Synovial tissues were either (1) disaggregated immediately followed by cryopreservation of dissociated cells or (2) cut into fragments that were cryopreserved for subsequent disaggregation at a central processing site. Dissociated synovial cells were resuspended in CryoStor® CS10 (BioLife Solutions) at ~ 2 million cells/ml to viably freeze them. Intact synovial tissue samples were divided into fragments as already described and transferred to a cryovial (1.5 ml; Nalgene) containing 1 ml of CryoStor® CS10 for viable freezing. Cryovials were then placed in an insulated container with isopropanol in the bottom chamber for slow freezing (Mr. Frosty; Nalgene), which comprised incubation at 4 °C for 10 min followed by 1 day at − 80 °C. The samples were then either shipped on dry ice or transferred into liquid nitrogen for long-term storage.
Synovial fragments were thawed by rapidly warming the cryovial in a 37 °C water bath. The preservation media was filtered out through a 70-μm strainer. The tissue was then rinsed through a series of incubations in a six-well culture plate: 10 min in 10% FBS/RPMI at room temperature with intermittent swirling, a quick rinse in 10% FBS/RPMI, and a final rinse in serum-free RPMI. Frozen synovial cells were thawed rapidly in a 37 °C water bath and transferred into 20 ml of 10% FBS/RPMI, centrifuged to pellet cells, and then resuspended in media for downstream analyses.
Synovial fragments were thawed by rapidly warming the cryovial in a 37 °C water bath. The preservation media was filtered out through a 70-μm strainer. The tissue was then rinsed through a series of incubations in a six-well culture plate: 10 min in 10% FBS/RPMI at room temperature with intermittent swirling, a quick rinse in 10% FBS/RPMI, and a final rinse in serum-free RPMI. Frozen synovial cells were thawed rapidly in a 37 °C water bath and transferred into 20 ml of 10% FBS/RPMI, centrifuged to pellet cells, and then resuspended in media for downstream analyses.
Free full text: Click here
