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When creating a microfluidic model using polydimethylsiloxane (PDMS), is PDMS used only as a stamp to lay proteins in coverslips or can cells be cultured in the channels or patterns created in PDMS?
PDMS in fact is established to be a reliable material for cell culture in many microfluidic devices. Here are several papers (Titles) including reviews on microfluidic devices using PDMS:
- Microfluidic devices for cell cultivation and proliferation Here
- Adhesion patterns in the microvasculature are dependent on bifurcation angle Here (Microvascular Research)
- Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices Here
- A physiological model of the tumor microenvironment for screening drug delivery systems Here (Cancer Research Proceedings)
- A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip Here (Plos One)
- A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction Here(JLB - Journal of Leukocyte Biology)
My research at Temple University is actually based on a microfluidic device to study cell-cell interaction in a real-time fashion. Majority of established protocols are using fibronectin or collagen, and in the case of tumor cells culture, matrigel for extracellular matrix. Devices are being coated with the ECM protein and then cells can be cultured in the device.
We have shown that in our microfluidic device, human umbilical vein endothelial cells (a.k.a. HUVECs) are forming a complete 3D lumen in the channels.
It is important to note that due to the elasticity of the PDMS, cells are needed to be under physiological flow conditions. Otherwise, it was shown that the cells start to detach from the PDMS. Under such physiological conditions (flow, media, gas exchange), cells can be alive and functional up to a week or two, depending on the design. In our system, the last two publications, cells are functional up to 2 weeks and 1 week respectively.
Plasma Treatment of PDMS for Microfluidics
PDMS (Polydimethylsiloxane) is by far the most popular material used in microfluidics research where it is deployed for rapid prototyping with minimal cost. PDMS however requires a surface treatment step in order to produce strong, conformal bonds between surfaces. Plasma treatment of PDMS increases the exposure of silanol groups (-OH) at the surface of the PDMS layers so that they form strong covalent bonds (Si– O–Si) when brought together with glass. These covalent bonds form the basis of a practically inseparable seal between the layers.
Polydimethylsiloxane (PDMS) is considered by many to be the material of choice for the fabrication of microfluidic devices 1–3 . Its wide adoption has been responsible in large part for the proliferation of microfluidics in the last two decades. Its attractiveness as a material is due to a wide and varied set of advantages that include low cost, chemical inertness, non-toxicity, and the ability to translate features in the micrometer range 3 . In addition, it is optically transparent and permeable to gases 4 . It is an elastic and physically robust material that is reversibly deformable, and it can be used to make components, including valves and pumps, in microfluidic devices 5 . The fabrication of devices using PDMS soft lithography is simpler, cheaper, and less time consuming than other competing techniques (e.g., silicon and plastic micromachining).
PDMS is a hydrophobic material (water contact angle >100°) 6 , which has consequences for droplet-based microfluidics. To successfully generate droplets, the continuous (i.e., external) phase needs to effectively wet the device walls therefore, PDMS is ideally suited for the generation of water-in-oil (w/o) droplets 7 . However, the hydrophobicity of PDMS prevents the production of oil-in-water (o/w) droplets with native, untreated PDMS. The generation of o/w droplets is key for various microfluidic applications, including the synthesis of advanced nano- and micro-materials in oil droplet microreactors 8,9 , organic functional group transformations 10,11 , encapsulation of single cells in double emulsions followed by flow cytometric sorting 12 , and the formation of vesicles 13 and other model membranes 14,15 . The incompatibility of PDMS in this regard has led researchers to use alternative, less desirable materials, such as glass and silicon 16–18 .
In response to this, there have been efforts to modify the PDMS surface to become hydrophilic. These efforts have included chemical vapor deposition of polymer coatings 19 , incorporation of an amphiphilic surfactant in the PDMS bulk 20 , deposition of glass-like layers on the substrate surface (sol-gel coating) 21 , and layer-by-layer (LbL) deposition of charged polyanions and polycations 22 . One well-established approach to produce hydrophilic PDMS surfaces is to oxidize the polymer surface with plasma or ultraviolet (UV) irradiation 23 . However, this effect is transient, and the hydrophobic nature of PDMS returns several minutes after plasma or UV exposure because of the migration of the uncured hydrophobic polymer chains to the surface. Methods to slow down or prevent this recovery from occurring include keeping the surface in water immediately after treatment and removing uncured polymers using solvent extraction 24,25 . However, the utility of these approaches for droplet microfluidics has not been established, and it is likely that exposure of the surface to oil will negate these effects. Another strategy to achieve surface modification is to coat substrates with polyvinyl alcohol (PVA a hydrophilic polymer). This strategy has primarily been used for non-PDMS surfaces, such as silica capillaries for biopolymer separation and DNA sizing applications 26–28 . PVA can also be irreversibly adsorbed onto hydrophobic polymer films 29 and gold substrates 30 . Others have demonstrated PVA adsorption onto PDMS via heat immobilization following plasma treatment 27 , via plasma oxidation and covalent attachment of an (3-aminopropyl)triethoxysilane (APTES) linker 31 , and via the synthesis of a PVA/PDMS copolymer microsuspension 32 . These procedures were developed to control the degree of biomolecule interaction with the PDMS substrate and not to modify the hydrophobicity of the surface with regards to droplet microfluidics. A combination of PVA and glycerol coating was immobilized on a plasma-treated PDMS microfluidic chip to encourage poly(L-lactic acid) microsphere generation 33 . However, this study relied on a continual supply of PVA during microsphere generation.
The generation process for lipid-stabilized droplets is the most challenging type of droplet generation because of the very specific favorable wettability between lipids and PDMS. There is one elegant example of using PVA-modified surfaces in droplet microfluidics for the controlled generation of lipid vesicles 34 . Although the aforementioned examples demonstrate the potential of the PVA deposition strategy for chemistry and biology, a systematic characterization and optimization of this technique for droplet microfluidics is lacking, specifically regarding the effects of the various process parameters on the surface properties. This has hindered the wide scale adoption of this versatile technique by the microfluidics community, particularly in the field of bottom-up synthetic biology for artificial cell manufacturing.
