CoChat
Sign Up
Cited by 55Viral Infectious Diseases and Gene Expression in InsectsRabies epidemiology and controlFluid Dynamics and Mixing

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Open access
Rourou, Samia, Ben Zakkour, Meriem, Kallel, Héla·2019·Vaccine
DOI 10.1016/j.vaccine.2019.05.092
Open this paper in CoChat — free accountOpen PDF

Abstract

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

AI snapshot

Written from the paper’s full text.

TL;DR

Vero cells were adapted to serum-free suspension growth, with the in-house IPT-AFM medium and a protocol starting from IPT-AFM-cryopreserved cells giving the best growth. The resulting VeroS cells supported LP-2061 rabies virus replication in shake flasks, reaching the highest titer of 5.2 ± 0.5 × 10^7 FFU/mL in IPT-AFM with reduced Ca²⁺ and Mg²⁺.

Key findings

  • Among three adaptation protocols, protocol 3 gave the highest cell density after 9 passages: 2.3 ± 0.14 × 10^6 cells/mL versus 1.4 ± 0.15 × 10^6 and 1.65 ± 0.12 × 10^6 cells/mL for protocols 1 and 2, respectively.
  • VeroS cells grown in IPT-AFM over 20 passages reached an average cell density of 2.1 ± 0.4 × 10^6 cells/mL, with cell division number 1.53 ± 0.3 and specific growth rate 0.014 ± 0.003 h⁻¹. After reducing Ca²⁺/Mg²⁺ and using mechanical dissociation, a maximum of 2.9 ± 0.1 × 10^6 cells/mL and μ around 0.018 h⁻¹ were reached within 4 days.
  • In commercial media, VeroS reached 2.9 ± 0.2 × 10^6 cells/mL in Hycell CHO, 2.5 ± 0.5 × 10^6 cells/mL in CD-U5, 1.9 ± 0.15 × 10^6 cells/mL in SFM4CHO, and 1.7 ± 0.2 × 10^6 cells/mL in CD293 and 293SFMII.
  • Rabies virus infection at MOI 0.1 yielded >10^7 FFU/mL in all tested media; the highest titer was 5.2 ± 0.5 × 10^7 FFU/mL in IPT-AFM with reduced Ca²⁺ and Mg²⁺, while CD-U5, SFM4CHO, and Hycell CHO reached 1.9, 2, and 2.4 × 10^7 FFU/mL, respectively. Table 1 reports VeroS/shake flask virus yield of 3.75 ± 1 FFU/cell/day versus 2.5 ± 0.8 FFU/cell/day for adherent/spinner Vero cells.

Methods

  • Vero cells at passage 131 (National Laboratory for Control of Biologicals, Tunisia; originally ATCC CCL-81) were grown adherently in MEM + 10% FBS or IPT-AFM, then adapted to suspension using three protocols; protocol 3 was selected and carried through 20 successive passages.
  • Suspension cultures used 125–500 mL vented nonbaffled shake flasks at 37 °C, 7% CO₂, 150 rpm, with 0.1% pluronic acid F-68; media tested included IPT-AFM, IPT-AFM with reduced Ca²⁺/Mg²⁺, CD293, Hycell CHO, CD-U5, 293SFMII, and SFM4CHO, with Cell Boost 5 supplement in some conditions.
  • Cells were infected with LP-2061 rabies virus at MOI 0.1 without medium exchange, shifted to 34 °C, and virus titers measured by a modified Rapid Fluorescence Focus Inhibition Test expressed as FFU/mL; growth was assessed by viable/nucleus counts and kinetic parameters Cd and μ.

