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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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| 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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| 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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| 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. |
- Cui, Yuan, 1995-, et al.
(författare)
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SlipO2Chip- single-cell respiration under tuneable environments
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In disciplines like toxicology and pharmacology, oxygen (O2) respiration is a universal metric for evaluating the effects of chemicals across various model systems, including mammalian and microalgal cells. However, for these cells the common practice is to segregate populations into control and exposure groups, which assumes direct equivalence in their responses and does not take into account cellular heterogeneity. This lack of resolution impedes our ability to precisely investigate differences among experimental groups in rare samples. To overcome this barrier, we introduce SlipO2Chip, an innovative microfluidic platform tailored for precisely quantifying single-cell O2 respiration in the coordinated absence and presence of chemical solutes. Constructed in glass, SlipO2Chip comprises a wet-etched channel plate on the top and a dry-etched microwell plate at the bottom. The microwells are coated with Pt(II) meso-Tetra(pentafluorophenyl)porphine (PtTFPP), an O2 sensing optode material and an O2-independent reference dye. A custom 3D-printed holder facilitates the controlled horizontal movement (‘slipping’) of the channel plate over the microwell plate, thereby establishing or disrupting the fluid path over microwells. Collectively, these design elements enable the immobilization of cells in microwells, their exposure to controlled fluid flows, the coordinated opening and closing of microwells and repeated measurements of single-cell O2 respiration. Uniquely, by sequentially executing opening and closing it becomes possible to measure single-cell respiration prior to and after exposure to chemical solutes. In a proof-of-concept application, we utilized SlipO2Chip to measure the impact of increasing exposures of the marine bacterial signal 2-heptyl-4-quinolone (HHQ) on the dark respiration of the diatom Ditylum brightwellii at single-cell resolution. Results revealed a dose-dependent decrease in per-cell O2 dark respiration, with a maximum reduction of 40.2% observed at HHQ concentrations exceeding 35.5 µM, and a half-maximal effective concentration (EC50) of 5.8 µM, consistent with that obtained via conventional bulk respiration methods. The ability of SlipO2Chip to sequentially assess the effects of chemical substances on single-cell O2 metabolism is advantageous for research where sample volumes are limited, such as clinical biopsies, studies involving rare microbial isolates, and toxicological studies wanting to address exposure effects while accounting for cell-to-cell variability.
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| 16. |
- 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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| 17. |
- 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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| 18. |
- 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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| 19. |
- 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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