Primary Human Liver Co-Culture With Flow and Kupffer Cell Integration On

Microfluidic Liver-On-A-Chip

Senior Thesis

Presented to

The Faculty of the School of Arts and Sciences Brandeis University

Undergraduate Program in Biology

Anthony Bahinski (Wyss Institute), Co-Sponsor Joan Press (Brandeis), Co-Sponsor

Payal Patel (Wyss Institute), Co-Mentor

In partial fulfillment of the requirements for the degree of Bachelor of Science

By

Marc Mazur

April, 2015

Thesis Sponsor

Name: Anthony Bahinski Signature: _____________________________

                                                                                     

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Abstract Primary human tissue cultures provide a more ethically sound and potentially effective alternative model to animal testing. It has been shown that culture of two or more cells

together provides improved epithelial polarization and differentiation in culture in vitro. Two non-parenchymal (NPC) liver cell types, Kupffer cells (KCs) and Liver Sinusoidal Endothelial Cells (LSECs), are located in close proximity in the liver sinusoid. As drug metabolism is a species- specific process, it is important to re-evaluate rat hepatic co-

culture experiments with human hepatocytes and NPCs. In order to accurately recapitulate liver responses in vitro, NPC such as KC must be incorporated into existing liver culture models. In an effort to incorporate KC into the Liver-On-A-Chip, I examined the relationship between human KCs and LSECs, and between hepatocytes and LSECs.

In addition to morphological study, I examined the viability and functionality of hepatocyte microfluidic co- and monocultures by looking at lactose dehydrogenase

(LDH) release, albumin secretion, and metabolic CYP3A4 activity. Hepatocyte-LSEC microfluidic co-culture revealed decreased LDH release after the first few days of flow

compared to hepatocyte static monoculture controls. Moreover, Liver-On-A-Chip cultures displayed comparable albumin synthesis when compared to in vivo data. KC-

LSEC co-culture maintained morphology and viability better than KC monoculture alone. Examination of KC-LSEC compatibility in static culture shows that further optimization is required to incorporate KC into liver tissue culture models in an

organized manner. Introduction

Modern drug development is burdened by a high cost, a lengthy approval process, and

potential adverse effects that may only be identified in late Phase clinical trials or post approval.

Despite technological advances, existing pre-clinical 2D and 3D static culture models often fail

to predict human clinical responses. Current models fail to fully recapitulate subtle organ-

specific cell makeup and mechanical forces, which may limit their ability to mimic physiological

and pathophysiological responses. As a result, animal models are commonly used. This practice

is not only costly but also ethically questionable. Moreover, they often do not accurately predict

human drug toxicity. It has repeatedly been shown that drugs, which were known to be toxic in

vivo, fail to model any hepatotoxicity also known as “silent” hepatotoxicity (Bale et al., 2014),

probably due to the lack of an organized-NPC presence in vitro. KC-dependent liver regeneration

and hepatotoxicity supports the crucial role KCs play for liver conditions such as hepatitis,

                                                                                     

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malaria, alcohol and acetaminophen (APAP) toxicity. Drug-toxicity is especially pertinent in an

organ through which all contaminants in the blood are filtered and metabolized; this is termed

Drug-Induced Liver Injury (DILI). Hepatocyte monoculture is ineffective, as the tissue quickly

degenerates, usually lasting less than a week. The collagen gel sandwich system (sandwich

method), in which Hepatocytes are cultured between layers of ECM, has been shown to improve

Hepatocyte functionality and viability (Kono, Yang, & Roberts, 1997). In the last decade,

scientists have devised many new methods of tissue culture in an effort to improve tissue

functionality: these include micro-patterned culture, Spheroids/3D culture, liver-slice technology

and various microfluidic devices like Liver-On-A-Chip. While these options all support

increased tissue viability (2-4 weeks), only some microfluidic devices address the matter of

mechanical forces, which directly affect the differentiations of cells in vivo (Huh, 2014). In light

of this we are investigating the effects of fluidic shear on liver cell viability and functionality

markers. The dual channel microfluidic organ chip also allows for more organized co-culture and

continuous perfusion, something that many current assays fail to recreate. In this paper, I will

take a short look at recent advances and other models. Each model has advantages and

disadvantages based on the experimental set up and each offers a different perspective on the

function of individual liver cell types. The Wyss organ chip, while similar to these models, takes

advantage of microfluidics to improve Hepatocyte differentiation and viable time-period.

