Microfluidic Cell Culture Devices Research Articles

Reversibly-bonded microfluidic devices for stable cell culture and rapid, gentle cell extraction.

Microfluidic chips have emerged as significant tools in cell culture due to their capacity for supporting cells to adopt more physiologically relevant morphologies in 3D compared with traditional cell culture in 2D. Currently, irreversible bonding methods, where chips cannot be detached from their substrates without destroying the structure, are commonly used in fabrication, making it challenging to conduct further analysis on cells that have been cultured on-chip. Although some reversible bonding techniques have been developed, they are either restricted to certain materials such as glass, or require complex processing procedures. Here, we demonstrate a simple and reversible polydimethylsiloxane (PDMS)-polystyrene (PS) bonding technique that allows devices to withstand extended operations while pressurized, and supports long-term stable cell cultures. More importantly, it allows rapid and gentle live cell extraction for downstream manipulation and characterization after long-term on-chip culturing, and even further subculturing. Our new approach could greatly facilitate microfluidic chip-based cell and tissue cultures, overcoming current analytical limitations and opening up new avenues for downstream uses of on-chip cultures, including 3D-engineered tissue structures for biomedical applications.

Rapid Prototyping of Thermoplastic Microfluidic 3D Cell Culture Devices by Creating Regional Hydrophilicity Discrepancy.

Microfluidic 3D cell culture devices that enable the recapitulation of key aspects of organ structures and functions in vivo represent a promising preclinical platform to improve translational success during drug discovery. Essential to these engineered devices is the spatial patterning of cells from different tissue types within a confined microenvironment. Traditional fabrication strategies lack the scalability, cost-effectiveness, and rapid prototyping capabilities required for industrial applications, especially for processes involving thermoplastic materials. Here, an approach to pattern fluid guides inside microchannels is introduced by establishing differential hydrophilicity using pressure-sensitive adhesives as masks and a subsequent selective coating with a biocompatible polymer. Optimal coating conditions are identified using polyvinylpyrrolidone, which resulted in rapid and consistent hydrogel flow in both the open-chip prototype and the fully bonded device containing additional features for medium perfusion. The suitability of the device for dynamic 3D cell culture is tested by growing human hepatocytes in the device under controlled fluid flow for a 14-day period. Additionally, the study demonstrated the potential of using the device for pharmaceutical high-throughput screening applications, such as predicting drug-induced liver injury. The approach offers a facile strategy of rapid prototyping thermoplastic microfluidic organ chips with varying geometries, microstructures, and substrate materials.

Special Issue on Bio-MEMS

A Micro Electro-Mechanical System (MEMS) is a micro-sized mechatronic device based on semiconductor integrated-circuit manufacturing technology. MEMS technology is inherently a very powerful tool for researchers to manipulate and measure the microscopic biological components of biological tissues since their basic constituent units, cells and intracellular microstructures, are also micron or even submicron in size. MEMS technology applied to medicine and life sciences is called Bio-MEMS. Many Bio-MEMS devices and technologies have been developed in recent years, including microfluidic devices for cell culture, manipulation, and measurement, as well as lab-on-a-chip devices for chemical reactions and analyses on MEMS devices. This special issue consists of 13 papers: 1 review article, 1 letter, and 11 research papers. These papers include studies on mechanical stress on cells using MEMS devices, the control of cell functions through microfabrication techniques, cell measurement and in vivo microenvironment simulation using microfluidic devices, and basic research on wearable devices and self-assembling microstructures using MEMS technologies. The editorial committee members are confident that this special issue will make a significant contribution to the further development of Bio-MEMS. We sincerely appreciate the excellent contributions of the authors and the time and effort of the reviewers. We would also like to thank the editorial board of the Journal of Robotics and Mechatronics for their support of this special issue.

Pumped and pumpless microphysiological systems to study (nano)therapeutics.

Fluidic microphysiological systems (MPS) are microfluidic cell culture devices that are designed to mimic the biochemical and biophysical in vivo microenvironments of human tissues better than conventional petri dishes or well-plates. MPS-grown tissue cultures can be used for probing new drugs for their potential primary and secondary toxicities as well as their efficacy. The systems can also be used for assessing the effects of environmental nanoparticles and nanotheranostics, including their rate of uptake, biodistribution, elimination, and toxicity. Pumpless MPS are a group of MPS that often utilize gravity to recirculate cell culture medium through their microfluidic networks, providing some advantages, but also presenting some challenges. They can be operated with near-physiological amounts of blood surrogate (i.e., cell culture medium) that can recirculate in bidirectional or unidirectional flow patterns depending on the device configuration. Here we discuss recent advances in the design and use of both pumped and pumpless MPS with a focus on where pumpless devices can contribute to realizing the potential future role of MPS in evaluating nanomaterials. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Development of Microphysiological Systems (MPSs) Based on Microfluidic Technology for Drug Discovery in Japan

