| 1. |
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| 2. |
- Barbe, Laurent, et al.
(författare)
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Toward a confocal subcellular atlas of the human proteome
- 2008
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Ingår i: Molecular and cellular proteomics. - 1535-9476 .- 1535-9484. ; 7:3, s. 499-508
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Tidskriftsartikel (refereegranskat)abstract
- Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
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| 3. |
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| 4. |
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| 5. |
- Cantoni, Federico, et al.
(författare)
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A carrier with an integrated perfusion and heating systems for long-term imaging of microfluidic chips
- 2021
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Konferensbidrag (refereegranskat)abstract
- Microfluidic chips offer many benefits for cell studies, including an accurate spatial and temporal control over the growth conditions1. Despite the large expansion of microfluidics in biological applications2, there have been only a few developments of devices to simplify microfluidic chip handling and imaging. Here, we present a carrier of well-plate format with integrated cell media recirculation and heating systems to provide a stable environment for the cell culture during the imaging outside the incubator. Moreover, the absence of external tubing reduces the risk of contamination and bubble formation during the carrier transfers and reagent injections enabling long-term experiment monitoring. Our system was validated by repeatedly (day 1, 3, 7 and 10) taking the cultured mouse endothelial cells (bEnd.3) out of the incubator for imaging.
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| 6. |
- Cantoni, Federico, et al.
(författare)
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A microfluidic chip carrier including temperature control and perfusion system for long-term cell imaging
- 2021
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Ingår i: HardwareX. - : Elsevier. - 2468-0672. ; 10, s. e00245-
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Tidskriftsartikel (refereegranskat)abstract
- Microfluidic devices are widely used for biomedical applications but there is still a lack of affordable, reliable and user-friendly systems for transferring microfluidic chips from an incubator to a microscope while maintaining physiological conditions when performing microscopy. The presented carrier represents a cost-effective option for sustaining environmental conditions of microfluidic chips in combination with minimizing the device manipulation required for reagent injection, media exchange or sample collection. The carrier, which has the outer dimension of a standard well plate size, contains an integrated perfusion system that can recirculate the media using piezo pumps, operated in either continuous or intermittent modes (50–1000 µl/min). Furthermore, a film resistive heater made from 37 µm-thick copper wires, including temperature feedback control, was used to maintain the microfluidic chip temperature at 37 °C when outside the incubator. The heater characterisation showed a uniform temperature distribution along the chip channel for perfusion flow rates up to 10 µl/min. To demonstrate the feasibility of our platform for long term cell culture monitoring, mouse brain endothelial cells (bEnd.3) were repeatedly monitored for a period of 10 days, demonstrating a system with both the versatility and the potential for long imaging in microphysiological system cell cultures.
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| 7. |
- Cantoni, Federico, et al.
(författare)
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A perfusable multi-hydrogel vasculature on-chip engineered by 2-photon 3D printing and scaffold molding to improve microfabrication fidelity in hydrogels
- 2024
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Ingår i: Advanced Materials Technologies. - : John Wiley & Sons. - 2365-709X. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Engineering vasculature networks in physiologically relevant hydrogelsrepresents a challenge in terms of both fabrication, due to the cell–bioinkinteractions, as well as the subsequent hydrogel-device interfacing. Here, anew cell-friendly fabrication strategy is presented to realize perfusablemulti-hydrogel vasculature models supporting co-culture integrated in amicrofluidic chip. The system comprises two different hydrogels to specificallysupport the growth and proliferation of two different cell types selected for thevessel model. First, the channels are printed in a gelatin-based ink bytwo-photon polymerization (2PP) inside the microfluidic device. Then, ahuman lung fibroblast-laden fibrin hydrogel is injected to surround the printednetwork. Finally, human endothelial cells are seeded inside the printedchannels. The printing parameters and fibrin composition are optimized toreduce hydrogel swelling and ensure a stable model that can be perfused withcell media. Fabricating the hydrogel structure in two steps ensures that nocells are exposed to cytotoxic fabrication processes, while still obtaining highfidelity printing. In this work, the possibility to guide the endothelial cellinvasion through the 3D printed scaffold and perfusion of the co-culturemodel for 10 days is successfully demonstrated on a custom-made perfusionsystem.
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| 8. |
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| 9. |
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| 10. |
- Cantoni, Federico, et al.
(författare)
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Round-robin testing of commercial two-photon polymerization 3D printers
- 2023
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Ingår i: Additive Manufacturing. - : Elsevier. - 2214-8604 .- 2214-7810. ; 76
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Tidskriftsartikel (refereegranskat)abstract
- Since its introduction in the 1980s, 3D printing has advanced as a versatile and reliable tool with applications in different fields. Among the available 3D printing techniques, two-photon polymerization is regarded as one of the most promising technologies for microscale printing due to its ability to combine a high printing fidelity down to submicron scale with free-form structure design. Recently, the technology has been enhanced through the implementation of faster laser scanning strategies, as well as the development of new photoresists. This paves the way for a wide range of applications, which has resulted in an increasing number of available commercial systems. This work aims to provide an overview of the technology capability by comparing three commercial systems in a round-robin test. To cover a wide range of applications, six test structures with distinct features were designed, covering various aspects of interest, from single material objects with sub-micron feature sizes up to multi-material millimeter-sized objects. Application-specific structures were printed to evaluate surface roughness and the stitching capability of the printers. Moreover, the ability to generate free-hanging structures and complex surfaces required for cell scaffolds and microfluidic platform fabrication was quantitatively investigated. Finally, the influence of the numerical aperture of the fabrication objective on the printing quality was assessed. All three printers successfully fabricated samples comprising various three-dimensional features and achieved submicron resolution and feature sizes, demonstrating the versatility and precision of two-photon polymerization direct laser writing. Our study will facilitate the understanding of the technology maturity level, while highlighting specific aspects that characterize each of the investigated systems.
