Microfluidic device flow field characterization around tumor spheroids with tunable necrosis produced in an optimized off-chip process

Tumor spheroids are a 3-D tumor model that holds promise for testing cancer therapies in vitro using microfluidic devices. Tailoring the properties of a tumor spheroid is critical for evaluating therapies over a broad range of possible indications. Using human colon cancer cells (HCT-116), we demonstrate controlled tumor spheroid growth rates by varying the number of cells initially seeded into microwell chambers. The presence of a necrotic core in the spheroids could be controlled by changing the glucose concentration of the incubation medium. This manipulation had no effect on the size of the tumor spheroids or hypoxia in the spheroid core, which has been predicted by a mathematical model in computer simulations of spheroid growth. Control over the presence of a necrotic core while maintaining other physical parameters of the spheroid presents an opportunity to assess the impact of core necrosis on therapy efficacy. Using micro-particle imaging velocimetry (micro-PIV), we characterize the hydrodynamics and mass transport of nanoparticles in tumor spheroids in a microfluidic device. We observe a geometrical dependence on the flow rate experienced by the tumor spheroid in the device, such that the “front” of the spheroid experiences a higher flow velocity than the “back” of the spheroid. Using fluorescent nanoparticles, we demonstrate a heterogeneous accumulation of nanoparticles at the tumor interface that correlates with the observed flow velocities. The penetration depth of these nanoparticles into the tumor spheroid depends on nanoparticle diameter, consistent with reports in the literature.     

Engineered 3D tumour model for study of glioblastoma aggressiveness and drug evaluation on a detachably assembled microfluidic device

3D models of tumours have emerged as an advanced technique in pharmacology and tumour cell biology, in particular for studying malignant tumours such as glioblastoma multiforme (GBM). Herein, we developed a 3D GBM model on a detachably assembled microfluidic device, which could be used to study GBM aggressiveness and for anti-GBM drug testing. Fundamental characteristics of the GBM microenvironment in terms of 3D tissue organisation, extracellular matrices and blood flow were reproduced in vitro by serial manipulations in the integrated microfluidic device, including GBM spheroid self-assembly, embedding in a collagen matrix, and continuous perfusion culture, respectively. We could realize multiple spheroids parallel manipulation, whilst, compartmentalized culture, in a highly flexible manner. This method facilitated investigations into the viability, proliferation, invasiveness and phenotype transition of GBM in a 3D microenvironment and under continuous stimulation by drugs. Anti-invasion effect of resveratrol, a naturally isolated polyphenol, was innovatively evaluated using this in vitro 3D GBM model. Temozolomide and the combination of resveratrol and temozolomide were also evaluated as control. This scalable model enables research into GBM in a more physiologically relevant microenvironment, which renders it promising for use in translational or personalised medicine to examine the impact of, or identify combinations of, therapeutic agents.     

Three-dimensional axisymmetric flow-focusing device using stereolithography

This paper describes a three-dimensional microfluidic axisymmetric flow-focusing device (AFFD) fabricated using stereolithography. Using this method, we can fabricate AFFDs rapidly and automatically without cumbersome alignment needed in conventional methods. The AFFDs are able to be fabricated reproducibly with a micro-sized orifice of diameter around 250 μm. Using this device, we are able to produce monodisperse water-in-oil (W/O) droplets with a coefficient of variation (CV) of less than 4.5%, W/O droplets with encapsulated microbes (CV < 4.9%) and oil-in-water (O/W) droplets (CV < 3.2%) without any surface modifications. The diameter of these droplets range from 54 to 244 μm with respect to the flow rate ratio of the fluids used; these results are in good agreement with theoretical behavior. For applications of the AFFD, we demonstrate that these devices can be used to produce double emulsions and monodisperse hydrogel beads. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acquisition of epithelial–mesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids

Cancer drug development has to go through rigorous testing and evaluation processes during pre-clinical in vitro studies. However, the conventional two-dimensional (2D) in vitro culture is often discounted by the insufficiency to present a more typical tumor microenvironment. The multicellular tumor spheroids have been a valuable model to provide more comprehensive assessment of tumor in response to therapeutic strategies. Here, we applied chitosan-hyaluronan (HA) membranes as a platform to promote three-dimensional (3D) tumor spheroid formation. The biological features of tumor spheroids of human non-small cell lung cancer (NSCLC) cells on chitosan-HA membranes were compared to those of 2D cultured cells in vitro. The cells in tumor spheroids cultured on chitosan-HA membranes showed higher levels of stem-like properties and epithelial–mesenchymal transition (EMT) markers, such as NANOG, SOX2, CD44, CD133, N-cadherin, and vimentin, than 2D cultured cells. Moreover, they exhibited enhanced invasive activities and multidrug resistance by the upregulation of MMP2, MMP9, BCRC5, BCL2, MDR1, and ABCG2 as compared with 2D cultured cells. The grafting densities of HA affected the tumor sphere size and mRNA levels of genes on the substrates. These evidences suggest that chitosan-HA membranes may offer a simple and valuable biomaterial platform for rapid generation of tumor spheroids in vitro as well as for further applications in cancer stem cell research and cancer drug screening.     

