Microfluidic cell culture

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Microfluidic cell culture integrates knowledge from biology, biochemistry, engineering, and physics to develop devices and techniques for culturing, maintaining, analyzing, and experimenting with cells at the microscale. [1] [2] It merges microfluidics, a set of technologies used for the manipulation of small fluid volumes (μL, nL, pL) within artificially fabricated microsystems, and cell culture, which involves the maintenance and growth of cells in a controlled laboratory environment. [3] [4] Microfluidics has been used for cell biology studies as the dimensions of the microfluidic channels are well suited for the physical scale of cells (in the order of magnitude of 10 micrometers). [2] For example, eukaryotic cells have linear dimensions between 10 and 100 μm which falls within the range of microfluidic dimensions. [4] A key component of microfluidic cell culture is being able to mimic the cell microenvironment which includes soluble factors that regulate cell structure, function, behavior, and growth. [2] Another important component for the devices is the ability to produce stable gradients that are present in vivo as these gradients play a significant role in understanding chemotactic, durotactic, and haptotactic effects on cells. [2]

Contents

Fabrication

Some considerations for microfluidic devices relating to cell culture include:

Fabrication material is crucial as not all polymers are biocompatible, with some materials such as PDMS causing undesirable adsorption or absorption of small molecules. [9] [10] Additionally, uncured PDMS oligomers can leach into the cell culture media, which can harm the microenvironment. [9] As an alternative to commonly used PDMS, there have been advances in the use of thermoplastics (e.g., polystyrene) as a replacement material. [11] [12]

Spatial organization of cells in microscale devices largely depends on the culture region geometry for cells to perform functions in vivo. [13] [14] For example, long, narrow channels may be desired to culture neurons. [13] The perfusion system chosen might also affect the geometry chosen. For example, in a system that incorporates syringe pumps, channels for perfusion inlet, perfusion outlet, waste, and cell loading would need to be added for the cell culture maintenance. [15] Perfusion in microfluidic cell culture is important to enable long culture periods on-chip and cell differentiation. [16]

Other critical aspects for controlling the microenvironment include: cell seeding density, reduction of air bubbles as they can rupture cell membranes, evaporation of media due to an insufficiently humid environment, and cell culture maintenance (i.e. regular, timely media changes). [17] [16] [18]

Cell's health is defined as the collective equilibrium activities of essential and specialized cellular processes; while a cell stressor is defined as a stimulus that causes excursion from its equilibrium state. Hence, cell health may be perturbed within microsystems based on platform design or operating conditions. Exposure to stressors within microsystems can impact cells through direct and indirect ways. Therefore, it is important to design the microfluidics system for cell culture in a manner that minimizes cell stress situations. For example, by minimizing cell suspension, by avoiding abrupt geometries (which tend to favor bubble formation), designing higher and wider channels (to avoid shear stress), or avoiding thermosensitive hydrogels. [19]

Advantages

Some of the major advantages of microfluidic cell culture include reduced sample volumes (especially important when using primary cells, which are often limited) and the flexibility to customize and study multiple microenvironments within the same device. [3] A reduced cell population can also be used in a microscale system (e.g., a few hundred cells) in comparison to macroscale culture systems (which often require 105 – 107 cells); this can make studying certain cell-cell interactions more accessible. [10] These reduced cell numbers make studying non-dividing or slow dividing cells (e.g., stem cells) easier than traditional culture methods (e.g., flasks, petri dishes, or well plates) due to the smaller sample volumes. [10] [20] Given the small dimensions in microfluidics, laminar flow can be achieved, allowing manipulations with the culture system to be done easily without affecting other culture chambers. [20] Laminar flow is also useful as is it mimics in vivo fluid dynamics more accurately, often making microscale culture more relevant than traditional culture methods. [21] Compartmentalized microfluidic cultures have also been combined with live cell calcium imaging, where depolarizing stimuli have been delivered to the peripheral terminals of neurons, and calcium responses recorded in the cell body. [22] This technique has demonstrated a stark difference in the sensitivity of the peripheral terminals compared to the neuronal cell body to certain stimuli such as protons. [22] This gives an excellent example as to why it is so important to study the peripheral terminals in isolation using microfluidic cell culture devices.

