|Home | About | Journals | Submit | Contact Us | Français|
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.
Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.
Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 1010 TU were recovered from a single HYPERFlask vessel.
The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.
LV vectors provide powerful tools for transgene delivery into dividing as well as nondividing cells and for long-term transgene expression in target cells in vitro and in vivo. Currently, the use of LV vectors is commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis have emerged . LV vectors are also being pursued in a number of clinical trials (see http://www.gemcris.od.nih.gov/). Despite significant progress, the production and concentration of high-titer, high-quality LV vectors for preclinical studies in animal models is still cumbersome and costly .
The production of LV vectors is typically carried out using transient transfection approaches involving tissue culture dishes or flasks , cell factories [4-6], or stirred-tank bioreactors . These protocols are cumbersome to scale up (dishes, flasks) or technically challenging (cell factories, bioreactors), preventing their routine use in a standard laboratory setting.
Typical LV vector titers involving the vesicular stomatitis virus (VSV) G glycoprotein range from 106 to 108 TU/ml . Higher titers can be achieved by physical concentration , including ultracentrifugation [3,9-11], or filtration approaches such as ultrafiltration [3,10,12-14], and diafiltration [4,6]. Vector production for large-scale in vivo applications in animal models requiring high-titer LV vector stocks is challenging due to the lack of simple procedures allowing rapid processing of large volumes of LV vector-containing cell culture supernatants. The traditional ultracentrifugation-based methods are limited in terms of their capacity to handle large volumes, thus making this procedure extremely tedious. Filtration approaches such as diafiltration are well suited for processing large volumes of vector supernatants. However, they are difficult to implement in a standard laboratory setting. Thus, there is an emerging need for simple and less laborious procedures that result in a rapid reduction of the volume of the cell culture supernatant to be processed without the need for a centrifugation step.
One problem with the centrifugation and filtration-based methods outlined above is that cell-derived components are concentrated along with the vector particles. These have the potential to cause immune and inflammatory responses . For example, concentrated VSV-G-pseudotyped LV vector preparations were shown to be contaminated with tubovesicular structures of cellular origin which carried nucleic acids, including the plasmid DNAs that were used to generate the LV vector stocks. DNA carried by these tubovesicular structures acted as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production . Thus, additional steps including chromatography-based methods such as anion exchange chromatography are needed in order to reduce host cell and cell culture-derived contaminants from LV vector preparations. Methods based on anion exchange column chromatography of LV vectors pseudotyped with VSV-G [17,18] or the baculovirus GP64 glycoprotein were previously described . However, the yields and purity of the LV vector stocks obtained in this way were not reported.
In an attempt to simplify the production and concentration of LV vectors and to make this approach more reproducible and cost-effective for preclinical studies in animals, we have worked out a facile LV vector production system based on HYPERFlasks. We also implemented a straightforward concentration procedure based on Mustang Q anion exchange membrane chromatography. Mustang Q anion exchange-based chromatography protocols for concentrating/purifying LV vectors were previously reported [4,20]. Such vector preparations displayed reduced toxicity compared to vectors concentrated using ultracentrifugation , as well as enhanced purity .
With a view toward improving high-titer LV vector production for preclinical studies in animals, we tested the usefulness of HYPERFlask vessels that have a total growth area of 1720 cm2, corresponding to ten standard T175 flasks. HYPERFlasks consist of ten interconnected growth surfaces each containing a membrane pretreated to allow improved cell adherence. The membrane is gas permeable, allowing exchange of oxygen and carbon dioxide through the base of the membrane, resulting in gas exposure to a large surface area within the flask .
