Aug 23, 2022
Single-Cell Isolation of Human Knee Meniscus
- 1Scripps Research
Protocol Citation: Hannah Swahn, Martin Lotz 2022. Single-Cell Isolation of Human Knee Meniscus. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj6k7blk5/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 10, 2022
Last Modified: August 23, 2022
Protocol Integer ID: 68487
Keywords: single cell isolation, meniscus
Abstract
This is a protocol that describes the process of isolating single cells from human knee meniscus for scRNA-seq.
Protocol materials
DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateFisher ScientificCatalog #MT-10-013-CV
Step 3
Collagenase Type IIWorthington Biochemical CorporationCatalog #L5004177
Step 3
Ethylenediamine tetraacetic acid [EDTA]Fisher ScientificCatalog #BP120-500
Step 10
Bovine Serum Albumin [BSA]Fisher ScientificCatalog #9048-46-8
Step 12
Dulbeccos Phosphate Buffered Solution [DPBS]Gibco - Thermo FisherCatalog #14190-144
Step 2
Bovine Calf Serum [CS]VWR InternationalCatalog #10158-358
Step 2
Antibiotic-Antimycotic 9100x0 [Anti-Anti]Thermo Fisher ScientificCatalog #15240062
Step 2
Penicillin-Streptomycin-Glutamine [PSG] CorningCatalog #30-009-CI
Step 2
~1g of tissue from healthy donor knees (Grades 0-1) is collected from the meniscus. For details regarding the tissue harvesting procedure please see
Meniscal tissue is washed with Room temperature Dulbeccos Phosphate Buffered Solution [DPBS]VWR InternationalCatalog #14190-144 supplemented with 10% Bovine Calf Serum [CS]VWR InternationalCatalog #10158-358 , 1% Antibiotic-Antimycotic 9100x0 [Anti-Anti]VWR InternationalCatalog #15240062 and 1% Penicillin-Streptomycin-Glutamine [PSG] VWR InternationalCatalog #30-009-CI .
The tissue is then finely minced with a #21 Feather disposable scalpel, and digested in 20mL DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateVWR InternationalCatalog #MT-10-013-CV supplemented with 1% Anti-Anti and 2% Collagenase Type IIVWR InternationalCatalog #L5004177 using 100 rpm shaking at 37 °C for 00:30:00 .
30m
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 00:05:00 at Room temperature
5m
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 01:00:00 and then 02:00:00 , for a total of 03:30:00 of digestion.
6h 30m
Combine all collected cells and spin down at 1200 rpm for 00:05:00 at Room temperature .
5m
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 Ethylenediamine tetraacetic acid [EDTA]VWR InternationalCatalog #BP120-500 .
Single cells are spun down at 1200 rpm for 00:05:00 at Room temperature .
5m
The supernatant is carefully removed, and the remaining cell pellet is delicately resuspended in 10 mL of DPBS supplemented with 0.04% Bovine Serum Albumin [BSA]VWR InternationalCatalog #9048-46-8 .
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.