Microfluidic Cell Culture Systems (Micro and Nano Technologies)

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Borenstein currently serves as Principal Investigator for projects involving the application of microsystems technology towards engineered tissue constructs for organ assist devices and drug discovery, as well as implantable drug delivery systems for hearing loss and other diseases. These programs are funded by the Department of Defense, the National Institutes of Health and several commercial sponsors. His research focuses on the design and testing of implantable microfluidic devices for drug delivery into the ear.

Her research interests lie in the areas of biomaterials, nanotechnology, regenerative medicine, drug delivery, BioMEMS, microfluidics and cell culture. Charest is director of in vitro model and organ-assist work at Draper Laboratory.

Beating heart on a chip

The work of his teams leverages micro- and nano-fabrication along with advanced machining techniques to create systems which recapitulate native tissue and organ architecture, morphology, and function in vitro. The systems span applications from medical devices to screening platforms for pharmaceuticals, and impact fields of use in various organ and tissue types such as tumor, kidney, vascular tissue and lung.

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Citations Publications citing this paper.

Microfluidic cell culture has entered the big league

Blain Christen. References Publications referenced by this paper. A self-contained microfluidic cell culture system Baoyue Zhang , M. Kim , Todd Thorsen , Zhanhui Wang. A gel-free 3D microfluidic cell culture system. A practical guide to microfluidic perfusion culture of adherent mammalian cells. Chandra Prakash , P. Figure 1 summarizes assay variables that can be changed among the different elements of cell chips to induce specific cell responses and understand cell adaptation. The most commonly controlled variables are soluble inputs, which can be added or removed from the culture medium.

They consist of standardized medium, metabolic substrates, vitamins, antibiotics, unspecified additives derived from animal serum, and of stimuli such as cytokines, growth factors, hormones, and putative therapeutic molecules added at various doses, combinations, and for different periods. Both assays require cell lysis, thus hindering precise and real-time space and time analysis.

The major limitation of these techniques is thus the fact that they do not allow time-course studies on the same biological sample. Multiple parallel cultures need to be set up and destructively analyzed at the desired time points, thus increasing sample-to-sample variability and potentially masking other relevant effects. It was used to estimate rates of gene expression [ 16 ], detect specific cells in vivo [ 17 ] or as biomarker or biosensor [ 18 ].

Even if the discovery of GFP can be considered as a breakthrough for the development of live-cell imaging, quantifications of time-lapse images still need corrections for the auto-fluorescence of culture medium, and methods to track objects and cell movement to identify individual cells without using endpoint nuclear staining or similar. As shown in Figure 1 , cells represent the central element of the sensor. Cells are extremely complex biological entities and incorporate a large number of variables.

Thus, particular attention must be paid to the identification of the best cellular response to measure and to the corresponding optimal sensor type capable of detecting it. Particular attention needs to be paid to the intrinsic heterogeneity of a cell population, determined by phenomena such as asynchronous cell division leading to the presence of cells at different cell cycle stages within the same population.

Although several methods allow the synchronization cells by morphological features, cellular metabolism or chemical compounds [ 25 ], chemical synchronization can potentially disrupt normal cell cycle regulatory processes [ 26 ] and the difficulty in elucidating the exact cellular target remains [ 27 ].

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An implicit assumption of conventional assays is that the measured average response is representative of a typical cell within the population. It is evident how one of the biggest drawbacks of conventional cell assays WB; ELISA is the fact that they measure average responses of large cell populations, with the aforementioned heterogeneity potentially obscuring cell-specific responses.

For these reasons a single-cell analysis approach is preferable to dissect cell-type specific behaviors avoiding misleading oversimplifications of averaged responses. Single cell analysis has already been used in gene expression [ 28 — 33 ] or genome analysis reviewed in [ 34 ]. Our group evidenced that in a complex tissue such as skeletal muscle, the analysis of purified single fiber significantly increased the resolution power of the assays [ 35 ]. Being a syncytium, the skeletal muscle fiber cannot be properly defined a single cell, nevertheless it represents the functional unit of the tissue.

