care and use of laboratory animals established by the Ethic Committee for Animal Experimentation of the University of Amsterdam in full compliance to the national legislation on animal research following the European directive 2010/63/EU for the use of animals for scientific purposes (ALC312).
1. Isolation of fetal small intestinal organoids
<ol>
	<li>Sacrifice the E18-E20 fetuses by decapitation with surgical scissors, according to the approved ethical regulations.</li>
	<li>Immediately after euthanasia, carefully cut open the lower abdomen of the fetus with intestinal scissors and remove the whole intestine. 
	NOTE: Isolation must be performed with intestines between E18 and E20 of gestation in order to achieve the proper suckling-to-weaning transition and maturation in vitro.</li>
	<li>With two small forceps, stretch the intestine. Using the stomach and the appendix as guides, cut apart the proximal and distal part of the small intestine. 
	NOTE: If dissection is done carefully, it is possible to isolate the colon as well. Cut it apart using appendix as guide (Figure 1).</li>
	<li>Make the intestine open longitudinally: fix the intestine using a razor blade placed lengthwise and pull the intestine by sliding forceps (one arm of the forceps on each side of the razor blade) along the razor blade. Subsequently, cut the opened intestine in 1 cm pieces.</li>
	<li>Prepare two 50 mL tubes with 10 mL of ice cold phosphate-buffered saline (PBS). Transfer the proximal and distal part of the small intestine (and the colon if applicable) separately to the tubes. Keep the tubes on ice while dissecting the intestines of additional mice. Collect all intestinal parts of one litter together in the same tube. 
	NOTE: One litter has usually 6-10 fetuses. The intestines of all fetuses must be combined. This amount should be enough to yield 4-8 wells with organoids, in a 48-well plate, per intestinal part.</li>
	<li>Proceed with organoid isolation as previously described11. In short, wash intestinal pieces with ice-cold PBS, incubate with 2 mM ethylenediaminetetraacetic acid (EDTA) for 30 minutes at 4 &#176;C, dissociate the crypts from the tissue by harshly washing the pieces with ice cold PBS + 10% fetal calf serum (FCS), filter using a 70 &#181;m cell strainer and centrifuge to collect the crypts at 150 x g for 5 min at 4 &#176;C.</li>
	<li>Plate 4 to 8 wells with crypts in extracellular matrix gel, depending on the pellet size, in a warm 48-well plate. Use 20 &#181;L of crypts suspended in extracellular matrix gel per well. Let extracellular matrix gel solidify in a 37 &#176;C incubator for 10 min. 
	NOTE: Considering that only 40% to 50% of isolated crypts form organoids, aim for a density of 250 to 300 organoids per well. First add less extracellular matrix gel than needed. Look under the microscope after platting the first well to analyze whether the density of the isolated crypts is ideal. If necessary, add more extracellular matrix gel.</li>
	<li>In the meantime prepare ENR medium: 14 mL of Advanced Dulbecco&#8217;s Modified Eagle Medium/Ham&#8217;s F-12 (DMEM/F12) 1:1 +++ (supplemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 1x, L-glutamine 0.01 M and 0.2U/mL Penicillin/Streptomycin), 4 mL of Noggin-conditioned media, 2 mL of Rspondin-conditioned media, 400 &#181;L of B27 supplement 1x, 200 &#181;L of N2 supplement 1x, 50 &#181;L of 1.25 mM n-Acetylcysteine, 20 &#181;L of 0.05 &#181;g/mL Epidermal Growth Factor (EGF). 
	NOTE: When culturing colon organoids, supplement with 50% Wnt conditioned media.</li>
	<li>Add 250 &#181;L of ENR medium per well.</li>
</ol>
2. Culturing of fetal organoids
<ol>
	<li>Change medium of the cultures 3 times per week. Maturation of the organoids is achieved after 1 month of culture.</li>
	<li>At day 3 after each passage, count the number of spheroids and organoids using an optical microscope. Quantify at least 3 wells per condition and all the organoids present in each well. 
	NOTE: Number of spheroids reduces with time, while the number of organoids increases (Figure 2).</li>
	<li>Passage the organoids once a week by mechanical dissociation as described below.
	<ol>
		<li>Remove medium and add 1 mL of ice-cold Advanced DMEM/F12 1:1 +++. Collect all extracellular matrix gel with organoids into a 15 mL tube. Use a 200 &#181;L tip on top of a 1000 &#181;L filter tip and pipette up and down 20 times to disrupt the organoids.</li>
