Isolation of Precise Number of Cells
In many applications, such as measuring enrichment rates or assessing sensitivity and specificity, a precise number of cells is required to create high-quality spike-ins for reference samples. Conventional cell sorting techniques like FACS or single cell printing cannot guarantee the deposition of such exact numbers of cells to a destination well/tube.
The CellCelector platform, combined with our nanowell plate technology, allows for an extremely precise and gentle isolation of a precise number of cells. For example, if a precise count of 1, 5, 10, 50 or 100 cells is necessary for your assay, the CellCelector can precisely retrieve exactly those 1, 5, 10, 50 or 100 intact cells.
Fig. 1. The CellCelector allows isolating cells to any standard or custom plates, tubes or vials.
The CellCelector platform for isolation of precise numbers of cells consists of (i) seeding cells in a CellCelector S200 ULA nanowell plate at a cell density corresponding to the target cell number per nanowell; (ii) automated scanning and counting of the cells in the nanowells; (iii) automated selection of the nanowells populated with the target cell number; (iv) validation of the list by the operator; (v) automated transfer of selected sets of cells to the destination tubes/wells with complete tracking of the whole process (Fig. 2).
Fig. 2. The CellCelector workflow for automated isolation of precise numbers of cells.
When seeding a single cell suspension into a nanowell plate the cells are randomly captured inside the nanowells. Ideally a uniform seeding of a perfect single cell suspension follows a standard Poisson distribution. Here the probability (p) to randomly capture a given number of cells (k) in a nanowell is described by the equation where λ is the average number of seeded cells per nanowell. Fig. 3 shows both the theoretical and measured cell distributions in one well of a S200 24-well nanowell plate for several cell seeding densities. This allows the calculation of the number of cells to be seeded into each well of a 24 -well plate that is needed in order to maximize the probability of capturing the required number of cells inside the nanowells.
Fig. 3. Theoretical (lines) and experimental (dots) Poisson distributions for seeded cell densities of 1, 5, 10 and 20 cells per nanowell. Total number of scanned and analyzed nanowells has been 2,500.
The seeded wells can be scanned in BF and any fluorescence channel (Fig. 3 and Fig. 4) if the cells are fluorescently labelled. Cell counting can be done both in BF and fluorescence modes. After cell counting the user can review the images of all nanowells in a gallery of nanowell images.
Fig. 4. Overview image of one well scanned in FITC channel. The circle visualizes the scan and analysis area.
Fig. 5. A typical cell distribution in 200 µm nanowells with an average cell density of 20 stained SK-BR-3 cells per nanowell. Left: bright field, right: FITC.
Fig. 6. Example of 12 nanowells with 5 cells automatically selected by the CellCelector software.
Once the operator has validated the list of nanowells to be recovered, the system automatically picks from all the selected nanowells. During picking the system automatically saves images before and after picking for quality control purposes. The system also allows pooling the content of several nanowells into one destination tube leading to a deposit of larger numbers of cells, e.g. 20 cells = 2 nanowells x 10 cells, 100 cells = 5 nanowells x 20 cells.
Fig. 7. Example of picking a set of 20 cells. Before and after picking images are saved automatically.
100% of the picked cells are deposited in the destination well. If the cells are transferred into a flat bottom plate or tube, the deposited cells can be easily visualized and counted using the CellCelector software (Fig. 8).
Fig. 8. Detection of 20 cells deposited in an optical flat bottom PCR tube.
The user can export all numerical data including full tracking information for source and destination plates as well as all images (scans, before/after picking, etc.) related to the picked nanowells.
Fig. 9. Example of a gallery of pre-picking nanowell images corresponding to sets of cells isolated to specific wells of a destination 96-well plate.
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