Human Knee Cartilage Collection Protocol for Single Cell RNAseq
molmer , Martin Lotz
Abstract
Cartilage from the medial femoral condyle is shaved for Single Cell RNAseq processing. The attached image indicates where the cartilage shavings are collected from.
Before start
Knee blocks are shipped on wet ice and received within 48 hours post-mortem from certified tissue banks. The average time from receiving the sample to harvesting cartilage from both knees is about 2 hours. Pictures are taken and macroscopic scoring of the cartilage is completed.
Steps
Tissue Collection for MERFISH
Three pieces of tissue are placed in 12mLs, per region, of
Pour off fixation solution and replace with
Prepare harvesting area with sterile drapes, tools and gauze. All harvesting is completed within an aseptic environment.
Wipe down the knee blocks with 95% ethanol prior to opening the joint capsule.
Once the joint capsule is opened, the femur and tibia are disarticulated. Wet a sterile gauze with DPBS+1% Anti-Anti and use it to wipe down the cartilage and place it on the cartilage surface to keep moist.
Full-thickness medial femoral condyle cartilage is shaved from the bone using a #21 scalpel; pieces typically measure maximum 10mmX25mm. 1.5g of shaved cartilage tissue is collected into a 50mL tube that is filled with 40mLs of DMEM + 1% Anti-Anti, then placed in wet ice until the cell isolation protocol for scRNAseq begins.
Procurement of Medial Femoral Cartilage With Subchondral Bone for MERFISH: Using an autoclaved 12” hacksaw with 24TPI blade, cut a 3-5mm slab from the middle region of the medial condyle.