By the use of isolated fibers, we demonstrated that most of information from blood, connective tissue, endothelial and neuronal cells associated to myofibers in the muscle is depleted. This approach can be useful for studies of pathology-altered muscle tissue where cellular heterogeneity is emphasized [ 36 ].

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Stem cells are unspecialized cells with the ability to self-regenerate and differentiate in many different cell types and this capacity, defined as pluripotency, is the main motivation supporting their intensive study. They play a central role in an organism allowing development, repair of damaged tissue, and cancer that results from stem cell division going awry. Among other research lines, the increased number of donations of cord blood along with the improvements in their storage and maintenance has enabled the possibility to explore new medical therapies based on stem cells.

Society could benefit even more from this with increases in donor availability and in hematopoietic stem cell transplantation applicability, thus raising the necessity for further knowledge about their biology and use. ESCs are pluripotent cells derived from the inner cell mass of the blastocyst. They grow relatively easily in culture but due to both technical and ethical clues, treatments based on ESCs are limited. An important issue favoring the use of ESCs in regenerative medicine is that they provide a more successful therapeutics than cells taken from older or less healthy donors.

This could be associated to longer telomeres [ 38 ]. The genetic manipulation required to obtain iPSC cells is the major drawback for their use in humans' treatment. ASCs are a small population of cells present in adult tissues that are able to differentiate in some particular cell types depending on their tissue of origin. Given their scarce number in adult tissues, they are extremely difficult to isolate. Many researchers are looking at mesenchymal stem cells MSC for the treatment of cardiovascular diseases [ 41 ] or at adipose derived stem cells [ 42 ] for their abundance in adipose tissue.

Hematopoeitic stem cells HSCs are the best characterized between all the adult stem cells identified. Stem cell plasticity allows bone marrow mesenchymal stem cells not only the function of forming the hematopoietic microenvironment but the ability of becoming neurons [ 43 ], or pancreatic islet cells that are capable of producing insulin [ 44 ]. In addition to general stem cell properties continuous cell cycle progression for self-renewal and the potential to differentiate into highly specialized cell types the International Society for Cellular Therapy ISCT [ 46 ] proposed a more specific panel of markers for the characterization of MSCs.

New tools are becoming available to perform controlled studies on stem cells under conditions that mimic some aspects of the developmental milieu. Here we will discuss some aspects of cell chips applied to stem cells. The components of the stem cell microenvironment that regulate their differentiation include cell—cell and cell—ECM interactions, soluble stimuli and gradients of soluble factors, and the three-dimensional architecture of the niche itself, which shapes and restricts the delivery of these cues.

A brief overview about microarray uses, companies involved in their market and microarray biosensors is available in [ 49 ], while the history of the microarray technology is available in [ 50 ]. Based on the key concepts of microarray production delivery of small volumes of solution, high miniaturization and high throughput assay , the cell microarray was developed. Cell microarrays can be divided in two types: those based on the delivery of cells [ 51 ] and those based on the delivery of components micropatterning [ 52 ] allowing cell attachment or their transformation depending on the cells position on the chip.

Cell microarrays inducing cells transformation are typically used to discover gene function [ 53 ]. Spotted cells on chips enable the determination of cellular states following exposure to chemical or genetic perturbations. Differently from living cell microarray, in spotted cell chips cells are first treated using standard cell culture conditions and then printed onto glass slides before being fixed. Technical challenges in the production of cell printed microarray are represented by the pins used to print and the capacity to maintain cells in suspension.

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  8. This last problem was solved by optimizing the viscosity of the suspension medium, while pins were adapted to have slots compatible with cell dimensions Figure 2 and to avoid clotting and cell shear stress [ 51 ]. Microarrays for cell delivery systems allow the analysis of multiple cell types and multiple growth and treatment conditions on a single slide; however, when compared to transfected cell array [ 53 , 54 ], they do not allow the high throughput screening of libraries e.

    Scheme of spotted-cell microarray spotting. Microarray spotter. Cell suspension is collected from a well plate by a microarray head supplied with 48 pins. Printing on glass slide.

