This method can help answer key questions in hepatotropic infections and liver disease fields. Such as the contribution of liver resident cell populations, to viral pathogenesis, longitudinal infection kinetics, mechanisms of viral replication, immune invasion, in a physiological model system and drug testing studies. The main advantage of this technique is the ability to recapitulate a hepatic microenvironment.
Which can be maintained for extended periods of time and is naturally susceptible to hepatitis B infection at physiological relevant levels. This has previously been a fundamental constraint in the hepatitis B field. The implications of this technique extend toward therapy or diagnosis of HBV infection because drug testing studies can be performed in a physiological platform for extended durations.
This allows the evaluation of sequential drug treatments alongside with analyzing the efficacy of existing and novel treatment strategies. This method can provide understanding into hepatitis B, and can also be applied to other studies of disease, including other hepatotropic infections, liver diseases, or drug metabolism studies. Generally, individuals new to this method will struggle, because it requires attention to particular details to allow the successful maintenance of liver tissue for long periods of time.
And also familiarization with the specialized tools and equipment. Visual demonstration of this method is critical. Including assembly of the plates, as well as thawing and seeding of the cells.
Since steps involved in the long-term culture of liver-derived cells are challenging when using a novel culture system. First, turn on both the compressor and the vacuum pump associated with the liver chip platform. Proceed to a glass tube cabinet to assemble and equilibrate the plates.
Place a sterile membrane on the plate base to begin aseptically assembling the microfluidic plates. Making sure that the sterile membrane rests smoothly on the two pins of the baseplate. Then add the well-containing top plate.
Add a sterile plate lid. And use an automated precision torque, set to 33 pounds. Using a spiral tightening sequence to tighten the screws at the base of the plate.
Using a manual torque, make sure that all screws are tightened to 35 pounds. Next, pre-warm the hepatocyte seeding medium to 37 degrees Celsius before priming. Place the completely-assembled plate in the washing dock.
And make sure the plate snaps in completely. Add 400 microliters of hepatocyte seeding medium to the reservoir side of each well to prime the plate. After this, initiate flow in the upward direction for 3.5 minutes at one microliter per second.
The red indicators at the side of the plate will indicate if the microfluidic circulation is functioning properly. Once the medium is pumped to the cell-growth side of the plate, add an additional 1.2 milliliters of hepatocyte seeding medium. Carefully transfer the plate into the docking station within a humidified incubator at 37 degrees Celsius and 5%CO2.
Initiate flow in the upward direction at a flow rate of one microliter per second for 16 hours. After this, transfer the plate to a washing dock. Gently pipette up and down to eliminate any bubbles.
Using a sterile forceps, add a sterile round filter paper to each well. Then add a cell attachment scaffold and a retaining ring to each well. Use a sterile plunger to push down each well and lock the retaining rings and scaffolds into place.
Next, aspirate all of the medium. And gently add 400 microliters of pre-warmed hepatocyte seeding medium over the scaffold. Initiate flow in a downward direction at one microliter per second for 3 1/2 minutes.
Aspirate all medium pumped out of the reservoir side of the plate. Add 1.4 milliliters of hepatocyte seeding medium to each well. Then return the plate to the dock to bring the total volume per well up to 1.6 milliliters.
First, pre-warm the hepatocyte thawing medium and hepatocyte seeding medium to 37 degrees Celsius. Next, thaw one vial of primary human hepatocytes according to the supplier's instructions. In a glass tube cabinet, resuspend the cells in one milliliter of hepatocyte seeding medium.
Then place the resuspended cells on ice. Using trypan blue, count the cells to ensure that the viability of the cells is above 90%Transfer the fully-assembled plate to the washing dock. And aspirate all medium from the wells.
Retrieve the cells from the ice, and add 600, 000 hepatocytes to each well in a 500-microliter volume of hepatocyte seeding medium. Initiate flow in the downward direction at a flow rate of one microliter per second. Add 900 microliters of hepatocyte seeding medium to each well to bring the total volume in each to 1.6 milliliters.
Transfer the plate to the docking station within a humidified incubator at 37 degrees Celsius and with 5%CO2. Initiate flow in the downward direction at a flow rate of one microliter per second for eight hours. After this, reverse the flow to the upward direction at a flow rate of one microliter per second for eight hours.
Then transfer the plate to the washing dock, and aspirate all medium from the wells. Add 400 microliters of hepatocyte maintenance medium to each well. And initiate flow in the downward direction at a flow rate of one microliter per second for 3 1/2 minutes.
Next, aspirate all medium from the reservoir, and add 1.4 milliliters of hepatocyte maintenance medium. Transfer the plate into the docking station within a humidified incubator at 37 degrees Celsius and 5%CO2. Initiate flow in the upward direction at a flow rate of one microliter per second for 48 hours.
After 48 hours, wash the plate by transferring it to the washing dock. Aspirate all medium from the wells. And add 400 microliters of maintenance medium.
And initiate flow in the downward direction at one microliter per second for 3.5 minutes. Then aspirate all medium appearing in the reservoir side of the wells. Add 1.4 milliliters of hepatocyte maintenance medium.
Then transfer the plate back to the docking station in the humidified incubator. Initiate flow in the upward direction at a flow rate of one microliter per second for 48 hours. Replace the medium using this wash-and-replace process every 48 hours.
Primary human hepatocytes are usually only stable for a limited amount of time when using conventional culture systems. However, using the protocol described here, they can be functionally maintained for extended time periods. Human albumin is secreted by functional hepatocytes, and is considered to be the best marker for evaluating hepatic functionality.
Albumin is seen to be stably and highly expressed by 3D cultures until at least day 40 post-seeding. For co-cultures, Kupffer cells'functionality and viability are evaluated by measuring the secretion of specific cytokines. As seen here, the measured levels of IL-6 and TNF alpha indicate that cells were functionally maintained and viable.
In addition to retaining their physiological cellular metabolism, these cultures became exceptionally susceptible to HBV infection. HBV DNA and other viral markers are readily detectable from day two post-infection. While conventional cultures require inoculation with at least 500 HBV genome equivalents supplemented with DMSO and PEG, these 3D cultures are infected with as little as 05 unsupplemented genome equivalents.
In addition to secreted markers of viral infection, hepatocyte-containing scaffolds are retrieved from these cultures. Immunofluorescence microscopy reveals that these scaffolds contain viral antigens. While attempting this procedure, it's important to remember to be gentle when handling the hepatocytes before seeding to avoid cell death.
Additionally, it's essential to ensure that the plates are correctly assembled and all the flow channels are allowing correct media circulation prior to seeding the hepatocytes. Following this procedure, other methods like immunofluorescence can be performed in order to answer additional questions. Including the location and frequency of infected cells within the tissue.
The physiological state of the hepatocytes can also be assessed by staining for albumin or hepatic structures including tight junction protein ZO-1. After this development, this technique paves the way for researchers in the field of virology to explore hepatitis B infection and immune responses in a physiological 3D model system. Don't forget that working with hepatitis B virus can be hazardous, and precautions such as previous vaccination, the use of appropriate PPE, and working under appropriate biosafety guidelines should always be taken while performing this procedure.
