Thermoplastic elastomer based microfluidic gradient generator for cell culture and drug testing.

Traditional drug testing via polystyrene or glass-based cell culture platforms exposes cells to static drug doses and mechanically rigid environments [stiffness in gigapascals (GPa)], which do not accurately replicate physiological conditions. To address these limitations, we developed a polydimethylsiloxane (PDMS)-based microfluidic concentration gradient generator (μCGG) with six integrated cell culture chambers, using a cost-effective and frugal micro-hydrogel molding-assisted technique that eliminates the need for cleanroom infrastructure, specialized equipment, or advanced expertise. This platform facilitates dynamic drug exposure to cells cultured in chambers with flexible PDMS bases [stiffness in kilopascal (kPa) range], providing a scalable and accessible approach for drug dose-response analysis under physiologically relevant conditions, thereby improving accuracy. μCGG utilized a pressure-driven flow design that repeatedly split, mixed, and recombined fluid streams owing to the presence of the mesh-like geometry of the microchannels. This generated a stable and predictable drug concentration gradient across six outlet chambers, as validated through COMSOL simulations, fluorescence microscopy, and UV-Vis spectroscopy using 5-fluorouracil (5-Fu) as a model drug. MDA-MB-231 breast cancer cells were then cultured in the outlet chambers and exposed to six distinct dynamically generated concentrations of 5-Fu. Cellular viability assessed via live/dead assays yielded an IC50 value of 41 ± 4 μM, closely matching the results from conventional multiwell plates using manually pipetted gradients under static conditions (IC50: 36 ± 3 μM). Additional validation was carried out using immunocytochemistry and flow cytometry to assess apoptotic markers and treatment responses. Overall, our study presents a simple, frugal, and scalable microfluidic platform that addresses the major limitations of traditional drug testing platforms by incorporating dynamic chemical gradients, physiologically relevant mechanical environments, and low-barrier fabrication methods, paving its way for broader adoption in preclinical drug evaluation and dose-response assays.

Microfluidic concentration gradient generators are useful in drug testing, drug screening, and other cellular applications to avoid manual errors, save time, and labor. However, expensive fabrication techniques make such devices prohibitively costly. Here, in the present work, we developed a microfluidic concentration gradient generator (μCGG) using a recently proposed non-conventional photolithography-less method. In this method, ceramic suspension fluid was shaped into a square mesh by controlling Saffman Taylor instability in a multiport lifted Hele-Shaw cell (MLHSC). Using the shaped ceramic structure as the template, μCGG was prepared by soft lithography. The concentration gradient was characterized and effect of the flow rates was studied using COMSOL simulations. The simulation result was further validated by creating a fluorescein dye (fluorescein isothiocanate) gradient in the fabricated μCGG. To demonstrate the use of this device for drug testing, we created various concentrations of an anticancer drug-curcumin-using the device and determined its inhibitory concentration on cervical cancer cell-line HeLa. We found that the IC50 of curcumin for HeLa matched well with the conventional multi-well drug testing method. This method of μCGG fabrication has multiple advantages over conventional photolithography such as: (i) the channel layout and inlet-outlet arrangements can be changed by simply wiping the ceramic fluid before it solidifies, (ii) it is cost effective, (iii) large area patterning is easily achievable, and (iv) the method is scalable. This technique can be utilized to achieve a broad range of concentration gradient to be used for various biological and non-biological applications.

3D printable acrylate polydimethylsiloxane resins for cell culture and drug testing.

A microphysiological system (MPS) is recently emerging as a promising alternative to the classical preclinical models, especially animal testing. A key factor for the construction of MPS is to provide a biomimetic three-dimensional (3D) cellular microenvironment. However, it still remains a challenge to introduce extracellular matrix (ECM)-like biomaterials such as hydrogels and nanofibers in a precise and spatiotemporal manner. Herein, we report a strategy to fabricate a MPS combining both electrospun nanofibers and hydrogels. The in situ formation of microsized hydrogel (microgel) array in MPS is realized by patterning electrospun poly(l-lactic acid) (PLLA)/Ca2+ nanofibers via a solvent-loaded agarose stamp and injecting an alginate solution to trigger the quick ionic cross-linking between alginate and Ca2+ released from patterned nanofibers. The one-on-one integration of electrospun nanofibers and microgels not only provides a 3D cellular microenvironment in designated regions in MPS but also improves the stability of these microenvironments under dynamic culture. In addition, due to the biocompatible properties of an ionic cross-linking reaction, patterned cell array can be achieved simultaneously during the microgel formation process. A breast cancer model is then built in MPS by coculturing human breast cancer cells and human fibroblasts in microgel array, and its application in drug (cisplatin) testing is evaluated. Our data prove that MPS-MA offers a more precise platform for drug testing to evaluate the drug concentration, duration time, cancer microenvironment, etc, mainly due to its successful construction of the biomimetic 3D cellular microenvironment.

