Vero Cells - From Viral Research to Vaccine Development

Vero cells constitute an immortalized cell line derived from the kidney epithelial cells of an African green monkey. This cell line is commonly applied in virology, microbiology, and cell and molecular biology research. It is used for vaccine production, drug screening purposes, and to study viruses and parasite infection, tumour immunology, and immunotherapy.

This article consists of almost all the crucial information about Vero cells you need to know for easy and efficient working with them. Specifically, it will cover:

Origin and general characteristics of the Vero cells
Culturing information
Advantages & Limitations of Vero cells
Applications of Vero cell line in research
Research publications
Resources for Vero cell line: Protocols, Videos, and More

1. Origin and general characteristics of the Vero cells

When we consider working with a cell line, many questions arise, such as: What are Vero cells? How was Vero cell line developed? Why are Vero cells called Vero? What is Vero cell meaning? This section of the article will help you learn about the origin and general characteristics of the Vero cells.

The Vero cell line was developed from the kidney epithelial cells of an African green monkey in 1962. It was established by two scientists, Y. Kawakita and Yasumura, at Chiba University in Japan [1]. The word “Vero” came from Verda reno, which denotes “green kidney” in the Esperanto language, whereas Vero originally means “truth”.
Vero cells grow as monolayers and represent an epithelial-like morphology. They are round and elongated in shape.
The approximate size of Vero cells is 17 µm in diameter.
The Vero cell line possesses a hypodiploid chromosome count. The modal chromosome number is 58, which occurs in almost 66% of the cell population. Higher ploidies may also exist in 1.7% of cells.

Clones of Vero cells

Many different Vero cell clones possess distinguishing attributes compared to the parent Vero cell line. Two well-known Vero cell clones are mentioned here.

Vero e6 cell line: Vero e6, also known as Vero C1008, is a clone of Vero 76 cells. It was cloned in 1979 by P.J. Price using a dilution method into microtiter plates. Vero E6 cells are appropriate for propagating slowly replicating viruses.
Vero 76 cells: Vero 76 cells were obtained from the kidney of a patient in 1968. These cells also exhibit an epithelial morphology.

Human kidney tissue under the microscope.

2. Culturing information

Vero cell culture is easily maintainable. However, to properly handle this Vero monkey cell line, you must know the following culturing information, i.e., doubling time, seeding density, and growth medium.

Key Points for Culturing Vero Cells

Population Doubling Time:	Vero cells have a population doubling time of approximately 24 hours.
Adherent or in Suspension:	Vero monkey cells are adherent and form monolayers.
Seeding Density:	1 x 10⁴ cells/cm² seeding density is recommended for Vero cell culture. Adherent Vero cells are washed with PBS and incubated with Accutase solution for detachment. Dissociated cells were added to fresh medium and centrifuged. Recovered cells were resuspended and dispensed into new flasks for culturing.
Growth Medium:	Ham’s F12 or DMEM medium can be used to propagate Vero cells. These media are usually added with 2.5 mM L-glutamine and 5% fetal bovine serum (FBS) for ideal cell growth. Vero cell media should be replaced 2 to 3 times per week.
Growth Conditions:	Vero cells grow at 37°C temperature, which is maintained in a humidified incubator with a 5% CO2 supply.
Storage:	Vero cells are stored below -150°C in an ultra-low temperature freezer or vapour phase of liquid nitrogen.
Freezing Process and Medium:	CM-1 or CM-ACF freezing media is recommended for freezing Vero cells using a slow freezing process (gradual 1°C drop in temperature).
Thawing Process:	Frozen Vero cells are thawed in a 37°C water bath by rapid agitation for 40 to 60 seconds. Afterwards, cells are added to fresh medium and centrifuged to eliminate freezing media components. Recovered cells are resuspended and added into a flask for growth.
Biosafety Level:	Vero cell cultures are maintained in a biosafety level 1 laboratory.

Vero cells two and one days after subculturing (10x magnification).

4. Applications of Vero cell line in research

The Vero cell line has numerous research applications in cell biology and virology field. Here, we have discussed some specific ones.

