Method Article

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

DOI:

10.3791/63821

⸱

November 4th, 2022

 , 
1Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 2Department of Medicine, Division of Nephrology, Duke University School of Medicine, 3Department of Cell Biology, Duke University, 4Center for Biomolecular and Tissue Engineering, Duke University

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery.

This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Organ-on-a-chip devices are dynamic 3D in vitro models that employ molecular and mechanical stimulation, as well as vascularization, to form tissue-tissue interfaces that model the structure and function of specific organs. Previously established organ-on-a-chip devices that aimed to recapitulate the kidney's glomerulus (glomerulus chips) consisted of animal cell lines1 or human primary and immortalized cell lines of heterogeneous sources2,3. The use of genetically heterogeneous cell sources present variations that significantly limit the studies of patient-specific respons....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Prepare basement membrane matrix solutions and coated substrates

  1. Thaw basement membrane matrix 1 overnight on ice at 4 °C. Aliquot according to the manufacturer's suggestion for dilution ratio. Using a 50 mL conical tube and pipette, thoroughly mix an appropriate amount of basement membrane matrix 1 into 25 mL of cold DMEM/F12 until completely thawed and dissolved.
    1. To dissolve a frozen aliquot, take ~200 µL from the 25 mL of cold DMEM/F12 and transfer it to the frozen aliquot tube. Pipette up and down until the matrix is thoroughly thawed and dissolved. Transfer the full tube content of basement membrane matrix....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Here we show that a functional 3D in vitro model of the glomerulus can be vascularized and epithelialized from an isogenic source of human iPS cells. Specifically, this protocol provides instructions on how to apply human iPS cell technology, particularly their ability to differentiate into specialized cell types, to generate kidney glomerular epithelium (podocytes) and vascular endothelium (viECs) that can be integrated with microfluidic devices to model the structure and function of the human kidney at the pat.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

In this report, we outline a protocol to derive vascular endothelium and glomerular epithelium (podocytes) from an isogenic human iPS cell line and the use of these cells to engineer a 3D organ-on-a-chip system that mimics the structure, tissue-tissue interface, and molecular filtration function of the kidney glomerulus. This glomerulus chip is outfitted with endothelium and glomerular epithelium that, together, provide a barrier to selectively filter molecules.

Researchers interested in adap.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