Alginate-based microfluidic system for tumor spheroid formation and anticancer agent screening

We demonstrate a microfluidic system for long-term tumor cell culture and drug testing. Three-dimensional cell culture is critical in characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Breast tumor cells were encapsulated within alginate which was gelled in situ within the microchannels. Tumor spheroid formation was observed several days after cell seeding, and various concentrations of doxorubicin were applied to the encapsulated cell aggregates. Drug effects on cell viability and proliferation were measured. In future, hydrogel-based microfluidic devices can comprise part of systems which replace labor intensive screening platforms currently implemented in the laboratory, and they address a need for improving preclinical testing of cancer cell sensitivity to anti-cancer drugs.     

Automatic microfluidic platform for cell separation and nucleus collection

This study reports a new biochip capable of cell separation and nucleus collection utilizing dielectrophoresis (DEP) forces in a microfluidic system comprising of micropumps and microvalves, operating in an automatic format. DEP forces operated at a low voltage (15 V_p–p) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected. In order to transport the cell samples continuously, a serpentine-shape (S-shape) pneumatic micropump device was constructed onto the chip device to drive the samples flow through the microchannel, which was activated by the pressurized air injection. The mixed cell samples were first injected into an inlet reservoir and driven through the DEP electrodes to separate specific samples. Finally, separated cell samples were collected individually in two outlet reservoirs controlled by microvalves. With the same operation principle, the nucleus of the specific cells can be collected after the cell lysis procedure. The pumping rate of the micropump was measured to be 39.8 μl/min at a pressure of 25 psi and a driving frequency of 28 Hz. For the cell separation process, the initial flow rate was 3 μl/min provided by the micropump. A throughput of 240 cells/min can be obtained by using the developed device. The DEP electrode array, microchannels, micropumps and microvalves are integrated on a microfluidic chip using micro-electro-mechanical-systems (MEMS) technology to perform several crucial procedures including cell transportation, separation and collection. The dimensions of the integrated chip device were measured to be 6 × 7 cm. By integrating an S-shape pump and pneumatic microvalves, different cells are automatically transported in the microchannel, separated by the DEP forces, and finally sorted to specific chambers. Experimental data show that viable and non-viable cells (human lung cancer cell, A549-luc-C8) can be successfully separated and collected using the developed microfluidic platform. The separation accuracy, depending on the DEP operating mode used, of the viable and non-viable cells are measured to be 84 and 81%, respectively. In addition, after cell lysis, the nucleus can be also collected using a similar scheme. The developed automatic microfluidic platform is useful for extracting nuclear proteins from living cells. The extracted nuclear proteins are ready for nuclear binding assays or the study of nuclear proteins.     

Three-dimensional modeling of transport of nutrients for multicellular tumor spheroid culture in a microchannel

The growth dynamics of avascular tumors in a microchannel bioreactor is investigated. A three-dimensional flow and nutrient transport model, incorporating the multicellular tumor spheroid (MTS) growth model, has been developed to study the influence of nutrients (oxygen and glucose) supply and distribution on the MTS growth. Numerical simulations based on the EMT6/Ro tumor cells show that the continuous-flow perfusion is more efficient to deliver nutrients to the MTS than the diffusion-only static culture. It is further demonstrated that as long as there is bulk flow, the growth of a single tumor spheroid at the early stage is insensitive to the flow velocity and the channel size. For multiple tumor spheroids in the same microchannel, however, increasing the perfusion velocity can improve the nutrient environment for the disadvantageous downstream tumor spheroid. The flow shear stress exerting on the MTSs in the current microchannel bioreactor is estimated to be far below the critical value to affect the MTS growth, which means that there is still much room for increasing perfusion velocity to satisfy the higher nutrient requirement by the growing tumor spheroids.     

A microfluidic cell culture platform for real-time cellular imaging

This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 μm, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.     

