Some tumor cell lines show genetic instability which can negatively affect historical data comparisons: what about HepaRG™?The karyotype of the cells has been tested regularly since 1999, and it has never changed over this time using the culture conditions that we use and recommend. The Master and Working banks which have been established contribute to retain this stability.
Since the cells came from resected tumor tissue, will they proliferate/pile up on each other, are tumorigenic, etc?At low density, HepaRG™ cells will proliferate until they reach confluence and stop proliferating due to exhibit contact inhibition. When growing, they produce fetal proteins and have an undifferentiated cell morphology. Once confluent, and when culture medium is changed in order to drive differentiation, they become terminally differentiated, such that they exhibit a well-differentiated hepatocyte morphology and a fully developed adult human hepatocyte phenotype. Differentiated cells at confluence stop growing but they may form very small clusters in which they pile up on themselves. These cells are not tumorigenic and even after several passages they do not grow on soft agar and do not induce tumor in nude mice.
What is the cellular material observed under the microscope, in between the hepatocyte colonies?There are normall two types of cells in the HepaRG™ cultures, mainly hepatocytes but primitive biliary cells are also present. We believe the presence of this second cell type assists with cell signaling and increases the general health of the hepatocytes.
Are primitive biliary cells contributing to hepatocyte metabolism quantification?We believe the contribution of the primitive biliary cells to metabolism is minimal since it has been clearly demonstrated that they do not express any hepatocyte functions. For instance, they are negative for CYP-specific stains. However, they do contribute to approximately half the total protein content of the culture, which leads to the under-estimatation of CYP activities if expressed per mg protein. The functions of these biliary cells is not well-studied, apart from those indicating their role in maintinaing hepatocyte health.
Is there a way to change the population to be all hepatocytes?We are unaware of a practical method to change the population to be all hepatocytes. When the HepaRG™ are undergoing differentiation, they naturally form both primitive biliary cells and hepatocytes. One method has been published using photodynamic light treatment but this is not generally practical. Therefore, reported DME activities are derived almost entirely from the hepatocyte population. Despite a mix of 50% hepatocytes and 50% primitive biliary cells, most DME, transporter and funtional activities are still well within the ranges found from population studies among human hepatocytes.
Why is the 2D6 activity relatively low?This might reflect the donor’s genetics, resulting in low activity of the polymorphic CYP2D6.
Some batches of plateable human hepatocytes show more fold induction (enzymatic activity) than the HepaRG™; is there something different about the HepaRG™?The responsiveness of primary human hepatocytes to CYP inducers is donor-dependent and the fold induction can vary considerably, depending on the CYP. The fold induction of the HepaRG™, using prototypical CYP inducers and concentrations recommended by the FDA, falls at approximately midway between the fold induction responses reported in primary human hepatocytes. Extraordinarily high fold induction responses (>50-fold) are often viewed with some skepticism because such induction rarely occurs in-vivo. The key point is that an induction study should allow for the discrimination between no/weak, moderate, and potent inducers, and such studies conducted using HepaRG™ comfortably demonstrate this requirement. Moreover, McGinnity et al. reported that the fold induction of HepaRG™, treated with prototypical inducers, closely resembled the human in-vivo findings with the same compounds.
HPR116. Since there are two different cell populations, are the imaging studies such as High Contents Screening (HCS) affected?It can, indeed: Mennecozzi, Whelan et al. reported that a reduction in seeding density, to 50 x 10³ cells/well (for 96-well plates) resulted in satisfactory performance using HepaRG™ in HCS. Pernelle, Guillouzo et al., came to similar conclusions. But for analytical procedures other than HCS imaging, we recommend to seed at a density of 72 x 10³cells/well (96 well plate).
What is the role of biliary cells in the culture ?The primitive biliary cells are not well characterized but we know that they are poorly differentiated. As a result, we prefer to refer to these cells as “primitive biliary cells” instead of “biliary cells”. In this respect, these cells share markers with progenitor cells and we believe that they contribute to the long-term maintenance of the line. This line of thinking is supported by evidence which shows exchanges between both differentiated hepatocyte-like cells and primitive biliary cells. The two cell types communicate with each other in order to maintain the ratio 50% hepatocytes and 50% primitive biliary cells.
With fully differentiated HepaRG™ cells, how can you explain the obtained ratio of 50% hepatocytes and 50% primitive biliary cells?The normal ratio of these cells is 1:1 (50% hepatocytes and 50% primitive biliary cells) and this is the best expected ratio when cultured according to our historically estalished culture conditions. However, the ratio readily varies with culture medium quality (and the key roles of serum and DMSO) and cell seeding density.
What is the amount of total RNA possible to extract from HepaRG™ cells ?It is possible to extract about 3-5 µg of total RNA from one well of a 24-well-plate.
How does the size of HepaRG™ cells compare with that of primary human hepatocytes?The differentiated HepaRG™ hepatocytes are slightly smaller (15-20µm) than primary human hepatocytes (20-25µm).
What are the donor details of the HepaRG™ cells?The original liver cells from which HepaRG™ were derived came from a female 66 year-old caucasian. She was treated for cholangiocarcinoma, and she was HCV positive (although HepaRG™ cells are HCV negative).
How do I use medium supplements?After thawing ADD6XX or ADD7XX, prepare the complete working HepaRG™ medium by adding the entire contents of the supplement bottle to 100 mL or 500 mL of Basal Medium, respectively.