		<li>Centrifuge at 150 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 20 &#181;L of extracellular matrix gel per well. Usually, fetal organoids can be expanded in a 1:2 ratio.</li>
		<li>Let extracellular matrix gel solidify for 5-10 min. Add 250 &#181;L of ENR medium per well.</li>
	</ol>
	</li>
</ol>
3. Maturation analysis at RNA and protein level
<ol>
	<li>Analyze culture every 3 days after each passage, for a period of 1 month (i.e., time in which complete maturation is achieved) (Figure 3). 
	NOTE: Fetal organoid culture is dynamic (Movie 1) and to avoid variation from the normal regeneration of organoids after mechanical disruption, it is necessary to always collect samples at the same day after passage.</li>
	<li>RNA isolation
	<ol>
		<li>Collect 3 wells of organoids using 200 &#181;L of RNA lysis buffer for each well supplemented with 2 &#181;L of &#946;-mercaptoethanol. After adding RNA lysis buffer+&#946;-mercaptoethanol to the well, make sure all extracellular matrix gel with organoids is transferred to a RNase-free 1.5mL tube.</li>
		<li>Vortex vigorously and keep at -80 &#176;C for no longer than 1 month. Isolate RNA using commercially available silica spin column kits.</li>
		<li>To increase RNA quality, after washing steps add 500 &#181;L of 80% EtOH and gently mix by inverting the column. Centrifuge for 2 min at 11,000 x g to dry the column completely. 
		NOTE: Make sure to wash the inside of the lid of the tube with the 80% EtOH by flicking the tube upside down three to five times to get rid of all traces of guanidine thiocyanate.</li>
		<li>To increase RNA yield, wait 1-2 min after applying RNase-free water before centrifuge. Re-elute RNA by applying the first flow-through eluate to the column a second time. 
		NOTE: Isolated RNA quality is sufficient for use in genome-wide expression analysis. Check whether RNA integrity number is above 8.</li>
	</ol>
	</li>
	<li>Protein isolation
	<ol>
		<li>Collect 5 wells of organoids using 250 &#181;L of ice-cold cell recovery solution into one 15 mL tube. Incubate for at least 30 min on ice to dissolve the extracellular matrix gel (this will reduce protein contribution from extracellular matrix gel).</li>
		<li>Wash with ice-cold PBS. Add 250 &#181;L of cell lysis buffer and store at -80 &#176;C. 
		NOTE: After sonication, samples can be used to detect enzyme activity or for Western Blots.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Collect 2 wells of organoids using 250 &#181;L of ice-cold cell recovery solution into a 15 mL tube. Incubate for at least 30 min on ice to dissolve the extracellular matrix gel (this will reduce staining background). Wash with ice-cold PBS.</li>
		<li>Fix the organoids using 500 &#181;L of of 4% paraformaldegyde (PFA) for 1 h at 4 &#176;C. Wash with ice-cold PBS. Proceed to whole-organoid immunofluorescence or to paraffin embedding, according to published protocols12. 
		NOTE: Use a plastic Pasteur pipette to handle the organoids. This will avoid disruption of its structure.</li>
	</ol>
	</li>
</ol>
4. Effect of extrinsic factor (dexamethasone as an example) on organoid maturation process
<ol>
	<li>On day one of culture, add 0.01M dexamethasone to the organoids. Incubate the organoids with dexamethasone during the whole month of culture by adding new dexamethasone every time medium is changed.</li>
	<li>Gene expression analysis
	<ol>
		<li>Isolate RNA as described above. Synthesize, at the same time, cDNA of all samples to be compared (treated and untreated). Proceed with preferred qRTPCR method.</li>
		<li>Use GeNorm to identify the two most stable reference gene every time a new treatment is used on the fetal organoids. Use the geometric mean of the two chosen reference genes for relative expression calculations. 
		NOTE: Suggestions of reference genes to test for mouse fetal organoids: Cyclophilin, Gapdh, &#946;actin, 36b4, Hprt, Rpl4, Rpl32, Ppib and Tbp.</li>
		<li>To investigate how a certain extrinsic factor affects postnatal mouse fetal maturation, all the following genes should be evaluated.
		<ol>