Calcitonin receptor-stimulated migration of prostate cancer cells is mediated by urokinase receptor-integrin signaling

Abundance of calcitonin (CT) and calcitonin receptor (CTR) mRNA in primary prostate tumors positively correlates with tumor grade, and exogenously added CT increases the invasion of prostate cancer cell lines. We examined acute and chronic actions of CT on migration of highly metastatic PC-3M cells and poorly invasive LNCaP cells on several extracellular matrices in a spheroid disaggregation/migration assay. While PC-3M spheroids displayed maximum disaggregation/migration on vitronectin (VN), LNCaP spheroids preferred collagen but also migrated significantly on VN. Up-regulation of CT significantly enhanced disaggregation/migration of PC-3M spheroids on VN, but not on fibronectin. In contrast, down-regulation of CT, CTR, protein kinase A or urokinase-type plasminogen activator receptor (uPAR) led to amelioration of PC-3M spheroid disaggregation/migration. CT selectively increased surface activity of αvβ3 or α6β5 integrins in PC-3M and LNCaP cell lines, respectively, and uPAR-integrin association. Finally, either CT or urokinase could completely restore migration of CT-knock-down PC-3M spheroids. But, only forced expression of urokinase receptor coupled with exogenous addition of urokinase restored migration of CTR-knock-down spheroids. These results support our hypothesis that up-regulation of CT biosynthesis and activation of CT–CTR axis in primary prostate tumors may have direct relevance in their progression to the metastatic phenotype.     

Rapid detection of pathogens using antibody-coated microbeads with bioluminescence in microfluidic chips

Detection of pathogens was demonstrated in a polydimethylsiloxane (PDMS)/glass microfluidic chip with which microbead-based immunoseparation platform and the bioluminescence technology were integrated. Escherichia coli (E. coli) O157:H7 was used as the model bacteria. The microchamber in microfluidic chip was filled with glass beads coated with antibodies which could capture specific organism, and the capture efficiency of the chip for the bacteria was about 91.75%∼95.62%. Then the concentration of bacteria was determined by detecting adenosine triphosphate (ATP) employing bioluminescence reaction of firefly luciferin-lucifera-ATP on chip. The method allowed reliable detection of E. coli O157:H7 concentrations from 3.2 × 10¹ cfu/μL to 3.2 × 10⁵ cfu/μL within 20 min. This research demonstrated excellent reproducibility, stability, and specificity, and could accurately detect the pathogenic bacteria in food samples. The microfluidic chip and the equipments used in this method are easy to miniaturize, thus the method has great potential to be developed to a portable device for rapid detection of pathogens.     

Tethered spheroids as an in vitro hepatocyte model for drug safety screening

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.     

A flexible polymer tube lab-chip integrated with microsensors for smart microcatheter

A flexible polymer tube lab-chip integrated with physical and biochemical sensor modules mounted on a flexible spiral structure for measuring physiological (temperature/flow rate) and metabolic data (glucose concentration) in a catheter application was designed, fabricated and characterized in this work. This new approach not only provides a unique way to assemble multiple sensors on both the inside and outside the flexible polymer tube using standard microfabrication methods while avoiding wiring and assembling problems associated with previous methods, but also maintains catheter inherent lumen potency for in situ drug delivery or insertion of medical tools. Three well-known sensors: temperature sensor (RTD), flow rate sensor (hot film anemometry) and glucose biosensor (amperometric sensor) have been successfully fabricated and fully integrated outside the spirally rolled polymer tube (ID = 500 μm, OD = 650 μm) of this demonstration device. The fabricated sensors showed good performances not only in a planar configuration but also in a spirally rolled configuration. This flexible micro tube lab-chip system provides a generic platform for developing patient-specific “smart” microcatheters that incorporate microsensors, microactuators, microfluidic devices and wireless signal communication modules that are tailored for the patients’ unique condition.     

A microperfused incubator for tissue mimetic 3D cultures

High density, three-dimensional (3D) cultures present physical similarities to in vivo tissue and are invaluable tools for pre-clinical therapeutic discoveries and development of tissue engineered constructs. Unfortunately, the use of dense cultures is hindered by intra-culture transport limits allowing just a few layer thick cultures for reproducible studies. In order to overcome diffusion limits in intra-culture nutrient and gas availability, a simple scalable microfluidic perfusion platform was developed and validated. A novel perfusion approach maintained laminar flow of nutrients through the culture to meet metabolic need, while removing depleted medium and catabolites. Velocity distributions and 3D flow patterns were measured using microscopic particle image velocimetry. The effectiveness of forced convection laminar perfusion was confirmed by culturing 700 μm thick neural-astrocytic (1:1) constructs at cell density approaching that of the brain (50,000 cells/mm³). At the optimized flow rate of the nutrient medium, the culture viability reached 90% through the full construct thickness at 2 days of perfusion while unperfused controls exhibited widespread cell death. The membrane aerated perfusion platform was integrated within a miniature, imaging accessible enclosure enabling temperature and gas control of the culture environment. Temperature measurements demonstrated fast feedback response to environmental changes resulting in the maintenance of the physiological temperature within 37 ± 0.2°C. Reproducible culturing of tissue equivalents within dynamically controlled environments will provide higher fidelity to in vivo function in an in vitro accessible format for cell-based assays and regenerative medicine.     