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    Types of printing pins: 1. Liquid load are 0. Bottom: enlargement of a printed area in the microarray slide. In A4 are showed nine spots of solution optimized for printing cells with a type 4 pin. B4 shows an enlargement of a spot from A4. C4 shows a spot of printed cells C2C12 cell line. High throughput screening of gene function is of primary importance, especially after the publication of the human genome [ 57 , 58 ].

    This event pushed the development of new sequencing technologies next-generation sequencing is reviewed in [ 59 ] making the whole genome analysis a more affordable task and extending gene identification also in non-model organisms. The ability to produce libraries of interfering RNA RNAi through chemical synthesis [ 60 ] or by enzymatic digestion of long double stranded molecules esiRNAs [ 61 ] allows the selective silencing of practically every gene of an organism. This ability, associated with fluorescence microscopy, provides a uniquely detailed phenotypic readout of cultured cells to discover gene function.

    RNAi using cell microarray reverse transfection , as opposed to RNAi on plate assay, has the advantage of miniaturization and therefore an enhanced throughput. Moreover, cell microarrays allow the delivery of complex stimuli such as concentration gradients, which are hard to generate in plate-based screenings. Furthermore, miniaturization allows the sparing use of RNAi reagents and rare cell lines such as adult stem cells.

    Gene function can be understood also by the transfection of libraries based on recombinant plasmids and viral vectors. In both cases gain-of-function can be tested as well. Cell microarray associated with cDNA printing was used for the identification of drug targets or to discover the gene function suggested by altered cellular physiology [ 54 ]; even if the overexpression of a specific gene may cause an altered phenotype confusing its real function in normal cell. Transfection of cDNA through cell microarray was also used in the characterization of the regulative elements of cAMP-dependent protein kinase [ 62 ] demonstrating the feasibility of high throughput approaches in transcriptional regulation.

    Loss-of-function was first used to select functional siRNA against MyoD gene [ 63 ], since not all RNAi sequences are equally efficient in the down regulation of the target gene. This technique was applied in many other studies to characterize different pathways p53 pathway [ 64 ], human proteasome [ 65 ] and NF-kB pathway [ 66 ] or in the D. Not only RNAi and recombinant plasmids were used in cell microarrays but also the spotting of small molecules was applied in order to monitor their effects.

    Spotting of small molecules to test with cell microarrays was obtained by embedding them into biodegradable materials to avoid diffusion [ 69 ]. In the next paragraphs we will discuss the advances of the cell microarray technology focusing on stem cells. Stem cell microarray Reverse transfection was used not only with somatic cells, but also with stem cells [ 70 ]. The increasing importance of these cells in regenerative medicine make essential the comprehension of molecular mechanisms involved in the maintenance of pluripotency and in the activation of differentiation.

    In this context, RNAi represent a powerful strategy for the discovery of gene function. Yoshikawa et al. Interestingly, transfection on solid surface is affected by the deposition of an ECM protein in conjunction with DNA to be transfected [ 70 ]. ECM proteins regulate cell signaling interacting with cell receptors and integrins [ 71 ], but they can also act as microenvironment determinant establishing availability and gradients of growth factors [ 72 ]. Moreover, domains in ECM fibrils e. The ECM degradation itself, obtained through lytic enzymes or metalloproteinases, results in the release of either EGF-like domains or of the ECM-linked growth factors making them available in a cell specific microenvironment.

    We previously proved that the cell microenvironment and the substrate elasticity are fundamental determinants for the behavior of adhering C2C12 cells an embryonic muscle cell line. Among the pioneering works in this field we should cite two publications from the Langer [ 76 ] and Bhatia groups [ 22 ], reporting important data for the comprehension of the ECM-cell interactions. They characterized the stem cell behavior in relation to different contact surfaces.

    In particular, Anderson et al. Moreover, they demonstrated that certain monomers inhibited ESC attachment or spreading, thus excluding their use in the production of ESCs-populated scaffolds. Flaim et al. Collagen IV best allowed the maintenance of primary rat hepatocyte phenotype, while a mixture of laminin, collagen I and fibronectin allowed a better differentiation of mouse ESCs toward a hepatic fate. This result dramatically increased the number of possible combinations of the ECM components that was recently used by Brafman et al.

