Culture Chip Research Articles

An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells’ autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.

The application of an optically switched dielectrophoretic (ODEP) force for the manipulation and assembly of cell-encapsulating alginate microbeads in a microfluidic perfusion cell culture system for bottom-up tissue engineering

This study reports the utilisation of an optically switched dielectrophoretic (ODEP) force for the manipulation and assembly of cell-encapsulating alginate microbeads in a microfluidic perfusion cell culture system for bottom-up tissue engineering. One of the key features of this system is the ODEP force-based mechanism, which allows a commercial projector to be coupled with a computer to manipulate and assemble cell-encapsulating microbeads in an efficient, manageable, and user-friendly manner. Another distinctive feature is the design of the microfluidic cell culture chip, which allows the patterned cell-encapsulating microbeads to be cultivated on site under culture medium perfusion conditions. For demonstrating its application in bottom-up cartilage tissue engineering, chondrocyte-encapsulating alginate microbeads varying in encapsulated cell densities were generated. The manipulation forces associated with operating the alginate microbeads were experimentally evaluated. The results revealed that the measured manipulation forces increased with increases in both the applied electric voltage and the number of cells in the alginate microbeads. Nevertheless, the observed manipulation force was found to be independent of the size of the cell-free alginate microbeads. It can be speculated that the friction force may influence the estimation of the ODEP force within the experimental conditions investigated. In this study, chondrocyte-encapsulating alginate microbeads with three different cell densities were manipulated and assembled in the proposed microfluidic system to form a compact sheet-like cell culture construct that imitates the cell distribution in the cross-section of native articular cartilage. Moreover, the demonstration case also showed that the cell viability of the cultured cells in the microfluidic system remained as high as 96 ± 2%. In this study, four sheet-like cell culture constructs were stacked to create a larger assembled cell culture construct. The cell distribution inside the cell culture construct was further confirmed by a confocal microscopy observation, which showed that the distribution was similar to that in native articular cartilage. As a whole, the proposed system holds great promise as a platform for engineering tissue constructs with easily tunable inner cell distributions.

On-chip CO2 incubation for pocket-sized microfluidic cell culture

We have developed an on-chip CO2 incubation system based on mass/heat transfer from aqueous solutions of bicarbonate source to cell culture media through a permeable poly(dimethylsiloxane) (PDMS) wall. Heating a carbonate-buffered bicarbonate solution successfully regulated CO2 generation without any feedback control. Because a microfluidic cell culture chip with the incubation system does not require an external chamber or gas supply, the entire microfluidic cell culture setup becomes pocket sized. Using 5 ml of 0.8 M sodium bicarbonate with 65 mM sodium carbonate as the water jacket, the chip maintained the temperature, osmolality, and pH of 750 μl cell culture medium within physiological levels when the chip was placed on a 37°C surface. The osmolality shift and pCO2 of the media reservoir stabilized within <5 mmol/kg and 5.0 ± 1.0% over at least 9 days. The incubation capabilities were demonstrated through microfluidic culture of COS-7 epithelial cells under an inverted microscope for 17 days.

액적의 균형공급에 의해 관류유량이 일정한 펌프 없는 세포배양 칩

본 논문에서는 액적의 균형공급에 의해 관류유량이 일정하게 유지되는 펌프 없는 세포배양 칩을 제안하였다. 기존의 펌프 없는 세포배양 칩은 유체 수위차가 시간에 따라 점차 감소하여 일정한 관류유량 유지가 어려웠다. 반면, 제안된 칩은 액적의 균형공급으로 유체 수위차를 일정하게 유지하여 일정한 관류유량의 세포배양이 가능하다. 제작된 세포배양 칩의 성능분석 결과, 펌프 없이 최대 9.96%와 6.92%의 편차 및 오차 내에서 0.1~0.3μl/min 의 관류유량, Q, 을 얻었다. H358 폐암 세포주 배양결과, Q=0.2μl/min 의 관류유량에서 최대 57.8±21.1%/일의 증식률을 보여, Q=0μl/min 의 정치배양보다 1.9 배 높은 값을 얻었으며 활성도 또한 정치배양보다 관류배양이 더 높은 값을 보였다. 제안된 펌프 없는 세포배양BR칩은 높은 증식률과 활성도의 좋은 배양환경을 제공하여 세포기반 바이오 분석에 응용 가능하다.

Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells

This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumping scheme, and the ITO glass microheater were experimentally evaluated. Results show that the medium delivery mechanism was able to provide pumping rates ranging from 15.4 to 120.0 μL·min−1. In addition, numerical simulation and experimental evaluation were conducted to verify that the ITO glass microheater was capable of providing a spatially uniform thermal environment, and precise temperature control with a mild variation of ±0.3 °C. Furthermore, a perfusion cell culture was successfully demonstrated, showing the cultured cells were kept at high cell viability of 95 ± 2%. In the process, the cultured chondrocytes can be clearly visualized microscopically. As a whole, the proposed cell culture system has paved an alternative route to carry out real-time microscopic observation of biological cells in a simple, user-friendly, and low cost manner.

Comparison of substance supply in static and perfusion cultures based on mass transport phenomena

Mass transport phenomena in cell culture can be formulated by using classical reaction-diffusion equations; however, in practice, it is difficult to solve these equations analytically. Here, we used computer simulation to solve these equations, and compared the time-dependent concentration profile of substances in conventional static culture with that in perfusion culture. The simulated glucose consumption in static culture agreed with that of actual culture conditions used in a general cell culture experiment. The simulation of perfusion culture revealed that the geometry of the chamber and its operating parameters are critical to obtaining a sufficient supply of substances. We also found that the previously reported perfusion chambers are well designed and operate under adequate conditions. Our kinetic model of time-dependent concentration profiles for general substances based on mass transport phenomena could, therefore, be used for the optimal design of a microfluidic perfusion culture chip.

Superior oxygen and glucose supply in perfusion cell cultures compared to static cell cultures demonstrated by simulations using the finite element method

Oxygen and glucose supply is one of the important factors for the growth and viability of the cells in cultivation of tissues, e.g., spheroid, multilayered cells, and three-dimensional tissue construct. In this study, we used finite element methods to simulate the flow profile as well as oxygen and glucose supply to the multilayered cells in a microwell array chip for static and perfusion cultures. The simulation results indicated that oxygen supply is more crucial than glucose supply in both static and perfusion cultures, and that the oxygen supply through the wall of the perfusion culture chip is important in perfusion cultures. Glucose concentrations decline with time in static cultures, whereas they can be maintained at a constant level over time in perfusion cultures. The simulation of perfusion cultures indicated that the important parameters for glucose supply are the flow rate of the perfusion medium and the length of the cell culture chamber. In a perfusion culture chip made of oxygen-permeable materials, e.g., polydimethylsiloxane, oxygen is hardly supplied via the perfusion medium, but mainly supplied through the walls of the perfusion culture chip. The simulation of perfusion cultures indicated that the important parameters for oxygen supply are the thickness of the flow channel and the oxygen permeability of the walls of the channel, i.e., the type of material and the thickness of the wall.

Analysis of Single Cell Metabolic Behavior in Controlled and High throughput Microfluidic Culture Array

The metabolic factor supply influences the cellular response. We have found based on bulk experiment that mouse embryonic stem (mES) cells have higher proliferation rates under lower metabolic factor supply while human carcinoma cells proliferate faster under higher metabolic factor supply. In terms of mES cells, the differentiation shows distinguished patterns of cell lines under different metabolic factor concentrations. These show the important role of extra-cellular metabolic factors on the fundamental modulation of cell growth and transcript gene expression. Single cell analysis shows the variation of individual cell response which could better demonstrate the true cellular phenomenon rather than the bulk experiments. However, which the conventional cell culture, the concentration effect cannot be addressed on a single cell level though the entire culture period, as well as the precise and time-dependent control of input doses, the immediate cellular responses cannot be obtained. Here we have developed a high throughput microfluidic chip for single cell trapping and long term culture with refined control of perfusions, which enables various data collection for the statistical trend of cell population upon metabolic gradient and variation of individual cells’ gene expression. With this microchip, we are able to capture isolated single cells in 70 % of 1.2K chambers, and provide various metabolic gradients with refined control of the concentration and rate, perfusion time and intervals. With the convenient time-lapse observation of fluorescence probes addressing the mitochondria metabolic rate and observation of proliferation, we are able to quantitatively address the immediate response of cells as well as their long term behavior in regard of concentration effects on a single cell level.

