Microphysiological Systems to Assess Nonclinical Toxicity

Kirk P. Van Ness1, Shih‐Yu Chang2, Elijah J. Weber1, Danielle Zumpano3, David L. Eaton2, Edward J. Kelly1

1 Department of Pharmaceutics, University of Washington, Seattle, Washington, 2 Department of Occupational and Environmental Health Sciences, University of Washington, Seattle, Washington, 3 Nortis, Inc. Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.18
DOI:  10.1002/cptx.27
Online Posting Date:  August, 2017
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Abstract

The liver and the kidney are key toxicity target organs during drug development campaigns, as they typically carry the burden of drug transport and metabolism. Primary hepatocytes and proximal tubule epithelial cells grown in traditional in vitro 2‐D culture systems do not maintain transporter and metabolic functions, thus limiting their utility for nonclinical toxicology investigations. We have developed a renal and hepatic microphysiological system (MPS) platform that uses a commercially available MPS device as the core cell culture platform for our methodologies. We describe protocols for isolating and propagating human proximal epithelial cells and how to seed and culture a renal MPS to recapitulate the human proximal tubule. We present two methods to culture hepatocytes within an MPS and the steps required to connect a renal MPS to a liver MPS. © 2017 by John Wiley & Sons, Inc.

Keywords: hepatocyte; proximal tubule; microphysiological systems; in vitro models

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation, Propagation, and Preservation of Primary Human Renal Tubular Epithelial Cells
  • Basic Protocol 2: MPS Collagen Filling Procedure
  • Basic Protocol 3: PTEC Seeding of MPS
  • Basic Protocol 4: Preparation and Maintenance of a Hepatic MPS
  • Basic Protocol 5: The Interconnected Hepatic:Renal MPS System
  • Alternate Protocol 1: Primary Human Hepatocyte Cultures in 120‐ and 450‐μm Wide‐Channel Lumens Formed in Type I Collagen
  • Alternate Protocol 2: Rat Hepatocyte Isolation and Propagation
  • Support Protocol 1: Immunocytochemistry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation, Propagation, and Preservation of Primary Human Renal Tubular Epithelial Cells

  Materials
  • Fresh human renal cortical tissue (∼1 cm3): access to fresh human renal cortical tissue is imperative for the successful cultivation of PTECs; ideally, close proximity to hospital‐generated tissue sections is recommended (renal cells may also be purchased from vendors such as Lifeline Cell Technology, ATCC, Cell Applications Inc., Lonza, ZenBio, CellSystems, and Sigma‐Aldrich; cells from these sources have not been characterized in respect to performance in the Nortis MPS device)
  • Collagenase Type IV solution (see recipe; typically, 50 ml of collagenase solution is prepared the day of each isolation procedure)
  • TRIzol reagent (Ambion, cat. no. 15596018)
  • 10% buffered formalin (4% formaldehyde; e.g., Thermo Fisher Scientific, cat. no. SF99)
  • Horse serum, heat‐inactivated (Invitrogen, cat. no. 26050‐088)
  • PTEC medium (see recipe)
  • 0.05% (1×) trypsin‐EDTA (Invitrogen, cat. no. 25300‐054)
  • PTEC medium (see recipe) containing 10% (v/v) FBS (VWR, cat.no. 10128‐194)
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes, sterile
  • Disposable polypropylene pestles and 1.5‐ml tubes (BioExpress, cat. no. C‐3452‐1)
  • 10‐ml glass scintillation vials (Fisherbrand Borosilicate Glass Scintillation Vials)
  • BioLite 100‐mm tissue culture dishes (Thermo Fisher Scientific)
  • Surgical instruments (sterile; prepare 5 to 10 of each instrument in advance of tissue processing):
    • Forceps (sterile)
    • Scissors (sterile)
    • Single–edge razor blades (sterile)
  • Temperature‐controlled orbital shaker
  • Humidified incubator set at 5% CO 2 and 37°C.
  • Benchtop centrifuge with buckets that can hold 15‐ml conical tubes
  • Inverted microscope
  • 25‐cm2 (T‐25) and 75‐cm2 (T‐75) tissue culture flasks
  • 1.5‐ or 2.0‐ml cryovials
  • Freezing chamber, e.g., Cryobaby (Diversified Biotech)
  • Dewar flask

