| 1. |
- 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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| 2. |
- Porras, Ana Maria, et al.
(författare)
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Cell chirality exhibition of brain microvascular endothelial cells is dependent on micropattern width
- 2022
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Ingår i: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 12:46, s. 30135-30144
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Tidskriftsartikel (refereegranskat)abstract
- Left-right asymmetry is a conserved property in nature and observed in the human body, a property known as cell chirality. Cell chirality is often studied using micropatterned in vitro models. However, micropattern geometry and size often varies across different studies, making it challenging to compare results. Here, we utilized micropatterned RGD-peptide lines on hyaluronic acid hydrogels to investigate the effect of the micropattern width on the exhibited cell chirality bias of brain microvascular endothelial cells. Overall, this cell type exhibited a negative chirality bias on micropatterned lines ranging from 10 mu m to 400 mu m in width, where the negative bias was most pronounced on the 100 mu m wide lines. We also observed that this exhibited chirality bias varied across the line width. This work serves as a guide to determine optimal micropattern width for further investigations on cell chirality bias and its prominence in e.g., disease states or upon exposure to toxic substances.
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| 3. |
- Porras Hernandez, Ana Maria, et al.
(författare)
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A simplified approach to control cell adherence on biologically derived in vitro cell culture scaffolds by direct UV-mediated RGD linkage
- 2020
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Ingår i: Journal of materials science. Materials in medicine. - : Springer Nature. - 0957-4530 .- 1573-4838. ; 31:10
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Tidskriftsartikel (refereegranskat)abstract
- In this work, we present a method to fabricate a hyaluronic acid hydrogel with spatially controlled cell-adhesion properties based on photo-polymerisation cross-linking and functionalisation. The approach utilises the same reaction pathway for both steps meaning that it is user-friendly and allows for adaptation at any stage during the fabrication process. Moreover, the process does not require any additional cross-linkers. The hydrogel is formed by UV initiated radical addition reaction between acrylamide groups on the hyaluronic acid backbone. Cell adhesion is modulated by functionalising the adhesion peptide sequence RGD (arginine-glycine-aspartate) onto the hydrogel surface via radical mediated thiol-ene reaction using the non-reacted acrylamide groups. We show that 10 x 10 µm2 squares could be patterned with sharp features and a good resolution. The smallest area that could be patterned resulting in good cell adhesion was 25 x 25 µm2 squares, showing single-cell adhesion. Mouse brain endothelial cells adhered and remained in culture for up to 7 days on 100 x 100 µm2 square patterns. We see potential for this material combination for future use in novel organ-on-chip models and tissue engineering where the location of the cells is of importance and to further study endothelial cell biology.
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| 4. |
- 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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| 5. |
- 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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| 6. |
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| 7. |
- Porras Hernández, Ana María
(författare)
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Micropatterning of hyaluronic acid hydrogels for in vitro models
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human body consist of a vast number of cells, and jointly, the cells, form tissues and organs. The cells interact and respond to their local microenvironment. The cellular microenvironment consists of a highly hydrated and compliant extracellular matrix, neighboring cells and circulating biochemical factors; and jointly, provide chemical and physical cues that regulate cell behaviour However, these cues are often not present in traditional in vitro models, where cells experience a stiff and unstructured environment. An approach to better mimic the in vivo microenvironment in vitro is to use hydrogels. Hydrogels are soft and highly hydrated polymers based on materials naturally found in the extracellular matrix of various tissues. Furthermore, these materials can be chemically functionalized to control the physical, chemical, and mechanical properties of the hydrogels. These functionalities can also be used to prepare micrometre sized cell adhesive regions, or micropatterns, on the hydrogel substrate. The micropatterns guide the cell shape and permit the study of the cell response to these changes in shape and function, which has been observed in e.g., endothelial cells from various origins. Taken all together, the aim of this work was to develop a hydrogel-based cell culture substrate that permits the control of the spatial adhesion of brain endothelial cells in order to study the morphological effects on these cells and contribute to the understanding of the function of brain endothelial cells in health and disease. This thesis demonstrates the functionalization of hyaluronic acid, a naturally occurring extracellular matrix polymer, to prepare photocrosslinkable hydrogels. Then, through photolithography, micropatterns of cell adhesive peptides were prepared on these hydrogels. Brain microvascular endothelial cells, a highly specialized type of endothelial cells, adhered to the micropatterns, and the effect on their alignment and cell chirality depending on the micropatterned sized was studied. Furthermore, changes in their alignment were also observed when exposed to different glucose concentration.
