• Applications & Products
    • Applications
    • Products
  • Media Formulation
  • About InVitria
  • News & Blog
  • Contact Us
  • Terms
  • Privacy
  • Sitemap
Performance. Defined.
  • 1-800-916-8311 or Email Us
  • iconMy Account
  • iconView Cart
  • Applications & Products
    • Applications
      • Gene Therapy
      • Cell Therapy
      • T-Cell Immunotherapy
      • Vaccine Production
      • Antibody Production
      • Final Formulation
      • Food Science
    • Product Types
      • Blood-Free Cell Culture Media
      • Cell Culture Components
      • Insulin-Transferrin Supplements
      • Recombinant Albumin
      • Recombinant Transferrin
      • Recombinant Human Lysozyme
      • Recombinant Human Interleukins
    • Products
      • Cellastim S® – Recombinant human albumin
      • Exbumin® – Recombinant human albumin excipient
      • Optibumin® 20 – Recombinant human albumin
      • Optibumin® 25 – Recombinant human albumin
      • Optiferrin® – Recombinant human transferrin
      • Lacromin® – Recombinant human holo lactoferrin
      • ITS AF – Blood-free cell culture media supplement
      • ITSE + A – Blood-free cell culture media supplement
      • ITSE Animal-Free® – Blood-free cell culture media supplement
      • Lysobac® – Recombinant human lysozyme
      • OptiLeukin 2 – Recombinant human IL-2
      • LIF – Leukemia Inhibitory Factor
      • OptiVERO® – Chemically-defined complete VERO media
  • About InVitria
  • News & Blog
  • Contact Us
  • Knowledge Base
  • Applications
  • Products
  • Contact Us
  • Home
  • OptiVERO – Virus Production Using VERO Cells in Flasks without Serum in Chemically Defined Cell Culture Media Application Note

OptiVERO – Virus Production Using VERO Cells in Flasks without Serum in Chemically Defined Cell Culture Media Application Note

Published on 15 October 2020

Atherly Pennybaker, Media Formulation and Product Applications Specialist, InVitria

Workflow Summary

Combine kit supplements of OptiVERO. Prepare complementary reagents. Wash cells to remove traces of FBS and harvest from growth surface. Seed in OptiVERO and grow to confluence. Add desired virus to be expanded in minimal basal EMEM. Incubate for 2 hours at 37°C. Add fresh OptiVERO. Allow virus expansion for 3 to 13 days, depending on virus strain and desired viral load. Quantify virus by desired method.

Introduction

VERO cells are one of the most common continuous cell lines used in laboratory and industrial settings. While they have many important applications in research, one of the most critical is as a preferred cellular substrate for virus production [1]. VERO cells are traditionally grown in 10% fetal bovine serum (FBS) and exhibit commercially acceptable doubling times and viability.  However, current guidance by regulatory bodies in addition to Good Manufacturing Practice (GMP) compliant manufacturing of cell-based, advanced therapy medicinal products (ATMPs) states that alternatives to FBS should be utilized due to poor reproducibility and safety concerns associated with FBS use [2,3]. Popular serum-free media (SFM), such as VP-SFM, contain high levels of hydrolysates that can cause batch-to-batch variability. OptiVERO was designed to eliminate these concerns and is a chemically defined and blood-free, serum-free media. Here, we compare VERO cell growth and virus production in OptiVERO vs FBS-containing media in 2D cell culture flasks.

Materials Needed

Growth Media Preparation

  • OptiVERO Components: Base Media, 50X Protein Supplement
  • Gentamicin/amphotericin (Life Technologies R01510)
  • L-Glutamine

Cell Passage

  • 75 cm2 tissue culture flask (Falcon 353136)
  • 150 cm2 tissue culture flask (Falcon 355001)
  • Soybean trypsin inhibitor (Life Technologies 17075-029)
  • TrypLE (Life Technologies A1217701)
  • 100 mM EDTA (Sigma Aldrich E6758)
  • Dulbecco’s phosphate buffered solution (VWR VWRL0119-1000)
  • 50 mL conical tubes (VWR 76211-286)

