Getting to grips with cell adhesion

Understanding how cells adhere and move is a rapidly growing area of research yet an assay to successfully replicate in-vivo conditions has, until now, proved elusive

Fibroblast adhering to a cover slip

BIOCHEMICAL, microbiological, chemical and many other assays are performed every day in laboratories. While a considerable amount of attention has naturally been placed on biological cell assaying for humans, this is also becoming more important in the field of animal welfare and plant production. 

A rapidly advancing research area in biology is the study of cell receptor-ligand interactions resulting in cell-substratum and cell-cell adhesion followed by subsequent cell migration. The pre-requisite to transendothelial migration of certain cell types into sites of infection is paramount to the study of inflammatory diseases. This can be briefly summarised as cell flow and rolling, tethering and activation of integrin receptors (which is a key recognition step), attachment to the endothelial ligands via activated integrins and finally transendothelial migration or diapedesis.  Unfortunately, to date, most of the assay techniques are not particularly successful for the study of these mechanisms. A microfluidic pump system, designed by Cellix has been developed to address this issue.

Currently, the majority of studies involving cell rolling and chemokine induced cellular arrest have utilised capillary systems wherein cell flow and shear stress are controlled utilising syringe pumps.  Such observations are constrained by a number of factors. Firstly, the relatively large (>100?m) size of the standard glass or plastic capillaries limits the physiological analogies to the proximal microvascular regions. Secondly, such studies can only be utilised to study single end-points and cannot be utilised to examine cell choices in migration. Thirdly, optical aberrations related to the spherical geometry of the glass capillary sections limit stage-related in situ (post-fixation) analysis of the intracellular structures (cytoskeleton and signalling molecules). Finally and most importantly, the usual observation periods lie between 5-30 minutes for rolling experiments.  Longer studies are required to study subsequent crawling steps on endothelial and extracellular matrix ligands. In this regard, studies relating to the effects of chemokines have largely been limited to cellular arrest on adhesion receptor ligands and have not been extended to the study of cell crawling. For example, specific chemokines have been shown to induce rolling arrest with enhanced binding of lymphocytes to ICAM-1.

The Cellix microfluidic platform circumvents a lot of the constraints mentioned above. The system uses polymer biochips that contain channels equivalent in size to smaller blood vessels, e.g. post-capillary venules. This is significant since the bulk of migratory processes occur in these blood vessels. Cells flowing through the biochip channels can be observed via a digital camera connected to a microscope. Depending on how the individual channel is coated (e.g. adhesion proteins, endothelial cells, etc) cells can be observed rolling, adhering, and/or migrating under flow conditions. Images can be captured of these events, using the digital camera, and can be analysed at a later date, thanks to the construction of the biochip allowing clear and defined images to be taken. Finally, the microfluidic platform can run experiments continuously for hours or even days where necessary and, due to the small volumes of materials required, consumable costs are kept to a minimum.

Presently accepted techniques for cell adhesion or binding assays involve the initial coating of a surface of a device with a substrate, typically a protein. Cells are deposited onto the substrate and allowed to settle. Following the settling of the cells, the device is placed on a heating stage at 37ºC, which is attached to an inverted microscope for visual analysis, or alternatively to a stand-alone heating stage and progression of cell binding can be checked at intervals with the inverted microscope. The duration of these assays may be varied depending on the cell line and choice of substratum. Following cell adhesion, free cells may be washed away and a subsequent cell count may be carried out. As mentioned, the Cellix platform uses polymer biochips whose channels can be coated with any desired protein e.g. VCAM-1, ICAM-11. Also, the microfluidic system is housed in an incubation chamber, kept at 37°C. This is a more efficient way of maintaining the required constant temperature, compared to a heating stage based in the microscope ledge. The assay can be altered easily to accommodate different flow conditions i.e. varying levels of and application times of shear stresses2,3.

The Cellix platform

Although current methods provide researchers with semi-quantitative information regarding a cell type’s affinity for a particular substratum, there is no simple method for quantitative characterisation of binding or methods enabling a prolonged study of cell rolling, the ensuing capture by the substratum and subsequent attachment. Furthermore, direct studies of changes in cell morphology, cell growth and biochemical changes cannot be provided easily with these techniques since, determining the kinetics of attachment and resulting morphological changes requires multiple replicated experiments being analysed at different times. In short, static assays do not have any consideration of cell flow and rolling. The Cellix microfluidic system allows assays to be completed under more physiologically relevant conditions i.e. under flow, giving more accurate data and a better understanding of in vivo events. The Ducocell software that accompanies the system produces highly detailed analyses of images taken during experiments, generating data pertaining to cell morphology and cell kinetics.