Basic Protocol 2: MPS Collagen Filling Procedure

  Materials
  • Ethanol, 200 proof
  • PTEC medium (see recipe), pre‐chilled on ice
  • Type I collagen (rat tail; Ibidi, cat no. 50204, or Corning cat. no. 354249; stock concentration ranges are between 5 to 10 mg/ml concentrations and vary by lot and manufacturer), pre‐chilled on ice
  • 10× Medium 199 (Gibco, cat no. 21180‐021), pre‐chilled on ice
  • 1 N NaOH, pre‐chilled on ice
  • Type IV collagen, mouse, natural (0.8 to 1.0 mg/ml stock solution; Corning, cat. no. 47743‐732)
  • Nortis SCC‐001 Microphysiological Device (MPS chip): aspirate or pour liquid (PBS) out of pouches used to store MPS during shipping from Nortis; remove MPS from packaging; wipe devices with Kimwipe and label devices as you see fit; store devices in 150 mm × 15 mm Petri dish; and place at 4°C until ready for use (each large Petri dish can hold two devices); before collagen filling, the chips should be wiped dry; fluid may accumulate between the chip and shell, if this occurs, use the edge of a Kimwipe to draw fluid from this space.
  • 1‐, 3‐, and 5‐ml luer‐lock syringes (Becton Dickinson)
  • Autoclave the following to have ready at beginning of the collagen‐filling procedure:
    • Luer‐lock blunt needles (22‐G × ½‐in. luer stubs; Instech Labs, cat. no. LS22; blue tip)
    • Metal couplers (blunt; Instech Lab, cat. no. SC 20/15)
    • Silicon tubing: C‐flex tubing (ID: 0.020 in., OD: 0.083 in.; Cole Parmer, cat. no. 06422‐00)
    • Single edge razor blade
    • Forceps
    • 1.5‐ml microcentrifuge tubes
  • 15‐ml conical tubes (e.g., Corning Falcon)
  • 10‐cm Petri dishes
  • Cole Parmer 74900 Series 10‐Syringe Infusion Pump or equivalent
  • Humidified incubator set at 5% CO 2 and 37°C

Basic Protocol 3: PTEC Seeding of MPS

  Materials
  • Type I and IV collagen‐filled Nortis MPS devices ( protocol 2)
  • Confluent T‐25 flask with human PTECs ( protocol 1, step 10)
  • PTEC medium (see recipe)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Inverted microscope
  • 5‐μl Hamilton syringe (microliter #65), sterile (Hamilton Company)
  • 22‐G small‐hub needle, 0.375 in. long, point style 3 (Hamilton Company, cat. no. 7804‐01)
  • 15‐ml conical tubes (e.g., Corning Falcon)
  • Humidified incubator set at 5% CO 2 and 37°C
  • Cole Parmer 74900 Series 10‐Syringe Infusion Pump or equivalent
  • 5‐ml luer‐lock syringes
  • Luer‐lock blunt needles (22‐G × ½‐in. luer stubs; Instech Labs, cat. no. LS22)
  • Silicon tubing: C‐flex tubing (ID: 0.020 in., OD: 0.083 in.; Cole Parmer, cat. no. 06422‐00)
  • Additional reagents and equipment for trypsinization ( protocol 1, steps 12 to 13)

