Innovating cell biology

Cell biology is one area in which technological developments are helping to drive ground-breaking advances in ourunderstanding, and Tecan has been at the forefront of this technicalinnovation.

MEDICALTECHNOLOGY: It is a universal truth that necessityis the mother of invention, and this has driven technological developmentin the life science sector since its earliest days. Recognizing and harnessingthis need to inspire new products and solutions has been key to Tecan’s successand, after 30 years as a leading globalprovider of laboratory instruments, innovation is still central to theCompany’s philosophy. Innovation takes place at many levels – from instrument andcomponent design to software and consumables development – and as the landscapeof life science research becomes increasingly specialized into focused areas ofstudy, so too must the solutions the industry provides to assist this research.Cell biology is one area in which technological developments are helping to driveground-breaking advances in our understanding, and Tecan has been at theforefront of this technical innovation. This article discusses some recent andemerging technologies designed to aid cell biology research, focusing on theneed for reliable, reproducible measurements to help scientists quantify livecells.

Fostering creativity

Tecan wasfounded on the premise that engineering solutions could accelerate scientificdiscovery by providing an easier way for researchers to work in the laboratory.Since those early days, Tecan has continued to innovate the laboratoryenvironment, developing analytical detection and laboratory automation devicesto meet the changing need of the scientific community. A great leap intechnology can start as the smallest of ideas, and most Tecan projects startwith somebody in the company recognizing a gap between what scientists aretrying to achieve in the laboratory and what existing products on the marketare capable of. Having a close working relationship with its customers is vitalto this process, due to the fast paced development currently being seen in manylife science sectors, as well as having a very strong research and developmentprogram.

Tecan pridesitself on the wealth of experience within the company, and combining thisknowledge with a continuous influx of fresh ideas and new people provides anexcellent environment for innovation. Creativity is nurtured and supported toensure that questioning the established way of doing things is not discouraged.i-control™ – the software interface for the Infinite^® series ofmultimode readers – is a good example of this, forgoing the traditionalmenu­based software architecture in favor of a workflow-oriented system (Figure 1), and was developed from observingthe way scientists in the laboratory used instruments. By understanding thatthere is a logical progression of experimental steps, Tecan engineers developeda straightforward, application-based system, allowing users to create theirworkflows by selecting and adapting pre-defined processing steps from a simple‘drag and drop’ menu.

Modularsystems offer tailored solutions

Understandingthe way users interact with existing technology is key to the development ofnew products, ensuring that these solutions perfectly match the requirements ofcustomers. However, the broad spectrum of potential applications for even asingle instrument – such as a microplate reader – requires highly versatilesystems that can be customized to meet individual users’ needs, and the Infinite200 PRO was designed from the ground up with this in mind (Figure 2). Buildingon the success of the previous Infinite 200 series instruments, the newplatform offers the user a choice of modules to reflect the intended use oftheir system, with the option to upgrade as their needs change. This latestreader is particularly suited to cell biology applications, incorporating arange of features designed specifically to aid fluorescence-based cellularstudies.

Simplifyingfluorescence measurements

Fluorescenceintensity (FI) measurements make up the majority of cell-based measurementsusing a microplate reader, and the Infinite 200 PRO has several featuresdesigned to maximize the reproducibility and reliability of FI measurements.For suspension cell cultures, and other applications where the fluorescenceemission is measured from above the microplate, the monochromator-basedInfinite M200 PRO offers adjustable, automated z-focusing for maximumflexibility and performance. The adjustable z-focus for FI top measurementsensures equally high sensitivity for almost any fill volume, as well as high orlow base 384-well plates, helping to reduce costs through assay miniaturizationwithout compromising the quality of results. The z­scan function also allowsthe user to determine the z-position providing maximum signal intensity whileminimizing background interference (Figure 3). Alternatively, the system canautomatically calculate the optimum z-position for measurement within eachwell, providing fully automated incubation and measurement.

In contrastto suspension cell cultures, fluorescence measurements for adherent cell typesare generally performed through the bottom of the well, in order to bring thecell monolayer as close as possible to the detector and avoid backgroundfluorescence from the overlying growth medium. It is vital to guaranteeillumination and reading of the entire well base for these applications, ascells are rarely distributed homogeneously over the growth surface. TheInfinite 200 PRO features an enlarged detection fiber for enhanced FI bottomreading, and has an Optimal Read (OR) function designed specifically formeasuring adherent cells in microplates. Enhanced FI bottom reading with ORprovides markedly improved well-to-well reading uniformity and bettermeasurement reproducibility compared with the previous generation of Infinite200 instrument, offering lower CVs through improved measurement homogeneity.

Uniqueenvironmental control

Manycell-based assays and investigations require time-dependent analysis ofcellular signals, with an experimental window ranging from several hours toseveral days. However, long-term incubation of cells requires rigorous controlof environmental conditions, as well as mechanical agitation to ensure mediumhomogeneity. Thanks to its soon to be launched Gas Control Module (GCM™), the Infinite 200 PRO is the onlymultimode reader on the market to offer rigorous environmental control,allowing the user to regulate CO₂ or O₂ partial pressurein addition to providing linear orbital shaking and temperature control.

