A Bioreactor Method to Generate High-titer, Genetically Stable, Clinical-isolate Human Cytomegalovirus

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Nov 2017



Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in transplant patients and a leading cause of congenital birth defects (Saint Louis, 2016). Vaccination and therapeutic studies often require scalable cell culture production of wild type virus, represented by clinical isolates. Obtaining sufficient stocks of wild-type clinical HCMV is often labor intensive and inefficient due to low yield and genetic loss, presenting a barrier to studies of clinical isolates. Here we report a bioreactor method based on continuous infection, where retinal pigment epithelial (ARPE-19) cells adhered to microcarrier beads are infected in a bioreactor and used to produce high-titers of clinical isolate HCMV that maintain genetic integrity of key viral tropism factors and the viral genome. In this bioreactor, an end-stage infection can be maintained by regular addition of uninfected ARPE-19 cells, providing convenient preparation of 107-108 pfu/ml of concentrated TB40/E IE2-EYFP stocks without daily cell passaging or trypsinization. Overall, this represents a 100-fold increase in gain of virus production of 100-times compared to conventional static-culture plates, while requiring 90% less handling time. Moreover, this continuous infection environment has the potential to monitor infection dynamics with applications for real-time tracking of viral evolution.

Keywords: Human cytomegalovirus (人巨细胞病毒), Bioreactor (生物反应器), Microbeads (Microcarrier beads) (微珠(微载体珠)), Viral tropism (病毒嗜性), Continuous infection culture (连续感染培养), Clinical isolate (临床分离)


Congenital CMV infection is a leading cause of birth defects with an annual direct cost of one billion dollars in the US alone, and represents a global unmet medical need (Bristow et al., 2011; Griffiths et al., 2013; Manicklal et al., 2013; Saint Louis, 2016). This would be preventable with an effective vaccine or therapeutic targeting women in their reproductive years. Human cytomegalovirus (HCMV) can infect a wide range of cell types, but a major barrier in the field is that extended passage of clinically derived HCMV strains in fibroblast cells leads to a loss of viral tropism for other cell types (Waldman et al., 1991; Sinzger et al., 1999). In the late 1960s, several laboratory-adapted strains of CMV were serially passaged in fibroblasts–including the HCMV AD169, Towne, and Davis strains, as well as the Smith strain of murine CMV–and became some of the first tools used to study the molecular biology of CMV (Plotkin et al., 1975). These laboratory-adapted strains–often created during unsuccessful attempts to generate a live attenuated vaccine–were found to have a number of mutations affecting (i) their ability to infect different cell types, (ii) rate of viral replication, and (iii) altering latency phenotypes (Albrecht and Weller, 1980; Yamane et al., 1983; Waldman et al., 1989; Kahl et al., 2000). Specifically, the HCMV open reading frames (ORFs) UL128, UL130, UL131, which comprise a viral glycoprotein entry complex, were found to accumulate mutations during passage in fibroblasts, leading to the loss of viral tropism for infection of epithelial cells, endothelial cells, macrophages, and dendritic cells (Sinzger et al., 1999, Hahn et al., 2004; Wang and Shenk, 2005; Adler et al., 2006). These lab-adapted HCMV strains were also shown to have lost several genes in the UL/b’ region of the viral genome, a region that confers immune evasion functions and replication dissemination in vivo (Cha et al., 1996). It is now known that sustained viral growth on fibroblast cultures removes the selection pressure to retain these sequences, resulting in genetic loss or rearrangement of sequences essential for replication and dissemination in other host cells and tissues. However, passage of HCMV clinical isolates (e.g., TB40/E and VR1814) in epithelial and endothelial cell settings maintains selection pressure to prevent loss of tropism for non-fibroblast cell types (Waldman et al., 1991; Hahn et al., 2002; Sinzger et al., 2008).

Differences between established laboratory-adapted strains of HCMV and clinical isolates of HCMV are important considerations when planning experiments, as the choice of virus strain used may influence results. Clinical isolates are much more similar to the viruses that replicate within patients, making them preferable for understanding clinical symptoms, as well as natural and drug-selected genetic variability of human CMV. These clinical isolates also maintain productive infection and latency phenotypes that best represent the wild-type virus population (Lee et al., 2015). Because clinical isolates spread through a cell-associated manner, yields from clinical isolates are significantly lower than those collected from laboratory-adapted virus strains, due in part to clinical strains being more limited to cell-associated spread. This contributes to time consuming and labor-intensive aspects of clinical virus stock preparation.

Here, we report a new, more efficient method for generating stocks of clinically derived HCMV isolates, represented by TB40/E IE2-EYEFP. This virus is genetically tagged with an EYEFP fusion to enable convenient monitoring of continuous infection in the bioreactor environment. Using a two-stage bioreactor system (Figure 1) with microcarrier beads and the advantage of the prolonged period of virus production characteristic of HCMV, we are able to maintain an end-stage infection and generate high titer virus stocks on primary adherent cell cultures that preserve genetic integrity of key viral tropism factors and the viral genome. We have used the TB40/E-IE2 EYFP tagged virus in development and characterization of the bioreactor infection. The fluorescent tag enables convenient monitoring of the virus in the bioreactor culture, by surveying aliquots of the infected samples with fluorescent microscopy.

Materials and Reagents

  1. 15 cm tissue culture dish (Corning, Falcon®, catalog number: 353025 )
  2. Vented screw caps (Corning, Falcon®, catalog number: 354639 ; Corning, catalog number: 3968 )
  3. 1.5 ml polypropylene microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  4. Conical sterile polypropylene centrifuge tubes, 50 ml (Fisher Scientific, catalog number: 05-539-12 )
  5. 96-well flat-bottom cell culture plates (Corning, catalog number: 3596 )
  6. 12-well reagent reservoirs (Corning, Costar®, catalog number: 4877 )
  7. 40 μm cell strainers (Corning, catalog number: 431750 )
  8. Conical sterile polypropylene centrifuge tubes, 15 ml (Fisher Scientific, catalog number: 06-443-18 )
  9. 0.45 μm sterile syringe filters (EMD Millipore, catalog number: SLHV033RS )
  10. Disposable sterile syringes with Luer-Lock tips (BD, catalog number: 309646 )
  11. Serological pipettes (50 ml, 25 ml, 10 ml, 5 ml, 2 ml)
  12. 0.2 or 0.1 μm filter system
  13. ARPE-19 cells (ATCC, catalog number: CRL-2302 )
  14. MRC-5 cells (ATCC, catalog number: CCL-171 )
  15. TB40/E IE2-EYFP bacmid cloned virus
    TB40/E IE2-EYFP is derived from the bacmid clone of TB40/E, a clinically derived HCMV isolate (Sinzger et al., 2008). The viral double stranded DNA genome has been cloned as a bacmid and can produce infectious virus encoding the tropism factors required for replication in multiple host cell types. This virus contains an EYFP fluorescent tag fused to the carboxyl terminus of a key viral transactivator, the IE2 gene, as previously reported (Teng et al., 2012). This bacmid clone was generated using the galK selection and counterselection recombination protocol described previously for bacmid cloning with HCMV (Murphy et al., 2008, Warming et al., 2005). Expression of this fluorescent tag occurs throughout the virus lytic infection cycle and enables convenient real time monitoring of continuous infection in the bioreactor environment.
  16. DPBS without calcium and magnesium, DPBS-CMF (Mediatech, catalog number: 21-031-CV )
  17. Trypsin-EDTA, 0.25% (Mediatech, catalog number: 25-053-CI )
  18. Trypsin-EDTA, 0.05% (Mediatech, catalog number: 25-052-CI )
  19. Dry ice pellets
  20. Isopropanol (Fisher Scientific, catalog number: A451-4 )
  21. DMEM with L-glutamine and sodium pyruvate (Mediatech, catalog number: 10-013-CV )
  22. DMEM/F-12 with L-glutamine and 15 mM HEPES (Mediatech, catalog number: 10-092-CV )
  23. Fetal bovine serum (FBS) (Mediatech, catalog number: 35-011-CV )
  24. Penicillin-streptomycin solution (Pen/Strep) (Mediatech, catalog number: 30-002-CI )
  25. Sigmacote (Sigma-Aldrich, catalog number: SL2-25ML )
  26. 1 M Tris, pH 7.5
  27. Magnesium chloride (MgCl2)
  28. Hydrochloric acid (HCl)
  29. Cytodex 1 microcarrier beads, 60-87 μm (Sigma-Aldrich, catalog number: C0646-5G )
  30. Culture media for titration assays (TCID50) (see Recipes)
  31. Culture media for bioreactor (see Recipes)
  32. Siliconizing glassware with SigmaCote (see Recipes)
  33. 20% sorbitol (see Recipes)
  34. Microcarrier beads ratio and preparation (see Recipes)
  35. Adherent cell ratio (see Recipes)