To test the usefulness of HYPERFlasks for LV vector production involving calcium phosphate-mediated transfection , we compared the titers of LV vector stocks prepared side-by-side using either ten 150-cm2 dishes or a single HYPERFlask. The data shown in Table Table11 represent the results of three independent productions each for the 150-cm2 dish system and the HYPERFlask system. These data indicate that the titers of unconcentrated LV vectors produced using HYPERFlasks were up to 2.3 × 108 TU/ml while the titers of LV vectors produced in 150-cm2 dishes were lower, up to 6.9 × 107 TU/ml. The total yields were up to 1.2 × 1010 TU from ten 150-cm2 dishes and up to 1.3 × 1011 TU for the HYPERFlask vessels. This corresponds to a productivity of up to 8 × 106 TU per cm2 for the 150-cm2 dishes. For the HYPERFlask, the productivity was about 10-fold higher, up to 0.75 × 108 TU per cm2. The higher productivity observed with HYPERFlasks may be related to better gas exchange during LV vector production. Overall, the 293T cells used for production appeared healthier and there was less cell debris from transfections carried out in HYPERFlaks compared to transfections carried out in 150-cm2 dishes (data not shown). We expect the HYPERFlask production protocol described here to be compatible with other DNA transfection formats such as polyethylenimine-mediated transfection .
The concentration and purification of LV vector stocks on a large scale presents a significant bottleneck and methods allowing rapid processing of large volumes of LV vector-containing supernatants are needed. Chromatography-based methods appear to be particularly attractive in this regard . For example, methods to concentrate/purify LV vectors based on anion exchange or affinity chromatography have been established [17-19]. Recently, Segura et al.  described an approach involving heparin affinity chromatography to concentrate LV vector particles directly from clarified supernatants. During this step, a recovery of 53% of infectious LV particles was achieved while 94% of the impurities were removed. This strategy may ultimately result in vector stocks of improved purity, increased infectivity and reduced toxicity. However, these approaches are cumbersome and difficult to implement in a standard laboratory setting.
We have recently described a facile membrane-based chromatography approach involving Mustang Q anion exchange chromatography cartridges (Acrodiscs) to purify adenoviral vectors . Mustang Q membranes resulted in high recoveries of infectious viral particles and allowed the processing of adenoviral vector particles from lysates in a fraction of the time required using the traditional cesium chloride-based ultracentrifugation method. Mustang Q membranes include a matrix that has a high dynamic binding capacity for adenoviral vectors and is capable of withstanding high flow rates. Other benefits of Mustang Q membrane chromatography cartridges include higher peak resolution at faster flow rates as compared to traditional ion exchange resins. Also, they can easily be adapted to bench-scale work and they are syringe-adaptable. Finally, there is no need for an HPLC setup or a column packing step. In work with LV vectors carried out by us [20,24] and by others , Mustang Q-based cartridges were used to concentrate/purify LV vectors. While these initial studies were encouraging, the capacity of Mustang Q membranes for crude LV vector supernatants was rather low , possibly caused by cell-derived proteins, serum proteins, or contaminating nucleic acids that bound to Mustang Q membranes during vector capturing.
To determine the binding capacity of Mustang Q membranes for LV vectors produced in HYPERFlask vessels compared to 150-cm2 dishes, a capture study was conducted. Figure Figure11 shows a typical elution profile of HYPERFlask-produced LV vectors following capturing onto a 0.18-ml Mustang Q Acrodisc and elution using a gradient ranging from 0.3 to 1.5 M NaCl. TU were determined by FACS analysis of transduced HOS cells. The results presented in Table Table22 show that the capacity of Mustang Q Acrodiscs at 10% breakthrough for LV vector supernatants produced in 150-cm2 dishes was up to 3.9 × 109 TU per ml of membrane while the capacity of Mustang Q membranes at 10% breakthrough for HYPERFlask vessel-derived LV vector samples was up to 9.6 × 1010 TU per ml of membrane. This is consistent with the observation that LV vector supernatants produced using HYPERFlasks contained fewer cellular proteins and nucleic acids compared to supernatants produced in 150-cm2 dishes (Table (Table3)3) that may have interfered with the binding of the LV vector particles. To remove excess NaCl, pooled Mustang Q fractions were subjected to a buffer exchange using Sepharose CL-4B. Table Table44 summarizes the recovery of LV vectors following processing of 500-ml aliquots of HYPERFlask vessel-derived supernatants. Up to 4.7 × 1010 TU were obtained after Mustang Q chromatography and up to 5.3 × 1010 TU after subsequent buffer exchange using Sepharose CL-4B (Table (Table44).