Microfluidic cell culture chip with multiplexed medium delivery and efficient cell/scaffold loading mechanisms for high-throughput perfusion 3-dimensional cell culture-based assays

This study reports a microfluidic cell culture chip consisting of 48 microbioreactors for high-throughput perfusion 3-dimensional (3-D) cell culture-based assays. Its advantages include the capability for multiplexed and backflow-free medium delivery, and both efficient and high-throughput micro-scale, 3-D cell culture construct loading. In this work, the microfluidic cell culture chip is fabricated using two major processes, specifically, a computer-numerical-controlled (CNC) mold machining process and a polydimethylsiloxane (PDMS) replication process. The chip is composed of micropumps, microbioreactors, connecting microchannels and a cell/agarose scaffold loading mechanism. The performance of the new pneumatic micropumps and the cell/agarose scaffold loading mechanism has been experimentally evaluated. The experimental results show that this proposed multiplexed medium-pumping design is able to provide a uniform pumping rate ranging from 1.5 to 298.3μl hr(-1) without any fluid backflow and the resultant medium contamination. In addition, the simple cell/agarose loading method has been proven to be able to load the 3-D cell culture construct uniformly and efficiently in all 48 microbioreactors investigated. Furthermore, a micro-scale, perfusion, 3-D cell culture-based assay has been successfully demonstrated using this proposed cell culture chip. The experimental results are also compared to a similar evaluation using a conventional static 3-D cell culture with a larger scale culture. It is concluded that the choice of a cell culture format can influence assay results. As a whole, because of the inherent advantages of a miniaturized perfusion 3-D cell culture assay, the cell culture chip not only can provide a stable, well-defined and more biologically-meaningful culture environment, but it also features a low consumption of research resources. Moreover, due to the integrated medium pumping mechanism and the simple cell/agarose loading method, this chip is economical and time efficient. All of these traits are particularly useful for high-precision and high-throughput 3-D cell culture-based assays.

A scalable microfluidic chip for bacterial suspension culture

Microfluidic systems could, in principle, enable high-throughput breeding and screening of microbial strains for industrial applications, but parallel and scalable culture and detection chips are needed before complete microbial selection systems can be integrated and tested. Here we demonstrate a scalable multi-channel chip that is capable of bacterial suspension culture. The key invention is a multi-layered chip design, which enables a single set of control channels to function as serial peristaltic pumps to drive parallel culture chamber loops. Such design leads to scalability of the culture chip. We demonstrate that E. coli growth in the chip is equivalent or superior to conventional suspension culture on shaking beds. The chip could also be used for suspension culture of other microbes such as Bacillus subtilis, Pseudomonas stutzeri, and Zymomonas mobilis, indicating its general applicability for bacterial suspension culture.

Asymmetric cancer-cell filopodium growth induced by electric-fields in a microfluidic culture chip

We combine a micro-fluidic electric-field cell-culture (MEC) chip with structured-illumination nano-profilometry (SINAP) to quantitatively study the variations of cancer cell filopodia under external direct-current electric field (dcEF) stimulations. Because the lateral resolution of SINAP is better than 150 nm in bright-field image modality, filopodia with diameters smaller than 200 nm can be observed clearly without fluorescent labeling. In the MEC chip, a homogeneous EF is generated inside the culture area that simulates the endogenous EF environment. With this MEC chip-SINAP system, we directly observe and quantify the biased growth of filopodia of lung cancer cells toward the cathode. The epidermal growth factor receptors around the cell edges are also redistributed to the cathodal side. These results suggest that cancer-cell filopodia respond to the changes in EFs in the microenvironment.