Probing of peripheral blood mononuclear cells anchoring on TNF-alpha challenged-vascular endothelia in an <Emphasis Type="Italic">in vitro</Emphasis> model of the retinal microvascular

Retinopathy is a complication of diabetes that affects the eyes; it stems from damage to the microvasculature of the retina and eventually compromises vision. The diagnosis of retinopathy is difficult to make because there are no early symptoms or warning signs. Dysfunction of the retina’s microvascular networks is believed to be associated with inflammatory cytokines and tumor necrosis factor alpha (TNF-α). To investigate the effect of these cytokines, such as TNF-α, a polydimethylsiloxane (PDMS)/glass hydride microfluidic device reflecting the physiological structure of the retina’s microvasculature was developed. In this model, the bifurcations and tortuosity of branch vessels were based on photographs of the fundus and an endothelial cell layer (EA.hy926 cells) were reconstructed within the microfluidic network. The adhesion, spreading, and growth of cells was ensured by optimizing the conditions for cell seeding and perfusion. Fluorescent staining was used to visualize the cytoskeleton and measurement of the nitric oxide (NO) level proved that the endothelial EA.hy926 cells had spread in the direction of flow perfusion system, forming artificial vascular networks. The endothelial layer was further challenged by TNF-α perfusion. Cytokine treatment increased the anchoring of peripheral blood mononuclear cells (PBMCs) on the endothelial layer. The microfluidic device developed in this study provides a low-cost platform reflecting the physiological structures of the retina’s microvasculature. It is anticipated that this device will be useful in evaluating the diseased retina as well as in drug screening.     

On-chip acidification rate measurements from single cardiac cells confined in sub-nanoliter volumes

The metabolic activity of cells can be monitored by measuring the pH in the extracellular environment. Microfabrication and microfluidic technologies allow the sensor size and the extracellular volumes to be comparable to single cells. A glass substrate with thin film pH sensitive IrO _x electrodes was sealed to a replica-molded polydimethylsiloxane (PDMS) microfluidic network with integrated valves. The device, termed NanoPhysiometer, allows the trapping of single cardiac myocytes and the measurement of the pH in a detection volume of 0.36 nL. For wild-type (WT) single cardiac myocytes an acidification rate of 6.45 ± 0.38 mpH/min was measured in comparison to 19.5 ± 0.38 mpH/min for very long chain Acyl-CoA dehydrogenase (VLCAD) deficient mice in 0.8 mM of Ca²⁺. VLCAD deficiency is a fatty acid oxidation disease leading to cardiomyopathy and arrhythmias. The acidification rate increased to 11.96 ± 1.33 mpH/min for WT and to 32.0 ± 4.64 mpH/min for VLCAD −/− in 1.8 mM of Ca²⁺. The NanoPhysiometer concept can be extended to study ischemia/reperfusion injury or disorders of other biological systems to identify strategies for treatment and possible pharmacological targets.     

Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds

In this study, we investigated the applicability of C3A--a human hepatocyte cell line--as a predicting tool for drug metabolism by applying tissue-engineering methods. Cultivation of C3A cells within alginate scaffolds induced the formation of spheroids with enhanced drug metabolism activities compared to that of two-dimensional (2-D) monolayer cultures. The spheroid formation process was demonstrated via histology, immunohistochemistry, and transmission electron microscope (TEM) analyses. The C3A spheroids displayed multilayer cell morphology, characterized by a large number of tight junctions, polar cells, and bile canaliculi, similar to spheroids of primary hepatocytes. Spheroid formation was accompanied by a reduction in P-glycoprotein (Pgp) gene expression and C3A cell proliferation was limited mainly to cells on the spheroid outskirt. The 3-D constructs maintained a nearly constant cell number according to MTT assay. Drug metabolism by the two most important cytochrome p-450 (CYP) enzymes in human liver, CYP1A2 and CYP3A4, was tested using preferred drugs. With CYP1A2, 3-fold enhancement in activity per cell was seen for converting ethoxyresorufin to resorufin compared to C3A cell monolayers. The spheroids responded to the inducer beta-naphthoflavone and to the inhibitor furafylline of CYP1A2. Enhanced metabolizing activity of CYP3A4, measured by the amount 6beta-testosterone formed from testosterone, and that of the phase II enzyme glucuronosyltransferases (UGT) further indicated that the tissue-engineered C3A spheroids may provide an efficient experimental tool for predicting drug activities by these CYPs. Moreover, the maintenance of constant cell number, as well as the elevated hepatocellular functions and drug metabolism activities, suggest that the tissue-engineered C3A may be applicable in replacement therapies.     

