Materials and Methods
Simulating and Validating model in COMSOL
MultiPhysics®
A 2D computational model was developed through COMSOL Multiphysics
(COMSOL Inc, Burlington, MA). Computational domains were generated using
COMSOL’s native geometry tools. To model the fluid flow in open and
porous domains, the Free and Porous Media Flow physics package was used
for porous membranes. Inlet, Outlet and No-Slip Wall conditions were
used to define the boundary conditions of the computational domain. A
symmetry boundary condition was used to reduce the computational load,
and domains were discretized using a combination of swept, tetrahedral
and boundary layer elements using native meshing tools. Using both
stationary and time-dependent solvers, the 2D model was used to optimize
tubing internal diameter (ID) and length (Cole-Parmer, Vernon Hills,
IL), and total flow rate from the peristaltic pump (Watson-Marlow,
Paramus, NJ). The primary design parameters were achieved a passive
fluid flow split at the glomerular filter T-junction (Eldon James,
Denver, CO), and obtaining a fluid shear stress of 0.65
dyn-s/cm2 across the top chamber of the proximal
tubule device (PCT), which mimics the physiological fluid shear stress
in the human proximal tubule.
Fabrication of the Microphysiological
System
Construction of the proximal tubule-on-a-chip has been previously
described (C. M. Sakolish & Mahler,
2017; C. M. Sakolish et al., 2019).
Detailed description of the fabrication method is in the supplementary
materials.
Cell Culture
All cell lines were adapted to Endothelial Serum Free Medium (ESFM,
Thermo Fisher Scientific, Waltham, MA) supplemented with 10 µg/mL of
human plasma fibronectin (Thermo Fisher Scientific, Waltham, MA), 20
ng/mL of human recombinant epidermal growth factor (EGF, Thermo Fisher
Scientific, Waltham, MA), and 10 ng/mL of human recombinant basic
fibroblast growth factor (bFGF, Thermo Fisher Scientific, Waltham, MA).
Conditionally immortalized human podocytes (CIHP-1) purchased from Dr.
Saleem Moin’s laboratory at the University of Bristol at passage 16
proliferated at 33°C in 5% CO2 and were passaged every
10-14 days. The human, renal proximal tubule cell line (HK-2) (passage
20-30) obtained from the American Type Culture Collection (ATCC,
Manassas, VA) was incubated at 37°C in 5% CO2 and
passaged every 5-7 days with 0.05% Trypsin-EDTA (Thermo Fisher
Scientific, Waltham, MA). Human umbilical vein endothelial cells
(HUVECs) purchased from Lonza Bioscience (Rockland, MD) were maintained
at 37°C in 5% CO2 and experiments were completed using
passages 3-7.
CIHP-1, HUVECs, and HK-2 adhesion on porous
membranes
30 nm pore size polyethersulfone
(PES) membranes (Sterlitech, Kent, WA) were coated with glomerular
extracellular matrix (GECM), which consist of tri-layer of 8
µg/cm2 rat-tail collagen 1 (BD Biosciences, Franklin
Lakes, NJ), 8 µg/cm2 heparin sulfate (Sigma-Aldrich
H7640), and 8 µg/cm2 collagen 1 (BD Biosciences,
Franklin Lakes, NJ). The selection of the GECM composition was adapted
from previous work (C. M. Sakolish &
Mahler, 2017; C. M. Sakolish et al.,
2019). Detailed description of the adhesion methods is in the
supplementary materials.
Assembly of housing unit and microfluidic
device
The experimental study was conducted in a sterile, biosafety cabinet
cleaned with 70% ethanol. All device components were autoclaved prior
to experimentation. The internal and external components of the
peristaltic pump were cleaned with 70% ethanol before stringing the
Pharmed BPT 0.25 mm ID tubing (Cole-Parmer, Vernon Hills, IL) into the
cassette with a conical tube of 1X phosphate buffered saline (PBS). All
tubing was subjected to an average flow of 40 µL/min with 1X PBS for 1
hour to eradicate bubble formation. Assembly of the glomerulus stainless
steel housing unit (Advantec MFS, Dublin, CA)
(C. M. Sakolish et al., 2016;
C. M. Sakolish & Mahler, 2017;
C. M. Sakolish et al., 2019), consisting
of a stainless steel mesh (Advantec MFS, Dublin, CA), pre-cut 14 mm in
diameter PES membrane (Sterlitech, Kent, WA), polytetrafluoroethylene
(PTFE) gasket (Advantec MFS, Dublin, CA), pre-cut 200 µL pipet tips (VWR
International, Radnor, PA), and leur locks (Cole-Parmer, Vernon Hills,
IL) was sealed with an autoclavable wrench (SteriTool, Jacksonville,
FL). While the assembled glomerulus housing unit was attached to the end
of the tubing to fill with 1X PBS, assembly of the T-junction and the
CNC machined, polycarbonate PCT device was completed. Within the PCT
device, a 0.4 µm pore size polycarbonate membrane and 0.01-in thick
silicone polymer gasket (McMaster-Carr, Elmhurst, IL) created anin vitro barrier system with an apical and basolateral sections.
