The overall goal of this procedure is to isolate primary cells from surgical human colon tumor, as well as from normal tissue, and to directly culture them on substrates with different elasticity for analysis. This is accomplished by first collecting surgical tissue from a human colon tumor and from a normal region. Next, enzymatic dissociation of minced tissue results in numerous single cells.
The final step is to culture the isolated cells on soft poly acrylamide hydrogels embedded with nanoscale, fluorescent beads, and on polystyrene dishes. Ultimately, traction cytometry is performed to obtain a spatial cellular traction map, and immunofluorescence microscopy is performed to visualize cytoskeleton organizations. The main advantage of this technique is that isolated primary human colon cells from surgical tissues can be directly cultured on substrates with different rigidity for traction cytometry and immunofluorescence microscope assays.
This method can help us answer the question of whether primary tumor cell contractility can be used as a potential marker for cancer prognosis. To begin, collect the tumor tissue sample as described in the text protocol. Then pour the tissue into a six well plate containing six to seven milliliters of Hank's balanced salt solution or HBSS using a pipette.
Keep the six well plate on an ice block and rinse the tissue in HBSS solution twice. Next, cut the tissue cleanly into separated sections with sterile scissors. Mince the tissue into smaller sections, no greater than one to two cubic millimeters in size with a sterile scalpel blade.
Weigh a labeled 0.1%tryin vial containing one milliliter of 0.1%tryin solution before transferring the tissue. Then transfer approximately 20 milligrams of tissue into each of the tripsin vials with a pipette. Weigh the trips and vials after tissue transfer and document the difference as a tissue mass.
Now pour approximately 20 milligrams of tissue and one milliliter of trypsin into each well of a 24 well plate. Put the 24 well plate on a shaker at four degrees Celsius for 16 to 20 hours. After 16 to 20 hours of incubation in the tryin, well use a pipette to transfer the tissue to two to three milliliters of complete growth media at four degrees Celsius.
Place the vial in a warm water bath at 37 degrees Celsius for 12 to 15 minutes. Next, transfer the tissue to a 15 milliliter vial containing HBSS solution. Using a pipette and shake gently repeat this step a second time.
Then remove the tissue from the HBSS solution and transfer to a vial containing one milliliter of 0.1%Collagenase solution. Incubate for 45 minutes at 37 degrees Celsius and 5%carbon dioxide environment in a humidified cell culture incubator During the weight period, warm growth media in 15 milliliter vials to 37 degrees Celsius. Next, pull out all tissue fragments into a pipette tip, minimizing collagenase, eject just the tissue fragments into the prewarm culture.
Media vials. Then place a 40 micron filter into each well of a six. Well plate moisten the filter with one milliliter of cell culture media.
Rerate the tissue in media with a 10 milliliter glass pipette several times until the tissue is completely dissociated. Maintain the pipette tip as close as possible to the base of the vial. Then deposit the solution onto the filter to remove debris and unassociated parts.
Centrifuge the filtered cell suspension in media at 150 times G for five minutes. At room temperature, remove the supernatant afterwards and resuspend the cell pellet with two milliliters of fresh culture media. Seed the isolated primary cells on extracellular matrix or ECM functionalized gels and polystyrene dishes at the desired concentration.
To perform the traction force microscopy assay, first warm 0.25%trips in EDTA 10%SDS solution to 37 degrees Celsius in a water bath for 10 minutes. Before traction experiments, start remove one gel at a time from the cell culture incubator and place on the microscope stage. After finding a single cell in the field of view, remove the Petri dish lid and take a phase contrast image of the cell.
Next, switch the imaging mode to fluorescence and select the appropriate filter. Don't move the microscope stage or sample during this time. Then take an image of the fluorescent beads displaced by cellular traction.
Now add one milliliter of trypsin SDS solution to the Petri dish to detach the cell from the gel. Take a reference image of the beads After the cell is removed, take the substrates to be immunostain to the laminar hood and remove the culture media. Then rinse the substrates with phosphate buffered saline or PBS and fix the cells with 4%para formaldehyde in PBS for 20 minutes At room temperature, wash the substrates with PBS for five minutes a total of three times.
Then incubate the substrates with 500 microliters of signal enhancer for 30 minutes before rinsing with PBS. Next, incubate the cells with monoclonal anti vanolin antibody at a one to 250 dilution in PBS for 45 minutes at room temperature. Then wash the substrates with PBS for five minutes a total of three times.
Incubate the samples with a secondary antibody LOR 4 88 goat anti mouse IgG at a one to 200 dilution in PBS at room temperature for 30 minutes. Then wash the substrates again with PBS for five minutes, a total of three times. To visualize the f actin structure, incubate the cells with tetraethyl rod domine Phin conjugates at a concentration of 50 microliters per milliliter for 45 minutes at room temperature.
Then wash the substrates again as before. Finally proceed to image the samples using a confocal scanning laser microscope phase contrast micrographs illustrate the representative morphology of primary human colon cancer and normal cells. As a function of substrate rigidity, it is evident that primary colon cells spread more on polystyrene dishes compared to the poly acrylamide gels.
Traction assays are performed on ECM coated soft two kilo pascal gels after confirmation of invasive adenocarcinoma from pathological hematin and eosin staining. Shown here is a snapshot of the nanoscale fluorescent beads embedded inside the gel from the displaced and reference bead images. The displacement fields are obtained using the image J particle image vilo symmetry plugin.
A representative bead displacement field generated by invasive colon tumor cells on a two kilo pascal gel is displayed with the traction stress obtained. Using the image J furrier transform traction cytometry plugin corresponding to the displacement field. Shown here is the f actin and cullin containing focal adhesions of primary human colon cells on soft two kilopascal gels and rigid polystyrene substrates.
No actin fiber was present in the less spread cells on soft gels. Also, punctate cullin containing focal adhesions are present on soft gels. Conversely, primary cells show well spread morphology, well-defined act, and stress fibers and discreet elongated focal adhesions on rigid polystyrene substrates.
Following this procedure, other methods like cell stiffness measurement using atomic force microscopy can be performed. After watching this video, you should have a good understanding of how to isolate colon cells from surgical tissue via in geomatic dissociation, and consequently cultured them directly on poly acrylamide hydrogels to assess cell contractility.
