ティッシュ・エンジニアリングと疾病調査に使用するための小児のヒト食道上皮細胞の条件付き再プログラミング

The overall goal of this procedure is to expand patient-derived esophageal epithelial cells from endoscopic biopsies to study esophageal diseases, or to create a 3D esophageal scaffold for downstream modeling or esophageal tissue replacement. This method can be used to better understand the role of esophageal epithelial cells in esophageal disease. They can also be used to see 3D scaffolds for modeling or implantation.

The advantage to this technique is it allows us to generate patient specific epithelial cells from the very small piece of tissue without the need for any genetic or permanent modifications. The implications of this technique extend toward understanding the pathogenesis of eosinophilic esophagitis, as well as developing treatments for esophageal atresia and eosinophilic esophagitis. Generally, individuals new to this technique will struggle with the ability to completely remove the feeder cells prior to harvesting human esophageal epithelial cells in order to maintain purity.

Demonstrating this technique will be Christopher Foster, a technician from our laboratory. At least three days before the patient samples are obtained, plate the 3T3 cells in 3T3 culture medium in tissue-treated T75 flasks. On the day of the experiment, just prior to obtaining the samples, detach the 3T3 cells with 5 milliliters of 0.25%Trypsin-EDTA per flask for about five minutes at 37 degrees Celsius.

When most of the cells are floating, add 5 milliliters of 3T3 medium to stop the reaction, and transfer the cell suspension into a 15 milliliter conical tube for centrifugation. Re-suspend the pellet in medium for counting. Then, transfer the appropriate number of cells for seeding into a 15 milliliter conical tube.

Next, add 25 milliliters of fresh 3T3 medium to the cells for irradiation at 3000 rads. Then, collect the cells by centrifugation, and re-suspend the pellet in conditional programming medium at five milliliters per 100 millimeter plate. After obtaining approximately six esophageal biopsies at the time of endoscopy, place six biopsies in a 15 milliliter conical tube containing complete serum-free keratinocyte medium, supplemented with Primocin on wet ice.

Place one biopsy in five milliliters of buffered Formalin on wet ice, and place one biopsy in a small biopsy cryomold containing embedding medium. Immediately immerse the mold in a beaker of ice or pentane inside a container of liquid nitrogen. Once the tissue is frozen, place the cryomold in a specimen bag on dry ice, in a polystyrene foam container.

Then, place the tubes of biopsies inside a sealable bag inside a shipping box with the biohazard label containing wet ice, and seal and label both containers with a biohazard sticker for transport to the laboratory. Upon entering the lab, immediately transfer the frozen biopsy to a minus 80 degree Celsius storage for downstream sectioning or RNA extraction. Place the sample in Formalin in a four degree Celsius refrigerator for overnight fixation and place the remaining biopsies in the centrifuge.

Replace the medium in each tube with 10 milliliters of dispase solution, and seal the tubes for a 15 minute incubation at 37 degree Celsius in a water bath. At the end of the incubation, spin down the biopsies again, and re-suspend the samples in five milliliters of 0.25%Trypsin-EDTA. Transfer the biopsies to a single 100 milliliter sterile plate, and use two sterilized razors to mince the tissues as finely as possible.

When all of the tissue has been cut, transfer the tissue fragments to a new 15 milliliter tube, and rinse the plate two times with five milliliters of 0.05%Trypsin-EDTA. Pool the washes in the tube, and return the samples to a 37 degree Celsius water bath. After 10 minutes, centrifuge the digested sample pieces and the feeder cells, and re-suspend the pellets in five milliliters of conditional reprogramming medium, each.

Then, combine the feeder and esophageal biopsy samples into one tube, and add rho kinase, or ROCK inhibitor, to the cells for their co-culture on a gelatin-coated tissue culture dish. After approximately five days, remove the medium and minced biopsy pieces by aspiration, and rinse the dish two to three times with five milliliters of sterile PBS. After the last wash, add 10 milliliters of fresh medium supplemented with ROCK inhibitor, to the plate, and return the co-culture to the incubator until the cells reach 70%confluency.

To expand the cells, remove the feeders with five milliliters of 0.05%Trypsin-EDTA at 37 degree Celsius for no more than two and a half minutes. Rinse the plate with five milliliters of fresh PBS to remove the floating feeder cells, and add five milliliters of 0.25%Trypsin-EDTA for a five to 10 minute incubation until the biopsy sample cells are loosened from the bottom of the plate. Then, stop the reaction with 10 milliliters of fresh medium, and transfer the cells into a 50 milliliter conical tube for centrifugation.

Re-suspend the pellet in fresh conditional reprogramming medium for counting, and, now, account one times 10 to the sixth human esophageal cells into a new conical tube. Add 1.2 times 10 to the sixth irradiated 3T3 cells to the esophageal cells, and bring the total volume in the tube to 15 milliliters with fresh conditional reprogramming medium. Then, add fresh ROCK inhibitor to the cells, and plate the co-culture onto a gelatin-coated 150 millimeter dish.

To freeze the esophageal cells for minus 80 degree Celsius storage, remove the feeders and detach the esophageal cells as just demonstrated. Next, transfer the cells into a new 50 milliliter conical tube for centrifugation, and re-suspend the pellet at a one times 10 to the sixth cells per two milliliter of freezing medium concentration. Then, transfer two milliliters of cells into each pre-labeled cryovial, and store the samples at minus 80 degree Celsius for at least 24 hours before moving them to long-term liquid nitrogen storage.

Epithelial cell colonies will form in approximately four to five days, and will be surrounded by fibroblast feeder cells. As these colonies expand, they will merge with other colonies to form larger colonies. Once the culture has become 70%confluent, they need to be expanded.

After the feeder cells have been removed, the adherent epithelial cells can be trypsonized and re-plated onto new irradiated feeder cells. Phenotypic characterizati6on of these cells via qRT-PCR reveals an up-regulation of P63, a progenitor cell marker at the end of passage one, compared to the initial biospy sample, indicating that cells have been reprogrammed to a more progenitor stem-like state. When these cells are transferred to normal tissue culture plates, the P63 expression decreases and tight junction protein one, or TJP1, and E-cadherin expressions increase, demonstrating a phenotype shift from progenitor cell to a more differentiated epithelial cell.

Flow cytometric analysis of the cells after passage one indicates that greater than 90%of the cells are also positive for EpCAM. Immunofluorescence staining demonstrates that the cells retain their epithelial marker, progenitor marker, proliferation marker, and epithelial tight junction protein expression. The doubling time of esophageal cells from a non-diseased patient has been calculated to be approximately 36 to 40 hours, although it is likely that the doubling time of diseased cells will be slower.

Once mastered, this technique should be completed in approximately one hour. While attempting this procedure, be sure to use sterile technique to prevent contamination, and be sure to work expeditiously to maintain cell viability. Following this procedure, other assays, such as transwell culture, can be performed in order to evaluate which antigens are causing esophageal inflammation, or pathogenesis.

After development, this assay will pave the way for researchers to identify triggering antigens causing eosinophilic esophagitis, as well as new treatment options for treating these patients. After watching this video, you should have a good understanding of how to isolate and expand esophageal epithelial cells from endoscopic biopsies for use in studying esophageal diseases, or generating 3D models. Don't forget that working with sharp instruments and biological tissues can be hazardous.

Be sure to dispose of sharp instruments properly, and use good biosafety practices.