media preparation for setting up the all-human 2d+ hepatic culture system

A Physiologically-Relevant All-Human 2D+ Hepatic System for Drug Discovery

Predicting therapeutic safety and efficacy is an important part of preclinical drug development. However, conventional preclinical in vitro hepatic models are limited in their ability to accurately mimic the in vivo liver cellular microenvironment and maintain hepatocyte functionality and morphology over time. There is a need for models that provide stable metabolic competence for more than 1 week to assess slow-turnover compounds or investigate outcomes associated with sub-acute or chronic exposure. In vivo animal tests often fail to predict drug efficacy and risk due to translational species differences in human hepatic clearance mechanisms1. Current in vitro 2-dimensional (2D) hepatic models, such as traditional primary human hepatocyte (PHH) monoculture or sandwich cultures, lack phenotypic stability and longevity in culture, leading to loss of key hepatocellular function and architectural integrity over time2. An alternative method involves the formation of 3-dimensional (3D) hepatocyte spheroids, offering a more relevant microenvironment versus 2D culture. However, this method is limited by the availability of raw materials, PHH donor-lot selection, reproducibility, and loss of viability with increasing spheroid size3,4,5,6. Multicellular platforms have been introduced whereby PHHs are seeded with feeder cells onto fixed-sized, micropatterned plates. Though these models may enable longer culture times, non-human feeder cells used in these platforms can alter experimental outcomes and limit their application due to innate background contribution to drug clearance and metabolic profiles1,7. The TruVivo all-human 2D+ hepatic system (TV2D+) recently described in Weaver, et al8, was developed to address some of the limitations of traditional, co-culture, and 3D culture methods of PHHs. The non liver feeder cells reduce the interspecies differences in metabolism and paracrine production and provide the support necessary for primary hepatocytes in a reproducible, robust fashion that cannot be provided by corresponding non-parenchymal liver cells from a single donor lot due to limitations in expansion, phenotype, and performance. The feeder cells chosen were able to be consistently expanded before use and lacked the need for transformation or differentiation. As described by Glicklis et al.4 and Khetani et al.5, 3D culture models like hepatocyte spheroids have challenges with reproducibility due to donor variability and maintaining consistency in spheroid size, which affects nutrient diffusion in spheroids greater than 200 &#181;m, leading to decreased viability and functionality. Like 3D spheroid formation, the TV2D+ system relies on self-assembly of PHHs; however, the PHH colonies formed are dispersed over the surface area of the well at a single-cell depth rather than compacted into a single aggregate. This method of culture may be useful to address donor variability, allowing PHHs to be plated, cultured, and maintain basic functionality at various seeding densities. The TV2D+ system may also increase the robustness of user handling due to loss during manipulation or specialized equipment required to perform extended culture in 3D spheroids.

The TV2D+ system combines standard 2D culture with the longevity and phenotypic stability that typically accompany 3D systems. The protocol described herein provides step-by-step directions for users with basic tissue culture skills in laboratories equipped with standard equipment such as biosafety cabinets, centrifuges, and CO2 incubators. Each step in the process is outlined in detail, including media preparation, thawing, plating, and maintenance of the resulting culture system. The protocol also contains a method for determining basic hepatocyte functionality outputs, albumin, and urea, as well as immunofluorescent image analysis for determining PHH attachment for normalization. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, mimicking the native hepatic morphology, and maintain extended viability, architectural integrity, and physiologically relevant levels of albumin and urea for at least 2 weeks8. Because the system can allow a range of PHH seeding densities, it may be useful in increasing the availability of less plateable PHH lots that have desirable donor characteristics. This combination of accessibility and functionality makes TV2D+ a suitable hepatic model for a variety of pharmacological and toxicological applications.

Author Spotlight: Developing a Simple and Robust Hepatic Model for Pharmacological and Toxicological Applications 

Ein rein humanes Leberkultursystem für Anwendungen in der Arzneimittelentwicklung

Watch this Scientific Journal Video about Media Preparation for Setting Up the All-Human 2D+ Hepatic Culture System at JoVE.com

Media Preparation for Setting Up the All-Human 2D+ Hepatic Culture System  

Medienvorbereitung für den Aufbau des rein humanen 2D+ Leberkultursystems

Zu Beginn wird eine Flasche jedes Auftaumediums der Zuführzelle, Ergänzung A, Ergänzung B und Ergänzung C, in einem 37 Grad Celsius warmen Wasserbad aufgetaut. Alternativ kannst du sie auch 16 bis 24 Stunden bei vier Grad Celsius in den Kühlschrank stellen. Füllen Sie den gesamten Inhalt jeder Flasche mit aufgetautem Medium in ein konisches Röhrchen mit einem Fassungsvermögen von 15 Millilitern oder 50 Millilitern.Um ein vollständiges Beschichtungsmedium herzustellen, nehmen Sie eine Flasche des Beschichtungsmediums und fügen Sie 4,5 Milliliter Nahrungsergänzungsmittel B hinzu, gefolgt von 11 Millilitern Nahrungsergänzungsmittel A in die Flasche. Mischen Sie in einer separaten Flasche 100 Milliliter Nährmedium, einen Milliliter Supplement C und 14 Milliliter Supplement A, um das komplette Nährmedium herzustellen. Lagern Sie das verbleibende Supplement C und Supplement A bis zur zweiten Woche bei minus 20 Grad Celsius.

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JoVE Journal

Biology

Media Preparation for Setting Up the All-Human 2D+ Hepatic Culture System

200 Views.  LifeNet Health. 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 int...

Video: Media Preparation for Setting Up the All-Human 2D+ Hepatic Culture System  

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

Recent technical advances enabled the scaled production of an in vitro platform for drug metabolism and toxicity applications. An all-human 2D+ hepatic system (TV2D+) provides physiologically relevant results using traditional two-dimensional culture methods. This protocol will support end-users in the setup, maintenance, and application of the system.

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...

Forschung

Biologie

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.

Plating Human Feeder Cells for Setting Up the All-Human 2D+ Hepatic Culture System

To begin, thaw the feeder cells in a 37 degree 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 1000 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 400 G for four minutes.Carefully discard the supernatant by aspirating or pipetting without disrupting the cell pellet. After resuspending 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.

Beschichtung menschlicher Feeder-Zellen für den Aufbau des rein humanen 2D+ Leberkultursystems

Plating Primary Human Hepatocytes for Setting Up the All-Human 2D+ Hepatic Culture System

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 polyethersulfone filter unit. Thaw the PHHs in a 37 degree 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 100 G 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 resuspend the cell pellet, and add five milliliters of complete plating medium. After counting the resuspended 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.