The quality of prepared single-cell suspension is crucial for obtaining high quality and reliable results for any single-cell omics methodology. And this protocol will show you how to isolate cells fast, gently, and robustly from even very challenging tissues. The main advantage of this protocol is the streamlined tissue processing which enables the tissue dissociation and isolation of cells from a large number of samples in a short time with perfect quality.
Although this method was developed for mouse and human teeth, it might be also applied for other extracellular matrix-rich tissues, including cartilage, bone, or dense connective tissue. The procedure of isolating dental pulps from human and especially mouse teeth is manually challenging. We advise everyone to test this before the real experiments begin.
To begin, hold the euthanized mouse behind its head looking on the ventral aspect of its head so that the tail points away. Using the small and sharp scissors, quickly remove the skin from the mandible to expose the mandibular arch, the soft tissue between each half of the mandibles and the adjacent facial muscles. For making a deep cut from each side of the mandible, first cut through the masseter along the buccal side of the mandible up to the temporomandibular joint and then cut along the inner part of each half of the mandible through the base of the oral cavity.
Cut all the muscles and ligaments along the mandible up to the temporomandibular joint from both outside and inside of the oral cavity. Grasp the mandible using the bent tip tweezers and remove it. Then with the scissors, cut through mandibular symphysis to split the dissected mandible into two halves.
Use an industrial low lint wipe to chafe the remaining soft tissue from each half of the mandible. After both parts of the mandible are cleaned, place them into a pre-prepared Petri dish with ice cold HBSS. For the dissection of mandibular incisors, remove of the alveolar ridge with all three molars and transversely crack the mandibular arch in the place corresponding to the position between the first and second molar.
Carefully pull the incisor out of the rest of the dental socket. If required, remove the remaining fragments of bone attached to the incisor with tweezers and a scalpel. Place the dissected incisors into fresh ice cold HBSS.
After dissecting the tissue of interest, place the dissected soft tissue into a droplet of fresh ice cold HBSS in the middle of a 10 centimeter Petri dish kept on ice. Using a round shaped number 10 scalpel blade, cut the tissue into the smallest possible pieces. After re-aggregating the tissue pieces in the middle of the droplet, repeatedly cut the tissue aggregates with the same scalpel blade and repeat this process until the material is sufficiently minced.
Transfer the shredded tissue pieces using a one milliliter pipette tip into a previously prepared digestion mixture. Then place the tube at an angle of 60 degrees and set the speed to 150 to 200 RPM to ensure constant suspension movements inside the tube and incubate for 15 to 20 minutes. After every three to four minutes during incubation, vigorously titrate the suspension using a one milliliter pipette tip to disintegrate all the clumps.
At the end of the incubation, after titrating the cell suspension for the last time, slowly add ice cold wash solution to a final volume of 12 milliliters. Centrifuge the cell suspension at 300 times G for five minutes at four degrees Celsius and then carefully remove the supernatant with a 10 milliliter serological pipette. Resuspend the pellet in the wash solution to achieve the final concentration of 700 to 1, 200 cells per microliter.
Pass the cell suspension through a 50 micrometer cell strainer to remove the remaining clumps or pieces of calcified tissue and keep the tube on ice until further processing. Then for fluorescently-activated cell sorting, load the cells into a prepared tube. Load the tube into the cell sorter and set a strict gating strategy to remove cell debris, doublets, and dead cells as described in the text manuscript.
First, the CD45 antibody staining and subsequent FACS analysis were used to clarify the number of the immune cells in the final single-cell suspension. As a complimentary method, the total number of CD45 positive immune cells in the single-cell RNA sequencing data were analyzed. Using FACS, 14.44%CD45 positive cells were identified.
Analysis of single-cell RNA sequencing data showed 10.9%of CD45-expressing cells and the decrease of cells in single-cell RNA sequencing data could be caused by additional thresholding during sequencing analysis. The key things are being fast and keeping this tissue or cells on ice whenever possible. The number of isolated cells can be very low especially during mouse molar isolation.
Avoid cell loss. Reducing the time of single-cell isolation is very important for the success of all single-cell experiments. And this specific method really allowed to reduce this time and pave the way for high-quality dental cell type for mouse and human systems.