Exploiting osmosis for blood cell sorting

Blood is a valuable tissue containing cellular populations rich in information regarding the immediate immune and inflammatory status of the body. Blood leukocytes or white blood cells (WBCs) provide an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Blood samples need to be processed to deplete contaminating erythrocytes or red blood cells (RBCs) and sorted into different WBC sub-populations prior to analysis. This is typically accomplished using immuno-affinity protocols which result in undesirable activation. An alternative is size based sorting which by itself is unsuitable for WBCs sorting due to size overlap between different sub-populations. To overcome this limitation, we investigated the possibility of using controlled osmotic exposure to deplete and/or create a differential size increase between WBC populations. Using a new microfluidic cell docking platform, the response of RBCs and WBCs to deionized (DI) water was evaluated. Time lapse microscopy confirms depletion of RBCs within 15 s and creation of > 3 μm size difference between lymphocytes, monocytes and granulocytes. A flow through microfluidic device was also used to expose different WBCs to DI water for 30, 60 and 90 s to quantify cell loss and activation. Results confirm preservation of ∼ 100% of monocytes, granulocytes and loss of ∼ 30% of lymphocytes (mostly CD3⁺/CD4⁺) with minimal activation. These results indicate feasibility of this approach for monocyte, granulocyte and lymphocyte (sub-populations) isolation based on size.     

In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids

In this study, we suggest in situ islet spheroid formation and encapsulation on a single platform without replating as a method for producing mono-disperse spheroids and minimizing damage to spheroids during encapsulation. Using this approach, the size of spheroid can be controlled by modulating the size of the concave well. Here, we used 300 μm concave wells to reduce spheroid size and thereby eliminating the central necrosis caused by large volume. As the encapsulation material, we used alginate and collagen-alginate composite (CAC), and evaluated their suitability through diverse in vitro tests, including measurements of viability, oxygen consumption rate (OCR), hypoxic damage to encapsulated spheroids, and insulin secretion. For in situ encapsulation, alginate or CAC was spread over a concave microwell array containing spheroids, and CaCl₂ solution was diffused through a nano-porous dialysis membrane to achieve uniform polymerization, forming convex structures. By this process, the formation of uniform-size islet spheroids and their encapsulation without an intervening replating step was successfully performed. As a control, intact islets were evaluated concurrently. The in vitro test demonstrated excellent performance of CAC-encapsulated spheroids, and on the basis of these results, we transplanted the islet spheroids-encapsulated with CAC into the intraperitoneal cavity of mice with induced diabetes for 4 weeks, and evaluated subsequent glucose control. Intact islets were also transplanted as control to investigate the effect of encapsulation. Transplanted CAC-encapsulated islet spheroids maintained glucose levels below 200 mg/dL for 4 weeks, at which they were still active. At the end of the implantation experiment, we carried out intraperitoneal glucose tolerance test (IPGTT) in mice to investigate whether the implanted islets remained responsive to glucose. The glucose level in mice with CAC-encapsulated islet spheroids dropped below 200 mg/dL 60 min after glucose injection and was stably maintained. In conclusion, the proposed encapsulation method enhances the viability and function of islet spheroids, and protects these spheroids from immune attack.     

Multiphysics simulation of a microfluidic perfusion chamber for brain slice physiology

Understanding and optimizing fluid flows through in vitro microfluidic perfusion systems is essential in mimicking in vivo conditions for biological research. In a previous study a microfluidic brain slice device (μBSD) was developed for microscale electrophysiology investigations. The device consisted of a standard perfusion chamber bonded to a polydimethylsiloxane (PDMS) microchannel substrate. Our objective in this study is to characterize the flows through the μBSD by using multiphysics simulations of injections into a pourous matrix to identify optimal spacing of ports. Three-dimensional computational fluid dynamic (CFD) simulations are performed with CFD-ACE + software to model, simulate, and assess the transport of soluble factors through the perfusion bath, the microchannels, and a material that mimics the porosity, permeability and tortuosity of brain tissue. Additionally, experimental soluble factor transport through a brain slice is predicted by and compared to simulated fluid flow in a volume that represents a porous matrix material. The computational results are validated with fluorescent dye experiments.     

Galactosylated cellulosic sponge for multi-well drug safety testing

Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 μm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell-cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to β-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.