The overall goal of this procedure is to develop a three-dimensional liver model to study viruses and viral vectors. This method allows for the study of biological processes in a vascularized three-dimensional system with human cells which differ in their physiology from rodent cells in a mouse or rat model. In addition, it is a step to improve animal welfare as it avoids experiments with living animals and is, on the long run, intended to fully replace animal components.
Though we developed this method to study viral vectors for gene transfer, it can also be applied to the study of infectious vital viruses and other research areas such as cancer research. Demonstrating the procedure will be Viola Rohrs, a technician from my laboratory. For the sake of animal welfare, only surplus animals were used for the present study that were sacrificed for other animal experiments, i.e.
no additional animals were needed to obtain the liver scaffolds. To begin, first expand the hepatic cell line Hep G2.Seed 15 million cells into T175 bottles in 30 milliliters of medium containing 10%fetal calf serum and two millimoles of gulatmine, penicillin, and streptomycin. After four days of culturing, use five minutes of trypsin solution exposure at culturing conditions to loosen the cells, and then harvest them.
Then spin down the cells at 300 Gs for three minutes. Then re-suspend them in four milliliters of PBS and get a cell count. Each bottle should yield about 45 million cells.
600 million cells are needed to recellularize a rat liver ECM. To recellularize the ECM, first set up the bioreactor system containing a liver profusion chamber, a profusion system, a reservoir of medium, and a bubble trap. There are two critical steps to the recellularization procedure.
One is to avoid trapping any air bubbles in the profusion system. The second is that the whole system must be kept sterile. First, sterilize these components of the bioreactor system at 121 degrees Celsius for 15 minutes.
Second, fill the tubings and the sterilized profusion chamber with medium. Then place the decellularized rat liver scaffold into the liver profusion chamber. The next step is to use tube clips to connect the canulated portal vein to the profusion system.
Now place the bioreactor system into the incubator and connect it to a peristaltic pump. Next, equilibrate the scaffold with 150 milliliters of supplemented medium over five days. Set the flow to 1.25 milliliters per minute.
After five days, disconnect the bioreactor system from the pump and transfer it to a sterile hood. Disconnect the liver scaffold from the media circuit. Then load a syringe with five milliliters of medium containing 300 million Hep G2 cells and inject the cells via the portal vein, avoiding the formation of air bubbles.
Then allow the cells to repopulate the ECM for an hour without running the pump. After an hour, start the pump at 1.25 milliliters per minute. After 10 minutes, increase the pressure to 2.5 milliliters per minute.
After 20 more minutes, set the pump to the operating speed of 3.75 milliliters per minute. After 30 minutes at full pump speed, turn off the pump, add another 300 million cells, and let the cells populate the ECM for an hour. After an hour, gradually increase the circulation by incremental adjustments to the pump speed as before.
Now let the culture recellularize the liver over two weeks. Every other day, replace 50 milliliters of the medium and measure the physiological parameters from a sample of the used medium. Production of AAV vectors is a complicated procedure involving many steps that are summarized in the text protocol.
In this section of the video, some of the crucial steps are demonstrated. First, produce, purify, and quantify the AAV vectors. Briefly produce the AAV vectors in roller bottles and purify them by iodixanol gradient centrifugation.
Remove the residual iodixanol by filtration over PD10 gel filtration columns. Then determine the AAV vector concentration by QPCR using genomic AAV DNA as a standard. A total of 27 trillion AAV vectors are needed per model.
Now transduce the liver model. First, get 27 trillion vectors in five milliliters of PBS loaded into a syringe. Phenol red may be added at 5 micrograms per milliliter.
Then disconnect the liver from the media circuit and inject the vectors into the system. Once injected, incubate the system for one hour without pumping. Then gradually increase the flow as previously described.
Later, as the transduced liver is incubated over six days, change 50 milliliters of the medium every other day. Using a scalpel, slice samples from each liver lobe that are about half a centimeter thick and 1.5 to two centimeters long. Incubate the slices in 4%paraformaldehyde with 4%sucrose for 90 minutes at four degrees Celsius.
Then wash the sample three times with PBS for one minute per wash. Follow the washes by incubating the samples overnight in 8%sucrose at four degrees Celsius. The next day, load the samples into cryomolds containing some fixing medium.
Avoid introducing any air bubbles. Once loaded, fully cover the samples with fixing medium and put them at negative 80 degrees Celsius. Once froze, prepare 10 micron cryosections from the samples using a cryotome.
Stain the samples as needed to confirm the recellularization. For gene expression analysis, take samples using a four millimeter biopsy punch. To assess the recellularization, cryosections were stained with hematoxylin and eosin.
Lobes from two transduced livers and one control liver that was recellularized but not transduced were all repopulated with Hep G2 cells. RNA expression and vector titre were quantified from 28 punch biopsies from each liver model. In the two transduced liver models, 55 and 90 vector genomes per cell were measured, which is enough vector to induce gene silencing.
No genomes were measured in the control, as expected. EmGFP expression was analyzed by RT-PCR and western blotting. Most of the biopsies showed mRNA expression of EmGFP and protein expression of EmGFP.
Immunostanding of the cryosections showed that, wherever the model was successfully recellularized, EmGFP expression could also be seen. This is indicative of good AAV gene expression. Next the AAV mediated knockdown of human cyclophilin B was analyzed by quantitative RT-PCR.
The average knockdown of human cyclophilin B was between 70 and 90%over all the lobes of the two transduced livers. After its development, this technique paved the way for researchers to study the distribution of viruses and viral vectors in the vascularized, three-dimensional organ system. Such studies will help to improve current techniques for gene transfer and help to develop new antiviral strategies.