    Differentiation of ESCs is typically obtained with the employment of embryoid bodies EBs [ 79 ], addition of cytokines to culture media [ 80 ] or co-culturing with feeder cells [ 81 , 82 ] for a review, see [ 83 ]. The co-culture method seems to be the most efficient [ 84 ], but it is dependent on feeder cells, among others. Cell microarrays were used for screening feeder cells for ESC differentiation [ 85 ]. These applications demonstrate the flexibility of cell microarray technology and the possibility to integrate different information recovering fundamental data in ESCs survival, duplication and differentiation.

    As stated before, cells live within a complex microenvironment that plays a crucial role in normal and pathologic conditions. In order to use cultured cells as models for tissue processes, researchers have to design complex patterns and structures able to mimic the in vivo microenvironment in an in vitro setting.

    Microfabrication technology helps in the production of these tools allowing the culture of cells on well-defined surfaces, patterning of cells into defined geometries, and measurements of force associated with cell-ECM interaction. Self-Assembled Monolayers SAM are used with surfaces such as gold [ 86 ] while metal evaporation with specialized masks, initially used in mids [ 87 , 88 ], allows the production of specific adhesive areas for living cells on non-fouling backgrounds. This method is however not easily adaptable for general uses in biological research. Some years later, the advent of microcontact printing and soft lithography allowed the production of chips competent for cell adhesion only in definite areas [ 89 , 90 ].

    Initially, through photolithography, a mold with an array of micrometer-sized features was designed and used to produce a complementary elastomeric stamp usually polydimethylsiloxane, PDMS. Stamps can be inked with silanes, alkanethiols covalently linked to gold or with ECM proteins [ 91 — 93 ] and used to transfer the inked material to the receiving surface Figure 3 A.

    Micropattering can define cell adhesive regions with a 50 nm resolution that is limited by the method used to generate the mold. These techniques have been largely used, and in our groups mainly to study skeletal muscle cells [ 75 , 94 — 96 ]. The production of functional cardiac and skeletal muscle tissues is certainly a challenging task, since they are composed in vivo by a complex aggregate of cells strictly associated and communicating through gap junctions in the heart [ 97 ] or forming a syncytium in skeletal muscle [ 98 ].

    We evidenced how microcontact printing of ECM proteins allows the orientation of single muscle cells and the formation of mature and functional myofibers [ 96 ] Figure 3 B. The tool can be used for pharmacological or biological studies at the single fiber level. Moreover, we demonstrated that electrical stimulation in association with cell orientation is able to improve differentiation of muscle cells [ 95 ].

    It is now widely assessed that the mechanical properties of the substrate where cells adhere greatly influence and guide cell proliferation and differentiation [ 99 , ]. To print adhesion proteins for myoblast cells adhesion we used a thin film of photo cross-linkable elastic poly-acrylamide hydrogel because of its physiological-like and tunable mechanical properties elastic moduli, E: 12, 15, 18 and 21 kPa.

    We demonstrated that substrate stiffness regulated the extent of myotubes formation and maturation, with higher percentages measured on substrates with in vivo -like stiffness [ 75 ]. We also modulated the spatial organization of cells demonstrating that wider adhesion lanes showed a decrease in murine myoblast proliferation while fusion index increased in narrower lanes. Our results underline the role of micropatterning in shaping the cellular niche through the accumulation of secreted factors [ 94 ].

    Patterned surfaces have also been used to investigate the effects of cell—cell contact in a well-controlled fashion. Traditional methods are based on seeding cells at different density, but this method produce cells with different sizes and shapes in function of the density. Using a bowtie-like pattern Nelson positioned two cells near each other to demonstrate that cell-cell contact lead to a decrease in cell spreading and proliferation, but not if cell spreading is kept constant [ ].

    Not only homotypic cell interactions were inspected using micropatterning but also heterotypic, being these latter critical for liver and breast functions.

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    For example, Collagen I was deposited in a controlled pattern using photolithography demonstrating that primary hepatocytes cultured in adjacent lines with fibroblasts increase their capacity for urea and albumin secretion [ ]. The soft lithography method. PDMS polydimethylsiloxane stamps are formed by replica molding onto a negative photoresist mold, generated via UV-mediated ultraviolet selective crosslinking through a photomask containing the desired features of photosensitive resins.