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| 8. |
- 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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| 9. |
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| 10. |
- 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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| 11. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite
- 2022
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Ingår i: Materials Today Bio. - : Elsevier. - 2590-0064. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.
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| 12. |
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Paloschi, Valentina, et al.
(författare)
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Organ-on-a-chip technology : a novel approach to investigate cardiovascular diseases
- 2021
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Ingår i: Cardiovascular Research. - : Oxford University Press. - 0008-6363 .- 1755-3245. ; 117:14, s. 2742-2754
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Tidskriftsartikel (refereegranskat)abstract
- The development of organs-on-chip (OoC) has revolutionized in vitro cell-culture experiments by allowing a better mimicry of human physiology and pathophysiology that has consequently led researchers to gain more meaningful insights into disease mechanisms. Several models of hearts-on-chips and vessels-on-chips have been demonstrated to recapitulate fundamental aspects of the human cardiovascular system in the recent past. These 2D and 3D systems include synchronized beating cardiomyocytes in hearts-on-chips and vessels-on-chips with layer-based structures and the inclusion of physiological and pathological shear stress conditions. The opportunities to discover novel targets and to perform drug testing with chip-based platforms have substantially enhanced, thanks to the utilization of patient-derived cells and precise control of their microenvironment. These organ models will provide an important asset for future approaches to personalized cardiovascular medicine and improved patient care. However, certain technical and biological challenges remain, making the global utilization of OoCs to tackle unanswered questions in cardiovascular science still rather challenging. This review article aims to introduce and summarize published work on hearts- and vessels-on chips but also to provide an outlook and perspective on how these advanced in vitro systems can be used to tailor disease models with patient-specific characteristics.
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| 17. |
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| 18. |
- Porras, Ana Maria, et al.
(författare)
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Chemical micropatterning of hyaluronic acid hydrogels for controlled cell adhesion
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The blood brain barrier is constituted by endothelial cells, astrocytes and pericytes; and are organized into well structured units [1]. Standard cell culture techniques cannot recapitulate this organized structure. Hydrogels are an attractive scaffold due to their mechanical and chemical properties similar to those in body tissue[2] We propose the use of a photo-crosslinkable hyaluronic acid hydrogel as cell culture scaffold. Furthermore, chemical cues can be added into the hydrogel matrix to promote and control cell adhesion using UV lithography.
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| 19. |
- Porras, Ana Maria, et al.
