| 1. |
- Frisk, Thomas, et al.
(författare)
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A concept for miniaturized 3-D cell culture using an extracellular matrix gel
- 2005
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:24, s. 4751-4758
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Tidskriftsartikel (refereegranskat)abstract
- This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber. Raising the temperature to 37 degrees C results in a gel, with cells embedded. The silicon pillars both stabilize and increase the surface to volume ratio of the gel. During polymerization the gel shrinks, thus creating channels, which enables perfusion through the chip. The pillars increase the mechanical stability of the gel permitting high surface flow rates without surface modifications. Within the structure cells were still viable and proliferating after 6 days of cultivation. Our method thus makes it possible to perform medium- to long-term cultivation of cells in a controlled 3-D environment. This concept opens possibilities to perform studies of cells in a more physiological environment compared to traditional 2-D cultures on flat substrates.
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| 2. |
- Frisk, Thomas, et al.
(författare)
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A microfluidic device for parallel 3-D cell cultures in asymmetric environments
- 2007
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 28:24, s. 4705-4712
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a concept for how a miniaturized 3-D cell culture in biological extracellular matrix (ECM) or synthetic gels bridges the gap between organ-tissue culture and traditional 2-D cultures. A microfluidic device for 3-D cell culture including microgradient environments has been designed, fabricated, and successfully evaluated. In the presented system stable diffusion gradients can be generated by application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cello. Culture for up to two weeks was performed showing cells still viable and proliferating. The cell tracer dye calcein was used to verify gradient formation as the fluorescence intensity in exposed cells was proportional to the position in the chamber. Cellular response to an applied stimulus was demonstrated by use of an adenosine triphosphate gradient where the onset of a stimulated intracellular calcium release also depended on cell position.
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| 3. |
- Frisk, Thomas, et al.
(författare)
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Cultivation of COS7-cells using extracellular matrix in 3D microfluidic surface enlarged structure
- 2005
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Ingår i: Micro Total Analysis Systems 2004. - 0854048960 ; , s. 261-263
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Konferensbidrag (refereegranskat)abstract
- This paper presents a novel method to cultivate cells in a controlled 3D surface enlarged micro-environment. The 3D environment is achieved by insertion of a gel-like extracellular matrix (ECM) mixed with cells into a micromachined silicon fluid structure. Shrinking of the gel enables further flow through the channel. Due to the structure design the shrinking is non-uniform, which results in an increased surface area. With the proposed design cells are alive and viable after 72 h of incubation within the chip.
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| 4. |
- Frisk, Thomas, et al.
(författare)
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Three dimensional asymmetric microenvironment for cell biological studies
- 2005
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Ingår i: Proceedings of µTAS 2005 Conference. ; , s. 915-917
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Konferensbidrag (refereegranskat)abstract
- We report on a method for three-dimensional cultivation of cells in a microstructured asymmetric environment. In the system an asymmetric environment is created by using diffusion through a gel of extra cellular matrix proteins surrounded by microfluidic flow channels. Individual cells embedded in the gel react on the concentration gradient. The system has been evaluated both for diffusion properties and based on the cellular response.
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| 5. |
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| 6. |
- Guldevall, Karolin, 1981-, et al.
(författare)
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Microchip screening platform for assessment of cytotoxic effector cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) or T cells within larger populations. Human primary NK cells or human Epstein-Barr virus (EBV)- specific T cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of effector and live or dead target cells in each well could be assessed at different time-points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic to the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12 hours long experiment. We demonstrate that this assay can be used to enumerate and characterize cytotoxic cells, something that could find clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.
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| 7. |
- Guldevall, Karolin, et al.
(författare)
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Microchip screening Platform for single cell assessment of NK cell cytotoxicity
- 2016
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (approximate to 75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (>= 3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.
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| 8. |
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| 9. |
- Rydholm, Susanna, et al.
(författare)
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Mechanical Properties of Primary Cilia Regulate the Response to Fluid flow
- 2010
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Ingår i: American Journal of Physiology - Renal Physiology. - : American Physiological Society. - 0363-6127 .- 1522-1466 .- 1931-857X. ; 298:5, s. 1096-1102
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Tidskriftsartikel (refereegranskat)abstract
- The primary cilium is a ubiquitous organelle present on most mammalian cells. Malfunction of the organelle has been associated with various pathological disorders, many of which lead to cystic disorders in liver, pancreas, and kidney. Primary cilia have in kidney epithelial cells been observed to generate intracellular calcium in response to fluid flow, and disruption of proteins involved in this calcium signaling lead to autosomal dominant polycystic kidney disease, implying a direct connection between calcium signaling and cyst formation. It has also been shown that there is a significant lag between the onset of flow and initiation of the calcium signal. The present study focuses on the mechanics of cilium bending and the resulting calcium signal. Visualization of real-time cilium movements in response to different types of applied flow showed that the bending is fast compared with the initiation of calcium increase. Mathematical modeling of cilium and surrounding membrane was performed to deduce the relation between bending and membrane stress. The results showed a delay in stress buildup that was similar to the delay in calcium signal. Our results thus indicate that the delay in calcium response upon cilia bending is caused by mechanical properties of the cell membrane.
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| 10. |
- Rydholm, Susanna, et al.
(författare)
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Microfluidic device for studies of primary cilium direction sensitivity
- 2005
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Ingår i: Proceedings of µTAS 2005 Conference. ; , s. 1416-1418
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Konferensbidrag (refereegranskat)abstract
- This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. It has previously been shown that bending the primary cilium by a fluid flow will give rise to a calcium signal, but the sensitivity for flow direction has so far not been studied. The microfluidic device presented here was designed for control of the local direction of fluid flow on the cellular level, and thus, enables studies of cellular response to a direction controlled cilium movement. Cells seeded on cover slips form cilia with the average length 2.9 μm after three days in culture and 4.3 μm after four days. Distinct calcium peaks were found after the initiation of flow in the channel. By using a microstructured flow system we have been able to study the sensitivity of confluent COS 7 cells expressing primary cilium to changes in fluid flow.
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