Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

Organoids and spheroids, being very fragile, get seriously damaged while handling. Our protocol ensures these 3D growing structures remain intact during various processes, thereby increasing accuracy, reproducibility, reliability, and repeatability. The researcher can culture, freeze, thaw, process, stain, label, and examine entire organoids and spheroids under various microscopes while they remain intact in a hydrogel within a single multi-purpose device.

It enables creation of a disease model and tracking of sequential steps in one multi-purpose device. The technology brings more accuracy when examining biomarkers for diagnosis and prognosis of diseases. This method can be applied to any 3D growing system, including organoids, spheroids, individual cells, or living organisms.

To begin the organoid or spheroid culture, place the properly thawed hydrogel on ice inside a laminar flow hood for 15 minutes. Also, keep the tube containing a pellet of the hepatocellular carcinoma, or HEPG2 cells, on ice. Next, plate 30 to 35 microliters of 100%hydrogel within the niche of the pre-warmed multipurpose device to create a gel drop, place 10, 000 HEPG2 cells in the center of the top of the hydrogel drop, and incubate the setup at 37 degrees Celsius for 15 minutes.

After incubation, cover the hydrogel drop with 200 microliters of Dulbecco's Modified Eagle Medium, or DMEM, containing 10%fetal bovine serum, or FBS. Place the lid on the device properly, allowing gas flow, before placing it in the incubator. Every other day, feed the cells with 200 microliters of DMEM containing 10%FBS.

Check the growth of the spheroids under an inverted microscope. Before starting immunofluorescence labeling of the organoids, warm up the required media and solutions to 37 degrees Celsius. Aspirate the cell culture medium surrounding the hydrogel drop before adding 200 microliters of 4%paraformaldehyde pre-warmed to 37 degrees Celsius and fix it by placing it in an incubator at 37 degrees Celsius for 15 to 30 minutes.

Then, aspirate the fixative and wash the solution for immunofluorescence labeling, or SIF, pre-warmed to 37 degrees Celsius three times for 10 minutes each. Add distilled water to the area surrounding the niche to provide humidity before proceeding with the next steps. Aspirate the SIF surrounding the hydrogel drop and add 100 microliters of the pre-warmed primary antibody solution in SIF to the hydrogel before incubating it at 37 degree Celsius for 30 to 60 minutes.

Then, aspirate the primary antibody solution and wash with SIF pre-warmed to 37 degrees Celsius three times for 10 minutes each. Following the wash, aspirate the remaining SIF and add 100 microliters of the pre-warmed secondary antibody solution in SIF to the hydrogel. Incubate the hydrogel at 37 degrees Celsius in the dark for 30 to 60 minutes.

After the incubation, aspirate the secondary antibody solution and wash the hydrogel drop with PBS at 37 degrees Celsius three times in the dark for 10 minutes each. Next, aspirate the residual PBS and incubate the hydrogel with 100 microliters of nuclear DNA stain containing mounting medium or glycerol at 37 degrees Celsius in the dark. Fill the niche with glycerol to avoid drying before tightly closing the multi-purpose device containing the hydrogel.

To examine the organoids by confocal microscopy, set the microscope parameters following the instructions given in the manuscript. To freeze the organoids, aspirate the cell culture media, add 200 microliters of the pre-warmed freezing solution, and incubate the sample at 37 degrees Celsius for one hour. Close the lid of the device tightly before placing it inside a foam box.

Place this foam box in another foam box and close both foam boxes tightly. Keep the box at minus 20 degrees Celsius for two hours, then leave it overnight in a minus 80 degree Celsius freezer. To store the organoids for longer than six months, remove the multi-purpose device containing the organoids from the boxes and transfer it to the liquid nitrogen tank.

To thaw the organoids, take out the multi-purpose device containing the sample from either the freezer or the liquid nitrogen tank and place it directly into an incubator at 37 degrees Celsius. Once thawed, gently aspirate the medium and freezing solution mixture before adding fresh medium and proceeding to image the whole-mount organoid hydrogel. In live imaging, the growing organoids inside the hydrogel became visible three to five days after inserting the cell pellet, especially at the periphery of the hydrogel dome.

The time series of images at different levels of a hydrogel containing spheroids under an inverted phase contrast microscope is shown here. Confocal microscopy images of the whole-mount living organoids after live-staining the cell membrane and the nucleus showed similar labeling density in the periphery and the center. Further, the immunofluorescence labeling of the whole-mount steroids could be successfully performed with one antibody, two antibodies, as well as three antibodies, eliminating any need for additional treatment steps.

The representative analysis demonstrates two immunofluorescent-labeled airway organoids in a device. SEM images of the entire spheroids in the multi-purpose device and 10 images of sectioned steroids indicated well-preserved 3D architecture of spheroids, cytoplasmic organelles, and other ultra-structural cellular features, demonstrating the effectiveness of the described protocol. Irregularity at the boundaries of the hydrogel dome was evident after freezing the samples.

However, the size and roundness of the spheroids remained similar. Cell migration on the culture surface was observed after thawing the frozen samples. The technique is very simple.

However, the researcher should be very gentle while aspirating or adding any liquids surrounding the hydrogel drop to prevent the damaging the sample. The researcher can look at the same sample in a single device using a high-tech microscope and conduct a correlative study.