Culture platforms

Traditional cell culture

Traditional two-dimensional (2D) cell culture is cell culture that takes place on a flat surface, e.g. the bottom of a well-plate, and is known as the conventional method. [1] While these platforms are useful for growing and passaging cells to be used in subsequent experiments, they are not ideal environments to monitor cell responses to stimuli as cells cannot freely move or perform functions as observed in vivo that are dependent on cell-extracellular matrix material interactions. [1] To address this issue many methods have been developed to create a three-dimensional (3D) native cell environment. One example of a 3D method is the hanging drop, where a droplet with growing cells is suspended and hangs downwards, which allows cells to grow around and atop of one another, forming a spheroid. [23] The hanging drop method has been used to culture tumor cells but is limited to the geometry of a sphere. [24] Since the advent of poly(dimethylsiloxane) (PDMS) microfluidic device fabrication through soft lithography [25] microfluidic devices have progressed and have proven to be very beneficial for mimicking a natural 3D environment for cell culture. [26]

Microfluidic cell culture

Microfluidic devices make possible the study of a single cell to a few hundred cells in a 3D environment. Comparatively, macroscopic 2D cultures have 104 to 107 cells on a flat surface. [10] Microfluidics also allow for chemical gradients, the continuous flow of fresh media, high through put testing, and direct output to analytical instruments. [10] Additionally, open microfluidic cell cultures such as "microcanals" allow for direct physical manipulation of cells with micropipettes. [27] Many microfluidic systems employ the use of hydrogels as the extracellular matrix (ECM) support which can be modulated for fiber thickness and pore size and have been demonstrated to allow the growth of cancer cells. [28] Gel free 3D cell cultures have been developed to allow cells to grow in either a cell dense environment or an ECM poor environment. [29] Although these devices have proven very useful, there are certain disadvantages such as cells sticking to the PDMS surface, small molecules diffusing into the PDMS, and unreacted PDMS polymers washing into cell culture media. [10]

The use of 3D cell cultures in microfluidic devices has led to a field of study called organ-on-a-chip. The first report of these types of microfluidic cultures was used to study the toxicity of naphthalene metabolites on the liver and lung (Viravaidya et al.). These devices can grow a stripped-down version of an organ-like system that can be used to understand many biological processes. [1] By adding an additional dimension, more advanced cell architectures can be achieved, and cell behavior is more representative of in vivo dynamics; cells can engage in enhanced communication with neighboring cells and cell-extracellular matrix interactions can be modeled. [1] [30] In these devices, chambers or collagen layers containing different cell types can interact with one another for multiple days while various channels deliver nutrients to the cells. [1] [31] An advantage of these devices is that tissue function can be characterized and observed under controlled conditions (e.g., effect of shear stress on cells, effect of cyclic strain or other forces) to better understand the overall function of the organ. [1] [32] While these 3D models offer better model organ function on a cellular level compared with 2D models, there are still challenges. Some of the challenges include: imaging of the cells, control of gradients in static models (i.e., without a perfusion system), and difficulty recreating vasculature. [32] Despite these challenges, 3D models are still used as tools for studying and testing drug responses in pharmacological studies. [1] In recent years, there are microfluidic devices reproducing the complex in vivo microvascular network. Organs-on-a-chip have also been used to replicate very complex systems like lung epithelial cells in an exposed airway and provides valuable insight for how multicellular systems and tissues function in vivo. [33] These devices are able to create a physiologically realistic 3D environment, which is desirable as a tool for drug screening, drug delivery, cell-cell interactions, tumor metastasis etc. [34] [35] In one study, researchers grew tumor cells and tested the drug delivery of cis platin, resveratrol, tirapazamine (TPZ) and then measured the effects the drugs have on cell viability. [36]

Applications of cells in microfluidic systems

Microfluidic systems can be used to culture several cell types.

