FAQs

General Questions
HepatoPac/HepatoMune Viabilility
Endpoints and Assays
    Tox
    DMPK
Kit Information

General Questions

What Products and Services does Hepregen Offer?

In which well formats is your model available, e.g. 24-well, 96-well?

What species are available?

  • HepatoPac: Human, Rat (Sprague Dawley or Wistar Han), Dog and Monkey (Cyno)
  • HepatoMune: Rat (Sprague Dawley), Human
  • Specification Sheets

Can I use HepatoPac in my own lab?

  • Yes, because HepatoPac plates meet industry standards for 96-well and 24-well plates, you can easily integrate HepatoPac into your work flow without the need for additional equipment. See Kit Information.
What are the culture volumes in HepatoPac? Do you have to use special media for HepatoPac?
  • Yes, we have our own formulations for media that are optimal for the various cultures and applications.

How do you account for hepatocyte-specific ATP and GSH responses with the presence of stromal cells?

  • In order to calculate hepatocyte-only responses (for ATP and GSH), cellular ATP and GSH levels are also determined in stromal-only cultures. ATP and GSH signals in stromal-only control cultures are then subtracted from those of HepatoPac co-cultures to obtain hepatocyte-specific effects.

How many different batches of human hepatocyte (or other cell type) donors have you tested in your model? 

  • Hepregen has tested over 50 individual human donors in its HepatoPac model.  Primary hepatocytes are acquired from many different sources including several commercial vendors licensed to sell primary hepatocytes, pharmaceutical companies and academic collaborators.  Hepregen has tested both fresh and cryo-preserved hepatocytes.

What quality control measures do you have in place to accept/reject certain hepatocyte batches? 

  • Hepregen certifies and validates each donor that goes into the manufacturing of HepatoPac.  The first step in the validation process is a donor selection.  Hepregen will acquire several small lots of various cryo-preserved primary hepatocytes from different vendors.  Each lot will be plated in HepatoPac and maintained for 30 days.  During that time, Hepregen’s manufacturing and quality assurance groups will monitor the plateability and the morphology of the hepatocytes over the 30-day period.  Acceptable donors must meet the following criteria:
    • An intact and stable micro-architecture consisting of islands of hepatocytes surrounded by supportive stromal cells
    • Hepatocyte islands void of defects, such as holes or incomplete hepatocyte coverage of islands
    • Viable hepatocytes with distinct nuclei, defined cell boundaries
    • Formation of bile canaliculi
    • Hepatocyte islands void of excessive particle/lipid accumulation
    • Sustained CYP3A activity 
    • Sustained and stable albumin and urea production over the 30 day period
  • Lots/Donors not meeting these criteria are rejected.  

HepatoPac® / HepatoMune™ Viability

How does HepatoPac compare with Primary Human Hepatocytes, HepG2, HepaRG cells?

How long are the cells viable under your standard culture conditions, e.g. days, weeks? 
  • Under Hepregen’s standard culture conditions, primary hepatocytes in the HepatoPac platform remain functional and viable for up to 4 weeks.

Can you demonstrate formation of bile canaliculi in your model, e.g. phase contrast, CDF excretion?

  • The formation of bile canaliculi can be reliably demonstrated in HepatoPac by fluorescent microscopy imaging of CDF secretion into bile canaliculi as shown below.                         

CDF_Uptake_052215

For which CYP isoforms have you demonstrated activity in your model?

  • Human: 3A4, 2A6, 2D6, 2B6, 2C9, 1A2, 2A2
  • Rat: 3A1/2, 2B2, 4A
  • Monkey: 2B17, 2C17, 3A5/8, 4A11

Which substrates have you used to demonstrate CYP activity for human?

Hepregen uses the following substrates to determine activity in its HepatoPac model:

  • 1A2 – phenacetin
  • 2A6 – coumarin
  • 2B6 – bupropion
  • 2C9 – diclofenac
  • 2C8 – amodiaquine
  • 2C19 – mephenytoin
  • 2D6 – dextromethorphan
  • 3A4 – testosterone

What method have you used to quantify activity, e.g. LC-MS, P540-Glo™ assay, substrate depletion, metabolite formation?

  • Hepregen quantifies enzyme activity by incubating HepatoPac cultures with specific enzyme substrates and monitoring metabolite formation via LC-MS.  For routine assessment of CYP3A4 activity we use the Promega P540-Glo™ kit.