Limitations

  • The paper states the results are preliminary and that further experiments are required to enhance cell population doubling; it also states that virus titer could be further enhanced by adapting the virus to VeroS cells through consecutive passages.
  • The paper states that the identity of the new VeroS cell line should be checked, its safety characterized according to international regulatory guidelines, and its tumorigenic potential studied in vivo; cell growth performance of clones obtained by limit dilution should also be studied to demonstrate homogeneity.
  • Evident from the text: virus production was tested only in batch shake flasks with one rabies strain, without medium exchange or perfusion, and glucose was not regulated; bioreactor process development and intensification remain future work.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.vaccine.2019.05.092

Cites

57 of 57 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 45 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    After 9 passages in shake flasks, consistent cell growth was noticed.

    “after 9 passages in shake flasks, a consistent cell growth was noticed.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Use of IPT-AFM free of Ca++ and Mg++ or with reduced amounts of these components next to mechanical dissociation allowed VeroS to grow as small aggregates (<10 cells) and/or single cells.

    “The use of an IPT-AFM free of Ca ++ and Mg ++ or with reduced amounts of these components next to mechanical dissociation, had allowed VeroS to grow as small aggregates (<10 cells) and/or single cells (Fig. 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Calcium is involved in cell to cell adhesion.
  • Findingown data

    Protocol 3 (starting from cells cryopreserved in IPT-AFM then moving to agitated culture within 2 passages in static culture) yielded higher cell density than protocols 1 and 2 after 9 passages in shake flasks.

    “higher cell density level was observed for cells adapted to suspension growth according to protocol 3. The highest cell density level reached 2.3 ± 0.14 Â 10 6 cells/mL versus 1.4 ± 0.15 Â 10 6 cells/mL and 1.65 ± 0.12 Â 10 6 cells/mL for protocols 1 and 2, respectively.”
    Quote in the paperSubstantial claim
  • Findingown data

    In SFM4CHO medium, cell density of 1.9 ± 0.15 ×10^6 cells/mL was achieved.

    “a cell density close to 2 Â 10 6 cells/mL (1.9 ± 0.15 Â 10 6 cells/mL) was achieved in the protein free medium SFM4CHO.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Elimination of Ca++ and Mg++ contributing to aggregate removal slightly reduced cell density level.

    “the elimination of Ca ++ , Mg ++ contributing in aggregate removal, had slightly reduced cell density level.”
    Quote in the paperSubstantial claim
  • May combine two claims. 19
  • Classified as probably not a substantial claim. 24

  • May combine two claims.

    Findingown data

    IPT-AFM with 20% Ca++ and Mg++, Hycell CHO, CD-U5, and SFM4CHO were suitable for VeroS growth as single cells and small aggregates, while CD293 and 293SFMII enhanced aggregation.

    “IPT-AFM with 20% Ca ++ , Mg ++ next to Hycell CHO, CD-U5 and SFM4 CHO media were suitable for VeroS growth as single cells and small aggregates while CD293 and 293SFMII media enhanced aggregation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VeroS cells reached 2.9 ± 0.2 ×10^6 cells/mL and specific growth rate 0.018 h^-1 ± 0.005 in Hycell CHO medium, 4 days after starting culture.

    “VeroS cells were able to reach 2.9 ± 0.2 Â 10 6 cells/ml and a specific growth rate (m) of 0.018 h À1 ± 0.005 in the chemically defined Hycell CHO medium, 4 days after starting the culture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Highest virus yield was obtained 2-3 days after cell infection, thereafter a progressive or sharp decrease of virus titer was noticed depending on the medium used.

    “The highest yield was obtained 2-3 days after cell infection, thereafter a progressive or a sharp decrease of virus titer was noticed depending on the medium used.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In IPT-AFM with 20% Ca++ and Mg++, maximum cell density of 2.9 ± 0.1 ×10^6 cells/mL and μ around 0.018 h^-1 were reached within 4 days; growth as single cells was noticed.

    “a maximum cell density level of 2.9 ± 0.1 Â 10 6 cells/mL and a m around 0.018 h À1 were reached within 4 days; furthermore growth as single cells was noticed in IPT-AFM with 20% Ca ++ , Mg ++ .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Highest virus titer was obtained in IPT-AFM 20% Ca++ and Mg++ and was equal to 5.22 ± 0.5 ×10^7 FFU/mL.