Furthermore, I will examine Hepatocyte-LSEC microfluidic co-culture, monoculture, KC-LSEC

co-culture and monoculture. KC & LSEC are two non-parenchymal cell types that have been

shown to have complementary paracrine activity in vivo and using rat cells (Ries et al., 2000).

This cellular relationship is understudied in humans and requires further investigation to

eliminate silent toxicity and improve liver models.

                                                                                     

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Review of Other Liver Models

Precision-Cut Tissue Explants Liver Slice Technology is an in vitro assay used for liver xenobiotic metabolism and

toxicity evaluation and represents a mini-model of the organ under study. Slices are prepared

from fresh liver by making a cylindrical core using a drill with a

hollow bit, from which slices are cut with a specially designed

tissue slicer (Fig. 1). Liver slices usually remain viable for up to

96 hours (de Graaf et al., 2010). The slices contain all cells of the

tissue in their natural arrangement, leaving intercellular and cell-

matrix interactions intact, and are therefore highly appropriate for studying multicellular

processes. Limitations of this model include limited tissue viability (maximum 5 days) and

cellular damage that affects the exterior of the tissue slices. Moreover, depending on the

thickness of the slice, there may be a differential perfusion of the interior and exterior of the

tissue slice (Worboys, Bradbury, & Houston, 1997). Most importantly, this model offers a

unique combination of in vitro and in vivo studies and retains the liver’s sinusoidal architecture.

Liver Slice Technology argues that primary human cells de-differentiate rapidly in vitro.

However, this model experiences inferior viability to primary human tissue culture.

Figure 1: Liver slice method shown via precise-a-sliceTM.

                                                                                     

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Microfluidic Devices Similar To Organs-On-A-Chip A microfluidic liver chip described by Frevert, contains monoculture and flow chambers,

separated by a micro fabricated baffle (Fig. 2 right), which shields the hepatocytes from fluid

shear to mimic the LSEC-hepatocyte interface (Space of Disse) and sinusoid of the liver. This is

mimicked by the sandwiching method as well. The geometry of the cell culture chamber

promotes linear alignment of hepatocytes in two lines, which facilitates the production of

functional bile canaliculi along hepatic-cord-like structures (Fig. 2 left). This method is great at

creating a physiologically relevant organization of hepatocytes. Conversely, it is a monoculture

and its metabolic, synthetic, and bio-transformative properties need to be documented (LeCluyse,

Witek, Andersen, & Powers, 2012). Hepatocytes are perfused semi-continuously using

microfluidics. The fenestrae-like barrier prevents hepatocytes from experiencing shear stress

while still exposing them to the contents of the microfluidic channel (D. Huh, Torisawa,

Hamilton, Kim, & Ingber, 2012). This chip attempts to model minimal hepatocyte exposure to

the non-parenchyma by mimicking LSEC fenestrae using a physical barrier. While this model

successfully gauges hepatocyte sensitivity to flow and organization, it underestimates the

interactions between hepatocytes and the non-parenchymal cell types.

Figure 2: Left, Classic model of liver sinusoid: displaying grey hepatocytes with smaller cholangiocytes in hepatic chords in the liver parenchyma. This is separated from the non-parenchyma (NPC) by the Space of Disse. Note the location of KCs and LSECs (non-parenchyma) (Frevert et al., 2005). Right, microfluidic, monoculture liver chip. A micro engineered porous ‘baffle’ simulates LSEC fenestrae (Huh, 2012).