  1. Personal protective equipment, PPE, required for working at BSL-2: Laboratory coats or gowns, eye and face protection, gloves
  2. Wheaton Celstir spinner flasks, 150 ml, 250 ml, and 500 ml (WHEATON, catalog numbers: 356879 and 356882 )
  3. Tissue culture incubator: 37 °C, 5% CO2
  4. 4 position slow-speed stir plate (Corning, catalog number: 440814 )
  5. Class II biosafety cabinets (The Baker)
  6. Vortex mixer
  7. Micropipette (1-1,000 μl capacity), multiple channel pipettes (1-200 μl capacity) (P1000 pipette and P200 pipette [Pipetteman])
  8. Hemocytometer (Hausser Scientific, catalog number: 1475 )
  9. Cell culture inverted epi-fluorescent microscope
  10. Tabletop centrifuge
  11. Water bath, 37 °C
  12. 100 ml autoclavable glass bottle (WHEATON, catalog number: W818012406 )
  13. Approved BSL-2 laboratory facilities
  14. Autoclave
  15. Pipette controller (Pipette-aid, Drummond Scientific, model: Pipet-Aid® XP2, catalog number: 4-000-501 )
  16. Freezers: -80 °C, -20 °C, 4 °C


Expanding ARPE-19 cells (step A1: 2 weeks)
Preparing the culture feeder flask and beads (steps A2-A6: 3 h to be performed the day before seeding)
Seeding ARPE-19 cells in feeder flask (step A7-A11: 6.5 h)
Replacing media in feeder bioreactor (step A12: 15 min every 3-4 days)
Preparing the samples for cell count (steps B1-B8: 25 min)
Incubating cells in the bioreactor until confluent (step C1: 1-2 weeks)
Infecting cells in bioreactor (steps C2-B4: 10 min)
Replacing media and cells in infected bioreactor (steps D1-D7: 25 min)
Preparing the 96-well plate for TCID50 (step E1: 15 min)
Setting up TCID50 infections (steps E2-E11: 25 min)
Determining titer from TCID50 (steps E12-E13: 10 min)
Preparation of virus collected from bioreactor (steps F1-F6: 25 min)
Concentrating virus stocks (step F7: 3 h)

See Figure 1 for bioreactor system setup for the experiment.

Figure 1. Bioreactor system setup for maintaining continuous virus production. A. An adherent cell line (ARPE-19) is added to a 500 ml silicon coated glass feeder flask containing 2.5 g hydrated microcarrier beads in 150 ml DMEM-F12 media. This feeder flask is placed in a 37 °C incubator for 4 h to allow ARPE-19 cells to adhere to microcarrier beads, mixing every half an hour. B. After 4 h, the volume of media is brought up to working concentration of 500 ml and put on a stirring plate set at 60 rpm back in the same incubator. Allow a week for the cells to divide on the microcarrier beads and reach confluence in the feeder flask. C. When confluence is reached, 250 ml of stirred microcarrier beads from the feeder flask are transferred to a smaller, 250 ml bioreactor. This smaller bioreactor is then infected with TB40/E IE2-EYFP at an MOI of 0.01. Both the small and the large bioreactors are placed back in the incubator on the stir-plate. D. When virus expansion reaches desired steady state, slurry (microcarrier beads/media, taken when spinning) and supernatant (media collected when stir plate is off and microcarrier beads settle) are collected from the infected bioreactor. Uninfected ARPE-19 cells are taken from the feeder flask and added into the infected bioreactor, along with fresh media. The rate of microcarrier bead and media replacement maintains end stage viral production of HCMV. The volume of the infected bioreactor and the microcarrier bead concentration remains constant. This induced steady state can be continued for as long as desired, and collected samples from infected reactor are concentrated to create virus stocks.

  1. Seeding Cytodex 1 microcarrier beads with ARPE-19 cells (Figure 1A)
    1. Culture sufficient ARPE-19 cells on 15 cm cell culture dishes such that you have 1 x 106 cells/ml final volume. For a 500 ml Celstir spinner flask (feeder flask), you need 5 x 108 cells.
    2. The day before seeding, siliconize 500 ml feeder flask and an autoclavable 100 ml glass container with Sigmacote according to manufacturer’s instructions (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/sl2pis.pdf).
      Note: This procedure has been optimized for greatest adherence of ARPE-19 cells to the prepared Cytodex 1 microcarrier beads during seeding. Sigmacote is used to prevent cells from adhering to the walls of the spinner flasks. Given that it can be toxic to cells, rigorous rinsing is needed after siliconizing.
    3. Rinse feeder flask and glass container thoroughly with MilliQ water, at least 10 times. Place a new vented screw-on caps onto each arm of the flask.
    4. Weigh the appropriate amount of dry Cytodex 1 microcarrier beads to reach 0.5 g/100 ml bioreactor final volume. For 500 ml feeder flask, measure out 2.5 g microcarrier beads.
    5. Pour dry Cytodex 1 microcarrier beads into a siliconized 100 ml glass container, and add 100 ml DPBS-CMF (DPBS without calcium and magnesium). Resuspend beads thoroughly. For more information refer to GE manual: http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/Microcarrier%20Cell%20Culture.pdf.
    6.  Autoclave feeder flask and resuspended microcarrier beads.
    7. Remove by pipetting 50 ml DPBS-CMF from settled beads in a 100 ml glass container, replace with 50 ml DMEM-F12 with 10% FBS. Resuspend by vortexing. Incubate the glass container with the beads at 37 °C until beginning step A9.
    8. Trypsinize 5 x 108 ARPE-19 cells, resuspend in 90 ml DMEM-F12 with 10% FBS, and place in feeder flask.
    9. Take the glass container with settled beads from the 37 °C incubator and remove by pipetting 60 ml (i.e., most of the liquid). Resuspend these microcarrier beads in the remaining 40 ml of DMEM-F12 with 10% FBS left in the glass container and transfer them to the feeder flask. Add another 20 ml of 100% FBS to enhance cell adhesion. Final volume in the feeder flask should be about 150 ml and the final FBS concentration should roughly be 25%.
    10. Place feeder flask in the 37 °C incubator to allow cells to adhere to beads for 4 h. Every 30 min, place the feeder flask on a slow-speed stir-plate set at 60 rpm within the incubator for 30 sec to gently agitate bioreactor and ensure even seeding of cells.
    11. After 4 h, bring the feeder flask to full 500 ml volume by adding 350 ml DMEM-F12 with 10% FBS.
    12. Place the feeder flask on 4 position slow-speed stir-plate set to 60 rpm in the incubator.
    13. Replace media within the feeder flask every 2-3 days to maintain cell density. To do this, let beads settle for 10 min, then remove 50-75% of the total media volume (250-375 ml), and replace it with the same amount of fresh media (DMEM-F12 with 5% FBS). Let cells expand across the microcarrier beads for a week, or until maximum cell density (step C1) is reached (monitor through cell counts in Procedure B and by eye).

