In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

The scope of our research is in this area of liver organogenesis. The question we're trying to answer is how does the liver get so big and use it in a reverse engineering approach to build liver tissue from stem cells. The most recent developments in our field of liver regenerative medicine are in the area of miniaturization.

We are using stem cells to make miniature tissues that replicate the actual organ, and this way, we can study organ formation and organ biology. Our lab has several significant findings within our field of liver regenerative medicine. By using some of these miniaturization techniques, we have identified key steps during liver organogenesis, including morphogenesis and cell migration.

And so that is one of our key findings in the field. The advantages of our protocol within this publication enable several different organoid techniques during miniaturization. So essentially we can now study processes like branching morphogenesis and other aspects of migration in a miniature liver tissue that no other technique can provide.

The research questions that we will ask in the future, because of the work we've done so far, relate to how does growth and migration of the cells, how is that coordinated? And also we will ask about mechanisms by which we can assemble tissues into larger tissues that resemble the liver. To begin, cultivate human hepatoblastoma HepG2 wild-type cells in a T75 flask at 37 degrees Celsius with 5%carbon dioxide, changing the medium daily.

Upon reaching 80%confluency, rinse the cells with PBS. After discarding the PBS, incubate the cells with five milliliters of 0.05%of trypsin EDTA. Then add equal amounts of cDMEM to wash the cells off the flask.

Once cells detach, transfer the mixture to a 15-milliliter sterile conical centrifuge tube and centrifuge the cell suspension at 300 G for five minutes at room temperature. After resuspending the cells in cDMEM, count the cells to determine the final concentration in cells per milliliter. To label the cells, spin them at 300 G for five minutes in a 15-milliliter conical tube.

Resuspend the pellet in a serum-free growth medium to achieve a final concentration of one times 10 to the power of six cells per milliliter and incubate them with five microliters of fluorescent cell labeling solution per milliliter of cell suspension on rotation. Next, centrifuge the cell suspension at 450 G for five minutes and resuspend the cell pellet in fresh cDMEM. For spheroid formation, mix the cell suspension well and dispense 100 microliters of cell suspension to each well of the agarose-coated 96-well plate.

Centrifuge the plate at 340 G for 10 minutes and incubate at 37 degrees Celsius with 5%carbon dioxide for five to nine days. Hepatic spheroids, regardless of size, compacted fully by day five, transitioning from translucent to opaque as they thickened. To begin, obtain hepatic and mesenchymal spheroids in 96-well plates.

Using a pipette, collect human foreskin fibroblasts or HFF MRC-5 spheroids individually into a 15-milliliter sterile conical centrifuge tube containing fresh cDMEM. Incubate the spheroids for five minutes on ice to let them settle, and then gently rinse them with warm cDMEM. Using a pipette, gently transfer a single HFF MRC-5 spheroid to a well containing a human hepatoblastoma G2 wild-type spheroid.

Incubate the spheroids with ice-cold growth factor reduced Matrigel in a 1:1 dilution. Then, add 75 microliters of fresh cDMEM to each well and incubate at 37 degrees Celsius with 5%carbon dioxide for two to three days. Fluorescence microscopy and image analysis confirmed that the assembloid formation was successful across different matrices and media, though slight morphological variations were noted.

Further analysis revealed closer proximity of spheroids led to quicker compaction times. To begin, obtain hepatic spheroids from human hepatoblastoma G2 wild-type cells. Collect the spheroids individually into a 15-milliliter sterile conical centrifuge, and place the tube on ice to let the spheroids settle.

Then, aspirate the medium from the tube carefully. In a separate 15-milliliter sterile tube, mix one milliliter of ice-cold growth factor reduced Matrigel or GFR MG and ice-cold control growth medium at a 1:1 dilution. Add the mix into the tube containing the spheroids, ensuring even spheroid distribution.

Using a 200-microliter pipette, collect 15 microliters of a single spheroid in the MG solution and slowly seed it onto a 60-millimeter Petri dish. Then, incubate the droplets at 37 degrees Celsius with 5%carbon dioxide for 60 minutes and slowly add five milliliters of the desired growth medium to the Petri dish. Hep spheroids developed thick migrating strands protruding towards the fibroblast clusters by day nine.

To begin, harvest human foreskin fibroblasts, or HFF MRC-5 cells, and seed them into a T75 tissue culture treated flask at a density of 5, 000 cells per square centimeter. Incubate the flask at 37 degrees Celsius with 5%carbon dioxide for 72 hours in 15 milliliters of cDMEM. Next, collect the mesenchymal-conditioned media, or M-CM, in a 15-milliliter sterile conical tube and centrifuge it at 290 G for five minutes to remove debris.

Filter the supernatant with a 0.2-micrometer filter for sterilization and retain the filtrate. Then, dilute the M-CM with complete growth medium at up to 1:7 dilution ratio for the desired experiment. Suspend the HFF MRC-5 cells in fresh cDMEM to achieve a final concentration of six times 10 to the power of five cells per milliliter and place the tube on ice.

Mix the cell suspension and ice-cold growth factor reduced Matrigel or rat tail collagen in 1:1 dilution. Transfer 100 microliters of HFF MRC-5 and Matrigel solution to a well containing a hepatic spheroid and incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 30 minutes. Finally, add 75 microliters of fresh cDMEM to each well and incubate for up to 12 days.

Collective migration was effectively induced in vitro using droplet formation of hepatic spheroids with M-CM. The concentration-dependent effects of M-CM fostered the emergence of cellular strands and branching from the spheroids. By day 11, the cellular protrusions from the spheroids became more pronounced, forming interconnected sheets.