In the context of droplet microfluidics, several criteria need to be considered when assessing the suitability of the surface modification technique. The surfaces should be sufficiently hydrophilic to generate o/w droplets. The treatment needs to be long lasting and irreversible, especially when the surface is in contact with both oil and water phases. The treatment should be versatile enough to yield droplets with a range of surfactants, including lipids, which are typically considered less effective for stabilizing droplets. Given the increased use of droplets for the construction of artificial membranes, the development of an adequate treatment is of great importance 13–15 . In addition, the modified channel should be biocompatible, and any deposited material should not interfere with cells and biological components. Furthermore, the technique should allow for modifications of different regions on the same device to create both hydrophobic and hydrophilic areas. This is especially relevant in droplet microfluidics, where the phase that is exposed to the channels (the external phase) is different in different regions of the chip (e.g., in double emulsions). This has been achieved by connecting together several devices with differing surface properties 35,36 and by selectively exposing regions to plasma using a scanning radial microjet 37 . The latter approach is problematic because it necessitates the use of expensive and specific equipment and suffers from hydrophobic recovery issues. A versatile method for selective modification is to use flow through solutions through defined regions of the device 22,36 and selectively modify these regions. Most importantly, the technique must be practical it should be simple, low cost, and not time consuming. Although existing surface modification techniques meet some of the criteria above, there is not a single technique that meets all of them. In this article, we developed and optimized an effective, simple, quick, versatile, and cheap method to make long-lasting, hydrophilic PDMS surfaces for droplet microfluidics. The method was based on coating channels with PVA immediately after plasma treatment. We characterized the effects of the procedure on the contact angle and hydrophilic surface lifetime and showed that the PVA-treated devices can generate droplets with traditional surfactants as well as phospholipids. The procedure can pattern specific regions of the device to exhibit defined wettability characteristics, which is further demonstrated in droplet microfluidics for the generation of emulsions, double emulsions, and inverse double emulsions using a single device design.
2. Fundamentals of microscale cell culture
Culturing cells in microfluidic devices requires an understanding of certain fundamental principles that span multiple disciplines, including biology, biochemistry, physics and engineering. First, it is necessary to have knowledge of key elements of the cellular microenvironment in order to help develop in vitro models that more closely mimic the conditions that exist in vivo. Second, it is imperative to have command of the techniques in cell culture to aid the translation of methods from macro- to microscale. Third, it is important to have a strong background in microfluidics, and an awareness of the state of the art in microscale technology so that tools can be designed appropriately for their intended applications. The ability to advance the area of microscale cell culture will depend on understanding these fundamental areas, which we review here in succession. We then integrate these basic ideas, and discuss how to control the microenvironment in vitro using microfluidics.
2.1. Cell microenvironment
Cells reside in a milieu composed of soluble factors, cell–matrix interactions, and cellll contacts, and do so while living within an environment with specific physicochemical properties (pH, oxygen tension, temperature, and osmolality) ( Fig. 1 ). These elements give the environment a distinct physiological character, and provide a set of extracellular cues that work in concert to regulate cell structure, function, and behavior, and ultimately influence the growth, development, and repair of neighboring tissue. The combination of these biochemical, physical, and physicochemical factors constitutes the cell microenvironment, (although the term tissue microenvironment is also used depending on the context of the work). For stem cells, the local microenvironment, or stem cell niche, holds the key to regulating stem cell survival, self-renewal, and differentiation. 6 In cancer biology, tumor and organ microenvironments can give rise to cancer cells that are conditioned for metastasis at ectopic locations. 7 In the context of microfluidic cell culture, we focus on microenvironments at the cell and local tissue level, which have physical scales amenable to microchannel dimensions. It is reasonable to assume that examining cell and tissue microenvironments will also help elucidate aspects of the microenvironment at the larger organ level.
The cell microenvironment consists of physical, biochemical, and physicochemical factors. For example, the endothelium that lines blood vessels is exposed to hemodynamic shear stress (external physical force) that stimulates a biochemical response, releasing nitric oxide (NO). NO diffuses to neighboring smooth muscle cells (SMCs), where it regulates cell contraction and relaxation. The gradient of diffused NO affects nearby SMCs more than distant SMCs. Endothelial cells are anchored to the basement membrane, while SMCs are anchored to the extracellular matrix of the interstitium, both via integrins that act as sensors and transducers of physical force. Local physicochemical properties ensure proper regulation of both physical and biochemical mechanisms.
Most cells in the body are non-circulating, and therefore depend on attachment to the surrounding extracellular matrix (ECM) for survival. Cells are anchored to the ECM via cell-surface integrins that are responsible not only for the physical attachment of cells to the matrix, but also for sensing and transducing mechanical signals from focal adhesion sites to the cytoskeletal machinery within the cell. 8 These signals are known to drive various cellular processes that include migration, proliferation, differentiation, and apoptosis. Some cell types such as endothelial cells also rely on cellll contacts via cadherins for additional physical support, allowing the endothelium to sense, transduce, and resist hemodynamic shear forces as a larger cellular monolayer. 9 Together, the forces exerted on the cell through mechanical attachments and external stimuli form a dynamic three-dimensional (3D) physical microenvironment that must be carefully considered when modeling cells and tissues in vitro.
The biochemical microenvironment consists of cytokines, growth factors, hormones and other biomolecules, which combine to form complex signaling pathways that contribute to deciding the fate of the cell. 10 Soluble factor signaling occurs mainly via autocrine and paracrine processes, which rely heavily on diffusion of molecules to neighboring cells either of the same or of a different type. Endocrine signaling also plays a role, but relies more on convective transport of hormonal signals from distant locations in the body to the local microenvironment. The effects of soluble factors on cell regulation depend on the concentration, half life, and receptor binding affinities of the ligand of interest. The majority of biological experiments revolve around determining these biochemical effects, and then proposing the mechanisms by which certain soluble factors regulate specific cell processes. For example, the success of certain drug candidates in drug screening tests depends on detailed pharmacokinetic analyses of turnover rates and dose-response curves that shed light on how the drug behaves in the biochemical microenvironment of the cell.
