On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

Hydrogel-based microfluidic incubator for microorganism cultivation and analyses

This work presents an array of microfluidic chambers for on-chip culturing of microorganisms in static and continuous shear-free operation modes. The unique design comprises an in-situ polymerized hydrogel that forms gas and reagent permeable culture wells in a glass chip. Utilizing a hydrophilic substrate increases usability by autonomous capillary priming. The thin gel barrier enables efficient oxygen supply and facilitates on-chip analysis by chemical access through the gel without introducing a disturbing flow to the culture. Trapping the suspended microorganisms inside a gel well allows for a much simpler fabrication than in conventional trapping devices as the minimal feature size does not depend on cell size. Nutrients and drugs are provided on-chip in the gel for a self-contained and user-friendly handling. Rapid antibiotic testing in static cultures with strains of Enterococcus faecalis and Escherichia coli is presented. Cell seeding and diffusive medium supply is provided by phaseguide technology, enabling simple operation of continuous culturing with a great flexibility. Cells of Saccharomyces cerevisiae are utilized as a model to demonstrate continuous on-chip culturing.     

Scaffold fabrication in a perfusion culture microchamber array chip by O(2) plasma bonding of poly(dimethylsiloxane) protected by a physical mask

Extracellular matrix (ECM) proteins are required for cell culture. In this paper, we report the use of O(2) plasma bonding to fabricate a perfusion culture microchamber array chip with identical-size ECM spots in the isolated microchambers. The chip was fabricated by assembly of two poly(dimethylsiloxane) (PDMS) layers, a microfluidic network layer, and an ECM array layer, which were aligned and then bonded by O(2) plasma oxidation with protection of the ECM microarray with a physical mask made from PDMS. We successfully cultivated Chinese hamster ovary K1 cells in the microchambers with fibronectin. In the fibronectin microchambers, the cells adhered and extended after 12 h of static culture and then grew over the course of 1 d of perfusion culture.     

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.     

A polystyrene-based microfluidic device with three-dimensional interconnected microporous walls for perfusion cell culture

In this article, we present a simple, rapid prototyped polystyrene-based microfluidic device with three-dimensional (3D) interconnected microporous walls for long term perfusion cell culture. Patterned 3D interconnected microporous structures were created by a chemical treatment together with a protective mask and the native hydrophobic nature of the microporous structures were selectively made hydrophilic using oxygen plasma treatment together with a protective mask. Using this polystyrene-based cell culture microfluidic device, we successfully demonstrated the support of four days perfusion cell culture of hepatocytes (C3A cells).     

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlate^TM (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocyte''s functions, Matrigel^TM-embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes'' responses to external stimuli.     

Metabolite profiling of microfluidic cell culture conditions for droplet based screening

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.     

Convenient surface functionalization of whole-Teflon chips with polydopamine coating

This paper presents a convenient strategy to modify the surface of whole-Teflon microfluidic chips by coating the channel walls with a thin layer of polydopamine (PDA) film, which is formed by oxidation-induced self-polymerization of dopamine in alkaline solution. Two coating strategies, static incubation and dynamic flow, are demonstrated and used for tuning the physical and chemical properties of the coated channel walls. The functionalized surfaces were investigated with the contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy measurements. The coating time was optimized according to the fluorescent intensity of the green fluorescent protein immobilized on the modified surface. Applications of the PDA-modified Teflon microchips in bioanalysis were demonstrated with a typical sandwich immunoassay. Moreover, long-term cell culture experiments on modified and native Teflon chips revealed that the chip biocompatibility can be greatly improved with PDA coating. The results indicate that the surface properties of the Teflon can be easily controlled by the PDA modification, thus greatly expanding the application scope of whole-Teflon chips for various chemical and biological research fields.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.     

A novel microfluidic platform for studying mammalian cell chemotaxis in different oxygen environments under zero-flow conditions

The cell''s micro-environment plays an important role in various physiological and pathological phenomena. To better investigate in vivo cellular behaviors, researchers have expended great effort in building controlled in vitro biophysical and biochemical environments. Because a cell''s gaseous environment affects properties such as its division, metastasis, and differentiation, we developed a zero-flow based platform for studying mammalian cell chemotaxis behavior in different oxygen environments. This platform can construct a linear range of oxygen tensions within one chip (i.e., from 1.4% to 3.6% or 5.5% to 14.5%). To study cell chemotaxis behavior under varying oxygen environments, the chemical gradient direction is established perpendicularly to oxygen change within an observation area. Because the observation area is not subject to flow, shear force is of no concern. In addition, water flow around the cell chambers greatly reduces evaporation and makes long-term microscope imaging possible. In this study, we precisely measure the chemotaxis velocity of MCF-7 human breast cancer cells under different oxygen tension conditions towards CXCL12, which is a stromal cell-derived factor. We find that cell migration rates are not equivalent, even under two close oxygen tensions. We also observed that cells move faster towards high concentrations of chemoattractant when the oxygen tension is below 3% due to the increased expression of HIF-1 (hypoxia-inducible factor 1), which promotes a transition to the amoeboid rather than mesenchymal mode of movement. Our experiments demonstrate that this new microfluidic platform is useful for the quantitative study of mammalian cell chemotaxis under different oxygen conditions in the absence of shear force. We also shed light on the study of chemotaxis under other gaseous environments.     

