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Microfluidic cell culture systems bettinger staffing

Автор: Fek | Category: Kraken crypto radar | Октябрь 2, 2012

microfluidic cell culture systems bettinger staffing

Hydrogel Scaffolds Containing Azobenzene as Dynamic Cell Culture Systems. as microfluidics, tissue engineering50 and optoelectronics In this thesis. initiate, in the host laboratory, the development a microfluidic system to perform continuous cell culture of anchorage-dependent mammalian cells. Modulation via cell culture density or micro-island size can both impact A study culturing MSCs on fibronectin-coated micropillars. HOW MUCH IS 0.05 BITCOIN

The consequences of this are considerable and have led to low success rates in terms of drug efficacy and toxicity in Phase II and III human clinical trials and have added significantly to the cost and time to develop new therapeutic compounds. In this Focus article, we summarize the major techniques involved in developing organs-on-chips along with their application in drug discovery and screening.

We discuss recent progress in four areas: 1 integrated micro-devices for cell culture; 2 three-dimensional 3D cell patterning and culture; 3 multi-layered microfluidic structures; and 4 perfusion-based micro-devices. Within each area, specific examples are provided to illustrate the rationale and characteristics of the individual techniques. In addition, an overview of ongoing efforts in this field and perspectives on future directions, opportunities, and challenges are presented.

Integrated micro-devices for cell culture During the preclinical phase, in vitro cell-based assays have been widely used as the standard method for high-throughput evaluation of new drug candidates due to its simple and low-cost nature in comparison with an in vivo approach. Then a drug-containing medium is added to the larger well, flooding all the smaller wells.

Khetanil et. Then the PDMS stencil was sealed against a polystyrene plate and collagen-I was adsorbed into the exposed polystyrene. The diameter of the through-holes in the PDMS stencils determined the size of the collagenous domains and thereby the ratio of the homotypic to the heterotypic interactions in these microscale cultures.

Semiconductor manufacturing techniques benefit from the compatibility with automated industrial processes, facilitating maximum resolution, accuracy and reproducibility. Conventional photolithography has been used to generate patterns of self-assembled monolayers SAMs and adhesion-promoting molecules, such as poly-D-lysine PDL for neural networking 35 and collagen for NRVM structuring In a recent study, commercial pMEAs were coated with poly ethylene glycol PEG silanes which were selectively removed by laser ablation In another study, custom-made MEAs were lithographically patterned with photoresist and subsequently coated with poly-D-lysine This method realized neuronal networks of different pattern geometries for electrophysiological studies.

When it comes to large-scale production of these platforms, the fabrication process needs to stop after the development of the photoresist, leaving the end-user to coat the MEAs with PDL, rinse with acetone and subsequently seed the cells. A major concern regarding this approach rises from the storage lifetime of the produced platforms, which is determined by the long-term stability of the photoresist pattern, which further depends on the storing conditions and environment.

Obviously the challenges of integrating structuring technologies with monitoring modalities are many; first, the development of scaffolding technologies that are based on biocompatible materials, support long-term and stable patterning and promote a more in vivo-like cellular morphology and physiology.

Second, the development of monitoring platforms that are compatible with state-of-art well-optimized read-out systems amplifiers, data acquisition systems, stimulus generators. This will enable the rapid employment of the developed tools in most research facilities and will bridge the gap between research and commercialization. Finally, the greatest challenge lies in the integration of the two aforementioned features, as it should be time and cost-effective, precisely controlled and reproducible.

We have previously developed a scaffolding technology to structure NRVM on a highly biocompatible material - Parylene C The Parylene scaffolds significantly induced cellular alignment and nuclear elongation. Here, we leverage this scaffolding technology in combination with commercially available MEAs to deliver an integrated and minimally invasive monitoring platform for electrophysiological studies of biomimetic cardiac tissue.

These application-specific MEAs are evaluated in terms of: i the production of anisotropic cardiac cultures, ii electrical performance, iii off-the-shelf storage lifetime and iv reusability. Activation time was extrapolated from microelectrodes 57 and 51 to ensure maximum distance and reliability of the calculations Fig. Four unprocessed standard MEAs served as the control group and six MEAs were micro-engineered to comprise the patterned group.