The components are assembled in their respective locations after filling
with 1X PBS, and placeholder porous membranes were replaced with the
cell seeded porous membranes. Afterwards, the inlet fluid was replaced
with the supplemented ESFM incubated at 37°C, and two outlet tubing was
connected to 50 mL conical tube for filtrate output and bloodstream
output. ESFM was changed every 2-3 days for a week.
MPS Cellular Models
Under dynamic conditions, two
cellular models (single and tri-cultures) were employed with the
glomerulus and PCT MPS components to assess the necessity of a
multi-culture model. The single culture contained only proximal tubule
cells (HK-2) adhered to the polycarbonate membrane in the PCT device.
The single culture MPS was operated with the T-Junction, stainless-steel
glomerulus housing unit with blank PES membrane, and seeded HK-2 cells
in the PCT device. There was no glomerular or endothelial cells (CIHP-1
and HUVECs) on the PES membranes. The tri-culture housed all three
cell-types on their respective membranes. The podocytes (CIHP-1) and
endothelial (HUVECs) cells were attached to the PES membrane in the
glomerulus unit in addition to the proximal tubule (HK-2) cells attached
to the polycarbonate membrane in the PCT device.
Volume Flow Assay
Filtrate output volume was collected daily in 50 mL conical tubes
(Corning, Corning, NY) and stored in -20°C for future assessment. In
post-experiment testing, the volumes were thawed, weighed, and
normalized to an empty conical tube. Based on the density of the volume
(1g/mL), total volume was calculated over time in mL/min.
FITC-human serum albumin flow
assay
After seven days of fluidic culture, the nephron-on-a-chip system was
subjected with 0.1 mg/mL of FITC-HSA (Abcam) diluted with 1X PBS by
replacing the inlet conical tube fluid. In two black 96 well plates,
samples (n =4) of the inlet and outlet fluid flow were collected at each
15-min intervals for 2 hrs. Fluorescence was assessed with Synergy Plate
Reader (Biotek, Winooski, VT) and Gen5 software with an
emission/excitation wavelength of 485/528 nm. Results were normalized to
the average maximum fluorescent intensity.
Fluorescent staining and image
processing
Following the system challenge with HSA, membranes were removed from the
devices and placed in a 4-well plate. The cells were fixed with 4%
paraformaldehyde (PFA) diluted in 1x PBS for 5 min at room temperature
and permeabilized with 0.1% Triton X-100 in 1x PBS for 15 min. The
cells were stained with phallodin 568 (Invitrogen) and counterstained
with Hoescht 33342 (Thermo Fisher Scientific, Waltham, MA) for 30 mins.
Membranes were then washed three times with 1X PBS for each 5 mins.
Samples were mounted onto 24 x 40 x 0.13 mm glass slides through ProLong
Gold and visualized with Zeiss Leica SP5 X confocal microscope. Images
were processed in FIJI ImageJ (Schindelin
et al., 2012) for directionality, confluence, and cell count. Z-stacks
were taken for the membranes to generate a three-dimensional (3D)
profile.
Infinity glucose assay
Glucose concentrations were calculated from the filtrate output volume
using the Infinity™ Glucose Hexokinase Liquid Stable Reagent assay
(Thermo Scientific, Waltham, MA). The reagent was diluted with filtrate
output volume at a ratio of 150:1, incubated in 37°C for three minutes,
and plated into a 96 well plate (n=4) for each day. Absorbance was
assessed with Synergy Plate Reader (Biotek, Winooski, VT) and Gen5
software with a primary/secondary wavelength of 340/380 nm. Standards
were established with both a glucose solution of 1 mg/mL of 0.1%
benzoic acid and supplemented ESFM.
Statistical Analysis
Nonparametric one-way ANOVA with post-hoc Dunn’s test, Mann-Whitney
test, and regression tests were conducted in
GraphPad Prism® (GraphPad, San
Diego, CA). All data from single- and tri-cultures were presented with
medians and ranges.