Culture of mammalian cells

Mammalian cell cultures can be seeded, grown for several weeks, detached, and passaged to a fresh culture medium ad nauseam by digital microfluidic (DMF) devices on a macro-scale. [37]

Culture of non-mammalian cells

Algae

Algae can be incubated, and their growth rate and lipid production can be monitored in a hanging-drop microfluidic system. For example, Mishra et al. developed a 25x75 mm, easily accessible microfluidic device. This design is used to optimize the conditions by changing well diameters, UV light exposure (causing mutagenesis), and light/no light tests for culturing Botryococcus braunii , which is one of the most common freshwater microalgae for biofuel production. [38]

Bacteria and yeast

Microfluidic systems can be used to incubate high volumes of bacteria and yeast colonies. [39] The two-layer microchemostat device is made to allow scientists to culture cells under chemostatic and thermostatic conditions without moving cells around and causing intercellular interaction. [39] Yeast cell suspension droplets can be placed on a plate with patterned hydrophilic areas and incubated for 24 hours; then the droplets are split the produced proteins from yeast are analyzed by MALDI-MS without killing the cells in the original droplets. [40]

Multi-culture in microfluidics

Compared to the highly complex microenvironment in vivo, traditional mono-culture of single cell types in vitro only provides limited information about cellular behavior due to the lack of interactions with other cell types. Typically, cell-to-cell signaling can be divided into four categories depending on the distance: endocrine signaling, paracrine signaling, autocrine signaling, and juxtacrine signaling. [41] For example, in paracrine signaling, growth factors secreted from one cell diffuse over a short distance to the neighboring target cell, [42] whereas in juxtacrine signaling, membrane-bound ligands of one cell directly bind to surface receptors of adjacent cells. [43] There are three conventional approaches to incorporate cell signaling in in vitro cell culture: conditioned media transfer, mixed (or direct) co-culture, and segregated (or indirect) co-culture. [44] The use of conditioned media, where the cultured medium of one cell type (the effector) is introduced to the culture of another cell type (the responder), is a traditional way to include the effects of soluble factors in cell signaling. [45] However, this method only allows one-way signaling, does not apply to short-lived factors (which often degrade before transfer to the responder cell culture), and does not allow temporal observations of the secreted factors. [46] Recently, co-culture has become the predominant approach to study the effect of cellular communication by culturing two biologically related cell types together. Mixed co-culture is the simplest co-culture method, where two types of cells are in direct contact within a single culture compartment at the desired cell ratio. [47] Cells can communicate by paracrine and juxtacrine signaling, but separated treatments and downstream analysis of a single cell type are not readily feasible due to the completely mixed population of cells. [48] [49] The more common method is segregated co-culture, where the two cell types are physically separated but can communicate in shared media by paracrine signaling. The physical barrier can be a porous membrane, a solid wall, or a hydrogel divider. [48] [49] [50] [51] [52] [53] If the physical barrier is removable (such as in PDMS or hydrogel), the assay can also be used to study cell invasion or cell migration. [49] [52] Co-culture designs can be adapted to tri- or multi-culture, which are often more representative of in vivo conditions relative to co-culture. [49] [50] [54] [55]

Closed channel multi-culture systems

The flexibility of microfluidic devices greatly contributes to the development of multi-culture studies by improved control over spatial patterns. Closed channel systems made by PDMS are most commonly used because PDMS has traditionally enabled rapid prototyping. For example, mixed co-culture can be achieved in droplet-based microfluidics easily by a co-encapsulation system to study paracrine and juxtacrine signaling. [56] Two types of cells are co-encapsulated in droplets by combining two streams of cell-laden agarose solutions. After gelation, the agarose microgels will serve as a 3D microenvironment for cell co-culture. [56] Segregated co-culture is also realized in microfluidic channels to study paracrine signaling. Human alveolar epithelial cells and microvascular endothelial cells can be co-cultured in compartmentalized PDMS channels, separated by a thin, porous, and stretchable PDMS membrane to mimic alveolar-capillary barrier. [51] Endothelial cells can also be co-cultured with cancer cells in a monolayer while separated by a 3D collagen scaffold to study endothelial cell migration and capillary growth. [57] When embedded in gels, salivary gland adenoid cystic carcinoma (ACC) cells can be co-cultured with carcinoma-associated fibroblast (CAF) in a 3D extracellular matrix to study stroma-regulated cancer invasion in the 3D environment. [58] If juxtacrine signaling is to be investigated solely without paracrine signaling, a single cell coupling co-culture microfluidic array can be designed based on a cellular valving principle. [59]

