Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant
© BioMed Central Ltd 2002
Received: 5 September 2000
Accepted: 16 January 2002
Published: 28 February 2002
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© BioMed Central Ltd 2002
Received: 5 September 2000
Accepted: 16 January 2002
Published: 28 February 2002
We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.
Synovial tissues isolated from patients with rheumatoid arthritis (RA) display biologic properties that differ from 'normal' synovium, and there is a rapidly expanding catalogue of biochemical and molecular changes that underlie this phenotype . We have investigated the feasibility of using Moloney murine leukemia virus (MoMLV) based vectors to constitutively express cloned genes in primary human fibroblast-like synovial cells (FLS), with the long-term objective of defining the contributions of specific signaling pathways and inflammatory mediators to the destructive phenotype of FLS in RA.
Prior studies have suggested that MoMLV-based vectors transduced FLS with relatively low efficiency [2–5]. We designed experiments to determine if viral titer influenced FLS transduction by concentration of retrovirus. In these experiments, we used a modified MoMLV vector (pRET2), designed to improve transcriptional stability in primary cells. We also employed the enhanced green fluorescent protein (EGFP) as a virally encoded transgene to optimize a rapid and efficient superspeed centrifugation technique for concentration of viral supernatant. Viral particles were concentrated to >108 colony forming units (cfu)/ml by superspeed centrifugation at 20,000 g for four hours. Up to 50% of primary human FLS were transduced in vitro following a single exposure to concentrated viral supernatant.
Murine fibroblast NIH 3T3 cells, amphotropic PA317 packaging cells, and Phoenix E ecotropic packaging cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-high glucose (GIBCO-BRL, Grand island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-BRL, Grand island, NY, USA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 200 mM L-glutamine. The FLS cultures were established from synovial tissues obtained during joint replacement surgery in RA patients . The FLS were cultured in DMEM plus 10% heat-inactivated human AB serum (BioWhittaker, Walkersville, MD, USA), 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine. The FLS were used between the third and tenth passage.
The EGFP cDNA was PCR amplified from pEGFP-1 (Clontech, Palo Alto, CA, USA) and subcloned into pRET2, a modified version of the MoMLV-based MFG retroviral vector, designed to optimize gene expression in primary cell lines. The pRET2 incorporates long-terminal repeats from the myeloproliferative sarcoma virus , and a point mutation in the primer binding site . A vector expressing the human cyclooxygenase-2 (COX-2) cDNA was constructed in the same backbone (pRET2.COX2). Amphotropic viral producers were established in PA317 cells (see Supplementary Material).
Fresh medium was added to subconfluent producer cell monolayers, collected 24 hours later, and filtered (0.45 μM) prior to use. Centrifugation was performed at 4°C in a Sorval RC-5B centrifuge, using SS-34 or GSA rotors. Following centrifugation, the supernatant was aspirated and saved for analysis. The viral pellet was resuspended in fresh medium by gentle pipetting.
Viral RNA was quantitated using a slot blot hybridization technique. See Supplementary Material for full details.
Titer (cfu/ml) = (2 × 105 target cells) × (% EGFP+ cells)/ volume of supernatant (ml).
See Supplementary Material for full details.
The FLS were plated in 6-well dishes at 2 × 105 cells/well. FLS were cultured with viral supernatant plus protamine sulfate (5 μg/ml) for 24 hours. Cells were analyzed for transgene expression 72 hours after infection.
Viral transduction of fibroblast-like synovial cells
10X Concentrated supernatant
100X Concentrated supernatant
Mean ± SD
0.2 ± 0.2%
14.2 ± 8.2%
41.3 ± 14.7%*
47.3 ± 14.8%**
We are characterizing molecular pathways involved in synovial pathophysiology by overexpression of biologically relevant transgenes and dominant negative inhibitors in FLS. The limited expansion potential of FLS, combined with the low efficiency of existing methods, stimulated a systematic examination of various transduction techniques to identify a rapid and efficient method for stable genetic modification of FLS. In this manuscript, we report a retroviral vector system and transduction protocol with the capacity to express a viral transgene in 50% or more of primary human FLS after a single exposure to virus. We have subsequently used this methodology to successfully express a panel of transgenes in FLS (L Crofford and K McDonagh, unpublished observations). We believe this approach will be of value to investigators addressing similar mechanistic questions in FLS.