Developing advanced biomaterials and precision techniques is critical for tissue engineering and drug testing. This study shows the design and development of a novel 3D-bioprinted gelatin methacryloyl (GelMA) scaffold embedded with RPMI-2650 nasal epithelial cells for nasal cell culture and drug testing applications. The CAD-designed scaffold presented a robust structure and biocompatibility. Studies of swelling behaviour displayed effective medium transport and retention, especially in cell-laden scaffolds. The potential of the GelMA scaffold to enable 3D cell growth was confirmed by the continuously high cell viability (>95%), spheroid formation and substantial cell proliferation. Biocompatibility was confirmed by live/dead cell imaging and coverage studies, which revealed a 5-fold increase in live cell coverage over two weeks. These findings highlight the potential of 3D-bioprinted GelMA scaffolds as a customisable, biocompatible, and robust platform for advancing drug screening, cell culture, and tissue engineering. Future studies will focus on optimising scaffold composition and incorporating dynamic culture conditions to further enhance physiological relevance and translational potential.

The biocompatibility of a wide variety of biomaterials was quantitatively assessed, in a physiologically normal environment" as to cytotoxicity induced in WI-38 cells by cell culture medium extracts. Materials tested included PVC plastic, rubber, silicone rubber, polyethylene, polypropylene, acetal, polyurethane, Teflon, nylon, epoxy, and polystyrene. Cell culture test results were correlated to U.S.P. animal tests. Potential test artifacts, lead, barium, cadmium, and endotoxin were tested for cytotoxicity in WI-38 cells. Cell culture methods yielded more positive tests, particularly rubber, PVC plastic and silicone rubber compounds, than observed in U.S.P. animal tests. Positivity in animal tests did not correlate quantitatively to cytotoxic titers in cell culture. Discrepancies between cell cultures tests and animal tests, specifically rubber compounds, were attributable, in some instances, to differentials in elution efficiency between saline, cottonseed oil, and complete MEM cell culture medium. In other instances, particularly PVC plastics, differences between cell culture and animal test results were due to an inherent difference in the two indicator systems to respond to specific toxic moieties.

Urethral Chlamydia trachomatis infection was diagnosed in 204 of 1,011 (20.2%) male patients by cell culture, in 219 (21.7%) by an antigen detection test consisting of a solid phase immunoassay, and in 247 (24.4%) patients by both methods combined. The positive results of the two methods agreed for 176 patients, and both positive and negative results of the tests agreed for 940 patients (93%). With cell culture as the reference method, the antigen detection test had a sensitivity of 86.3%, a specificity of 94.7%, a positive predictive value of 80.4% and a negative predictive value of 96.5%. It gave false negative results in 28 patients. In 43 patients the antigen detection test gave a positive result, whereas culture was negative. Thirty-nine of these males were treated with antibiotics (tetracycline or erythromycin), 19 because their consorts had a proven Chlamydia trachomatis infection, and 20 for obvious clinical and/or microscopic findings of urethritis requiring treatment. According to this analysis there were 19 probable misses by cell culture test and four true false-positives by the antigen detection test, i.e. less than 0.4% of all patients examined. Since one-third of males with a final diagnosis of Chlamydia trachomatis infection were clinically asymptomatic efforts to control genital chlamydial infections must identify this reservoir. The antigen detection test provides an alternative diagnostic method to the more laborious and time-consuming cell culture procedure.