			<li>Check whether fetal markers lactase (Lct), argininosuccinate synthase 1 (Ass1), B lymphocyte-induced maturation protein 1 (Blimp-1) and neonatal Fc receptor (FcRn) decrease in expression during the first two weeks of culture and are absent for the remaining culture time (Figure 4C). Analyze whether this pattern is altered.</li>
			<li>Check whether adult markers sucrase-isomaltase (Sis), arginase 2 (Arg2), trehalase (Treh) and lysozyme (Lyz) increase in expression after two weeks of culture (Figure 4D). Analyze whether this pattern is altered by the external factor. 
			NOTE: Dexamethasone is an external factor that can accelerate the maturation of the fetal organoids and can be used as a positive control in all experiments aimed to test other extrinsic factors.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Enzyme activity analysis
	<ol>
		<li>Isolate protein as described above. Detect intestinal disaccharidases activity according to protocols published by Dahlqvist and Messer13,14.</li>
		<li>Prepare 0.625 M maleic-buffer pH 6.0 (keep for 3 months at 4 &#176;C). Using this buffer, prepare 0.05 M lactose (add p-hydroxymercuribenzoate sodium as stabilizer to inhibit lysosomal p-galactosidase activity); 0.05 M maltose; 0.05 M sucrose and 0.05 M trehalose. 
		NOTE: All these solutions can be kept for 5 days at 4 &#176;C. Keep on ice while preparing assay.</li>
		<li>Prepare assay standards by diluting 5.56 M glucose solution with ultrapure water to obtain solutions with the following concentrations: 0.125 M; 0.1 M; 0.075 M; 0.05 M and 0.025 M. 
		NOTE: solution stable for at least 3 months at 4 &#176;C.</li>
		<li>Incubate in a 96-well plate for 60 min at 37 &#176;C: 
		- 30 &#181;L of organoid lysate with 30&#181;L of lactose 
		- 30 &#181;L of organoid lysate with 30&#181;L of sucrose 
		- 30 &#181;L of ten times diluted organoid lysate with 30 &#181;L of maltose 
		- 30 &#181;L of five times diluted organoid lysate with 30 &#181;L of trehalase 
		- 30 &#181;L of undiluted organoid lysate with 30 &#181;L of maleic acid, as sample background 
		- 30 &#181;L of each glucose standard 
		- 30 &#181;L of ultrapure water, as blank 
		- 30 &#181;L of positive control (optimize dilution; lysed fetal intestinal tissue can be used as control for lactase activity while lysed adult intestinal tissue can be used as control for sucrase, maltase and trehalase) 
		NOTE: Dilution of samples should be made with cell lysis buffer. Keep the samples and plate on ice while preparing the assay.</li>
		<li>To determine the amount of glucose produced by the enzymes present in the organoid lysate after incubation with their respective substrates, add quickly 200 &#181;L of PGO-color solution and measure absorbance at 450 nm every 5 min for 30 min at 37 &#176;C. 
		NOTE: Make PGO-color solution fresh. Use 10 U/mL glucose-oxidase, 2 U/mL peroxidase and 7.88 mmol/L o-dianisidine in 0.5mol/L Tris-HCl buffer at pH 7.0. Solution should be at room temperature when added to the plate.</li>
		<li>Calculate activity according to glucose standard and correct for total amount of protein (determined by bicinchoninic acid assay (BCA)). Enzyme activity should be expressed as &#181;M glucose/&#181;g protein&#183;min-1 (Figure 5B). 
		NOTE: Measure arginase activity using a commercially available arginase activity assay kit.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Henning, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+development:+coordination+of+feeding,+digestion,+and+metabolism.">Postnatal development: coordination of feeding, digestion, and metabolism.</a> American Journal of Physiology. 241 (3), 199-214 (1981).</li><li>Krasinski, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+intestinal+hydrolase+biosynthesis+during+postnatal+development+in+rats.">Transcriptional regulation of intestinal hydrolase biosynthesis during postnatal development in rats.</a> American Journal of Physiology. 267 (4), 584-594 (1994).</li><li>Hurwitz, R., Kretchmer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+arginine-synthesizing+enzymes+in+mouse+intestine.">Development of arginine-synthesizing enzymes in mouse intestine.</a> American Journal of Physiology. 251 (1), 103-110 (1986).</li><li>Rath, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunologic+functions+of+the+neonatal+Fc+receptor+for+IgG.">The immunologic functions of the neonatal Fc receptor for IgG.</a> Journal of Clinical Immunology. 33, 9-17 (2013).