What factors influence cell attachment and growth?Several factors may influence cell attachment and growth on cell culture vessels, including the type of cell, the age and condition of culture, culture environment, nutrients, toxins, cell culture protocol, handling and selection of an appropriate surface. The age of the cell line and/or the passage number are also important parameters. Some cell lines may exhibit morphological changes after several passages.Our protocols and media are optimised for HepaRG™ cultures such that they reproducibly attach to the surface of multiwell plates and flasks and achieve the expected morphology consistent with that on the Certificate of Analysis provided with the cells. In order to ensure a consistently high quality of HepaRG™ cultures, we strongly recommend not using the cells beyond passage 20 (as advised in our SOPs).NOTE: Passaging proliferating HepaRG™ cells (HPR101) is only allowed when MTA has been established for the use of the HepaRG™ cell line. However passaging the HPR116 cells is forbidden.
Is Percoll separation recommended or suggested?Percoll separation is commonly used to eliminate dead and dying cells from the healthy ones, especially since it has been shown that necrotic or apoptotic cells may disturb the functions of the healthier ones. Percoll separation can be used for cryopreserved primary hepatocytes to improve post-thaw viabilities of some poorer quality batches; however, since HepaRG™ behave so predictably, we have identified a cryopreservation process that consistently results in post-thaw viabilities of >85%. In conclusion: Percoll separation would only add expense and complexity for only a negligible, if any, benefit.
HPR116: Can I use fewer cells per well when I seed differentiated HepaRG™ in multiwell plates?The differentiated HepaRG™ need to maintain cell-cell contact in order to obtain optimum cell-cell signaling, viability, and an adult phenotype. If they are seeded such that confluence is lost or compromised, this will adversely affect many types of studies e.g. they are less reposnive to CYP inducers. Our recommended procedures are designed to allow for proper cell-cell contact, among other important factors which are needed for different assays.
HPR116: Why do the User Instructions for metabolism studies indicate to either use the cells the day of thawing, or to wait until at least the fourth day after thawing to use them?HepaRG™ behave like primary human hepatocytes in that they are intially fully functional and responsive after thawing, but then they undergo a reorganization phase which lasts 6-7 days. During the reorganization period, the cell morphology changes somewhat and their metabolic competence declines, making them unsuitable for metabolism studies during this time. With this in mind, if the study design is not compatible with same-day thawing, treatment and analysis, then the cells need to be allowed to adjust their differentiation status before they can be used for metabolism studies. The culturing for the reorganization period involves simply replenishing the medium.
Is a collagen coating of the culture support beneficial?Biopredic has performed many different types of analysis with and without collagen coating and observed benefit only with HPR116 cells. The collagen coating helps the cells to attach faster to the support thus allowing the use of the cells as little as 4hours after seeding.
After I use the cells for an experiment, can I re-use them?The cells are licensed for single-use applications. In addition, the cells are likely be altered by test compounds, thus introducing a risk of misleading results if they are re-used. Having said this, the cells are ideal for assays in which they are exposed to serial treatments, or measuring of some endpoints over several days (i.e., in-vitro chronic toxicity).
Since proliferating HepaRG™ cells reach confluency after 5-7 days of culture in growth medium, is it really necessary to wait 14 days before proceeding to subculture ?HepaRG™ cells are different from other cell lines. For other cell lines, it is indeed generally advised to subculture the cells before they reach confluence. This is not true for HepaRG™ cells, which must reach confluence and start differentiating before subculturing them. After seeding, HepaRG™ cells will reach confluence after 5 to 7 days. At this point they should be kept in the HepaRG™ growth medium for an additional 7 days (a total of 14 days after seeding in HepaRG™ growth medium). The cells will have initiated the differentiation program and will be ready for subculture. If you subculture before this period, the cells will completely lose their capacity to differentiate.
Biopredic's protocols omit the removal of DMSO (after thawing) or neutralised trypsin (when passaging) from the cells before seeding them. Do DMSO and/or trypsin affect the attachment efficiency or growth of the cells?The presence of DMSO or trypsing In the seeding medium does not affect the attachemt or growth of the cells. After seeding thawed cells, the medium is changed after 6h, thus removing the remaining DMSO. After trypsinisation, the trypsin is diluted twice: once for neutralisation and a second time in the seeding step. The medium contains 10% serum, which inactivates any trypsin present.
Can we use smaller volumes for culturing cells in flasks than those recommended by Biopredic?We do not recommend reducing the medium volume because this may affect the growth and phenotype of the cells. We have shown that 5ml and 14ml are optimal for T25 and T75 flasks, respectively.
Can HepaRG™ cells be cultured in sandwich format using Matrigel or collagen gel overlays? If so, what are Biopredic's recommendations for overlaying the cells?HepaRG™ cells (HPR116) attach well to the collagen coated plates and once in sandwich format, these cells reorganize themselves. Therefore, if these cells are tested in sanwich format, we recommend adding the overlay on day 5 to cells cultured in 24-well plates. Biopredic does not use sandwich formats because HepaRG™ cells can form the canalicualr network without an overlay.
When the Differentiation medium is added on the HepaRG cell line, a strange odor appears from the cell culture. is it normal?Yes this is normal. The Differentiation medium contains a high % of DMSO.
In contact with the cells, an odor appears. The intensity of the odor is much stronger that there are plates and/or flasks in the incubator.