Whether the microenvironment is physical or biochemical in nature, an important aspect of the environment is the presence of gradients that can persist in the vicinity of the cell, often acting as signals themselves to regulate cell function and behavior. Chemical gradients exist naturally due to diffusion, whereas gradients in matrix stiffness or ECM composition are intrinsically built into the heterogeneity of the tissue structure. Since gradients have such an important role in many processes, including migration, angiogenesis, and tumorigenesis, more and more studies are incorporating gradients into their assays, resulting in improved understanding of chemotactic (cell motility in presence of chemical gradient), durotactic (cell motility in presence of substrate stiffness gradient), and haptotactic (cell motility in presence of gradient of surface-bound ligands) effects on cells. More importantly, microfluidics is expected to play a significant role in the design and implementation of such gradient assays because of the ability for microfluidic geometries to establish stable gradients of various forms. 11
Because of the obvious coupling between biochemical and physical cues, studies are beginning to surface that examine the combinatorial effects of soluble factor signaling and cell–matrix interactions on cell behavior. 12 These types of studies are especially important for drug screening tests and for optimization of microenvironments in tissue engineering applications where long-term performance of tissue constructs are dependent on many factors that act synergistically. It is not surprising that these studies are appearing at the same time that microscale technologies are beginning to integrate into the biological research community, and it is likely that microfluidics will also contribute in a significant way to how cellular microenvironments are designed and studied in vitro.
While the physical and biochemical aspects of the microenvironment receive the bulk of the attention in experimental biology, the effects of physicochemical properties on cell behavior have not been studied as extensively. This is understandable given that the physical and biochemical cues are considered the main factors that determine cell processes in normal and pathological development, while properties such as pH, temperature, and osmolality of the surrounding milieu are considered inherent to the in vivo environment, and are expected to remain unperturbed throughout the normal development and life of an organism. However, given reports that abnormal levels of pH and oxygen tension are associated with the development of various pathologies, such as cancer within tumor microenvironments, 13 it is important to ensure these properties are not overlooked when designing and using in vitro microenvironments. In particular, physicochemical properties are critical to the maintenance of cell cultures (see section 2.2 below), and this issue is magnified for cultures at the microscale.
2.2. Cell culture
It is rather remarkable that only a century ago the idea of cultivating a living cell outside of a living organism was met with enormous skepticism and resistance. 14 Today, cell culture is part of a huge biotechnology industry that relies on it for mass production of proteins and vaccines, and preparation of cell-based assays for drug screening applications. Moreover, cell culture techniques are an integral part of fundamental and applied cell biology research. Much of our current understanding of biology stems from in vitro experimentation with cells in Petri dishes and well plates, and biology laboratories spend significant amounts of effort and resources on designing and performing experiments based on the in vitro methods that are available to them. Proponents of in vivo methodology often cite as a major weakness of in vitro techniques the inability of a Petri dish to fully capture nuances of the in vivo cellular microenvironment. Yet, there is little doubt that cell culture has had a major impact on modern biology, and will continue to do so as pressures rise to reduce animal testing (particularly in Europe 15 ) and we continue to make further advances in microfluidic cell culture.
Cell culture allows the researcher to isolate specific factors for experimentation outside the complex in vivo microenvironment. By doing so, scientists can make logical hypotheses of the effects of those factors, and through controlled experimentation elucidate the mechanisms that regulate cell function. The goal in cell culture is twofold: to recapitulate as closely as possible the cellular microenvironment while also maintaining enough simplicity so that experimental replicates can be performed to achieve statistically significant results in a reasonable amount of time. Often, there is a tradeoff between these two aspects, and model accuracy is sacrificed for higher throughput, or vice versa. This is where microfluidics is likely to have its largest impact: it has the potential to improve both model accuracy and throughput simultaneously, by giving scientists the freedom to tailor the microenvironment while also reducing the scale of the experimental platform. Here, we briefly summarize the main considerations when performing cell culture in hopes that it will provide insight on the needs of microscale cell culture platforms, and ultimately facilitate the integration between cell culture and microfluidics. Readers interested in a more thorough treatment of basic cell culture techniques are referred to the popular manual by Freshney. 16
The basic elements of cell culture have changed little in the past fifty years. The culture medium serves as the biochemical microenvironment of the culture, and consists of essential amino acids, vitamins, salts, carbohydrates, and other components in aqueous solution. The composition of essential components were discovered in a rigorous set of experiments by Eagle more than fifty years ago (see Freshney 16 ), and is still used today as the main source for the components of some basal media. For cells to proliferate in culture, basal media must be supplemented with factors that promote cell growth and division, and this is typically achieved by adding fetal bovine or calf serum. While sera contain the necessary growth factors and hormones for cells to proliferate, the composition of sera can also vary considerably from batch to batch, leading to variations in results from one experiment to another. Considerable progress has been made in the development of serum-free media where all components including growth factors and hormones are well defined so that experimental variations can be reduced.
For the most part, the physicochemical properties of the culture system are expected to remain unchanged throughout an experiment (as well as between separate experiments), unless the properties themselves are being tested. The pH and osmolality of the media can be measured with standard laboratory equipment (pH meter and osmometer) prior to use in culture experiments to ensure media has not deteriorated. Temperature and CO2 levels are usually monitored directly from the incubator controls, while the ambient air within the incubator provides normal oxygen tension levels for the cultures. To maintain a relatively constant and physiological pH of between 7.2 and 7.4, the media can be buffered with sodium bicarbonate, and in certain situations where CO2 cannot be supplied, with additional buffering agents such as HEPES.
In terms of cultureware, the majority of cell cultures are performed on two-dimensional flat surfaces in commercial plasticware such as polystyrene Petri dishes, flasks and well plates. Hydrophobic polystyrene surfaces are typically plasma-treated to render it hydrophilic, which facilitates cell adhesion. For certain cell types, it may be necessary to provide a coating of matrix proteins on the surface to further promote adhesion, growth, and proliferation. The key factors to healthy, viable cells during regular maintenance of the cultures include appropriate cell seeding density, regular changes of culture media, monitoring of cell growth rates, and timely subculturing of the cells. These aspects are critical to maintaining proper cell phenotype and function, and apply to both macroscale and microscale culture. Each cell type is unique, and it is still routine procedure for the biologist to fine-tune their culturing protocols to suit the needs of their cell type.