</li><li>Martin, M. G., Wu, S. V., Walsh, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogenetic+development+and+distribution+of+antibody+transport+and+Fc+receptor+mRNA+expression+in+rat+intestine.">Ontogenetic development and distribution of antibody transport and Fc receptor mRNA expression in rat intestine.</a> Digestive Diseases and Sciences. 42 (5), 1062-1069 (1997).</li><li>Bry, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cell+differentiation+in+the+developing+intestine+of+normal+and+transgenic+mice.">Paneth cell differentiation in the developing intestine of normal and transgenic mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (22), 10335-10339 (1994).</li><li>Muncan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blimp1+regulates+the+transition+of+neonatal+to+adult+intestinal+epithelium.">Blimp1 regulates the transition of neonatal to adult intestinal epithelium.</a> Nauret Communications. 2, 452 (2011).</li><li>Beaulieu, J. F., Calvert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influences+of+dexamethasone+on+the+maturation+of+fetal+mouse+intestinal+mucosa+in+organ+culture.">Influences of dexamethasone on the maturation of fetal mouse intestinal mucosa in organ culture.</a> Comparative Biochemistry and Physiology A Comparative Physiology. 82 (1), 91-95 (1985).</li><li>Nanthakumar, N. N., Henning, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+sucrase-isomaltase+gene+expression+in+rat+intestine:+responsiveness+to+glucocorticoids.">Ontogeny of sucrase-isomaltase gene expression in rat intestine: responsiveness to glucocorticoids.</a> American Journal of Physiology. 264 (2), 306-311 (1993).</li><li>Navis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fetal+intestinal+organoids:+new+model+to+study+epithelial+maturation+from+suckling+to+weaning.">Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.</a> EMBO Reports. 20 (2), (2019).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Van Lidth de Jeude, J. F., Vermeulen, J. L., Montenegro-Miranda, P. S., Van den Brink, G. R., Heijmans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+lentiviral+transduction+and+downstream+analysis+of+intestinal+organoids.">A protocol for lentiviral transduction and downstream analysis of intestinal organoids.</a> Journal of Visualized Experiments. (98), e52531 (2015).</li><li>Dahlqvist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+intestinal+disaccharidases.">Assay of intestinal disaccharidases.</a> Scandinavian Journal of Clinical and Laboratory Investigation. 44 (2), 169-172 (1984).</li><li>Messer, M., Dahlqvist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one-step+ultramicro+method+for+the+assay+of+intestinal+disaccharidases.">A one-step ultramicro method for the assay of intestinal disaccharidases.</a> Anal Biochemistry. 14 (3), 376-392 (1966).</li><li>Fordham, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+expanded+fetal+intestinal+progenitors+contributes+to+colon+regeneration+after+injury.">Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury.</a> Cell Stem Cell. 13 (6), 734-744 (2013).</li><li>Mustata, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Lgr5-independent+spheroid-generating+progenitors+of+the+mouse+fetal+intestinal+epithelium.">Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.</a> Cell Reports. 5 (2), 421-432 (2013).</li><li>Holscher, H. D., Bode, L., Tappenden, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Milk+Oligosaccharides+Influence+Intestinal+Epithelial+Cell+Maturation+In+Vitro.">Human Milk Oligosaccharides Influence Intestinal Epithelial Cell Maturation In Vitro.</a> Journal of Pediatric Gastroenterology and Nutrition. 64 (2), 296-301 (2017).</li><li>Solomon, N. S., Gartner, H., Oesterreicher, T. J., Henning, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+glucocorticoid-responsiveness+in+mouse+intestine.">Development of glucocorticoid-responsiveness in mouse intestine.</a> Pediatric Research. 49 (6), 782-788 (2001).</li><li>Kedinger, M., Simon, P. M., Raul, F., Grenier, J. F., Haffen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+dexamethasone+on+the+development+of+rat+intestinal+brush+border+enzymes+in+organ+culture.">The effect of dexamethasone on the development of rat intestinal brush border enzymes in organ culture.</a> Developmental Biology. 74 (1), 9-21 (1980).</li></ol>