S.M. is an inventor on a patent related to podocyte differentiation from human iPS cells. The other author has nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This work was supported by the Pratt school of Engineering at Duke University, the Division of Nephrology at Duke Department of Medicine, a Whitehead Scholarship in Biomedical Research, and a Genentech Research Award for S. Musah. Y. Roye is a recipient of the Duke University-Alfred P. Sloan Foundation Scholarship and William M. "Monty" Reichert Graduate Fellowship from Duke University's Department of Biomedical Engineering. The DU11 (Duke University clone #11) iPS cell line was generated at the Duke iPSC Core Facility and provided to us by the Bursac Lab at Duke University. The authors thank N. Abutaleb, J. Holmes, R. Bhattacharya, and Y. Zhou for technic....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Antibodies
Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodiesThermo/Life TechnologiesA32744; A32754; A-11076; A32790; A21203; A11015
Collagen IVThermo/Life Technologies14-9871-82
NephrinProgenGP-N2
PECAM-1R&D SystemsAF806
PodocinAbcamab50339
VE-CadherinSanta Cruzsc-9989
Basement membrane matrices
Corning Fibronectin, HumanCorning356008Basement membrane (3)
iMatrix-511 Laminin-E8 (LM-E8) fragmentIwai North AmericaN8922012Basement membrane matrix (2)
Matrigel hESC-qualified matrix, 5-mL vialBD Biosciences354277Basement membrane matrix (1); may show lot-to-lot variation
Cells
DU11 human iPS cellsThe DU11 (Duke University clone #11) iPS cell line was generated at the Duke iPSC Core Facility and provided to us by the Bursac Lab at Duke University. The line has been tested and found to be free of mycoplasma (last test in November 2021) and karyotype abnormalities (July 2019)
Culture medium growth factors and media supplements
0.5M EDTA, pH 8.0Invitrogen15575020
2-MercaptoethanolThermo/Life Technologies21985023
Albumin from Bovine serum, Texas Red conjugateThermo/Life TechnologiesA23017
All-trans retinoic acid (500 mg)Stem Cell Technologies72262
B27 serum-free supplementThermo/Life Technologies17504044
B-27 supplement (50x) without Vitamin AThermo/Life Technologies12587010
Bovine serum albuminSigma-AldrichA9418
CHIR99021Stemgent04-0004May show lot-to-lot variation
Complete medium kit with CultureBoost-RCell Systems4Z0-500-RPodocyte maintenance media
DMEM/F12Thermo/Life Technologies12634028
DMEM/F12 with GlutaMAX supplementThermo/Life Technologies10565042DMEM/F12 with glutamine
Forskolin (Adenylyl cyclase activator)Abcamab120058
GlutaMAX supplementThermo/Life Technologies35050061glutamine supplement
Heat-inactivated FBSThermo/Life Technologies10082147
Heparin solutionStem Cell Technologies7980
Human Activin AThermo/Life TechnologiesPHC9544
Human BMP4Preprotech120-05ET
Human BMP7Thermo/Life TechnologiesPHC9544
Human VEGFThermo/Life TechnologiesPHC9394
Inulin-FITCSigma-AldrichF3272
mTeSR1 mediumStem Cell Technologies05850Human iPS cell culture media (CCM). Add 5x supplement according to the manufacturer. Human iPS CCM can be stored for up to 6 months at -20 °C.
N-2 Supplement (100x)Thermo/Life Technologies17502048
Neurobasal mediaThermo/Life Technologies21103049Lateral mesoderm basal media
PBS (Phosphate-buffered saline)Thermo/Life Technologies14190-250
Penicillin-streptomycin, liquid (100x)Thermo/Life Technologies15140-163
ROCK inhibitor (Y27632)Tocris1254
StemPro-34 SFMThermo/Life Technologies10639011Endothelial cell culture medium (CCM). Add supplement according to manufacturer. Endothelial CCM can be stored for up to two weeks at 4 °C or -20 °C for up to 6 months.
TGF-Beta inhibitor (SB431542)Stem Cell Technologies72234
Enzymes and other reagents
AccutaseThermo/Life TechnologiesA1110501Cell detachment buffer
Dimethyl Suloxide (DMSO)Sigma-AldrichD2438
Ethanol solution, 70% (vol/vol), biotechnology gradeVWR97065-058
Paraformaldehyde (PFA)Thermo/Life Technologies28906
Sterile distilled waterThermo/Life Technologies15230162
Triton X-100VWR97062-208
Equipment
Trypsin EDTA, 0.05%Thermo/Life Technologies25300-120
(Orb) Hub moduleEmulateORB-HM1
100mm x 15 mm round petri dishFisherbrandFB087579B
120 x 120 mm square cell culture dishVWR688161
Accuri C6BD Biosciences
Aspirating pipettes, individually wrappedCorning29442-462
Aspirating UnitSP Bel-ArtF19917-0150
Avanti J-15R CentrifugeBeckman CoulterB99516
Conical centrifuge tube, 15 mLCorning352097
Conical centrifuge tube, 50 mLCorning352098
EVOS M7000Thermo/Life TechnologiesAMF7000Fluorescent microscope to take images of fixed and stained cells.
HemocytometerVWR100503-092
Heracell VIOS 160i CO2 incubatorThermo/Life Technologies51030403
Inverted Zeiss Axio Observer equipeed with AxioCam 503 cameraCarl Zeiss Micrscopy491916-0001-000(microscope) ; 426558-0000-000(camera)
Kimberly-Clark nitrile glovesVWR40101-346
Kimwipes, largeVWR21905-049
Leoca SP8 Upright Confocal Microscope
Media reservoir (POD Portable Module)EmulatePOD-1
Microplate shakerVWR12620-926
Organ-chipEmulateS-1 Chip
Organ-chip holderEmulateAK-CCR
P10 precision barrier pipette tipsDenville ScientificP1096-FR
P100 barrier pipette tipsDenville ScientificP1125
P1000 barrier pipette tipsDenville ScientificP1121
P20 barrier pipette tipsDenville ScientificP1122
P200 barrier pipette tipsDenville ScientificP1122
Plasma AsherQuorum techK1050X RFThis Plasma Etcher/Asher/Cleaner was used as a part of Duke University's Shared Materials Instrumentation Facility (SMiF).
Round bottom polystyrene test tube with cell strainer snap capCorning352235
Serological pipette, 10 mL, indivdually wrappedCorning356551
Serological pipette, 25 mL, indivdually wrappedCorning356525
Serological pipette, 5 mL, indivdually wrappedCorning356543
Steriflip, 0.22 µm, PESEMD MilliporeSCGP00525
Sterile Microcentrifuge tubesThomas Scientific1138W14
T75cm2 cell culture flask with vent capCorning430641U
Tissue culture-treated 12 well platesCorning353043
Tissue culture-treated 6 well platesCorning353046
Vacuum modulator and perstaltic pump (Zoe Culture Module)EmulateZOE-CM1Organ Chip Bioreactor
VE-Cadherin CD144 anti-human antibody - APC conjugatedMiltenyi Biotec130-126-010
Wide-beveled cell lifterCorning3008
MACS
CD144 MicroBeads, humanMiltenyi Biotec130-097-857
CD31 MicroBead Kit, humanMiltenyi Biotec130-091-935
LS columnsMiltenyi Biotec130-042-401

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Wang, L., et al. A disease model of diabetic nephropathy in a glomerulus-on-a-chip microdevice. Lab on a Chip. 17 (10), 1749-1760 (2017).
  2. Musah, S., et al. Mature induced-pluripot....

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Kidney Glomerulus ChipInduced Pluripotent Stem CellsIsogenic Organ ChipGlomerular Filtration BarrierPodocyte DifferentiationVascular Endothelial CellsMicrofluidic ChipNephrotoxicity TestingPrecision MedicinePatient Specific Disease Modeling

Related Articles