A Novel, All-Human Hepatic Triculture System
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In this webinar, Edward LeCluyse, PhD, presents a novel, all-human hepatic triculture system and discusses how it addresses the need for a convenient and human-relevant long-term hepatic culture. The use of primary human hepatocytes in preclinical pharmacological and toxicological applications has been restricted in part by the lack of a suitable culture platform that is convenient, supports a multitude of donor lots, and maintains structural and functional properties of the cells over prolonged periods of time. The sandwich system and previous co-cultures have addressed some of these issues; however, some limitations remain including the short- to mid-term loss of cell polarity and metabolic functions. Animal models are an option, but there are marked species differences in hepatic metabolic and receptor-signaling pathways. The new triculture system from LifeNet Health LifeSciences was developed to offer researchers a convenient all-human hepatic system that maintains both hepatocellular morphology and metabolic function over several weeks. This new system offers a superior combination of human relevance, reproducibility, longevity, and convenience. It contains 100% all-human cryopreserved hepatocytes and feeder cells with optimized media. Hepatocytes are pre-qualified in the system to meet minimum specifications for morphology, architecture, and basic hepatic functionality for at least two weeks. The system gives researchers control over experimental timelines and workflow in a standard plate format. It can be set up in a matter of hours and tested in multiple applications. These appealing features make this new triculture a suitable model for hepatocyte and liver studies – including metabolic clearance, drug-induced liver injury, risk assessment, disease modeling, and high content imaging – in addition to distinguishing it significantly from current alternative hepatocyte culture systems. Key Topics Include: - The LifeNet Health LifeSciences all-human hepatic triculture model accurately and consistently mimics native liver tissue architecture and key hepatocellular functions over several weeks. - Hepatocytes are pre-qualified to reduce variability and enable a broad range of pharmacological and toxicological applications. - This hepatic system gives researchers the flexibility to choose from a selection of hepatocyte lots that enables a broad population diversity. - The system is convenient, offering the simplicity of a 2D model, along with the robust levels of data that typically accompany 3D models.
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A Novel, All-Human Hepatic Triculture System
- 1. A Novel, All-Human Hepatic Tri-Culture System Ed LeCluyse, PhD LifeNet Health LifeSciences Chief Scientist Paul Gallant LifeNet Health LifeSciences Global Sales Director
- 2. Copyright 2022. All Rights Reserved. Contact Presenter for Permission A Novel, All-Human Hepatic Tri-Culture System Paul Gallant LifeNet Health LifeSciences Global Sales Director
- 3. Visit us at Booth #2204 3 HEPATIC TRICULTURE SYSTEM Paul Gallant - Global Sales Director Edward LeCluyse, PhD - Chief Scientist
