Opto-magnetic Selection and Isolation of Single Cells

[Abstract] Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or intracellular protein localization. We previously introduced a method termed Cell Labeling via Photobleaching (CLaP) for the efficient tagging of cells chosen for visual criteria. Here we describe a new protocol for capturing such cells using ferromagnetic beads termed single-cell magneto-optical capture (scMOCa). This technology is especially useful when the number of target cells represents an extremely low fraction of the total population (potentially one single cell), a situation in which conventional sorting techniques like fluorescent or magnetic activated cell sorting (F/MACS) cannot provide satisfactory results in terms of capture efficiency and specificity. scMOCa uses the lasers of a confocal microscope to photobleach and crosslink biotin-4-fluorecein molecules to cell membranes. Streptavidin coated magnetic beads then adhere to biotin moieties and a magnet allows the capture of illuminated cells. By precisely controlling liquid volumes and spacing between the different parts of a simple setup, high cell selectivity and capture efficacy can be achieved. scMOCA allows visual selection and isolation of any number of cells in a microscopy field and captured cells remain viable to generate new colonies of chosen phenotypes for downstream analyses.

. Once tagged, these cells may still need to be isolated, which can be done using cytometry approaches. Unfortunately, FACS or MACS cannot sort these cells since they are sensitive to the total signal intensity rather than its localization. Some rare label free techniques are based on size selection but have very limited applications (Khojah et al., 2017;Zhao et al., 2017). New technologies based on microfluidics and droplets encapsulation reach extremely low capture rates (less than 10%) (Salomon et al., 2019). A microfluidic chip can only capture a given number of cells that depends on the size of the chip (for instance 96 capture sites, or 384…) which is necessarily smaller than the total number of cells in the sample (which may reach millions). Because most cells are lost, chances to extract a cell population that is already rare in the original population is low.
Finally, some situations involve cells for which there is a marker, but that are too rare to be efficiently extracted with FACS or MACS (Leary, 2000). For instance, rare mutated metastatic or transfected cells. scMOCA is optimized for low cell numbers and as such is an ideal technique in these situations.
Our protocol allows manual selection of single cells one by one based on imaging, regardless of their biochemistry. A sample can be imaged or filmed with a microscope to identify fast migrating cells, or cells with different expression patterns (such as the localization of a 53bp1-GFP ).
Chosen cells are then tagged by crosslinking biotin on their membrane using photobleaching: a laser is used to photobleach biotin-4-fluorescein in the close proximity of the membrane of the cell of interest.
Upon photobleaching, a reactive free radical is created, which will bind to the cell membrane, hence biotinylating the cell of interest (Binan et al., 2016;Binan et al., 2019). Because this reaction does not depend on the surface chemistry of the cell, any cell type can be labeled. Using scMOCA, as few as one single cell can be extracted as shown in Figure 1 to generate highly pure samples.

Materials and Reagents
1. N35 Neodymium Disc Magnet 3/8'x1/16" Rare Earth Disc Magnets, CMS magnetics, store away from hard drives and cell phones as it may damage them 2. Nails from a hardware store. Nails should be made of iron (not steel) and have a 1 mm diameter head. This nail will channel magnetic field lines to the center of the collection chamber   Figure 2 and the setup is visible in Video 1. 3. The coverslip with the collection chamber will be inserted just above the long bricks from Step 7 in Figure 2. The long transversal bars from Step 8 in Figure 2 will be used to pinch it in position. Reagents list above) is hollow and two magnets are inserted inside. They allow holding the rest of the pile of magnets on the side of the brick, as visible in Figure 3 and Video 1. vary from a cell type to another. Repeat this step for each cell that needs to be captured.

Rinse thoroughly in PBS.
The medium has to be well rinsed as remaining free biotin-4-fluorescein will block binding sites on the beads. It is key that the chamber never dries, which can happen very fast due to its extremely small total volume. In our hands, dipping the culture chamber in a PBS beaker several times provides excellent results whereas pipetting tends to detach some cells, and the tip may scratch the bottom of these small chambers.
6. Replace liquid in the chamber with medium containing 3 µl of beads washed in PBS as per manufacturer instructions, without further dilution.
7. Use a magnet to pull beads down towards the cells.
When strongly adherent cells are used, the magnet can be used to pull beads across the whole area to ensure maximal attachment of the beads. When sensitive cells are manipulated, beads can be pulled down with the magnet, then resuspended either by pipetting the solution up and down a few times or by placing the magnets above the sample, then pulled back down with the magnets three times. The ideal strategy has to be determined for each different cell type and culture substrate as it is dependent on adhesion strength.

Notes
1. Before its first use on any setup and with any new biological system, the procedure's parameters should be optimized. The adequate laser power that permits efficient cell labeling without killing has to be determined for each experiment and is highly dependent on the alignment of the laser with the microscope objective but should not vary with the cell type. The ideal laser power is the lowest power that allows efficient cell labeling on the setup used. We usually use around 50 µW.
2. The appropriate surface preparation for the culture chambers depends on the cell type. Most sensitive cell types may require collagen coating, but this option makes following steps more difficult. When cells are more resistant, one may prefer to choose gelatin or poly-ornithine, or even bare glass when working with resistant cells. where the cells will be attracted by the magnets. The distance between the two chambers substrates should be kept at 6 mm, which is close enough for the magnets to efficiently pull up positive cells, but also far enough to maintain negative cells far from the collection chamber.

Conditioned medium
When passaging cells, the day prior to the experiment, keep old medium and filter it through 0.22 µm filter to remove debris (with syringe and syringe filters). This medium is rich in secreted factors and helps culture cells at very low density.