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| 11. |
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| 12. |
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| 13. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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Exploring microfluidics as a tool to evaluate the biological properties of a titanium alloy under dynamic conditions
- 2020
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Ingår i: Biomaterials Science. - : Royal Society of Chemistry (RSC). - 2047-4830 .- 2047-4849. ; 8, s. 6309-6321
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Tidskriftsartikel (refereegranskat)abstract
- To bring novel biomaterials to clinical use, reliable in vitro models are imperative. The aim of this work was to develop a microfluidic tool to evaluate the biological properties of biomaterials for bone repair. Two approaches to embed medical grade titanium (Ti6Al4V) on-chip were explored. The first approach consisted of a polydimethylsiloxane microfluidic channel placed onto a titanium disc, held together by an additively manufactured fixture. In the second approach, a titanium disc was assembled onto a microscopic glass slide, using a double-sided tape microfluidic channel. Both approaches demonstrated potential for maintaining MC3T3-E1 preosteoblast-like cell cultures on-chip, as was shown by the vast majority of living cells after 1 day. In addition, the cells cultured on-chip showed a more elongated morphology compared to cells grown under static conditions and a tendency to align to the direction of the flow. For longer-term (i.e. 10 days) studies, the glass-based chip was selected. Assessment of cell viability showed a high number of living cells during the entire experimental period. Cell proliferation and differentiation studies revealed an increase in cell proliferation on-chip, suggesting that proliferation was the dominating process at the detriment of differentiation in this micrometric dynamic environment. These results illustrate the importance of optimizing in vitro cell culture conditions and how these may affect biomaterial testing outcomes. Overall, this work provides a step towards more in vivo-like microfluidic testing platforms, which are expected to provide more reliable in vitro screening of biomaterials.
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| 14. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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On-chip evaluation of the biological properties of medical-grade titanium
- 2020
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Konferensbidrag (refereegranskat)abstract
- On-chip evaluation of the biological properties of medical-grade titaniumSarah-Sophia D. Carter1, Laurent Barbe1, Maria Tenje1 and Gemma Mestres1,*1 Department of Materials Science and Engineering, Science for Life Laboratory, Uppsala University, Uppsala, Sweden*E-mail: gemma.mestres@angstrom.uu.seIntroductionBefore entering the clinic, biomaterials need to be thoroughly evaluated, which requires accurate in vitro models. However, it has been shown that the currently used models correlate poorly with in vivo results [1]. In this work, microfluidic chips that integrate medical grade titanium (Ti6Al4V) were fabricated and subsequently used to study the biological properties of this biomaterial on-chip. The overall goal of this project is to develop on-chip platforms to evaluate novel biomaterials for bone regeneration.Theory and Experimental procedureA glass coverslip (175 µm thick) was laser cut to fit a Ti6Al4V disc (⌀ = 12 mm) and assembled onto a microscopic glass slide (1 mm thick) using double-sided tape (140 µm thick), the latter shaping the microfluidic channel. To ensure a tight seal between the glass coverslip and the Ti6Al4V disc, a UV adhesive was used. MC3T3-E1 pre-osteoblast cells were seeded at 45,000 cells/cm2 and allowed to adhere for 4 hours prior to starting the perfusion. After 1, 5 and 10 days, cell proliferation and cell differentiation were evaluated by the lactate dehydrogenase (LDH) assay (used as an indirect method to quantify the cytosolic enzyme LDH of cells previously adhered to the biomaterial) and the alkaline phosphatase (ALP) assay. As a static control, MC3T3-E1 cells were seeded on Ti6Al4V discs in a well plate.Results and DiscussionMC3T3-E1 cells were successfully grown on Ti6Al4V on-chip, as was confirmed by an increase in cell proliferation over time, which became significantly elevated compared to the static condition from day 5 onwards (Figure 1A). Cell differentiation increased over the studied period for both on-chip and static conditions (Figure 1B). However, in the static condition, ALP activity reached much higher levels compared to on-chip. All together, these results correlate well with the fact that cell proliferation is the dominating process on-chip during the experimental period.ConclusionMicrofluidic chips that integrate medical grade Ti6Al4V were fabricated and used to evaluate the biological properties of this biomaterial under dynamic conditions. Cell proliferation and differentiation studies indicate that MC3T3-E1 cells cultured on Ti6Al4V on-chip remain in a proliferative state during the time period of 10 days.AcknowledgmentsGM acknowledges Formas, Vetenskapsrådet and Göran Gustafsson´s Foundation for financial support.References[1] G. Hulsart-Billström et al., European Cells and Materials 31, 312-322 (2016).