    Note: If seeding is too low, the maximum cell density in the spinner flask will also remain low over time. Once seeding is completed, if there are any microcarrier beads without cells, they will always remain uncoated. After the spinner flask is placed permanently on the stir-plate, cells are unable to bind to new cells, and the only method of cell spreading is through cell division.

  1. Monitoring ARPE-19 cell density on microcarrier beads (Figure 1B)
    Note: In the procedure, we are obtaining accurate cell counts for the feeder reactor. To do this, we take a sample of the slurry, perform a wash to remove debris, and then trypsinize the cells off of the microcarrier beads. Because the microcarrier beads settle much more quickly than the cells, we are able to easily separate the two without centrifuging.
    1. Remove the feeder flask from the incubator and place on a stir-plate set at 60 rpm inside a class II biosafety cabinet.
    2. Pipette 1 ml slurry (mixed cells/microcarrier beads/media) from the feeder flask while it stirs on stir-plate and dispense into a 1.5 ml microcentrifuge tube. Put the feeder flask back into the incubator on stir-plate, you will not need to use it for the remainder of this procedure.
    3. Let microcarrier beads in the microcentrifuge tube settle for 2 min. The microcarrier beads can be observed visually settling to the bottom of the microcentrifuge tube by gravity, no centrifugation is necessary.
    4. Wash by pipetting out 0.8 ml of media from the microcentrifuge tube, and resuspending with 0.8 ml of DPBS-CMF. Resuspend beads by gently vortexing on vortex mixer or by pipetting up and down, then let microcarrier beads settle again.
    5. Remove 0.8 ml of DPBS-CMF from the microcentrifuge tube and replace with 0.8 ml of 0.25% trypsin. Resuspend cells by gently vortexing.
    6. Place microcentrifuge tube in the 37 °C incubator and let cells trypsinize for 15-20 min, resuspending every 5 min by gently vortexing or flicking to ensure even trypsinization.
    7. Place microcentrifuge tube back in the biosafety cabinet and pipette vigorously to resuspend using P1000 pipette. This step also helps to remove all of the cells from the microcarrier beads and break up aggregates. Let beads settle for 30 sec by gravity.
    8. Pipette 10 μl media containing the detached cells from the microcentrifuge tube, avoiding taking up any microcarrier beads. Transfer this 10 μl to a hemocytometer, and determine the cell concentration.

    Note: Cell counts should be performed regularly (every 2 or 3 days) to ensure that cells are healthy. Cell density in the feeder flask should increase until max density (1 x 106 cells/ml) is reached and then remains constant. It is also possible to view the cells adhered to the beads under a microscope. If the cells appear to be dissociating from the beads or cell counts are suddenly dropping, it is probably because the media is not being replaced frequently enough.

  1. Infection of ARPE-19 cells on microcarrier beads in the bioreactor with TB40/E-IE2-EYFP (Figure 1C)
    Note: Our EYFP fusion tagged IE2 virus was used to track viral dissemination in epithelial cells within the bioreactor setting by eye using an inverted epi-fluorescent microscope.
    1. Once the cell concentration in the feeder flask reaches roughly 1 x 106 cells/ml, as measured through cell counts (Procedure B), transfer 250 ml of slurry to a smaller, siliconized, rinsed, and autoclaved 250 ml WHEATON spinner flask (referred herein as bioreactor). Continue maintaining cell growth in the feeder flask by replacing media (DMEM-F12 with 5% FBS) regularly (see step A13).
    2. Once roughly 1 x 106 cells/ml is reached, as measured through cell counts, transfer 250 ml of slurry to a smaller, siliconized and autoclaved 250 ml WHEATON spinner flask (bioreactor). Continue maintaining what remains in the feeder flask by replacing media regularly (refer to step C1).
    3. Inoculate the cells in the new bioreactor with TB40/E-IE2-EYFP at a multiplicity of infection, MOI, of 0.01 based on the viable cell counts and TCID50 of the virus stock.
    4.  Place the bioreactor (250 ml) and the feeder flask (500 ml) on the same 4 position stir-plate set to 60 rpm within the 37 °C incubator.
    5. Monitor virus expansion by taking 200 μl aliquots from the stirred bioreactor, and visualizing EYFP spread through a fluorescent microscope. Expansion should also be monitored through regular TCID50.

    Note: The infected cells will fall off of the beads.

  1. Continuous high-titer viral production by maintenance of steady-state TB40/E IE2-EYFP infection (Figure 1D)
    Note: In order to maintain an ongoing CMV infection and avoid a crash of the infection, we need to regularly provide uninfected cells from the feeder flask, which newly produced viruses will then infect. Fresh media is also added to prevent cell death. The rate at which media and cells need to be replaced depends on the particular virus’s kinetics. For TB40/E IE2-EYFP, we determined that replacing media and cells every 48 h resulted in highest viral production over time. Maintaining as regular of a feeding schedule as possible is ideal for sustaining steady viral production. Additionally, maintaining healthy naïve cells in the feeder flask is essential for continued viral production. Media should be replaced, and the cell density should remain constant in this feeder flask (see step A13).
    1. Regular visual monitoring can follow the health of the infected culture in the bioreactor. When 80% of the cells are killed (and detach from the beads), which usually occurs at 5-7 days post infection, start regular feeding with cells and media as followed.
    2. Every 48 h, remove 37.5 ml (15% of total bioreactor volume) slurry from the infected bioreactor as it is stirred on a stir-plate in the biosafety cabinet. Transfer this slurry into a 50 ml conical tube, and centrifuge for 5 min at 300 x g (to removed non-adhered cells). Aliquot 3 supernatant samples of 500 μl (to be used for TCID50). Freeze the three aliquots and the 50 ml conical tubes of media at -80 °C to be used later (for step E2).
    3. Set aside an aliquot of 500 μl for TCID50 determination (for step E2) and freeze the rest at -80 °C for later.
    4. Removed the bioreactor from the stir-plate, and let beads settle for 5-10 min. Remove another 25 ml (10% of total bioreactor volume) of supernatant from the top of the infected bioreactor. Do not pipette the settled beads.
    5. From the uninfected feeder flask, pipette 37.5 ml of the slurry while spinning and transfer it to the infected bioreactor. Be sure not to cross contaminate your feeder flask.
    6. Pipette 25 ml DMEM-F12 with 5% FBS into the infected bioreactor. This media should have been warmed to 37 °C.
    7. Place both spinner flasks back onto the stir-plate in the incubator.