Basic Protocol 4: Preparation and Maintenance of a Hepatic MPS

  Materials
  • Type I collagen (rat tail; Ibidi, cat. no. 50204, ∼10 mg/ml, or Corning, ∼4.1 mg/ml); dilute Type I collagen to 50 to 100 µg/ml in 0.02 M glacial acetic acid
  • Cryopreserved human hepatocyte plating medium (TRL/Lonza, cat. no. MP250; can be replaced by human hepatocyte plating medium prepared as described in Reagents and Solutions)
  • Cryopreserved human hepatocytes, plateable, induction qualified (TRL/Lonza, cat. no. HUCPI); alternatively use rat hepatocytes ( protocol 7)
  • Cryopreserved human hepatocyte thawing medium (TRL/Lonza, cat. no. MCHT50)
  • Human hepatocyte culture/washing medium (see recipe)
  • Matrigel (Corning Life Science)
  • Cryopreserved human hepatocyte maintenance medium (TRI/Lonza, cat. no. MM250; can be replaced by human hepatocyte culture/washing medium)
  • Live/Dead Viability/cytotoxicity Kit (Molecular Probes, cat. no. L3224)
  • 15‐cm Petri dishes
  • 50‐ml conical tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • Nortis SCC‐001 Microphysiological Device (MPS chip): aspirate or pour liquid (PBS) out of pouches used to store MPS during shipping from Nortis; remove MPS from packaging; wipe devices with Kimwipe and label devices as you see fit; store devices in 150 mm × 15 mm Petri dish; and place at 4°C until ready for use (each large Petri dish can hold two devices); before collagen filling, the chips should be wiped dry; fluid may accumulate between the chip and shell, if this occurs, use the edge of a Kimwipe to draw fluid from this space.1‐, 5‐, 10‐, 20‐ml luer‐lock tip syringes (Becton Dickinson)
  • Luer‐lock blunt needles (20‐G × ½‐in. luer stubs; Instech Labs, cat. no. LS20; pink tip)
  • Luer‐lock blunt needles (22‐G × ½‐in. luer stubs; Instech Labs, cat. no. LS22)
  • Inverted microscope
  • Humidified 37°C, 5% CO 2 incubator
  • Metal coupler (blunt; Instech Lab, cat. no. SC 20/15)
  • Silicon tubing: C‐flex tubing (ID: 0.020 in., OD: 0.083 in.; Cole Parmer, cat. no. 06422‐00)
  • Cole Parmer 74900 Series 10‐Syringe Infusion Pump or equivalent
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (Phelan & May, )

Basic Protocol 5: The Interconnected Hepatic:Renal MPS System

  Materials
  • Hepatic:renal MPS medium: mixture of hepatocyte culture medium (see recipe) and PTEC medium (see recipe) at 50:50 volume ratio (stable 1 month at 4°C)
  • Nortis 3D microphysiological Device (SCC‐001) seeded with primary hepatocytes ( protocol 4)
  • Nortis 3D microphysiological Device (SCC‐001) seeded with PTECs ( protocol 3)
  • 70% ethanol
  • 15‐cm Petri dishes
  • 3‐ml luer‐lock tip syringes (Becton Dickinson)
  • Silicon tubing: C‐flex tubing (ID: 0.020 in., OD: 0.083 in.; Cole Parmer, cat. no. 06422‐00)
  • Luer‐lock blunt needles (22‐G × ½‐in. luer stubs; Instech Labs, cat. no. LS22)
  • Metal coupler (blunt; Instech Lab, cat. no. SC 20/15)
  • Sterile single‐edged razor blades
  • Cole Parmer 74900 Series 10‐Syringe Infusion Pump or equivalent

Alternate Protocol 1: Primary Human Hepatocyte Cultures in 120‐ and 450‐μm Wide‐Channel Lumens Formed in Type I Collagen

  Additional Materials (also see Basic Protocols protocol 22 and protocol 44)
  • PTEC medium (see recipe)
  • Human hepatocyte Matrigel medium (see recipe)
  • Cryopreserved Human Hepatocyte Maintenance Medium (TRI/Lonza, cat. no. MM250; optional)

Alternate Protocol 2: Rat Hepatocyte Isolation and Propagation

  Materials
  • Rat pre‐perfusion medium (see recipe)
  • Rat perfusion medium (see recipe)
  • Rat 100 Dex medium (see recipe)
  • Rat hepatocyte wash medium (see recipe)
  • Collagenase Type IV (Worthington Biochemical Corp., cat. no. CLS4)
  • 70% and 75% ethanol
  • 50 mg/ml Avertin in normal saline
  • Rat hepatocyte plating medium (see recipe)
  • Rat hepatocyte maintenance medium (see recipe)
  • Matrigel (Corning Life Science)
  • Gilson Minipuls 2 perfusion pump or equivalent
  • Pump tubing
  • Dissecting tray
  • Surgical instruments:
    • Scalpel
    • Clip forceps
    • Clamps
  • Sutures
  • 18‐G catheter
  • 250‐ and 500‐ml beakers, sterile
  • End‐over‐end rotator
  • Nylon gauze (100‐µm, TETKO Inc., cat. no. 3‐100/35; 200 µm, Tetko, cat. no. 3‐200/35)
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • Additional reagents and equipment for injection of rats (Donovan & Brown, ) and counting viable cells by trypan blue exclusion (Phelan & May, )

