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. 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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. 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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&#181;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 &#62;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>&#946;-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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.9K 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

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...

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

Summary

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Chapters in this video

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Transuterine Fetal Tracheal Occlusion Model in Mice