Organotypic tissue slices have been generated from various tissues. Due to the preserved tissue integrity and the development of cultivation techniques, they have been suitable for modeling complex physiological mechanisms. Organotypic slice cultures from pancreatic carcinomas closely recalculate the multicellular architecture of the tumor and its microenvironment ex vivo.
They can be used for various downstream applications, such as drug response testing. They can be cultured immediately after surgical resection of the tumor, and are inexpensive and less time-consuming compared to most other primary culture techniques. Demonstrating the procedure will be Olha Lapshyna, a technical assistant from the University of Lubeck.
To begin, prepare 100 milliliters of low-melting 8%agarose by dissolving eight grams of agarose in 100 milliliters of pre-warmed ringer solution, and store it at four degrees Celsius until needed. Upon the announcement of tumor resection, melt the agarose in a microwave. Place the agarose in a preheated water bath, allowing it to cool to physiological temperatures prior to the preparation.
Place a razor blade into the holder of the Vibratome and perform an automated angle adjustment according to the manufacturer's instructions. Cool down the jacket of the cutting chamber using a cooling unit or wet ice. Fill the cutting chamber with approximately 100 milliliters of ringer solution, then place the mounted razor blade into the pre-chilled cutting solution, allowing the razor blade to cool down.
Wash the tissue specimen with cooled PBS and place it into a large 14-centimeter Petri dish on ice. Remove macroscopically visible excess connective tissue on ice using a scalpel. Place the tissue into a small Petri dish.
Adjust the tissue orientation, so that the remaining macroscopically visible connective tissue has the same orientation as the plain of the bottom of the Petri dish. Pour the prepared low-melting agarose into the small Petri dish, readjusting the orientation of the tissue. If needed, using forceps, then place the Petri dish on wet ice for faster hardening of the agarose.
Carefully cut the tissue using a scalpel, leaving at least five millimeters of surrounding agarose on each side of the tissue. Add glue to the sample holder, spread it, and transfer the embedded tissue and glue it on the sample holder using superglue. After a few seconds, place the sample holder into the cutting chamber and adjust the orientation of the tissue toward the razor blade if needed.
Define the outer limits of the cutting range according to the size of the tissue specimen. Adjust the blade toward the top of the tissue block. Set the cutting speed to 0.04 millimeters per second, cutting amplitude to one millimeter and slice thickness to 300 micrometers.
Carefully cut the first slices and transfer the slices to a separate container with pre-chilled ringer solution on wet ice. Prepare a six-well plate with one milliliter of the appropriate cultivation medium per well. Place the six-well plate with the medium into an incubator, allowing temperature and pH to adjust prior to cultivation.
Place slices onto cell culture inserts using a gaze filter, then remove any excess ringer solution by placing the loaded filter onto a sterile cloth. Place the loaded filter into the prepared six-well plate without adding any additional medium to the insert, then place the six-well plate in an incubator and change the medium every two days. Place the cultivation filter with the mounted slice on a Petri dish.
With a scalpel, carefully cut out the filter membrane with the mounted tissue slice. Transfer the filter membrane with the mounted slice into a biopsy nylon bag and place it in an embedding cassette. Subsequently, transfer plastic embedding cassettes in a container with pre-chilled 4.5%formalin for a minimum of 24 hours or until further use.
Cautiously rinse the formalin fixed slice culture with running tap water for 1.5 hours. Dehydrate the formalin fixed tissue slice by incubation in 70%95%and absolute ethanol. Then, clear the formalin fixed tissue slice with two three-hour incubations in xylene.
Immerse the tissue with paraffin at 60 degrees Celsius overnight and then again for two hours. Embed the tissue in a paraffin block in a tissue embedding mold and place it on ice to cool the sample faster. Section the paraffin-embedded tissue block at four-micrometer thickness with a Microtome and in a 40-degree-Celsius water bath containing distilled water.
Transfer the sections onto glass slides. Incubate paraffin sections for one hour at 60 degrees Celsius to bond the tissue to the glass. Then, incubate the slides overnight at 37 degrees Celsius.
Deparaffinize sections by incubation in xylene three times for five minutes each. Then, rehydrate by successive incubations in absolute alcohol, 95%alcohol, and 70%alcohol. Stain the sections in Mayer Hematoxylin solution for five minutes and rinse with running tap water for 10 minutes.
Counterstain in 0.5%ESN solution for 40 seconds and rinse with distilled water, then repeat the dehydration in ethanol. Clear the tissue in three changes of xylene for few seconds each, then place a drop of mounting medium and cover the slides with a cover slip. The macroscopic morphology of each OTSC decreased during cultivation for six days.
The in viability is saved for OTSCs from two representative primary tumors in two different media showed a decrease in viability after day zero due to the sectioning procedure and adjustment to culture conditions, as well as an increase in viability of the tumor specimen in the right panel. H&E stain sections showed that the overall structure of the tissue was preserved over the entire time of cultivation ex vivo, and that no gross changes of proliferation and apoptosis were detected during the culture period of six days. Microscopic histopathologic evaluation of H&E sections did not reveal a substantial increase in necrosis of all cultivated tissues during cultivation.
There was an increase in cleaved Caspase-3-positive cells after cultivating a primary pancreatic ductal adenocarcinoma, or PDAC, for 15 days. The histopathology of a peritoneal metastasis of a PDAC demonstrated a high intratumoral heterogeneity between individual slices, as well as naturally-occurring apoptosis measured by cleaved Caspase-3 staining. H&E staining and immunohistological characterization for Cytokeratin 7 in a PDAC metastasis of the abdominal wall showed that the derived OTSCs did not contain any tumor cells, but only consisted of connective tissue, which was partially necrotic.
Each cultured slice can be profiled individually, depending on the specific research question, for example, by RNA sequencing for transcriptomics or mild imaging for spatially resolved proteomics. In the future, the combination with non-destructive analytical methods has great potential for predicting patient-specific responses to different therapeutic regimes.