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| 15. |
- Fornell, Anna, et al.
(författare)
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A microfluidic platform for SAXS measurements of liquid samples
- 2022
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Konferensbidrag (refereegranskat)abstract
- Small-angle X-ray scattering (SAXS) is a technique that can measure the size and shape of small particles such as proteins and nanoparticles using X-rays. At MAX IV, we are developing a microfluidic sample delivery platform to measure liquid samples containing proteins under flow using SAXS. One of the main advantages of using microfluidics is that the sample is continuously flowing, thus minimizing the risk of radiation damage as the sample is continuously refreshed. Other advantages include low sample volume and the possibility to study dynamic processes, e.g. mixing. To obtain good SAXS signals, the X-ray properties of the chip material are essential. The microfluidic chip must have low attenuation of X-rays, low background scattering, and high resistance to X-ray-induced damage, and preferably be low cost and easy to fabricate. In this work, we have evaluated the performance of two different polymer microfluidic chips for SAXS measurements.
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| 16. |
- Fornell, Anna, et al.
(författare)
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A Microfluidic Platform for Synchrotron X-ray Studies of Proteins
- 2021
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Konferensbidrag (refereegranskat)abstract
- New tools are needed to allow for complex protein dynamics studies, especially to study proteins in their native states. In the AdaptoCell project a microfluidic platform for academic and industrial users at MAX IV Laboratory is being developed. MAX IV is a Swedish national laboratory providing brilliant synchrotron X-rays for research. Due to the high photon flux, sensitive samples such as proteins are prone to rapid radiation damage; thus, it is advantageous to have the liquid sample underflow to refresh the sample continuously. This, in combination with small volumes, makes microfluidics a highly suitable sample environment for protein studies at MAX IV. The AdaptoCell platform is being integrated at three beamlines:Balder (X-ray absorption/emission spectroscopy), CoSAXS (small angle x-ray scattering) and Micromax (serial synchrotron crystallography). Currently, the platform is fully available atBalder, under commissioning at CoSAXS and being developed for MicroMAX.
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| 17. |
- Fornell, Anna, et al.
(författare)
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AdaptoCell : Microfluidics at MAX IV Laboratory
- 2022
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Ingår i: 25th Swedish Conference on Macromolecular Structure and Function.
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Konferensbidrag (refereegranskat)abstract
- The AdaptoCell project at MAX IV has developed a microfluidic sample delivery platform for academic and industrial users to enable studies of protein samples in solution and in microcrystals underflow. The platform is compatible with various X-ray techniques and has so far been integrated onto two beamlines at MAX IV: the CoSAXS beamline for small angle X-ray scattering studies and the Balder beamline for X-ray absorption spectroscopy studies. Initial implementation of the platform for serial crystallography sample delivery is ongoing and will be integrated onto the BioMAX and MicroMAX beamlines once commissioned. With this platform, we aim to meet the demand from our user community for studying proteins at physiologically relevant temperatures and give the ability to follow dynamical processes in situ as well as decreasing sample volumes and radiation damage.To determine the optimized flow rates and components for mixing etc. using different microfluidic chips, a dedicated off(beam)line test station with a microscope has been established at the Biolab. The Biolab also provides a number of characterization techniques, such as Dynamic Light Scattering, UV-Vis spectrophotometry, for quality control of the samples; as well as an anaerobic chamber for preparation and characterization of metalloproteins. The microfluidic flows are controlled via syringe pumps or a pressure-driven system. Channel design varies, depending on the needs of the experiment, from straight channel, cross-junction to herringbone micromixers etc. On-chip mixing of buffers with different viscosity, pH, ion strength and protein concentrations has been demonstrated successful and will be presented.
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| 18. |
- Fornell, Anna, et al.
(författare)
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AdaptoCell – Microfluidic Platforms at MAX IV Laboratory
- 2021
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Konferensbidrag (refereegranskat)abstract
- In the AdaptoCell project, we are developing microfluidic platforms for X-ray studies of liquid samples. Microfluidics is a suitable technology for samples that are prone to radiation damage, such as proteins. By having the sample underflow, the sample is continuously refreshed, and the risk of radiation damage is reduced. The technology is also suitable for investigating dynamic events such as in situ mixing. The microfluidic platforms are being integrated at three beamlines at MAX IV Laboratory: Balder (X-ray absorption/emission spectroscopy), CoSAXS (small angle x-ray scattering) and MicroMAX (serial synchrotron crystallography). Currently, the platforms are available for users at Balder and CoSAXS, which is under development at MicroMAX. In addition, we also provide a microfluidic offline test station where users can test their samples and optimise their devices before the beam time. The main components of the microfluidic setup are the pressure-driven flow controller and the microfluidic chip. We mainly use commercially available polymer microfluidic chips made of COC (cyclic olefin copolymer). COC is used as a chip material as it has high X-ray transmission and high resistance to radiation damage. There are several different chip designs available such as straight channel chips, droplet generator chips and mixing chips. We believe the AdaptoCell platforms will be useful and versatile sample environments for academic and industrial users at MAX IV Laboratory who want to perform experiments with liquid samples under flow.