    Satellite cells cultured on patterned hydrogel. The inset in III. Interference microscope image shows aligned satellite cells after 7 days in culture IV. Newly formed myotubes express desmin V. Organization in regular and uniform striations of mhc is highlighted on the two insets VI. Cell nuclei were counterstained with Hoechst blue. Modified from [ 96 ]. Stem cell pattering Micropatterning allows precise control of the shape of cell-adhesive islands on a substrate, a known important determinant of stem cell fate.

    An example was provided by Wan's work [ 93 ]. Human adipose stem cells were cultured in differently shaped micropatterned adhesive surfaces, demonstrating a correlation between proliferative condition for more spread cells and differentiative for smaller and elongated cells.

    Microfluidic Cell Culture Systems - 2nd Edition

    We confirmed a similar behavior for human murine satellite cells mSC. Seeding multipotent muscle cells onto organized rectangular micropatterned polyglycolic acid scaffold allowed a better myoblasts differentiation [ ], holding great importance for cell therapy for skeletal muscle disorders. Like cell microarrays, stamp-based micropattering has also proven to be an important technique to examine how cell-substrate interactions influence stem cell proliferation and differentiation, among other phenomena.

    Cell shape and topographical features dictate cell behavior allowing stem cell lineage commitment Table 2. Effect of micropattern shape in cell behavior. Modified from [ ]. In summary, to test how topological features influence cell behavior soft lithography is particularly advantageous thanks to its flexibility in creating patterns with different geometries.

    A drawback of this technique is that it has to be used with adherent cells. Moreover, 2D micropatterns tends to deteriorate over time [ ]. Understanding signals that define stem cell niche can be improved by 3D culture approaches. Exploiting bioengineered scaffolds and nanoscale devices mimicking the mechanical properties of natural tissue would offer new tools for approaching cells spatial organization, differentiation and tissue synthesis.

    Ref.07.18.34 - Research Fellow - Microfluidic Cell Culture – Gut-on-a-chip

    Recent reviews addressed new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidic approaches to create cell culture microenvironments that both support tissue differentiation and recapitulate the tissue—tissue interfaces, spatial-temporal chemical gradients, and mechanical microenvironments of living organs [ 10 ]. Here we will focus our attention on the 3D stem cell culture not discussed in [ 10 ].

    Among other materials, hydrogels can be used for the formation of 3D structures, even though the bigger the scale, the more difficult it gets to control 3D architecture and cell-cell interactions. Moreover, it is hard to replicate the actual complexity of in vivo tissues. Microscaled hydrogels have none of these limitations and in contrast allow minimizing diffusion limitations while maintaining tissue-like microarchitectures [ ].

    Microgels can be manufactured by micromolding, emulsification, photolithograpy and microfluidic techniques. Advantages and disadvantages of each method are summarized in Table 3. The association of monomers composing the gel and crosslink agent determines the mechanical, physical and biochemical characteristics that in turn influence stem cells behavior [ , ]. Yeh et al. Cells spatial distribution was controlled via the micromolded stamps shape, and the technique was used for fabricating 3D microcultures.

    Their constructs are compatible with most immunofluorescence methodologies and most microscopy detection techniques. Significant improvements in the field of stem cell culture and tissue regeneration should include innovative culture systems that integrate sophisticated monitoring platforms to ensure continuous culture evaluations at a cellular level. For this purpose micro- and nano-biosensors constitute promising solutions. Once integrated in the bioreactors they would be able to regulate cell culture parameters closing the feedback loop between measured values and corresponding variations in culture conditions.

    In living tissues the microvascular system modulates the concentration of soluble molecules such as metabolites, gases, therapeutics, and anti-fouling agents. To mimic this functional structure in vitro , microfluidic gels can be used. Moreover, microfluidic flow of ECM precursors and cell suspensions within the hydrogel bulk phase allows the formation of stable patterns of different 3D extracellular matrices interfaced with cell cultures [ ] Figure 4. An alternative for a better alignment of micropatterned protein structures and cells is dielectrophoresis [ ].