Finally, we wanted to compare the titers and total yields of our concentrated LV vectors with those reported by other groups. To do this, we carried out a side-by-side titer comparison of a HYPERFlask-derived LV vector stock concentrated by Mustang Q anion exchange chromatography using HOS cells, 293T cells and HeLa cells. Table Table55 shows that the titers of fresh (unfrozen) vector stocks were similar in HOS cells and 293T cells and slightly lower in Hela cells. Upon freezing and thawing of Mustang Q-concentrated vector stocks there was a 40% drop in vector titers on average (data not shown).
Overall, the titers obtained using the HYPERFlask system were as high or higher compared to those obtained using 150-cm2 dishes and ultracentrifugation-based concentration approaches . The yield (total TU) from a single HYPERFlask (cell growth area: 1720 cm2) was above 1011 TU (Table (Table1).1). This is about two orders of magnitude higher than the yields reported by Karolewski et al.  who used a cell factory system (total cell growth area: 6320 cm2), and at least one order of magnitude higher than the yields reported by Geraerts et al. 2005  who also used Cell Factories.
In conclusion, we present here a simple protocol for LV vector production and concentration involving HYPERFlasks in conjunction with Mustang Q anion exchange membrane cartridges. These protocols are easy to implement in a standard laboratory setting and allow high-titer LV vector production without the need for multiple tissue culture dishes or flasks or ultracentrifugation-based concentration procedures.
293T cells (CRL-11268), human osteosarcoma (HOS) cells (CRL-1543) and HeLa cells (CCL-2) were obtained from the American Type Culture Collection (ATCC) and propagated in DMEM (high glucose), 10% fetal bovine serum (FBS, Invitrogen-GIBCO), 1% GlutaMAX (Invitrogen), 1% Pen/Strep (Invitrogen).
LV vectors were generated by calcium phosphate-mediated transfection of 293T cells as described [10,25], with modifications. 293T cells were plated in 150-cm2 dishes at a density of 8 × 106 cells per dish in 25 ml DMEM medium supplemented with 10% FBS, 1% Pen/Strep, 0.3% HyQ CelPro-LPS (HyClone). Twenty four h later, chloroquine (Sigma-Aldrich) was added to the medium at a final concentration of 25 μM. LV vector, packaging (helper), and envelope plasmid DNAs were combined in 3 ml of 0.25 M CaCl2 and then mixed with 3 ml of 2 × HEPES-buffered saline (2 × HBS)  using gentle vortexing. The DNA/CaCl2/HBS mixture was then added to medium. The amount of plasmid DNA used per dish was 21 μg of the pNL-EGFP/CMV/WPREΔU3 LV vector plasmid DNA  (Addgene plasmid 17579), 14 μg of the pCD/NL-BH*ΔΔΔ packaging plasmid DNA  (Addgene plasmid 17531) and 7 μg of the VSV-G-encoding pLTR-G plasmid DNA  (Addgene plasmid 17532). The medium was removed 16 h later and replaced with 17 ml of fresh DMEM/10% FBS/1% GlutaMAX per plate. Forty eight h later, the vector-containing medium was collected and spun at 500 × g for 5 min, filtered through a 0.45-μm pore size filter (Corning) and stored at -80°C.