                                                                                     

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Micro-Patterned Cultures In 2014, Hepregen, a biotech company in Medford, MA, released HepatopacTM, a

revolutionary model involving the micropatterning of hepatocytes with stromal cells in a static

culture (Bale et al., 2014). Micropatterning has become increasingly popular means controlling

tissue organization. (Mi, Chan, Trau, Huang, & Chen, 2006). In this model the hepatocytes

differentiate to form a confluent monolayer with consistent cell density and 2-4 week viability.

The pattern is reminiscent of the hepatic acinus (Fig. 3.3 & 3.5), and maintains 3 cell types but

does not have any microfluidic flow. While bile cannuliculi form in this model1, there is limited

vascularization of the tissue as fibroblasts are used instead of endothelial cells.

In 2015, HepatomuneTM, essentially HepatopacTM with KC, was released to model liver

inflammation and KC-dependent cytotoxicity2. The limitations of this model are that it includes a

non-liver cell type (stromal) in great concentration, has no endothelial cells, and the KC are in

direct contact with the hepatocytes, which is not in vivo organization. These cultures do reach

impressive metabolic activity. Hepregen’s website motto is “Architecture is Important” and yet

they seem to ignore the importance of LSECs in hepatic tissue culture. While very effective at

increasing functionality and organization, this model does not reconstruct a liver ‘sinusoid’.

                                                                                                               1 This occurs in other models to varying degrees as well including the original “Sandwich method”. 2 This information is courtesy of Hepregen’s website. http://www.hepregen.com/

Figure 3: (from left to right) 1) Full well in a 96 well plate with spherical hepatocyte islands 2) close up of an island 3) Distance was found to be very important for signaling between islands. 4) There is a defined and constant boarder to the hepatocyte monolayer. It seems the fibroblasts ‘trap’ the cells into confined area. 5) Liver acinus is a diamond shaped area bounded by two portal triads. Within the liver acinus are 3 physiologically distinct zones. http://fblt.cz/en/skripta/ix-travici-soustava/5-jatra-a-biotransformace-xenobiotik (multimedia textbook)

                                                                                     

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3D Culture & Spheroids Insphero, a company in Switzerland has developed a brand new method of 3D culture to

form a microtissue (Bale et al., 2014). Hepatocytes and NPCs are suspended in a droplet for 3

days, the tissue forms in a 3D environment before being transferred to a spheroid compatible

plate (Fig. 4 left3). This suggests that this group feel that KC might be partly responsible for

tissue modeling during hepatogenesis. While the microtissue is a spectacular example of the

power of tissue engineering, once mature the spheroid is difficult to perfuse because of its size

and cell composition (Fig. 4 right). This is possibly due to LSECs being responsible for

organizing the vasculature of liver tissue. In the Liver-On-A-Chip hepatocytes are confined to

the apical side of the membrane, but they can communicate through pores with LSECs on the

other side. In a recent rat cell culture experiment, LSEC’s have been shown to be responsive to

inflammatory signaling, and act as scavengers in the sinusoid (Wang et al., 2012). A 2013 review

found that LSECs are responsible for liver regeneration, but only after upregulation via liver

injury markers (Deleve, 2013). These studies support the pivotal role of LSECs regular liver

function and regeneration after liver injury. As the liver is a 3D organ, it is safe to assume that

3D tissue culture will become increasingly popular for modeling the liver in vitro. Insphero has                                                                                                                3 Images courtesy of http://www.insphero.com/

Figure 4: Left, details the manipulation of physics, mainly surface tension, to create a pseudo-amnion of culture media for the spheroid. Right, Immunofluorescence 3D reconstruction of a liver spheroid made of

hepatocytes and LSECs

                                                                                     

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displayed the ability of liver cell types to self-organize but their microtissue lacks the cell

diversity of the liver in vivo. Furthermore, while organization occurs it does not fully mimic in

vivo organization. Again, this could be due to a lack of comparable diversity of cellular

composition in vitro.