    Cells are moved in a heterogeneous electrical field across the hydrogel allowing their accurate positioning inside the 3D structure. Drawbacks of dielectophoresis are related to the use of buffers that are potentially toxic and to the presence of relatively strong electrical fields that induce heating of the solution [ ].

    Here we don't extensively discuss the production of 3D hydrogels for more details see [ — ] , instead we focus on their applications in stem cells analysis. Schematic diagram of a construct consisting of multiple 3D matrices: a microfluidically patterned phase and a bulk microfluidic hydrogel phase. Magnified view of the interface boxed region in A showing the formation of each phase. I The bulk phase is formed by doping collagen into an alginate solution and allowing a collagen fibers network to form by increasing temperature. II The alginate is gelled by ionic crosslinking around the collagen fiber network to complete formation of the bulk matrix.

    As temperature is increased, collagen precursors in the second ECM nucleate and assemble from exposed collagen fibers at the interface to integrate the two matrices. IV Formation of the patterned ECM is completed on gelling of fibrin in this example by diffusion of a thrombin solution into the construct to cleave fibrinogen into fibrin in situ.

    Time-lapse differential interference contrast imaging of collagen fibers assembly at the phase interface. Collagen fibers in the patterned ECM assemble from the collagen-doped bulk phase interface into the polymerizing ECM solution left panel , but do not nucleate from a pure alginate bulk phase interface right panel. HUVECs red are localized to the channel pattern, whereas the fibroblasts green are distributed uniformly throughout a pure alginate bulk phase.

    D and E. Confocal reflectance microscopy. In D , the 3D reconstruction of microfluidically patterned collagen green seeded with HUVECs red in a bare alginate bulk phase confirms that HUVEC-seeded collagen completely filled the channels as opposed to coating the walls and that the phases were separated by the intended sharp boundaries. In E , the 3D reconstruction of the confocal z series through a collagen—alginate bulk phase before microfluidic patterning of collagen.

    We demonstrated that micro-patterned scaffolds seeded with murine satellite cells and implanted in injured mouse skeletal muscle allow a better deliver of satellite cells than direct cell injection [ ] and that constant bioreactor-driven perfusion of nutrients improves cell density and distribution throughout the scaffold [ ].

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    These advantages derive from the improved spatial cell organization and dense cellularization within the scaffold combined with the effect of fresh medium perfusion mimicking blood circulation. Other important applications are the ability to differentiate stem cells in pancreatic islets [ ], neuronal cells [ ], and vascular grafts [ ]. Cardiovascular diseases are one of the major problems in the developed society i. As previously discussed, one of the characteristics that make stem cells research of paramount importance is their ability to recapitulate a diseased or injured condition.

    In this sight, it is fundamental to be able to expand them while preserving their ability to differentiate. The obtainment of high cell numbers is especially difficult for adult stem cells, so their efficient expansion become a crucial step for therapy. Microelectronic cell-based biosensors have the potential of providing rapid, sensitive, low-cost measurement technology. Cells are naturally equipped with a host of receptors that can transduce chemical and biological signals into electrical ones. The on-off behavior of cellular receptors and ion channels induces the migration of charged proteins and ions on both sides of cellular membrane, which could be in turn coupled with microelectronic devices.

    These sensors can be applied to measure extracellular action potentials, impedance, and transmission paths of ionic channels detecting, for example, the transmission velocity of biological signals along layers of neurons. However, successful culture of cells on microelectronic devices is still a challenging issue. The main problem is that the material itself is not attractive to cells in terms of roughness, hydrophilicity, surface functional groups, and stiffness. Further work is needed to improve the surface characteristics of transducers. According to the transduction method, microelectronic cell-based biosensors can be of different nature: microelectrode arrays MEA , electric cell-substrate impedance sensor-based ECIS , field-effect transistors-based FET , light addressable potentiometric sensors-based LAPS , patch clamp chips, surface plasmon resonance chips SPR , and quartz crystal microbalance chips QCM.