Previous studies exploring the use of recombinant MoMLV vectors concluded that FLS were relatively resistant to transduction [2–5], limiting enthusiasm for this approach. The basis for this resistance was unclear, but could be attributable to many factors including vector design, viral titer, or biologic features inherent to FLS. Our experiments differ from prior studies of retroviral gene transfer to FLS in several important respects that may impact on the observed results. First, our viral backbone is a modified MoMLV vector that incorporates genetic elements (myeloproliferative sarcoma virus long-terminal repeats and B2 mutation) associated with resistance to transcriptional silencing following proviral integration in primary cells [7, 8]. While we did not perform a detailed comparison of EGFP expression in FLS using the modified and unmodified vector backbones, preliminary experiments suggested that the modified vector was superior (J Yang, unpublished observations). A similar, modified MoMLV vector has been used to stably express EGFP in human marrow stromal cells , another fibroblast-like primary cell type. A second distinction is the use of EGFP as a transgene, whereas prior studies relied on lacZ or beta-galactosidase. The expression of EGFP is readily detectable in living cells by fluorescence microscopy or flow cytometry, and expression can be monitored serially over time in a single culture. In contrast to staining for lacZ, which is often complicated by background staining from endogenous galactosidase activity, there is no significant background staining with EGFP. We do not know if analysis of EGFP expression is more or less sensitive than analysis for lacZ expression, although we believe it provides more reproducible and quantitative data due to the absence of background staining.
Using this vector system, we observed a low ex vivo transduction efficiency (14.2 ± 8.2%) of FLS with unconcentrated supernatant (titer of 106 cfu/ml) that was roughly comparable to prior reports. Centrifugal concentration of viral supernatant by 10- to 100-fold significantly increased the efficiency of viral transduction, with 50% or more of FLS expressing EGFP in several independent experiments using FLS lines from separate donors. Concentration of supernatant to viral titers exceeding 107 cfu/ml appeared to have the greatest quantitative impact on improving transduction efficiency. Increasing viral titer to 108cfu/ml yielded an additional increase in transduction efficiency in some, but not all experiments. This observation suggests that factors in addition to viral titer may limit the maximum number of transduced FLS observed using these culture conditions. Lentiviral vectors have the capacity to transduce nonreplicating cells , and may represent an alternative to MoMLV-based vectors for some applications.
We report a retroviral vector system and transduction methodology that achieve stable transgene expression in primary human FLS with efficiencies of approximately 50%. These results establish the feasibility of using widely available retroviral gene transfer techniques to study the biologic impact of overexpression of specific regulatory and inflammatory molecules in primary FLS.
Synovial cells play a central role in the pathophysiology of inflammatory arthritis. Much of our understanding of this biology has been derived from the study of primary fibroblast like synovial cells cultured from arthritic joints after arthroscopic biopsy or surgery. Stable genetic modification of primary synovial cells is an approach that may be useful in defining the roles that specific signaling pathways or inflammatory mediators play in the joint destruction associated with rheumatoid arthritis. As our understanding of this biology improves, investigators have also proposed that gene transfer to primary synovial cells could be developed as a therapeutic approach to the treatment of patients with inflammatory arthritis [2, 3].
Recombinant retroviral vectors are widely used in the laboratory, and in experimental clinical applications, to introduce new genetic material into the host genome in a stable form. Retroviral packaging cells routinely yield viral supernatants with titers in the range of 105 to 106 cfu/ml or higher, and titers of up to 107 cfu/ml may be achieved in some cases. Physical methods to concentrate viral supernatants have been pursued with mixed results. Ultracentrifugation can be used to physically concentrate MoMLV-based retroviral particles, but viral infectivity is impaired secondary to damage to the envelope protein. Pseudotyped retroviruses containing the vesicular stomatitis virus G protein are more robust, and can be concentrated more than 100-fold by ultracentrifugation without significant loss of viral infectivity. However, because of the toxicity of the vesicular stomatitis virus G glycoprotein, only transient methods of virus production have been described [S1, S2]. Bowles et al. previously reported a superspeed centrifugation technique for concentration of recombinant retrovirus . A MoMLV based recombinant retrovirus was concentrated over 100-fold by centrifugation at 6000g for 16 hours.
The murine fibroblast NIH 3T3 cell line (CCL 92) and the amphotropic retroviral packaging cell line PA317 (CRL 9078) were obtained from the American Type Culture Collection (Rockville, MD, USA). The Phoenix E ecotropic packaging cell line was obtained from Dr Gary Nolan (Stanford University, USA).
A transinfection technique was used to rapidly establish a polyclonal amphotropic producer line of moderate to high titer. The pRET2.EGFP or pRET2.COX2 plasmids were transfected into ecotropic Phoenix E packaging cells by the calcium phosphate precipitation method, using the ProFection kit (Promega, Madison, WI, USA). Retroviral supernatant was collected 48 hours after transfection, filtered through a 0.45 μM filter (Nalgene, Rochester, NY, USA), supplemented with 5 μg/ml protamine sulfate (Elkins-Sinn, Inc. Cherry Hill, NJ, USA), and incubated with amphotropic PA317 packaging cells for 24 hours. The transinfection procedure was repeated twice. Following transinfection with ecotropic viral supernatant, 100% of the PA317 cells were transduced with the pRET2.EGFP vector, as determined by fluorescence microscopy. The successful transinfection of pRET2.COX2 into PA317 was confirmed by G418 selection. These polyclonal populations of PA317 producer cells were used as the source of viral supernatant for subsequent viral transduction and concentration experiments. The presence of replication competent retrovirus was excluded by PCR for viral envelope coding sequence in genomic DNA isolated from virally transduced NIH 3T3 target cells (primers: 5'-AAG-GTGGTAAACCAGGGGGATC-3' and 5'-TGAGCAGCT-TCATGCCGCTATC-3').