Optional

  • VP-SFM (Thermo Fisher 11681020)
  • Minimum essential medium (MEM) with Earle’s salts and L-glutamine (Corning 10-010-CV)
  • Fetal Bovine Serum (FBS)

Equipment

  • 5% CO2 incubator at 37°C, saturating relative humidity
  • Class A2 biological safety counter
  • Centrifuge
  • Cell counter or hemocytometer

Protocol:

Growth Media Preparation

  1. To generate 1 liter of complete OptiVERO, thaw one OptiVERO Protein Supplement (20 mL) in a 37°C water bath. Do not subject the Protein Supplement to multiple freeze-thaw cycles.
  2. Once the supplement is completely thawed, gently stir each supplement by pipetting up and down. Do not vortex.
  3. Add 20 mL from the OptiVERO Protein Supplement directly to the OptiVERO Base.
  4. Add 20 mL of OptiVERO Protein Supplement directly to the OptiVERO Base Media.
    • This medium requires the addition of L-Glutamine to a final concentration of 4 mM. If desired, add gentamicin/amphotericin to 0.1-0.5x final. Do not add to a 1x final concentration.
    • Avoid repetitive heating and cooling of the complete medium by withdrawing and pre-warming only the volume needed in the final culture vessel to be used.

Stock-Reagent Preparation

50x Soybean trypsin inhibitor

  1. To a 1 g container of soybean trypsin inhibitor, add 8 mL of DPBS and allow the powder to reconstitute at room temperature for at least 3 hours prior to use. Avoid the excessive formation of bubbles during reconstitution by allowing the vial to sit undisturbed.
  2. Upon successful reconstitution, pipette the solution up and down with a 10 mL pipette to achieve adequate mixing.
  3. Pass the solution through a 0.22 µm PES syringe filter into a sterile 15 mL tube.
  4. Aliquot solution into (12) 650 µL aliquots in 1.5 sterile tubes and store at -20°

100mM EDTA

  1. Weigh 1.86 g of dry EDTA on an analytical balance and bring the solution up to 50 mL with DPBS.
  2. Once the EDTA has dissolved into solution, sterile-filter the solution using a 0.22 µm PES syringe filter into a sterile 50 mL tube.
  3. Store the solution at 4°C for up to 6 months.

1x TrypLE + 1 mM EDTA

  1. To a sterile 250 mL media bottle, aseptically add 222.5 mL of DPBS followed by 25 mL of 10x TrypLE and 2.5 mL of 100 mM EDTA.
  2. Store the solution at 4°C for up to 6 months. This solution may be heated and cooled repeatedly without affecting the activity of the enzyme.

Working Reagent Preparation

  1. Dilute 50X soybean trypsin inhibitor to 1x in dPBS (0.05 mL/cm2 volume needed).

Culture Adaptation to Serum-Free Medium

 For additional information about VERO cell growth and maintenance in OptiVERO, please refer to the Application Note: “Expansion of VERO Cells Without Serum in Chemically Defined Cell Culture Media”  

  1. Aliquot 0.2 mL/cm2 OptiVERO into the desired culture vessel and allow the media to equilibrate in the incubator for at least 30 minutes. Prewarm the diluted soybean trypsin inhibitor and 1x TrypLE in a 37°C water bath.
  2. Wash VERO cells with 0.5 mL/cm2 dPBS twice to remove all traces of serum.
  3. Remove cells from the growth surface using 0.05 mL/cm2 TrypLE/EDTA (NOTE: do not expose cells to the TrypLE/EDTA for longer than 6 minutes)
  4. Tap the side of the vessel to ensure all cells have been removed from the growth surface.
  5. After cell detachment is observed, add an equal volume of soybean trypsin inhibitor followed by 0.2 mL/cm2 DPBS to dilute out the enzyme. Collect the cells in a 50mL conical tube.
  6. Wash the surface and collect any remaining cells with 5-10 mL prewarmed media.
  7. Pellet by centrifugation at 1000 RPM for 5 minutes.
  8. Resuspend cells in a minimal volume of prewarmed OptiVERO and count.
  9. Seed cells at an initial cell density of 10,000-20,000 cells/cm2. VERO cells should be sub-cultured every 3 to 5 days.
  10. At time of subculture, repeat the procedure above.