Authors declare nothing to disclose.

This protocol describes culturing of late fetal intestinal organoids for prolonged time to mimic suckling-to-weaning transition in vitro. The process of maturation equals the pace in vivo and is completed after one month in culture. Downstream analysis of this culture using quantitative RNA and protein techniques are detailed.
In this protocol, primary intestinal cells from E18-E20 mouse embryos are used. The developmental stage of primary mouse cells used to generate organoids for this protocol is particularly important. Using earlier developmental stage will result in generation of intestinal spheroids that maintain their specific fetal gene expression over a prolonged period of time with limited transition to adult organoids15,16. Only late fetal stage spheroids are capable in transiting to adult organoids in vitro10. To maximize the window of opportunity with respect to impact of extrinsic factors on gut maturation, intestines from late fetal stage are recommended and not intestines from born pups that have been exposed to microbes and mother milk. It is reported that certain bacterial metabolites and milk components can act as modifiers of the maturation process17.
To obtain sufficient amounts of cells to maintain the culture for one month to study the whole maturation from birth up to adulthood, while collecting the samples for downstream analyses, intestines from 6-8 embryos should be used as starting material. It is preferred to use embryos from the same developmental stage for generating the culture. We do not recommend pooling different litters as slight differences in developmental stage can influence expression of the maturation genes.
The protocol described here accounts for organoid generation from the proximal and distal small intestine to maintain developmental features of different segments of the gut. As an alternative, whole intestine can be used to investigate overall maturation with respect to the increase/decrease expression of the specific markers. In the latter case, fewer embryos could be used to isolate intestinal cells for starting culture.
This protocol is developed using three-dimensional organoid cultures. As organoids undergo dynamic growth in the culture, it is important to collect samples for downstream analyses at the same time point after passaging. In this protocol, we have selected day 3 after passage, as it represents the medium time between two splits at which organoids contain robust buds and little to no cell death. An alternate time point after passaging can be used, but it should be consistent during the whole experiment. We do not recommend growing organoids for more than 7 days after a passage, as increase of death cells in the organoid lumen can affect the results.
In our experiments, we have used dexamethasone as an example of an extrinsic factor that is shown and best studied in literature to accelerate intestinal maturation in vivo9,18. Dexamethasone exerts its effects via both genomic and non-genomic routes. For example, on the level of genomic regulation, a precocious increase of Sis mRNA levels can be observed. On a non-genomic level, we observe alterations in the activity of digestive enzymes such as trehalase. Both are in accordance with described specific aspects of dexamethasone on sucrase gene activation and non-genomic activating effect on intestinal brush border enzymes observed in vivo19. The fact that extrinsic factors, like synthetic glucocorticoids, can modulate certain aspects of suckling-to-weaning transition in the organoid culture, similarly to that described in vivo, further establishes the mouse fetal intestinal organoids as a model for the investigation of different kind of modulators of gut maturation.
Even though the morphological maturation of human intestinal epithelium is completed in utero at gestational stage of 22 weeks, the intestinal barrier function matures till childhood in a close relationship with the type of feeding, development of microbiota and immune response. Due to the limited availability of human tissues at these developmental stages, the translational value of in vitro murine model lies in the possibility of high throughput screens of factors capable of modulating intestinal maturation, a process conserved among suckling mammals.
Importantly, with respect to animal ethics in research, this model can contribute to refinement of animal experiments as it does not include interventions performed on animals. The number of animals can be further reduced by redesigning research questions to one or two time points of culture which will allow testing of multiple components within one culture.

