Alteration of 3D Matrix Stiffness Regulates Viscoelasticity of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material against osteoporosis. Although interrogation of directing effect on lineage specification by physical cues has been proposed, how mechanical stimulation impacts intracellular viscoelasticity during osteogenesis remained enigmatic. Cyto-friendly 3D matrix was prepared with polyacrylamide and conjugated fibronectin. The hMSCs were injected with fluorescent beads and chemically-induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. Inverted epifluorescence microscope was exploited to capture the Brownian trajectory of hMSCs. Mean square displacement was calculated and transformed into intracellular viscoelasticity. Two different stiffness of microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. hMSCs possessed equivalent mechanical traits initially in the first week, while cells cultured in rigid matrix displayed significant elevation over elastic (G') and viscous moduli (G") on day 7 (p < 0.01) and 14 (p < 0.01). However, after two weeks, soft niches no longer stiffened hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course. Stiffness of matrix impacted the viscoelasticity of hMSCs. Detailed recognition of how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.

Quantitative mechanical analysis of indentations on layered, soft elastic materials.

Atomic force microscopy (AFM) is becoming an increasingly popular method for studying cell mechanics, however the existing analysis tools for determining the elastic modulus from indentation experiments are unable to quantitatively account for mechanical heterogeneity commonly found in biological samples. In this work, we numerically calculated force-indentation curves onto two-layered elastic materials using an analytic model. We found that the effect of the underlying substrate can be quantitatively predicted by the mismatch in elastic moduli and the homogeneous-case contact radius relative to the layer height for all tested probe geometries. The effect is analogous to one-dimensional Hookean springs in series and was phenomenologically modeled to obtain an approximate closed-form equation for the indentation force onto a two-layered elastic material which is accurate for up to two orders of magnitude mismatch in Young's modulus when the contact radius is less than the layer height. We performed finite element analysis simulations to verify the model and AFM microindentation experiments and macroindentation experiments to demonstrate its ability to deconvolute the Young's modulus of each layer. The model can be broadly used to quantify and serve as a guideline for designing and interpreting indentation experiments into mechanically heterogeneous samples.

Lis1 dysfunction leads to traction force reduction and cytoskeletal disorganization during cell migration.

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

A biomimetic honeycomb-like scaffold prepared by flow-focusing technology for cartilage regeneration.

A tissue engineering chondrocytes/scaffold construct provides a promise to cartilage regeneration. The architecture of a scaffold such as interconnections, porosities, and pore sizes influences the fates of seeding cells including gene expression, survival, migration, proliferation, and differentiation thus may determine the success of this approach. Scaffolds of highly ordered and uniform structures are desirable to control cellular behaviors. In this study, a newly designed microfluidic device based on flow-focusing geometry was developed to fabricate gelatin scaffolds of ordered pores. In comparison with random foam scaffolds made by the conventional freeze-dried method, honeycomb-like scaffolds exhibit higher swelling ratio, porosity, and comparable compressive strength. In addition, chondrocytes grown in the honeycomb-like scaffolds had good cell viability, survival rate, glycosaminoglycans production, and a better proliferation than ones in freeze-dried scaffolds. Real-time PCR analysis showed that the mRNA expressions of aggrecan and collagen type II were up-regulated when chondrocytes cultured in honeycomb-like scaffolds rather than cells cultured as monolayer fashion. Oppositely, chondrocytes expressed collagen type II as monolayer culture when seeded in freeze-dried scaffolds. Histologic examinations revealed that cells produced proteoglycan and distributed uniformly in honeycomb-like scaffolds. Immunostaining showed protein expression of S-100 and collagen type II but negative for collagen type I and X, which represents the chondrocytes maintained normal phenotype. In conclusion, a highly ordered and honeycomb-like scaffold shows superior performance in cartilage tissue engineering. Biotechnol. Bioeng. 2014;111: 2338-2348. © 2014 Wiley Periodicals, Inc.

Calcium-dependent cAMP mediates the mechanoresponsive behaviour of endothelial cells to high-frequency nanomechanostimulation.

The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho-ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably-without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1-Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.

Dexamethasone accelerates muscle regeneration by modulating kinesin-1-mediated focal adhesion signals.

