Bioprinting of Hydrogel Tumor Slices as a 3D Model for Mantle Cell Lymphoma

We developed a hydrogel-based bioprinted model to replicate the conditions of mantle cell lymphoma in patients, so we can better study how it survives and responds to treatments. The challenge is to figure out the best way to bioprint, culture, and analyze 3D models specifically designed for lymphomas. Due to drug resistance and relapse, mantle cell lymphomas are still not curable.

Our model can help to understand how tumor heterogeneity and the microenvironment influence drug responses. Compared to other 3D models of mantle cell lymphoma, our model mimics the network of extracellular matrix fibers within the lymph node by using a bioink containing collagen and Matrigel. The presented model enhances physiological relevance compared to traditional 2D cell culture systems.

It has the potential to advance both biological and therapeutic studies of mantle cell lymphomas. To begin, gather materials to prepare the bioink adapted for the cells of interest. For mantle cell lymphoma or MCL Jeko-1 cells, mix alginate and collagen in a suitable buffer to obtain one milliliter of bioink.

Transfer the MCL cell suspension from the culture flask into a 50 milliliter conical tube. Mix the cells with 0.4%trypan blue solution in a 1:1 ratio. Using a cell counter or Neubauer chamber, count the viable unstained cells and calculate the volume of cell suspension corresponding to 14 million cells for one milliliter of bioink.

Transfer the calculated volume into a fresh 50 milliliter conical tube and centrifuge the tube at 340 g for five minutes. After centrifugation, remove the supernatant, and resuspend the cell pellet in the prepared bioink to generate the final cell-laden bioink. Switch on the bioprinter and connect it to the 3D printer software.

Open the STL file containing the hydrogel tumor slice blueprint using the software. Ensure each hydrogel slice has a diameter of eight millimeters and a height of 1.5 millimeters. Then set the fill density to 85%and home the printer head.

Now, transfer the gelatin slurry into a 35 millimeter Petri dish until it is half full. Use disposable precision wipes to remove excess water and eliminate any air gaps in the gelatin, forming a stable support bath. Load the cell-laden bioink into a 2.5 milliliter glass syringe and attach a 0.8 millimeter blunt nozzle.

Invert the syringe and slowly eject air bubbles, and then insert the prepared syringe into the bioprinter. Using the 3D printer software, extrude a small amount of bioink to check the flow. Wipe off the extruded drop with a disposable precision wipe to avoid nozzle clogging.

Next, place the gelatin support bath beneath the syringe nozzle and lower the printer head until the nozzle is two millimeters above the bottom of the gelatin support bath. Start the printing process using the 3D printer software. Once printing is complete, remove any excess gelatin slurry from the nozzle using a disposable precision wipe to prevent clogging.

Take out the Petri dish containing the gelatin support bath with the printed hydrogel tumor slices and cover it with a sterile lid. Place the covered support bath in an incubator set to 37 degrees Celsius to allow the gelatin to melt and release the printed hydrogel tumor slices. Then warm the wash buffer and cell culture medium to 37 degrees Celsius.

While the gelatin is melting, prepare a six-well plate by filling two wells with 10 millimolar HEPES and 14.4 millimolar calcium chloride wash buffer, and two wells with pre-warmed cell culture medium. Once the gelatin has melted, use a sterile spatula to carefully transfer the hydrogel tumor slices into the first of the wash buffer-filled wells. After washing for one minute in each of the wells, transfer them into the medium-containing wells, washing for one minute in each.

To culture the hydrogel tumor slices, place them onto a 0.4 micrometer pore size filter support positioned inside a six-well plate. Add 1.5 milliliters of cell culture medium below the filter support, followed by a small drop of medium directly on top of each hydrogel tumor slice to prevent drying. For drug treatment, add the desired amount of drug to the culture medium.

Finally, place the six-well plate into a cell culture incubator set to 37 degrees Celsius and 5%carbon dioxide for the desired cultivation period. The hydrogel tumor slices retained their structure over three days of culture, with air bubbles present after printing that disappeared during cultivation. The viability of Jeko-1 cells cultured in hydrogel slices for three days was 80%which was not significantly different from cells cultured in 2D suspension at 88%Immediately after printing, Jeko-1 cells were evenly distributed in the hydrogel slice and were predominantly TMRM positive, indicating viability.

Only a few cells were positive for caspase-3 and PicoGreen, markers of apoptosis and death respectively. After three days of culture, Jeko-1 cells formed clusters and remained mostly TMRM positive. The hydrogel tumor slices maintained their structural integrity after three days of doxorubicin treatment.

Jeko-1 cells cultured in hydrogel slices showed a dose-dependent decrease in viability in response to doxorubicin treatment. The half-maximal inhibitory concentration of doxorubicin was higher for Jeko-1 cells in hydrogel slices at around 5.8 micromolar compared to suspension culture at two micromolar, indicating reduced sensitivity in 3D culture. Live fluorescence imaging of hydrogel slices treated with doxorubicin showed reduced TMRM signal, indicating loss of mitochondrial membrane potential, and increased PicoGreen signal, indicating enhanced cell death.

After four days of culture, primary MCL cells in hydrogel slices maintained a viability of 43.6%which was significantly higher than the 19.6%observed in suspension culture. Fluorescence imaging indicated that cells in hydrogel were majorly TMRM positive.