Open channel multi-culture systems

Although closed channel microfluidics (discussed in the section above) offers high customizability and biological complexity for multi-culture, the operation often requires handling expertise and specialized equipment, such as pumps and valves. [49] [53] In addition, the use of PDMS is known to cause adverse effects to cell culture, including leaching of oligomers or absorption of small molecules, thus often doubted by biologists. [60] Therefore, open microfluidic devices made of polystyrene (PS), a well-established cell culture material, started to emerge. [60] The advantages of open multi-culture designs are direct pipette accessibility and easy fabrication (micro-milling, 3D printing, injection molding, or razor-printing – without the need for a subsequent bonding step or channel clearance techniques). [49] [53] [61] [62] [63] They can also be incorporated into traditional cultureware (well plate or petri dish) while remaining the complexity for multi-culture experiments. [49] [53] [62] [63] For example, the "monorail device" which patterns hydrogel walls along a rail via spontaneous capillary flow can be inserted into commercially available 24-well plates. [62] Flexible patterning geometries are achieved by merely changing 3D printed or milled inserts. The monorail device can also be adapted to study multikingdom soluble factor signaling, which is difficult in traditional shared media co-culture due to the different media and culture requirements for microbial and mammalian cells. [62] Another open multi-culture device fabricated by razor-printing is capable of integrating numerous culture modalities, including 2D, 3D, Transwell, and spheroid culture. [49] It also shows improved diffusion to promote soluble factor paracrine signaling. [49]

Controlling cell microenvironment

Microfluidic systems expand their ability to control the local cell microenvironment beyond what is possible with conventional culture systems. Being able to provide different environments in a steady, sustainable and precise manner has a significant impact on cell culture research and study. Those environmental factors include physical (shear stress), biochemical (cell-cell interactions, cell-molecule interactions, cell-substrate interactions), and physicochemical (pH, CO2, temperature, O2) factors. [64]

Oxygen concentration control

Oxygen plays an essential role in biological systems. [65] Oxygen concentration control is one of the key elements when designing the microfluidic systems, whether the aerobic species or when modulating cellular functions in vivo , such as baseline metabolism and function. [65] Multiple microfluidic systems have been designed to control the desired gas concentrations for cell culture. For example, generating oxygen gradients was achieved by single-thin-layer PDMS construction within channels (thicknesses less than 50 μm, diffusion coefficient of oxygen in native PDMS at 25 °C, D= 3.55x10−5 cm2 s−1) without using gas cylinders or oxygen scavenging agents; thus the microfluidic cell culture device can be placed in incubators and be operated easily. [66] However, the PDMS may be problematic for the adsorption of small hydrophobic species. [67] Poly(methyl pentene) (PMP) may be an alternative material, because it has high oxygen permeability and biocompatibility like PDMS. [68] [69] In addition to the challenges of controlling gas concentration, monitoring oxygen in the microfluidic system is another challenge to address. There are numerous different dye indicators that can be used as optical, luminescence-based oxygen sensing, which is based on the phenomenon of luminescence quenching by oxygen, without consuming oxygen in the system. [70] This technique makes monitoring oxygen in microscale environments feasible and can be applied in biological laboratories. [70]