During differentiation, skeletal muscle develops mature multinucleated muscle fibers, which could contract to exert force on a substrate. Muscle dysfunction occurs progressively in patients with muscular dystrophy, leading to a loss of the ability to walk and eventually to death. The synthetic glucocorticoid dexamethasone (Dex) has been used therapeutically to treat muscular dystrophy by an inhibition of inflammation, followed by slowing muscle degeneration and stabilizing muscle strength. Here, in mice with muscle injury, we found that Dex significantly promotes muscle regeneration via promoting kinesin-1 motor activity. Nevertheless, how Dex promotes myogenesis through kinesin-1 motors remains unclear. We found that Dex directly increases kinesin-1 motor activity, which is required for the expression of a myogenic marker (muscle myosin heavy chain 1/2), and also for the process of myoblast fusion and the formation of polarized myotubes. Upon differentiation, kinesin-1 mediates the recruitment of integrin ß1 onto microtubules allowing delivery of the protein into focal adhesions. Integrin ß1-mediated focal adhesion signaling then guides myoblast fusion towards a polarized morphology. By imposing geometric constrains via micropatterns, we have proved that cell adhesion is able to rescue the defects caused by kinesin-1 inhibition during the process of myogenesis. These discoveries reveal a mechanism by which Dex is able to promote myogenesis, and lead us towards approaches that are more efficient in improving skeletal muscle regeneration.

Spherical microwell arrays for studying single cells and microtissues in 3D confinement.

Microwell arrays have emerged as three-dimensional substrates for cell culture due to their simplicity of fabrication and promise for high-throughput applications such as 3D cell-based assays for drug screening. To date, most microwells have had cylindrical geometries. Motivated by our previous findings that cells display 3D physiological characteristics when grown in the spherical micropores of monodisperse foam scaffolds (Lee et al 2013 Integr. Biol. 5 1447-55 and Lin et al 2011 Soft Matter 7 10010-6), here we engineered novel microwells shaped as spherical caps with obtuse polar angles, yielding narrow apertures. When used as bare substrates, these microwells were suitable for culturing cell spheroids; the narrow apertures sterically hindered unattached cultured cells from rolling out of microwells under agitation. When only the walls of the microwell were conjugated with extracellular matrix proteins, cells remained confined in the microwells. Epithelial cells proliferated and burst out of the aperture, and cell polarity was oriented based on the distribution of extracellular matrix proteins in the microwells. Surprisingly, single fibroblast cells in spherical wells of various diameters (40-100 µm) underwent cell-cycle arrest, while cells in circular cylindrical microwells continued to proliferate. Spatial confinement was not sufficient to cause cell-cycle arrest; however, confinement in a constant negative-curvature microenvironment led to cell-cycle arrest. Overall, these investigations demonstrate that this spherical microwell substrate constitutes a novel basic research tool for elucidating how cells respond to dimensionality and microenvironment with radii of curvature at the cellular length scale.

Imaging through the Whole Brain of Drosophila at λ/20 Super-resolution.

Recently, many super-resolution technologies have been demonstrated, significantly affecting biological studies by observation of cellular structures down to nanometer precision. However, current super-resolution techniques mostly rely on wavefront engineering or wide-field imaging of signal blinking or fluctuation, and thus imaging depths are limited due to tissue scattering or aberration. Here we present a technique that is capable of imaging through an intact Drosophila brain with 20-nm lateral resolution at ∼200 µm depth. The spatial resolution is provided by molecular localization of a photoconvertible fluorescent protein Kaede, whose red form is found to exhibit blinking state. The deep-tissue observation is enabled by optical sectioning of spinning disk microscopy, as well as reduced scattering from optical clearing. Together these techniques are readily available for many biologists, providing three-dimensional resolution of densely entangled dendritic fibers in a complete Drosophila brain. The method paves the way toward whole-brain neural network studies and is applicable to other high-resolution bioimaging.

Assembly of microspheres with polymers by evaporating emulsion droplets.

We study the packing of colloidal microspheres mixed with polymers in oil-in-water emulsion droplets by evaporation. The addition of polymers produces non-unique configurations of final clusters when the number of particles N inside the droplet is larger than 4. The cluster configurations are classified into three categories based on symmetry. Stablized colloidal clusters of spherical packings are observed. Our observations on packing process suggest the mechanisms which cause different and nonunique structures. The osmotic pressure and the interparticle interaction due to polymers changes the force balance between microspheres and result in different structures.