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| 19. |
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| 20. |
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| 21. |
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| 22. |
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| 23. |
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| 24. |
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| 25. |
- Kadekar, Sandeep, et al.
(författare)
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Effect of the Addition Frequency of 5-Azacytidine in Both Micro- and Macroscale Cultures
- 2021
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Ingår i: Cellular and Molecular Bioengineering. - : Springer Nature. - 1865-5025 .- 1865-5033. ; 14, s. 121-130
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments.Methods: hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 mu M) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPAR gamma (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated.Results: Daily delivering 5-AzaC caused a higher upregulation of PPAR gamma, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPAR gamma (tenfold-change) could indicate priming of hMSCs towards adipocytes.Conclusions: Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.
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| 26. |
- Li, Shiyu, et al.
(författare)
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Dynamics of DNA Clogging in Hafnium Oxide Nanopores
- 2020
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 124:51, s. 11573-11583
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Tidskriftsartikel (refereegranskat)abstract
- Interfacing solid-state nanopores with biological systems has been exploited as a versatile analytical platform for analysis of individual biomolecules. Although clogging of solid-state nanopores due to nonspecific interactions between analytes and pore walls poses a persistent challenge in attaining the anticipated sensing efficacy, insufficient studies focus on elucidating the clogging dynamics. Herein, we investigate the DNA clogging behavior by passing double-stranded (ds) DNA molecules of different lengths through hafnium oxide(HfO2)-coated silicon (Si) nanopore arrays, at different bias voltages and electrolyte pH values. Employing stable and photoluminescent-free HfO2/Si nanopore arrays permits a parallelized visualization of DNA clogging with confocal fluorescence microscopy. We find that the probability of pore clogging increases with both DNA length and bias voltage. Two types of clogging are discerned: persistent and temporary. In the time-resolved analysis, temporary clogging events exhibit a shorter lifetime at higher bias voltage. Furthermore, we show that the surface charge density has a prominent effect on the clogging probability because of electrostatic attraction between the dsDNA and the HfO2 pore walls. An analytical model based on examining the energy landscape along the DNA translocation trajectory is developed to qualitatively evaluate the DNA–pore interaction. Both experimental and theoretical results indicate that the occurrence of clogging is strongly dependent on the configuration of translocating DNA molecules and the electrostatic interaction between DNA and charged pore surface. These findings provide a detailed account of the DNA clogging phenomenon and are of practical interest for DNA sensing based on solid-state nanopores.
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| 27. |
- Liu, Zhenhua, 1992-, et al.
(författare)
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Long-term droplet cell culture enabled by droplet acoustofluidics
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Droplet microfluidics can be used to encapsulate cells for biochemical applications, such as single-cell analysis and drug screening. However, the concentration of nutrients and growth factors decreases over time, while the concentration of catabolic byproducts increases, that make droplet hard for long-term cell culture. Here, the cells encapsulated in droplets continued to divide in the first 8 hours and then stopped to grow. We developed a droplet acoustofluidic chip that can exchange cell medium in droplets by the combination of the pico-injection and the droplet split with acoustophoresis. After running droplets through this chip, cell medium in droplets got exchanged and the cells in droplets started to grow again. By this way, long-term droplet cell culture can be achieved.
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| 28. |
- Liu, Zhenhua, 1992-, et al.
(författare)
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On-chip background dilution in droplets with high particle recovery using acoustophoresis
- 2019
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Droplet microfluidics has shown great potential for on-chip biological and chemical assays. However, fluid exchange in droplet microfluidics with high particle recovery is still a major bottleneck. Here, using acoustophoresis, we present for the first time a label-free method to achieve continuous background dilution in droplets containing cells with high sample recovery. The system comprises droplet generation, acoustic focusing, droplet splitting, picoinjection, and serpentine mixing on the same chip. The capacities of the picoinjection and the droplet split to dilute the background fluorescent signal in the droplets have been characterized. The sample recovery at different droplet split ratios has also been characterized. The results show a maximum of 4.3-fold background dilution with 87.7% particle recovery. We also demonstrated that the system can be used to dilute background fluorescent signal in droplets containing either polystyrene particles or endothelial cells.
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| 29. |
- Lucchetti, Mara, et al.