While most biologists continue to use conventional two-dimensional cultures as their main format for in vitro experimentation, the past quarter century has also seen the major development of 3D cultures that provide a more realistic model of the physical microenvironment that exists in vivo. Bissell at the Lawrence Berkeley National Laboratories has been a pioneer in the area of 3D culture systems where cells are cultured and tested in in vivo-like tissue architectures. Using techniques developed in her lab, Bissell and colleagues have made novel discoveries in breast cancer development and the mammary gland physical microenvironment, 17 and have also begun to incorporate microscale technology in the form of combinatorial microenvironment microarrays to tease out the synergistic effects of combined soluble and ECM factors on stem cell fate. 18
Although culture techniques have remained unchanged for the bulk of its history, recent progress in microfluidics and other microscale technologies, as evidenced in the work of Bissell described above, suggests that cell culture practices are beginning to evolve. Research laboratories will likely adopt microscale techniques at an ever-increasing pace because of their ability to create physiological microenvironments, as well as their promise to provide high-throughput solutions for intensive biological studies. Although tissue culture in industrial settings will likely follow that trend given enough time, it will perhaps require significant progress both in high-throughput capabilities and in the marketability of microfluidic systems before a massive industry such as biotechnology will change its current course.
2.3. Microfluidics: tools and techniques
Microfluidic devices designed specifically for cell culture have certain requirements that distinguish them from microscale systems used for other applications in chemistry or physics. Design considerations of particular importance to microfluidic cell culture include: (1) the choice of material for device fabrication, (2) the geometry and dimensions of the culture region, and (3) the method of pumping and controlling fluid flow. The latter consideration ultimately dictates how the microfluidic device is connected to external components of the overall system. While it is clear that microfluidics offers the engineer𠅊nd the biologist—ultimate flexibility over system and experimental design given the plethora of options, it also implies that those involved with the experiment must be aware of all the available choices so that designs can be optimized according to the application. We highlight here the types of materials and options for control that have been largely accepted as the major classes within the microfluidics community, and discuss some of the new directions being pursued.
2.3.1. Device materials and fabrication
Similar to other niche areas within microfluidics, cell-based studies made the largest strides after the introduction of soft lithography. Soft lithography was popularized by Whitesides and his group at Harvard in the late 1990s, and comprises a set of fabrication techniques similar in concept to photolithography, but with significant benefits for biochemistry and biology. 19 The most popular material used in soft lithography is poly(dimethylsiloxane), or PDMS, a silicon-based elastomeric material with a number of physical and practical properties that make it desirable for experimentation. PDMS is fairly cheap and easy to mold, making it ideal for rapid prototyping of microfluidic designs and for transferring micropatterns with high fidelity via stamping techniques. PDMS is suitable for cell experiments because it is non-toxic to cells, is gas permeable, and has excellent optical properties, including low autofluorescence and optical transparency for imaging applications. Furthermore, the elastomeric properties of PDMS allow it to readily deform when subjected to local displacements, allowing the integration of built-in valves and pumps via multilayer soft lithography. 20 To make enclosed microchannels, PDMS can be bonded to different materials (e.g., PDMS, glass, polystyrene) quite easily using various methods such as oxygen plasma treatment and additional curing.
While the many advantages of PDMS have established its well-known popularity among microfluidics researchers, a growing number of reports are beginning to reveal some unfavorable characteristics of PDMS that may limit its future use for microscale cell culture. Kim and co-workers alluded to these challenges in a previous review on microfluidic perfusion systems. 21 More recently, however, work from our group has revealed that PDMS further confounds cell culture results by sequestering small hydrophobic molecules such as estrogen, and by leaching out uncrosslinked oligomers from the PDMS bulk during culture, which then bind to cell membranes. 22 Also, as cells are exposed to PDMS for longer durations, cell metabolism and proliferation are affected, possibly as a result of the presence of PDMS. 23 This growing awareness of the potential artifacts and biases associated with PDMS is providing an impetus for the microfluidics community to consider other options for materials.
The most logical choice for an alternative material for devices is polystyrene because it is the most common plastic used for traditional cell cultureware. Polystyrene microfluidic devices for cell culture applications have recently surfaced in the commercial market (e.g., Bellbrook Labs, Integrated BioDiagnostics), illustrating that the industry has already recognized a need to conform to the needs of biologists who are accustomed to certain materials. In academic research laboratories, micromolding techniques for fabrication of thermoplastic microfluidic devices has also been well-documented. 24 A recent report from the Takayama group has employed a hot embossing technique using epoxy molds to fabricate polystyrene-based microfluidic devices for cell culture, with potential to incorporate soft substrates such as polyurethane or PDMS as a bonded surface. 25 In yet another example, rapid prototyping of polystyrene microfluidic devices has been achieved by the use of “Shrinky-Dinks” thermoplastic sheets. 26 Shrinky Dinks plastics are an arts and crafts toy for children with the property that drawn artwork on the plastic surface can be shrunk in size after heating the material. This property was recently exploited by Khine and co-workers to produce positive relief masters for microfluidic devices. Together, these developments are revealing a trend toward microfluidic devices made with more common bioware materials, as well as a trend against further investments into materials with undesirable and unknown effects on cultured cells. For the foreseeable future, however, PDMS will continue to provide an affordable rapid prototyping option for most research laboratories, and will work in concert rather than in competition with other materials such as polystyrene.
Lithographic techniques allow for infinite possibilities when it comes to geometry, but for cell culture applications, some important considerations must be recognized. First, microchannel dimensions for cell culture are typically at the larger end of the spectrum for channel sizes, ranging from 100 to 1000 microns. Smaller microchannel cross-sections, on the order of tens of microns, are common in chemical applications such as electrophoretic separations, and are more suitable for single cell analyses or for chips designed for cell sorting and cell manipulation. These applications have been reviewed elsewhere. 4 , 5 , 27 For biological experiments, unless single cell analysis is coupled with high throughput methods for measuring endpoints, enough cells will need to be cultured in the microchannels to permit population-based analyses, and this implies a need for larger channel culture regions.
