Ryo Sudo

Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

Microfluidic platforms for studies of angiogenesis, cell migration, and cell-cell interactions. Sixth International Bio-Fluid Mechanics Symposium and Workshop March 28-30, 2008 Pasadena, California.

Recent advances in microfluidic technologies have opened the door for creating more realistic in vitro cell culture methods that replicate many aspects of the true in vivo microenvironment. These new designs (i) provide enormous flexibility in controlling the critical biochemical and biomechanical factors that influence cell behavior, (ii) allow for the introduction of multiple cell types in a single system, (iii) provide for the establishment of biochemical gradients in two- or three-dimensional geometries, and (iv) allow for high quality, time-lapse imaging. Here, some of the recent developments are reviewed, with a focus on studies from our own laboratory in three separate areas: angiogenesis, cell migration in the context of tumor cell-endothelial interactions, and liver tissue engineering.

Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments.

Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory.

In vitro 3D collective sprouting angiogenesis under orchestrated ANG-1 and VEGF gradients

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.

Surface‐Treatment‐Induced Three‐Dimensional Capillary Morphogenesis in a Microfluidic Platform

Angiogenesis and capillary morphogenesis denote the growth of microvessel sprouts, which has an essential role in development, reproduction, and repair, but also occurs in tumor formation and in a variety of diseases. Since the first in vitro angiogenesismodel waspresentedbyFolkman&Haudenschild,manyhaveattemptedto develop assays that bettermimic the true in vivo situation. Recent research efforts that are described in review references have provided new insights into the cellular andmolecularmechanisms ofangiogenesis,asforexample,intermsoftheroleofrecombinant humanvascularendothelialgrowthfactor(VEGF)andmechanical stresses. However, there remains a need for quantitative angiogenic assays and 3D models providing a more physiologically relevant microenvironment of living tissues in vivo compared to previous 2D culture models. Tissue engineering applicationsusedtorestore,maintainorenhancetissuesandorgans further require a better understanding of 3D structures and responses of cells. To meet the needs for 3D models, assays adapting hydrogel scaffolds, stack-up method or endothelial cell plated beads were introduced. However, these studies could not produce well controlled microenvironments with dimensions similar to the relevant tissue structures in vivo. The continuing need for technologies that recognize, quantify andperturb localized cellular morphogenesis was also expressed in a recent study of cell migration. Griffith and Swartz suggested using microfluidic approaches for capturing complex 3D tissue physiology and mimicking in vivo conditions in vitro. Microfluidics offer the possibility of applying spatially and temporary controlled gradients of chemical factors in culture and quantifying their responses. Many research groups have presented robust assays to investigate migration of neutrophils, leukemia cells, stem cells, bacteria and cancer cells under varying chemical gradients in microfluidic channels. These studies applied controlled chemical gradients to the cells in a microfluidic channel, reporting the responses of cells migrating on the channel surface or suspended in medium. In other studies, hydrogel scaffolds were cast in microfluidic channels in order to mimic the extracellular matrix (ECM). Molecular diffusion across these scaffolds was analyzed under flow conditions and cells, i.e., neutrophils, hepatocytes, carcinoma cell lines, astrocytes, and macrophage-like cells, were cultured within the hydrogel scaffold under linear chemical gradients to study their migration. Kim et al. and Toh et al. also reported that hepatocytes formed 3D structures when they were mixed and cultured within a hydrogel scaffold. Three-dimensional capillary morphogenesis into these scaffolds mimicking in vivo behavior, however, has not yet been realized in a microfluidic platform, nor has the sprouting of endothelial cells into hydrogel scaffold forming in vivo like 3D capillary structures. (The different modes of cell migration previously observed in microfluidic platforms are categorized in Supporting Information Table 1.) Microfluidic devices made of hydrogel can be expected to induce 3D responses of the cells seeded in the channel into the surrounding hydrogel. However, they reported limited cellular morphogenesis into the hydrogel due to handling difficulties and the need for the hydrogel to serve as the primary structural material of the device. We previously reported the formation of vascular networks inside a collagen scaffold between microfluidic channels. One aspect of these structures, however, was non-physiologic. Rather than forming vessels directly, the endothelial cells plated on the walls of the channel or gel often migrated into the gel as a sheet and initially remained attached to the side walls of the gel chamber (Supporting Information Table 1; 2.5D, migrating cells). These planar structures eventually formed lumens and bridged across the gel region but the process by which these networks formed, initially as sheets adherent to an artificial channel surface, differs from the process of capillary network formation in vivo. Here we introduce a new surface treatment for the microfluidic platform to induce physiologically relevant 3D capillary morphogenesis. (see Fig. 1a for a schematic of the developed microfluidic concept and Supporting Information Fig. 1 for a schematic of a three-channel microfluidic device used as an example. Device preparation details are provided in the Experimental section and Supporting Information Fig. 2.) Endothelial cells were seeded and cultured in one microfluidic channel (cell channel) in direct contact with hydrogel scaffolds. After 12 h, a continuous endothelial monolayer (EC monolayer) formed in the cell channel (Supporting Information Fig. 3) and a growth factor gradient was established from the condition channel to induce the cells to migrate toward the opposing channels through the scaffolds, more on the condition side than on the control side. We show here that surface treatment on the

