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.
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.
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.
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.
Background: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale.
Results: Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms.
Conclusion: Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.
Na, K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappa B activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na, K-ATPase/ IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na, K-ATPase and IP3R. We identify the N terminus tail of the Na, K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na, K-ATPase and IP3R, reduced the number of cells responding to pM doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappa B. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na, K-ATPase and no effect on the distribution and apparent mobility of Na, K-ATPase in the plasma membrane.
In this work we present the fabrication and testing of a thermally actuated one-shot liquid dispenser, which actuation is based on highly expandable microspheres. We show an uncomplicated, fully functional, low cost device for use in medical disposables, e.g. transdermal systems based on microneedles. All device components are made out of low cost materials and fabrication processes have the potential for high volume batch manufacturing. The device utilizes the properties of the expandable microspheres to form a heat insulating layer to the delivered liquid. Moreover, it does not require any feed-back or complicated flow metering. The device was successfully tested showing a mean dispensed liquid of 101 mul with a relative standard deviation of 3.2% and with a maximum temperature of 59 degreesC in the liquid during actuation. No back-flow was observed for the device.
This work presents a new low-cost liquid dispenser for the dispensing of microliters to milliliter volumes. The dispensing mechanism is based on a thermal actuator where highly expandable microspheres expand into a liquid reservoir consequently displacing any stored liquid. All device components are made out of low-cost materials and the fabrication process has the potential for high volume batch manufacturing. The device utilizes the property of the expandable microspheres to form a heat insulating layer between the heat source and the delivered liquid. Moreover, it does not require any feed back or complicated flow metering. The device was successfully tested showing a mean dispensed volume of 101 mu 1 with a standard deviation of 3.2% and with a maximum temperature of 59 degrees C in the liquid during actuation. It was shown that the dispenser is strong enough to deliver against counter pressures as high as 75 kPa. The device can also function as a low flow rate dispenser as demonstrated in a microfluidic dye laser application. The flow rate can be controlled between 1 mu 1 h(-1) and 2400 mu 1 h(-1) by adjusting the actuation power.
This thesis presents methods to combine 3D cell culture, microfluidics and gradients on a controlled cellular scale. 3D cultures in biological extracellular matrix gels or synthetic gels bridge the gap between organ-tissue cultures and traditional 2D cultures. A device for embedding, anchoring and culturing cells in a controlled 3D flow through micro-environment was designed and evaluated. The device was realized using an etched silicon pillar flow chamber filled with gel mixed with cells. The pillars anchor and stabilize the gel as well as increase the surface to volume ratio, permitting higher surface flow rates and improving diffusion properties. Within the structure cells were still viable and proliferating after six days of cultivation, showing that it is possible to perform medium- to-long term cultivation of cells in a controlled 3D environment.
This concept was further developed to include controllable and time stable 3D microgradient environments. In this system stable diffusion gradients can be generated by the application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cells. 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 fluorescent 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 an intracellular calcium release also depends on cell position.
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.
We present the first microfabricated microfluidic devices designed specifically for studies of primary cilium mediated cellular response to dynamic flow. The primary cilium functions as a mechano-sensor in renal tubular epithelium, sensing the extracellular fluid flow. Malfunction of cilia has been implicated in e.g. polycystic kidney disease and other pathological conditions. Bending of the primary cilium by fluid flow has been shown to give rise to an intracellular calcium signal, however little is known about the sensitivity to flow duration, magnitude and direction. This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. The microfluidic devices presented here were designed for a dynamic control of the local fluid flow on a cellular level, and thus, enables studies of cellular responses to an amplitude, frequency and direction controlled cilium movement.
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.
With confocal microscopy new knowledge in cell physiology is acquireddaily. However, most cell assays today are carried out either as multiwellplate assays, or in standard petridish assays. These two methods havedifferent features and foci, but they have in common the large amount ofcells submitted for treatment and imaging. In order to study only a few cellson a more detailed level[1, 2] in a relevant context, we have designed, built,and evaluated a microfluidic system. It features 1) immobilization of cells inthree dimensions, 2) transportation of cell nutrients and treatments as wellas removal of residual products, 3) an extremely stable and physiologicallyrelevant gradient of chemical concentration distribution around the cell.Previous efforts in this field by our group revealed a few very importantissues, indicating that microfabrication would be the enabling technologyfor experiments on cells in asymmetricenvironments.
We present an improved device and method for studying cellular response onmicrofluidic controlled stimuli of primary cilia. The primary cilium functions as amechano-sensor in renal tubular epithelium. Malfunction of cilia has been implicated inpolycystic kidney decease as well as other kidney abnormalities. Bending of cilia willgive rise to an intracellular calcium signal [1,2,3], but little is known about theimportance of flow direction, magnitude and duration to the calcium response. Ourpreliminary results indicate flow speed sensitivity.