Vaccine Production: Vero cell line is a widely used continuous cell line for vaccine manufacturing. Several studies have been conducted to produce human vaccines in Vero cells. A study published in 2019 used Vero cells to make the yellow fever virus to manufacture inactivated viral vaccine [2].
Drug testing: Vero cells are used to evaluate the efficacy and safety of drugs. Many studies have taken Vero as a normal kidney cell line to explore the cytotoxicity of drugs or other therapeutic agents, such as research examined and compared the cytotoxic effect of Terminalia avicennioides plant root extracts in a cancer cell line HepG2 and monkey kidney epithelial Vero cells. The study outcomes showed that extracts were more toxic to cancer cells than normal cells [3].
Virology research: Vero cells are suitable for virus production. They are commonly used in viral infection studies, such as a study conducted in 2020 infected Vero cells with different isolates of the SARS-CoV-2 virus to study the growth properties of the virus [4]. Similarly, another research investigated cell responses to SARS-CoV-2 infection using Vero cell culture [5].

5. Research publications

The following are some recent and most cited research publications featuring Vero cells.

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

This study published in the Vaccine Journal (2019) adapted Vero cells to grow in suspension cultures to produce rabies virus at high titer using various serum-free media.

Susceptibility of Toxoplasma gondii to Ethanolic Extract of Tinospora crispa in Vero Cells

This paper was published in the Evidence-Based Complementary and Alternative Medicine journal in 2019. This study proposed that ethanolic extract of the plant Tinospora crispa exerts a detrimental effect on the Toxoplasma gondii parasite. However, it is safe to host cells (Vero cell line).

Effects of Colchicum baytopiorum leaf extract on cytotoxicity and cell death pathways in C-4 I and Vero cell lines

This article was published by the Journal of the Balkan Union of Oncology in 2021. In this research, Ozlem Dagdeviren Ozsoylemez and Gul Ozcan explored the cytotoxic effect of Colchicum baytopiorum leaf extract on C-4I and Vero cell lines.

Resveratrol inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in cultured Vero cells

This study in the Phytotherapy Research (2021) used SARS-CoV-2 infected Vero cells to investigate the therapeutic effect of Resveratrol on virus replication.

Lipophilic statins inhibit Zika virus production in Vero cells

This paper in Nature scientific reports (2019) proposed that lipophilic statins, i.e., cerivastatin, lovastatin, fuvastatin, simvastatin, and mevastatin, can impede the production of Zika virus in Vero cells.

6. Resources for Vero cell line: Protocols, Videos, and More

Vero is a famous cell line. The available resources featuring Vero cell culturing and transfection protocols are mentioned here.

Transfection of Vero cells: This link will guide you about the transfection protocol of the adherent Vero monkey kidney cell line.
Transfecting Vero cell line: This video shows the transfection protocol for the Vero cell line easily and effectively.
Subculturing a Cell line: This video describes a general subculturing method for adherent cell lines.

Cell culture protocols

Here are some resources describing cell culture protocol for Vero cells.

Culturing of Vero cells: This website contains a well-explained protocol for culturing Vero cells.
Vero cell Culture: This document may help you learn the protocol for propagating, maintaining, and freezing Vero cells.

References

Ammerman, N.C., M. Beier‐Sexton, and A.F. Azad, Growth and maintenance of Vero cell lines. Current protocols in microbiology, 2008. 11(1): p. A. 4E. 1-A. 4E. 7.
Pato, T.P., et al., Purification of yellow fever virus produced in Vero cells for inactivated vaccine manufacture. Vaccine, 2019. 37(24): p. 3214-3220.
Aliyu-Amoo, H., et al., Antiproliferative effect of extracts and fractions of the root of Terminalia avicennioides (Combretaceae) Guill and Perr. on HepG2 and Vero cell lines. Clinical Phytoscience, 2021. 7(1): p. 1-7.
Yao, P., et al., Isolation and growth characteristics of SARS-CoV-2 in Vero cell. Virologica Sinica, 2020. 35(3): p. 348-350.
Park, B.K., et al., Differential signaling and virus production in Calu-3 cells and Vero cells upon SARS-CoV-2 infection. Biomolecules & Therapeutics, 2021. 29(3): p. 273.