(författare)
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Photopatterning of hyaluronic acid hydrogels for cell culture scaffolds
- 2016
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Konferensbidrag (refereegranskat)abstract
- Organs-on-chips technologies require the development of micro engineered devices to represent functional units of human organs. These devices use cell culture scaffolds to give support and structure for the cultured cells. Hydrogels are attractive scaffold materials, due to their high water content and because they are derived from natural polymers found in the extracellular matrix of different tissues in the human body. Hyaluronic acid can form hydrogels when functionalized with chemo-selective groups. These chemically cross-linked hydrogels can be modified with adhesion motifs, such as RGD peptides, to increase their biocompatibility and promote cell adhesion. In this work we use a photolithographic method to pattern the RGD peptide into distinct areas of the hyaluronic acid hydrogel with the aim to spatially control the cell attachment on the HA hydrogel scaffolds
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| 20. |
- Porras Hernández, Ana Maria
(författare)
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Chemical micropatterning of hyaluronic acid hydrogels for brain endothelial in vitro cell studies
- 2022
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The building blocks of human tissues are cells. The cells interact and respond to the characteristics of their local microenvironment. The cellular microenvironment is formed by three main components, the extracellular matrix, neighbouring cells and signalling molecules. Particularly, the extracellular matrix and neighbouring cells impose boundary conditions that limits the cell volume and cell spreading. However, these characteristics are often not present in traditional in vitro models, where cells experience a stiff and vast environment. An approach to improve in vitro models is to use hydrogels, soft and highly hydrated polymers. Through chemical modifications, polymers naturally found in the extracellular matrix can be functionalized to form crosslinked hydrogels. Moreover, these functionalities can also be used to prepare micropatterns, micrometre sized cell adhesive areas on the hydrogels. These micropatterns guide the cell shape and permit the study of the cell response to these changes in shape, which has been observed in e.g. endothelial cells from various origins. Taken all together, the aim of this work was to develop a hydrogel-based cell culture scaffold that permits the control of the spatial adhesion of brain endothelial cells in order to study the morphological effects on these cells and contribute to the understanding of the function of brain endothelial cells in health and disease. This thesis demonstrates the functionalization of hyaluronic acid, a naturally occurring extracellular matrix polymer, to prepare photocrosslinkable hydrogels. Furthermore, through photolithography, micropatterns of cell adhesive peptides were prepared on these hydrogels. Brain microvascular endothelial cells, a highly specialized type of endothelial cells, adhered to the micropatterns, and the effect on their alignment depending on the micropatterned sized was studied. Furthermore, changes in their alignment were also observed when exposed to different glucose concentration.
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| 21. |
- 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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| 22. |
- 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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| 23. |
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| 24. |
- Tenje, Maria, et al.
(författare)
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Acoustophoretic removal of proteins from blood components
- 2015
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Ingår i: Biomedical microdevices (Print). - : Springer Science and Business Media LLC. - 1387-2176 .- 1572-8781. ; 17:5
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Tidskriftsartikel (refereegranskat)abstract
- This work presents the development of a miniaturized system for removing plasma proteins and other low-molecular-weight compounds from red blood cell (RBC) concentrate in a simple one-step-process using integrated ultrasound. The technology utilizes the principles of acoustophoresis to transfer the RBCs from the original plasma-containing solution into a protein-free SAG-M additive solution in a continuous flow process. The preparation of protein free RBC concentrate is important for blood transfusion to patients suffering from immunoglobulin A (IgA)-deficiency and developing antibodies against IgA. We show a nearly complete removal of both albumin and IgA from concentrated RBCs via this one-step-processes in samples obtained from RBC concentrate. The cell recovery of our technology is close to 97 %, compared to just above 90 % of the current procedure of repeated dilution and centrifugation steps. This work clearly shows the potential of integrated acoustophoresis in a miniaturized system for clinical applications.
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| 25. |
- Wolff, Anette, et al.
(författare)
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In vitro blood-brain barrier models : An overview of established models and new microfluidic approaches
- 2015
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Ingår i: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 104:9, s. 2727-2746
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Tidskriftsartikel (refereegranskat)abstract
- The societal need for new central nervous system (CNS) medicines is substantial, because of the global increase in life expectancy and the accompanying increase in age-related CNS diseases. Low blood-brain barrier (BBB) permeability has been one of the major causes of failure for new CNS drug candidates. There has therefore been a great interest in cell models, which mimic BBB permeation properties. In this review, we present an overview of the performance of monocultured, cocultured, and triple-cultured primary cells and immortalized cell lines, including key parameters such as transendothelial electrical resistance values, permeabilities of paracellular flux markers, and expression of BBB-specific marker proteins. Microfluidic systems are gaining ground as a new automated technical platform for cell culture and systematic analysis. The performance of these systems was compared with current state-of-the-art models and it was noted that, although they show great promise, these systems have not yet reached beyond the proof-of-concept stage. In general, it was found that there were large variations in experimental protocols, BBB phenotype markers, and paracellular flux markers used. It is the author's opinion that the field may benefit greatly from developing standardized methodologies and initiating collaborative efforts on optimizing culture protocols.
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