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 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.
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.
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
2. Culturing of fetal organoids
3. Maturation analysis at RNA and protein level
4. Effect of extrinsic factor (dexamethasone as an example) on organoid maturation process
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).
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. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
Movie 1: Representative video showing fetal organoid culture dynamics, from day 4 to 6 of culture. Please click here to view this video. (Right-click to download.)
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
None.
Advanced DMEM/F12 1:1 | Invitrogen | 12634-028 | |
Arginase Activity Assay Kit | Sigma-Aldrich | MAK112-1KT | |
B27 supplement | Invitrogen | 17504-044 | 100x |
BCA protein assay Kit | Fisher | 10741395 | |
Cell lysis buffer | Cell Signaling Technology | 9803S | |
Cell Recovery Solution | Corning B.V. | 354253 | |
Cell strainer 70µM | BD/VWR | 734-0003 | |
Dexamethasone | Sigma-Aldrich | D4902 | |
EDTA | Merck | 10,84,18,250 | EDTA Titriplex III |
EGF | Invitrogen | PMG8045 | |
Ethanol | Merck | 1,00,98,31,000 | |
Glucose solution | Sigma-Aldrich | G6918 | |
Glutamax | Invitrogen | 35050-038 | 100x |
Hepes | Invitrogen | 15630-056 | 1M |
Isolate II RNA Mini Kit | Bioline | BIO-52073 | |
Lactose | Sigma-Aldrich | L3625 | a-Lactose monohydrate |
Maleic Buffer | Sigma-Aldrich | M0375 | Maleic acid |
Maltose | Sigma-Aldrich | M5885 | D-(+)-Maltose monohydrate >99% |
Matrigel | Corning B.V. | 356231 | Growth Factor Reduced Basement Membrane Matrix |
Millipore water | N.A. | ||
N2 supplement | Invitrogen | 17502-048 | 100x |
n-Acetylcystein | Sigma-Aldrich | A9165 | |
Noggin-conditioned media | Homemade | ||
o-dianisidine | Sigma-Aldrich | 191248 | |
PBS | Homemade | ||
Penicillin/Streptomycin | Invitrogen | 15140-122 | 0,2 U/mL |
PGO-enzyme preparation | Sigma-Aldrich | P-7119-10CAP | capsules with Peroxidase/ Glucose Oxidase |
p-hydroxymercuribenzoate sodium | Sigma-Aldrich | 55540 | |
Rspondin-conditioned media | Homemade | ||
Sucrose | Sigma-Aldrich | 84097 | |
Trehalose | Sigma-Aldrich | T5251 | D-Trehalose dihydrate |
Tris-HCl Buffer | Homemade | ||
β-mercaptoethanol | Sigma-Aldrich | M3148 |