layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Massachusetts General Hospital, Harvard Medical School

Shriners Hospitals for Children-Boston

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Supercooling as a viable non-freezing cell preservation method of rat hepatocytes.

We demonstrate that a polymer confined to a narrow channel migrates towards the center when driven by an external force parallel to the channel walls. This migration results from asymmetric hydrodynamic interactions between polymer segments and the confining walls. A weak pressure-driven flow, applied in the same direction as the external force, enhances the migration. However, when the pressure gradient and the external force act in opposite directions the polymer can migrate towards the boundaries. Nevertheless, for sufficiently strong forces the polymer always migrates towards the center. A dumbbell kinetic theory explains these results qualitatively. A comparison of our results with experimental measurements on DNA suggests that hydrodynamic interactions in polyelectrolytes are only partially screened. We propose new experiments and analysis to investigate the extent of the screening in polyelectrolyte solutions.

Transverse migration of a confined polymer driven by an external force.

Using computational modeling, we simulate the motion of compliant microcapsules on patterned surfaces. The microcapsules, which consist of an elastic shell and an encapsulated fluid, model biological cells or polymeric particles. We focus on a surface that is decorated with a Y-shaped pattern. As compared to the stem of the Y, one branch is relatively soft, and the other branch is relatively sticky. The capsules are driven to move over this substrate by an imposed fluid flow. Upon reaching the junction point, we find that deformable capsules preferentially move onto the sticky branch and stiffer capsules move onto the soft branch. Thus, through their inherent interactions with the patterned domains, the microcapsules are driven to "make decisions" about their path along the surface. Such surface patterning provides a facile means of routing particular capsules to specified locations in microfluidic devices and can form a fundamental component in creating fluidic circuits where microcapsules carry out simple logic operations.

Fork in the road: patterned surfaces direct microcapsules to make a decision.

Using computational modeling, we simulate the fluid-driven motion of microcapsules on patterned surfaces to establish guidelines for creating simple microfluidic devices for bioassays and multistage chemical reactions. The microcapsules, which consist of an elastic shell and an encapsulated fluid, model biological cells or polymeric particles. We focus on patterned substrates that encompass chemically adhesive and mechanically compliant domains. By probing the interactions between the microcapsules and these patterned surfaces, we determine the factors that control the movement of the capsules along the substrates. Using this information, we optimize the arrangement of the adhesive and compliant surface domains to create robust systems that effectively discriminate between various soft particles moving through the microchannels and "autonomously" direct certain species to specific locations. These findings could facilitate the fabrication of low-cost, portable microfluidic devices for sorting cells or performing fundamental chemical studies.

Designing patterned substrates to regulate the movement of capsules in microchannels.

Using simulation and theory, we demonstrate how nanoparticles can be harnessed to regulate the interaction between two initially stationary microcapsules on a surface and promote the self-propelled motion of these capsules along the substrate. The first microcapsule, the "signaling" capsule, encases nanoparticles, which diffuse from the interior of this carrier and into the surrounding solution; the second capsule is the "target" capsule, which is initially devoid of particles. Nanoparticles released from the signaling capsule modify the underlying substrate and thereby initiate the motion of the target capsule. The latter motion activates hydrodynamic interactions, which trigger the signaling capsule to follow the target. The continued release of the nanoparticles sustains the motion of both capsules. In effect, the system constitutes a synthetic analogue of biological cell signaling and our findings can shed light on fundamental physical forces that control interactions between cells. Our findings can also yield guidelines for manipulating the interactions of synthetic microcapsules in microfluidic devices.

Modeling microcapsules that communicate through nanoparticles to undergo self-propelled motion.

We investigate the effects of polymer chains and nanoparticles on the deformation of a droplet in shear and extensional flow using computational modeling that accounts for both the solid and fluid phases explicitly. We show that under shear flow, both the nanoparticles and the encapsulated polymers reduce the shear-induced deformation of the droplet at intermediate capillary numbers. At high capillary numbers, however, long polymer chains can induce the breakup of the droplet. We find that the latter behavior is dependent on the nature of the imposed flow. Specifically, under extensional flow, long polymers inhibit the droplet breakup and reduce deformation. Overall, the findings provide guidelines for tailoring the stability of filled droplets under an imposed flow, and thus, the results can provide useful design rules in a range of technological applications.

Shear and extensional deformation of droplets containing polymers and nanoparticles.