- 4. Visit us at Booth #2204 Regulatory Guidelines FDA EMA EPA Animal Ethics 3Rs In Vitro to In Vivo Translation High Rate of Failure in Clinical Trials Gold Standard All-Human Cells & Tissues
- 5. Visit us at Booth #2204 NON- PROFIT GLOBAL 40 YEAR HISTORY
- 6. Visit us at Booth #2204 ORGAN TRANSPLANT BIOLOGICS LIFESCIENCES
- 7. Visit us at Booth #2204
- 8. Visit us at Booth #2204 ORGAN TRANSPLANTS 600+/year TISSUE ALLOGRAFTS 850,000+/year DONOR FAMILIES 12,000/year DONOR TISSUES AND CELLS 150+/year
- 9. Visit us at Booth #2204
- 10. Visit us at Booth #2204 Cells Preserved Tissue Cell Models Tissues For Research Liver Thyroid Lung Brain Gut Other …
- 11. Visit us at Booth #2204
- 12. Visit us at Booth #2204 LIVER PORTFOLIO
- 13. Visit us at Booth #2204 EXTENDS THE VIABILITY AND FUNCTIONALITY OF HEPATOCYTES FOR GREATER THAN 2 WEEKS COMPRISED OF ALL HUMAN CELLS HIGH REPRODUCIBILITY
- 14. Copyright 2022. All Rights Reserved. Contact Presenter for Permission A Novel, All-Human Hepatic Tri-Culture System Ed LeCluyse, PhD LifeNet Health LifeSciences Chief Scientist
- 15. Edward LeCluyse, PhD 25+ Years of Experience Academic and Industry Leadership Research Focus: Human Liver Key Opinion Leader >120 Publications 15 Copyright 2022 LifeNet Health® All rights reserved. Chief Scientist LifeNet Health
- 16. Visit us at Booth #2204 16 HEPATIC TRICULTURE SYSTEM Edward LeCluyse, PhD
- 17. System Features All human-derived cells Self-assembled organization Native cell-cell interactions Stable morphology & hepatic function Sustained metabolic activity 17 Copyright 2022 LifeNet Health® All rights reserved. CD31 / ALB / DAPI (Day 28)
- 18. All-Human Hepatic Triculture Kit: 24-well Format Copyright 2022 LifeNet Health® All rights reserved. Stabilized Phenotype Triculture Assembly Kit Components Feeder Cells Feeder Cells + Hepatocytes
- 19. Specifications PHH lots with high attachment, colony formation and desired cell morphology Optimal seeding density ratio is determined between PHHs and FCs All-human hepatic triculture system stabilizes by day 5 and continues to be stable for greater than 14 days Pre-Qualified Benefits • Guaranteed performance and longevity • Multiple healthy adult lots available to select from for both 24 and 96 well formats 19 Copyright 2022 LifeNet Health® All rights reserved. Performance Specifications Performance Test Pass Criteria Long-term Plateability ≥ 14 Days Morphology 1. Multicellular Cluster Formation 2. Mix of multinucleated & Mononucleated Cuboidal Cells Albumin Levels ≥ 37 ug/day/M cells Urea Levels ≥ 56 ug/day/M cells Pre-qualified hepatocytes and feeder cells for 24- and 96-well formats
- 20. Protocol at a Glance Copyright 2022 LifeNet Health® All rights reserved.
- 21. All-Human Hepatic Triculture 24-well System Performance and Application Data
- 22. Longevity 22 Copyright 2022 LifeNet Health® All rights reserved. Day 4 Day 7 Day 11 Day 14 Mono Triculture D7 D14 D28 D21 D7 D28 D21 D14 same field of view over 4 weeks in culture Histotypic 3D architecture – more native cell to cell interaction and cell polarity
- 23. Morphology 23 Copyright 2022 LifeNet Health® All rights reserved. Tight Junction Marker: ZO-1 Gap Junction Marker: Cx-32 Junctional complexes resemble in vivo architecture and cell polarity (Day 7)