How long is this metabolic phenotype maintained in HepatoPac? 

  • The metabolic phenotype for almost all CYPs monitored is maintained over the 4-week culture period as shown above.  Activity was monitored by the formation of metabolites for various CYPs across three donors and analyzed by LC-MS/MS.
CYP_Activity_over_Time_052215

Have you demonstrated presence of other metabolic pathways, e.g. Phase 2 (UGT, SULT, GST) or other Phase I activity in your model? 

  • Hepregen also routinely monitors Phase II activity of its donors in HepatoPac.  The figure below shows sustained Phase II activity (glucuronidation and sulfation) in three human donors over 24 days in culture.
  • Aldehyde oxidase (AO) activity has been shown to be active in HepatoPac co-cultures and species differences in AO activity is represented in HepatoPac cultures.  For example, the AO metabolite of carbazeran (4-oxo-carbazeran) is produced in human and monkey HepatoPac co-cultures (as expected) but not in rat and dog HepatoPac (as expected).

Phase_II_Activity_over_Time_052215

Duration of treatment/incubation: for how many days do you typically dose the cells?

  • For toxicity assessment, Hepregen’s typical dosing regimen involves treatment of the HepatoPac cultures with 4 (repeat) doses of compound(s) over 9 days. 
  • For metabolic stability and metabolite identification, cultures are treated with one dose and continuously incubated for up to seven days.

What endpoints are typically measured to assess general liver functionality? 

  • Hepregen routinely performs an assessment of albumin and urea production over a 28 day period for each of its validated donors during the characterization process.  Typical albumin production and urea synthesis levels for Human HepatoPac (labeled as Micropatterned co-cultures) are seen below.

Albumin_and_Urea_052215

End Points and Assays

Toxicology End Points

What toxicology end points can be run?

  • Current endpoints include:  ATP, GSH, urea production, albumin secretion, ALT leakage, MTT assay, and gene expression.  Hepregen is developing other endpoint assays such as High Content Imaging (HCI).  Hepregen quantifies the effect of compounds in HepatoPac co-cultures by estimating TC50 values.

How many DILI/Non DILI compounds have you used to validate your model? 

  • Hepregen has evaluated >100 compounds for validation of the model.  Data from 64 compounds used in the validation studies have been published (Khetani, et. al. Toxicol. Sci. 132 (1): p 107-117, (2013)).  54 compounds in this compound set were DILI positive while 10 were non-DILI compounds.

Dosing regimen for toxicity assessments:  How many compound concentrations do you test and what is your typical concentration range?

  • Hepregen usually suggests a minimum of 6 concentrations of compound (plus a vehicle control) to enable accurate calculation of TC50 values.  Typical concentration range is 0 to 100*Cmax.  When the Cmax of compound is not known Hepregen assumes a therapeutic Cmax of 1µM and uses a top concentration of 100µM for the compound.  (See p100-102 of Xu et. al. Toxicol. Sci 105(1): 97-105, 2008 for rationale.)

Dosing regimen for toxicity assessments:  How often do you re-administer compound? 

  • Compound is re-administered every 2-3 days.

Does your model detect P450 mediated toxicity, e.g. toxicity prevented by a CYP inhibitor?

  • Yes.  Hepregen has shown reduction of compound toxicity when pre-treated with various CYP inhibitors such as 1-aminobenzotriazole (ABT), ketoconazole, or ritonavir. 

Does your model detect reactive metabolite toxicity, e.g. toxicity enhanced with GSH depletion?

  • Yes.  Hepregen has evaluated a number of bioactivated compounds (aflatoxin, tienilic acid, cyclophosphamide, acetaminophen etc.) in HepatoPac co-cultures and have successfully shown that depletion of cellular GSH using buthionine sulfoximine (BSO) exacerbates toxicities of these compounds.  In a recent publication (Ukairo, et. alJournal of Biochemical and Molecular Toxicology, Vol. 27(10), p 471-478,(2013)), addition of 200µM L-buthionine (S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, to Rat HepatoPac co-cultures potentiated acetaminophen-induced hepatotoxicity in these cultures irrespective of culture age (over 4 weeks).

Does your model detect mitochondrial toxicity?

  • Hepregen has been able to reproduce the toxicity of fialuridine (a mitochondrial toxicant) in Human HepatoPac cultures, which is difficult to replicate in conventional 2D culture systems.  Fialuridine produces mitochondrial toxicity by causing mtDNA depletion with reduction of mtDNA-encoded proteins.  Using MTT assay as marker for mitochondrial health Hepregen has shown dose- and time-dependent toxicity of fialuridine in Human HepatoPac cultures.  