Microphysiological systems (MPSs) based on microfluidic devices are attracting attention as an alternative cell assay platform to animal experiments in drug discovery. When we use microfluidic devices for cell culture, it is possible to experiment with various culture conditions that are difficult with conventional cell culture methods, such as fabrication of microstructures for cell placement, temporal and spatial control of liquid factors and adhesive conditions, and physical stimulation by flow and expansion/contraction. MPSs, which use microfluidic technology to construct the structure and function of physiological biological tissues and organs, are being commercialized and put to practical use worldwide with the entry of venture companies and pharmaceutical companies. Although research on the practical application of MPS in Japan has lagged far behind the efforts of Western countries, the Japan Agency for Medical Research and Development (AMED) launched the MPS Development and Research Project in FY2017 and established a system for MPS commercialization through industry-government-academia collaboration. The project is characterized by the formation of a consortium involving many researchers not only from academia but also from manufacturing and pharmaceutical companies with the aim of commercializing MPS devices. By FY2021, the final year of this project, several MPSs were successfully positioned in various stages of commercialization. This paper introduces two MPSs that the author was involved in commercializing in collaboration with domestic companies within the project.

KTeXpand – a novel microfluidic device for advanced cell based cross matching

Abstract Antibody-mediated rejection after kidney transplantation is a major limitation for long-term survival of donated kidneys within the recipient allograft. With the existing HLA cross-matching test only complement activating donor specific antibodies are evaluated. Other measurements are more sensitive, but cannot assert the clinical relevance of the donor specific antibodies. Thus, novel methods that address the more complex nature of cellular interactions with serum components are needed to improve antibody-mediated rejection prediction. Recent studies suggest, that the interaction of natural killer cells of the recipient with endothelial cells of the donor is of major interest. Here we present a microfluidic cell culture device and a corresponding assay to test for the relevance of natural killer cell activated antibody-mediated rejection in vitro. Therefore, a protocol to co-cultivate both cell types and track natural killer cell activated killing within a fluorescence-based assay was established. To automate this approach, the prototype of a well plate sized microfluidic device was developed, that enables staining of the two cell types and repeatable and timedependent co-cultivation of the cells with serum. This automated approach enables further evaluation of the clinical relevance of the established assay paving the way to additional information on HLA cross-matching that helps to quickly estimate the probability of Antibody-mediated rejection and the clinical relevance of donor specific antibodies.

Novel Fabrication Technique to Confine Hydrogels with Different Patterns inside Microfluidic Devices without Pillars

In the field of microtechnologies applied to the simulation of controlled biological environments are the so-called organ-on-a-chip, microfluidic cell culture devices. The gradient model plays an indispensable role in this technology. Here, we present a novel microfabrication process for pillarless microfluidic platforms which enables the creation of gradients inside them.

Aspiration-mediated hydrogel micropatterning using rail-based open microfluidic devices for high-throughput 3D cell culture

Microfluidics offers promising methods for aligning cells in physiologically relevant configurations to recapitulate human organ functionality. Specifically, microstructures within microfluidic devices facilitate 3D cell culture by guiding hydrogel precursors containing cells. Conventional approaches utilize capillary forces of hydrogel precursors to guide fluid flow into desired areas of high wettability. These methods, however, require complicated fabrication processes and subtle loading protocols, thus limiting device throughput and experimental yield. Here, we present a swift and robust hydrogel patterning technique for 3D cell culture, where preloaded hydrogel solution in a microfluidic device is aspirated while only leaving a portion of the solution in desired channels. The device is designed such that differing critical capillary pressure conditions are established over the interfaces of the loaded hydrogel solution, which leads to controlled removal of the solution during aspiration. A proposed theoretical model of capillary pressure conditions provides physical insights to inform generalized design rules for device structures. We demonstrate formation of multiple, discontinuous hollow channels with a single aspiration. Then we test vasculogenic capacity of various cell types using a microfluidic device obtained by our technique to illustrate its capabilities as a viable micro-manufacturing scheme for high-throughput cellular co-culture.

3D printed microfluidic devices for cell culture

A successful application of the 3D printed materials in the biomedical field requires extensive studies to ensure their biocompatibility at every step of the process. Here, different components suitable for cell applications, including a microfluidic device, were 3D printed using common resins and a deep analysis of their biocompatibility and post printed protocols was conducted.