293T cells (2 × 108 cells) were seeded into a HYPERFlask Cell Culture Vessel (Corning) in 550 ml of DMEM medium supplemented with 10% FBS, 1% GlutaMAX, 1% Pen/Strep, 0.3% HyQ CelPro-LPS. Twenty four h later, the medium was removed from the HYPERFlask vessel. Sixty ml of the medium were discarded, and chloroquine was added to the remaining medium at a final concentration of 25 μM. LV vector, packaging, and VSV-G plasmid DNAs were combined in 30 ml 0.25 M CaCl2 and mixed with 30 ml of 2 × HBS using gentle vortexing. The plasmid DNA/CaCl2/HBS mixture was combined with the remaining medium and transferred back into the HYPERFlask vessel. The total amount of plasmid DNA used was 210 μg of the pNL-EGFP/CMV/WPREΔU3 LV vector plasmid, 140 μg of the pCD/NL-BH*ΔΔΔ packaging plasmid, and 70 μg of the pLTR-G plasmid. The medium was removed 16 h later and replaced with 550 ml of fresh DMEM/10% FBS/1% GlutaMAX. Forty eight h later, the vector-containing medium was collected and filtered through a 0.45-μm pore size filter and stored at -80°C. Protein and DNA concentrations in vector supernatants were determined using a Qubit kit (Invitrogen) as recommended by the manufacturer.
To determine the binding capacity of Mustang Q Acrodiscs (PALL) for LV vectors, 75-ml aliquots of 150-cm2 tissue culture dish-derived or HYPERFlask-derived vector supernatants were adjusted to 25 mM Tris-HCl, pH 8.0, 0.3 M NaCl and loaded onto a Mustang Q Acrodisc (bed volume 0.18 ml) at a flow rate of 10 ml/min for 8 min, using an AKTA purifier HPLC system and Unicorn 4.0 Software (Amersham Pharmacia). After loading, the Acrodisc's membrane was washed with 2 ml of 25 mM Tris-HCl, pH 8.0, 0.3 M NaCl. LV vectors were eluted with a 10 ml gradient ranging from 0.3 to 1.5 M NaCl in 25 mM Tris-HCl, pH 8.0. Flow through, wash, and eluate fractions were collected using a FRAC 950 collector (Amersham Pharmacia) and analyzed for vector TU.
For concentrating LV vectors prepared using HYPERFlask vessels, 500-ml aliquots of vector-containing supernatants were adjusted to 25 mM Tris-HCl, pH 8.0, 0.6 M NaCl and loaded onto a Mustang Q Acrodisc (bed volume 0.18 ml) attached to an AKTA purifier HPLC system using the system's pump at a flow rate of 10 ml/min for 50 min. The Acrodisc's membrane was washed with 2 ml of 25 mM Tris-HCl, pH 8.0, 0.6 M NaCl, and vector particles were eluted with a step gradient ranging from 0.3 to 1.5 M NaCl in 25 mM Tris-HCl, pH 8.0. The flow through, wash, and eluted fractions (1 ml per fraction) were collected using a FRAC 950 collector and immediately analyzed for TU. To desalt the vector samples, the eluted fractions showing the highest TU levels were pooled, loaded onto a 2.0-ml Sepharose CL-4B column (Amersham Pharmacia) equilibrated with Tris-HCl, pH 7.4, and 150 mM NaCl. The column was spun at 500 × g for 1 min and the excluded volume was immediately analyzed for TU. The vectors were stored at -80°C.
To determine vector titers (TU), HOS cells, HeLa cells or 293T cells were plated in 6-well plates at a density of 5 × 104 cells per well in DMEM high glucose medium supplemented with 10% FBS, 1% GlutaMAX, 1% Pen/Strep. The next day, the medium was removed and 0.5 ml of medium containing 8 μg/ml polybrene was added. Aliquots of concentrated LV vector preparations diluted 1:500 or of unconcentrated preparations were added. After incubation overnight, the medium containing the vector samples was removed and 2 ml of fresh medium was added to each well. Seventy two h after vector addition, cells were processed for FACS analysis .
RK, SP, MM and JR conceived and designed the experiments. SP and RK performed the LV vector production and RK and MM optimized the Mustang Q anion exchange membrane chromatography steps. JR and RK wrote the manuscript. All authors read and approved the final manuscript.
Support for this work was provided by NIH grants R01 NS044832 and P01 HL076100.