The GCM is abreakthrough for microplate-based cell biology applications, and isparticularly suited to eukaryotic cells (Figure 4). Proliferationand survival of eukaryotic cells is strictly dependent on the environmentaltemperature and on the culture medium conditions. The pH of eukaryotic culturemedia is typically maintained using a bicarbonate buffer system in conjunctionwith sensor-based control of atmospheric CO₂ partial pressure tostabilize the buffer system. This requires an incubator equipped withsensor-based CO₂ control to avoid pH shifts which might adverselyaffect proliferation or even cause cell death. The patent pending GCMeliminates the need for an external CO₂ incubator, by providingdynamic control of CO₂ partial pressure from 0 to 10percent within the reader’s measurement chamber. This allows long terminvestigation of cellular activity without repetitive transfer of platesbetween the reader and incubator, improving reliability and reproducibility ofdata, and ensuring complete data sets without overnight gaps in results.

Liquidhandling expertise

Althoughprecise regulation of environmental conditions is crucial to ensure reliableand reproducible results, careful handling and manipulation of cultured cellsduring processing and analysis is equally important, particularly for adherentcell lines. Many protocols require gentle yet thorough washing of adherentcultures to remove medium components and other reagents, and this can bedifficult to achieve reproducibly, particularly for commonly used looselyadherent cell lines such as P815 (mouse mastocytoma)and HEK293 (human embryonic kidney) cells. Automation of washing proceduresoffers improved consistency for cell-based assays, by eliminating handlingerrors and variations between users, however loosely adherent cellspresent a significant challenge for typical plate washers. Excessive dispensespeeds can cause detachment of cells and holes in the cell layer and this, coupledwith loss of cells during poorly controlled aspiration, can lead toreproducibility issues and high CVs, resulting in failed experiments, repeattesting and a loss of productivity.

With over three decades of experience inliquid handling for life science applications, Tecan is well placed to developadvanced solutions for automated washing of microplate-based cell cultures. TheHydroFlex™ microplate washer combines application flexibility and performanceto offer a 3-in-1 solution for washing cells, beads and ELISAs, and has anumber of ‘gentle washing’ options to improve reproducibility of cell-basedassays. Advanced liquid handling is at the heart of this platform, offeringindividual control of critical wash parameters such as the aspiration anddispense speeds and position of the wash needle within the well. This allowsthe user to fine-tune the wash protocol to their specific cell type and assay,ensuring minimal disturbance to the cell layer during washing (Figure 5).

Tecan’s soon to be released new washer – theHydroSpeed™ – has been designed to better serve the cell biology sector,offering even greater performance for simultaneous washing of loosely adherentcells in 96- and 384-well formats (Figure 6). With tuneable Cell Protection™wash parameters, the HydroSpeed combines the same drop-wise dispensing andaspiration vacuum control as the HydroFlex with intelligent needle positioningthat continuously optimises the wash position during aspiration, and starts dispensing at the sameposition aspiration stopped, minimizing the risk of damaging the cell layer.

Cellbiology meets immunology

Althoughculturing cells in a microplate format is now commonplace, particularly forcompound screening applications, other microplate-based cell biology techniquesare growing in popularity, due to the reduced costs and time savingsminiaturization offers. One such technique is the ELISPOT (enzyme-linked immunosorbent spot) assay, which is similar inprinciple to ELISA methods but allows visualization ofindividual cells exhibiting surface markers of interest (Figure 7). Due to its ultra­high sensitivity, ELISPOT assays areused to study small populations of active cells, such as those found inspecific immune responses, making them highly suited to investigation of variousdiseases, including infections and cancer. Precise, reproducible washing ofELISPOT plates is vital to the sensitivity of the assay, particularly for frequency analysis studies, as manual washing or inappropriate washparameters are likely to lead to artefacts and false positive results. Thetuneable wash parameters of Tecan washers make them ideally suited to ELISPOTwashing applications, allowing the user to determine the optimum conditions forwashing, based on both cell type and target binding affinity (Figure 8).

As with high throughput ELISA processing using a microplate washer, crystallizationof wash buffer within the aspiration/dispense needles is a problem forautomated ELISPOT washing. This causes clogging of the needles while the washeris idle between plates, and can lead to loss of entire runs as a result ofoverflowing wells. Cleaning of wash heads is laborious and time consuming, andcan significantly affect productivity if washers are integrated into automatedliquid handling systems. To address this issue, the new HydroSpeed washer hasan Anti­Clogging™ function that actively prevents needle clogging, byautomatically rinsing and soaking the wash head when the washer is idle. Thissimple innovation simplifies laboratory processes and increases productivitywhile avoiding periodic maintenance, and was born of an in depth understandingof customers’ needs.

Summary

Laboratory automation is changing the face of cell biology, allowingmore experiments to be completed faster and more cost effectively than everbefore. Miniaturization is a vital part of this process yet, as the capacity ofindividual experiments decreases, the drive towards large scale, parallelstudies continues to grow, especially for compound screening and drug discoveryapplications. Advanced laboratory automation is vital to the success of theseinvestigations, providing a high level of consistency and reproducibility toallow meaningful comparison of large data sets, and Tecan continuous strives toremain at the forefront of this development.

Byunderstanding that the same solution is not right for every application, andclosely collaborating with its customers and industry partners, Tecan is ableto offer advanced, modular readers and washers that can be tailored to eachlaboratory’s needs. Putting the user in control of instrument settings andparameters is a key aspect of achieving high quality, reproducible results, yetit is also important to understand the way these systems are used in thelaboratory and the level of programming experience amongst users. By developingintuitive, application driven software interfaces for its instruments, Tecan isable to offer the user optimized control of critical technicalparameters, without the need for extensive automation experience and training.