Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device

Our lab develops nanoparticles as tools to study biological barriers and to overcome these barriers at mucosal surfaces to improve drug delivery and immunotherapy. One of the most notable developments is the COVID-19 vaccines by Pfizer, BioNTech, and Moderna that use lipid nanoparticle formulations to deliver mRNA for vaccination. There are a lot of ongoing efforts to improve immunotherapies like vaccines and cancer treatments using nanoparticles as these formulations could improve target delivery to immune cells and reduce the systemic side effects of immunotherapies.

Single cell analysis is an essential component of immunology in biomedical research, one of the most common techniques being flow cytometry. Performing such cell isolations from tissues of interest requires reliable tissue dissociation. Making laboratory research more accessible without compromising quality and accuracy can be challenging.

Automating processes makes technical and labor intensive steps easier to learn and allows trainees to gain experience doing more sophisticated analyses without affecting the reliability of results. The main advantage of this protocol and accompanying device design is its customizability and cost effectiveness. The ability to process up to 12 tissues simultaneously reduces processing times.

This low cost alternative makes this technology more accessible in lower resource research settings. To begin, place the dissected mouse tissue in a cold cell culture medium containing 5%FBS. Using sharp dissection scissors, chop the tissue until approximately five millimeters fragments are obtained.

Transfer the chopped fragments to a C-tube and add one milliliter of cell culture media. Load the C-tube onto the motorized device and fit the tube holder plate over the D-shaped tube bottoms. Latch the tension arms into the acrylic plate to secure the tubes to the motor plate.

To set a custom program on the motorized device, from the main menu, press the blue button to choose custom mode. In the first custom mode menu, press the green button to specify the duration of forward rotation to 30 seconds. Then press the blue button to select the onscreen value.

In the second custom mode menu, press the green button to specify the duration of reverse rotation to 10 seconds. Then press the blue button to select the onscreen value. In the third custom mode menu, press the red button to specify the selection loops to four times.

Then press the blue button to select the onscreen value. Adjust the voltage control dial to 200 RPM and start the custom program. Next, add digestion enzyme and four milliliters of cold culture media containing dissected tissues.

Again, load the C-tube onto the device and repeat the custom program as demonstrated previously. After the run, transfer the device with loaded tubes to a 37 degree Celsius incubator for 45 minutes. Next, load the C-tube onto the device and set the custom program at 50 RPM with forward rotation to 270 seconds, reverse rotation to 30 seconds, and looping nine times.

Add EDTA to a five millimolar final concentration to the sample. Set the custom program at 100 RPM with forward rotation to 30 seconds, reverse rotation to 10 seconds, and looping twice. Next, pass the cell suspension through a 70 micrometer cell strainer and collect the filtrate in a 50 or 15 milliliter tube.

Centrifuge the collected cell suspension at 300g for five minutes at four degrees Celsius and discard the supernatant. Resuspend the pellet in one milliliter of red blood cell lysis buffer, and incubate for one minute. Neutralize the lysis buffer with nine milliliters of cold PBS.

Again, centrifuge the cells and resuspend the pellet in the desired buffer or media. Cell suspension prepared with motorized device and manual dissociation exhibited comparable cell viability and yields across mouse lung, kidney, and heart tissues. Immune cell populations like T cells and dendritic cells were not significantly affected by the isolation protocol.

Surface marker expression also showed similar frequencies of isolated T cells, and mean fluorescence intensity for the antigen presentation marker MHC-II in dendritic cells between manual and device isolation.