Support Protocol 1: Immunocytochemistry

  Materials
  • 10% Buffered formalin acetate (4% w/v formaldehyde; Thermo Fisher Scientific, cat. no. SF99‐4)
  • DPBS++ (Invitrogen, cat. no. 14190)
  • PTB solution (see recipe)
  • Primary antibodies (Table 14.18.2)
  • Secondary antibody appropriate for primary antibody
  • Nortis MPS devices containing cells (see protocols above)
  • Luer‐Lock blunt needles 20‐G × ½‐in. stubs (pink; Instech Lab., cat . no. LS20)
  • Silicon tubing: C‐flex tubing (ID: 0.020 in., OD: 0.083 in.; Cole Parmer, cat. no. 06422‐00)
  • ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, cat. no. P‐36931)
  • Cole Parmer 74900 Series 10‐Syringe Infusion Pump or equivalent
Table 4.8.2   MaterialsPrimary Antibodies Used for Immunocytochemistry

Epitope Host Dilution Commercial source
GGT Mouse 1:100 Abcam, cat. no. ab551136
Heme oxygenase 1 Rabbit 1:100 Abcam, cat. no. ab52947
HNF4α Rabbit 1:100 Abcam cat. no. ab8987
KIM‐1 Mouse 1:100 R&D Systems, MAB1750
Na+/K+ ATPase Mouse 1:100 Abcam, cat. no. ab7671
SGLT2 Rabbit 1:100 Abcam, cat. no. ab37296
ZO‐1 Mouse 1:100 Invitrogen, cat. no. 339194

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Figures

Videos

Literature Cited

Literature Cited
  Chang, S. Y., Weber, E. J., Van Ness, K. V., Eaton, D. L., & Kelly, E. J. (2016). Liver and kidney on chips: Microphysiological models to understand transporter function. Clinical Pharmacology and Therapeutics, 100(5), 464–478. doi: 10.1002/cpt.436.
  Donovan, J., & Brown, P. (2006). Parenteral injections. Current Protocols in Immunology, 73, 1.6.1–1.6.10. doi: 10.1002/0471142735.im0106s73.
  Esch, E. W., Bahinski, A., & Huh, D. (2015). Organs‐on‐chips at the frontiers of drug discovery. Nature Reviews. Drug Discovery, 14(4), 248–260. doi: 10.1038/nrd4539.
  Griffith, L. G., Wells, A., & Stolz, D. B. (2014). Engineering liver. Hepatology, 60(4), 1426–1434. doi: 10.1002/hep.27150.
  Halldorsson, S., Lucumi, E., Gómez‐Sjöberg, R., & Fleming, R. M.. (2015). Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosensors and Bioelectronics, 63, 218–31. doi: 10.1016/j.bios.2014.07.029.
  Huh, D., Hamilton, G. A., & Ingber, D. E. (2011). From 3D cell culture to organs‐on‐chips. Trends in Cell Biology, 21(12), 745–754. doi: 10.1016/j.tcb.2011.09.005.
  Phelan, K., & May, K. M. (2016). Basic techniques in mammalian cell tissue culture. Current Protocols in Toxicology, 70, A.3B.1–A.3B.22. doi: 10.1002/cptx.13.
  Wang, J.‐D., Douville, N. J., Takayama, S., & ElSayed, M. (2012). Quantitative analysis of molecular absorption into PDMS microfluidic channels. Annals of Biomedical Engineering, 40, 1862–73. doi: 10.1007/s10439‐012‐0562‐z.
  Weber, E. J., Chapron, A., Chapron, B. D., Voellinger, J. L., Lidberg, K. A., Yeung, C. K., … Kelly, E. J. (2016). Development of a microphysiological model of human kidney proximal tubule function. Kidney International, 90, 627–637. doi: 10.1016/j.kint.2016.06.011.
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