Single-Cell Isolation of Human Knee Meniscus

~1g of tissue from healthy donor knees (Grades 0-1) is collected from the meniscus. For details regarding the tissue harvesting procedure please see

dx.doi.org/10.17504/protocols.io.6qpvr614zvmk/v1

Meniscal tissue is washed with Room temperature supplemented with 10% , 1% and 1% .

The tissue is then finely minced with a #21 Feather disposable scalpel, and digested in 20mL supplemented with 1% Anti-Anti and 2% using 100 rpm shaking at 37°C for 0h 30m 0s.

Cells are gently passed through a 100 µm filter into a 50 mL centrifuge tube followed by gentle passage through a 40 µm filter into a fresh 50 mL centrifuge tube.

Filtered cells are spun down at 1200 rpm for 0h 5m 0sat 37Room temperature

Carefully remove and collect the collagenase supernatant; the collagenase is reused in subsequent steps.

Cells are then resuspended in DMEM supplemented with 10% CS, 1% Anti-Anti and 1% PSG and stored at 37°C.

Repeat the digestion, spin down and filtration steps for 1h 0m 0s and then 2h 0m 0s, for a total of 3h 30m 0s of digestion.

Combine all collected cells and spin down at 1200 rpm for 0h 5m 0s at Room temperature.

The supernatant is carefully removed, and the remaining cell pellet is delicately resuspended in 10mL of Room temperature DPBS supplemented with 5% CS and 5 mM .

Single cells are spun down at 1200 rpm for 0h 5m 0s at 37Room temperature.

The supernatant is carefully removed, and the remaining cell pellet is delicately resuspended in 10 mL of DPBS supplemented with 0.04% .

The Invitrogen Countess II FL automated cell counter is used to quantify single cells and determine cell viability. Live cells are determined by trypan blue staining. If >70% cell viability is confirmed, the single cell suspension is diluted to a concentration of 1x10⁶cells/mL for single cell RNA-seq library preparation.