This protocol describes a culture of mouse fetal intestinal cells that is the best suited to achieve maturation to adult stage in vitro. This in vitro approach can be used to study molecular mechanisms of suckling-to-weaning transition as well as the modulators of this process and to achieve more efficient use of experimental animals. It is essential for this in vitro model of gut maturation to use primary intestinal cells from late developmental stage, between embryonic day 18 and 20.
With two small forceps, stretch the intestine. Using the stomach and appendix as guides, cut apart the proximal and distal parts of the small intestine. Using the appendix as a guide, cut the colon apart.
To make the intestine open longitudinally, place a razor blade lengthwise along the intestine and pull the intestine by sliding forceps along the razor blade. Subsequently, cut the opened intestine into one centimeter pieces. Prepare three 50-milliliter tubes with ten milliliters of ice cold PBS.
Transfer the proximal and distal parts of the small intestine and the colon 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.
After washing the intestinal pieces with ice cold PBS, incubate with two millimolar EDTA for 30 minutes. To dissociate crypts from the tissue, partially wash the pieces with ice cold PBS and ten percent calf serum and filter through a 70 micron cell strainer into a new tube. Repeat washing the tissue two additional times and combine the filtered solution.
Centrifuge the filtered solution containing the crypts at 150 times G for five minutes at four degrees Celsius to collect the crypts as a pellet. Transfer the crypts into a 15 milliliter tube and centrifuge again. To plate wells, add the total amount of extracellular matrix gel needed into the tube and dissolve the pellet.
Add 20 microliters of the dissolved crypts into each well of a warm 48-well plate. After plating the first well, look under the microscope to confirm the density of around 250 to 300 crypts per well. Place the plate in a 37 degree Celsius incubator for ten minutes to let the extracellular matrix gel solidify.
Then, prepare ENR medium. Add 250 microliters of the ENR medium into each well onto the gel. Change the medium three times per week and passage the organoids once a week.
For one month, every three days after each passage, analyze the culture. Three days after each passage, isolate RNA from one well by adding 200 microliters of RNA lysis buffer supplemented with two microliters of beta mercaptoethanol to the well. Then, transfer the sample from the well into an RNAse-free 1.5 milliliter tube.
In our in vitro experiments, we have used dexamethosone as an example of an external factor that is known to accelerate intestinal maturation in vivo. To isolate protein, add 250 microliters of ice cold cell recovery solution per well to five wells of organoids and collect them all in a 15 milliliter tube. Incubate for at least 30 minutes on ice to dissolve the extracellular matrix gel.
Wash with ice cold PBS, add 250 microliters of cell lysis buffer, and store at minus 80 degrees Celsius. To analyze enzyme activity, first prepare 0.625 molar maleic buffer at pH six and 0.05 molar solutions of lactose, sucrose, maltose, and trehalase and store them on ice. Prepare assay standards by diluting 5.56 molar glucose solution with ultra-pure water to obtain solutions with 5 different concentrations.
Then, into a 96-well plate, add eight different groups of organoid lysate with their respective substrates including standards and control according to the manuscript. Incubate for 60 minutes at 37 degrees Celsius. To determine the amount of glucose produced by the enzymes present in the organoid lysate, quickly add 200 microliters of freshly prepared PGO color solution and measure absorbance on the spectrophotometer at 450 nanometers every five minutes for thirty minutes at 37 degrees Celsius.
In this protocol, proximal and distal intestine were isolated from E18 to E20 mouse fetuses. Upon isolation, epithelial cells were seeded in extracellular matrix gel domes. After approximately 28 to 30 days of culture, fetal organoids were developed to the mature adult state.
Proximal markers Onecut2 and Gata4 were mainly expressed in the proximal organoid culture, while distal markers Fabp6 and Guca2a were mostly expressed in the distal organoid culture. Fetal markers lactase, Ass1, Blimp-1 and neonatal Fc receptor decreased in their relative expression and adult markers sucrase isomaltase, argenase 2, trehalase and lysozyme increased over time in both proximal and distal organoid cultures. The effects of extrinsic factors do not necessarily ought to be genomic.
Gene expression of fetal marker Blimp-1 was decreased at day 12 for the dexamethasone-treated organoids compared to the control organoids. However, both gene expression and enzyme activity of adult markers sucrase isomaltase were increased. When using this protocol, it is important to realize that only late fetal stage organoids are capable of transiting to adult organoids in vitro.
The number of six to ten fetuses as a starting point of this culture can be further reduced by using the whole intestine to study the overall maturation. The translational value of this model lies in the possibility of high group extremes effectors are capable of modulating intestinal maturation which is a process conserved between suckling mammals.