Experimental Design of Liver-on-a-Chip Microchip Design (Liver Chip V2)

The assembled microfluidic organ chip (Fig. 5 right) is elegant and may appear to be

simple, but it is extremely complex in its fabrication and manipulation of microfluidics4. The

chip used was an optimized and adapted version of the Lung-On-A-Chip microchip (D. D. Huh,

2015). Soft lithography was used to etch a 1000 µm x 200 µm basal or endothelial channel and

1000 µm x 1000 µm tall apical or epithelial channel from polydimethylsiloxane (PDMS;

Sylgard, Dow Corning) polymer. This size difference between channels is to reduce the shear

stress for Hepatocytes. A 50 µm thick PDMS membrane, containing 7 µm diameter circular

pores with 40 µm spacing, separates the channels (Fig. 5 left). Due to its biocompatibility and its

ability to be stretched the membrane was made from PDMS. Vacuum channels located on either

side of the main channels can be depressurized, deforming the membrane and the cells on it.

                                                                                                               4 Wyss chips are manufactured in house by the microfluidic engineering team.

Figure 5: Left, The chip is fabricated from 3 PDMS parts: bottom channel (200µm), top channel 1000 µm, and the flexible 50 µm porous membrane which is plasma bonded according to (D. Huh et al., 2013). Right, Wyss Organ Chip overview. Top left port is the top channel. Blue ports on the side indicate this chip’s membrane is stretchable (not positively indicated for hepatocyte culture).

                                                                                     

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Stretch was not used in this experiment, as it is not physiologically relevant. However, in the

Lung-On-A-Chip cell culture, the membrane is rhythmically deformed effectively stretching the

cells, simulating the stretching of the lungs in vivo.

Cell Culture (LSEC Expansion & Hepatocyte Co-Culture) Human primary LSECs (ACBRI 566; Cell-Systems Corporation CSC, Kirkland, WA),

were expanded in CSC Complete Medium (4Z0-500; CSC) supplemented with CultureBoost

(4CB-500; CSC), 1% Penicillin/Streptomycin (Pen/Strep; Gibco), and Fungin (ant-fn-1;

Invivogen, San Diego, CA).

Upon chip bonding, the tubing, reservoirs, and microfluidic channels were sterilized by

exposure to plasma for 10 min. 20 µg/mL Rat type I collagen5 (354236; Thermo Fisher Scientific

Inc., Waltham, MA) in channel-specific culture media was introduced into the channels and

incubated at 37 oC for 2 hours, after which the channels were washed with fresh medium to

remove excess collagen. Human primary hepatocytes (ACBRI 3716; CSC) were rapidly

defrosted from liquid nitrogen, centrifuged to remove cryogenic, and re-suspended in William’s

E Medium (WEM; Gibco) containing 10% Fetal Bovine Serum (FBS; Gibco), GlutaMAX-I

(A12860-01; Gibco), L-Ascorbic Acid, Insulin-Transferrin-Sodium Selenite (ITS; Thermo

Fisher), Dexamethasone, and Fungin. The hepatocytes (~35,000 cells/chip) were pipetted into

the top channel, forming a monolayer or simple epithelium. Hepatocytes sunk to the membrane

in 2-3 hours, but required 24-hour incubation at 37oC and 5% CO2 to form the monolayer. One

day later, the Hepatocytes were overlayed with a thin layer of Matrigel6 (Becton Dickinson,

Franklin Lakes, NJ) at 250 µg/ml in WEM without serum and incubated for 2-3 hours in the

                                                                                                               5 Rat collagen provides a cheap and more rigid alternative to human collagen. The ECM used should be considered a ‘starting point’ for cellular ECM manipulation; Cells will start secreting their own ECM once attached. While Liver ECM is complex, collagen I consistently achieves Hepatocyte attachment. 6 Matrigel is a commercially available formulated ECM. http://bd.com/resource.aspx?IDX=17841

                                                                                     

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incubator. LSECs were harvested at 90% confluence with 0.25% trypsin/EDTA (R001100;

Gibco) and subsequently seeded7 (100,000 cells/chip) in the bottom channel producing a

monolayer or simple endothelium. Effort was made to avoid cell aggregation and superposition,

by thoroughly mixing and carefully introducing cells to limit microfluidic turbulence or

obstruction. To ensure that the LSECs attached on the porous membrane, the organ chips were

flipped upside down and incubated at 37oC for 2 hours.