A nylon transfer membrane (Micron Separations Inc. Westborough, MA, USA) was soaked in 10X SSC for 10 min and inserted into a BRL convertible filtration manifold system (BRL Life Technologies Inc. Gaithersburg, MD, USA). Each well was washed twice with 200 μl of 10X SSC immediately before sample loading. Retroviral supernatant samples were directly loaded onto the membrane without further preparation. After application of the sample to the membrane, the wells were washed three times with 200 μl of 10X SSC. The membrane was cross-linked with UV light (Stratalinker 1800, Stratagene, La Jolla, CA, USA) and stored for analysis by hybridization. An EGFP probe fragment (~800 base pairs) was prepared by PCR and labeled with 32P-dCTP (Amersham Life Science Inc., Arlington Heights, IL, USA) using a kit (Prime-It RmT, Stratagene, La Jolla, CA, USA). The membrane was prehybridized for 2 hours at 42°C in 10 ml of hybridization buffer (final concentrations: 50% formamide, 5X Denhardt's solution, 0.1% SDS, 5X SSPE, 150 μg/ml denatured herring sperm DNA), and hybridized with the denatured probe overnight in 5 ml of hybridization buffer at 42°C. The membrane was washed twice with 2X SSPE at room temperature for 10 min, three times with 0.1X SSPE/0.5% SDS at 55°C for 30 min, and twice with 0.1X SSPE at room temperature for 10 min. The autoradiograph was visualized by exposing the membrane to X-ray film at -80°C with an intensifying screen.
The NIH 3T3 cells were plated in 6-well tissue culture dishes at a density of 105 cells per well. The following day, the medium was replaced with 2 ml of fresh medium containing a defined volume of viral supernatant, supplemented with protamine sulfate (5 μg/ml). After exposure to viral supernatant for 24 hours, the medium was replaced with fresh, virus-free medium and the cells were cultured for an additional 48 hours. At the conclusion of the experiment, the cells were trypsinized and analyzed by flow cytometry on an EPICS XL (excited by 488 nm light, using a 530 ± 15 nm bandpass filter to detect the signal on FL1) to determine the percentage of cells expressing EGFP. In all cases, serial dilutions of viral supernatant were tested.
Supernatant collected from the RET2.EGFP producer cells was centrifuged at 6000 g for time periods varying between 1 and 20 hours. After centrifugation, the supernatant was collected and saved for quantitation of residual viral particles. The viral pellets were resuspended in a thirtieth of the original volume of the supernatant. As measured on NIH 3T3 cells by flow cytometry, viral titer increased 14-fold after four hours of centrifugation, and appeared to plateau after 12 hours of centrifugation at 1.34 × 107 cfu/ml (Supplementary Fig. 1). There was a proportional decline in the viral titer of the postcentrifugation supernatant. Even following concentration for as long as 20 hours, the infectivity of the recombinant virus was preserved.
To confirm the viral titer derived by expression analysis, we performed slot blot hybridization analysis on viral RNA in the postcentrifugation supernatant and the resuspended viral pellet (Supplementary Fig. 2). Following centrifugation at 6000 g for four hours, most retroviral RNA was concentrated in the viral pellet. Almost no retroviral RNA remained in the postcentrifugation supernatant after centrifugation for 12 hours.
To further optimize the concentration procedure, we examined a range of relative centrifugal force (RCF). The time of centrifugation was fixed at four hours and the RCF was varied in a range from 6000 to 30,000 g. Following centrifugation, the viral pellet was resuspended in a hundredth of the original volume. Viral titer was quantitated by expression studies in NIH 3T3 cells (Supplementary Fig. 3) and slot blot hybridization analysis (Supplementary Fig. 4). We observed a progressive rise in viral titer as RCF was increased from 6000 to 20,000 g. At a RCF of 20,000 g, the titer of the resuspended pellet reached a plateau value of 1.3 × 108cfu/ml. Further concentration of viral particles was not achieved by increasing RCF above 20,000 g. Viral particles were not detectable by expression assay or by slot blot hybridization analysis in the post-centrifugation supernatant at an RCF of 20,000 g or higher. The expression data also suggested that centrifugation at a RCF as high as 30,000 g for four hours did not affect viability of the recombinant retrovirus.
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  and endothelial cells . 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, S7]. 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, S9]. 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].
colony forming units
Dulbecco's modified Eagle's medium
enhanced green fluorescent protein
fluorescence-activated cell sorting
fibroblast-like synovial cells
Moloney murine leukemia virus
polymerase chain reaction
relative centrifugal force
This work was supported in part by NIH grants DK02349 (KTM), CA77219 (KTM), AR01943 (LJC), by the University of Michigan Multi-purpose Arthritis and Musculoskeletal Disease Center (P60 AR20557), and by the University of Michigan General Clinical Research Center (M01-RR00042).