Measurement of respiration and acidification rates of mammalian cells in thermoplastic microfluidic devices

• Fast prototyping strategy for non-PDMS based microfluidic structures. • Integration of miniaturized optical pH and O 2 sensors into microfluidic structures. • Thermoplastic chip material enabling respiration measurements. • Investigating cell metabolism via 2-minute-long stop/flow experiments. • Measurement of respiration and acidification rates within perfused cell cultures. Luminescent chemical sensors have been proven to be a valuable asset in cell cultures and generally in microbiological studies. We present microfluidic cell culture devices with integrated optical chemical sensors. The integrated sensors offer the possibility to monitor dissolved oxygen and pH levels and enable the measurement of respiration and acidification rates. The cell metabolism was observed using this method by temporarily stopping the incubation flow, barely influencing the cell culture. A thermoplastic polymer is used as oxygen impermeable chip material to enable the measurement of respiration rates, which is not possible with widely used PDMS-based microfluidics. Hence, an innovative fast prototyping strategy is utilized to enable the replication of small series of thermoplastic microfluidic chips. We were able to demonstrate the suitability of this measurement method by monitoring the metabolic response of a human lung carcinoma epithelial-like cell line (A549) to the exposure of FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone), a drug known for its up-regulating effect on respiration and acidification rates. This universal measurement approach can potentially be deployed in all sorts of microfluidic devices and help to retrieve valuable data from inside those systems beyond classical end-point detection methods. It therefore complements modern 3D cell cultures and organ-on-chip research with a powerful analytical technology for gathering information about ongoing cell metabolism.

Microfluidic Devices as Process Development Tools for Cellular Therapy Manufacturing.

Cellular therapies are creating a paradigm shift in the biomanufacturing industry. Particularly for autologous therapies, small-scale processing methods are better suited than the large-scale approaches that are traditionally employed in the industry. Current small-scale methods for manufacturing personalized cell therapies, however, are labour-intensive and involve a number of 'open events'. To overcome these challenges, new cell manufacturing platforms following a GMP-in-a-box concept have recently come on the market (GMP: Good Manufacturing Practice). These are closed automated systems with built-in pumps for fluid handling and sensors for in-process monitoring. At a much smaller scale, microfluidic devices exhibit many of the same features as current GMP-in-a-box systems. They are closed systems, fluids can be processed and manipulated, and sensors integrated for real-time detection of process variables. Fabricated from polymers, they can be made disposable, i.e. single-use. Furthermore, microfluidics offers exquisite spatiotemporal control over the cellular microenvironment, promising both reproducibility and control of outcomes. In this chapter, we consider the challenges in cell manufacturing, highlight recent advances of microfluidic devices for each of the main process steps, and summarize our findings on the current state of the art. As microfluidic cell culture devices have been reported for both adherent and suspension cell cultures, we report on devices for the key process steps, or unit operations, of both stem cell therapies and cell-based immunotherapies.

Surface Modification Techniques for Endothelial Cell Seeding in PDMS Microfluidic Devices.

Microfluidic lab-on-a-chip cell culture techniques have been gaining popularity by offering the possibility of reducing the amount of samples and reagents and greater control over cellular microenvironment. Polydimethylsiloxane (PDMS) is the commonly used polymer for microfluidic cell culture devices because of the cheap and easy fabrication techniques, non-toxicity, biocompatibility, high gas permeability, and optical transparency. However, the intrinsic hydrophobic nature of PDMS makes cell seeding challenging when applied on PDMS surface. The hydrophobicity of the PDMS surface also allows the non-specific absorption/adsorption of small molecules and biomolecules that might affect the cellular behaviour and functions. Hydrophilic modification of PDMS surface is indispensable for successful cell seeding. This review collates different techniques with their advantages and disadvantages that have been used to improve PDMS hydrophilicity to facilitate endothelial cells seeding in PDMS devices.

From cells-on-a-chip to organs-on-a-chip: scaffolding materials for 3D cell culture in microfluidics.

It is an emerging research area to integrate scaffolding materials in microfluidic devices for 3D cell culture (organs-on-a-chip). The technology of organs-on-a-chip holds the potential to obviate the gaps between pre-clinical and clinical studies. As accumulating evidence shows the importance of extracellular matrix in in vitro cell culture, significant efforts have been made to integrate 3D ECM/scaffolding materials in microfluidics. There are two families of materials that are commonly used for this purpose: hydrogels and electrospun fibers. In this review, we briefly discuss the properties of the materials, and focus on the various technologies to obtain the materials (e.g. extraction of collagen from animal tissues) and to include the materials in microfluidic devices. Challenges and potential solutions of the current materials and technologies were also thoroughly discussed. At the end, we provide a perspective on future efforts to make these technologies more translational to broadly benefit pharmaceutical and pathophysiological research.

Automated microfluidic cell culture of stem cell derived dopaminergic neurons

Parkinson’s disease is a slowly progressive neurodegenerative disease characterised by dysfunction and death of selectively vulnerable midbrain dopaminergic neurons and the development of human in vitro cellular models of the disease is a major challenge in Parkinson’s disease research. We constructed an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity. Using this system, we continuously monitored the differentiation of Parkinson’s disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol. This system is the first example of an automated Organ-on-a-Chip culture and has the potential to enable a versatile array of in vitro experiments for patient-specific disease modelling.