Reconstruction of 3D stacked-up structures by rat small hepatocytes on microporous membranes

The three‐dimensional (3D) culture of hepatocytes is essential for the reconstruction of functional hepatic tissues in vitro. In the present experiment, we developed a 3D‐culture method in order to reconstruct hepatic cordlike structures by stacking up two‐dimensional (2D) tissues composed of rat small hepatocytes (SHs), which are hepatic progenitor cells. Pairs of membranes were prepared and the cells were separately cultured on each membrane. After the SH colonies had developed, one membrane was inverted on top of the other to form an SH bilayer. Thereafter, we investigated whether the stacked cells were organized into differentiated tissues. In the 3D stacked‐up structures, bile canaliculi (BC) started to form and gradually developed into anastomosing networks. Transmission electron microscopy revealed that the SHs of the upper and lower layers adhered to one another, and that BC formed between them. Bile canalicular proteins localized on the lumina of the tubular structures. Furthermore, the cells within the structures exhibited mRNA transcription of the hepatic‐differentiation markers and maintained a relatively high level of albumin secretion. We conclude that highly differentiated 3D tissues, including functional BC, can be reconstructed by stacking up layers of SHs. This 3D stacked‐up culture is useful for the reconstruction of tissue‐engineered livers.

Intra-aneurysmal hemodynamic alterations by a self-expandable intracranial stent and flow diversion stent: high intra-aneurysmal pressure remains regardless of flow velocity reduction

Object Little is known about how much protection a flow diversion stent provides to a non-thrombosed aneurysm without the adjunctive use of coils. Methods A three-dimensional anatomically realistic computation aneurysm model was created from the digital subtraction angiogram of a large internal carotid artery-ophthalmic artery aneurysm which could have been treated with either a neck bridging stent or a flow diversion stent. Three-dimensional computational models of the Neuroform EZ neck bridging stent and Pipeline embolization device were created based on measurements with a stereo-microscope. Each stent was placed in the computational aneurysm model and intra-aneurysmal flow structures were compared before and after placement of the stents. Computational fluid dynamics were performed by numerically solving the continuity and Navier–Stokes momentum equations for a steady blood flow based on the finite volume method. Blood was assumed as an incompressible Newtonian fluid. Vessel walls were assumed to be rigid, and no-slip boundary conditions were applied at the lumens. To estimate the change in the intra-aneurysmal pressures we assumed that, at the inlets, the intra-arterial pressure at peak systole was 120 mm Hg both before and after stent placement Results Without any stent, the blood flow entered into the aneurysm dome from the mid to proximal neck area and ascended along the distal wall of the aneurysm. The flow then changed its direction anteriorly and moved along the proximal wall of the aneurysm dome. In addition to the primary intra-aneurysmal circulation pattern, a counterclockwise vortex was observed in the aneurysm dome. The placement of a Neuroform EZ stent induced a mean reduction in flow velocity of 14% and a small change in the overall intra-aneurysmal flow pattern. The placement of a Pipeline device induced a mean reduction in flow velocity of 74% and a significant change in flow pattern. Despite the flow velocity changes, Neuroform EZ and Pipeline devices induced reductions in intra-aneurysmal pressure of only 4 mm Hg and 8 mm Hg, respectively. Conclusions The flow diversion effects of both stents were limited to flow velocity reduction. In a non-thrombosed aneurysm or an aneurysm with delayed thrombosis, the intra-aneurysmal pressure remains essentially unchanged regardless of the level of the intra-aneurysmal flow velocity reduction induced by the stents.

