Apr 28, 2022

Public workspaceHuman Knee Cartilage Collection Protocol for Single Cell RNAseq

DOI
dx.doi.org/10.17504/protocols.io.14egn7996v5d/v1
  • 1Scripps Research
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Merissa Olmer
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DOI: dx.doi.org/10.17504/protocols.io.14egn7996v5d/v1
Protocol Citation: molmer , Martin Lotz 2022. Human Knee Cartilage Collection Protocol for Single Cell RNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7996v5d/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: March 31, 2022
Last Modified: April 28, 2022
Protocol Integer ID: 60103
Keywords: medial femoral condyle, Cartilage
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.
Materials
Sterile drapes
Sterile gauze
Autoclaved tools
Disposable #21 scalpels
DPBS
DMEM
Antibiotic-Antimycotic
50mL conical tubes
Vice

Protocol materials
Reagent1x DPBSGibco - Thermo Fisher ScientificCatalog #14190144
ReagentAntibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
ReagentDMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateFisher ScientificCatalog #MT-10-013-CV
ReagentAntibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
ReagentPAXgene Tissue FIXQiagenCatalog #765312
ReagentPAXgene Tissue STABILIZERQiagenCatalog #765512
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.
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. Reagent1x DPBSGibco, ThermoFisherCatalog #14190144 ReagentAntibiotic-Antimycotic (100X)Gibco, ThermoFisherCatalog #15240062

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. ReagentDMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateGibco, ThermoFisherCatalog #MT-10-013-CV ReagentAntibiotic-Antimycotic (100X)Gibco, ThermoFisherCatalog #15240062
Note
See Description Section for image of medial condyle area of interest.