The flexibility in geometry permits the generation of stable gradients in both soluble and surface-bound factors. 11 This is particularly useful for controlling the cell microenvironment in chemotaxis studies (as well as durotaxis and haptotaxis studies) where spatial and temporal concentration gradients can induce cell responses such as migration. 28
An important geometric consideration in microfluidic channels is the height-to-width aspect ratio, especially for PDMS-based devices. While the deformability of PDMS was beneficial for fabricating multilayer devices with built-in valves and pumps, the same property leads to undesirable sagging and bulging of microchannel walls when (1) the PDMS layer is thin, (2) pressure is substantial, and (3) maintaining channel cross-sectional shape is important for analysis, such as in shear flow experiments. 29 This issue would be less important for devices made of stiffer materials, such as glass or polystyrene.
2.3.3. Pumps and valves: going tubeless
Within microfluidic cell culture devices, fluid volumes must be transported and displaced from region to region, using valves and pumps that are either externally connected to the device, or directly built into the system. Inlet and outlet ports of the system serve as points of interaction between the culture region and the external world. The majority of systems employ external pumps (e.g., syringe pumps for non-recirculatory flow, and peristaltic roller pumps for recirculatory flow) that can be hooked up to the access ports via tubing. This is the method of choice for perfusion systems that rely on constant fluid flow to replenish nutrients and remove waste products in a timely manner. Using multilayer soft lithography, it is possible to incorporate pneumatic valves into the system to produce fully automated, high-throughput culture systems ( Fig. 2 ). 30 Such a system provides a concrete example of the many benefits associated with microfluidic cell culture. The major concerns with this type of system are the large number of connections required, the potential for leakages at those connections, the technical expertise necessary for proper operation, and the need for elastic materials that possess properties with some confounding issues (see section 2.3.1). These concerns will likely prohibit the widespread use of such complex integrated systems in the research community at large.
A fully automated PDMS-based microfluidic cell culture system consisting of 96 individually addressable cell culture chambers (blue dye), on-chip peristaltic pumping (lower right inset), and multiplexing capabilities that allow different mixtures of reagents to be formulated. Reprinted with permission from Gomez-Sjoberg et al 30 Copyright 2007 American Chemical Society.
A particularly interesting method for pumping and valving fluids in microfluidic channels was developed by the Takayama group, and employs Braille pin displays to deform thin PDMS layers into microchannels in specific sequences to generate peristaltic flow. 31 While the design and concept are both novel, it also has not been widely used in research circles, likely stemming from the challenges associated with the complexity of the equipment, and from the unconventional nature of the platform.
An alternative method of fluid replacement, which has potential for widespread acceptance because of its simplicity and compatibility with existing techniques in biology, is passive pumping. First developed by the Beebe group in the early 2000s, passive pumping relies on the surface tension of different-sized droplets placed at the inlet and outlet ports to drive fluid from one port to the other ( Fig. 3 ). The difference in droplet volumes induces a differential pressure between ports that generates flow in the microchannel. 32 The major advantage of passive pumping is that it can be performed without connecting to an external pump, eliminating the need for tubing and interconnections at the ports. Passive pumping can be performed simply by pipetting the appropriate volumes of droplets at the ports, and is therefore amenable to automated liquid handling systems that are commonly used in major biology research laboratories. 33 The ability to do experiments with “tubeless” microfluidics is likely to attract an increasing number of biologists to the microscale techniques that are currently being developed. In fact, other researchers have recently begun to study the physical phenomena related to passive pumping in their own microfluidic systems, 34 , 35 demonstrating that the technique is receiving support. While passive pumping is an effective method for many applications, it is limited to low volume flows and low pressures. Steady continuous perfusion of microchannels is better achieved with external pumps, even though droplets can theoretically be pipetted continuously in a passive pump cycle that simultaneously adds droplets at the inlet while removing droplets at the outlet.
Passive pumping relies on surface tension of small droplets to pump fluid from inlet to outlet. A smaller drop of radius Ri has an internal pressure Pi greater than the pressure in a larger drop of radius Ro because of Laplace’s law (ΔP = 2γ/R), where ΔP is the pressure difference across the liquid𠄺ir interface of the droplet, and γ is the surface tension at the interface.
Certain on-chip accessories can be incorporated into microfluidic devices to add extra functionality to the cell culture systems. Three-dimensional networks are achievable by incorporating commercially available track-etched membranes (polycarbonate or polyethylene terephthalate) into the device during fabrication. The membrane serves as a semi-permeable barrier that separates microchannels on different horizontal planes, allowing communication only at locations where microchannels intersect. Several groups have begun using this geometry for cell-based studies. 36 – 38 The most notable is the Takayama group, who have employed membrane-PDMS devices to study lung epithelial cell rupture, and more recently, the adhesion of cancer cells on a microfluidic endothelium ( Fig. 4 ). 36 , 39 The usefulness of this arrangement lies in the in vivo-like organization of the endothelium into luminal and abluminal compartments separated by a membrane that mimics the basal lamina. Such compartmentalization is potentially useful for coculture studies that involve communication between endothelial cells and neighboring cells from the stromal or smooth muscle layers.
PDMS-based microfluidic device containing a porous polyester membrane for supporting growth of endothelial cells. Device was used to study adhesion of cancer cells on the endothelium in the presence of chemokines released on the basolateral side of the endothelium. (Open access: Song et al., PLoS One, 2009, 4(6) 5756.)
What’s it all about? Microfluidics
This installment of “What’s it all about?” will attempt to demystify the exciting miniature world of microfluidic technology, particularly as it pertains to cell biology and chip-sized cell culture systems.
What are microfluidics?
The term microfluidics can be used for both the physics-based study of how fluids move through small spaces and for the application of micro-sized fluid-filled devices generally thought of as “labs on a chip.” Here we will focus on the latter and how these microfluidic devices function in cell biology research. Generally, microfluidics for cell biology represent a mini cell culture system where a single cell or a few cells are seeded into a device with input and output channels. These cells are exposed to dynamic fluid flow, often accompanied by live imaging. The devices are engineered to fit the experimental conditions—meaning, what flows in, what flows out, flow rates, mixing, and extracellular microenvironment, are all controlled very precisely. This has created a field in which microfluidic devices can vary to a large degree.
