Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture

Technical limitations and evolution of therapeuticapplications for cell culture-derived products haveaccelerated elimination of animal-derived constituentsto minimize inadvertent introduction of adventitiousviral or prion agents. Practical considerationsdemand adequate emphasis both on design of theserum-free/protein-free culture environment and onnutrient media manufacturing process controls. Protein components may be acceptable, given adequateattention to synthetic process, sourcing (e.g.,geographic location and endemicity, species andtissue/organ) and validated treatment method. Variousoptions exist for re-engineering of traditionalserum-free formulations (containing insulin,transferrin and other protein factors) withnon-protein substitutes. Caution must also beexercised with sourcing of non-protein additives,particularly amino acids and lipids, to avoidintroducing adventitious contaminants. Simpleguidelines facilitate adaptation, cryopreservation andrecovery of many cell types within a protein-freeculture environment. Scrupulous maintenance offacility and equipment and monitoring of processwater, air handling systems and technical personnelare required to ensure that approved raw materials arecorrectly formulated and dispensed. Validatedsanitization processes provide additional assuranceagainst cross-contamination from previous batches ina multi-use facility.     

Detection of contaminants in cell cultures,sera and trypsin

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.     

Prevention of mycoplasma contamination in leukemia-lymphoma cell lines.

The contamination of cell lines with mycoplasmas is certainly one of the major problems occurring in cultured cells. Analyzing more than 460 human leukemia-lymphoma (LL) cell lines, we found that 28% of the cultures were mycoplasma-positive. Mycoplasmas can produce extensive changes, growth arrest and cell death in the infected cultures. While mycoplasma-infected cell lines can be truly cleansed from the contaminants, all the efforts would be in vain when the cells return to a mycoplasma-infested environment or are handled with unsuitable culture practices. Hence, the main focus of mycoplasma control should be on preventing cell culture contamination. Mycoplasmas can be introduced through several routes including culture reagents and laboratory personnel. Cross-contamination from infected cell cultures within one laboratory continues to be the major source for the spread of mycoplasma. Specific technical protocols and cell culturing guidelines may be followed in order to minimize the risk of mycoplasma contamination of cell lines. This "good culture practice" is of utmost importance as faulty cell culture techniques appear to be also the main reason for the high incidence of cross-contaminated LL cell lines which according to our experience using DNA fingerprinting of some 500 LL cell lines is about 15%.     

Torque teno sus virus (TTSuV) in cell cultures and trypsin

Torque teno sus virus (TTSuV), a member of the family Anelloviridae, is a single-stranded, circular DNA virus, widely distributed in swine populations. Presently, two TTSuV genogroups are recognized: Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). TTSuV genomes have been found in commercial vaccines for swine, enzyme preparations and other drugs containing components of porcine origin. However, no studies have been made looking for TTSuV in cell cultures. In the present study, a search for TTSuV genomes was carried out in cell culture lineages, in sera used as supplement for cell culture media as well as in trypsin used for cell disaggregation. DNA obtained from twenty-five cell lineages (ten from cultures in routine multiplication and fifteen from frozen ampoules), nine samples of sera used in cell culture media and five batches of trypsin were examined for the presence of TTSuV DNA. Fifteen cell lineages, originated from thirteen different species contained amplifiable TTSuV genomes, including an ampoule with a cell lineage frozen in 1985. Three cell lineages of swine origin were co-infected with both TTSuV1 and TTSuV2. One batch of trypsin contained two distinct TTSuV1 plus one TTSuV2 genome, suggesting that this might have been the source of contamination, as supported by phylogenetic analyses of sequenced amplicons. Samples of fetal bovine and calf sera used in cell culture media did not contain amplifiable TTSuV DNA. This is the first report on the presence of TTSuV as contaminants in cell lineages. In addition, detection of the viral genome in an ampoule frozen in 1985 provides evidence that TTSuV contamination is not a recent event. These findings highlight the risks of TTSuV contamination in cell cultures, what may be source for contamination of biological products or compromise results of studies involving in vitro multiplied cells.     

Best practices for media selection for mammalian cells

Cell culture medium is a complex mixture of nutrients and growth factors that, along with the physical environment, can either help or destroy your experiment or production run. Nutritional requirements differ with different cell types and functions, as do optimal pH and osmolality. As cell growth proceeds, different cells will utilize amino acids and other components at different rates. By controlling for ammonia, free radicals, heavy metal toxicity, pH shifts, fluctuations in osmolality, nutrient depletion, and chemical and biological contaminants, you will optimize the chances of success. The contribution of each component of the medium is essential for the maintenance of the cell type of interest. While some cell types, such as established human cancer cell lines, may be quite able to tolerate a range of media and supplements, many normal cells and stems cells are not. Optimization of each component may be required to successfully maintain the latter cell types. The procedures for selecting and optimizing cell culture media and supplements are presented.     

Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements

The establishment of cell lines from marine invertebrates has been encountered with obstacles. Contrary to insects and arachnids where the development of a variety of cell lines has become routine, there is no single established cell line from marine invertebrates. This review examines the activity in the field of marine invertebrate cell cultures within the last decade (1988–1998). During this period, attempts (90 peer reviewed studies in addition to many other abstracts, chapters in books, symposia presentations and reports) were limited to a few species within only six phyla (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, Urochordata; in addition to freshwater/terrestrial annelids and platyhelminths). These studies which are summarized here, on one hand indicated ubiquitous problems and on the other, unique characterizations to each phylum studied. Only one-third of the studies revealed cultures of 1 month or longer but most of these were long-term cultures found or suspiciously considered to be contaminated by other unicellular eukaryotic organisms, mainly by thraustochytrids. Three unique approaches/obstacles for marine invertebrate cell cultures (source of cell, cryopreservation and eukaryotic contaminants) are further discussed. The overall impact of recent improvements and developed protocols raises the suggestion for testing different, novel routes in the establishment of cell cultures from marine invertebrates.     

Elimination of Mycoplasma Contaminants from Virus Stocks by Treatment with Nonionic Detergents

Five nonionic detergents (Tweens 20, 40, 60, and 80, and Triton WR-1339) were tested for their ability to inactivate four Mycoplasma species which are common contaminants of animal cell cultures. Tween 20 was found to be the most effective, in that a concentration of 2.5 mg/ml completely inactivated cultures of M. hominis, M. hyorhinis, and Acholeplasma laidlawii within 1 hr and a culture of M. orale within 3 hr. The other detergents exhibited various degree of activity against the different mycoplasmas, with Triton WR-1339 being the least effective. The virucidal activity of the detergents was determined for six viruses. All four Tween compounds were highly virucidal for herpes simplex virus. Tween 20 also exhibited virucidal effects against vesicular stomatitis virus, California encephalitis virus, and Newcastle disease virus, and Tween 80 was found to be active against California encephalitis and Newcastle disease viruses. Detergent treatment procedures were effective in two instances in eliminating mycoplasma contaminants from virus preparations while the preparations retained most of the viral infectivity. The limitations of this technique for routine use are discussed.     

Effect of lectins and sugars on primary sperm attachment in the horseshoe crab, Limulus polyphemus L

The in vitro differentiation of quail neural crest cells into serotoninergic neurons is reported. Serotoninergic neurons were identified by two independent methods, formaldehyde-induced histofluorescence and indirect staining with antiserotonin antibodies. Serotonin-positive cells first appeared on the third day in culture, simultaneously, or slightly prior to the first pigmented cells and adrenergic neurons. Comparable numbers of serotoninergic cells were found in crest cell cultures derived from vagal, thoracic/upper lumbar, and lumbosacral levels of the neuraxis. The neural crest origin of the serotonin neurons was further corroborated by the demonstration that cultures of somites, notochords, and neural tubes (three tissues adjacent to the neural crest and thus the most likely contaminants of crest cell cultures) did not contain serotonin-producing cells, and that mast cells were absent in crest cell cultures. The identification of serotoninergic neurons in quail neural crest cell cultures makes an important addition to the number of neural crest derivatives that are capable of differentiating in culture. Furthermore, it suggests that the in vitro culture system will prove a valid approach to the elucidation of the cellular and molecular mechanisms that govern neural crest cell differentiation.     

Compraison of mycoplasmas associated with human tumors, leukemia, and tissue cultures

Leach, R. H. (Wellcome Research Laboratories, Beckenham, Kent, England), and M. Butler. Comparison of mycoplasmas associated with human tumors, leukemia, and tissue cultures. J. Bacteriol. 91:934-941. 1966.-Mycoplasmas originally isolated by various workers from tissue cultures prepared from or inoculated with tumor or leukemic cells fell into four groups; each related to existing species or serotypes. These were Mycoplasma pulmonis, M. fermentans, M. hominis, and the GDL serotype, the last two being well known as contaminants of uninoculated cell lines. All the test strains were able to grow well in certain tissue cultures, and some caused cytopathic effects and acidity. These observations are discussed in terms of the relationship of these strains to the malignant tissues with which they were originally associated. The variable results obtained in certain biological tests on these strains emphasized the need for standardization of the conditions under which such tests may be employed to assist in identification of Mycoplasma species.     

