The FLS are the principal cell type of sublining synovial tissue. Proliferation of FLS is observed in RA, a debilitating condition that affects as many as 1–2% of adult individuals worldwide. Primary FLS cultures can be established following arthroscopic biopsy or surgical resection of synovium from the joint. Protease digested synovial tissues placed in culture rapidly yield fibroblast-like cells. After three passages, these primary cultures are depleted of macrophage-like type A synoviocytes [S3
]. Doubling time is stable between the third and the tenth passages, but marked reduction in proliferation rate occurs in later passage cells [S4
Retroviral mediated gene transfer is a commonly used technique to stably introduce genes into primary cells. The titer of retroviral supernatant is one of several factors that influence transduction efficiency. A variety of strategies have been employed to physically concentrate retroviral particles in an attempt to further increase viral titer and improve the efficiency of target cell transduction. Centrifugation of retroviral supernatant is a potentially attractive approach to viral concentration because of the wide availability of centrifuge equipment, the simplicity of the technique, and the theoretical potential for rapid processing of large sample volumes.
Concentrated recombinant retrovirus, generated by super-speed centrifugation of retroviral supernatant, has been used to improve the transduction efficiency of primary cells, including hepatocytes [9
] and endothelial cells [11
]. In these prior reports, concentration was accomplished by centrifugation for 16 hours at a RCF of 6000 g
. We used a recombinant retrovirus encoding the green fluorescent protein to optimize a protocol to rapidly and efficiently concentrate retrovirus by superspeed centrifugation. Our studies indicate that the time necessary to recover essentially all viral particles can be reduced to four hours by increasing the RCF to 20,000 g
. The protocol does not appear to adversely affect the infectivity of the viral preparation, as the functional viral titer on NIH 3T3 cells closely matched the titer that was predicted by the degree of concentration. Although it has been reported that centrifugation may result in concurrent concentration of noninfectious viral particles or inhibitors of viral transduction [S5
], we have been able to substantially increase the transduction efficiency of primary FLS using concentrated viral supernatant produced by our protocol. This optimized technique may be useful in generating high titer retroviral supernatants from production lots of relatively modest titer. We anticipate that this method will be effective in concentrating other pseudotyped MoMLV vectors and lentivirus based vectors, though additional testing will be required to evaluate its suitability for each vector system.
While our studies were not initiated with the objective of developing a therapeutic protocol, these results may also have implications for clinical studies. The ex vivo
genetic modification of FLS has been proposed as a potential approach to the treatment of arthritis [S6
]. In these studies, FLS are cultured from synovial tissue obtained by synovectomy, transduced with retroviral supernatant ex vivo
, and injected into another joint of the same individual. Approval for these clinical studies was based on ex vivo
transduction data in preclinical animal models [S8
]. Essentially, all data on transduction efficiency of FLS was derived using retroviral vectors that express lacZ or beta-galactosidase. Although most authors have obtained ex vivo
transduction efficiencies of cultured FLS in the range of 1–5%, some have reported transduction efficiencies up to 20%. Preactivation of FLS with tumor necrosis factor α, however, may increase transduction efficiency levels to over 30% [S8