  2. Monitoring viral production in TB40/E IE2-EYFP infected bioreactor using TCID50 (Figure 2)
    1. Seed MRC-5 fibroblast cells in a 96-well flat-bottom plate by pipetting 100 μl per well of a cell suspension at 6,000 cells/ml. Incubate overnight at 37 °C to allow cell adhesion and growth.
    2. Take one aliquot of 500 μl of supernatant (prepared in step D2), and freeze-thaw 3 times by alternating incubation in a dry ice/isopropanol bath and, once frozen, transfer the tube to a 37 °C water bath to thaw. Make sure to vortex between each freeze-thaw. This step releases the virions that remain within the infected cells.
    3. Centrifuge 500 μl aliquot for 3 min at 300 x g.
    4. Add 1 ml DMEM with 10% FBS to 6 wells of a 12-well reagent reservoir.
    5. To the first well, pipette 110 μl of prepared viral supernatant into media. Using P1000, mix thoroughly while avoiding bubbles. The viral dilution in the first well is 1:10.
    6. Pipette 110 μl media from well #1 to well #2 to obtain 1:100 dilution of the viral preparation. Mix thoroughly using a P1000 Pipetman and avoid bubbles.
    7. Repeat serial dilutions of 1:10 through the remaining wells to reach in the last well a dilution of 1:1,000,000.
    8. Take prepared 96-well plate with adhered MRC-5 cells out of the incubator and aspirate media.
    9. Using a multichannel P200 pipette, transfer 100 μl of each serial dilution onto the MRC-% fibroblast plate. Repeat the transfer 12 times into successive rows to make sure that 8 wells of infected MRC-5 are being infected with each viral dilution.
    10. Place plate in a 37 °C incubator and monitor using fluorescent microscope over the next week.
    11. At day 7, score the eight wells within each dilution as either positive or negative for cytopathic effect.
    12. Calculate TCID50 using score (Reed Muench, 1938). TCID50 is the infectious virus titer at which pathological changes in 50% of inoculated cell cultures occur. This is determined by inoculating the series of diluted virus to susceptible cells. An Excel file from ResearchGate is a simple way to complete this calculation: https://www.researchgate.net/file.PostFileLoader.html?id=58dad730f7b67ea37125593f&assetKey=AS%3A476999471898624%401490736944531.

      Figure 2. TB40/E IE2-EYFP virus infection can be reproducibly held at end-stage for long periods of time enabling high-titer TB40/E IE2-EYFP production. A. Adherent ARPE-19 cells infected at an MOI of 0.01 show a characteristic eclipse phase one day after infection, where the concentration of virus drops to low levels. By day two, the viral concentration begins to increase until reaching its peak end stage viral production by day 7. After this point, viral concentrations begin to decline when using static culturing methods, as there are no more cells to infect. This same viral kinetics curve is observed in the bioreactor setting. With this system, however, it is possible to maintain peak viral production stage by regular replacements of microcarrier beads coated with naïve ARPE-19 cells. B. We demonstrate that 100-times more TB40/E IE2-EYFP virus can be generated in comparison to the static culture plates in a three-week timeframe. Virus stocks taken at each time-point are cumulated, and compared with the average titration from collecting virus from one 15 cm cell culture dish, estimated at 7.5E6 total pfu per plate. This estimates how many 15 cm plates infected with TB40/E IE2-EYFP it would take to obtain the same concentration of virus as the cumulative samples collected from the bioreactor. Once steady state viral production is reached, and regular feedings begin, the number of plates it would take to reach the same total pfu increases quickly. On day 23 it would take 105 plates to have the same pfu/ml as the reactor system. C. To show reproducibility of the maintained end-stage infection, a bioreactor containing microcarrier beads coated with ARPE-19 was infected with TB40/E IE2-EYFP at an MOI of 0.01. Once peak viral production was reached, steady state was established and maintained. After a period of time, the infected bioreactor was split equally into 4 smaller 100 ml bioreactors, in which the steady states were maintained equivalently by regularly changing the same percentage of the slurry (15%) and of the supernatant (10%) as before the split. D. TCID50 titrations were completed using the supernatant samples from each 48 h time point of all four bioreactors. Each day’s results for the four bioreactors were averaged and standard deviations were determined. The results show that the maintained viral production state is reproducible. The longer the bioreactors are carried, the more consistent their viral production becomes.

  3. Preparing stocks of virus for use in other experiments
    Note: Depending on your TCID50 results (determined from Procedure E), virus collected from the bioreactor during the regular feedings may be at a high enough concentration for certain experiments. If you require a higher concentration, however, standard ultracentrifugation using a sorbitol underlay can be performed. Supernatant and slurry samples need to be processed differently before use. Frozen supernatant samples (no microcarrier beads) simply need to be thawed and filtered. Slurry samples (frozen samples with microcarrier beads) require multiple freeze thaws to isolate the virus from within the cells.
    1. Supernatant samples
      1. Thaw frozen conical tube samples of virus from days that have been titered with TCID50 in a water bath.
      2. Supernatant samples can be used directly (prepared in step D7). Before using, however, filter media using a cell strainer to remove any remaining microcarrier beads. After, media should also be filtered using a 40 μm filter.
    2. Slurry samples
      1. Thaw frozen conical tube samples of virus from days that have been titered with TCID50 in a water bath.
      2. For slurry samples, centrifuge thawed conical tubes for 5 min and pipet off all media except for 5 ml that contain pelleted microcarrier beads. Media removed can be filtered and used as explained in step F2.
      3. Freeze thaw the remaining 5 ml of slurry in the conical tube 3 times by placing it in a dry ice/isopropanol bath and then transferring to a water bath once frozen. In between each freeze-thaw, vortex all samples. This step releases the virions that remain within the infected cells.
      4. Centrifuge 5 ml for 3 min at 300 x g.
      5. Remove remaining media from pellet, and pipette through cell strainer. This can now be used for infections or frozen for later.
    3. Optional: To concentrate the virus stock further, the protocol developed by Stinski et al. (1976) should be used. Briefly, on 23 ml viral preparation, underlay 7 ml of 20% sorbitol. After 1.5 h of centrifuge at 7,200 x g (20,000 rpm) at 18 °C, remove carefully the supernatant and resuspend the viral pellet in 2 ml of DMEM with no FBS.

Data analysis

We report a bioreactor system (Figure 1) for producing continuous high-titer stocks of clinically derived isolates of CMV that also maintains the integrity of the virus genome and tropism factors. Infection in the bioreactor system exhibits kinetics similar to infections in static culture, but maintains a reproducible production of virus for extended periods of time (Figure 2A) at the steady state that is reproducible (Figures 2C-2D). This bioreactor approach enables preparation of clinical isolate TB40/E IE2-EYFP HCMV virus at 107-108 pfu/ml over a 23-day period without requiring daily cell passaging or trypsinization, and the virus maintains cell-tropism factors. Overall, this system increased TB40/E IE2-EYFP virus production by more than 100 times in comparison to conventional static-culture plates (Figure 2B), while requiring substantially less time and manpower. Although we have used TB40/E IE2-EYFP to clearly portray the procedure and benefits of this system, this bioreactor method can be used to generate stocks of any clinically derived CMV strain. The end-stage steady-state infection exhibits minimal variation (Figure 2C) indicating that this system may potentially be useful for other assays such as real-time tracking of viral evolution.
Expected results

  1. Given that an average of 7.5 x 106 pfu can be collected from a single 15 cm dish of TB40/E IE2-EYFP-infected cells, we can relate the amount of HCMV viruses produced in our bioreactor to the number of plates that would have been needed. In our bioreactor, once steady state viral production is reached, and regular feedings begin, the virus production increases very rapidly. By day 8, it would take 8 plates to obtain the same amount of total viral particles, and by day 22, it would take 32.5 plates. On day 23, when the full bioreactor volume of 250 ml was collected, it would take 105 plates to obtain the amount of virus reached in our bioreactor system (Figure 2B).
  2. The sample collected at each time point, can be frozen, pooled and concentrated to achieve high concentrated HCMV stocks (i.e., obtaining up to 3 ml of a viral preparation at more than 2 x 107pfu/ml).