Using theory and simulation, we design a "train" of N microcapsules that undergoes self-sustained, directed motion along an adhesive surface in solution. The motion is initiated by the release of nanoparticles from a single "signaling" capsule at one end of the train. The released nanoparticles can bind to the underlying surface and thereby induce an adhesion gradient on the substrate. Through the combined effects of the self-imposed adhesion gradient and hydrodynamic interactions, the N microcapsules autonomously move in single file toward the region of greatest adhesion. At late times, this train reaches a steady-state velocity U, which decreases with train length as N(-1/2). We calculate the maximum length for which the train maintains this cooperative, autonomous motion.

Self-sustained motion of a train of haptotactic microcapsules.

We use computational modeling to capture the three-dimensional interactions between oscillating, synthetic cilia and a microscopic particle in a fluid-filled microchannel. The synthetic cilia are elastic filaments that are tethered to a substrate and are actuated by a sinusoidal force, which is applied to their free ends. The cilia are arranged in a square pattern, and a neutrally buoyant particle is initially located between these filaments. Our computational studies reveal that, depending on frequency of the beating cilia, the particle can be either driven downward toward the substrate or driven upward and expelled into the fluid above the cilial layer. This behavior mimics the performance of biological cilia used by certain marine animals to extract suspended food particles. The findings uncover a new route for controlling the deposition of microscopic particles in microfluidic devices.

Designing oscillating cilia that capture or release microscopic particles.

We model the transport of a microscopic particle via a regular array of beating elastic cilia, whose tips experience an adhesive interaction with the particle's surface. At optimal adhesion strength, the average particle velocity is maximized. Using simulations spanning a range of cilia stiffness and cilia-particle adhesion strength, we explore the parameter space over which the particle can be "released", "propelled", or "trapped" by the cilia. We use a lower-order model to predict parameters for which the cilia are able to "propel" the particle. This is the first study that shows how both stiffness and adhesion strength are crucial for manipulation of particles by active cilia arrays. These results can facilitate the design of synthetic cilia that integrate adhesive and hydrodynamic interactions to selectively repel or trap particulates. Surfaces that are effective at repelling particulates are valuable for antifouling applications, while surfaces that can trap and, thus, remove particulates from the solution are useful for efficient filtration systems.

Propulsion and trapping of microparticles by active cilia arrays.

The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug-induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information, and their shortcomings have resulted in "silent" hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that (a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells, and endothelial cells; and (b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion, and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.

In vitro platforms for evaluating liver toxicity.

The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or in 'sandwich' configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretions and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures show formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete a higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain a well-connected cellular network and a differentiated function.

Dynamic interplay of flow and collagen stabilizes primary hepatocytes culture in a microfluidic platform.

The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen "double gel" or "overlay". To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.

A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture.

The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.

Supercooling enables long-term transplantation survival following 4 days of liver preservation.

Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

Article	Year
Supercooling as a viable non-freezing cell preservation method of rat hepatocytes. PloS one\| PubMed ID: 23874947	2013
Transverse migration of a confined polymer driven by an external force. Physical review letters\| PubMed ID: 17359205	2007
Fork in the road: patterned surfaces direct microcapsules to make a decision. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 17880118	2007
Designing patterned substrates to regulate the movement of capsules in microchannels. The Journal of chemical physics\| PubMed ID: 18570531	2008
Modeling microcapsules that communicate through nanoparticles to undergo self-propelled motion. ACS nano\| PubMed ID: 19206572	2008
Shear and extensional deformation of droplets containing polymers and nanoparticles. The Journal of chemical physics\| PubMed ID: 19548754	2009
Self-sustained motion of a train of haptotactic microcapsules. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 19705878	2009
Designing oscillating cilia that capture or release microscopic particles. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 19799457	2010
Propulsion and trapping of microparticles by active cilia arrays. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 22233228	2012
In vitro platforms for evaluating liver toxicity. Experimental biology and medicine (Maywood, N.J.)\| PubMed ID: 24764241	2014
Dynamic interplay of flow and collagen stabilizes primary hepatocytes culture in a microfluidic platform. Lab on a chip\| PubMed ID: 24770663	2014
A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture. Technology\| PubMed ID: 24932459	2014
Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nature medicine\| PubMed ID: 24973919	2014
Polyethylene glycol protects primary hepatocytes during supercooling preservation. Cryobiology\| PubMed ID: 25936340	2015

O. Berk Usta

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