(författare)
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Emulating the gut-liver axis : Dissecting the microbiome’s effect on drug metabolism using multi-organ-on-chip models
- 2021
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Over the past few years, several studies have shown that the gastrointestinal microbiome plays a key role in the processing of exogenous pharmaceutical compounds [1]. Additionally, previous work on the gut-liver axis has shown that the interplay between the intestinal microbiome, the gut barrier and the liver is fundamental for regulating these drug metabolism processes [2]. Disruption of these processes may lead to gut barrier dysfunction. This might result in the induction of inflammatory signaling pathways in the liver and ultimately modify drug metabolism by hepatocytes. Modeling the highly variable luminal gut environment and understanding how gut microbes can modulate drug availability or induce liver toxicity remains a challenge. Hence, there is a significant need to develop new platforms that enable the co-culture of human and microbial cells in a patient-specific manner. Microfluidics-based technologies such as organ-on-chips (OoC) could overcome current challenges in drug toxicity assessment assays as these technologies are designed to be more physiologically relevant than conventional in vitro and in vivo models. The main objective of this work is to interconnect our microfluidics-based Human Microbial Crosstalk (HuMiX) model [3] with the Dynamic42 liver-on-chip [4] in order to create a stem-cell based multi-organ platform to predict the effect of the gut microbiome on pharmacokinetics [5]. The improvement of current OoC platforms could lead to a powerful alternative to stepwise replace the use of animal experimentation regarding the microbiome and its role on drug metabolization for individual patients.[1] Zimmermann M et al., Mapping human microbiome drug metabolism by gut bacteria and their genes. Nature 2019;570:462–7. [2] Khalsa J et al., Omics for Understanding the Gut‐Liver‐Microbiome Axis and Precision Medicine. Clin Pharm Drug Dev 2017;6:176–85. [3] Shah, P.et al. A microfluidics-based in vitro model of the gastrointestinal human–microbe interface. Nat. Commun.7:11535 (2016)[4] Maurer M et al., A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies. Biomaterials 2019;220:119396. [5] Lucchetti M et al., Emulating the gut-liver axis: Dissecting the microbiome’s effect on drug metabolism using multi-organ-on-chip models, Current Opinion in Endocrine and Metabolic Research.
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| 30. |
- Lucchetti, Mara, et al.
(författare)
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Integration of multiple flexible electrodes for real-time detection of barrier formation with spatial resolution in a gut-on-chip system
- 2024
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Ingår i: MICROSYSTEMS & NANOENGINEERING. - : SPRINGERNATURE. - 2055-7434. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- In healthy individuals, the intestinal epithelium forms a tight barrier to prevent gut bacteria from reaching blood circulation. To study the effect of probiotics, dietary compounds and drugs on gut barrier formation and disruption, human gut epithelial and bacterial cells can be cocultured in an in vitro model called the human microbial crosstalk (HuMiX) gut-on-a-chip system. Here, we present the design, fabrication and integration of thin-film electrodes into the HuMiX platform to measure transepithelial electrical resistance (TEER) as a direct readout on barrier tightness in real-time. As various aspects of the HuMiX platform have already been set in their design, such as multiple compressible layers, uneven surfaces and nontransparent materials, a novel fabrication method was developed whereby thin-film metal electrodes were first deposited on flexible substrates and sequentially integrated with the HuMiX system via a transfer-tape approach. Moreover, to measure localized TEER along the cell culture chamber, we integrated multiple electrodes that were connected to an impedance analyzer via a multiplexer. We further developed a dynamic normalization method because the active measurement area depends on the measured TEER levels. The fabrication process and system setup can be applicable to other barrier-on-chip systems. As a proof-of-concept, we measured the barrier formation of a cancerous Caco-2 cell line in real-time, which was mapped at four spatially separated positions along the HuMiX culture area.
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| 31. |
- Mestres, Gemma, 1984-, et al.
(författare)
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Advantages of microfluidic systems for studying cell-biomaterial interactions : focus on bone regeneration applications
- 2019
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Ingår i: Biomedical Engineering & Physics Express. - : IOP Publishing. - 2057-1976. ; 5:3
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Forskningsöversikt (refereegranskat)abstract
- The poor correlation between in vitro and in vivo studies emphasises the lack of a reliable methodology for testing the biological properties of biomaterials in the bone tissue regeneration field. Moreover, the success of clinical trials is not guaranteed even with promising results in vivo. Therefore, there is a need for a more physiologically relevant in vitro model to test the biological properties of biomaterials. Microfluidics, which is a field concerning the manipulation and control of liquids at the submillimetre scale, can use channel geometry, cell confinement and fluid flow to recreate a physiological-like environment. This technology has already proven to be a powerful tool in studying the biological response of cells in defined environments, since chemical and mechanical inputs as well as cross-talk between cells can be finely controlled. Moving a step further in complexity, biomaterials can be integrated into microfluidic systems to evaluate biomaterial-cell interactions. The biomaterial- microfluidics combination has the potential to produce more physiologically relevant models to better screen the biological interactions established between biomaterials and cells. This review is divided into two main sections. First, several possible cell-based assays for bone regeneration studies in microfluidic systems are discussed. Second, and the ultimate goal of the review, is to discuss how the gap between in vitro and in vivo studies can be shortened by bridging the biomaterials and microfluidics fields.
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| 32. |
- Micheal Raj, Pushparani, et al.