In many mammalian species, including mice and men, the neonatal intestine has several features that are distinct from the fully mature intestinal epithelium. These features facilitate neonatal enterocytes to digest and absorb milk, which contains high fat and low carbohydrates, with lactose as the major carbohydrate. The brush border of the neonatal intestinal epithelial cells express the disaccharidase lactase-phlorizin hydrolase (Lct)1 to digest the milk disaccharide lactose. After the suckling period, enterocytes adapt to digest solid food that is rich in complex carbohydrates and low in fat. This is manifested by a switch in brush border disaccharidase expression from lactase to sucrase-isomaltase (Sis) and trehalase (Treh), which can digest more complex carbohydrates present in solid food2. Another metabolic switch is related to the low concentration of arginine in milk. To provide for the need for arginine, neonatal enterocytes express the rate limiting enzyme in arginine biosynthesis, argininosuccinate synthase-1 (Ass1), to synthetize arginine3. In contrast, adult enterocytes express arginase 2 (Arg2), an enzyme capable of catabolizing arginine that is abundant in solid foods. Furthermore, the neonatal intestinal epithelium expresses the neonatal Fc receptor for immunoglobulins (FcRn), which mediates absorption of the maternal IgG from the milk into the circulation/bloodstream4. The expression of FcRn declines significantly during the suckling-to-weaning transition5. In mice, maturation of Paneth cells occurs postnatally, coincidently with the formation and maturation of crypts, and is characterized by expression of antimicrobial peptides lysosome (Lyz) and defensins6.
All these changes are part of the suckling-to-weaning transition, a process occurring gradually after birth to one month of age in mice, when the intestinal epithelium reaches its mature adult state. Suckling-to-weaning transition is intrinsically regulated and developmentally set in the gut tube. Transcription factor B lymphocyte-induced maturation protein-1 (Blimp-1) plays a key role in this intrinsic maturation process7. Blimp-1 is highly expressed in neonatal epithelium, while its expression decreases and is lost during the suckling-to-weaning transition and therefore can serve as a reliable marker of neonatal intestinal epithelium. Despite being an intrinsic process, the suckling-to-weaning transition can be modulated by external factors. For example, the synthetic analogue of cortisol, dexamethasone, is known to accelerate gut maturation in vivo8,9.
Current in vitro models used to study intestinal epithelial maturation including the suckling-to-weaning transition, utilize adult epithelial cell lines and/or adult organoids which bear characteristics of adult intestinal epithelium. We have recently demonstrated that primary intestinal epithelial cells isolated from the late fetal intestine mature and recapitulate the suckling-to-weaning transition when growing in vitro as organoids10. We further showed that this gut maturation process in vitro occurs at the same pace as in vivo. Finally, we used dexamethasone to accelerate the maturation process in the same fashion described for in vivo studies.
Here, we outline a precise protocol for the isolation and culture of mouse late fetal intestinal organoids. We describe the preferred way of collecting samples for prolonged organoid culture and methods to monitor suckling-to-weaning transition in vitro. This protocol can be used for in vitro studies of intestinal epithelial maturation and modulators of this process and results in higher throughput, increased quality and translational value of the data and a reduction of animal use.