Synchronous protein cycling in batch cultures of the yeast Saccharomyces cerevisiae at log growth phase

The assumption that cells are temporally organized systems, i.e. showing relevant dynamics of their state variables such as gene expression or protein and metabolite concentration, while tacitly given for granted at the molecular level, is not explicitly taken into account when interpreting biological experimental data. This conundrum stems from the (undemonstrated) assumption that a cell culture, the actual object of biological experimentation, is a population of billions of independent oscillators (cells) randomly experiencing different phases of their cycles and thus not producing relevant coordinated dynamics at the population level. Moreover the fact of considering reproductive cycle as by far the most important cyclic process in a cell resulted in lower attention given to other rhythmic processes. Here we demonstrate that growing yeast cells show a very repeatable and robust cyclic variation of the concentration of proteins with different cellular functions. We also report experimental evidence that the mechanism governing this basic oscillator and the cellular entrainment is resistant to external chemical constraints. Finally, cell growth is accompanied by cyclic dynamics of medium pH. These cycles are observed in batch cultures, different from the usual continuous cultures in which yeast metabolic cycles are known to occur, and suggest the existence of basic, spontaneous, collective and synchronous behaviors of the cell population as a whole.     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

Best practices for the use and evaluation of animal serum as a component of cell culture medium

Animal serum is a common additive for cell culture medium and is often required at 5 to 10% (v/v) for the attachment and growth of primary and continuous anchorage-dependent (monolayer) cultures. The use of animal serum in cell culture medium confers several advantages and also some risks. This article discusses the use of animal serum as a component of cell culture medium. The best practices associated with the sourcing, storage, thawing, testing, and mitigation of risk associated with the use of animal sera are among the topics described in this article.     

Comparative evaluation of different preservation methods for cyanobacterial strains

Maintaining pure cultures using preservation methods is of high importance for biotechnological purposes. However, preservation does not necessarily guarantee the genetic stability of these cultures. Therefore, preservation methods are currently needed to assure viability as well as genetic, physiological, and morphological integrity across storage periods. In this study, preservation of five isolates from the microalgae and cyanobacteria collection of the Plant Biology Department, Federal University of Viçosa, Minas Gerais, Brazil was investigated via monthly analyses of cell viability, biomass recovery, and contaminant concentrations over a period of 120 days. Lyophilization was adequate for both heterocystous cyanobacteria and other strains that were able to differentiate hormogones or to synthesize thick layers of exopolysaccharides. Lyophilization was also able to maintain cultures with low levels of contaminants. Dimethyl sulfoxide was relatively efficient, though some of the strains were susceptible to its cytotoxic effects. Our results demonstrated that cryopreservation with glycerol was the most efficient method. The ability to routinely preserve cyanobacterial strains reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. The results obtained in this study are therefore discussed in the context of the efficiency of the methods and the current need to develop suitable methods for maintenance of cyanobacterial collections.     

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.     

A Vessel for the Replicate Culture of Cells in Monolayer

Modification of a commercially available Carrel-type of disposable plastic flask with 10 compartments, by removing the central junctions of the two middle divisions to create 6 intercommunicating chambers, provides a vessel for easy preparation and handling of exact replicate cell cultures on coverglasses. Periodic sampling, fluid changes, attainment of identical inoculation and culture conditions, and microscopic observation are greatly facilitated in radio-isotope, virological and pharmacological experiments.     

Reemergence of covert bacteria Bacillus pumilus and Brevibacillus sp. in microbe-freed grape and watermelon stocks attributable to occasional autoclaving-defying residual spores from previous cycles