(författare)
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Fabrication and characterisation of a silicon-borosilicate glass microfluidic device for synchrotron-based hard X-ray spectroscopy studies
- 2021
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Ingår i: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 11:47, s. 29859-29869
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Tidskriftsartikel (refereegranskat)abstract
- Some of the most fundamental chemical building blocks of life on Earth are the metal elements. X-ray absorption spectroscopy (XAS) is an element-specific technique that can analyse the local atomic and electronic structure of, for example, the active sites in catalysts and energy materials and allow the metal sites in biological samples to be identified and understood. A microfluidic device capable of withstanding the intense hard X-ray beams of a 4th generation synchrotron and harsh chemical sample conditions is presented in this work. The device is evaluated at the K-edges of iron and bromine and the L-3-edge of lead, in both transmission and fluorescence mode detection and in a wide range of sample concentrations, as low as 0.001 M. The device is fabricated in silicon and glass with plasma etched microchannels defined in the silicon wafer before anodic bonding of the glass wafer into a complete device. The device is supported with a well-designed printed chip holder that made the microfluidic device portable and easy to handle. The chip holder plays a pivotal role in mounting the delicate microfluidic device on the beamline stage. Testing validated that the device was sufficiently robust to contain and flow through harsh acids and toxic samples. There was also no significant radiation damage to the device observed, despite focusing with intense X-ray beams for multiple hours. The quality of X-ray spectra collected is comparable to that from standard methods; hence we present a robust microfluidic device to analyse liquid samples using synchrotron XAS.
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| 33. |
- Porras, Ana Maria, et al.
(författare)
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Brain microvasculature endothelial cell orientation on micropatterned hydrogels is affected by glucose level variations
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- This work reports on an effort to decipher the alignment of brain microvasculature endothelial cells to physical constrains generated via adhesion control on hydrogel surfaces and explore the corresponding responses upon glucose level variations emulating the hypo- and hyperglycaemic effects in diabetes. We prepared hydrogels of hyaluronic acid a natural biomaterial that does not naturally support endothelial cell adhesion, and specifically functionalised RGD peptides into lines using UV-mediated linkage. The width of the lines was varied from 10 to 100 µm. We evaluated cell alignment by measuring the nuclei, cell, and F-actin orientations, and the nuclei and cell eccentricity via immunofluorescent staining and image analysis. We found that the brain microvascular endothelial cells aligned and elongated to these physical constraints for all line widths. In addition, we also observed that varying the cell medium glucose levels affected the cell alignment along the patterns. We believe our results may provide a platform for further studies on the impact of altered glucose levels in cardiovascular disease.
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| 34. |
- Porras Hernandez, Ana Maria, et al.
(författare)
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Alignment of brain endothelial cells on patterned hyaluronic acid hydrogels
- 2020
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Endothelial cells (ECs) line the blood vessel walls and present an elongated characteristic morphology. While the effect of micropatterning on different endothelial cells have been extensively studied, the effects on brain endothelial cells, which are highly specialized cells, have been overlooked [1]. Moreover, it has been shown that brain ECs do not elongate and align in response to shear stress, as e.g. HUVECs do. [2]. Hence, we set out to conclude how brain endothelial cells would behave on micropatterned lines. I We fabricated an RGD micropatterned photocrosslinked hyaluronic acid (HA-am) hydrogel substrate, with lines of controlled dimensions. These substrates were used to study cell elongation and alignment of the cell nuclei when adhering to lines raging from 10 µm to 100 µm in width.
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| 35. |
- Porras Hernández, Ana Maria, et al.
(författare)
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Confocal imaging dataset to assess endothelial cell orientation during extreme glucose conditions
- 2022
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Ingår i: Scientific Data. - : Springer Nature. - 2052-4463. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Confocal microscopy offers a mean to extract quantitative data on spatially confined subcellular structures. Here, we provide an imaging dataset of confocal z-stacks on endothelial cells spatially confined on lines with different widths, visualizing the nucleus, F-actin, and zonula occludens-1 (ZO-1), as well as the lines. This dataset also includes confocal images of spatially confined endothelial cells challenged with different glucose conditions. We have validated the image quality by established analytical means using the MeasureImageQuality module of the CellProfilerTM software. We envision that this dataset could be used to extract data on both a population and a single cell level, as well as a learning set for the development of new image analysis tools.
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| 36. |
- Raj, Pushparani, et al.
(författare)
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Fabrication and characterisation of a silicon-borosilicate glass microfluidic device for synchrotron-based hard X-ray spectroscopy studies
- 2021
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Ingår i: RSC Advances. - Cambridge : RSC Publishing. - 2046-2069. ; 11:47, s. 29859-29869
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Tidskriftsartikel (refereegranskat)abstract
- Some of the most fundamental chemical building blocks of life on Earth are the metal elements. X-ray absorption spectroscopy (XAS) is an element-specific technique that can analyse the local atomic and electronic structure of, for example, the active sites in catalysts and energy materials and allow the metal sites in biological samples to be identified and understood. A microfluidic device capable of withstanding the intense hard X-ray beams of a 4th generation synchrotron and harsh chemical sample conditions is presented in this work. The device is evaluated at the K-edges of iron and bromine and the L3-edge of lead, in both transmission and fluorescence mode detection and in a wide range of sample concentrations, as low as 0.001 M. The device is fabricated in silicon and glass with plasma etched microchannels defined in the silicon wafer before anodic bonding of the glass wafer into a complete device. The device is supported with a well-designed printed chip holder that made the microfluidic device portable and easy to handle. The chip holder plays a pivotal role in mounting the delicate microfluidic device on the beamline stage. Testing validated that the device was sufficiently robust to contain and flow through harsh acids and toxic samples. There was also no significant radiation damage to the device observed, despite focusing with intense X-ray beams for multiple hours. The quality of X-ray spectra collected is comparable to that from standard methods; hence we present a robust microfluidic device to analyse liquid samples using synchrotron XAS.
