An All-Human Hepatic Culture System for Drug Development Applications

TruVivo was developed to fill the need for a convenient, yet robust in-vitro platform that maintains phenotypically stable and functional primary human hepatocytes. Because of these qualities, the system serves as a basic platform for answering a wide range of questions pertaining to preclinical drug development. One challenge is the need to create a multicellular system that has the biological complexity required to answer today's questions, yet is still easy to use.

Current systems often fail to maintain hepatocyte morphology and function long term or present technical challenges with establishing routine, reproducible results. With TruVivo, users can still rely on the simplicity and flexibility of traditional 2D cell culture techniques. Additionally, there is no special equipment required and the system is amenable to traditional analytical methods.

With TruVivo, we are now able to predict metabolic clearance, major metabolites, key pathways of elimination, other pharmacokinetic properties of compounds more accurately with long half-lives prior to preclinical evaluation. To begin, thaw one bottle of each feeder cell thawing media, supplement A, supplement B, and supplement C, in a 37 degrees Celsius water bath. Alternatively, refrigerate them at four degrees Celsius for 16 to 24 hours.

Transfer the entire contents of each bottle of thawed medium into a 15-milliliter or 50-milliliter conical tube. To prepare complete plating medium, take a bottle of the plating medium and add 4.5 milliliters of supplement B, followed by 11 milliliters of supplement A into the bottle. In a separate bottle, combine 100 milliliters of culture medium, one milliliter of supplement C, and 14 milliliters of supplement A to prepare the complete culture medium.

Store the remaining supplement C and supplement A at minus 20 degrees Celsius until week two media preparation. To begin, thaw the feeder cells in a 37 degrees Celsius water bath for one to two minutes. Immediately after thawing, place the feeder cells on ice.

Using aseptic techniques within a biosafety cabinet, add the freshly thawed cells to 10 milliliters of feeder cell thawing medium. Then with a 1, 000-microliter pipette, take one milliliter of the resultant cell suspension in the thawing medium, wash the empty cell vial with it, and combine it with the rest of the cell suspension. Centrifuge the cells at room temperature and 400G for four minutes.

Carefully discard the supernatant by aspirating or pipetting without disrupting the cell pellet. After re-suspending the pellet in one milliliter of complete plating medium, count the feeder cells. Then dilute the cell suspension using the complete plating medium.

Next, plate 500 microliters of the diluted cell suspension per well in a collagen-coated 24-well plate. Agitate the plate in a north, south, east, west motion by employing a back and forth shaking in the north-south direction two times, followed by the same motion in the east-west direction. Finally, incubate the plate before seeding the primary human hepatocytes in it.

Examine the feeder cell attachment before thawing the hepatocytes for seeding. Proceed to perform the plating of primary human hepatocytes or PHHs after almost one hour of culturing the feeder cells. To do so, after 30 minutes of feeder cell culture, filter the pre-warmed hepatocyte thawing medium through a 0.2-micron polyether-sulphone filter unit.

Thaw the PHHs in a 37 degrees Celsius water bath for one to two minutes. Immediately after thawing, place the thawed cells on ice. Then, working in a biological safety cabinet, transfer the cells into the hepatocyte thawing medium.

Using a 1, 000-microliter pipette, transfer one milliliter of the hepatocyte suspension in the thawing medium back into the vial and wash the vial three to four times by gentle pipetting. Combine the washing with the rest of the cell suspension in the tube. Cap and gently invert the thawed PHH suspension five times.

Spin it at 100G for eight minutes at room temperature. Carefully discard the supernatant by vacuum aspiration or pipetting without disturbing the cell pellet. Then add three milliliters of complete plating medium to the wall of the conical tube containing the pellet.

Rock the conical tube side to side to re-suspend the cell pellet and add five milliliters of complete plating medium. After counting the re-suspended hepatocytes, adjust the cell suspension to the desired seeding density using complete plating medium. Next, obtain the collagen-coated 24-well plate containing the previously plated feeder cells, and using a pipette or vacuum aspiration, remove the medium from the feeder cells.

Immediately plate 500 microliters of the diluted hepatocyte suspension in each well containing the feeder cells. Shake the plate in a north, south, east, west motion by performing a back and forth motion in the north-south direction two times, followed by the same motion in the east-west direction. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for two to four hours.

For the first 60 minutes of culture, shake the plate every 15 minutes, as demonstrated previously. Following incubation, transfer the plate from the incubator to the biological safety cabinet. After shaking the plate, use pipetting or vacuum aspiration to remove the complete plating medium.

Immediately add 500 microliters of pre-warmed complete culture medium to each well. Images of PHHs from two donors cultured at various seeding densities with feeder cells were obtained on day seven and day 14 of the culture. The various seeding densities showed visual differences of confluence, but maintained the typical hepatic cuboidal morphology.