Yi-Chin Toh

A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

A practical guide to microfluidic perfusion culture of adherent mammalian cells

Culturing cells at microscales allows control over microenvironmental cues, such as cell-cell and cell-matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.

A gel-free 3D microfluidic cell culture system

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction

Shear stress is a ubiquitous environmental cue experienced by stem cells when they are being differentiated or expanded in perfusion cultures. However, its role in modulating self‐renewing stem cell phenotypes is unclear, since shear is usually only studied in the context of cardiovascular differentiation. We used a multiplex microfluidic array, which overcomes the limitations of macroperfusion systems in shear application throughput and precision, to initiate a comprehensive, quantitative study of shear effects on self‐renewing mouse embryonic stem cells (mESCs), where shear stresses varying by >1000 times (0.016–16 dyn/cm2) are applied simultaneously. When compared with static controls in the presence or absence of a saturated soluble environment (i.e., mESC‐conditioned medium), we ascertained that flow‐induced shear stress specifically up‐regulates the epiblast marker Fgf5. Epiblast‐state transition in mESCs involves heparan sulfate proteoglycans (HSPGs), which have also been shown to transduce shear stress in endothelial cells. By disrupting (with sulfation inhibitors and heparinase) and partially reconstituting (with heparin) HSPG function, we show that mESCs also mechanically sense shear stress via HSPGs to modulate Fgf5 expression. This study demonstrates that self‐renewing mESCs possess the molecular machinery to sense shear stress and provides quantitative shear application benchmarks for future scalable stem cell culture systems.—Toh, Y.‐C., Voldman, J. Fluid shear stress primes mouse embryonic stem cells for differentiation in a self‐renewing environment via heparan sulfate proteoglycans transduction. FASEBJ. 25, 1208–1217 (2011). www.fasebj.org

Cellular responses to a nanofibrous environment

Cells respond profoundly to the mechanical rigidity and three-dimensional nanotopology of substrates, as well as the spatial and temporal arrangements of extracellular cues. We summarize the latest developments in probing and engineering biocompatible nanofibrous extracellular environments at the cell and molecular level for applications in tissue engineering and biological research. This will, in turn, guide further development of three-dimensional nanofibrous scaffolds in order to elicit specific cellular responses for relevant applications.

The controlled presentation of TGF-β1 to hepatocytes in a 3D-microfluidic cell culture system

3D-microfluidic cell culture systems (3D-microFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100-200 pg/ml TGF-beta1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-beta1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-microFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled released TGF-beta1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-beta1 or without TGF-beta1. The controlled presentation of TGF-beta1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications.

Microfabricated silicon nitride membranes for hepatocyte sandwich culture

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.

Scalable alignment of three-dimensional cellular constructs in a microfluidic chip

There have been considerable efforts to engineer three-dimensional (3D) microfluidic environments to enhance cellular function over conventional two-dimensional (2D) cultures in microfluidic chips, but few involve topographical features, such as micro/nano-grooves, which are beneficial for cell types of cardiac, skeletal and neuronal lineages. Here we have developed a cost-effective and scalable method to incorporate micro-topographical cues into microfluidic chips to induce cell alignment. Using commercially available optical media as molds for replica molding, we produced large surface areas of polydimethylsiloxane (PDMS) micro-grooved substrates and plasma-bonded them to multiple microfluidic chips. Besides aligning a 2D monolayer of cells, the micro-grooved substrate can align 3D cellular constructs on chip. C2C12 mouse myoblasts were cultured three-dimensionally in a microfluidic chip with incorporated PDMS micro-grooved substrate remodeled into an aligned 3D cellular construct, where the actin cytoskeleton and nuclei were preferentially oriented along the micro-grooves. Cells within the 3D cellular constructs can align without being in direct contact with the micro-grooves due to synergism between topography and fluid shear stress. Aligned C2C12 3D cellular constructs showed enhanced differentiation into skeletal muscles as compared to randomly aligned ones. This novel method enables the routine inclusion of micro-topographical cues into 2D or 3D microfluidic cultures to generate relevant physiological models for studying tissue morphogenesis and drug screening applications.

Modulation of integrin and E-cadherin-mediated adhesions to spatially control heterogeneity in human pluripotent stem cell differentiation

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here, we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions, resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions, which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity.

Development of Dual-Compartment Perfusion Bioreactor for Serial Coculture of Hepatocytes and Stellate Cells in Poly(lactic-co-glycolic acid)-Collagen Scaffolds

An important challenge in liver tissue engineering is to overcome the rapid loss of hepatocyte functions. In vivo, hepatocytes are compact polyhedral cells with round nuclei; however, they readily loss many of their differentiated functions in vitro. To overcome this challenge, we have established a new perfusion bioreactor that consists of two compartments which enabled the serial coculture of hepatocytes and hepatic stellate cells-T6 without direct contact between each other. Three dimensional scaffolds were utilized in the bioreactor as physical anchors for cells. The scaffolds consist of collagen grafted poly(lactic-co-glycolic acid) microfibers and cross-linked collagen sponges between microfibers for additional cellular support and adhesion. The advantages of this new bioreactor are enabling cell culture in three dimensional organization and controlling the culture parameters of the supporting cells independently from the hepatocytes. The results showed that the hepatocytes exhibited much higher levels of the differentiated functions such as albumin secretion, urea synthesis, and cytochrome P450 enzymatic activity when compared with the monoculture system where hepatocytes alone were cultured. This perfusion bioreactor system has potential applications in the development of bioartificial liver devices or cell-based tissue constructs transplantation therapies.