Covert bacteria-harboring long-term micropropagated culture of grape that was sanitized of associated microbes as assessed through the indexing of tissue and medium for several passages (Thomas and Prakash (2004) In Vitro – Plant 40:603–607) showed reemergence of bacteria in a section of cultures (3/40) during the subsequent screening. Similarly, long-term propagated triploid watermelon cultures showed bacterial reentry in some sub-stocks (2/14) during their extended monitoring post-antibiotic treatment (Thomas et al. (2006) Plant Cell Tiss Org Cult, 85:317–329). Each of the three grape sub-stocks showed single bacterium, identified as Bacillus pumilus (×2) or Brevibacillus sp. (×1) based on 16S rDNA sequence analysis with 100% similarity to ‘ARBG2’ and ‘ARBG1’ isolates respectively, retrieved originally from grape stocks as long-term alcohol-surviving spore-forming covert contaminants. The two isolates from watermelon included Brevibacillus sp. (ARBG1) and B. pumilus (AuRB-WM1), the latter distinct from the ‘ARBG2’ isolate. The chance of bacterial re-entry through endophytic survival or during culture handling appeared remote on account of the tissue indexing employed and the care taken. Monitoring the autoclaves for proper functioning using chemical, physical and biological indicators, sealing the vessels to protect from microscopic vectors and scrutinizing the autoclaved medium for microbial growth all pointed the contaminant source to occasional vessel-adhering residual spores from the previous covertly contaminated cycle(s) that were not recognized as contaminated. Such spores withstood the standard autoclaving either due to their high heat resistance or some conditions typical to tissue culture vessels, and the proportion of vessels showing carry-over inoculum increased with the number of passages of culturing with the organism. The problem due to these hardy spore-forming bacteria was circumvented through double autoclaving of culture vessels with the first one a day prior to the medium preparation     

Investigating the establishment of primary cultures of hemocytes from <Emphasis Type="Italic">Mytilus edulis</Emphasis>

Anthropogenic influences on the environment have been become a focal point for many social and political endeavors. With an ever-increasing rate of new contaminants being introduced into the environment every year, regulatory policies have begun to shift to prevention rather than mitigation. However, current in vivo testing strategies, in addition to ethical considerations, are too expensive and time consuming to adequately screen potential contaminants within a realistic timeframe. As a result, in vitro testing on cell cultures has been identified as an ideal alternative testing strategy for emerging contaminants. In the context of ecotoxicology, in vitro testing has had limited use particularly with marine invertebrates like the marine mussel Mytilus edulis mainly due to difficulties in establishing longer term cell cultures and cell lines. The aim of this study was to define an optimal technique (extraction and maintenance) for establishing a primary cell culture on M. edulis hemocytes that could be used for screening contaminants.     

Forensic Ecotoxicology: Establishing Causality between Contaminants and Biological Effects in Field Studies

Twelve papers in this series were derived from two conference sessions focusing on causality in field studies. Eight of these papers involve case studies examining biological effects of chemical contaminants in field situations. Using a weight-of-evidence approach, these case studies were evaluated against seven proposed criteria for establishing causality. The seven criteria were: strength of association; consistency of association; specificity of association; time order; biological gradient; experimental evidence; and biological plausibility. One of these seven criteria, ‘specificity of association’ was found to be of little utility for establishing causality in these field studies. The case studies are presented in approximate order of increasing levels of biological organization (i.e., going from endpoints at the suborganismal level to endpoints at the population or community level). In case studies examining higher levels of biological organization, it appears that the ‘biological gradient’ criterion was also not useful in establishing causality. These results, together with suggestions from other papers in the series, are used to recommend a set of modified criteria for establishing causality in field studies of the biological effects of chemical contaminants.     

The use of rotifers for the maintenance of monoalgal mass cultures of Spirulina

Zooplankton was successfully used for the biological control of unicellular algal contaminants in Spirulina mass cultures even under conditions adverse to the growth of Spirulina (maximal winter daily temperature of approximately 10 degrees C and very low bicarbonate concentration). Brachionus plicatilis (Rotifera) was the most successful species of zooplankton used. The interrelationships between Spirulina, green unicellular contaminant, and B. plicatilis were studied under various conditions. Two species of unicellular contaminant were used; Monoraphidium minutum was isolated from local cultures and Chlorella vulgaris, obtained from contaminated Spirulina cultures in Israel. The rotifer B. plicatilis successfully controlled the population size of both contaminants whether they were introduced in a single addition or as a daily dose. The biological control of the unicellular contaminants allows Spirulina to be cultured in a medium low in bicarbonate, thereby reducing the cost of the medium and increasing the quantity of CO(2) that may be freely absorbed from the atmosphere at the optimal pH for Spirulina cultivation.     

On-line monitoring of cell mass in mammalian cell cultures by acoustic densitometry

Acoustic resonance densitometry (ARD) provides a highly reproducible and stable method for on-line measurement of culture biomass density. The technique provides a direct determination of changes in relative density of culture medium and cell mass. At cell concentrations higher than 10(6) cells mL(-1)this method can replace cell counts and provide a continuous measure of total cell mass. In cultures of hybridomas or U937 human lymphoma cells, the ARD value correlates well with cell number except when the average cell size changes during culture. It is argued that cell mass determined by ARD rather than cell number should be used as the basis for measurements of specific biological activity.