Temperature control

Temperature can be sensed by cells and influences their behavior, such as biochemical reaction kinetics. [71] However, it is hard to control high-resolution temperature in traditional cell culture systems; whereas, microfluidic systems are proven to successfully reach the desired temperature under different temperature conditions through several techniques. [71] For example, the temperature gradient in the microfluidic system can be achieved by mixing two or more inputs at different temperatures and flow rates, and the temperature is measured in the outlet channels by embedding polymer-based aquarium thermocouples. [72] Also, by installing heaters and digital temperature sensors at the base of the microfluidic system, it has been demonstrated that a microfluidic cell culture system can continuously operate for at least 500 hours. [73] The circulating water channels in the microfluidic system are also used to precisely control temperatures of the cell culture channels and chambers. [39] Furthermore, putting the device inside a cell culture incubator can also easily control the cell culture temperature. [74]

See also

Related Research Articles

Microfluidics refers to a system that manipulates a small amount of fluids using small channels with sizes ten to hundreds micrometres. It is a multidisciplinary field that involves molecular analysis, molecular biology, and microelectronics. It has practical applications in the design of systems that process low volumes of fluids to achieve multiplexing, automation, and high-throughput screening. Microfluidics emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips, lab-on-a-chip technology, micro-propulsion, and micro-thermal technologies.

<span class="mw-page-title-main">Digital microfluidics</span>

Digital microfluidics (DMF) is a platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets are dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.

<span class="mw-page-title-main">Surface acoustic wave</span> Sound wave which travels along the surface of an elastic material

A surface acoustic wave (SAW) is an acoustic wave traveling along the surface of a material exhibiting elasticity, with an amplitude that typically decays exponentially with depth into the material, such that they are confined to a depth of about one wavelength.

A lab-on-a-chip (LOC) is a device that integrates one or several laboratory functions on a single integrated circuit of only millimeters to a few square centimeters to achieve automation and high-throughput screening. LOCs can handle extremely small fluid volumes down to less than pico-liters. Lab-on-a-chip devices are a subset of microelectromechanical systems (MEMS) devices and sometimes called "micro total analysis systems" (µTAS). LOCs may use microfluidics, the physics, manipulation and study of minute amounts of fluids. However, strictly regarded "lab-on-a-chip" indicates generally the scaling of single or multiple lab processes down to chip-format, whereas "µTAS" is dedicated to the integration of the total sequence of lab processes to perform chemical analysis.

<span class="mw-page-title-main">Polydimethylsiloxane</span> Chemical compound

Polydimethylsiloxane (PDMS), also known as dimethylpolysiloxane or dimethicone, is a silicone polymer with a wide variety of uses, from cosmetics to industrial lubrication.

<span class="mw-page-title-main">Plasma cleaning</span>

Plasma cleaning is the removal of impurities and contaminants from surfaces through the use of an energetic plasma or dielectric barrier discharge (DBD) plasma created from gaseous species. Gases such as argon and oxygen, as well as mixtures such as air and hydrogen/nitrogen are used. The plasma is created by using high frequency voltages to ionise the low pressure gas, although atmospheric pressure plasmas are now also common.

<span class="mw-page-title-main">Bio-MEMS</span>

Bio-MEMS is an abbreviation for biomedical microelectromechanical systems. Bio-MEMS have considerable overlap, and is sometimes considered synonymous, with lab-on-a-chip (LOC) and micro total analysis systems (μTAS). Bio-MEMS is typically more focused on mechanical parts and microfabrication technologies made suitable for biological applications. On the other hand, lab-on-a-chip is concerned with miniaturization and integration of laboratory processes and experiments into single chips. In this definition, lab-on-a-chip devices do not strictly have biological applications, although most do or are amenable to be adapted for biological purposes. Similarly, micro total analysis systems may not have biological applications in mind, and are usually dedicated to chemical analysis. A broad definition for bio-MEMS can be used to refer to the science and technology of operating at the microscale for biological and biomedical applications, which may or may not include any electronic or mechanical functions. The interdisciplinary nature of bio-MEMS combines material sciences, clinical sciences, medicine, surgery, electrical engineering, mechanical engineering, optical engineering, chemical engineering, and biomedical engineering. Some of its major applications include genomics, proteomics, molecular diagnostics, point-of-care diagnostics, tissue engineering, single cell analysis and implantable microdevices.

Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intracellular protein expression. The homogeneous cell population obtained after sorting can be used for a variety of applications including research, diagnosis, and therapy.

<span class="mw-page-title-main">Centrifugal micro-fluidic biochip</span>

The centrifugal micro-fluidic biochip or centrifugal micro-fluidic biodisk is a type of lab-on-a-chip technology, also known as lab-on-a-disc, that can be used to integrate processes such as separating, mixing, reaction and detecting molecules of nano-size in a single piece of platform, including a compact disk or DVD. This type of micro-fluidic biochip is based upon the principle of microfluidics; to take advantage of noninertial pumping for lab-on-a-chip devices using noninertial valves and switches under centrifugal force and Coriolis effect to distribute fluids about the disks in a highly parallel order.

An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture, integrated circuit (chip) that simulates the activities, mechanics and physiological response of an entire organ or an organ system. It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS. The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context. By acting as a more sophisticated in vitro approximation of complex tissues than standard cell culture, they provide the potential as an alternative to animal models for drug development and toxin testing.

Microfluidics in chemical biology is the application of microfluidics in the study of chemical biology.

A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.

<span class="mw-page-title-main">Off-stoichiometry thiol-ene polymer</span>

An off-stoichiometry thiol-ene polymer is a polymer platform comprising off-stoichiometry thiol-enes (OSTE) and off-stoichiometry thiol-ene-epoxies (OSTE+).

Droplet-based microfluidics manipulate discrete volumes of fluids in immiscible phases with low Reynolds number and laminar flow regimes. Interest in droplet-based microfluidics systems has been growing substantially in past decades. Microdroplets offer the feasibility of handling miniature volumes of fluids conveniently, provide better mixing, encapsulation, sorting, sensing and are suitable for high throughput experiments. Two immiscible phases used for the droplet based systems are referred to as the continuous phase and dispersed phase.

Paper-based microfluidics are microfluidic devices that consist of a series of hydrophilic cellulose or nitrocellulose fibers that transport fluid from an inlet through the porous medium to a desired outlet or region of the device, by means of capillary action. This technology builds on the conventional lateral flow test which is capable of detecting many infectious agents and chemical contaminants. The main advantage of this is that it is largely a passively controlled device unlike more complex microfluidic devices. Development of paper-based microfluidic devices began in the early 21st century to meet a need for inexpensive and portable medical diagnostic systems.

<span class="mw-page-title-main">Alvéole Lab</span>

Alvéole is a French company based in Paris and founded in 2010 by Quattrocento, a business accelerator company in the life science field, in collaboration with researchers from the French National Center for Scientific Research with expertise in bioengineering and cell imaging.

Microfluidics refers to the flow of fluid in channels or networks with at least one dimension on the micron scale. In open microfluidics, also referred to as open surface microfluidics or open-space microfluidics, at least one boundary confining the fluid flow of a system is removed, exposing the fluid to air or another interface such as a second fluid.

Mingming Wu is a professor at Cornell University within the Department of Biological and Environmental Engineering, and associate editor of Physical Biology.

Open microfluidics can be employed in the multidimensional culturing of cell types for various applications including organ-on-a-chip studies, oxygen-driven reactions, neurodegeneration, cell migration, and other cellular pathways.

Intestines-on-a-chip are microfluidic bioengineered 3D-models of the real organ, which better mimic physiological features than conventional 3D intestinal organoid culture. A variety of different intestine-on-a-chip models systems have been developed and refined, all holding their individual strengths and weaknesses and collectively holding great promise to the ultimate goal of establishing these systems as reliable high-throughput platforms for drug testing and personalised medicine. The intestine is a highly complex organ system performing a diverse set of vital tasks, from nutrient digestion and absorption, hormone secretion, and immunological processes to neuronal activity, which makes it particularly challenging to model in vitro.

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