Aug 17, 2022
Human Knee Meniscus Collection Protocol for scRNA-seq
- molmer1,
- Martin Lotz1
- 1Scripps Research
Protocol Citation: molmer, Martin Lotz 2022. Human Knee Meniscus Collection Protocol for scRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr614zvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 15, 2022
Last Modified: August 17, 2022
Protocol Integer ID: 68682
Keywords: Meniscus, knee
Abstract
Meniscus is taken from the medial knee compartment is collected for scRNA-seq processing. The attached image indicates where the meniscal tissue is collected from.
Materials
Sterile drapes
Sterile gauze
Autoclaved tools
Disposable #21 scalpels
DPBS
DMEM
Antibiotic-Antimycotic
50mL conical tubes
Protocol materials
Antibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
Step 4
PAXgene Tissue FIXQiagenCatalog #765312
Step 0.9
PAXgene Tissue STABILIZERQiagenCatalog #765512
Step 0.9
DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateFisher ScientificCatalog #MT-10-013-CV
Step 4
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 of knee tissues and macroscopic scoring of the cartilage is completed.
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 and menisci are resected.
A full-thickness (sagittal) piece of medial meniscus is cut out using a #21 scalpel; the piece measures about 1.60cmX15mmx8mm and weighs around 1g. This is collected into a 50mL tube that is filled with 40mLs ofDMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateFisher ScientificCatalog #MT-10-013-CV + 1%Antibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 , then placed in wet ice until the cell isolation protocol for scRNAseq begins.
Note
See Description Section for image for the area of interest.