A pumpless cell culture chip with the constant medium perfusion-rate maintained by balanced droplet dispensing

This paper presents a pumpless cell culture chip, where a constant-rate medium perfusion is achieved by balanced droplet dispensing. Previous pumpless cell culture chips, where the gravity-driven flow is induced by gradually decreasing the hydraulic-head difference, Δh, between source and drain reservoirs, result in a decreasing perfusion-rate. However, the present pumpless cell culture chip, where autonomous droplet dispensers are integrated on the source reservoirs, results in a constant perfusion-rate using a constant Δh maintained by balanced droplet dispensing between the source-inlet and the drain-outlet. In the experimental study, constant perfusion-rates of 0.1, 0.2, and 0.3 μl min(-1) are obtained by Δh of 38, 76, and 114 mm, respectively. At the constant perfusion-rate (Q=0.2 μl min(-1)), H358 lung cancer cells show the maximum growth-rate of 57.8 ± 21.1% d(-1), which is 1.9 times higher than the 30.2 ± 10.3% d(-1) of the static culture. At a perfusion-rate varying between 0.1-0.3 μl min(-1) (average=0.2 μl min(-1)), however, the H358 cells show a growth-rate of 46.9 ± 8.3% d(-1), which is lower than that of the constant Q of 0.2 μl min(-1). The constant-rate perfusion culture (Q=0.1, 0.2, and 0.3 μl min(-1)) also results in an average cell viability of 89.2%, which is higher than 75.9% of the static culture. This pumpless cell culture chip offers a favorable environment to cells with a high growth-rate and viability, thus having potential for use in cell-based bio-assays.

3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body

This paper proposes a microfluidic device for the on-chip differentiation of an embryoid body (EB) formed in a microwell via 3-dimensional cultures of mouse embryonic carcinoma (EC) cells. The device adjusted the size of the EB by fluid volume, differentiated the EB by chemical treatment, and evaluated its effects in EC cells by on-chip immunostaining. A microfluidic resistance network was designed to control the size of the embryoid body. The duration time and flow rate into each microwell regulated the initial number of trapped cells in order to adjust the size of the EB. The docked cells were aggregated and formed a spherical EB on the non-adherent surface of the culture chip for 3 days. The EC cells in the EB were then differentiated into diverse cell lineages without attachment for an additional 4 days; meanwhile, retinoic acid (RA) was applied without serum to direct the cells into early neuronal lineage. On-chip immunostaining of the EB in the microwell with a neuronal marker was conducted to assess the differentiation-inducing ability of RA. The effect of RA on neuronal differentiation was analyzed with confocal microscopic images of the TuJ1 marker. The RA-treated cells expressed more neuronal markers and appeared as mature neuronal cells with long neurites. The fluorescence intensity of the TuJ1 in the RA-treated EB was twice that observed in the non-treated EB on day 5. It was demonstrated that the pre-screening of inducing chemicals on the early neuronal differentiation of EC cells in a single microfluidic chip was indeed feasible. This chip is expected to constitute a useful tool for assessing the early differentiation of ES cells without attachment, and is also expected to prove useful as an anti-cancer drug test platform for the cytotoxicity assay with cellular spheroids.

Analysis of the paracrine loop between cancer cells and fibroblasts using a microfluidic chip

We use a microfluidic cell culture chip equipped with pneumatic microvalves to analyze the paracrine loop between lung cancer cells and fibroblasts. In order to assess the cellular responses in the paracrine loop, we measure the migration speeds of cancer cells and the aspect ratios of fibroblasts which reflect the phenotype of myofibroblasts. With well-controlled interaction sequences between these two types of cells, we verify that the cytokines from cancer cells effectively stimulate the fibroblasts into myofibroblasts. The cytokines from myofibroblasts, rather than fibroblasts, increase the migration speeds of cancer cells. We confirm that the transforming growth factor-β1 (TGF-β1) is involved in the interaction between cancer cells and fibroblasts, and we also interrupt this paracrine loop in the cell culture chip by inhibiting the TGF-β1 receptors on fibroblasts.