Study on the regulation of focal adesions and cortical actin by matrix nanotopography in 3D environment.

Matrix nanotopography plays an important role in regulating cell behaviors by providing spatial as well as mechanical cues for cells to sense. It has been proposed that nanoscale topography is possible to modulate the tensions which direct the formation of cytoskeleton and the organization of the membrane receptor within the cell, which in turn regulate intracellular mechanical and biochemical signaling. With current studies on this topic being performed mainly in 2D platforms, the question on how nanotopography can influence cell bahaviors in 3D environments has yet to be addressed. In this paper, we explored this question by placing cells in 3D hollow spherical polydimethylsiloxane scaffolds. After culturing rat embryonic fibroblast cells in two kinds of scaffold, one with smooth surface and the other with numerous nano-spikes, we observed that cells in the smooth scaffold have more anchoring sites and more focal adhesions than in the etched scaffold. Moreover, we found the presence of correlation between cortical actin, the important component for supporting cell attachment, and local cell geometry.

Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells.

The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 µm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs' ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues.

Matrix dimensionality and stiffness cooperatively regulate osteogenesis of mesenchymal stromal cells.

Osteogenic potential of mesenchymal stromal cells (MSCs) is mechanosensitive. It's affected by the mechanical properties of the cellular microenvironment, particularly its mechanical modulus. To explore the effect of mechanical modulus on osteogenesis in the third dimension (3D), this study used a novel polyacrylamide (PA) scaffold whose pores are monodisperse and spherical, the mechanical moduli of which can be tuned across a wide range. It was found that MSCs have similar proliferation rates in PA scaffolds independent of the matrix stiffness. The contractile force exerted by MSCs inside PA scaffolds was strong enough to deform the pores of scaffolds made of more compliant PAs (whose shear modulus, G'scaffold<4 kPa). Only scaffolds of the highest stiffness (G'scaffold=12 kPa) can withhold the contraction from MSCs. After osteogenic induction for 21 days, the expression profiles of marker genes showed that PA scaffolds of G'scaffold=12 kPa promoted osteogenesis of MSCs. Confocal image analysis demonstrated that there are more F-actin cytoskeletons and bundled stress fibers at higher matrix moduli in 2D and 3D. Moreover, the 3D porous structure promotes osteogenesis of MSCs more than 2D flat substrates. Together, the differences of cellular behaviors when cultured in 2D and 3D systems are evident. The PA scaffolds developed in the present study can be used for further investigation into the mechanism of MSC mechanosensing in the 3D context. STATEMENT OF SIGNIFICANCE: Mechanical properties of the microenvironment affect cellular behaviors, such as matrix stiffness. Traditionally, cell biological investigations have mostly employed cells growing on 2D substrates. The 3D porous PA scaffolds with the same topological conformation and pore sizes but different stiffness generated in this study showed that the differences of cellular behaviors in 2D and 3D systems are evident. Our 3D scaffolds provide insights into tissue engineering when stem cells incorporated with 3D scaffolds and support the future studies of cellular mechanobiology as well as the elucidation the role mechanical factor plays on the physiology and fate determination of MSCs in the 3D context.

Expandable Scaffold Improves Integration of Tissue-Engineered Cartilage: An In Vivo Study in a Rabbit Model.

One of the major limitations of tissue-engineered cartilage is poor integration of chondrocytes and scaffold structures with recipient tissue. To overcome this limitation, an expandable scaffold with a honeycomb-like structure has been developed using microfluidic technology. In this study, we evaluated the performance of this expandable gelatin scaffold seeded with rabbit chondrocytes in vivo. The chondrocyte/scaffold constructs were implanted into regions of surgically introduced cylindrical osteochondral defects in rabbit femoral condyles. At 2, 4, and 6 months postsurgery, the implanted constructs were evaluated by gross and histological examinations. As expected, the osteochondral defects, which were untreated or transplanted with blank scaffolds, showed no signs of repair, whereas the defects transplanted with chondrocyte/scaffold constructs showed significant cartilage regeneration. Furthermore, the expandable scaffolds seeded with chondrocytes had more regenerated cartilage tissue and better integration with the recipient tissue than autologous chondrocyte implantation. Biomechanical tests revealed that the chondrocyte/scaffold group had the highest compressive strength among all groups at all three time points and endured a similar compressive force to normal cartilage after 6 months of implantation. Histological examinations revealed that the chondrocytes were distributed uniformly within the scaffolds, maintained a normal phenotype, and secreted functional components of the extracellular matrix. Histomorphometric assessment showed a remarkable total interface of up to 87% integration of the expandable scaffolds with the host tissue at 6 months postoperation. In conclusion, the expandable scaffolds improved chondrocyte/scaffold construct integration with the host tissue and were beneficial for cartilage repair.

