Results
A glomerular and proximal tubular microphysiological
system
Filtration and reabsorption of the glomerulus and PCT were assessed by
two main devices in the construction of the MPS (Figure 1C-D ).
Within the stainless steel housing unit (Figure 1C ), the
glomerulus enclosed a single porous PES membrane (porosity = 60) seeded
with the differentiated CIHP-1 and HUVECs. The differentiated podocytes
were cultured onto the GECM and EECM coated PES membranes 14 days prior
to initiation of flow. The negatively charged GECM and EECM generates a
charge-selective filtration barrier of the glomerulus
(C. M. Sakolish et al., 2016). During
differentiation, podocytes were observed for flattened morphological
characteristics with finger-like projections in areas surrounding PES
membranes. On day 15, PES membranes were flipped and seeded with the
endothelial cells for 4 hours. Once the HK-2 cells were seeded onto a
fibronectin-coated polycarbonate membrane (porosity = 20) for 4-hours,
it was sealed in the machined bi-layer, polycarbonate microfluidic
device described by previous work (Figure 1D )
(C. M. Sakolish & Mahler, 2017;
C. M. Sakolish et al., 2019). Its
physical dimensions (30 mm long x 15 mm wide x 50 μm thick) is
comparatively similar to reported in vivo PCT dimensions in the
human kidney (14 mm long x 40 μm thick)
(Lote & Lote, 1994). The fully
constructed MPS was connected to a T-Junction (Figure 2A-B ), a
splitter of fluid flow to recycle 90% of flow into the bloodstream and
10% into the filtrate output. While the peristaltic pump maintained the
MPS flow rate for the seven-day operation, additional modeling was
required to optimize tubing length, diameter, and to safeguard
physiological flow rates and shear rates (0.4 -1.5
dynes/cm2) within both glomerular housing unit and PCT
microfluidic device.
Flow characterization of microphysiological
system
To mediate the MPS microenvironment, a two-dimensional (2D) numerical
simulation of porous media transport was performed using a finite
element model of the device via COMSOL MultiPhysics®. A series of
parametrizations (Table ) was performed for the velocity flow
(m/s) (Figure 3A-B ) and shear rate (1/S) (Figure 3C-D )
based on the assumptions of steady state, constant fluid flow, viscosity
and density, incompressible flow, minimal fluid evaporation, and
negligible gravity. Conversions into physiological measurements were
adopted using the following shear stress (Equation 1 ).
\(\tau=\frac{6Q\mu}{bh^{2}}\ \) (Equation 1 )
where μ is the endothelial serum free culture medium viscosity at
37 °C; \(\tau\) is the shear stress (dyn-s/cm2);Q is the volumetric flow rate (cm3/s); bis the channel width; and h is the channel height (Q = 7.6
x 10-4 cm3/s, μ = 8.9 ×
10−3 dyn-s/cm2, h = 0.05 mm,b = 30 mm)
Based on the conversions, the optimal tubing length of filtrate output
and bloodstream were ~508 mm and 734 mm respectively.
The tubing length ranges were selected based on peristaltic pump and
incubator sizing constraints. The inlet mass flow rate was 7.6 x
10-7 kg/s (7.6 x 10-4cm3/s) to generate a shear stress of 0.5
dyne-s/cm2 across the apical layer of the PCT device.
Means of 1-D velocity profiles from filtrate output and the bloodstream
had a ratio of 83/81 (Figure 3 ). Concentration profiles of 0.1
mg/mL FITC-HSA were computed using its diffusivity coefficient (61
μm2/s) in Transport by Diluted Species with a
time-dependent solver from 0-120 minutes (Figure 3E-F ). The
concentration 1-D profile was characterized with an exponential increase
until it plateaued and approached steady state around 105-120 minutes.
While these simulations are effective in determining the optimal
geometry for the desired shear stress, the model was idealistic and
reproduced without the inclusion of cellular effects that may alter the
overall outcome.
Inlet mass flow rate (kg/s) was experimentally validated by using a
single inlet 0.25 ID mm tubing at 4 -16 rpm pump rate (0.19 – 0.72
dyne-s/cm2) (Figure 4A-B ). Flow rates
(μL/min) and shear stress (dyne-s/cm2) was determined
with pumping 1X phosphate buffered saline (PBS) over the course of 2
hours, and collecting samples at 15-minute intervals (n= 4 for each
rpm). At 12 rpm pump rate, the flow inlet (Qin) achieved
an average of 45.3 ± 3.0 μL/min with a shear rate of 0.5 ± 0.04
dyne-s/cm2. With the assembled MPS, 1X PBS was
cyclically pumped for the same duration, and filtrate formation
(Qprimary filtrate) and bloodstream recycling
(Qblood) rates were an average of 16.2± 4.5 μL/min and
29.0± 4.5 μL/min, respectively. Filtrate output and shear stress across
the PCT for the single culture and tri-culture conditions
(Figure 4C-D ) were compared with the simulated and experimental
settings to characterize the system under non-ideal conditions. Single
culture devices contained only the proximal tubule (HK-2) cells seeded
into the PCT device, while the tri-culture was seeded with CIHP-1 and
HUVECs in the glomerulus housing unit along with the HK-2 cells in the
PCT. Implementation of single culture is representative of
pre-established PCT devices (Homan et al.,
2016; Jang et al., 2013;
Jansen et al., 2016;
Ng et al., 2012;
Raghavan, Rbaibi, Pastor-Soler, Carattino,
& Weisz, 2014; Sabbisetti et al., 2014;
C. Sakolish et al., 2020;
C. Sakolish et al., 2018;
Weber et al., 2016), whereas tri-cultures
reflect the combination of the human glomerulus and PCT. While the
90.0% of fluid was not recycled into the bloodstream as shown in the
simulation, approximately 36.0% was outputted into the waste collection
and 64.0% was recycled into the MPS.