Though there are many variations of microfluidic devices ranging in complexity, they do have one unifying feature. They are small (Figure 1). Their micro size confers many advantages, probably the most obvious being the reduced volume needed for reagents. Typically microfluidic devices work in the nanoliter range of fluid volume, meaning fewer cell media components and less of the expensive experimental compounds. It also means fewer cells, which can be of particular importance if you are working with a precious cell population, like primary patient-derived cells. Not only are the volumes inside the device small, but making a microfluidic device also requires a micro amount of materials (material costs reviewed here). Using less stuff means using less money, something most labs can get behind in the current state of public funding availability. In addition to the small size of microfluidics being a reagent savings advantage, it may also make more sense biologically. Traditional cell culture techniques have not changed for over a century now: the classic large, round dish or flask filled with 8–15 ml of red media. This is obviously not how cells live in an intact organism. Downsizing to smaller, tighter-packed, mini-tissues may give us a better replica of an in vivo situation (Figure 2).
Adapted from https://www.sciencedirect.com/science/article/pii/S0958166913006794?via%3Dihub
In addition to their size providing unity to the field, microfluidic devices are also generally made using the same technique, soft lithography. This technique is about making things out of elastic-type or “soft” materials using stamps and molds. The most commonly used material is poly-dimethylsiloxane (PDMS). Though PDMS is still used the most, new materials are constantly being tested. In general, scientists are seeking something more biocompatible because PDMS can be toxic and must be coated with extracellular matrix proteins to make it adhesive for cells. Some recent advancements have been made using different kinds of plastic, hydrogels and even using paper!
What are the applications in cell biology research?
So what are people actually using these tiny devices for? There seem to be two broad classes of applications in academic cell biology research: 1) the high-throughput testing of single cells in specific microenvironments and 2) tissue engineering approaches at making better in vitro models. I’ll go into more detail of each below.
Somewhat similar to what you see when looking at a traditional hemocytometer, thousands of cells can fit in one microfluidic device, but they can now be separated into personalized microenvironments via multiple chambers. These multi-chamber devices allow you to analyze thousands of replicates or thousands of different conditions. Introducing active flow allows you to expose the cells to gradients of different compounds—a technology particularly applied by those studying cell migration and chemotaxis. In addition, the soft nature of the device material allows you to also introduce gradients of stiffness, mainly utilized by those who study durotaxis. Combined with live imaging, precisely and dynamically controlling thousands of cells’ microenvironments and watching what they do in response is a major application of microfluidics. You are also not just limited to measures of cell behavior. Systems have also been developed to lyse each individual cell to get at what is going on inside. Combined with single-molecule detection techniques, you can measure the levels of mRNA, protein, etc., in response to changing the microenvironment. For more details on microfluidics for high-throughput single cell analysis check out this review.
Here, the goal is not to just increase the speed, efficiency, and quality of data obtained by traditional in vitro cell culture approaches, but instead to better replicate an in vivo situation. Intact tissues are under constant flow and also exist in 3D. Microfluidic tissue engineering devices don’t have repeating chambers generally, but have sophisticated 3D set-ups where often multiple cell types are seeded. Flow is then used to control the supply of nutrients, as it is in vivo. It has been particularly effective in replicating vascular tissues and how cancer cells are disseminated by extravasation into the bloodstream. For more details on microfluidics and 3D culture see this review.
What’s happening now and where is this field going?
Microfluidics in academic research remains an interdisciplinary field of collaboration between cell biologists and engineers. These collaborations are continuing to find better ways to do cell culture experiments. On the other hand, the biomedical industry is just starting to vastly expand its use of microfluidics. Some of the major new developments have utilized microfluidic devices for medical diagnostics and research and development of new therapeutic drugs. On the medical diagnostic side, for example, using a microfluidic device for a simple blood test means using less blood and vastly improves processing times. Remarkably, a recent study has been able to demonstrate the usefulness of an “in the field” microfluidic device for the successful detection of HIV. This could have huge implications for emergency and low resource-type medical settings. Research and development teams working on testing new drugs also are beginning to implement microfluidic approaches. Here they aim to use the tissue engineering-type devices to more accurately test new drugs on an in vivo-like system before moving to costly animal and clinical trials. If you can test for compatibility and side effects on an organ-on-a-chip model (reviewed here) ahead of time, the speed at which useful drugs make it to market could be improved.
Data presented from https://www.ncbi.nlm.nih.gov/pubmed/?term=microfludics
Using microfluidics in your own research
The ease with which you can implement microfluidics into your research will greatly depend on the strength of your collaborations. This technique is still deeply rooted in engineering the materials and the resources out there for a lone, starting cell biologist are few. You can view this international list of companies producing microfluidic products or, if you really just want to try it, consider this starter kit. Most labs that are using microfluidics routinely are appointed in engineering and proficient at soft lithography. Find one of these labs, talk to them about what you want to do, and be willing to work with them on it with hands-on training on these techniques. Starting something new in the lab is always a challenging endeavor, but you can make it happen!
We can be sure of one thing: microfluidic systems have already accomplished a great deal for cell culture, especially for improving the physiological relevance and giving unprecedented momentum to 3D cell culture. Microfluidic cell culture has proved its worth but needs to make more of an effort to leave the comfortable academic home that has done so much to nurture it. Indeed, there seems to be a disconnect between academia and industry 18,25 , typical of other fields too, whereby the pressure to publish drives “certain academics to try to do the next big fancy thing there is a need to listen to the voice of the customers”, as Prof. Toner points out. In other words, much of academic research in microfluidic cell culture is pushing the technology it might be up to companies to instead find the market that pulls the technology. Companies might also be better incentivized to focus energy on improving the practical aspects of device design and fabrication that might have fallen by the wayside in academia, and adapt microfluidic cell culture to robust industrial workflows which cannot be as finnicky as that often found in academic labs 25 . At any rate, while the field of microfluidics is ready to leave the academic nest it will not cut ties: it might just be time to learn to be marketable outside of academia while keeping strong links with it. After all, who says adulthood should be entered alone?