The role of band 3 protein in oxygen delivery by red blood cells

The synergistic effects of hemoglobin, carbonic anhydrase and the band 3 protein make red blood cells the ideal vehicle for oxygen delivering to the tissues. As long as oxygen is supplied by these ideal vehicles, oxygen intoxication of the tissues is precluded. Band 3 protein mediates the “Chloride-Shift”, i.e., the anion exchange of Cl⁻/HCO₃ ⁻. Because of the Chloride-Shift, red blood cells are able to recognize metabolically active tissues and to supply the minimum amount of oxygen to the tissues. Investigation into the molecular mechanisms of the anion exchange mediated by the band 3 protein was introduced.     

A microfluidic device for uniform-sized cell spheroids formation,culture, harvesting and flow cytometry analysis

Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.     

Towards plug and play filling of microfluidic devices by utilizing networks of capillary stop valves

Robust bubble-free priming of complex microfluidic chips represents a critical, yet often unmet prerequisite to enable their practical and widespread application. Towards this end, the usage of a network of capillary stop valves as a generic design feature is proposed. Design principles, numerical simulations, and their application in the development of a microfluidic cell culture device are presented. This chip comprises eight parallel chambers for the assembly and cultivation of human hepatocytes and endothelial cells. The inlet channel divides into cell chambers, after which the flows are reunited to a single chip outlet. Dimensions and geometry of channels and cell chambers are designed to yield capillary burst pressures sequentially increasing towards the chip outlet. Thus, progress of liquid flow through the device is predefined by design and enclosure of air bubbles inside the microfluidic structures is efficiently avoided. Capillary stop valves were designed using numerical simulations. Devices were fabricated in cyclic olefin polymer. Pressure during filling was determined experimentally and is in good agreement with data obtained from simulation.     

Microfluidic device capable of medium recirculation for non-adherent cell culture

We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells.     

A pump-free membrane-controlled perfusion microfluidic platform

In this article, we present a microfluidic platform for passive fluid pumping for pump-free perfusion cell culture, cell-based assay, and chemical applications. By adapting the passive membrane-controlled pumping principle from the previously developed perfusion microplate, which utilizes a combination of hydrostatic pressure generated by different liquid levels in the wells and fluid wicking through narrow strips of a porous membrane connecting the wells to generate fluid flow, a series of pump-free membrane-controlled perfusion microfluidic devices was developed and their use for pump-free perfusion cell culture and cell-based assays was demonstrated. Each pump-free membrane-controlled perfusion microfluidic device comprises at least three basic components: an open well for generating fluid flow, a micron-sized deep chamber/channel for cell culture or for fluid connection, and a wettable porous membrane for controlling the fluid flow. Each component is fluidically connected either by the porous membrane or by the micron-sized deep chamber/channel. By adapting and incorporating the passive membrane-controlled pumping principle into microfluidic devices, all the benefits of microfluidic technologies, such as small sample volumes, fast and efficient fluid exchanges, and fluid properties at the micro-scale, can be fully taken advantage of with this pump-free membrane-controlled perfusion microfluidic platform.     

Dielectrophoretic field-flow method for separating particle populations in a chip with asymmetric electrodes

This paper presents a field-flow method for separating particle populations in a dielectrophoretic (DEP) chip with asymmetric electrodes under continuous flow. The structure of the DEP device (with one thick electrode that defines the walls of the microfluidic channel and one thin electrode), as well as the fabrication and characterization of the device, was previously described. A characteristic of this structure is that it generates an increased gradient of electric field in the vertical plane that can levitate the particles experiencing negative DEP. The separation method consists of trapping one population to the bottom of the microfluidic channel using positive DEP, while the other population that exhibits negative DEP is levitated and flowed out. Viable and nonviable yeast cells were used for testing of the separation method.     

Optofluidic tweezer on a chip

A novel method to realize an optical tweezer involving optofluidic operation in a microchannel is proposed. To manipulate the optical tweezer, light from an optical fiber is passed through both PDMS (polydimethylsiloxane)-air surface lenses and an optofluidic region, which is located in a control channel. Two liquids with different refractive indices (RIs) are introduced into the control channel to form two different flow patterns (i.e., laminar and segmented flows), depending on the liquid compositions, the channel geometry, and the flow rates. By altering the shapes of the interface of the two liquids in the optofluidic region, we can continuously or intermittently control the optical paths of the light. To demonstrate the functionality of the proposed method, optical tweezer operations on a chip are performed. Changing the flow pattern of two liquids with different RIs in the optofluidic region results in successful trapping of a 25 μm diameter microsphere and its displacement by 15 μm.     

Continuously perfused microbubble array for 3D tumor spheroid model

Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.