M3P.073
LIVER ON A CHIP: ENGINEERING THE LIVER SINUSOID Y.B. Kang1*, T.R. Sodunke1, J. Cirillo1, M.J. Bouchard2, and H. Noh1 1 Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA, USA 2 Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, USA The liver lobule consists of operational units termed the liver sinusoid (Figure 1), where most of the liver activities take place. The hepatic sinusoid is composed of hepatocytes, LSEC, Kupffer cells, stellate cells, blood sinusoid and bile duct [10,11]. Our goal is to generate an accurate human liver model that closely mimics the liver sinusoid. For this purpose we have designed, fabricated, and tested three microchannel configurations (Figure 1).
ABSTRACT We have developed a novel microfluidic device using poly-dimethylsiloxane (PDMS) microchannels for long term, layered co-culture of primary rat hepatocytes (PRH) and endothelial cells (EC) to mimic the liver sinusoid. Three microfluidic configurations were investigated as shown in Figure 1. In configuration 1, where PRH and rat adrenal medullary endothelial cells (RAMEC) were cocultured with matrigel in layers in a single PDMS microchannel, cells remained viable for 30 days. In configuration 3, where PRH and liver sinusoidal endothelial cells (LSEC) were cultured on the opposite sides of the microporous membrane between two microchannels under static and dynamic conditions, PRH retained their viability and normal morphology up to 35 days and 63 days, respectively. Thus, our novel liver models that closely mimic the liver sinusoid have been proven to facilitate long-term layered co-cultures of PRH and EC.
The Liver Sinusoid
KEYWORDS
Microfluidic Platforms
Liver model, liver on a chip, liver co-culture, long term culture, primary hepatocytes, microfluidic platform.
LSEC
INTRODUCTION
Matrigel
Liver biology/disease studies and drug discovery/ screening research predominantly rely on cell culture models [1-3]. While much progress has been made during the past two decades in prolonging liver cell viability and maintaining liver functions in vitro, there are still no authentic liver models that accurately represent the architecture and functions of human liver tissue, thereby limiting advances in liver-related research and drug development. Recently, microfabrication and microfluidic technologies have been applied to the development of in vitro liver models that can allow control of the cellular micro-environment. Micropatterned elastomeric PDMS stencils in industry-standard multiwell format have been presented for long term co-culture of primary human hepatocytes and fibroblasts under static condition [4]. Powers et al developed a micro-array bioreactor with a micro-filter for three-dimensional culture of liver cells that enables the formation of hepatocellular aggregates reminiscent of liver structures [5]. Lee et al reported a microfluidic device that mimics the structure of the endothelial–epithelial interface that forms the liver sinusoid and used this device to replace the need for endothelial cells [6]. Lastly, a flat-plate microchannel with a groove pattern was presented in order to minimize shear stress exerted on the cells exposed to continuous perfusion [7]. However, none of the previously reported liver models have shown long term co-culture of primary liver cells in an in vivo-like organizations [4-10].
978-1-4673-5983-2/13/$31.00 ©2013 IEEE
Mini-liver Bioreactor
Configuration 1 Micro filter
Pump
Hepatoctyes
Configuration 2
Membrane Configuration 3
Bile
Bile collection
Pump
Micro filter
Flow rate (oxygen, shear stress) Medium Kupffer LSEC Hepatocyte Bile
Figure 1: The liver sinusoid (from www.akaikelab.bio.titich.ac.jp) and microfluidic platforms that mimic the liver sinusoid. Three configurations have been designed, fabricated, and tested. The key elements of these models are: 1) layered coculture of hepatocytes and LSEC and 2) microchannels that simulate blood sinusoid and bile duct. Configuration 1 is the simplest configuration in which primary hepatocytes and EC are co-cultured in layers in a single channel. A thin layer of matrigel is deposited between two cell layers. Configuration 2 adds a bile duct to Configuration 1. A microporous membrane is placed between two microchannels and cells are cultured in layers in the top channel. In Configuration 3, the two cells are physically separated by culturing them on the opposite sides of the microporous membrane. In configuration 1 and 3, we achieved the layered
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Transducers 2013, Barcelona, SPAIN, 16-20 June 2013
long term co-colculture of cells using PDMS microchannels under static condition. We further developed a dynamic culture model (continuous perfusion) based on configuration 3. PRH retained a stable level of urea synthesis up to 63 days. We believe that this liver model more closely mimics the liver sinusoid than previously reported liver models [4-10].