The fabrication process is presented in Fig. Representative bright field microscopy images are provided in Supplementary Fig. Figure 2 Micro-engineered MEAs. Full size image Electrical performance of the micro-engineered MEAs When an electrogenic cell generates an action potential above an electrode, ions flowing across the cell membrane induce a charge redistribution on the electrode.

The frequency of these signals for a cardiomyocyte has been reported to be within a bandwidth of 16 Hz-6 kHz 37 , Impedance measurements for electrodes before and after processing are presented here within this frequency bandwidth. Typically, manufacturing companies provide impedance references for microelectrodes around an intermediate frequency, usually 1 kHz.

According to the manufacturing company these MEAs usually have an impedance of approximately 30 kOhms at this frequency 29 , however, brand new MEAs are often hydrophobic and require a plasma treatment before first use. Therefore, the impedance of the electrodes is initially higher than referenced. Average electrode impedance has increased almost 5-fold after processing Supplementary Table 1. The introduction of Parylene C areas on top of a metal electrode results both in an extra resistance, which is responsible for an increase in the electrode thermal noise, but also to an extra capacitance.

Despite the rise of the impedance levels, recording and stimulation was still possible using bipolar pulses of larger amplitude — mV instead of — mV peak-to-peak. At 1 kHz the average absolute phase angle of the micro-engineered electrodes is 0. The noise levels of the MEA depend on the electrode size and material, which further depend on the electrode impedance. The smaller the electrode, the higher the impedance and, therefore, the higher are the noise levels.

Besides, the magnitude of the acquired electrical signal is such that enables recordings even after introducing Parylene C stripes on top of the gold electrodes. NRVM seeded on collagen-coated micro-engineered MEAs aligned sufficiently to the direction of the lines throughout the electrode grid, as demonstrated in Fig.

Supplementary Fig. In four analyzed isotropic monolayers the corresponding velocity propagation profile revealed circular isochrones, showing uniform, smooth propagation throughout the culture, which agrees with previously reported studies Cell elongation and coalignment on the micro-engineered MEAs induced by oriented cell growth was followed by the fastest impulse propagation along the direction of the pattern, resulting in elliptical activation profiles best represented by Fig.

Figure 3 Isochrones of action potential propagation. Full size image We also tested the shelf-life of the micro-engineered MEAs by storing them for 60 days before culturing them with NRVM through standard cell culturing protocols. Corresponding conduction velocity studies indicate that the micro- engineered MEAs were still able to promote anisotropic cardiac cultures in terms of cell alignment and CV profiles Supplementary Fig.

Conduction velocity along the four distinct directions on control MEAs was at similar levels no statistically significant difference Fig. Figure 4c presents the r values for distinct MEA dishes. Figure 4 Statistics of conduction velocity study. Error bars indicate standard error of means.

Data derived from two isolations. Reusability of the micro-fabricated platforms will ensure a low-cost and versatile tool for the biologically-oriented researchers, serving as an off-the shelf component for electrophysiological studies. The proposed technique is advantageous, as the MEAs can be recycled after every use; the Parylene membrane is easily peeled-off, the MEAs are cleaned thoroughly according to standard procedures and the fabrication process is repeated.

However, here the effectiveness of using the micro-engineered MEAs in two consecutive cultures is validated. By the end of the first experimental week, the MEAs were washed thoroughly with water to remove the cells from their surface Supplementary Fig.

The reusability of the MEAs was quantified in terms of i impedance, ii cellular alignment and iii conduction velocity similarly as previously see Methods. First, impedance was measured to evaluate the electrical functionality of the electrodes. Figure 5a presents average impedance measurements of brand-new MEAs blue , after they were processed red and after their first use with cells green. The measured electrode impedance is lower than the initial average impedance of the electrodes after processing and before cell culturing.

Another possible reason for the noticeable decrease in impedance is the fact that the MEAs were maintained in water-based environment cell medium for one week during cell culturing, which increases the hydrophilicity of the exposed electrode areas according to the manufacturer 29 and gradually lowers their impedance. Figure 5 Reusability of micro-engineered MEAs. Error bars indicate standard error of mean. Full size image Cell alignment was quantified using immunofluorescent staining and confocal microscopy.