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What is PDMS used for in microfluid models? - BiologyChristian G. M. van Dijk, /> a Maarten M. Brandt, b Nikolaos Poulis, a Jonas Anten, a Matthijs van der Moolen, a Liana Kramer, a Erik F. G. A. Homburg, c Laura Louzao-Martinez, /> a Jiayi Pei, a Merle M. Krebber, a Bas W. M. van Balkom, a Petra de Graaf, /> d Dirk J. Duncker, b Marianne C. Verhaar, /> a Regina Luttge c and Caroline Cheng* ab
a Department of Nephrology and Hypertension, University Medical Center Utrecht, PO Box 85500, 3584 CX Utrecht, The Netherlands
E-mail: [email protected]
Fax: +31 (0) 88 7556283
Tel: +31 (0) 88 7557329
b Experimental Cardiology, Department of Cardiology, Thorax Center Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands
c Department of Mechanical Engineering, Microsystems Group, Materials Technology Institute (MaTe) and, ICMS Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands
d Department of Urology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands
Microfluidic organ-on-a-chip designs are used to mimic human tissues, including the vasculature. Here we present a novel microfluidic device that allows the interaction of endothelial cells (ECs) with pericytes and the extracellular matrix (ECM) in full bio-matrix encased 3D vessel structures (neovessels) that can be subjected to continuous, unidirectional flow and perfusion with circulating immune cells. We designed a polydimethylsiloxane (PDMS) device with a reservoir for a 3D fibrinogen gel with pericytes. Open channels were created for ECs to form a monolayer. Controlled, continuous, and unidirectional flow was introduced via a pump system while the design facilitated 3D confocal imaging. In this vessel-on-a-chip system, ECs interact with pericytes to create a human cell derived blood vessel which maintains a perfusable lumen for up to 7 days. Dextran diffusion verified endothelial barrier function while demonstrating the beneficial role of supporting pericytes. Increased permeability after thrombin stimulation showed the capacity of the neovessels to show natural vascular response. Perfusion of neovessels with circulating THP-1 cells demonstrated this system as a valuable platform for assessing interaction between the endothelium and immune cells in response to TNFα. In conclusion: we created a novel vascular microfluidic device that facilitates the fabrication of an array of parallel soft-channel structures in ECM gel that develop into biologically functional neovessels without hard-scaffold support. This model provides a unique tool to conduct live in vitro imaging of the human vasculature during perfusion with circulating cells to mimic (disease) environments in a highly systematic but freely configurable manner.
Polymers are widely used in the fabrication of microfluidic devices because of their good bio-chemical performance and low-cost. Among them, one of the most used is PDMS. The acronym PDMS indicates the Polydimethylsiloxane, a mineral-organic polymer of the siloxane family. This material can be found in food as an additive (E900), in cosmetic products and lubricating oils. The reasons that make PDMS a very good material for chip fabrication are several, the main are here summarized:
- Transparency: micro-channels and their content can be seen directly
- Elasticity: PDMS is quite elastic and this propriety can be used for various applications, e.g. valve integration through channel deformation. Furthermore its elasticity can be “tuned” using cross-linking agents
- Cost : PDMS is much more less expensive than other materials used for microfluidic chip fabrication
- Permeability: PDMS is gas permeable which can be used in cell culture, gas sensors, etc..
Image courtesy of A. Håti and J. Ribe (NUST)
Obviously, there are also shortcomings in using PDMS for microfluidic chip fabrication. Some examples are: the aging of the material which limits the performance of the chip during the years and the poor chemical compatibility with many organic solvents which makes PDMS suitable mainly for aqueous applications.
Furthermore, PDMS absorbs hydrophobic molecules and water vapor that can be then accidentally emitted during the experiment. Another drawback of PDMS microfluidic devices is the impossibility to implement electrodes within the chip, even if this can be worked around by putting the electrodes in the glass cover slide instead of the chip itself. More can be found on PDMS and its application to microfluidics at Elvesys site. As mentioned above, while PDMS allows fast and easy microfluidic chip fabrication, it has various shortcomings that prevent its use in some application. In order to come over these drawbacks, but still benefit of inexpensive and fast production, other polymers can be used to build microfluidic devices. One example of another polymer used for microfluidic chip is Polystyrene (PS) which is a polymer of standard use in drug research for cell-culture dishes. PS is optically transparent, biocompatible, inert, rigid, and its surface can be readily functionalized.
Furthermore, its hydrophobic surface can be easily made hydrophylic by various physical and chemical means, including:
corona-discharge, gas plasma and irradiation. Others often used polymers for microfluidic devices are Polymethylmethacrylate (PMMA) and Polycarbonate (PC). PC presents a better thermal resistance than PMMA and thus it can be used in a wider range of temperature. PMMA is an elastomer with little deformation in comparison with PDMS and hence is employed when rigidity is required, e.g. for construction of canalization of microvalves.
Microfluidics and Biological Research: The perfect partners?
For years, microfluidics technology has promised a revolution in biological and biomedical research. Opinion leaders predicted that microfluidics would allow analysis of biological structures at an unprecedented level of efficiency and accuracy and would change how everyday research was conducted. Whilst that paradigm shift has not yet occurred, microfluidics has proved its use in biological research. What is microfluidics? What roles can it fulfil at the bench, and what obstacles remain in the path of a technology that seems to offer so much?
Microfluidics, at its core, is the study of fluids at a microlitre volume or below, flowing through spaces that are micrometres or less in size. The basic microfluidic devices used in biological research consist of miniscule microchannels etched onto a base material. The miniaturisation of electronics was a key influence in the development of microfluidics technology, and, as with microelectronic chips, the original materials used in microfluidics were silicon and glass, although these have now been replaced by less rigid substrates such as poly(dimethylsiloxane) (PDMS) that work better alongside biological samples. These etched channels combine to form a system where tiny volumes of liquid can be channelled and mixed.
The tiny dimensions used in microfluidic analysis produce interesting physical quirks. The flow of the liquids will be largely laminar, meaning that two parallel liquid flows will largely not mix. This facilitates prediction of liquid flow and makes control of the sample environment far easier. In addition, reaction times are faster and reagent consumption is far lower than for conventional macroscale reactions.
Microfluidic analysis allows high-throughput analysis of samples with minute volumes, using assays with streamlined experimental procedure. This marries well to microbiological studies, which use complex assays and very small samples.