- 24. Bile Canaliculi 24 Copyright 2022 LifeNet Health® All rights reserved. CDFDA fluorescent staining (Day 15) 100X 40X Native Liver Functional anastomosing canalicular networks
- 25. NTCP Expression 25 Copyright 2022 LifeNet Health® All rights reserved. NTCP/DAPI (Day 21) NTCP highly expressed with sinusoidal localization
- 26. Albumin Production and Urea Synthesis 26 Copyright 2022 LifeNet Health® All rights reserved. Albumin Urea * * * Sustained production of albumin (>37 ug/106 cells/day) and urea (>56 ug/106 cells/day) Multiple lots - >16 days
- 27. Albumin Production and Urea Synthesis 27 Copyright 2022 LifeNet Health® All rights reserved. Albumin and urea levels in sandwich (SC) monoculture vs. All-Human Hepatic Triculture (TCS) 40 30 20 10 0 70 60 50 SC Monoculture Triculture µg Urea / day / 10 6 PHHs *** 10 30 0 20 40 50 SC Monoculture Triculture µg Alb / day / 10 6 PHHs *** Sustained production of albumin and urea over time compared to SC system (Day 16)
- 28. Metabolic Activity 28 Copyright 2022 LifeNet Health® All rights reserved. Sustained phase 1 and 2 metabolic pathways over 2-week period 15 mM midazolam @ 60 min 100 mM 7-ethoxycoumarin @ 30 min Day 5 Day 7 Day 10 Day 14 0 10 20 30 1'-hydroxymidazolam formation, pmol/min/million cells
- 29. Cytochrome P450 Induced Gene Expression 29 Copyright 2022 LifeNet Health® All rights reserved. CYP2B6 CYP3A4 Consistent induction response to reference compounds across donor lots (Day 10)
- 30. Phase II Enzyme Induced Gene Expression 30 Copyright 2022 LifeNet Health® All rights reserved. UGT1A1 UGT2B4 Consistent induction response to reference compounds across donor lots (Day 10)
- 31. Cytochrome P450 Induced Activity 31 Copyright 2022 LifeNet Health® All rights reserved. Robust and stable CYP induced activity over 2-week period 48hr treatment with Omeprazole (100µM), CITCO (100nM), and Rifampicin (25µM) using P450-Glo Assays (Promega) 0 10000 20000 30000 40000 Lot E Lot F Log G pmol CYP3A4 / 10 6 / min CYP3A4 16 days in the TCS with uninduced (grey, horizontal strip, and white bars) and induced CYP3A4 (black, vertical strip, and checkered bars) enzyme activity being measured on days 4 (grey and black bars), 10 (striped bars), and 16 (white and checkered bars)
- 32. Metabolic Clearance Study: Reference Compounds 32 Copyright 2022 LifeNet Health® All rights reserved. SUBSTRATE T1/2 (min) Range Clinical In Vivo Clsys * (mL/min/kg) Midazolam 114 108-384 11.9 Dextromethorphan 250** 180-360 8.6** Tolbutamide 870 240-1500 1.6 Lorazepam 12,840 NA 1.1 *Values reported in Goodman and Gilman 11th ed. (2006) PM = poor metabolizers; EM = extensive metabolizers ** PM: 3.9 ± 1.4 EM: 1575 ± 658 ** PM: 1770 ± 504 EM: 204 ± 30
- 33. Metabolic Clearance Study 33 Copyright 2022 LifeNet Health® All rights reserved. t1/2 = 190.5 min Clint = 6.1 mL/min/M t1/2 = 908 min Clint = 1.3 mL/min/M t1/2 = 1908 min Clint = 0.61 mL/min/M t1/2 = 2893 min Clint = 0.4 mL/min/M Midazolam Tolbutamide Lorazepam Dextromethorphan
- 34. Metabolic Clearance Study 34 Copyright 2022 LifeNet Health® All rights reserved. SUBSTRATE T1/2 (min) Range Clinical In Vivo Clsys * (mL/min/kg) All-Human Hepatic Triculture Calc Clsys Midazolam 114 108-384 12 15.3 Dextromethorpha n 250** 180-360 8.6** 3.2** Tolbutamide 870 240-1500 1.6 1.5 Lorazepam 12,840 ND 1.1 1.0 *Values reported in Goodman and Gilman 11th ed. (2006) PM = poor metabolizers; EM = extensive metabolizers ** PM: 3.9 ± 1.4 EM: 1575 ± 658 ** PM: 1770 ± 504 EM: 204 ± 30 Metabolic clearance predictions consistent with clinical values