  1. Culture media for titration assays (TCID50)
    Dulbecco’s modified eagle medium (DMEM/F-12) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin
    Store at 4 °C
    Bring to 37 °C before use
  2. Culture media for bioreactor
    DMEM/F12 with 5% FBS and 1% penicillin-streptomycin
    Store at 4 °C
    Bring to 37 °C before use
  3. Siliconizing glassware with Sigmacote
    Clean glassware (stir flask, 100 ml containers) with deionized water and dry
    Apply 3 ml of Sigmacote in the glass flask and swirl such that liquid covers all of the inside surface of the glassware for 10 sec. Remove excess solution, and save for reuse
    Rinse the glass flask with deionized water thoroughly to ensure that excess Sigmacote is removed
    Note: Store Sigmacote at 4 °C.
  4. 20% sorbitol
    1. Combine:
      100 g sorbitol
      25 ml 1 M Tris, pH 7.5
      0.5 ml 1 M MgCl2 (30.5 g MgCl2 + 150 ml MilliQ water)
    2. Add enough water to bring to 350 ml
    3. Add HCl dropwise to bring to pH 7.2
    4. Add enough MilliQ water to bring to 500 ml
    5. Filter stock with 0.2 or 0.1 μm filter system
  5. Microcarrier beads ratio and preparation
    0.5 g Cytodex 1 microcarrier beads per 100 ml final bioreactor volume (dry weight)
    Store dry microcarrier beads at room temperature
  6. Adherent cell ratio
    1 x 105 cells per 100 ml final bioreactor volume (cells should be low-passage)


This work was supported by funding from NIH grants, DP1DE024408 and DP2OD006677 (to L. Weinberger).
Conflict of Interest Disclosure: L. Weinberger is a co-founder of Autonomous Therapeutics Inc. Other authors declare no competing financial interest.


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  3. Bristow, B. N., O'Keefe, K. A., Shafir, S. C. and Sorvillo, F. J. (2011). Congenital cytomegalovirus mortality in the United States, 1990-2006. PLoS Negl Trop Dis 5(4): e1140.
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人类巨细胞病毒(HCMV)感染是移植患者发病率和死亡率的主要原因,并且是先天性先天缺陷的主要原因(Saint Louis,2016)。接种疫苗和治疗性研究通常需要以临床分离物为代表的野生型病毒的可扩展的细胞培养生产。获得充足的野生型临床HCMV库存往往是劳动密集型的,由于产量低和遗传损失效率低下,对临床分离物的研究构成障碍。在这里,我们报告了一种基于连续感染的生物反应器方法,其中在生物反应器中感染粘附到微载体珠上的视网膜色素上皮(ARPE-19)细胞并用于产生维持关键病毒嗜性因子的遗传完整性的高滴度临床分离株HCMV和病毒基因组。在该生物反应器中,通过定期加入未感染的ARPE-19细胞可以维持末期感染,提供了10 7 -10 -8 pfu / ml浓缩的方便的制备TB40 / E IE2-EYFP库存无需日常细胞传代或胰蛋白酶消化。总的来说,与传统的静态培养板相比,这代表病毒产量增加了100倍的100倍,同时需要90%的处理时间。此外,这种连续的感染环境有可能监测感染动态,并可以实时跟踪病毒的进化。

【背景】先天性巨细胞病毒感染是导致出生缺陷的主要原因,仅在美国,每年的直接成本就高达10亿美元,并代表了全球未得到满足的医疗需求(Bristow等人,2011; Griffiths 2013; Manicklal等人,2013; Saint Louis,2016)。这是可以预防的有效的疫苗或治疗针对妇女在其生育年。人巨细胞病毒(HCMV)可以感染多种细胞类型,但是该领域的主要障碍是临床衍生的HCMV毒株在成纤维细胞中的延伸通路导致其他细胞类型的病毒嗜性丧失(Waldman等人,1991; Sinzger等人,1999)。在20世纪60年代后期,几种实验室适应的CMV毒株连续传代于成纤维细胞(包括HCMV AD169,Towne和Davis毒株,以及鼠CMV的Smith毒株),并成为用于研究CMV的分子生物学(Plotkin等人,1975)。这些实验室适应的菌株通常在不成功尝试产生减毒活疫苗时被发现具有多种影响(i)它们感染不同细胞类型的能力,(ii)病毒复制速率,和(iii)改变潜伏表型(Albrecht和Weller,1980; Yamane等人,1983; Waldman等人,1989; Kahl等人, 2000)。具体而言,发现包含病毒糖蛋白进入复合物的HCMV开放阅读框(ORF)UL128,UL130,UL131在成纤维细胞中传代期间积累突变,导致病毒向上感染上皮细胞,内皮细胞,巨噬细胞和树突状细胞(Sinzger等人,1999,Hahn等人,2004; Wang和Shenk,2005; Adler等人, ,2006)。这些实验室改造的HCMV毒株也显示在病毒基因组的UL / b'区丢失了几个基因,该区域赋予体内免疫逃逸功能和复制扩散 et al。,1996)。现在已知在成纤维细胞培养物上持续的病毒生长消除了保留这些序列的选择压力,导致在其他宿主细胞和组织中复制和传播所必需的序列遗传丢失或重排。然而,HCMV临床分离株(例如,TB40 / E和VR1814)在上皮细胞和内皮细胞环境中的传代保持选择压力以防止非成纤维细胞类型向性的丧失(Waldman等人。1991; Hahn等人,2002; Sinzger等人,2008)。


在这里,我们报告了一种新的,更有效的方法来产生临床派生的HCMV分离株,由TB40 / E IE2-EYEFP代表。该病毒通过EYEFP融合基因标记,可以方便地监测生物反应器环境中的连续感染。使用具有微载体珠粒的两阶段生物反应器系统(图1)和HCMV特征性病毒生产的延长周期的优势,我们能够维持末期感染并在原代贴壁细胞培养物上产生高滴度病毒储液保护关键病毒嗜性因子和病毒基因组的遗传完整性。我们使用TB40 / E-IE2 EYFP标记的病毒来开发和表征生物反应器感染。荧光标签通过荧光显微镜检查等分的感染样品,可以方便地监测生物反应器培养中的病毒。