    Here we will discuss their structure and applications in particular related to stem cells. MEAs are fabricated by depositing Au, Ir, Pt, or other metals on silicon substrates or glass to form electrodes, connecting leads, passivation layers, and forming electrode sites where the cells or tissues contact Figure 5 A. Given their relatively simple fabrication and good biocompatibility, they have been used in many applications such as cell pattering [ 23 ], drug screening, observing signal transfer of cardiac myocytes [ ] or to evaluate ion signals in neuronal cells [ ].

    For instance, we employed a microelectrode array to perform single cell experiments. Using an on-chip-single-cell electroporation protocol, we transformed cells adherent to electrode with specific molecules [ ]. We were able to modulate the permeability of the cell membrane, which represents a step towards a high throughput gene analysis on single cells. MEA technology still faces some problems. For example, substrate surface is easily eroded when dipped in the culture solution for a long time and the gap between cells and electrodes is difficult to control during cell seeding also in case of cell movements after adhesion to the MEA.

    Cell positioning with respect to the electrodes affects measures. Other than planar microelectrodes, also 3D electrodes were used Figure 5 B , and electrodes shaped as microtips allowed to record signals deeper in the cell layer [ ]. A Schematic diagram of MEA cell-based biosensor. In yellow the electrode and in blue the insulator. It is composed of 60 tip-shaped protruding platinum electrodes. Communication between cells in the nervous system is fundamental for all the complex functions that are provided by this tissue.

    Pathologies of the neuronal cells are particularly debilitating and understanding the regenerative capacity of neuronal cells is challenging. Using a co-culture approach onto MEA chip, Stephens et al. Their chip allowed monitoring a network of cells studying what happens when new stem cells are added and how many cells will be needed to restore brain function.

    The growth of human neural networks of stem cells on a MEA was studied also by Pizzi et al. The chip can be used both to stimulate cells and to record responses to stimuli, as in drug discovery screenings. For example human embryonic stem cell derived neuronal networks were used in neurotoxicological screening during drugs exposure [ ]. Of great relevance is the issue of cardiac and hepatic drug toxicities. To address this problem, Mummery's lab implemented the use of patch clamp analyses and MEAs on human cardiomyocytes derived from hESCs, used as a renewable and scalable cell source more closely resembling functional cardiomyocytes of the human heart [ ].

    Their system was validated for the capacity of performing reliable cardiac safety pharmacological assays. Field potential duration FPD values following exposure to different drugs could be recorded, and drug-induced QT changes in response to selective ion channel blockers were measured, highlighting adverse effects of the tested drugs with greater confidence than standard in vitro assays. Since mesenchymal stem cells MSC can differentiate into multiple tissue-specific cells adipose, bone, tendon, cartilage, muscle, and marrow stroma [ ] , their high throughput characterization would be of a great benefit for use in regenerative medicine.

    Cho et al. Stem cell differentiation was also studied using EICS sensors Figure 6 A , which allow investigating bioelectrical properties of cells. The most important components of EICS sensors are the frequency characteristics and sensitivity that can be attained; in this sight, potential problems might be related to the obtainment of sufficient sensitivities though optimized design of the electrodes. For an exhaustive dissertation about these problems see [ ].

    When cells are stimulated to change morphology or proliferate, the capacity to cover the electrode changes with the electrode impedance. Measuring the current and voltage across a small empty electrode, the impedance, can be calculated. When cells cover the electrode the measured impedance changes because the cell membranes block the current flow. B Time-course measurement of mean impedance at 64 kHz. Clear differences in impedance can be observed between all groups.

    Also, adipose-derived stem cells have been intensively studied for their ease of isolation in high concentration from lipoaspirates [ ] and for being a realistic source of autologous stem cells. Bagnaninchi et al. Other analyses were based on EICS to verify the adhesiveness of stem cells, for example in response to paracrine stimulation [ ].

    EICS is a label-free and noninvasive monitoring technique, a characteristic of paramount importance in stem cells characterization since most other tools end up being invasive and precluding their therapeutic potential. Field effect transistors were first patented in by Julius Edgar Lilienfeld. The device consists of an active channel through which charged carriers flow from the source to the drain Figure 7 A.