<table><tbody><tr><td>Advanced DMEM/F12 1:1</td><td>Invitrogen</td><td>12634-028</td><td></td></tr><tr><td>Arginase Activity Assay Kit</td><td>Sigma-Aldrich</td><td>MAK112-1KT</td><td></td></tr><tr><td>B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td>100x</td></tr><tr><td>BCA protein assay Kit</td><td>Fisher</td><td>10741395</td><td></td></tr><tr><td>Cell lysis buffer</td><td>Cell Signaling Technology</td><td>9803S</td><td></td></tr><tr><td>Cell Recovery Solution</td><td>Corning B.V.</td><td>354253</td><td></td></tr><tr><td>Cell strainer 70&amp;micro;M</td><td>BD/VWR</td><td>734-0003</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>EDTA</td><td>Merck</td><td>10,84,18,250</td><td>EDTA Titriplex III</td></tr><tr><td>EGF</td><td>Invitrogen</td><td>PMG8045</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1,00,98,31,000</td><td></td></tr><tr><td>Glucose solution</td><td>Sigma-Aldrich</td><td>G6918</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-038</td><td>100x</td></tr><tr><td>Hepes</td><td>Invitrogen</td><td>15630-056</td><td>1M</td></tr><tr><td>Isolate II RNA Mini Kit</td><td>Bioline</td><td>BIO-52073</td><td></td></tr><tr><td>Lactose</td><td>Sigma-Aldrich</td><td>L3625</td><td>a-Lactose monohydrate</td></tr><tr><td>Maleic Buffer</td><td>Sigma-Aldrich</td><td>M0375</td><td>Maleic acid</td></tr><tr><td>Maltose</td><td>Sigma-Aldrich</td><td>M5885</td><td>D-(+)-Maltose monohydrate &gt;99%</td></tr><tr><td>Matrigel</td><td>Corning B.V.</td><td>356231</td><td>Growth Factor Reduced Basement Membrane Matrix</td></tr><tr><td>Millipore water</td><td>N.A.</td><td></td><td></td></tr><tr><td>N2 supplement</td><td>Invitrogen</td><td>17502-048</td><td>100x</td></tr><tr><td>n-Acetylcystein</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Noggin-conditioned media</td><td>Homemade</td><td></td><td></td></tr><tr><td>o-dianisidine</td><td>Sigma-Aldrich</td><td>191248</td><td></td></tr><tr><td>PBS</td><td>Homemade</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td>0,2 U/mL</td></tr><tr><td>PGO-enzyme preparation</td><td>Sigma-Aldrich</td><td>P-7119-10CAP</td><td>capsules with Peroxidase/ Glucose Oxidase</td></tr><tr><td>p-hydroxymercuribenzoate sodium</td><td>Sigma-Aldrich</td><td>55540</td><td></td></tr><tr><td>Rspondin-conditioned media</td><td>Homemade</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>Trehalose</td><td>Sigma-Aldrich</td><td>T5251</td><td>D-Trehalose dihydrate</td></tr><tr><td>Tris-HCl Buffer</td><td>Homemade</td><td></td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr></tbody></table>