    “The highest titer was obtained in IPT-AFM 20% Ca ++ , Mg ++ and was equal to 5.22 ± 0.5 Â 10 7 FFU/mL.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Maximum cell density in IPT-AFM without Ca++ and Mg++ was 1.95 ± 0.05 ×10^6 cells/mL versus 2.23 ± 0.07 ×10^6 cells/mL in standard IPT-AFM.

    “The maximum cell density reached 1.95 ± 0.05 Â 10 6 cells/mL versus 2.23 ± 0.07 Â 10 6 cells/mL in IPT-AFM (standard composition).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In CD-U5 medium, maximum cell density was 2.5 ± 0.5 ×10^6 cells/mL and μ around 0.017 h^-1.

    “CD-U5 which is also a chemically defined medium, showed promising results; a maximum cell density of 2.5 ± 0.5 Â 10 6 cells/mL and m around 0.017 h À1 were obtained.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Enrichment of diluted IPT-AFM with cell boost 5 at 5% slightly improved cell growth; highest cell density around 2.3 ± 0.07 ×10^6 cells/mL, comparable to standard IPT-AFM.

    “The enrichment of diluted IPT-AFM with cell boost 5 at 5% slightly improved cell growth; the highest cell density level was around 2.3 ± 0.07 Â 10 6 cells/mL which was comparable to that obtained in the standard IPT-AFM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Enrichment of IPT-AFM with cell boost 5 supplement at 5% did not improve virus titer, which was about 2.4 ×10^7 FFU/mL.

    “The enrichment of IPT-AFM with cell boost 5 supplement at 5% did not improve virus titer which was about 2.4 Â 10 7 FFU/mL.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Virus titer levels reached 1.9, 2, and 2.4 ×10^7 FFU/mL in CD-U5, SFM4CHO, and Hycell CHO media, respectively.

    “Virus titer levels reached 1.9, 2 and 2.4 Â 10 7 FFU/mL in CD-U5, SFM4CHO and Hycell CHO media, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Average cell density of VeroS cells in IPT-AFM was 2.1 ± 0.4 ×10^6 cells/mL; Cd reached 1.53 ± 0.3; specific growth rate was 0.014 ± 0.003 h^-1.

    “An average cell density of 2.1 ± 0.4 Â 10 6 cells/mL was obtained; C d reached 1.53 ± 0.3 and the specific growth rate (m) was 0.014 ± 0.003 h À1 .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The adaptation procedure lasted 60 days, which is shorter than the 120 days reported by Paillet et al. for adapting Vero E6 cells to suspension growth in serum-free home-made medium using stepwise method.

    “The adaptation procedure lasted 60 days which is shorter than the period reported by Paillet et al. [23] who needed 120 days to adapt Vero E6 cells to suspension growth in serum-free home-made medium using stepwise method.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    VeroS cells were able to reach 2.9 × 10^6 cells/mL in IPT-AFM with 20% Ca++, Mg++ 4 days after culture inoculation, although cell density level at inoculation was high (around 10^6 cells/ml).

    “In our hands, VeroS cells were able to reach 2.9 Â 10 6 cells/mL in IPT-AFM with 20% Ca ++ , Mg ++ 4 days after culture inoculation, although cell density level at inoculation was high (around 10 6 cells/ml).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Once adapted to suspension growth, Vero cells no longer adherent, named VeroS cells, were able to grow in suspension as single cells and small aggregates.

    “Once adapted to suspension growth, Vero cells no longer adherent, were named VeroS cells and they were able to grow in suspension as single cells and small aggregates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The use of cell lines adapted to suspension growth contributes to reducing vaccine manufacturing costs and making products more accessible to low- and middle-income countries.