    Source and drain terminal conductors are connected to the semiconductor through ohmic contacts that allow the formation of a linear and symmetric current—voltage I-V curve. The conductivity of the channel is a function of the potential applied across the gate and source terminals. The open-gate area of the FET is completely covered by one cell as indicated in the schematics.

    S and D designate the built-in source and drain connections, while B the bulk. B Schematics of 3D device fabrication I. Highlight of extracellular IV. For a review of the development of FET in biological area and specific discussion of cellular signaling see [ ] and [ ], respectively. FET are also used to quantify the extracellular potential of electrogenic cells e. The advantages deriving from the use of FET to monitor cell behavior are: fast response of the sensor, low cost and non-invasive long-term recording processes.

    Similarly to MEA, the distance between the detector and the cells has a strong impact on the sensitivity of FET-based detection. Rather than forcing the cell to adapt to the substrate, Tian et al. We evidenced how the implementation of the third dimension to integrated circuits technology improved performance and functionality [ ]. In fact, being able to distribute on different chips the sensitive low noise analog circuits in low-noise operation from the digital circuits, would lead to improved sensitivity performance and space exploitation.

    Recently, we addressed the issues related to processing and material solutions to accomplish the robustness requirements towards prolonged contact with electrolyte solution and surface cleaning processes [ , ]. Nano-objects such as nanowires [ ] and carbon nanotubes [ , ] have received increasing attention. Nanowires represent a class of inorganic materials that are surface-passivated by thin oxide layer and serve as electrodes or connecting bridges between micro- and nano-electronic devices. Carbon nanotubes exhibit useful properties such as mechanical strength, enormous surface area and large-scale high density.

    However, the extreme sensitivity of nanowires- and nanotubes-based field-effect sensors is hampered by their sensitivity to impurities and other ionic species in the analyte solution. Nonetheless, 3D structures with nanowires will allow fast drug discovery in a more suitable cell environment Figure 7 B. As an example, Tian et al. LAPS were first proposed in by Hafeman et al. Most researchers using LAPS adopted the commercial microphysiometer produced by Molecular Device Corporation [ ] and use them also in the analysis of single cell response [ ].

    LAPS was used in different studies to analyze cells' electrophysiological properties [ ], signaling mechanisms [ ], ligand-receptor binding [ ], and drug analysis [ ]. Here we will address some applications with stem cells. Liu et al. This study is important because is setting the ground for the development of a platform to evaluate cardiotoxicity of new drugs [ ]. Mouse embryonic stem cells cultured on the surface of LAPS were induced to differentiate into synchronized and spontaneously beating cardiomyocytes.

    Since changes of extracellular potentials and cell shape during contractions induce modulation of photocurrents in the LAPS system, it was possible to record the prolongation of ventricular action potentials induced by drugs and correlated it with cardiotoxicity [ ]. Moreover, the sensing of intracellular biomolecules, enzyme activity and pH in real time easily allowed by LAPS can contribute for a better understanding of biological processes in stem cells leading to the development of strategies to control and use them therapeutically.

    Schematic set-up of a LAPS device with living cells and light sources. Mechanisms involved in cell attachment can be analyzed through the use of SPR technique, an optical-electrical phenomenon arising from the interaction of light with a metal surface, enabling the detection of the presence of a biopolymer on chemically modified gold surfaces. The working principle is the change in the local refraction index upon adsorption of light.

    SPR could be used in association with electrochemistry EC-SPR where the thin metal film on the substrate is used not only to excite surface plasmons, but also acts as a working electrode for electrochemical detection or control. SPR, in association with different surface functionalization, may be used to obtain distinct spectra for specific cell types.

    This technique was used to analyze mesenchymal stem cells [ , ]. Kuo et al. The quartz crystal microbalance QCM is a very sensitive sensor capable of detecting small mass changes based on the piezoelectric effect. Some properties of cultured cells had been successfully monitored with QCM, such as cell attachment, proliferation, and cell-substrate interaction.

    Although this technique is not widely applied for stem cells studies [ , ], Pirouz et al. This paper is important for its role in assessing the issue of stem cell-substrate interaction, which as discussed above is fundamental for the understanding of cell differentiation processes. Pirouz et al.

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