In this work, a simple and versatile microfluidic cell density gradient generator was successfully developed for cytotoxicity of quantum dots (QDs) assay. The microfluidic cell density gradient generator is composed of eight parallel channels which are respectively surrounded by 1-8 microwells with optimized length and width. The cells fall into microwells by gravity and the cell densities are obviously dependent of microwell number. In a case study, HepG2 and MCF-7 cells were successfully utilized for generating cell density gradients on the microfluidic chip. The microfluidic cell density gradient generator was proved to be easily handled, cell-friendly and could be used to conduct the subsequent cell-based assay. As a proof-of-concept, QD cytotoxicity was evaluated and the results exhibited obvious cell density-dependence. For comparison, QD cytotoxicity was also investigated with a series of cell densities infused by pipette tips. Higher reproducibility was observed on the microfluidic cell density gradient generator and cell density was demonstrated to be a vital factor in cytotoxic study. With higher efficiency, controllability and reproducibility, the microfluidic cell density gradient generator could be integrated into microfluidic analysis systems to promote chip-based biological assay.

Oxygen tension plays an important role in regulating various cellular functions in both normal physiology and disease states. Therefore, drug testing using conventional in vitro cell models under normoxia often possesses limited prediction capability. A traditional method of setting an oxygen tension in a liquid medium is by saturating it with a gas mixture at the desired level of oxygen, which requires bulky gas cylinders, sophisticated control, and tedious interconnections. Moreover, only a single oxygen tension can be tested at the same time. In this paper, we develop a microfluidic cell culture array platform capable of performing cell culture and drug testing under various oxygen tensions simultaneously. The device is fabricated using an elastomeric material, polydimethylsiloxane (PDMS) and the well-developed multi-layer soft lithography (MSL) technique. The prototype device has 4 × 4 wells, arranged in the same dimensions as a conventional 96-well plate, for cell culture. The oxygen tensions are controlled by spatially confined oxygen scavenging chemical reactions underneath the wells using microfluidics. The platform takes advantage of microfluidic phenomena while exhibiting the combinatorial diversities achieved by microarrays. Importantly, the platform is compatible with existing cell incubators and high-throughput instruments (liquid handling systems and plate readers) for cost-effective setup and straightforward operation. Utilizing the developed platform, we successfully perform drug testing using an anti-cancer drug, triapazamine (TPZ), on adenocarcinomic human alveolar basal epithelial cell line (A549) under three oxygen tensions ranging from 1.4% to normoxia. The developed platform is promising to provide a more meaningful in vitro cell model for various biomedical applications while maintaining desired high throughput capabilities.

This paper reviews the development and application of concentration gradient generators based on microfluidics. Generating solutions of varying concentrations is a frequent requirement in numerous experiments and applications. Traditional approaches involve the preparation of solutions with varying concentrations or the dilution of highly concentrated solutions. Nevertheless, these methods are intricate, time-consuming, and susceptible to errors. The advent of microfluidic concentration generators presents a novel concept for the realms of chemistry, biology, and medicine, allows biological or chemical experiments to be performed on a device with a measurement size of a few square centimeters or even smaller, thus greatly reducing the number of basic routine biological or chemical laboratory operations, such as sample preparation, reaction, separation, and analysis. In this review, we discuss the development and application of microfluidic concentration gradient generators through observation, introduce the development history of microfluidic concentration gradient generators and how they are used, summarize their wide range of applications in various fields, and discuss the possible future development direction of microfluidic concentration gradient generators.

Microfluidic concentration gradient generators (µ-CGGs) have been utilized to identify optimal drug compositions through antimicrobial susceptibility testing (AST) for the treatment of antimicrobial-resistant (AMR) infections. Conventional µ-CGGs fabricated via photolithography-based micromachining processes, however, are fundamentally limited to two-dimensional fluidic routing, such that only two distinct antimicrobial drugs can be tested at once. This work addresses this limitation by employing Multijet-3D-printed microchannel networks capable of fluidic routing in three dimensions to generate symmetric multidrug concentration gradients. The three-fluid gradient generation characteristics of the fabricated 3D µ-CGG prototype were quantified through both theoretical simulations and experimental validations. Furthermore, the antimicrobial effects of three highly clinically relevant antibiotic drugs, tetracycline, ciprofloxacin, and amikacin, were evaluated via experimental single-antibiotic minimum inhibitory concentration (MIC) and pairwise and three-way antibiotic combination drug screening (CDS) studies against model antibiotic-resistant Escherichia coli bacteria. As such, this 3D µ-CGG platform has great potential to enable expedited combination AST screening for various biomedical and diagnostic applications.

