Cell counting is a fundamental process for determining cellular density, viability, and proliferation rate in life science workflows aimed at monitoring and controlling cells as they grow. This routine process in research and biomanufacturing can now benefit from a new, stain-free and automated cell counter: CytoDirect.
METTLER TOLEDO offers stain-free, automated cell counting in a portable device, CytoDirect – that can be easily sterilized and used under the hood. CytoDirect leverages non-invasive technologies: digital holographic microscopy, and machine learning algorithms.
CytoDirect’s technology, together with an extra-large measurement area (31.2 mm2) and the elimination of cell staining, ensures measurement reliability in a short measurement time: 15 seconds. A high degree of automation decreases human error, improves process standardization, and eliminates inter-operator variability.
Take it to your sample
CytoDirect is a portable device that enables semi-operator-independent cell counting, viability tests, and cell size distribution assessments at the actual work location (i.e. laminar flow hood, lab bench, in production). CytoDirect also follows the researcher anywhere in the lab thanks to its small footprint, Wi-Fi connection, and integrated battery. The device’s portability (Figure 1) helps to further reduce the risk of cell culture contamination and displacements across the workspace.
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Figure 1. The CytoDirect Cell Counter. |
Say bye to trypan blue
Measuring cell viability typically involves additional biochemical procedures such as staining to probe membrane intactness. This classical method involves extra incubation and handling steps with cytotoxic reagents, which increases error potential, experimental time, and operator exposure risk.
The unique combination of high-resolution digital holographic microscopy analyzed by a convolutional neural network allows reliable and reproducible cell analysis in a fully automated way [1] and without the need for staining. CytoDirect uses the morphological and compositional differences between dead and living cells to determine culture viability. The digital holographic microscope can quantitatively assess changes in composition e.g., reflect protein turnover, making the percentage of viable cells directly accessible. This eliminates staining steps and reduces operation intervention to a minimum. Therefore, CytoDirect preserves the sample integrity and removes any cytotoxicity bias during the measurement [2, 3].
The machine learning algorithm is trained to reliably evaluate a variety of cell lines, making it a useful tool for many applications in biopharma, biotech, and academic research.
More information about digital holographic microscopy can be found in the application note “Holographic Microscopy and Machine Learning: The Revolution of Cell Analysis”.
Ensured reliability
CytoDirect uses an extra-large measurement area – 30 times bigger than a standard hemocytometer – maximizing statistical significance in every single measurement. Moreover, CytoDirect is robust and maintenance-free. It avoids wear-affected moving parts and does not require calibration. The cell counting performance was assessed for many different cell types, including HeLa, PBMCs and yeast, comparing linearity, accuracy, and reproducibility against manual counting using a hemocytometer. The experiments showed a high degree of linearity across the evaluated cell types.
Therefore, it is shown that CytoDirect reliably detects and counts cells across a wide linear operating range, proving that it can be successfully applied in a variety of cell-based experimental settings, ranging from established mammalian lines through primary cells to eucaryotic cells with walls. Moreover, CytoDirect shows higher precision in comparison to manual counting (Figure 2).
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Figure 2. Precision comparison of CytoDirect and the hemocytometer in different cell concentrations. |
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Figure 3. User inserting CytoDirect sample carrier into the measurement slot. |
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Figure 4. CytoDirect with the sample carriers. The black arrows on the sample carriers guide the user for a correct sample loading. |
Keep it simple
Operating CytoDirect is straightforward: The user pipettes 20 μL of a cell suspension without prior staining into the glass-made sample carrier (Figure 4). Then, the user can select an application (cell count or cell count and viability measurement). Guided by on-screen instruction, the user inserts the sample carrier in the adapter slot (Figure 3). The measurement starts automatically. Results are displayed after 15 seconds. The microscopic image can be displayed with a click, providing direct visual insight into the automatic detection of viable and dead cells, as shown in Figure 5. Moreover, CytoDirect easily recognizes cells vs. debris and other contamination, thanks to an integrated debris filter, making the results even more reliable. The user still has the option to further filter the results by size by selecting the appropriate size range as shown in Figure 5.
Connecting CytoDirect to LabX™ software will provide user management, store all measurements and metadata, and enable full 21 CFR Part 11 compliance in regulated environments such as biopharma.
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Figure 5. CytoDirect results display. |
References
[1] Holographic Microscopy and Machine Learning – The Revolution of Cellular Assays, UserCom 28.
[2] Riss, Terry L., et al. ”Cell viability assays.“ Assay Guidance Manual [Internet]. Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2016.
[3] Aslantürk, Özlem Sultan. In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. Vol. 2. InTech, 2018.