Recapitulation of glomerulus and proximal tubule
functions
Key characteristics of a functional nephron are its blood serum protein
filtration, glucose resorption, and filtrate formation capacity. Two
culture conditions were established to demonstrate system characteristic
differences, which was denoted as a single (n=3) and tri-culture (all
cell-types) (n=3). Throughout the seven-day operation, primary filtrate
formation was collected every 24 hours in conical tubes and immediately
stored at -20°C before additional assessment. Volumes of waste for each
culture type were weighed to measure velocity flow rate (mL/min)
(Figure 5A-B ). The primary filtrate formation in the
single-culture maintained a median of approximately 30 μL/min, while the
tri-culture had 15.0 μL/min. The introduction of the glomerular cells
and endothelial cells created a barrier effect, reducing the flow rate
within the apical PCT of the MPS to generate a shear stress from 0.3 to
0.2 dyne-s/cm2. Results of the simulated,
experimental, single, and tri-culture filtrate output and shear stress
across the apical region of the PCT were shown (Figure 4C-D ).
While the simulated model exhibited a linear behavior based on its
regression (R2=1.0), the shear stress under
experimental conditions significantly decreased in the presence of
cells, suggesting that simulations provided only ideal conditions and
required further parametrizations.
Since the proximal tubule is responsible for nearly 90.0% of glucose
reabsorption in the kidney, glucose secretion (mg/mL) in both single and
tri-culture devices were evaluated using the Infinity Glucose Assay
(Figure 5C-D ). Filtrate output volumes were sampled three times
into a 96 well plate and compared to a 1 mg/mL glucose solution diluted
with 0.1% benzoic acid standard curve. Samples were read with the plate
reader at an absorbance of 340 nm. Both culture conditions lacked
statistically significant difference due to the large volume of daily
filtrate output (~30 - 40 mL/day), although there was a
downward trend in the amount of glucose present in the filtrate output
over the course of the 7 day culture, indicating some proximal tubule
function. No significant change in both velocity and glucose
concentrations (Figure 5B and D ) demonstrated that the MPS was
stable during the seven days.
On Day 7 of MPS operation, 0.1 mg/mL of fluorescently tagged human serum
albumin (FITC-HSA, Abcam #ab8030) diluted with 1X PBS was circulated
into the MPS to assess glomerulus function and were analyzed for the
amount of unfiltered serum proteins in both the filtrate outlet and
blood stream circuit reservoir (McQuarrie,
Shakerdi, Jardine, Fox, & Mackinnon, 2011). Samples were drawn from
the filtrate outlet and blood stream circuit reservoirs every 15 minutes
for 2 hrs and placed in a black, 96-well plate. Samples were analyzed
for fluorescence with a Synergy 2 plate reader against a standard curve
(Figure 5E-G ). Protein concentration in the bloodstream
minimally declined from 0.1 mg/mL of FITC-HSA for both case conditions.
In the tri-culture conditions, there was a statistically significant
difference (p< 0.001) between filtrate output and bloodstream
circuit output (Figure 5F ). FITC-HSA concentration in
tri-culture in the blood circuit output (0.083 mg/mL) was 8 times
greater than the filtrate output (0.011 mg/mL), suggesting that there is
a functional filtration process in the tri-culture MPS. This behavior
was not exhibited in the single culture conditions. Single-cell culture
exhibited an increase in protein concentrations in the filtrate output,
while the tri-culture devices had little protein reach the filtrate
output (Figure 5G ). Absence of protein in the waste or filtrate
stream suggests that the glomerular cells prevent the FITC-HSA from
passing through the glomerular membrane, similar to the function of the
glomerulus in vivo .
Fluorescent stains provided insight into the cellular phenotype
following dynamic culture. The membranes, PCT (polycarbonate) and
glomerular (PES) membranes from the MPS were removed, fixed with 4%
paraformaldehyde (PFA), stained with phallodin 568) and
Hoescht 33342, and imaged using a
Zeiss confocal microscope at 40x magnification. F-actin cytoskeleton
restructuring was imaged with phallodin 568, whereas DNA in the nuclei
was counterstained with Hoescht 33342. To determine the cellular
confluence, qualitative 2D orthogonal views and three-dimensional (3D)
views (Figure 6A-F ) were obtained. Qualitatively, the HK-2
cells on the PCT membrane (Figure 6A-B ) demonstrated a
monolayer behavior as previously shown (C.
M. Sakolish & Mahler, 2017), and quantitatively represented by the
75.3% confluence and significantly greater cell count in comparison to
podocytes and endothelial cells (Figure 6G-H ). The CIHP-1 cells
on the glomerular membrane appeared flatter, fewer in cell count, and
fewer F-actin across the plane due to a larger average cell area (831.1
μm2) compared to HK-2 (183.6 μm2)
and HUVECs (353.7 μm2). From the 3D view ofFigure 6C , finger like projections also validated full
differentiation (day 14). The endothelial cells were highly directional
in the presence of flow, but attachment was limited by day 7
(Figure 6E-F ). This may be related to HUVECs poor ability to
adapt to the dynamic crossflow culture conditions.