Three-dimensional (3D) cell culture provides an effective way over conventional two-dimensional (2D) monolayer culture to more closely imitate the complex cellular organization, heterogeneity, and interactions as well as tissue microenvironments in vivo. Here we present a novel droplet-based 3D cell culture method by using droplet array attached on the sidewall of a PDMS piece. Such an arrangement not only avoids cells from adhering on the chip surface for achieving 3D cell culture in nanoliter-scale droplets, but also facilitates performing multiple operations to cells in droplets, including cell suspension droplet generation, drug treatment, and cell staining with a capillary-based liquid handling system, as well as in situ observation and direct scanning with a confocal laser scanning microscope. We optimized the system by studying the effects of various conditions to cell culture including droplet volume, cell density and fabrication methods of the PDMS pieces. We have applied this system in the 3D culture of HepG2 cells and the stimulation testing of an anticancer drug, doxorubicin, to 3D cell spheroids.

A microfluidic device capable of precise chemical control is helpful to mimic tumor microenvironments in vitro, which are closely associated with malignant progression, including metastasis. Cancer cells under a concentration gradient of oxygen and other sustenance materials inside a tumor in vivo have recently been reported to increase the probability of metastasis. The influence of glucose concentration on cancer cells has not been measured well, whereas that of oxygen concentration has been thoroughly examined using microfluidic devices. This is because glucose concentrations can be controlled using microfluidic concentration gradient generators, which trade off temporal stability of the glucose concentration and shear stress on the cells; by contrast, oxygen concentration can be easily controlled without microfluidic device-induced shear stresses. To study cell division and migration responses as a function of glucose concentration, we developed a microfluidic device to observe cell behaviors under various chemical conditions. The device has small-cross-section microchannels for generating a concentration gradient and a large-cross-section chamber for cell culture. With this design, the device can achieve both a cell culture with sufficiently low shear stress on cell activity and a stable glucose concentration gradient. Experiments revealed that a low glucose concentration increased the total migration length of HeLa cells and that HeLa cells under a glucose concentration gradient exhibit random motion rather than chemotaxis.

To the extent possible, artificial organs should have characteristics that match those of the in vivo system. To this end, microfabrication techniques allow us to create microenvironments that can help maintain cell organization and functionality in in vitro cultures. We present three new microbioreactors, each of which allows cells to be cultured in a perfused microenvironment similar to that found in vivo. Our microbioreactors use new technology that permits integration onto the chip (35mm × 20mm) of an electrical sensor, in addition to one or more pumping systems and associated perfusion circuitry. The monitoring of Caco-2 cell cultures using electrical impedance spectroscopy (EIS) has allowed us to measure the effects of cell growth, cellular barrier formation and the presence of chemical compounds and/or toxins. Specifically, we have investigated the ability of the electrical sensor to maintain appropriate sensitivity and precision. Our results show that the sensor was very sensitive not only to the presence or the absence of the cells, but also to changes in cell state. Our perfused microbioreactors are highly efficient miniaturized tools that are easy to operate. We anticipate that they will offer promising new opportunities in many types of cell culture research, including drug screening and tissue engineering.

Modular microfluidics offer the opportunity to combine the precise fluid control, rapid sample processing, low sample and reagent volumes, and relatively lower cost of conventional microfluidics with the flexible reconfigurability needed to accommodate the requirements of target applications such as drug toxicity studies. However, combining the capabilities of fully adaptable modular microelectromechanical systems (MEMS) assembly with the simplicity of conventional microfluidic fabrication remains a challenge. A hybrid polydimethylsiloxane (PDMS)-molding/photolithographic process is demonstrated to rapidly fabricate LEGO®-like modular blocks. The blocks are created with different sizes that interlock via tongue-and-groove joints in the plane and stack via interference fits out of the plane. These miniature strong but reversible connections have a measured resistance to in-plane and out-of-plane forces of up to >6000× and >1000× the weight of the block itself, respectively. The LEGO®-like interference fits enable O-ring-free microfluidic connections that withstand internal fluid pressures of >120 kPa. A single layer of blocks is assembled into LEGO®-like cell culture plates, where the in vitro biocompatibility and drug toxicity to lung epithelial adenocarcinoma cells and hepatocellular carcinoma cells cultured in the modular microwells are measured. A double-layer block structure is then assembled so that a microchannel formed at the interface between layers connects two microwells. Breast tumor cells and hepatocytes cultured in the coupled wells demonstrate interwell migration as well as the simultaneous effects of a single drug on the two cell types.