In cell biology research, it is vital that cell lines under study be monoclonal, proliferating from a single cell. This is important for all applications in this field, such as the production of knock-down cells or clones which highly express some selected trait.
This is difficult to achieve in academic laboratories, because of the need for automated microscopy, which requires complex and expensive systems. The other and more usual alternative is therefore manual verification of monoclonality using microscopy.
In this setting, the new Spark® platform has multiple reading modes and a bright-field imaging setup which allows cells to be counted as well as the analysis of cell viability using the disposable Cell Chips™ modality.
The result is a real-time cell count without the use of labels, within microplate wells. The areas covered by cells are first detected and then the relative confluence ratios are calculated, in any plate format ranging from 6 to 96 wells.
This is followed by a user-friendly application in the SparkControl™ software built into the reader, which gives accurate measurements and patterns that lend themselves to user definitions, as shown in Figure 1.
This tool is appropriately used for applications with low to medium throughput. Both end-point and kinetic modes of confluence measurements may be performed, and it takes less than 40 minutes of total time to measure cell counts and do an analysis of a 96-well plate.
The user has the option of selecting one or more areas within the well, as per individual experimental requirements, as shown in Figure 2. It can thus produce one image of the center of each well, or an array of side-by-side images compiled to create a picture of the whole well. The well borders are automatically detected by the software, to make up for any non-uniformity of well size.
This article describes the use of the Spark platform to count cells and produce images of the wells in a microplate with 96 wells, using automated imaging and semi-automated analysis of the image and other data, so as to verify cell monoclonality in a cost-effective manner.
Materials and methods
Cell seeding and growth
In this setup, HEK293 cells were used. They were first grown to the point of confluence of 70-90% of cells, following which trypsin was added. The separated cells were again suspended in normal culture medium at a concentration of 10 cells/ml. The Spark was then used to count the cells and determine the viability.
After this was done, 100 μl of suspended cells was added to each well of a flat-bottomed 96-well plate which was treated for cell culture. 1E+04 cells were also seeded simultaneously to each well to act as positive growth controls. Nutrients were added at intervals of 3-4 days. The cells were allowed to grow until they attained a 20-30% confluence in each well, which took about 17 days on average.
Imaging and analysis
Following seeding, the first image acquisition was at four hours, allowing this time for cells to begin to adhere to the floor of the wells. Thereafter weekly imaging was performed until the target confluence was achieved.
The SparkControl software was set to include Whole-well imaging, Well border detection, and Data analysis included modes. 20 images were acquired per well. These were then combined by tiling. The selection of Whole-well imaging and Well border detection meant that even cells lying singly on the bottom of the well, at the very boundary, were also imaged. Both raw and processed images are then saved because of the Data analysis included mode.
The reader also has facilities to set environmental controls using which the measurement chamber was kept at a temperature of 37 °C and 5% CO2 (in the present case), to keep the cells viable at an optimal level while they are being imaged.
The Humidity Cassette from Tecan was also employed to keep the cell media from being depleted by evaporation, using the automated Lid Lifter which can take off the lid of the cassette but keep the lid of the culture plate on while measurements are proceeding. The culture plate has a transparent lid. The Lid Lifter thus keeps the plate sterile until the completion of the experiment.
On Day 17, manual screening was performed on whole-well images which showed a confluence of over 10% on automated analysis, so that cell growth could be picked up.
These wells, which were positive for growth, were then traced back manually to the first day, and the raw images were screened without assessing the confluence, to ensure the monoclonal origin of these cells. It must be noted that this is made possible by the high quality of the imaging, ensuring easy identification of single cells.
As seen in Figure 3, a single cell clone of HEK292 may be identified in a representative well.
Discussion and conclusions
The results of this experiment validate the concept of semi-automated verification of monoclonality of a cell culture using semiautomated image acquisition, in applications with low to medium throughput, using the Spark to count cells and acquire images of the wells and its SparkControl software for data analysis.
The quality of imaging was found to be very high, producing only one error in autofocus in over 1000 microplate wells which were analyzed during the period of the experiment. The confluence level which was automatically generated was used to detect the colonies on Day 17. The immediate next step was manual inspection of the raw images, backtracking to day 1 to ensure that the cultures had been generated by a single clone.
This approach was very efficient and proves the value of the Spark reader platform in providing a cost-effective and useful alternative to time-consuming and wearisome manual verification using microscopic images to validate monoclonality.
Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.
In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.
They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.
They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.
In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world
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