Layered co-culture of primary rat hepatocytes and endothelial cells To create the layered co-culture of PRH and EC in configuration 1 and 2, PRH were first plated in collagen coated PDMS microchannels. 30% (v/v) growth factor reduced matrigel (BD Biosciences) that was mixed with PRH culture media was placed on the PRH layer. 2 hours after matrigel coating EC were plated on top of matrigel to create a co-culture of PRH and EC separated by matrigel. Cells were incubated at 37°C in 5% CO2. For a layered co-culture of PRH with EC in configuration 3, both sides of PDMS microchannel membrane were first coated with rat-tail collagen. We then cultured PRH on one side of the membrane for a minimum of 3 hours. After the attachment of PRH, EC were then plated on the other side of the membrane. To visualize PRH and EC in the layered co-culture, the PRH were infected with recombinant adenoviruses that express Green Fluorescent Protein (Ad-GFP).
MATERIAL AND METHOD Construction of microfluidic cell culture platforms PDMS microchannesl were fabricated using replica molding technique. Master molds were fabricated by either SU-8 photolithography or stereolithography techniques (Figure 2). PDMS Channels were 80-1000 µm in height, 1 mm in width, and 15-17 mm in length. For configuration 1, PDMS channel was placed on collagencoated culture dish. For configuration 2, PDMS channel was placed on transwell (microporous membrane). For configuration 3, two PDMS channels were bonded together with a microporous membrane in the middle after oxygen plasma treatment. For dynamic culture, the fabricated PDMS dual microchannel was connected to a syringe pump that can feed medium to cells with a flow rate of 30-40 µl/hr as shown in Figure 1. In order to maintain sterile conditions, a 0.2 µm sterile filter was placed upstream of the channel inlets. Microchannel inlets were designed to feed medium in a counter direction for each channel. The outlet of microchannel was connected to a waste bottle with a silicon tube. Multiple valves were located in the middle of the outlet line for sampling the medium.
Assessment of hepatocellular function Hepatocyte function was evaluated by assessing the level of urea synthesis [13-15]. Medium samples from cell cultures were collected every 2-3 days and stored at 80oC until assayed. Urea concentration was quantified using a colorimetric endpoint assay utilizing diacetylmonoxime with acid and heat (Urea Nitrogen, Stanbio Labs). The urea concentration in the medium was calculated by subtracting the urea concentration value of control medium from the urea concentration value measured from the medium sample from the hepatocytes culture.
RESULTS AND DISCUSSION Primary rat hepatocytes and endothelial cells cocultured under static condition in configuration 1 In the first configuration, a layered co-culture of PRH with EC separated by matrigel was created in a PDMS microchannel. Figure 3 shows PRH and RAMEC cocultured in configuration 1 under static condition. Configuration 1 LSEC Matrigel RAMEC
PRH (Day 30)
Hepatoctyes Ad-GFP Positive PRH
Figure 2: Construction of microfluidic cell culture platforms.
Primary cells isolations
Figure 3: Co-culture in Configuration 1. PRH and RAMEC cultured in layers in a microchannel.
PRH and LSEC were isolated from 6-12 weeks old Sprague-Dawley rats as previously described [11-12]. Established cell lines such as RAMEC and bovine aortic endothelial cells (BAECs) were also used because they were more readily available. Cells were cultured in both static and dynamic culture conditions. For static culture, medium was replaced every 24 hrs.
In order to verify the viability of PRH that were cocultured with RAMEC, we used adenoviruses that were expressed green fluorescent proteins to infect PRH. PRH retained normal morphology and remained viable for more than 30 days. Expression of GFP indicates that these cells are viable. However in this configuration; it was
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the PRH were separated from the EC layer by a rigid microporous membrane. Consequently, configuration 3 was more suitable than configuration 1 for long term coculture of PRH and EC.
difficult to observe whether a homogenous thin layer of matrigel is formed between the PRH and EC, separating the two cell types.
Primary rat hepatocytes and endothelial cells cocultured under static condition in configuration 2
Primary rat hepatocytes and endothelial cells coculture under dynamic condition in configuration 3
Co-culture with PRH and EC under static condition was attempted in the PDMS microchannel using configuration 2. However, similar to configuration 1, it was difficult to observe whether the monolayer formed by matrigel was of uniform thickness. Furthermore, the EC on the matrigel layer invaded into the hepatocytes layer plated on the bottom [12].