MTT_Over_Time_052215

Figure: Toxicity profile of FIAU in human HepatoPac (using 5 different human hepatocyte donors) and rat HepatoPac co-cultures. FIAU was able to recapitulate the greater sensitivity in human vs. rat HepatoPac.  HepatoPac cultures were treated with varying concentrations of FIAU (4 doses over 9 days) and assessed for hepatocyte function and viability.

Does your model detect innate immune-mediated toxicity, e.g. enhanced toxicity with trovafloxacin? 

  • We have demonstrated proof-of-concept utility of our system in the evaluation of immune-mediated toxicity using trovafloxacin as model compound.  When co-administered with LPS, trovafloxacin toxicity was potentiated in both the rat and human HepatoMune.  The LPS-induced exacerbation of trovafloxacin toxicity was reversed by pre-treatment of the co-cultures with pentoxifylline (an inhibitor of TNF-α transcription) consistent with literature evidence that trovafloxacin toxicity is, in part, mediated by TNF-α).  (See this Hepregen poster for more information.)

hepatomune-trovafloxacin-fig4-lg

Figure: Stimulation of HepatoMune tri-culture with Lipopolysaccharide (LPS) exacerbated trovafloxacin-induced toxicity as seen above where there’s a leftward shift (to lower TC50 values) in the concentration-response curve for ATP content.

Does your model detect biliary transporter toxicity, e.g. enhanced toxicity in presence of bile acids, inhibition of transport activity? 

  • Yes.  A common mechanism of cholestasis is the inhibition of bile salt excretory pump (BSEP) leading to disruption of bile acid secretion.   HepatoPac co-cultures are characterized by extensive canalicular networks (“bile pockets”) and longer-term retention of transporter functions, and can be used to successfully identify cholestasis- mediated hepatotoxins.  Treatment of human HepatoPac with the cholestasis inducing hepatotoxin, troglitazone, for 5 days adversely affected hepatocyte health in these cultures.   ATP and albumin levels were decreased to less than 10% of control values.  Furthermore, at low micromolar concentrations (IC50 ~7.5µM), troglitazone inhibited the biliary excretion index (BEI) of radiolabeled taurocholate in human HepatoPac, indicating an inhibitory effect on the bile salt excretory pump (BSEP).  In human MPCCs, troglitazone was much more acutely toxic than its structural analog, rosiglitazone (Avandia), a drug approved by the FDA.  The ability to distinguish between toxic troglitazone and the non- hepatotoxic rosiglitazone shows that human MPCCs can be used reliably to detect hepatotoxic adverse reactions such as cholestasis.  The hepatotoxicity of other cholestasis inducing hepatotoxins such as bosentan and sitaxsentan has been observed in human HepatoPac as well (data not shown).

DMPK Endpoints

Can you assess transporter activity in HepatoPac?

  • Hepregen has demonstrated activity of BSEP, OATP, NTCP, MRP2, P- gP.
  • Hepregen is the only next-generation liver model licensed to assess transporter activity using the patented B-CLEAR® technology from Qualyst Transporter Solutions.  Learn More.

Which substrates have you used to demonstrate transporter activity in HepatoPac? 

  • Taurocholate (TCA), E217bG, rosuvastatin, pravastatin and digoxin

How long is transport phenotype maintained?

  • The transport phenotype is maintained in HepatoPac cultures for up to 4 weeks.
How do you perform your clearance/metabolic stability and metabolite identification assays using HepatoPac?
  • HepatoPac co-cultures are incubated with compound in serum-free media for several days (up to 7 days) without media change and aliquots of culture supernatant are collected at select time intervals and evaluated for metabolite generation (Met ID) or parent compound depletion (clearance/metabolic stability) using mass spec.

Kit Questions

Can I use HepatoPac in my own lab?

  • Yes, because HepatoPac plates meet industry standards for 96-well and 24-well plates, you can easily integrate HepatoPac into your work flow without the need for additional equipment.  Learn More.
How long does it take to receive HepatoPac once I've ordered it?
  • Approximately 2 weeks.

What do I get when I order HepatoPac?

  • A kit with the correct media components, an expert guide, and the specific directions and licensing for the application you want to run.