The deviation of the nuclear angles from the direction of the pattern was assessed with appropriate image processing software see Methods and results are presented in Fig. This result is influenced to a great extent by the presence of unstructured cells sitting on top of the electrodes, which is well-justified by the electrowetting on-dielectric effect previously mentioned.

CV along the pattern direction was still the fastest impulse propagation Figure 5e demonstrates representative conduction velocity profiles of the same MEA before and after the first use with cells. Discussion Parylene C is a highly biocompatible material with a long history of use in the encapsulation of implantable microdevices 40 , 41 , Our work also takes into account manufacturing, yield and endurance challenges in exploiting such platforms more widely.

For example, the proposed platforms can be stored for at least two months post fabrication without compromising their capability to induce anisotropic cultures. Several existing approaches based on lithographic patterning of adhesion molecules 17 , 35 require the fabrication and ECM coating to take place one after the other, limiting the storage lifetime of the platforms.

Other approaches such as micro-stamping and microfluidic patterning, require special consideration when aligning the pattern with the active electrode array and usually require special equipment alignment stage to ensure precise and repeatable patterning of the MEA surface Finally, the proposed methodology yields MEA platforms that are easy and simple to use, as the ECM compounds fibronectin or collagen and cells self-assemble after random plating.

This enables the biologically oriented user to efficiently culture these platforms through standard cell culturing protocols. The electrical functionality of the micro-engineered MEAs was assessed in terms of electrode impedance and noise levels and was found not to affect the performance of the MEAs. Conduction velocity was used to demonstrate the effectiveness of this platform for producing anisotropic cardiac cultures.

The anisotropy ratios achieved with our platform 1. The average longitudinal conduction velocity of the cells on the micro-engineered MEAs In addition, average conduction velocity for isotropic monolayers The MEAs were reused in two consecutive cultures and were still able to reliably produce anisotropic cardiac cultures at the expense of a Considering the high cost of customized MEA platforms and their typical average performance, this error is small and can often be compared with errors produced due to cell culturing protocol variability.

Despite this observation, the proposed micro-engineering approach facilitates the recycling of the MEA platforms, as the Parylene film can be easily peeled-off and the fabrication process can be repeated to produce freshly engineered MEAs. Evaluation of the CV in a cardiac cell culture can reveal important information during drug side effect screening. First of all, a thorough study of the QT interval can shed light on the mechanisms by which drugs cause arrhythmias and potentially enable the distinction between arrhythmic and anti-arrhythmic effects.

Furthermore, a CV investigation can contribute to a better understanding of the pharmacological modulation of cardiac gap junction channels, since the expression and distribution of connexins changes under the presence of certain cardiovascular diseases 45 , which further change the CV of the culture.

Our platforms facilitate the oriented spread of excitation similarly to native heart tissue while they perform minimally invasive and stable electrophysiological monitoring, enabling more reliable CV measurements. We thus anticipate that our technology achieves anisotropism independently of the nature of the bathing solution, nonetheless, further research needs to be undertaken to elucidate this.

The development of platforms that have high predictive value in vitro has significant scientific and commercial impact on drug efficacy and optimization, as it could reduce the cost, time and failure rates of current drug assays It is hoped that the application of these platforms with cardiac myocytes derived from promising cell lines, such as human induced pluripotent stem cells and embryonic stem cells, can yield human-relevant assays for screening and characterizing novel drug candidates 47 , 48 , 49 , Our promising initial results encourage the continuation of this work in combination with human cardiomyocytes towards the development of a functional drug screening platform.

The fabrication process is outlined in Fig. To achieve anisotropic etching of Parylene C and optimize vertical etching, oxygen plasma was performed at a very low pressure 10 mTorr , while oxygen ions were adequately accelerated and directed perpendicular to the surface using a radio frequency power of 20 W, which corresponds to a bias voltage of approximately V between the electrodes.

After plasma etching the remaining photoresist was removed through immersing the MEAs into acetone, isopropyl alcohol and deionized water. For the control group standard non-processed MEAs were used. Sample preparation and electrode impedance measurements The electrical functionality of the MEAs before and after processing was assessed through measurements of the impedance of the micro-electrodes. A four-electrode configuration is provided by the system working, sensing, counter and reference electrode , but in this study a two-electrode configuration was employed, connecting the working with the sensing electrode and the reference with the counter electrode.

Before measuring, the MEAs were washed with ethanol and left to dry.

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