Laminin α5 mediates ectopic adhesion of hepatocellular carcinoma through integrins and/or Lutheran/basal cell adhesion molecule

Laminins are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta and three gamma chains; they are major components of all basal laminae (BLs). One laminin chain that has garnered particular interest due to its widespread expression pattern and importance during development is laminin alpha 5. Little is known, however, about the expression and function of laminins containing the alpha 5 chain in human hepatocellular carcinoma (HCC). Here, using a specific antibody, we examined the expression of laminin alpha 5 in normal liver and in HCCs. In normal liver, although laminin alpha 5 was observed in hepatic BLs underlying blood vessels and bile ducts, it was absent from the parenchyma, which may be the origin of HCC. On the other hand, laminin alpha 5 deposition was observed throughout all HCCs tested, regardless of tumor grade. In well-differentiated HCCs, it localized along the trabecules of the tumor. In poorly-differentiated HCCs, it was present in surrounding tumor nodules. In HCC cell lines, laminin alpha 5 heterotrimerized with beta and gamma chains and was secreted into the culture media. To attempt to understand the function of laminins containing alpha 5, the expression of its receptors in HCCs was also determined. In this regard, alpha 3 beta 1/alpha 6 beta 1 integrins and Lutheran/basal cell adhesion molecule (Lu/B-CAM) were expressed in HCC cells. In vitro studies showed that HCC cells readily attached to laminin containing the alpha 5 chain, more so than did primary hepatocytes. In addition to alpha 3 beta 1/alpha 6 beta 1 integrins and Lu/B-CAM, laminin alpha 5 was recognized by integrin alpha 1 beta 1, which also was expressed in HCC cells. These results suggest that laminins containing alpha 5 serve as functional substrates regulating progression of HCC.

Reconstruction of 3D stacked hepatocyte tissues using degradable, microporous poly(d,l-lactide-co-glycolide) membranes.

There is great demand for constructing well-organized three-dimensional (3D) tissues in vitro. Here, we developed a 3D stacked culture method using biodegradable poly(d,l-lactide-co- glycolide) (PLGA) membranes with defined topography. Pore size and porosity of the membranes can be controlled by changing the moisture content during fabrication. The optimized membrane served as a scaffold to manipulate small hepatocyte (SH) layers when they were stacked, while it degraded after stacking, resulting in the reorganization of the cells into a 3D stacked structure. Immunofluorescent staining for domain markers of cell polarity and electron microscopy confirmed that the cells in the 3D stacked structures recovered polarity. Furthermore, the cells exhibited improved liver-specific function as compared with cells in a monolayer. This 3D stacked culture may enable reconstruction of multilayered hepatic tissues with highly differentiated functions in vitro.

Concentration gradients in microfluidic 3D matrix cell culture systems

Microfluidic technology enables the creation of well-defined cell culture environments, which integrate the control of multiple biophysical and biochemical cues for designing novel in vitro assays. Growth-factor concentration gradients play a critical role in a wide range of biological processes ranging from development to cancer, guiding cell migration and influencing cell signaling. We present a microfluidic device capable of generating stable concentration gradients in a 3D matrix, while allowing for direct imaging of cellular behavior. The design consists of polydimethylsiloxane microchannels interconnected through 3D matrices. Optical access of the 3D matrix permits direct observation of invasive properties of cells seeded inside the channels or embedded in the matrix. An important characteristic of the microfluidic platform is the capability to generate reproducible, stable and quantifiable concentration gradients that are essential for systematic studies of soluble factor signaling in chemotaxis as...