关键字:人巨细胞病毒, 生物反应器, 微珠(微载体珠), 病毒嗜性, 连续感染培养, 临床分离


  1. 15cm组织培养皿(Corning,Falcon ,目录号:353025)
  2. 通风螺帽(Corning,Falcon ,目录号:354639; Corning,目录号:3968)
  3. 1.5ml聚丙烯微量离心管(Corning,Axygen,目录号:MCT-150-C)
  4. 锥形无菌聚丙烯离心管,50ml(Fisher Scientific,目录号:05-539-12)
  5. 96孔平底细胞培养板(Corning,目录号:3596)
  6. 12孔试剂池(Corning,Costar ,目录号:4877)
  7. 40μm细胞过滤器(Corning,目录号:431750)
  8. 锥形无菌聚丙烯离心管,15毫升(Fisher Scientific,目录号:06-443-18)
  9. 0.45μm无菌注射器过滤器(EMD Millipore,目录号:SLHV033RS)
  10. 一次性无菌注射器,带鲁尔锁头(BD,目录号:309646)
  11. 血清移液管(50毫升,25毫升,10毫升,5毫升,2毫升)
  12. 0.2或0.1微米过滤系统
  13. ARPE-19细胞(ATCC,目录号:CRL-2302)
  14. MRC-5细胞(ATCC,目录号:CCL-171)
  15. TB40 / E IE2-EYFP杆粒克隆病毒
    TB40 / E IE2-EYFP来源于临床获得的HCMV分离物TB40 / E(Sinzger等人,2008)的杆粒克隆。病毒双链DNA基因组已被克隆为杆状病毒,并且可以产生编码在多种宿主细胞类型中复制所需的趋向性因子的感染性病毒。如先前报道的,该病毒含有与关键病毒反式激活因子IE2基因的羧基末端融合的EYFP荧光标签(Teng等人,2012)。使用之前描述的用HCMV进行杆粒克隆的galK选择和反选择重组方案产生该杆粒克隆(Murphy等人,2008,Warming等人, 2005年)。这种荧光标签的表达贯穿于病毒裂解感染周期,并且能够方便地实时监测生物反应器环境中的连续感染。
  16. 不含钙和镁的DPBS,DPBS-CMF(Mediatech,目录号:21-031-CV)
  17. 胰蛋白酶-EDTA,0.25%(Mediatech,目录号:25-053-CI)
  18. 胰蛋白酶-EDTA,0.05%(Mediatech,目录号:25-052-CI)
  19. 干冰粒
  20. 异丙醇(Fisher Scientific,目录号:A451-4)
  21. 含有L-谷氨酰胺和丙酮酸钠的DMEM(Mediatech,目录号:10-013-CV)
  22. 带有L-谷氨酰胺和15mM HEPES的DMEM / F-12(Mediatech,目录号:10-092-CV)
  23. 胎牛血清(FBS)(Mediatech,目录号:35-011-CV)
  24. 青霉素 - 链霉素溶液(Pen / Strep)(Mediatech,目录号:30-002-CI)
  25. Sigmacote(Sigma-Aldrich,目录号:SL2-25ML)
  26. 1M Tris,pH 7.5
  27. 氯化镁(MgCl 2)
  28. 盐酸(HCl)
  29. Cytodex 1微载体珠,60-87微米(Sigma-Aldrich,目录号:C0646-5G)
  30. 用于滴定测定的培养基(TCID50)(参见食谱)
  31. 生物反应器的培养基(见食谱)
  32. 用SigmaCote硅化玻璃器皿(见食谱)
  33. 20%山梨醇(见食谱)
  34. 微载体珠比例和准备(见食谱)
  35. 贴壁细胞比例(见食谱)


  1. 在BSL-2工作所需要的个人防护装备PPE:实验室外套或长袍,眼睛和脸部保护,手套
  2. Wheaton Celstir旋转瓶,150ml,250ml和500ml(WHEATON,目录号:356879和356882)
  3. 组织培养的培养箱:37℃,5%CO 2
  4. 4位慢速搅拌盘(Corning,目录号:440814)
  5. 第二类生物安全柜(贝克)
  6. 涡旋混合器
  7. 微量移液器(1-1,000μl容量),多通道移液器(1-200μl容量)(P1000移液器和P200移液器[Pipetteman])
  8. 血细胞计数器(Hausser Scientific,目录号:1475)
  9. 细胞培养倒置epi荧光显微镜
  10. 台式离心机

  11. 水浴,37°C
  12. 100毫升可高压灭菌的玻璃瓶(WHEATON,目录号:W818012406)
  13. 批准BSL-2实验室设施
  14. 高压灭菌器
  15. 移液器控制器(Drum-aid,Drummond Scientific,型号:Pipet-Aid XP2,目录号:4-000-501)
  16. 冰柜:-80°C,-20°C,4°C


准备TCID 50的96孔板(步骤E1:15分钟)
设置TCID <50>感染(步骤E2-E11:25分钟)
确定来自TCID 50的滴度(步骤E12-E13:10分钟)


图1.用于维持连续病毒生产的生物反应器系统设置A.将粘附的细胞系(ARPE-19)加入到500ml硅涂覆的玻璃加料器烧瓶中,烧瓶中含有在150ml中的2.5g水合微载体珠DMEM-F12媒体。将此进料瓶置于37℃培养箱中4小时以使ARPE-19细胞粘附至微载体珠,每半小时混合一次。 B. 4小时后,将培养基的体积升至500毫升的工作浓度,并将其放置在60转/分钟的搅拌板中,放回相同的培养箱中。让细胞在微载体珠粒上分开一周,并在供料瓶中达到汇合。当达到汇合时,将来自进料瓶的250ml搅拌的微载体珠粒转移到更小的250ml生物反应器中。然后用MOI为0.01的TB40 / E IE2-EYFP感染此较小的生物反应器。小型和大型生物反应器都放回到搅拌盘上的培养箱中。 D.当病毒扩展达到期望的稳定状态时,从感染的生物反应器收集浆液(在旋转时取得的微载体珠粒/培养基)和上清液(当搅拌板关闭并且微载体珠粒沉降时收集的培养基)。将未感染的ARPE-19细胞从进料烧瓶中取出,并与新鲜培养基一起加入感染的生物反应器中。微载体珠和培养基置换的速率维持HCMV的末期病毒生产。感染的生物反应器的体积和微载体珠浓度保持恒定。这种诱导的稳定状态可以持续所需的时间,从感染的反应器收集的样品被浓缩以产生病毒储备。

  1. 用ARPE-19细胞接种Cytodex 1微载体珠(图1A)
    1. 在15cm细胞培养皿中培养足够的ARPE-19细胞,使得您有1×10 6个细胞/ ml终体积。对于500ml Celstir旋转瓶(进料瓶),需要5×10 8个细胞。
    2. 在播种前一天,根据制造商的说明硅化500ml进料烧瓶和可Sigmaocote的可高压灭菌的100ml玻璃容器( http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/sl2pis.pdf )。
      注意:该程序已经被优化,以在播种期间将ARPE-19细胞最大程度地粘附到制备的Cytodex 1微载体珠上。 Sigmacote用于防止细胞粘附在旋转瓶的壁上。鉴于对细胞有毒性,硅化后需要进行严格的漂洗。
    3. 用MilliQ水彻底冲洗饲养瓶和玻璃容器至少10次。将一个新的通风螺旋盖放在烧瓶的每个臂上。
    4. 称量适量的干燥的Cytodex 1微载体珠子,以达到0.5g / 100ml生物反应器最终体积。对于500毫升的饲养瓶,量出2.5克微载体珠。
    5. 将干燥的Cytodex 1微载体珠粒倒入硅化的100ml玻璃容器中,并加入100ml DPBS-CMF(不含钙和镁的DPBS)。彻底重新悬浮珠子。有关更多信息,请参阅GE手册: http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/Microcarrier%20Cell%20Culture.pdf
    6. &nbsp;高压灭菌器供料瓶和重新悬浮的微载体珠。
    7. 通过从100ml玻璃容器中的沉降珠移出50ml DPBS-CMF移除,用50ml含10%FBS的DMEM-F12代替。通过涡旋重悬。孵化玻璃容器与珠在37°C,直到开始步骤A9。
    8. 将5×10 8 ARPE-19细胞胰蛋白酶消化,用含有10%FBS的90ml DMEM-F12重悬,并置于饲养瓶中。
    9. 从37°C培养箱中取出带有沉降珠的玻璃容器,并通过移液60毫升(即,大部分液体)来移除。将剩余的40ml DMEM-F12(含有10%FBS)的微载体珠粒重新悬浮在玻璃容器中,并将它们转移到进料瓶中。再加入20ml的100%FBS以增强细胞粘附。补料瓶中的最终体积应为约150毫升,最终的FBS浓度应大致为25%。
    10. 将供料瓶置于37℃培养箱中以使细胞粘附至珠4小时。每隔30分钟,将饲养瓶放置在培养箱内60rpm的慢速搅拌盘上30秒,以缓慢搅拌生物反应器,并确保细胞接种。
    11. 4小时后,通过加入含有10%FBS的350ml DMEM-F12,使进料瓶充满500ml体积。
    12. 将孵化器中的饲料瓶置于4位置的慢速搅拌盘中,转速设置为60rpm。
    13. 每2-3天更换饲养瓶内的媒体,以保持细胞密度。要做到这一点,让珠定居10分钟,然后删除50-75%的总媒体量(250-375毫升),并用相同数量的新鲜媒体(含5%FBS的DMEM-F12)取代它。让细胞在微载体珠上扩展一周,或者直到达到最大细胞密度(步骤C1)(通过程序B中的细胞计数和眼睛监测)。