What are the different applications of microfluidics in biological research? The miniaturisation and streamlining of complex biological assays has long been a goal of microfluidic research. The term “lab-on-a-chip” refers to the integration of several lab-based assays onto a microfluidic chip. DNA amplification and detection using Polymerase Chain Reaction (PCR) is one widely-used biological technique that has benefitted from the use of microfluidic technology. Usually, PCR requires several shifts in temperature, called thermal cycling, that facilitate the separation and recoupling of DNA strands. These changes in temperature take far less time in the small space of a microfluidic chip, which means microfluidic devices perform PCR in a fraction of the normal time for example, in 2014, Farrar and Wittwer were able to perform 35 cycles of PCR, which normally take a few minutes each, in just 14.7 seconds using a customised microfluidic device. Assay specific devices, such as heaters to change sample temperature, can be integrated onto the chip, meaning the entire assay can be conducted using a single device.
Microfluidic miniaturisation has been applied to a huge range of macroscale assays, including ELISA, chemotaxis assays and cell cultures. As for PCR, these assays often exploit the intrinsic properties of analysis at minute dimensions to improve the techniques beyond what could be accomplished in traditional assays. Control of culture environment for individual cells is inexact in a petri-dish, but can be tightly controlled in a microfluidic culture chip. These innovations can lead to analyses that would simply not be possible in a larger setting and move beyond simply miniaturising larger experiments.
The pharmaceutical industry has been plagued in recent years by spiralling costs, forcing it to develop new techniques to reduce overheads. Microfluidic innovation is playing a role in these changes the promising “organ-on-a-chip” technology seeks to reduce cost and increase efficiency in drug development as “lab-on-a-chip” assays have done in microbiology. Microfluidic chips have been designed to replicate in vivo organ structures, increasing the therapeutic relevance of findings produced by model studies. The complex microenvironments experienced by cells in the human body are replicated to a degree of accuracy not achievable with other in vitro techniques the “artery-on-a-chip” developed by Axel Gunther and colleagues at the University of Toronto places a segment of artery into a loading chamber in the centre of a chip. From there, the artery can be kept at a constant pressure and temperature and perfused with drugs to allow pharmacological analysis.
Microfluidic applications in biological research are not confined to chips. Flow cytometry is an analytical technique that allows observation of single cells. To isolate single cells from a complex mix of cell types, a sample is introduced into a tube where two streams of “sheath fluid” are present. The two sheath streams form into a parallel laminar flow, and the sample is forced into a single file cell stream in a process called hydrodynamic focusing.
Microfluidics can improve on existing techniques, produce new analytical possibilities, and has potential both on and off chip. Why, therefore, has it not become ubiquitous in research labs worldwide?
Knowledge transfer between the engineers developing microfluidic technology and the biologist end-users is certainly a limiting factor. A review by Sackmann and colleagues in 2014 identified that 85% of recent microfluidic publications were in engineering journals. Increasing interaction between these groups of scientists will surely speed up the adoption process. Simplifying the design further will also be helpful – current technologies still often use external pumping equipment that would be difficult for anyone not well versed in fluid physics to understand. Nevertheless, the field is simplifying the cheap production costs and versatility of microfluidic technology means it can be easily adapted to create diagnostic tests for clinicians in developing countries using paper as a substrate in place of PDMS. Simple assays using these paper-based devices have already been developed that can detect glucose and protein in urine. The exciting advances in medical microfluidics will be explored in a future article.
Microfluidics has immense potential, and whilst the slow rate of uptake may be frustrating for researchers in the field, the potential rewards of developing this technology means they should not be deterred.
What is PDMS used for in microfluid models? - Biology
Microfluidics is the science of manipulating and controlling fluids, usually in the range of microliters to picoliters, in networks of channels with dimensions from tens to hundreds of micrometers. The behavior of fluids at this scale is significantly different from “macrofluidic” behavior. For example, surface tension and capillary forces are more dominant at microscale, and flow is almost always in the laminar regime. These phenomena enable microfluidics to be used for creating exceptional flow control, formation of monodisperse droplets, and passive fluid pumping.
Laminar flow in microfluidics
Surface tension phenomena – droplet generation
Microfluidics is a unique technology that allows experiments to be performed in an environment that mimics the inside of the human body. For example, the smallest capillaries measure only five micrometers and can be modeled by microfluidic devices. An average sized pulmonary alveolus in an adult human being is around 100 microns and alveolar-capillary interface can be modeled by a microfluidics cell culture device replicating air-liquid interface.
The smallest blood vessels measure only five micrometers
Glomerular capillary bundles in the kidney
There are numerous advantages for using microfluidics in scientific research. Notably, microfluidics allow researches to improve the precision of experiments, lower limits of detection, run multiple analyses simultaneously, and reduce sample and reagent consumption.
Microfluidics is a continuously growing field with applications in cell biology research, drug discovery, point-of-care diagnostics and precision medicine, and lab-on-a –chip systems. Some examples of applications include:
- Organ-on-a-chip models
- Single cell isolation and analysis
- Droplet generation and manipulation
- Microbubbles production (for medical ultrasound and drug delivery)
- Cell sorting, cytometry
- Pattern substrates for cells to grow
Microfabrication refers to cleanroom fabrication processes used for fabricating micron scale structures on solid flat substrates. Historically, it has been developed for micro-electronic circuit fabrication, but today it is also widely used for micro-electromechanical systems (MEMS) and microfluidics devices fabrication.
Photolithography is a key technique used in microfabrication to transfer patterns onto the substrates. Fundamentally, it is an optical printing process where light is used to pattern a layer of photosensitive material called photoresist.
Soft lithography is related set of techniques to create microstructure based on molding, embossing, and printing. The terms soft comes from the fact that it utilizes soft elastomers or polymers other than silicon or glass traditionally used in semiconductor industry. The most common substrate is PDMS (polydimethylsiloxane which is rubber like polymer that is exceptionally conformable elastomer). The soft lithography has become popular in cell biology research because it is versatile, simple and relatively inexpensive while providing compatibility with biological materials (cells, gels, polymers). PDMS is biocompatible, chemically inert, transparent to light, permeable to gases, inexpensive and easy to prepare. Soft lithography is an essential method for microfluidics device fabrication.