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| 37. |
- Searle, Sean, 1991-, et al.
(författare)
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Hyaluronic acid based hydrogel droplets: A potential injectable cell culture scaffold
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- IntroductionCell culture scaffolds such as hydrogels give support and structure for cultured cells in 3D environments that better mimic in vivo conditions [1]. Hyaluronic acid (HA) derived hydrogels are particularly attractive scaffold materials, due to their high water content, and its high presence in the extracellular matrix of a multitude of tissues in the human body [2]. Adequate diffusion of oxygen and nutrients however, is generally limited to a depth of 200 µm in bulk hydrogels [3], heavily limiting their applicability to relatively large size constructs. We propose the use of droplet-based microfluidics to produce monodisperse HA-derived injectable microgel droplets which could enable the diffusion of nutrients and metabolites, while maintaining a size in which encapsulating sufficient cells to allow cell-cell interactions and proliferation would be possible. Experimental resultsHyaluronic acid acrylamide (HA-am) was synthesized by partially modifying high molecular weight sodium hyaluronan with a N-(2-aminoethyl)acrylamide linker. Degree of modification was confirmed by NMR to be of 20%. HA-am bulk hydrogels were formed by exposing a solution of HA-am and photoinitiator Irgacure 2959 (0.4 % w/v) to a UV light source of 365 nm wavelength. Gel droplets were produced in a PDMS microfluidic device designed in a flow focusing geometry. In order to simulate cell encapsulation in the microgel, hydrogel precursor mixtures were prepared as for bulk hydrogels with the addition of polystyrene beads (10µm in diameter) at a concentration of 10 million beads ml-1. For the oil phase, a fluorinated oil (Novec 7500, 3M) with 0.5% surfactant (PicoSurf 1) was used. The flow rates for the oil phase and aqueous phase were adjusted to 15 and 5 µl min-1, respectively to produce highly monodisperse droplets of 151 µm in average diameter. Collected droplets were polymerized by exposing to UV light, washed and transferred to an aqueous solution. ConclusionHighly monodisperse microgels containing microbeads were obtained. We demonstrate that photocrosslinkable hydrogel droplets can be produced from HA-am in a microfluidic flow-focusing chip which could enable the encapsulation of cells and the use of the droplets as injectable cell culture scaffolds. References[1] G. D. Nicodemus and S. J. Bryant, “Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications,” Tissue Eng. Part B Rev., vol. 14, no. 2, pp. 149–165, Jun. 2008.[2] J. A. Burdick and G. D. Prestwich, “Hyaluronic acid hydrogels for biomedical applications,” Adv. Mater., vol. 23, no. 12, pp. 41–56, Mar. 2011.[3] H. Huang, Y. Yu, Y. Hu, X. He, O. Berk Usta, and M. L. Yarmush, “Generation and manipulation of hydrogel microcapsules by droplet-based microfluidics for mammalian cell culture,” Lab Chip, vol. 17, no. 11, pp. 1913–1932, 2017.
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| 38. |
- Searle, Sean, 1991-, et al.
(författare)
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Production of hyaluronic acid-acrylamide microgels as potential cell culture scaffolds
- 2018
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Ingår i: Micronano System Workshop, May 13-15, 2018. ; , s. 24-24
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Konferensbidrag (refereegranskat)abstract
- Hyaluronic acid (HA) derived hydrogels give support and structure for cultured cells in 3D environments that better mimic in vivo conditions 1. Adequate diffusion of oxygen and nutrients however, is generally limited to a depth of 200 µm in bulk hydrogels 2, limiting their applicability to larger size constructs. Through droplet-based microfluidics we produced monodisperse HA-derived microgel droplets. Hyaluronic acid acrylamide (HA-am) was synthesized by partially modifying high molecular weight sodium hyaluronan with a N-(2-aminoethyl)acrylamide linker to a 20% degree.Gel droplets were produced in a PDMS microfluidic device designed in a flow focusing geometry. In this setup polystyrene beads were added to simulate cell-encapsulation into a matrix that would better reflect in vivo conditions. The hydrogel precursor mixtures were prepared with 2% solution of HA-am and a photoinitiator with the addition of polystyrene beads (10µm in diameter) at a concentration of 10 million beads per milliliter. A fluorinated oil (Novec 7500, 3M) with 0.5% surfactant (PicoSurf 1) was used as the continuous phase. Highly monodisperse droplets of 151 µm in average diameter were produced and later polymerized by exposing to a long-wave UV light source (365 nm). We demonstrate that photocrosslinkable hydrogel droplets can be produced from HA-am. These microgels could enable the diffusion of nutrients and metabolites, while maintaining a size in which encapsulating sufficient cells to allow cell-cell interactions and proliferation would be possible.[1] J. A. Burdick and G. D. Prestwich, Adv. Mater., 2011, 23, 41–56.[2] H. Huang, Y. Yu, Y. Hu, X. He, O. Berk Usta and M. L. Yarmush, Lab Chip, 2017, 17, 1913–1932.