- 35. All-Human Hepatic Triculture System: 96-Well Format
- 36. 96-well System 36 Copyright 2022 LifeNet Health® All rights reserved. Established optimal conditions for retention of morphology and function for >14 days Lot A Lot C Lot B Lot A Lot B Lot C Day 7 Day 14 Day 10 CK18 staining
- 37. 96-well System 37 Copyright 2022 LifeNet Health® All rights reserved. Lot A Lot B Day 14 Day 7 Extensive functional anastomosing canalicular networks
- 38. 24-well vs. 96-well 38 Copyright 2022 LifeNet Health® All rights reserved. Urea Synthesis Albumin Production Comparable performance for both 24- and 96-well formats
- 40. All-Human Hepatic Triculture System All human cells Self-assembled organization Native cell-cell interactions Stable morphology & hepatic function Sustained metabolic activity 40 Copyright 2022 LifeNet Health® All rights reserved. CD31 / ALB / DAPI (Day 28) Advantages Additional Benefits Pre-Qualified Cells Multiple Donor Lots Broad Applications Ready to Use
- 41. All-Human Hepatic Triculture Advantages 41 Copyright 2022 LifeNet Health® All rights reserved. Potential Applications Challenge/Opportunity All-Human Hepatic Triculture Solution Metabolic Clearance of low turn-over compounds Valuable dosing information for clinical trials New drugs require more stable assays to assess their metabolism Investigative Toxicology Determine the potential or mechanism for liver injury Prolonged exposure, re- exposure, reactive metabolites & conjugates Disease Modeling Discovery groups require human cell-based systems Improve fidelity of disease phenotypes and drug efficacy De-risking Chemicals Risk assessment Stable, reliable, robust platform
- 42. Future Development/Partnership Opportunities Donor matched cells (NPCs + PHHs) Translate to 384-well format Animal models Pooled hepatocytes Disease models iDILI - immune responses High-content imaging Other? 42 Copyright 2022 LifeNet Health® All rights reserved.
- 43. Visit us at Booth #2204 2D Simplicity with 3D Cellular Biology All Human Cells High Predictability High Reproducibility Simplicity All-Human Hepatic Triculture System Open to Evaluation Partners: LNHLifeSciences.org
- 44. Thank you for participating! CLICK HERE to learn more and watch the webinar
Editor's Notes
- Hello everyone, thanks for joining us today. Before I hand off to Ed, I would like to give you an overview of LifeNet Health and our newest division of Lifesciences
- THERE IS AN INCREASING NEED AND DEMAND FOR ACCESS TO HUMAN TISSUES, CELLS AND CELL MODELS FOR PRECLINICAL RESEARCH THERE ARE A NUMBER OF DRIVERS FOR THIS INCLUDING REGULATORY GUIDELINES, EFFORTS TO REDUCE ANIMAL USE, POOR IN VITRO TO INVIVO TRANSLATION AND A HIGH FAILURE RATE AS COMPOUNDS MOVE INTO CLINICAL TRIALS I BELIEVE THAT LIFENET HEALTH LIFE SCIENCES IS UNIQUELY POSITIONED TO PROVIDE THESE TOOLS….
- WE ARE A NON-PROFIT ORGANIZATION BEEN IN BUSINESS FOR OVER 40 YEARS WE ARE BASED IN VIRGINIA BUT WE MULTIPLE SITES IN US AND EUROPE AND WE ARE DISTIBUTING OUR PRODUCTS GLOBALLY