  1. 监测微载体珠上的ARPE-19细胞密度(图1B)
    1. 从培养箱中取出饲养瓶,置于II级生物安全柜内60rpm的搅拌盘上。
    2. 在搅拌盘上搅拌1ml浆液(混合细胞/微载体珠/培养基),并分装到1.5ml微量离心管中。将饲料瓶放回搅拌盘的培养箱中,在此过程的其余部分不需要使用它。
    3. 让微量离心管中的微载体珠粒沉降2分钟。可以通过重力在微量离心管的底部观察微载体珠,不需要离心。
    4. 通过从微量离心管吸取0.8ml培养基来洗涤,并用0.8ml DPBS-CMF再悬浮。通过在涡旋混合器上轻轻涡旋或通过上下移液重悬珠,然后使微载体珠再次沉降。
    5. 从微量离心管中取出0.8ml的DPBS-CMF,用0.8ml的0.25%胰蛋白酶代替。轻轻涡旋重悬细胞。
    6. 将微量离心管置于37°C培养箱中,让细胞胰蛋白酶消化15-20分钟,轻轻振荡或甩动以保证胰蛋白酶消化每5分钟重新悬浮。
    7. 将微量离心管放回生物安全柜中,用P1000移液管强力吸取重悬。这一步也有助于从微载体珠粒中去除所有的细胞并分解聚集体。让珠子靠重力沉降30秒。
    8. 移取10微升介质,从微量离心管中取出分离的细胞,避免吸收任何微载体珠粒。转移这10μL血细胞计数器,并确定细胞浓度。

    注意:应定期(每2或3天)进行细胞计数,以确保细胞健康。饲养瓶中的细胞密度应该增加,直至达到最大密度(1×10 6细胞/ ml),然后保持恒定。在显微镜下观察粘附在珠上的细胞也是可能的。如果细胞似乎从珠粒中解离出来,或者细胞数量突然下降,这可能是因为介质没有被足够频繁地更换。

  1. 在具有TB40 / E-IE2-EYFP的生物反应器中的微载体珠子上感染ARPE-19细胞(图1C)
    1. 一旦通过细胞计数(程序B)测量,在进料瓶中的细胞浓度达到大约1×10 6细胞/ ml时,将250ml浆液转移至更小的硅化,漂洗和高压灭菌250毫升WHEATON旋转瓶(在此称为生物反应器)。通过定期更换培养基(含有5%FBS的DMEM-F12)继续维持饲养瓶中的细胞生长(参见步骤A13)。
    2. 一旦达到大约1×10 6个细胞/ ml,通过细胞计数测量,将250ml浆液转移到更小的硅化和高压灭菌的250ml WHEATON旋转瓶(生物反应器)中。定期更换培养基,继续保持饲养瓶内的残留物(参见步骤C1)。
    3. 以TB40 / E-IE2-EYFP为基础,以感染复数MOI为0.01,根据病毒储存的活细胞计数和TCID50,接种新生物反应器中的细胞。
    4. 将生物反应器(250毫升)和饲料瓶(500毫升)放置在37°C培养箱内设置在60转/分的同一4位搅拌盘上。
    5. 通过从搅拌的生物反应器中取200μl等分试样监测病毒扩增,并通过荧光显微镜观察EYFP扩散。扩展也应该通过定期的TCID 50 进行监控。


  1. 通过维持稳态TB40 / E IE2-EYFP感染持续高滴度病毒生产(图1D)
    注意:为了保持正在进行的CMV感染并避免感染的崩溃,我们需要定期提供未感染的细胞,然后感染新生成的病毒。新鲜的媒体也被添加到防止细胞死亡。媒体和细胞需要被替换的速度取决于特定病毒的动力学。对于TB40 / E IE2-EYFP,我们确定每48小时更换一次培养基和细胞,随着时间的推移,病毒产量最高。尽可能保持饲养日程的规律是维持稳定的病毒生产的理想选择。另外,在饲养瓶中保持健康的幼稚细胞对持续的病毒生产是必不可少的。应该更换培养基,细胞密度应该保持恒定在这个饲料瓶(见步骤A13)。
    1. 定期视觉监测可以跟踪生物反应器中感染培养物的健康状况。当80%的细胞被杀死(并从珠粒脱落),通常发生在感染后5-7天,开始定期喂养与细胞和媒体如下。
    2. 每48小时,从生物安全柜内的搅拌板上搅拌,从感染的生物反应器中移出37.5毫升(占总生物反应器体积的15%)淤浆。将该浆液转移到50ml锥形管中,并在300×g下离心5分钟(去除未粘附的细胞)。等分3份500μl(用于TCID 50)的上清液样品。在-80℃冷冻三个等分试样和50ml锥形的培养基管以供以后使用(对于步骤E2)。
    3. 放置一个500μl的等分试样用于TCID 50测定(对于步骤E2),并将其余的在-80℃冻结以备后用。
    4. 从搅拌板上取下生物反应器,让珠粒沉降5-10分钟。从被感染的生物反应器的顶部除去另外25ml(总生物反应器体积的10%)上清液。不要移动沉降的珠子。
    5. 从未感染的饲养瓶中,吸取37.5ml浆液,同时旋转并将其转移到感染的生物反应器中。一定不要交叉污染你的饲料瓶。
    6. 吸取25毫升含5%FBS的DMEM-F12到感染的生物反应器中。这个媒体应该被加热到37°C。
    7. 将两个旋转瓶放回培养箱中的搅拌板上。

  2. 使用TCID 50(图2)监测TB40 / E IE2-EYFP感染的生物反应器中的病毒生产
    1. 通过以6,000个细胞/ ml的细胞悬浮液每孔移取100μl,在96孔平底板中种入MRC-5成纤维细胞。在37°C孵育过夜,使细胞粘附和生长。
    2. 取一份500μl上清液(步骤D2中制备),并通过在干冰/异丙醇浴中交替温育来冻融3次,一旦冷冻,将管转移至37℃水浴解冻。确保在每个冻融之间进行涡旋。这一步释放了感染细胞内的病毒颗粒。
    3. 在300μgxg离心500μl等分试样3分钟。

    4. 添加1毫升含10%FBS的DMEM到12孔试剂池的6个孔中
    5. 向第一个孔中,移取110μl制备的病毒上清液到培养基中。使用P1000,彻底混合,避免气泡。第一口井的病毒稀释度为1:10。
    6. 吸取110#培养基从#1井到#2#培养液,以获得1:100稀释的病毒制剂。
    7. 重复1:10的系列稀释液至最后一个孔,稀释度为1:1,000,000。