Using a Microfluidic Device for Culture and Drug Toxicity Testing of 3D Cells.

Microfluidic devices provide convenient assays tools for testing cell cultures in three-dimensional (3D) formats. These devices have significant potential for establishing assays that are better at predicting drug toxicity and efficacy effects compared to assays in conventional two-dimensional (2D) cultures. Microfluidic cell culture devices consist of perfused cell culture chambers with inlets and outlet for seeding, culturing, sampling, and assaying the cells. This protocol describes how to prepare and seed cells in a microfluidic cell culture device for drug toxicity testing on cells in a 3D structure. The protocol exemplifies the use of a basic microfluidic device with HepG2 hepatoma cells but can be transferred and optimized for other cells and cell types, including iPSC-derived tissue and organ cells.

Fabrication of a Microfluidic Cell Culture Device Using Photolithographic and Soft Lithographic Techniques.

Photolithography and soft lithography are two common methods for fabrication of microfluidic cell culture devices. Well-defined microstructures are created by exposing a photoresist to UV-light under a photolithographic mask in which the intended patterns are transparent. The subsequent cross-linking of UV-exposed photoresist generates a reusable master that serves as a template for an elastomer, commonly polydimethylsiloxane (PDMS), that reciprocally recaptures the structures of the master in an optically clear and oxygen-permeable rubber-like material. Connections to the cell culture-containing channels of the device for perfusion of culture medium can be established by inserting tubing through the elastomer. In this protocol, the basic steps for making a standard microfluidic device for cell-based assays from photolithography and soft lithography techniques are outlined.

Reconstruction of Hepatic Tissue Structures Using Interstitial Flow in a Microfluidic Device.

Construction of three-dimensional (3D) hepatic tissue structures is important for in vitro tissue engineering of the liver, because 3D culture of hepatocytes is critical for the maintenance of liver-specific functions. Although conventional 3D culture methods are useful for constructing 3D hepatic tissue structures, the precise control of culture microenvironments is required to construct more physiological tissues in vitro. Recent advances in microfluidics technologies have allowed us to utilize microfluidic devices for hepatic cell culture, which opened the door for creating more physiological 3D culture models of the liver. Here, we describe the method for the construction of hepatic tissue structures using a microfluidic device which has a 3D gel region with adjacent microchannels. Primary rat hepatocytes are seeded into a microchannel in a microfluidic device. The cells are then cultured in interstitial flow conditions, which leads to the construction of 3D tissue structures.

Determination of the rheological properties of Matrigel for optimum seeding conditions in microfluidic cell cultures

Hydrogels are increasingly used as a surrogate extracellular matrix in three-dimensional cell culture systems, including microfluidic cell culture. Matrigel is a hydrogel of natural origin widely used in cell culture, particularly in the culture of stem cell-derived cell lines. The use of Matrigel as a surrogate extracellular matrix in microfluidic systems is challenging due to its biochemical, biophysical, and biomechanical properties. Therefore, understanding and characterising these properties is a prerequisite for optimal use of Matrigel in microfluidic systems. We used rheological measurements and particle image velocimetry to characterise the fluid flow dynamics of liquefied Matrigel during loading into a three-dimensional microfluidic cell culture device. Using fluorescence microscopy and fluorescent beads for particle image velocimetry measurements (velocity profiles) in combination with classical rheological measurements of Matrigel (viscosity versus shear rate), we characterised the shear rates experienced by cells in a microfluidic device for three-dimensional cell culture. This study provides a better understanding of the mechanical stress experienced by cells, during seeding of a mixture of hydrogel and cells, into three-dimensional microfluidic cell culture devices.

Quantitative analysis of sensitivity to a Wnt3a gradient in determination of the pole-to-pole axis of mitotic cells by using a microfluidic device.

Proper determination of the cell division axis is essential during development. Wnt3a is a known regulator of the cell division axis; however, the sensitivity of cells to Wnt3a signalling and its role in determining the cell division axis have not been measured to date. To address this gap, we took advantage of the asymmetric distribution of outer dense fibre 2 (ODF2/cenexin) proteins on centrosomes in dividing cells. To precisely quantify the sensitivity of cells to Wnt3a signalling, we developed a microfluidic cell culture device, which can produce a quantitative gradient of signalling molecules. We confirmed that mitotic SH‐SY5Y neuroblastoma cells could detect a 2.5 ~ 5 × 10−3 nm·μm−1 Wnt3a concentration gradient and demonstrated that this gradient is sufficient to affect the determination of the pole‐to‐pole axis of cell division during the later stages of mitosis.

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.