The overall goal of this procedure is to culture miniature guts, called enteroids, in the laboratory. Enteroids are treated with mouse breast milk extracted in the laboratory to elucidate the effects of breast milk on proliferation in vitro. This method can help answer key questions related to various gastrointestinal diseases, such as necrotizing enterocolitis, which affects premature infants.
The main advantage of this technique is that enteroids require little maintenance, can be established quickly, and exhibit many of the functional properties of the gastrointestinal epithelium. This technique of establishing enteroids is a highly effective model of the human small intestine. Though this method can provide insight into the small intestine, it can also be applied to other organ systems in other regions of the GI tract.
To begin, thaw solubilized basement membrane extract on ice. Then, use scissors and forceps to make a vertical incision down the entire abdominal mid-line of a euthanized mouse. Using forceps, excise the small intestine and remove the mesentery.
To prepare the small intestine for crypt isolation and enteroid creation, position the intestine straight and use scissors to cut longitudinally along its entire length. Remove any stool by gently shaking the intestine with forceps in a Petri dish filled with PBS antibiotic mixture. Then, add 30 milliliters of cell disruption media number one to a 50 milliliter conical tube.
Place half centimeter segments of intestine into the tube and set the tube into an ice bucket on a shaker. In a hood, add 9 microliters of basement membrane to the bottom of each well in an eight well chamber slide. Quickly spread the membrane with a pipette tip and allow the basement membrane to polymerize in an incubator.
It is important to to quickly spread the basement membrane so that it forms a thin layer on the bottom of the well. The crypts will not attach if there are air bubbles present or if the basement membrane is too thick. After agitating the conical tube, use a 100 micrometer strainer to filter the membrane tissue and discard the flow-through.
Add 30 milliliters of cell disruption media number two to a new 50 milliliter conical tube, and then, add the intestinal tissue to the tube. After agitating the tube for 15 minutes, filter the tissue through a 100 micrometer strainer. Keep the flow-through from this step.
Add 15 milliliters of TMEM with 4.5 grams per liter glucose and L-glutamine to a new conical tube. Remove tissue from the 100 micrometer strainer and add it to the new conical tube. Then, manually shake the tube for 10 seconds and filter through a 100 micrometer strainer.
In the hood, use a 70 micrometer strainer to filter the tissue into a new conical tube. After centrifuging the tube for 8 minutes, place it in an ice bucket in the hood. Use a pipette to carefully remove the supernatant without disturbing the formed pellet.
Re-suspend the pellet with an appropriate amount of crypt culture media without growth factors. After incubating the chamber slide for 30 minutes, plate the crypt medium mixture directly onto the basement membrane and incubate at 37 degrees Celsius for 3 hours while the crypts adhere. After the incubation period, use a P200 pipette to carefully remove any media and unattached crypts.
Another critical step is to make sure to slowly remove the media and excess crypts that failed to attach using a P200 pipette. Do not disturb the basement membrane, as the attached crypts can become dislodged. Slowly add 250 microliters of crypt culture media with growth factors to each well.
Anesthetize a lactating dam with isoflurane delivered via nose cone and administer the appropriate dose of subcutaneous oxytocin. Clean teats with 70%ethanol and allow them to dry before initiating the milking procedure. Using an electric human breast pump outfitted with silicone tubing sized to fit mice and a 5 milliliter tube, extract milk from the teats one at a time.
Finally, aliquot the milk immediately and store at minus 80 degrees Celsius. Treat the enteroids on day five with stored mouse breast milk for 24 hours at 37 degrees Celsius. On the first day of staining, use PBS to remove experimental treatments and wash the enteroids.
Add 250 microliters of 4%PFA to each well and place the slide on a rotator for 1 to 2 hours at 4 degrees Celsius. Use a pipette to remove the PFA and then gently wash each well four times with 250 microliters of PBS. Place the chamber slide on a rotator for 10 minutes at room temperature.
Add 250 microliters of 0.1%Triton X-100 to each well and incubate for 1 hour at room temperature. After incubating, remove the Triton X-100 with a pipette and wash four times with 250 microliters of PBS in each well. Place the slide on a rotator for 15 minutes at room temperature.
Add 250 microliters of 10%NDS/PBST to each well and incubate for 45 minutes at room temperature. After incubation, remove the NDS/PBST with a pipette. Finally, add 250 microliters of diluted primary antibody to each well and incubate overnight at 4 degrees Celsius.
On the second day of staining, wash each well at least five times with 250 microliters of PBST and place the slide on a rotator at 4 degrees Celsius between each wash. Add 250 microliters of diluted secondary antibody in 1%NDS/PBST to each well. Wrap the slide in foil and incubate overnight at 4 degrees Celsius.
On the final day of staining, add 250 microliters of nuclear stained DAPI to each well and allow it to sit for 15 minutes. Then, wash each well six times with 250 microliters of PBST. Place the slide on a rotator for 10 minutes at 4 degrees Celsius between each wash.
Finally, prepare the slide for microscopic imaging by mounting a cover slip on the chamber slide with mounting media and allowing it to dry for 24 hours. In this study, small intestinal enteroids derived from neonatal mice were treated with breast milk collected from lactating mice. As shown in this figure, mouse breast milk increased proliferation in mouse enteroids.
To investigate species-specific effects of breast milk, human breast milk and the pasteurized donor breast milk were also applied to the mouse enteroids. As shown in this figure, human and pasteurized donor breast milk increased proliferation of mouse enteroids. While attempting this procedure, it's important to keep the crypts on ice during the isolation steps.
Slowly and gently perform all methods after the enteroids are in culture. Following this procedure, other methods like treatments with immune cells, microbiota, or various drugs can be performed in order to answer additional questions to better recapitulate the microenvironment of the intestine. After watching this video, you should have a good understanding of how to culture, treat, and perform immunofluorescence on the enteroids.
In addition, you should be able to efficiently extract breast milk from a lactating mouse. Don't forget that working with human tissue and bodily fluids can be extremely hazardous, and personal protective equipment should always be worn while performing these procedures.