Virus Production in Tissue Culture Flasks

  1. Prior to infection studies, VERO cells should be allowed to adapt to OptiVERO for at least 3 passages. VERO cells should be grown to confluence in the desired flatware.
  2. For viral inoculation, 75% of the growth media should first be removed
  3. The virus to be expanded should be inoculated at 0.001 – 0.01 PFU/cell in basal medium (0.06 mL/cm2).
  4. To ensure uniform infection over the entire monolayer, VERO cells should be incubated at 37°C for 90 minutes after inoculation, with gentle rocking of the flask every 15 minutes.
  5. Add 0.7 mL/cm2 of fresh, prewarmed OptiVERO to each flask
  6. Specific protocols for virus propagation are dependent on virus type. For the attenuated Dengue strain used here, for example, culture glucose (2 g/L) and neutral pH were maintained by the addition of 45% glucose and 7.5% sodium bicarbonate solutions, respectively.
  7. Samples should be removed daily for virus quantification

Results and Discussion

Here, a blood- and serum-free, chemically defined virus-production media known as OptiVERO is evaluated for its ability to support VERO virus production, compared to media supplemented with FBS and to VP-SFM.

 

OptiVEROAppNoteGraph_DengueVirusProduction copy
Figure 1 Dengue virus production in Vero cells cultured in 10% FBS, OptiVERO, and VP-SFM

 

An attenuated Dengue strain was used as a model virus to demonstrate the virus production capabilities of each medium (Figure 1). Dengue was propagated for 12 days, with samples removed for plaque titration every other day. The Dengue virus growth plateaued at day8 and was determined to be 7.25E+7, 7.5E+6, 6.00E+7 PFU/mL for EMEM + 10% FBS, VP-SFM + 4 mM Glutamine, and OptiVERO, respectively. These data show that VERO cells grown in OptiVERO showed virus production capabilities similar to those grown in 10% FBS and exhibited superior virus production compared to hydrolysate-containing medium, VP-SFM.

View PDF Here

Footnotes

  1. Ammerman NC, Beier-Sexton M, Azad AF. Growth and maintenance of Vero cell lines. Curr Protoc Microbiol 2008;Appendix 4:Appendix 4E.
  2. Note for guidance on minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products (EMA/410/01 rev.3). Official Journal of the European Union 2011;C73:1-18.
  3. van der Valk J, Bieback K, Buta C et al. Fetal Bovine Serum (FBS): Past – Present – Future. ALTEX 2018;35:99-118.
Start New Search

Quick Search Links

Application:

Antibody Production

Cell Therapy

Final Formulation

Food Science

Gene Therapy

T-Cell Immunotherapy

Vaccine Production

Product Category:

Cell Culture Supplements

Albumin

Cell Culture Media

Interleukins

Lactoferrin

LIF

Lysozyme

Single Components

Transferrin

Product:

Exbumin® – Recombinant human albumin excipient

ITS AF™ – Blood-free cell culture media supplement

ITSE + A™ – Blood-free cell culture media supplement

ITSE Animal-Free® – Blood-free cell culture media supplement

Lacromin® – Recombinant human holo-lactoferrin

OptiLeukin™ 2 – Recombinant human interleukin-2

OptiVERO®

Cellastim® S – Recombinant human albumin

Optibumin ® 20 – Recombinant human albumin

Optibumin ® 25 – Recombinant human albumin

Optiferrin® – Recombinant human transferrin

Leukemia Inhibitory Factor

Lysobac® – Recombinant human lysozyme

Document Type:

Application Notes

Blog

Catalog

Data Sheets

Interviews

Journal Articles (DOI Links)

News

Posters

Product Brochures

Product Specifications

SDS

Use Guidelines

Webinar

White Papers

Cell Line:

B Lymphocyte

CHO

ESC

HEK293

Hematopoetic Stem Cells

Hybridoma

iPSC

Mesenchymal Stem Cells

Natural Killer Cells

Neural Stem Cells

T Lymphocyte

VERO

  • 2718 Industrial Drive • Junction City, KS 66441
  • 800-916-8311
  • © 2025 InVitria

    • Terms
    • Privacy
    • Sitemap