    “the use of cell lines adapted to suspension growth is highly encouraged.This is because suspension culture offers easier scale-up and higher cell densities.Consequently, cheaper products more accessible to Low and Middle Income Countries (LMICs) can be obtained [3,16,17].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [16]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [17]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • Rests on an assumption: There is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Vero cells were the first approved continuous cell line for human viral vaccines production.

    “they were the first approved continuous cell line for human viral vaccines production [36,46].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [36]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [46]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Adaptation of anchorage-dependent cells to suspension cultures solves scale-up limitations associated with monolayer cultures and is considered an attractive tool for industrial applications.

    “Since the adaptation of anchorage-dependent cells to suspension cultures solves scale-up limitations associated with monolayer cultures, it is considered as an attractive tool for industrial applications [17,23].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [17]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [23]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Vero cells have an excellent safety profile and show permissiveness to various viruses.

    “Vero cells have an excellent safety profile and show permissiveness to various viruses.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [36]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [46]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Various cell lines such as MDCK, HEK-293, BHK-21, CHO, and AGE1.CR have been successfully adapted to suspension growth for biopharmaceuticals production.

    “Various cell lines such as MDCK [25,47], HEK-293 [48,49], BHK-21 [21,[50][51][52], CHO [53] and AGE1.CR [27,54] have been successfully adapted to suspension growth for biopharmaceuticals production.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [25]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [47]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [48]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [49]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [53]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Suspension culture offers easier scale-up and higher cell densities.

    “suspension culture offers easier scale-up and higher cell densities.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [16]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [17]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lohr et al. also reported that suspension adapted MDCK (MDCK.SUS2) cells grown in suspension in a chemically defined medium gave similar performances to MDCK cells regarding cell proliferation and influenza virus production.

    “Lohr et al. [47] also reported that suspension adapted MDCK (MDCK.SUS2) cells grown in suspension in a chemically defined medium gave similar performances to MDCK cells regarding cell proliferation and influenza virus production.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    During the adaptation process, a long lag period of between 20 and 30 days was required to reach cell density levels over 10^6 cells/mL even though these cells had been adapted to a SFM in anchorage-dependent cultures.

    “They also demonstrated that during the adaptation process, a long lag period of between 20 and 30 days was required to reach cell density levels over 10 6 cells/mL even though these cells had been adapted to a SFM in anchorage-dependent cultures.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    Vero cells are widely used for rabies vaccine manufacturing.

    “Vero cells are widely used for rabies vaccine manufacturing [36].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Vero cells at low passage have been approved by WHO since 1986 to manufacture different viral vaccines for human use.

    “Vero cells at low passage have been approved by World Health Organization since 1986 to manufacture different viral vaccines for human use [14,15].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [15]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Merten et al. developed MDSS2, a serum free medium sustaining the growth of BHK-21 cells as aggregates (BHK21-BRS).

    “Merten et al. [57] developed MDSS2, a serum free medium sustaining the growth of BHK-21 cells as aggregates (BHK21-BRS)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [57]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    New cell lines growing in suspension like PER.C6, HEK293, AGE1.CR and CAP cells were designed as new cell substrate candidates for biopharmaceuticals manufacturing.

    “new cell lines growing in suspension like PER.C6, HEK293, AGE1.CR and CAP cells were designed as new cell substrate candidates for biopharmaceuticals manufacturing [27].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The resulting virus yields obtained by Van Wielink et al. for eight influenza virus strains (H1N1, H3N8, H5N2, H5N7, H5N9, H7N1, H7N7 and H9N2) were comparable to those obtained in adherent cells.

    “For example, the resulting virus yields obtained by Van Wielink et al. [25] for eight influenza virus strains (H1N1, H3N8, H5N2, H5N7, H5N9, H7N1, H7N7 and H9N2) were comparable to those obtained in adherent cells.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Virus titer can be further enhanced once it is adapted to VeroS cells through several consecutive passages.

