We are integrating single-cell transcriptomics and organoid technology to better understand the biology of the small intestine. These technologies are allowing us deeper insight into the cellular heterogeneity that exists within that tissue. The tools that improve the throughput, sensitivity, and the resolutions are the keys that advance research in the field.
Some examples are improved computational methods and the technological innovations. Cost reduction is also a major focus. The process of preparing and sequencing the libraries or samples can cause transcriptional variations, which is one challenge.
For organoids, specific challenges include limited spatial context or information about temporal dynamics. Single-cell analysis of the human intestinal organoids provides previously unattainable information into the cell types that make up the intestinal epithelium. This thereby opens the window to the transcriptional landscape of those cells.
We are using these technologies to identify biomarkers and mechanisms of human intestinal diseases. These biomarkers and mechanisms will advance our ability to utilize personalized medicine-based approaches to treat human disease and improve human health overall. To begin, thaw the enzymatic cell digestion reagent at 37 degrees Celsius for 30 minutes.
Under a brightfield microscope at 10x magnification, observe the differentiated three-dimensional human intestinal organoids to ensure cell health. Replace the media from the wells of a 24-well plate containing differentiated organoids with 500 microliters of cold cell recovery solution. Pipette up and down to release the cells from the basement membrane.
Transfer the cell suspension to a 1.5-milliliter microcentrifuge tube. Incubate the tube on ice for 10 minutes, pipetting up and down every five minutes. Centrifuge the cell suspension at 400g for three minutes at four degrees Celsius.
Remove the supernatant from the tube without disturbing the pellet. Resuspend the pellet in 500 microliters of prewarmed enzymatic cell digestion reagent, pipetting up and down 10 times. Place the organoids for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator.
Every 10 minutes, pipette the entire volume 10 times using a P1000 pipette to help dissociate the cells. Centrifuge the cell suspension at 400g for three minutes at four degrees Celsius, and resuspend the pellet in one milliliter of differentiation media. Rapidly pipette the cells up and down 50 times on ice to dissociate organoids into single cells.
Filter the entire volume through a 70-micron tip strainer and collect the eluate in a fresh 1.5-milliliter tube. Then filter the eluate obtained through a 70-micron strainer on a 40-micron tip strainer. Add five microliters of 0.4%trypan blue to five microliters of cell suspension and count viable single cells using trypan blue exclusion.
Dilute the sample with cell culture media to obtain 1, 000 cells per microliter. A total of 4, 402 single cells were isolated from differentiated ileal three-dimensional human intestinal organoids representing expected cell types, including absorptive enterocytes and secretory cells. To begin, place the 15-milliliter tube containing prewarmed enzymatic cell digestion reagent in a 37 degree Celsius incubator until use.
Aliquot 500 microliters of freshly prepared PBS EDTA into the wells of a 24-well plate. Under a brightfield microscope, observe the growth of differentiated three-dimensional human intestinal organoids and transwells. Transfer the cell culture inserts from the growth media to the PBS EDTA solution.
Remove the culture media from the apical side and replace it with 100 microliters of PBS EDTA without disturbing the cell layer. After discarding PBS EDTA, remove the insert from the well, blot it gently onto the edge of a paper towel, and invert it on the bench top. Using a sharp scalpel blade, cut around the inner circumference of the membrane to remove the membrane from the insert.
Transfer the membrane with forceps to the 1.5-milliliter tube containing 200 microliters of prewarmed enzymatic cell digestion reagent. Place the membrane for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator. Every 10 minutes, pipette the entire volume five times using a P200 pipette.
Transfer one to two microliters of cell suspension to a glass slide every 10 minutes to assess dissociation by qualitatively assessing the percentage of single cells. After dissociation, combine three replicate wells per condition in a single 1.5-milliliter microcentrifuge tube. Add 400 microliters of cell culture differentiation medium containing 10 micromolar ROCK inhibitor and mix gently by pipetting.
Filter the entire volume through a 70-micron tip strainer, collecting the flow-through in a fresh 1.5-milliliter tube. Then filter the flow-through obtained through a 70-micron strainer on a 40-micron tip strainer. Add five microliters of 0.4%trypan blue to five microliters of cell suspension and count viable single cells.
Dilute the sample with WRNE growth medium to obtain 1, 000 cells per microliter. A total of 5, 623 single cells were isolated from differentiated jejunal human intestinal organoids grown on membrane cell culture inserts. To begin, prepare enzymatic cell digestion reagent and PBS EDTA for human intestinal organoids'digestion.
Under a brightfield microscope, confirm the growth of differentiated three-dimensional human intestinal organoids as monolayers. Discard the cell culture media from monolayer culture and replace it with 100 microliters of PBS EDTA. After removing PBS EDTA, add 100 microliters of warm enzymatic cell digestion reagent to each well.
Place the plate at 37 degrees Celsius in 5%carbon dioxide incubator and pipette the entire volume five times every 10 minutes for 20 to 30 minutes. Transfer one to two microliters of cell suspension to a glass slide every 10 minutes to assess dissociation by observing the percentage of single cells. After dissociation, combine three replicate wells per condition in a single 1.5-milliliter microcentrifuge tube.
Add 700 microliters of cell culture differentiation medium containing 10 micromolar RCOK inhibitor, and mix gently by pipetting. Filter the entire volume through a 70-micron tip strainer, collecting the flow-through in a fresh 1.5-milliliter tube. Filter the flow-through obtained through a 70-micron strainer on a 40-micron tip strainer.
Add five microliters of 0.4%trypan blue to five microliters of cell suspension and count viable single cells. Dilute the sample with cell culture media containing RCOK inhibitor to obtain 1, 000 cells per microliter. A total of 9, 952 single cells were isolated from differentiated jejunal human intestinal organoids grown in 96 well plates.