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| 39. |
- Shi, Qian, et al.
(författare)
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Mapping the acoustic properties of two-phase systems for use in droplet acoustofluidics
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The emergence of droplet microfluidics as a powerful tool for on-chip biological assays has prompted the development of a variety of intra-droplet particle manipulation techniques, such as droplet acoustofluidics. Previous study has shown that the acoustic properties between the continuous and dispersed phase must match for high-quality intra-droplet particle focusing. As a follow up, this study investigates the acoustic properties, i.e., speed of sound and density, of a selection of non-polar fluids that can be used as the continuous phase in droplet microfluidic systems. Our experimental results show that within our collection, linseed oil is the non-polar phase that most closely matches the acoustic properties of water and the fluorinated oil HFE-7500 is the one that least matches the acoustic properties compared to water. We believe this collection of data will serve the community by providing results that aid in the selection of continuous phase in future droplet acoustofluidic studies and data for performing acoustofluidic simulations.
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| 40. |
- Tenje, Maria, et al.
(författare)
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A practical guide to microfabrication and patterning of hydrogels for biomimetic cell culture scaffolds
- 2020
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Ingår i: Organs-on-a-Chip. - The Netherlands : Elsevier. - 2666-1020. ; 2
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Forskningsöversikt (refereegranskat)abstract
- This review article describes microfabrication techniques to define chemical, mechanical and structural patterns in hydrogels and how these can be used to prepare in vivo like, i.e. biomimetic, cell culture scaffolds. Hydrogels are attractive materials for 3D cell cultures as they provide ideal culture conditions and they are becoming more prominently used. Single material gels without any modifications do however have their limitation in use and much can be gained by in improving the in vivo resemblance of simple hydrogel cell culture scaffolds. This review article discusses the most commonly used cross-linking strategies used for hydrogel-based culture scaffolds and gives a brief introduction to microfabrication methods that can be used to define chemical, mechanical and structural patterns in hydrogels with micrometre resolution. The review article also describes a selection of literature references using these microfabrication techniques to prepare organ and disease models with controlled cell adhesion, proliferation and migration. It is intended to serve as an introduction to microfabrication of hydrogels and an inspiration for novel interdisciplinary research projects.
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| 41. |
- Werr, Gabriel, 1991-, et al.
(författare)
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Fabrication of electrodes on flexible substrates for OoC integration
- 2021
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Konferensbidrag (refereegranskat)abstract
- Integration of sensors in OoC is advantageous to monitor cell proliferation and differentiation, for example via TEER measurements. The established method to pattern electrical sensors is thin-film deposition of metals such as Au or Pt, often structured on stiff substrates like glass or polycarbonate. There is however a need to be able to define electrodes on flexible substrates for integration in more complex OoCs including multiple layers, such as the HuMiX system [1]. Handling and metal deposition on thin flexible substrates is challenging, due to poor adhesion under bending stress. Here, we present a robust method where electrodes can be patterned on flexible substrates for OoC integration. Using polyimide substrate and carrier foils allows for integration of electrodes also into system designs that are themselves too thick to fit into common sputter machines. This method also allows for integration of electrodes over uneven surfaces and steps, that are otherwise difficult to handle.[1] P. Shah et al., “A microfluidics-based in vitro model of the gastrointestinal human–microbe interface,” Nat. Commun., vol. 7, no. 1, p. 11535, Sep. 2016.
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| 42. |
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| 43. |
- Wu, Lulu, et al.
(författare)
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Microfluidic system with integrated nanocellulose cell culture substrate to study alignment of human umbilical vein endothelial cells in relation to external physical cues
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In this work, aligned cationic cellulose nanofibrils (c-CNF) were integrated into a microfluidic channel to guide cell attachment of human umbilical vein endothelial cells (HUVECs) and the effect of different mechanical cues on cell orientation was investigated. The on-chip cultured cells were exposed to external stimuli by the c-CNF topography and a fluid flow-induced shear stress, either separately or combined. Fluorescent images of the c-CNF pattern stained with calcofluor white and HUVECs stained for F-actin fibers and cell nuclei were obtained and used to quantify orientation of the CNFs, the F-actin fibers and the cell nuclei together with the eccentricity of the nuclei. Compared to the control, where the cells were cultured on a smooth surface in static conditions, cells cultured on the c-CNF pattern alone showed a clear alignment to the underlying microtopography. Cells cultured on a smooth surface responded slightly to the external mechanical stimuli indicating that the cell orientation was more strongly affected by c-CNF topography than the shear stress. With these results, we established a platform that can de-couple external mechanical stimuli originating from surface topography and shear stress to increase our understanding of how cells react to these factors when cultured in microfluidic in vitro systems.
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