    “For instance, virus titer can be further enhanced once it is adapted to VeroS cells through several consecutive passages.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Suspension adapted MDCK cells preserved their susceptibility to influenza strains.

    “It was shown that suspension adapted MDCK cells preserved their susceptibility to influenza strains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Vero cells at low passage (not higher than 150) are widely used for human vaccine manufacturing owing to their safety and permissiveness to different viruses.

    “Vero cells at low passage (not higher than 150) are widely used for human vaccine manufacturing owing to their safety and permissiveness to different viruses [9,13].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [13]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cell culture derived rabies vaccines are considered among the safest and most efficient vaccines for human rabies prevention.

    “Cell culture derived rabies vaccines are considered among the safest and most efficient vaccines for human rabies prevention [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisecitation

    All studies described in the literature performed with various cell lines adapted to suspension growth indicated that cell behaviour is medium dependent.

    “All studies described in the literature performed with various cell lines adapted to suspension growth indicated that cell behaviour is medium dependent.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

    Premisecitation

    The development of safe and cheaper vaccines can significantly reduce the economic burden of rabies vaccination programs worldwide leading to eventual success of rabies control globally.

    “The development of safe and cheaper vaccines can significantly reduce the economic burden of rabies vaccination programs worldwide leading to eventual success of rabies control globally [58].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [58]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    The use of this new cell line combined with IPT-AFM will highly contribute to reduce the vaccine cost.

    “The use of this new cell line combined with IPT-AFM an in-house developed animal-component free medium will highly contribute to reduce the vaccine cost.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: There is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Litwin demonstrated that suspension culture of Vero cell aggregates might be an alternative method for large-scale cultivation for vaccine production.

    “He demonstrated that suspension culture of Vero cell aggregates might be an alternative method for large-scale cultivation for vaccine production.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Generally, cell adaptation to suspension growth results in an enhancement of the virus titer as reported in several studies described in the literature.

    “Generally, cell adaptation to suspension growth results in an enhancement of the virus titer as reported in several studies described in the literature.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Perrin et al. demonstrated that the adaptation of the virus strain to BHK-21 cell aggregates by serial passages had significantly increased the virus titer from 10^5 to 8.5 × 10^8 FFU/mL.

    “This is in line with the data described by Perrin et al. [50] who demonstrated that the adaptation of the virus strain to BHK-21 cell aggregates by serial passages had significantly increased the virus titer from l0 5 to 8.5 Â 10 8 FFU/mL.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Paillet et al. obtained sVero cells growing as individual cells in suspension.

    “They obtained sVero cells growing as individual cells in suspension.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nikolay et al. recently obtained BHK-21 cells grown as individual cells in suspension (BHK-21SUS).

    “Nikolay et al. [51] recently obtained BHK-21 cells grown as individual cells in suspension (BHK-21SUS).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The first work describing Vero cells adaptation to suspension growth was reported in 1992 by Litwin.

    “the first work describing Vero cells adaptation to suspension growth was only reported in 1992 by Litwin [22].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The results pave the way to develop a VeroS cell bioreactor process for rabies vaccine production.

    “pave the way to develop VeroS cells bioreactor process for rabies vaccine production.”
    Quote in the paperSubstantial claim
41 of 41

  • The paper relies on this without support.

    Vero cells are anchorage dependent, which complicates upstream processing and process scale-up.

    “However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up.”

  • The paper relies on this without support.

    There is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide.

    “there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide.”

  • The paper relies on this without support.

    No published work describes the use of Vero cells suspension culture for rabies virus replication.

    “no published work describes the use of Vero cells suspension culture for rabies virus replication.”

  • The paper relies on this without support.

    Only few studies describing the adaptation of Vero cells to suspension growth are published.

    “only few studies describing the adaptation of Vero cells to suspension growth are published.”

  • The paper relies on this without support.

    Calcium is involved in cell to cell adhesion.