Entropic interactions in suspensions of semiflexible rods: short-range effects of flexibility.

We compute the entropic interactions between two colloidal spheres immersed in a dilute suspension of semiflexible rods. Our model treats the semiflexible rod as a bent rod at fixed angle, set by the rod contour and persistence lengths. The entropic forces arising from this additional rotational degree of freedom are captured quantitatively by the model, and account for observations at short range in a recent experiment. Global fits to the interaction potential data suggest the persistence length of the fd virus is about two to three times smaller than the commonly used value of 2.2 microm.

Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes.

We developed a microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM). We cocultured human umbilical vein endothelial cells (HUVECs) within the spheroids formed by hepatocellular carcinoma cells, and studied the distributions of the HUVECs over time. We observed that the migration of HUVECs depended on the size of spheroids. In the spheroids of ∼200 µm diameters, HUVECs migrated outwards to the edges within 48 h; while in the spheroids of ∼250 µm diameters, there was no outward migration of the HUVECs up to 72 h. In addition, we studied the effects of pro-angiogenic factors, namely, vascular endothelial growth factor (VEGF) and fibroblast growth factor (ß-FGF), on the migration of HUVECs in the carcinoma cell spheroid. The outward migration of HUVECs in 200 µm spheroids was hindered by the treatment with VEGF and ß-FGF. Moreover, some of the HUVECs formed hollow lumen within 72 h under VEGF and ß-FGF treatment. The combination of SPIM and microfluidic devices gives high resolution in both spatial and temporal domains. The observation of HUVECs in spheroids provides us insight on tumor vascularization, an ideal disease model for drug screening and fundamental studies.

Differentiation of lung stem/progenitor cells into alveolar pneumocytes and induction of angiogenesis within a 3D gelatin--microbubble scaffold.

The inability to adequately vascularize tissues in vitro or in vivo is a major challenge in lung tissue engineering. A method that integrates stem cell research with 3D-scaffold engineering may provide a solution. We have successfully isolated mouse pulmonary stem/progenitor cells (mPSCs) by a two-step procedure and fabricated mPSC-compatible gelatin/microbubble-scaffolds using a 2-channel fluid jacket microfluidic device. We then integrated the cells and the scaffold to construct alveoli-like structures. The mPSCs expressed pro-angiogenic factors (e.g., b-FGF and VEGF) and induced angiogenesis in vitro in an endothelial cell tube formation assay. In addition, the mPSCs were able to proliferate along the inside of the scaffolds and differentiate into type-II and type-I pneumocytes The mPSC-seeded microbubble-scaffolds showed the potential for blood vessel formation in both a chick chorioallantoic membrane (CAM) assay and in experiments for subcutaneous implantation in severe combined immunodeficient (SCID) mice. Our results demonstrate that lung stem/progenitor cells together with gelatin microbubble-scaffolds promote angiogenesis as well as the differentiation of alveolar pneumocytes, resulting in an alveoli-like structure. These findings may help advance lung tissue engineering.

Three-dimensional fibroblast morphology on compliant substrates of controlled negative curvature.

Traditionally, cell biological investigations have mostly employed cells growing on flat, two-dimensional, hard substrates, which are of questionable utility in mimicking microenvironments in vivo. We engineered a novel scaffold to achieve cell culture in the third dimension (3D), where fibroblasts lose the strong dorsal-ventral asymmetry in the distribution of cytoskeletal and adhesion components that is induced by growth on flat substrates. The design principle of our new 3D substrate was inspired by recent advances in engineering cellular microenvironments in which rigidity and the patterning of adhesion ligands were tuned on two-dimensional substrates; the engineered substrates enable independent control over biochemical and mechanical factors to elucidate how mechanical cues affect cellular behaviours. The 3D substrates consisted of polyacrylamide scaffolds of highly ordered, uniform pores coated with extracellular matrix proteins. We characterized important parameters for fabrication and the mechanical properties of polyacrylamide scaffolds. We then grew individual fibroblasts in the identical pores of the polyacrylamide scaffolds, examining cellular morphological, actin cytoskeletal, and adhesion properties. We found that fibroblasts sense the local rigidity of the scaffold, and exhibit a 3D distribution of actin cytoskeleton and adhesions that became more pronounced as the pore size was reduced. In small pores, we observed that elongated adhesions can exist without attachment to any solid support. Taken together, our results show that the use of negatively curved surfaces is a simple method to induce cell adhesions in 3D, opening up new degrees of freedom to explore cellular behaviours.

Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds.

In this paper, we report a new method to incorporate 3D scaffold with electrotaxis measurement in the microfluidic device. The electrotactic response of lung cancer cells in the 3D foam scaffolds which resemble the in vivo pulmonary alveoli may give more insight on cellular behaviors in vivo. The 3D scaffold consists of ordered arrays of uniform spherical pores in gelatin. We found that cell morphology in the 3D scaffold was different from that in 2D substrate. Next, we applied a direct current electric field (EF) of 338 mV/mm through the scaffold for the study of cells' migration within. We measured the migration directedness and speed of different lung cancer cell lines, CL1-0, CL1-5, and A549, and compared with those examined in 2D gelatin-coated and bare substrates. The migration direction is the same for all conditions but there are clear differences in cell morphology, directedness, and migration speed under EF. Our results demonstrate cell migration under EF is different in 2D and 3D environments and possibly due to different cell morphology and/or substrate stiffness.

Cartilage regeneration in SCID mice using a highly organized three-dimensional alginate scaffold.

Tissue engineering for cartilage regeneration provides an alternative to surgery for degenerative osteoarthritis. Recently, a highly organized three-dimensional (3D) alginate scaffold was prepared using a microfluidic device; this scaffold is effective for chondrocyte culture in vitro. The performance of this scaffold was further demonstrated; an alginate scaffold seeded with porcine chondrocytes was implanted in the dorsal subcutaneous site of SCID mice. The recipients were sacrificed at 2, 4, and 6 weeks after transplantation. The grafted implants retrieved from the subcutaneous site were analyzed with histologic examinations. Real-time PCR was used to identify the gene expression patterns of the chondrocytes. The hematoxylin and eosin staining showed that the chondrocytes survived normally in SCID mice; cartilage-like structures were formed after 4 weeks implantation. Immunohistochemical staining revealed cells secreted type II collagen, produced glycosaminoglycans (proved by alcian blue stain), and maintained the expression of S-100. On the other hand, the cells were negative for type I and type X collagen staining. PCR showed that the mRNA expressions of aggrecan and type II collagen were up-regulated at weeks two and four, while type I and type X collagen were down-regulated during the study period. In summary, this highly organized 3D alginate scaffold provided a suitable environment and maintained functional phenotypes for chondrocytes in this animal study.

A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology.

Osteoarthritis is a degenerative disease and frequently involves the knee, hip and phalangeal joints. Current treatments used in small cartilage defects including multiple drilling, abrasion arthroplasty, mosaicplasty, and autogenous chondrocyte transplantation, however, there are problems needed to be solved. The standard treatment for severe osteoarthritis is total joint arthroplasty. The disadvantages of this surgery are the possibility of implant loosening. Therefore, tissue engineering for cartilage regeneration has become a promising topic. We have developed a new method to produce a highly organized single polymer (alginate) scaffold using microfluidic device. Scanning electron microscope and confocal fluoroscope examinations showed that the scaffold has a regular interconnected porous structure in the scale of 250 µm and high porosity. The scaffold is effective in chondrocyte culture; the cell viability test (WST-1 assay), cell toxicity (lactate dehydrogenase assay), cell survival rate, extracellular matrix production (glycosaminoglycans contents), cell proliferation (DNA quantification), and gene expression (real-time PCR) all revealed good results for chondrocyte culture. The chondrocytes can maintain normal phenotypes, highly express aggrecan and type II collagen, and secrete a great deal of extracellular matrix when seeded in the alginate scaffold. This study demonstrated that a highly organized alginate scaffold can be prepared with an economical microfluidic device, and this scaffold is effective in cartilage tissue engineering.