recapitulating suckling-to-weaning transition in vitro using fetal intestinal organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

Prolonged culture of fetal intestinal epithelial cells 
The protocol for mimicking suckling-to-weaning transition in vitro depends on correct handling of fetal organoids during prolonged culture. Proximal and distal intestine isolated from E18-E20 mouse fetuses are separated as presented in (Figure 1). Upon isolation, epithelial cells are seeded in extracellular matrix gel domes (Figure 2). It typically takes four passages and approximately 28-30 days of culture for fetal organoids to mature to the adult state. During this time, cells at various stages of maturation can be collected (Figure 3).
Representative downstream analysis of suckling-to-weaning transition in vitro 
To confirm that isolated fetal organoids are distinctly proximal or distal, compare the expression level of proximal markers One cut domain family member 2 (Onecut2) and GATA binding protein 4 (Gata4) and distal markers Fatty acid binding protein 6 (Fabp6) and Guanylate Cyclase Activator 2A (Guca2a) between both proximal and distal organoid cultures (Figure 4A,B). Suckling-to-weaning transition in vitro can be monitored by two sets of genes: fetal (Figure 4C) and adult markers (Figure 4D). Fetal markers should gradually decrease during the course of the culture, while the expression of adult markers should gradually increase (Figure 4C,D).
Using extrinsic factor as a modifier of sucking-to weaning transition in vitro 
In this protocol dexamethasone a synthetic glucocorticoid, was used as an example of extrinsic factor capable of modifying suckling-to-weaning transition in vitro. Representative data in Figure 5 suggests that effects of extrinsic factors are best to be determined by multiple assays, as they do not necessarily ought to be genomic. For example, in the case of sucrase-isomaltase both RNA and protein expression are induced with dexamethasone (Figure 5A) whereas trehalase expression is only changed at the protein level. (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60470/60470fig01.jpg" /> 
Figure 1: Isolation of mouse fetal small intestine. (A) Photograph of dissected and stretched fetal gut, including stomach, proximal and distal small intestine, appendix and colon. Black line indicates where gut should be cut to divide the proximal and the distal small intestine. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60470/60470fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60470/60470fig02.jpg" /> 
Figure 2: Representative microscopic images of proximal and distal fetal organoid culture at day 3, day 17 and day 28 of culture. All images were obtained at day 3 after passage and show the decrease in the number of spheroids overtime. Scale bar: 500 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60470/60470fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1: Representative video showing fetal organoid culture dynamics, from day 4 to 6 of culture. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60470/culture_dynamics.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60470/60470fig03.jpg" /> 
Figure 3: Schematic representation of organoid collection for analysis of gut maturation over time. Proximal and distal fetal organoid cultures should be cultured for one month and passaged every week. Samples for maturation analysis should be collected 3 days after isolation and every 3 days after each passage. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60470/60470fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60470/60470fig04.jpg" /> 
Figure 4: Representative qRTPCRs of gut maturation markers in proximal and distal fetal organoids. (A) Proximal markers Onecut2 and Gata4 are mainly expressed in the proximal organoid culture while (B) distal markers Fabp6 and Guca2a are mostly expressed in the distal organoid culture. (C) Fetal markers Lct, Ass1, Blimp-1 and FcRn decrease and (D) adult markers Sis, Arg2, Treh and Lyz increase over time in both proximal and distal organoid cultures. Error bars represent SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60470/60470fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60470/60470fig05.jpg" /> 
Figure 5: External factor dexamethasone can modulate the maturation of fetal organoids. (A) Gene expression of fetal marker Blimp-1 is decreased at day 12 of culture in dexamethasone treated organoids compared to control organoids, while both gene expression (B) and enzyme activity (C) of adult marker sucrase-isomaltase (Sis) is increased. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60470/60470fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids at JoVE.com

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

recapitulating-suckling-to-weaning-transition-vitro-using-fetal

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.0K Views.  University of Amsterdam. This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

Video: Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Medicine

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Bioengineering

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Biology

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

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.

Author Spotlight: Generation and Manipulation of Rat 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.

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

Summary

Explore More Videos

Chapters in this video

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Innervation of Human Intestinal Organoids

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Generation and Manipulation of Rat Intestinal Organoids