Elsevier

Biologicals

Volume 41, Issue 6, November 2013, Pages 407-414
Biologicals

Detection of contaminants in cell cultures, sera and trypsin

https://doi.org/10.1016/j.biologicals.2013.08.005Get rights and content

Abstract

The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials.

Introduction

Contamination of cell cultures is not easily eliminated and causes delays, financial loss and requires effort to detect and eliminate such contamination [1]. The discovery of contaminants can place past or current results in question and cause cell cultures to be lost [2]. Scientific credibility may become threatened by reports of contamination, which is capable of invalidating results and devaluing products and medicines [3]. Moreover, vaccines produced in infected cell cultures can lead to seroconversion or disease in vaccinated humans and animals [4], [5].

Vaccines against classical swine fever virus, infectious bovine rhinotracheitis, bovine respiratory syncytial virus infected with the bovine viral diarrhea virus (BVDV) have been responsible for infection in pigs vaccinated or by the origin of disease outbreaks in cattle [6]. There are reports of vaccines against Aujeszky's disease virus produced in cells from sheep infected with Border disease virus, causing infection and consequently producing antibodies that are cross-reactive for Classical Swine Fever Virus (CSFV), generating false-positive results in serological diagnosis of classical swine fever [7].

Classical Swine Fever (CSF) in Brazil presents controlled without vaccination. According to the World Organization for Animal Health (OIE), the final report of this disease was in 2009. The BVDV is one of differential virus in the diagnosis of CSF, which assigns high importance to it. In this sense, it is of great importance to use of cell cultures and fetal bovine serum free of contaminants, mainly for BVDV, thus avoiding false-positive results in the diagnosis of diseases of major impact as the CSF.

Recently, the porcine circovirus type 1 (PCV1) was detected in a commercial vaccine against human rotavirus and it was confirmed that it was present in the cell stock used for vaccine production [8]. The case of vaccine contamination with PCV1 reinforces the need for continued efforts to reduce the likelihood of introducing viruses of animal origin in materials used in the manufacture of biological products [9].

Cell culture contamination is caused by biological and chemical agents. The biological contaminants most commonly found in cells are mycoplasma, viruses, bacteria and yeasts. In most cases, contamination can occur without the knowledge of the analyst [2], [10]. Contamination with mycoplasma and viruses may go unnoticed, causing changes in infected cells, such as a reduction in growth rate, as well as alterations in cell morphology, chromosomes and metabolism of amino acids and nucleic acids [11].

Mycoplasma and viruses can originate from contaminated cultures or supplies, such as serum and trypsin that are commonly used in the maintenance of cell cultures [12] or from inadequate handling procedures [13].

Methodologies used to monitor cell culture contamination include the PCR, virus culture and measuring the reactivity of antibodies against specific agents [5], [10].

Because many infectious agents are not easily cultivable [5] PCR has been shown to be an efficient methodology for the detection of biological contaminants in cell culture and its supplies. This technique has attracted much attention in the detection of cell culture contaminants because it is fast, robust, highly sensitive and specific compared to traditional techniques [14], [15], [16].

The importance of working with the detection of contaminants has been demonstrated by studies in Brazil [17], [18], [19], [20], [21], however, the laboratorial practice of cell contaminants is not carried out regularly.

Noting the relevance of the effects caused by cell culture contaminants in virological diagnosis and vaccine production, and the fact that PCR may be a useful tool in the detection of these contaminants, the present study aimed to develop methods for routine use to detect mycoplasma, PCV1, BVDV and BLV and evaluate the occurrence of these agents in cell culture, trypsin and bovine serum samples of government laboratories and, research and teaching institutions.

Section snippets

Cell cultures, sera and trypsin

We analyzed 88 cell culture samples from different species (cattle, pig, monkey, hamster, rat, mouse, rabbit, cat, sheep, canine, human, equine and insect) belonging to 32 cell cultures in suspension. The samples were obtained from eight laboratories, five of which were government laboratories (coded GL1 to GL5) where they were used in the diagnosis of viral diseases that affect animals and also for evaluation of vaccines and three of which were research and teaching institutions which were

Analytical specificity

The PCR protocol to detect mycoplasma amplified the DNA from M. bovigenitalium, M. hyopneumoniae and M. mycoides subspecies mycoides small colony. PCR for PCV1 did not amplify PCV2 DNA. The RT-PCR for BVDV amplified both strains of BVDV1 (NADL and W2 strains) but did not detect CSFV RNA. There was no amplification of bovine or swine DNA or RNA by any PCR used to detect the contaminants.

Comparison of the sequences of amplified products from the positive controls to others available in GenBank

Discussion

The PCR protocols used in this study were specific to the targets of interest. Furthermore, when assessing the specificity of PCR for BVDV, special attention was given to the use of RNA from another pestivirus (CSFV) due to the great importance of differential diagnosis between viruses of this genus.

Standard PCR protocols for the detection of cellular contaminants had high sensitivity and were considered specific based on sequence analysis of the amplified products. The PCRs for the two

Acknowledgments

M.B.H. is indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for Fellowships received. Research was supported by CNPq, Fapemig and Pro-Reitoria de Pesquisa da Universidade Federal de Minas Gerais.

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