Generation of human liver spheroids using autologous sources can contribute to various research area like toxicology, cancer, and drug discovery. The main advantage of this technique is to use BDPC-derived human hepatocytes to engineer human liver spheroids, circumventing the shortage of primary human hepatocytes or PHH. Autologous human spheroids can be extended to regenerative medicine and therapy, especially in patient with acute liver failure.
Demonstrating the procedure will be Dr.Anne-Katherin Schott, project leader in my laboratory. Begin by preparing 500 milliliters of hepatoblast KnockOut serum replacement dimethyl sulfoxide or KSR DMSO medium and hepatocyte maturation medium. Aliquot the medium from the stock and add fresh hepatocyte growth factor or HGF and oncostatin M or OCM at final concentrations of 10 or 20 nanograms per milliliter for each medium change.
Transfer three times 10 to the fifth blood-derived pluripotent stem cells or BD-PSCs cells into a biolaminin coated four-well plate and incubate the plate in an incubator at 37 degrees Celsius and 5%carbon dioxide for five days. To support the endodermal differentiation, culture the cells for five days in KSR DMSO hepatoblast medium and change the medium every 48 hours. On day five, add hepatocyte maturation medium and culture the cells for an additional seven to 10 days in the incubator at 37 degrees Celsius and 5%carbon dioxide, ensuring changing of the medium every 48 hours.
Count cells using a counting chamber and centrifuge BD de-differentiated cell suspension at 300 G for 10 minutes at room temperature. After removing the supernatant, resuspend BD de-differentiated cells in KSR DMSO medium at two times 10 of the six cells per milliliter concentration. After ensuring the single cell suspension, remove any additional debris by passing the cells through a 40 micrometer cell strainer.
Again, count the cells using a counting chamber and prepare a sufficient volume of each cell seeding density to dispense the required volume per well. Prepare a gradient with top seeding of one million cells to a low seeding density of 4, 000 cells. Next, dispense 100 microliters of KSR DMSO medium in 96-well low attachment plates and add 100 microliters of cell seeding dilution.
Incubate the low attachment plate for five days. Change 50%of the medium on the third or fourth day after seeding when the spheroids are sufficiently compact. On day five, change the medium to hepatocyte maturation medium and culture the cells in it for an additional seven to 10 days, changing the medium every 48 hours.
After culturing the cells for 4, 8, 15, and 24 days using the differentiation method described earlier, remove the media. Incubate the cells with pre-warmed fixatives containing 4%paraformaldehyde in PBS for 10 minutes. Next, discard the fixative and wash the cells with PBS two times for five minutes each.
Immediately add 0.1%Triton X-100 solution and permeabilize the cells for five minutes before two washes of PBS. Add a blocking solution made of PBS and 5%bovine serum albumin or BSA before placing the cells on a rocker plate for one hour at room temperature. Dilute primary antibodies in dilution buffer 1%BSA PBS and add 50 microliters of the antibody dilution per well.
Discard the antibody solution after a one-hour incubation at room temperature and give three washes of five minutes each using PBS. Add 50 microliters of secondary antibodies diluted in dilution buffer in each well and incubate the cells for 30 minutes at room temperature. After washing the cells three times with PBS for five minutes each, mount the coverslips with mounting media containing DAPI for microscopic analysis.
Carefully discard culture media without touching the spheroids and add freshly prepared PBS with 0.1%Triton X-100 solution and incubate for five minutes to permeabilize the cells. Wash the spheroids two times with the medium by slowly pipetting the medium. Next, incubate the spheroids with 50 microliters of the primary antibodies for one hour.
Once done, carefully remove the excess antibody solution and give three washes of medium. Prepare the corresponding secondary antibodies like goat anti-mouse IgG Cy3, goat anti-mouse IgG 488, and rabbit anti-chicken IgG Texas Red in PBS with 1%BSA and add 50 microliters of antibody dilution per well before 20 minutes of incubation in the carbon dioxide incubator. Again, wash thrice with the medium before 30 minutes of incubation in the incubator.
Switch on the fluorescence light source 10 minutes before use. Turn on the computer and open the imaging software. Use 4X magnification objective by clicking on the 4X button in the toolbar to select the correct scale bar.
Then place the 96-well plate on the stage center plate. Turn on the LED light source, use the brightfield filter, and position the plate to the well of interest using the XY axis stage adjustment knob. Change to camera light path and click the live button in the imaging software to visualize the image on the screen.
Ensure the spheroid is centered using the XY axis knobs and focus using the coarse or fine focus knob. Next, choose the brightfield observation method in the toolbar, put exposure settings to automatic, and click the snapshot button in the camera control panel to take a picture. Then save the picture as vsi file using the appropriate name in the folder of interest.
Place ambient light shielding plate to turn off the LED light and change the filter for B excitation. Then choose 488 observation method. Open the shutter, take a picture by clicking the snapshot button and close the shutter and save the file as a vsi.
Repeat the process with a filter for G excitation to reiterate the process for each well of interest. Morphological changes during the hepatic differentiation process showed that the BD-PSCs differentiated into hepatocytes through three stages. The first stage represented the differentiation into endodermal cells.
The second differentiation into hepatic progenitor cells exhibiting a typical polygonal morphology. And the third, the maturation to hepatocytes. Immunofluorescence analysis showed the strong expression of endoderm human liver progenitor markers like the alpha fetoprotein or APF and transthyretin or TTR in the cells at the first stage of hepatic differentiation process at L4 to L8 day.
However, their expression decreased at L15 day. The expression of albumin or ALB and hepatocyte nuclear factor four alpha or HNF4 alpha appeared firstly at L4, increased throughout the differentiation time L4 to L15, and reached a strong and stable expression during the maturation time L15 to L24. Spontaneous aggregation of cells was observed in low attachment plates containing hepatocyte induction or maturation medium initiated spheroid formation.
A consistent correlation was observed between steroid spheroid and the variable number of cells. The spheroids formed and differentiated at L14 and live stained with antibodies revealed the potential hepatic functional activity of BD-PSCs derived spheroids. The preparation of mononuclear cells for reprogramming is the most important step in this procedure.
Generating human liver spheroids is the first step to creating organoids in vitro simplified version of the organ in 3D with micro anatomy similar to that in vivo. Organoids created in the lab are used to study organogenesis, disease development, and nutrients.