The ability of T-cells circulating in the bloodstream to adhere to the endothelium, switch to a motile phenotype and penetrate through the endothelial layer is recognised as a necessary requirement for the effective in vivo trafficking of specific lymphocyte sub-populations. Motility assays are done in combination with attachment assays since following adhesion, cells are expected to switch to the motile phenotype. Motility assays are assessed by estimating the ratio of cells undergoing cytoskeletal rearrangements and the formation of uropods (extension of the trailing tail). One of the major disadvantages of this and the previous adhesion assays is the geometrical design (microscope slides and multiple well chambers), which does not at all resemble the in vivo situation. The Cellix system was designed to resemble in vivo conditions as closely as possible, from the range of shear stresses achievable by the microfluidic pump, to the dimensions of the channels in the Vena8 biochip.

The most commonly used cell transmigration assay is a modified Boyden chamber assay. This involves assessing the crossing of a quantity of cells through a microporous membrane under the influence of a chemoattractant. Here the diameter of the micropores are less than the diameter of the cells under investigation, such that the cells must deform themselves in order to squeeze through the pores thereby constructing an analogy to the transendothelial migration of cells in physiological circumstances. Once cells are deposited onto the membrane, the chamber can be incubated for intervals. Following this, the bottom chamber or opposite side of the top chamber may be analysed for cells that have squeezed through the microporous membrane.

The main disadvantage of such assays is that the biological process of transmigration through the micropores is difficult to observe due to the geometrical configuration of the apparatus involved.  The lens of the optically inverted microscope must be able to focus through the lower chamber and the microporous membrane. This obviously leads to difficulties due to optical aberrations. In effect, the study of the cells morphology changes while transmigrating across the membrane and their subsequent cytoskeletal changes reverting to their former state is a process which is difficult to monitor and record due to limitations with current techniques. The Cellix platform’s Vena8 biochip allows clear and detailed images of cells undergoing rolling, adhesion, and/or migration to be obtained. 

In addition, with protocols like the Boyden Chamber assays, although it is possible to alter experiment parameters following the initiation of the experiment - such as the introduction of a second chemoattractant at some specified time after commencing the experiment, it is not possible to distinguish separate effects from each said chemoattractant. Cellix’s microfluidic system enables to user to observe cell behaviour in the biochip channels, via a digital camera connected to the microscope. Also, it is very easy to introduce other parameters, such as another chemoattractant, either before, during, or even after the assay has been completed. This means a greater amount for data can be obtained from just one experiment.

In addition to cell biology studies, the pharmaceutical industry has major problems in the drug screening process and while high throughput screening (HTS) has been extremely successful in the elimination of the large majority of unsuitable drugs, it has not progressed beyond that. Usually, after a successful HTS assay, a pharmaceutical company may still have several thousand possible drugs requiring assessment. This requires laborious bench-testing and animal trials and anything that can be done to reduce the amount of animal trials is to be desired.  Thus, there is a need for new techniques for drug testing in the pharmaceutical industry. The Cellix microfluidic system closely mimics in vivo conditions in an in vitro system, allowing a large number of assays to be completed that, currently, are done using animals (e.g. pharmacokinetic studies, drug efficacy and potency studies).

The current proposals are to screen the physiological response of cells to biologically active compounds. This again, unfortunately, is still a static test. Since the cells are spatially confined with the drug, there may be a reaction but it may not necessarily take place when the cells are free to flow relative to the drug as in, for example, the microcapillaries of the body. There are other disadvantages such as the transport and subsequent reaction of the drug following its injection into the animal. Probably the most important disadvantage is that it does not in any way test, in a real situation, drug efficacy. The Cellix platform has been shown to give accurate data with respect to drug efficacy and potency, due to assays being under flow conditions2,3.

Finally, there are no widely known techniques at the present moment for performing assays to test the interaction of a large number of chosen compounds with living cells while the cells or compounds mimic the in vivo situation of continuous flow. The Cellix system aims to rectify this situation.

References
1. WILLIAMS, V., KASHANIN, D., SHVETS, I.V., MITCHELL, S., VOLKOV, Y., & KELLEHER, D. (2002). Microfluidic enabling platform for cell-based assays. J. Assoc. Lab. Automation, 7, 135-141.
2. WU, P., MITCHELL, S., & WALSH, G.M. (2005). A new antihistamine levocetirizine inhibits eosinophil adhesion to vascular cell adhesion molecule-1 under flow conditions. Clin. Exp. Allergy, 35, 1073-1079.
3. ROBINSON, A.J., MITCHELL, S., KASHANIN, D., WILLIAMS, V., & WALSH, G.M. (2006). Montelukast inhibits both resting and GM-CSF-stimulated eosinophil adhesion to VCAM-1 under flow conditions. Eur. Resp.  J., 28(Suppl) 50, P2527.

Jaquie Finn
Jaquie has worked in the life science industry for over 16 years. She is currently Marketing Manager for Stratech Scientific.