- WE ARE ORGANIZED IN 3 DIVISIONS We are an Organ Procurement Organization for Virginia – Facilitating life-saving organ transplants Our Biologics Division develops Human Tissue Allografts – We process human tissues for clinical use for surgical implantation in sports medicine, spine, trauma, wound management, dental and cardiovascular applications Lastly, Lifesciences – This is a newer division focused on leveraging LNH’s vertically integrated infrastructure to supply the highest quality of human tissues, cells and models for pre-clinical research
- WE ARE A MISSION DRIVEN ORGANIZATION FOCUSED ON AGGRESSIVELY INNOVATING TECHNOLOGY TO ADVANCE CLINICAL AND PRECLINICAL RESEARCH OUR MISSION, LIKE YOURS, IS TO SAVE LIVES, RESTORE HEALTH, AND GIVE HOPE
- WE ARE PROUD OF OUR ACCOMPLISHMENTS AND ANNUALLY WE ARE FACILITATING OVER 600 ORGAN TRANSPLANTS WE DISTRIBUTE OVER 850,000 TISSUE ALLOGRAFTS GLOBALLY EACH YEAR WE WORK VERY CLOSELY OUR DONOR FAMILIES TO SUPPORT THEM THROUGHOUT THE DONATION PROCESS AND BEYOND LASTLY, OUR LIFESCIENCES DIVISION HAS BUILT AN IMPRESSIVE INVENTORY OF LIVER CELLS AND TISSUES FROM OVER 15O DONORS
- LifeSciences division is leveraging LifeNet Health’s infrastructure, relationships, reputation and vertical integration to provide the highest quality tissues and cells for research. We see this as a key advantage. WHAT DO I MEAN BY VERTICAL INTEGRATION? - WE HAVE CONTROL OVER EACH STEP OF THE PROCESS FROM DONATION TO RECOVERY TO HANDLING AND PROCESSING ENSURING THE HIGHEST QUALITY OF STANDARDS ARE MET
- WE HAVE AN EXPANDING PORTFOLIO WE INITIALLY HAVE FOCUSED ON LIVER BUT NOW HAVE A NUMBER OF PRODUCTS IN DEVELOPMENT I WILL NOW GIVE YOU A BRIEF OVERVIEW OF OUR CURRENT PORTFOLIO OF SOLUTIONS AND FUTURE DIRECTIONS
- WE OFFER A PROSPECTIVE HUMAN TISSUE RECOVERY SERVICE WE WORK DIRECTLY WITH CLIENT TO DEFINE DONOR AND TISSUE SPECIFICATIONS USING OUR NETWORK, WE IDENTIFY A DONOR, RECOVER THE TISSUE AND DELIVER DIRECTLY TO THE CLIENT FOR RESEARCH APPLICATIONS YEARS OF EXPERIENCE WITH 100s OF ESTABLISHED PROTOCOLS – DELIVERING 100s OF HIGH QUALITY TISSUES ANNUALLY
- WE RECOVER AND PROCESS BOTH HEALTHY AND DISEASED LIVERS ~80/YR ISOLATE, CRYO AND CHARACTERIZE; PHH, KUPPFER, LECS AND STELLATE CELLS WE ALSO PRESERVE MATCHED TISSUE SAMPLES, SNAP FROZEN AND FORMALIN FIXED FOR GENOMIC AND HISTOLOGICAL INVESTIGATION WE DEEPLY CHARACTERIZE EACH CELL TYPE AND PROVIDE DONOR MEDICAL/SOCIAL HISTORY SO RESEARCHERS CAN SELECT THE BEST LOT FOR THEIR APPLICATIONS OUR GOAL IS TO BUILD THE LARGEST AND HIGHEST QUALITY INVENTORY IN THE INDUSTRY AND WE ARE WELL ON OUR WAY
- TODAY….WE ARE EXCITED TO PRESENT OUR ALL HUMAN HEPATIC TRI-CULTURE MODEL. AND NOW ITS MY PLEASURE TO INTRODUCE TODAY’S SPEAKER – DR ED LECLUYSE
- ED IS A CHIEF SCIENTIST IN OUR LIFESCIENCES DIVISION HE HAS OVER 25 YEARS EXPERIENCE AND HAS HELD SENIOR POSITIONS IN BOTH ACADEMIA AND INDUSTRY HIS RESEARCH FOCUS IS LIVER…SPECIFICALLY THE DEVELOPMENT OF IN VITRO HUMAN LIVER MODELS – WHICH IS THE TOPIC OF TODAYS PRESENTATION. ED…..
- Hello everyone, thanks for joining us today. Before I hand off to Ed, I would like to give you an overview of LifeNet Health and our newest division of Lifesciences
- CK18 – hepatocytes CDFDA –illustrates functional canicular networks
- There is a transparent “box” over the “Click here” button where you can link the webinar LP.