After the LSECs were fully adhered, a syringe pump (BS-8000; Braintree Scientific Inc.,

Braintree, MA) was used to continuously perfuse culture medium8 through both channels at a

constant flow rate (Fig. 6). Two syringe sizes, 1mL and 3mL (BD; Bedford, MA) for the apical

and basal channels respectively,

were used to create different shear

stresses across the membrane that

recapitulates the shear exposure in

the liver sinusoid. This was

calculated to be 0.0001 dyne/cm2 for

the top-channel and 0.008 dyne/cm2

for the bottom channel. Chips were

maintained by refreshing reservoirs

every other day until loss of viability.

A control study was carried out using static mono-cultures of primary human hepatocytes

in 24-well Collagen I plates. At 24 hours, Hepatocytes were ‘sandwiched’ with Matrigel.

                                                                                                               7 LSEC seeding is approximately 80% of monolayer confluence since this cell type is proliferative. 8 Hepatocyte maintenance culture (post day 1) medium contained no FBS (serum free). LSEC remained perfused with medium with serum.

Figure 6: Typical syringe-pump set up in incubator to withdraw media from the microfluidic devices (up to 8chips/shelf).

                                                                                     

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Cultures were maintained by refreshing 500µL of serum-free WEM every day. For consistency

this study was seeded with serum but maintained in serum-free medium as well.

KC Mono & Co-Culture Each human liver cell type has a unique media formulation (RPMI, WEM, CSC). KC-

LSEC media optimization was necessary to determine the compatibility/toxicity of the media

with both cell types before introducing the co-culture to microfluidic flow. Theoretically, since

they are very proximal in vivo in the non-parenchyma, they should exhibit the same

compatibility in vitro. Expanded LSECs were plated (100,000 cells/well) in 96-well plates and

cultured until a confluent monolayer was present. KC were rapidly defrosted from liquid

nitrogen, centrifuged to remove cryogenic, and re-suspended in cold 9 RPMI-1640 w/ L-

glutamine (R8758; Gibco) containing 10% Fetal Bovine Serum (Gibco), and 5% Pen/Strep

(Gibco), and plated at 1.5 x 105 cells/mL on top of the LSEC monolayer. KC-LSEC co-cultures

were cultured in 50:50 (RPMI 1640:CSC Complete Medium) that was refreshed every 24 hours.

Cultures were monitored via phase contrast microscopy for morphology.

Cell Viability & Functionality Test (LDH, Albumin, P450) Apoptosis and necrosis are two major forms of cell death. Although there are many

assays for detection of apoptosis, few assays measure necrosis. Permeabilization of the plasma

membrane is a key signature for necrosis (Chan, Moriwaki, & De Rosa, 2013). This can be

quantified by measuring the release of the intracellular enzyme lactate dehydrogenase (LDH).

An increase in tissue necrosis will correlate with decreased tissue viability overall. Waste media

was collected from hepatocyte-LSEC chip outlets and plates every 24 hours and analyzed for

LDH content using a cytotoxicity kit (Promega, WI). Positive (1:5000; LDH control in medium)

                                                                                                               9 KC are super ‘sticky’ and must be seeded in cold media to avoid clumping and sticking to the pipets and falcon tubes

                                                                                     

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and negative (Blank Media) controls were run and all samples were duplicated. Wells and

microchips were selected and perfused with lysis buffer 1X (Promega, WI) to simulate 100%

hepatotoxicity LDH for standardization.