    8. 从培养箱中取出贴有MRC-5细胞的96孔板,吸出培养基。
    9. 使用多通道P200移液器,将100μl的每个连续稀释液转移到MRC-%成纤维细胞平板上。重复转移连续12次,以确保感染的MRC-5的8个孔被每种病毒稀释物感染。
    10. 将板置于37℃培养箱中,并在下一周使用荧光显微镜进行监测。
    11. 在第7天,将每个稀释度内的八个孔评分为细胞病变效应的阳性或阴性。
    12. 使用得分计算TCID 50(Reed Muench,1938)。 TCID 50是在50%的接种细胞培养物发生病理变化时的感染性病毒效价。这是通过将一系列稀释的病毒接种至易感细胞来确定的。从研究之门Excel文件是一个简单的方式来完成这个计算: https://www.researchgate.net/file.PostFileLoader.html?id=58dad730f7b67ea37125593f&assetKey=AS%3A476999471898624%401490736944531

      图2. TB40 / E IE2-EYFP病毒感染可以在终末阶段长期重复保存,从而实现高效价的TB40 / E IE2-EYFP生产。 A.以0.01的MOI感染的粘附的ARPE-19细胞在感染后一天显示特征性的食期,其中病毒浓度下降至低水平。到第二天,病毒浓度开始增加,直到第7天达到其高峰期病毒产量。在此之后,当使用静态培养方法时,病毒浓度开始下降,因为没有更多的细胞被感染。在生物反应器设置中观察到相同的病毒动力学曲线。然而,使用该系统,可以通过定期更换涂有初始ARPE-19细胞的微载体珠来维持峰值病毒生产阶段。 B.我们证明,与三周的静态培养板相比,可以产生100倍以上的TB40 / E IE2-EYFP病毒。累积在每个时间点采集的病毒库,并与从一个15cm细胞培养皿中收集病毒的平均滴度进行比较,估计为每个平板7.5E6总pfu。这估计了用TB40 / E IE2-EYFP感染多少15cm平板以获得与从生物反应器收集的累积样品相同浓度的病毒。一旦达到稳定状态的病毒产量,并且开始定期喂食,达到相同总pfu将需要的平板数量迅速增加。在第23天,需要105个平板具有与反应器系统相同的pfu / ml。为了显示维持的末期感染的再现性,用MOI为0.01的TB40 / E IE2-EYFP感染包含用ARPE-19包被的微载体珠的生物反应器。一旦达到高峰病毒产量,建立并保持稳定状态。经过一段时间后,感染的生物反应器被等分成4个较小的100ml生物反应器,其中稳定状态通过定期改变相同比例的浆液(15%)和上清液(10%)而保持相同分裂。 D.使用来自全部四个生物反应器的每个48h时间点的上清液样品完成TCID 50滴定。每天对四种生物反应器的结果进行平均,并确定标准偏差。结果显示维持的病毒生产状态是可重现的。
  3. 准备用于其他实验的病毒库存
    1. 上清液样品
      1. 从TCID 50在水浴中滴定的天内解冻病毒的冷冻锥形管样品。
      2. 上清液样品可以直接使用(在步骤D7中制备)。然而,在使用之前,使用细胞过滤器的过滤介质去除任何剩余的微载体珠子。之后,介质也应该使用40微米的过滤器进行过滤。
    2. 泥浆样品
      1. 从TCID 50在水浴中滴定的天内解冻病毒的冷冻锥形管样品。
      2. 对于浆液样品,将解冻的锥形管离心5分钟,并移除除了包含粒状微载体珠粒的5ml之外的所有培养基。
      3. 通过将其置于干冰/异丙醇浴中冷冻剩余的5ml锥形管中的浆液3次,然后一旦冷冻转移到水浴中。在每次冻融之间,涡旋所有样品。这一步释放了感染细胞内的病毒颗粒。

      4. 在300×g离心5分钟3分钟。
      5. 从颗粒中去除剩余的培养基,并通过细胞过滤器移液。这现在可以用于感染或以后冻结。
    3. 可选:为了进一步浓缩病毒储存,由Stinski等人开发的方案应该使用(1976)。简言之,在23ml病毒制剂中,底层7ml的20%山梨糖醇。在18℃以7200×g(20,000rpm)离心1.5小时后,小心地取出上清液并将病毒沉淀重悬于2ml不含FBS的DMEM中。


我们报道了一种生物反应器系统(图1),用于生产持续的高滴度CMV临床分离株,同时保持病毒基因组和趋向因子的完整性。生物反应器系统中的感染表现出与静态培养中的感染类似的动力学,但是在可重现的稳定状态(图2C-2D)下维持了可再现的病毒长时间生产(图2A)。该生物反应器方法能够在23天时间内以10 7 -10 -8 pfu / ml制备临床分离株TB40 / E IE2-EYFP HCMV病毒,而不需要每日细胞传代或胰蛋白酶化,病毒维持细胞取向因子。总体而言,与传统静态培养板(图2B)相比,该系统使TB40 / E IE2-EYFP病毒产量增加了100倍以上,同时需要大量的时间和人力。尽管我们已经使用TB40 / E IE2-EYFP来清楚地描述该系统的程序和益处,但是这种生物反应器方法可用于产生任何临床衍生的CMV毒株。末期稳态感染表现出最小的变异(图2C),表明该系统可能潜在地用于其他测定,诸如病毒进化的实时追踪。

  1. 鉴于平均7.5×10 6 pfu可从单个15厘米的TB40 / E IE2-EYFP感染细胞中收集,我们可以将生物反应器中产生的HCMV病毒的量与本来需要的板数。在我们的生物反应器中,一旦达到稳定状态的病毒生产,并且定期喂食开始,病毒产生增加非常迅速。到第8天,需要8个平板来获得相同数量的总病毒颗粒,到第22天,需要32.5个平板。在第23天,当收集250ml的全生物反应器体积时,需要105个平板来获得在我们的生物反应器系统中达到的病毒量(图2B)。
  2. 在每个时间点收集的样品可以被冷冻,汇集并浓缩以获得高浓度的HCMV储液(即,获得高达3ml的病毒制剂,超过2×10 7




  1. 用于滴定测定的培养基(TCID50) 带有10%胎牛血清(FBS)和1%青霉素 - 链霉素的Dulbecco改良的Eagle培养基(DMEM / F-12)
  2. 生物反应器的培养基
    含有5%FBS和1%青霉素 - 链霉素的DMEM / F12
  3. 用Sigmacote
    硅化玻璃器皿 清洁玻璃器皿(搅拌烧瓶,100毫升容器)与去离子水和干燥

    用去离子水彻底冲洗玻璃烧瓶,确保过量的Sigmacote被清除 注意:将Sigmacote储存在4°C。
  4. 20%山梨糖醇
    1. 合并:
      25毫升1M Tris,pH 7.5
      0.5ml 1M MgCl 2(30.5g MgCl 2 + 150ml MilliQ水)。
    2. 添加足够的水,以达到350毫升
    3. 滴加HCl使pH值达到7.2
    4. 添加足够的MilliQ水,以达到500毫升
    5. 用0.2或0.1微米过滤系统过滤原料
  5. 微载体珠比和制备
    每100毫升最终生物反应器体积(干重)0.5克Cytodex 1微载体珠子
  6. 贴壁细胞比例
    每100毫升最终生物反应器体积为1×10 5个细胞(细胞应该是低通的)


这项工作得到了美国国立卫生研究院拨款DP1DE024408和DP2OD006677(给L. Weinberger)的资助。
利益冲突披露:L. Weinberger是Autonomous Therapeutics Inc.的联合创始人。其他作者声明没有竞争性的经济利益。


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引用:Saykally, V. R., Rast, L. I., Sasaki, J., Jung, S., Bolovan-Fritts, C. and Weinberger, L. S. (2017). A Bioreactor Method to Generate High-titer, Genetically Stable, Clinical-isolate Human Cytomegalovirus. Bio-protocol 7(21): e2589. DOI: 10.21769/BioProtoc.2589.