    “As calcium is involved in cell to cell adhesion”

  • The paper relies on this without support.

    The new VeroS cell line generated in this study is indeed Vero cells.

    “Nevertheless, the identity of the new VeroS cell line generated in this study should be checked.”

  • The paper relies on this without support.

    VeroS cells are safe for use as a cell substrate for human vaccine manufacturing.

    “The safety of this new cell line should also be characterized according to the international regulatory guidelines before its acceptance as a cell substrate for human vaccine manufacturing.”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Vero cells are widely used in the production of viral vaccines due to their suitability for large-scale culture and sensitivity to various virus strains.

    “Vero cells are widely used in the production of viral vaccines due to their suitability for large-scale culture and sensitivity to various virus strains [15,16].”

    From Evaluation of the Efficacy of the Vaccine Production Process in Removing Residual Host Cell DNA from the Vero Cell Rabies Vaccine (2024) · cites this paper as [16]

    The Introduction says low-passage Vero cells are widely used for human vaccine manufacturing because they are permissive to different viruses. But the Discussion describes scale-up limitations of conventional adherent Vero cultures; suspension adaptation is presented as a way to overcome them. The source therefore does not support unqualified suitability for large-scale culture as a reason Vero cells are widely used. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Reference 43 does not describe IPT-AFM adaptation

    Minor

    The text cites Smith et al. [43] for prior adaptation of Vero cells to IPT-AFM, but reference 43 is a paper on a rabies neutralizing-antibody test.

  • Conflicting CO2 levels for shake-flask cultures

    Minor

    The methods specify 7% CO2 for shake-flask cultures, but the virus-production results say VeroS cells were initially grown in shake flasks at 5% CO2.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. Yasumura, 1963, Study of SV40 in tissue culture, Nippon Rinsho, 21, 1201
  2. World Health Organization, 1987, Requirements for continuous cell lines used for biological substances, WHO Tech Rep Ser, 745, 99
  3. World Health Organization, 1987, Requirements for rabies vaccine (inactivated) for human use produced in continuous cell lines, WHO Tech Rep Ser, 760, 167
  4. Owens, 1953, A new method for the cultivation of mammalian cells suspended in agitated fluid medium, Proc Am Assn Cancer Res, 1, 41

Cited by

Sorted by citations

Related

Authors
Rourou, S., Ben Zakkour, M., & Kallel, H.
Affiliations
Institut Pasteur de Tunis (TN)
Corresponding author
Kallel, Héla
ORCID
Venue
Vaccine
Publisher
Elsevier BV
Published
2019-11
Volume
37
Issue
47
Pages
6987-6995
ISSN
0264-410X (ISSN-L)
DOI
10.1016/j.vaccine.2019.05.092
OpenAlex
W2950344390
Semantic Scholar
10017c762ba91c61a62d6085fcdc8ef912673580
Cited by
53
References
56
Citations by year
2026: 7 · 2025: 9 · 2024: 12 · 2023: 5 · 2022: 9 · 2021: 6 · 2020: 2 · 2019: 3
FWCI
1.64
Type
journal article
Language
en
Open access
Licence
Locations
  • Vaccine (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by-nc-nd)
Funders
Ministère de l’Enseignement Supérieur et de la Recherche Scientifique
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Immunology and Microbiology › Virology › Rabies epidemiology and control
  • Physical Sciences › Engineering › Biomedical Engineering › Fluid Dynamics and Mixing
Keywords
Vero cells, suspension culture, serum-free medium, chemically defined medium, cellular adaptation, cell aggregation, mechanical dissociation, microcarriers, vaccine production
MeSH terms
Adaptation, Physiological, Animals, Cell Count, Cell Line, Chlorocebus aethiops, Culture Media, Rabies, Rabies Vaccines, Rabies virus, Vero Cells, Virus Cultivation, Culture Media, Serum-Free, Cell Culture Techniques, Bioreactors, Viral Load

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-28.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review