Hepatic functionality was assessed in liver chips using Albumin production, and

metabolism of Testosterone, a P450 substrate. Albumin is the most abundantly produced serum

hepatic protein and contributes to oncotic10 pressure. Synthesis was analyzed11 in microfluidic

co-culture media using Albumin human ELISA Kit (ab108788; Abcam, Cambridge, UK), which

is specific for human Albumin12. Testosterone added to culture medium and activated hepatic

metabolism by cytochrome P450 (CYP) 3A4. P450 enzymes are a broad superfamily of mono-

oxygenases that are involved in drug and food metabolism. Specifically, CYP3A4 oxidizes small

foreign organic molecules (xenobiotics), such as toxins or drugs. This enzyme has been found to

metabolize roughly 55% of all pharmaceutical drugs, making it essential to study for drug

toxicity. Testosterone is metabolized to 6-betahydroxytestosterone by hepatocytes once

administered. Metabolite production was examined by performing liquid chromatography–mass

spectrometry on collected effluent at various time points during the two-week viability and on a

lot of fresh hepatocytes to simulate cellular metabolism post-isolation.

Morphological Analysis Cell images were recorded during culture using a Moticam 2500 camera (Motic China

Group Co., Ltd.) with imaging software (Motic images plus 2.0; Motic China Group Co., Ltd.)

on a Zeiss Axiovert 40CFL phase contrast microscope. Hepatocytes and KC are quite sensitive

to temperature and were handled minimally to produce the best viability.                                                                                                                10 Oncotic (colloid osmotic) pressure, is exerted by proteins, notably albumin, in a blood vessel's plasma (blood/liquid) and tends to pull water into the circulatory system. 11 Following the protocol found on http://www.abcam.com/albumin-human-elisa-kit-ab108788.html 12 FBS contains bovine Albumin, which has no cross-reactivity in this ELISA. ITS does contain Albumin but this is very small amount (negligible).

                                                                                     

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Results & Discussion Cell Viability Study Evaluation of cell viability was achieved via LDH assay. Plated monoculture hepatocytes

dropped from 12% LDH release to less than 5% LDH release (Fig. 7 right), indicating that the

culture stabilized after cellular isolation and seeding. This plate control is usually run to control

for lot variety, since some donors cells do not plate well. The plates jumped up to 15-20% LDH

release for the rest of the time course. This indicates increasing cellular stress on day 7 of

culture, as more LDH indicates more cellular membranes leeching cytosolic molecules. This has

commonly been accepted as the limit of culturing hepatocytes in a static monoculture.

Hepatocytes, co-cultured with LSECs, in organ chips (V2) were similarly assessed for

LDH release, and showed much greater LDH (>50% LDH release) directly after seeding (Fig. 7

left). On Day 4 the cultures had dropped to 4% LDH release, which did not increase until Day

11. This data supports similar culture stabilization as in the hepatocyte plates. The LDH values

are higher in the chips during the initial stabilization period due adaptation of the tissue in

response to microfluidic shear. The co-cultured LSECs consistently expressed low levels of LDH

(~5-20%). Since they are a proliferative cell type this LDH release may be due to tissue turnover

Figure 7: Cell Viability/Toxicity – Left, Liver-On-A-Chip V2 11 Day LDH time-course. Right, 10-Day LDH time-course with monoculture human primary hepatocytes in a 12-well plate.

                                                                                     

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in microfluidic culture. The cells must actively differentiate since they are seeded at 80%

confluence, and encounter more shear stress than hepatocytes. LDH is not actively transported

and with the layers of ECM there is essentially no crossing-over of LDH molecules between

channels13. In this study, the chips displayed a longer duration and better viability than the

hepatocyte monocultures indicating that microfluidic flow and co-culture with LSECs aided in

maintaining hepatocyte differentiation and tissue viability.