PRH was placed on the bottom of the membrane and LSEC was placed on the top of membrane of PDMS dual microchannel as shown in configuration 3. After PRH and LSEC were allowed to adhere to the surface, the PDMS microchannel was connected to syringe pump and waste collection device. In the dynamic culture condition with a flow rate of 30-40 µl/hr, hepatocytes formed a confluent monolayer and remained viable up to 63 days (Figure 5). PRH retained normal morphology for up to 63 days.
Primary rat hepatocytes and endothelial cells cocultured under static condition in configuration 3 PRH were placed on the bottom of the membrane and LSEC were placed on the top of the membrane of the PDMS microchannel in configuration 3 under static condition. Figure 4 shows the morphology of PRH infected with AdGFP at Day 20. PRH retained their viability and normal morphology up to 35 days. However, it was observed that the PRH layer peeled off from the edge of cell monolayer with long culture time. The shear stress induced during medium replacement with a micropipette may be the reason for this phenomenon.
PRH (Day 21)
PRH (Day 40)
PRH (Day 63)
Configuration 3
Figure 5: Co-culture in Configuration 3. PRH and LSEC cultured in a dual channel device under dynamic condition. PRH (Day20)
Ad-GFP PRH (Day 20)
Analysis of hepatocytes function Urea synthesis function of hepatocytes was analyzed in order to confirm hepatocyte-specific functions. Urea synthesis in single culture of hepatocytes in microchannel initially increased but then decreased to a very low amount of urea for the remaining two weeks (data not shown); we were unable to assess urea synthesis beyond two weeks due to extensive cell death of hepatocytes single culture system.
Figure 4: Co-culture in Configuration 3. PRH and LSEC cultured in a dual channel device under static condition for 20 days (LSEC labeled with mito-tracker red and PRH with Ad-GFP). From the experimental results of PRH co-cultured with EC under static condition, we achieved long term layered co-culture in configuration 1 and 3. Configuration 1 has one inlet and one outlet for medium supply of both PRH and EC in a single microchannel (fig. 1). In that case, the analyzed PRH medium samples could be affected by EC because secretion from PRH will be mixed with the secretion of EC into the same medium. In addition, it is difficult to isolate PRH from co-culture with EC owing to the co-existence of PRH and EC without rigid segregation in these single microchannels. Finally, EC intrusion into PRH layer also remains a potential problem. On the other hand, configuration 3 has the separated two inlets and two outlets for medium supply of each channel (fig. 1). The samples from PRH secretion are therefore segregated from the secretion of EC. Finally, PRH can be harvested from co-culture with EC because
Figure 6: Urea synthesis test for long term co-culture hepatocytes with EC in the PDMS microchannel under dynamic condition. The level of urea synthesis in the PRH-EC co-culture system increased at early time points but eventually
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Sah, R. L., DeLong, S., West, J. L., Bhatia, S. N., FASEB J. 21, 790–801 (2007). [16] Nicola J. Hewitt, Drug Metabolism Reviews, 39: 159–234, 2007. [17] D. Runge, D. M. Runge, D. Ja¨ger, K. A. Lubecki, D. B. Stolz, S. Karathanasis, T. Kietzmann, S. C. Strom, K. Jungermann, W. E. Fleig, and G. K. Michalopoulos, Biochemical and Biophysical Research Communications 269, 46–53 (2000). [18] Jing Shan, Kelly R. Stevens, Kartik Trehan, Gregory H. Underhill, Alice A. Chen, Sangeeta N. Bhatia, Hepatic tissue engineering.
remain stable for up to 35 days in static culture (data not shown) and 61 days in dynamic culture in configuration 3 (figure 6). PRH co-cultured with EC retained their morphology and viability for over 30 days, which is difficult to achieve when hepatocytes are cultured in the absence of other liver cells [3,16-18]. This demonstrates that EC is essential for long term culture of PRH.
CONCLUSION In order to mimic the liver sinusoid in vitro, PRH were co-cultured with EC in PDMS microchannels according to three different configurations. Long term, layered co-culture of PRH and RAMEC was successfully achieved for 30 days in configuration 1, where a thin layer of matrigel is placed between two cell layers in a single PDMS microchannel under static condition. In configuration 3, where PRH were co-cultured with LSEC on the opposite sides of the microporous membrane between two microchannels under static and dynamic conditions, PRH retained their normal morphology up to 35 days and 63 days, respectively. Thus, we have developed novel liver models that closely mimic the liver sinusoid and demonstrated longterm co-cultures of primary hepatocytes and endothelial cells. These engineered liver sinusoids can find numerous applications in liver-related research and drug development.
CONTACT * Young Bok(Abraham) Kang, tel: +1-215-895-2174;
[email protected]
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