Development of high throughput microfluidic cell culture chip for perfusion 3-dimensional cell culture-based chemosensitivity assay

This study reports a microfluidic cell culture chip encompassing 36 microbioreactors for high throughput perfusion 3-dimensional (3D) cell culture-based chemosensitivity assays. Its advantages include the capability for multiplexed medium delivery, and the function for both efficient and high throughput micro-scale 3D culture construct preparation and loading. The results showed that the proposed medium pumping mechanism was able to provide a uniform pumping rates ranging from 1.2 to 3.9 μl h −1. In addition, the simple cell/hydrogel loading scheme has been proven to be able to carry out 3D cell culture construct preparation and loading precisely and efficiently. Furthermore, a chemosensitivity assay was successfully demonstrated using the proposed cell culture chip. The results obtained were also compared with the same evaluation based on a conventional 2D monolayer cell culture. It can be concluded that the choice of cell culture format can result in different chemosensitivity evaluation results. Overall, because of the nature of miniaturized perfusion 3D cell culture, the cell culture chip not only can provide stable, well-defined and more biologically relevant culture environments, but it also features low consumption of research resources. All these traits are found particularly useful for high-precision and high-throughput 3D cell culture-based assays.

On‐chip cell culture on a microarray of extracellular matrix with surface modification of poly(dimethylsiloxane)

Microfluidic cell culture chips allow to perform assays of small-volume samples rapidly and reproducibly. Most of these chips are made of poly(dimethylsiloxane) (PDMS), which is a flexible, durable, transparent and inexpensive polymer that can be easily applied to fabrication of microstructures by photolithography and replica molding. However, not many cells are able to grow on unmodified PDMS because the cells need appropriate scaffolds on the surface. Here we report surface modification of a PDMS substrate with a microarray of extracellular matrix (ECM) for on-chip cell culture. The ECM proteins collagen and fibronectin were covalently immobilized on an 8 x 8 microarray format by micropatterned UV-induced graft polymerization through a photomask and dehydration-condensation reaction through a microfabricated stencil. Identical spots of ECMs were successfully formed and the geometry of the spots accurately corresponded to the micropattern of the photomask and stencil. We demonstrate the culture of CHO-K1 cells on the ECM microarray chip. Cells proliferated on the fibronectin spots during the 2-day culture.

BioSpotlight

BioSpotlight

Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

High-resolution microcontact printing and transfer of massive arrays of microorganisms on planar and compartmentalized nanoporous aluminium oxide

Handling microorganisms in high throughput and their deployment into miniaturized platforms presents significant challenges. Contact printing can be used to create dense arrays of viable microorganisms. Such "living arrays", potentially with multiple identical replicates, are useful in the selection of improved industrial microorganisms, screening antimicrobials, clinical diagnostics, strain storage, and for research into microbial genetics. A high throughput method to print microorganisms at high density was devised, employing a microscope and a stamp with a massive array of PDMS pins. Viable bacteria (Lactobacillus plantarum, Esherichia coli), yeast (Candida albicans) and fungal spores (Aspergillus fumigatus) were deposited onto porous aluminium oxide (PAO) using arrays of pins with areas from 5 x 5 to 20 x 20 microm. Printing onto PAO with up to 8100 pins of 20 x 20 microm area with 3 replicates was achieved. Printing with up to 200 pins onto PAO culture chips (divided into 40 x 40 microm culture areas) allowed inoculation followed by effective segregation of microcolonies during outgrowth. Additionally, it was possible to print mixtures of C. albicans and spores of A. fumigatus with a degree of selectivity by capture onto a chemically modified PAO surface. High resolution printing of microorganisms within segregated compartments and on functionalized PAO surfaces has significant advantages over what is possible on semi-solid surfaces such as agar.

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.