Second Cell Viability & Functionality Study I was able to assist in a second viability study conducted for DARPA14. Hepatocytes15

were co-cultured with LSECs, in organ chips (V2) were incubated in the same manner described

previously. Hepatocytes displayed extremely consistent LDH values (<5%) for the entire 14-day

time course. LSECs were similarly consistent displaying less than 1% LDH release during the

entire period (Fig. 8 left). This validates the results of the first experiment in which the culture is

stabilized by microfluidic flow and co-culture. The culture does not experience the variability

                                                                                                               13 LDH will pass through gaps in the cell monolayers, which can occur due to necrosis or mechanical (bubbles) or handling (seeding) variability. 14 Defense Advanced Research Projects Agency (DARPA) 15 This experiment was conducted with new different hepatocyte and LSEC lot from study in (Fig. 7).

Figure 8: Cell Viability & Synthesis – Left, 14 Day Albumin synthesis time course with co-culture V2 liver chips. Right, 14 Day LDH time course with co-culture V2 liver chips.

                                                                                     

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previous experiment due to the hepatocytes and LSECs coming from different donors. This

stability can be attributed to variability in plating between commercial cell lots or seeding

technique.

Albumin was assessed to determine the functionality and synthetic ability of the

hepatocytes in V2 liver chips. Albumin production started low in the chips but increases steadily

during the first week of culture. While dropping in the second week the synthesis remains about

10 µg/day/million cells (Fig. 8 right). A 1997 study, using human blood and liver samples found

human Albumin production to be 109 +/- 21 µg/day/g body weight (Barle et al., 1997). If the

average gram of liver tissue produces 5-12 million hepatocytes then a million cells should

produce ~6-15 µg/day (Rudo, Meyers, Dauterman, & Langenbach, 1987). While this calculation

is simple and may not be entirely accurate, it is an indicator that the Liver-On-A-Chip produces a

comparable Albumin production per cell to hepatocytes in vivo.

Figure 9: Cell Functionality – 14 Day comparison of new and old liver chips (for optimization) and a culture of fresh hepatocytes.

                                                                                     

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Lastly, the newly optimized liver chip (V2) was compared with the original version16

(V1) for CYP3A4 activity using Testosterone as a P450 substrate (Fig. 9). Fresh hepatocytes,

analyzed directly after isolation/storage, produced 750 pmol/min/million cells, which was used

as a control. The liver chip V1 produced far less metabolite <400 pmol/min/million for the first

week and <600 pmol/min/million cells in the second week of culture. The optimized (V2) liver

chip produced close to 600 pmol/min/million cells in the first week, and reached >1200

pmol/min/million cells in the second week of culture indicating that the optimized chip is much

better at increasing CYP3A4 expression. The V1 chip has a 10x difference in channel size,

having drastic effects on the shear stress the cells see in the channel. Liver chip V1 decreased

performance may be interpreted that the levels of shear stress exerted on the hepatocytes may

have been above an optimal level. As a particularly sensitive cell type, hepatocytes require less

shear stress than LSEC’s in vitro. Increasing channel volume and coating the hepatocytes with

ECM both serve to decrease the hepatocyte exposure to fluidic shear.

Experimental Limitations While the microchip is robust, displaying LSEC fenestrae basally and bile canaliculi

apically, issues can arise including but not limited to bubbles, misalignment and delamination.

These can lead to failure of the device and loss of tissue viability. The impurity of primary

human cell lots is another issue that makes studying these cultures difficult. A 2005, study found

that human hepatocyte isolates were consistently contaminated by low number of NPCs (Łaba,

Ostrowska, Patrzałek, Paradowski, & Lange). This means that most studies that have looked at

hepatocytes have unknowingly cultured KC and other NPCs. Similar results were observed in

this experiment by finding moderate amounts of KC contamination in hepatocyte monocultures

                                                                                                               16 Liver chip V1 – epithelial channel was 100 µm tall (V2 = 1000 µm), producing much higher shear stress.

                                                                                     

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and microfluidic co-cultures. Furthermore, when primary human KC monoculture culture was

attempted, LSECs appeared to be present in culture as well indicating that these cultures cannot

be considered complete monocultures. Day 1 KC-LSEC co-culture in 96-well plates revealed

that KC maintained a “balled-up” morphology. On Day 2, while some KC had detached, many

remained and would not flatten out (Fig. 12 middle). Although the desirable concentration was

achieved in culture, KC morphology must be assessed to determine their level of activation.

Between isolation and tissue analysis in vitro, cells are exposed to additional cellular stress.

Moreover, while human primary cells are commercially available now, expanding, culturing, and

differentiating them takes time. This experiment attempted to look at a very complex question

about intricate liver physiology. KC integration to liver tissue culture models requires much

more experimentation before any benefit should be considered conclusive.

Conclusion

The human Liver-On-A-Chip microdevice provides a controlled environment to examine

and challenge critical hepatic functions in the presence of relevant physiological cues, fluidic

shear, and co-culture with LSECs. Comparison of the device with static cultures revealed that

Figure 12: Left, KC cultured in static monoculture change morphology and ‘flatten out’ on the rigid plate surface. Middle, D1 KC-LSEC co-culture in 96-well plates revealed that KC maintained a “balled-up” morphology. LSEC monolayer remained intact during culture although ‘clumping’ was observed. Right, D2 KC-LSEC co-culture in 96-well plates reveals much KC detachment, and the attached ones are still ‘balled-up’.

                                                                                     

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recapitulating fluid flow experienced in the liver in vivo is sufficient to promote longer organ

level function in vitro. The improved function may be due to removal of waste products or

maintenance of nutrient delivery to these highly metabolically active hepatocytes. A comparative

study of V1 liver chip and V2 Liver chip found that sheer stress is good but too much sheer

stress is definitely bad for Hepatocytes. The chip effectively models complex organization such

as separation of Hepatocytes and LSECs by the Space of Disse (membrane). In order to account

for the variability of LSEC co-culture this experiment must be repeated using LSECs and

Hepatocytes in co-culture Trans-well static culture models. This control will more accurately

reveal the effect of shear.

In the future, it is necessary to determine the purity of commercial human primary cell

isolates via immunofluorescence. It is also possible, that the composition of the liver in vivo

could be used advantageously to create more accurate tissue in vitro, by allowing the tissue to

self-organize17. This could be done in a method similar to PCTS (Fig. 1) to produce a sinusoid

and comparable cellular composition. To better interpret the co-culture results of this experiment,

it is necessary to detail inflamed/non-inflamed KC phenotypes. If KC can be introduced in a

controlled fashion the device may become an essential platform not only for drug screening and

toxicology testing but also for inflammation and chronic infections (malaria & hepatitis). KC-

LSEC co-culture was somewhat beneficial to retaining a healthy tissue in culture, as monoculture

experienced rapid dedifferentiation and necrosis. Further media optimization is necessary to

improve this co-culture. It is vital to continue exploring whether KC can benefit from exposure

to microfluidic sheer stresses in the Liver-On-A-Chip as LSEC do. Once established, Liver-On-

A-Chip could prove useful for modeling chronic and acute inflammatory diseases of the liver.

                                                                                                               17 This is somewhat visible in Insphero’s 3D spheroid culture method (Fig. 3 left)

                                                                                     

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Acknowledgements

I would like to thank Don Ingber, PI and Founding Director of the Wyss, for the

wonderful opportunity to work in his lab and for providing all the resources and funding that

made this project possible. I would like to thank all of my mentors and friends from the last 3

years18: at Wyss Lori McPartlin, Jake Fraser, Carol Luccesi, Kambez Benam, Joshua Resnikoff,

Payal Patel, and Tony Bahinski for their guidance and insight. I thank my past professors at

Brandeis University for inspiring my curiosity and enlightening me over the past 4 years. I also

thank my family and friends who have supported me at home and abroad.

Ultimately, I sincerely thank my thesis committee, Joan Press, Neil Simister, and Tony

Bahinski for their patience, understanding, and interest in my project.

                                                                                                               18 Worked on Kidney-On-A-Chip (Summer 2012), Lung-On-A-Chip (2012-2014), Liver-On-A-Chip (2014-2015)

                                                                                     

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