Best practices for detecting and mitigating the risk of cell culture contaminants

A variety of biological and chemical contaminants can adversely impact cells in culture, ranging from outright destruction of the culture, mutation, phenotypic changes to relatively minor changes in morphology, or growth rate. There are various approaches to detecting and mitigating the risk of biological or microbial contaminants in cell cultures, and these are discussed in this article. Chemical contaminants typically arise from improper handling or sourcing of cell culture reagents, glassware, or other types of consumables. These and other sources of chemical contaminants of cell cultures are discussed. The occurrence of chemical contamination is mitigated through adherence to best practices in sourcing and handling of such materials and by avoiding the use of volatile solvents within incubators that are used for maintaining cell cultures.     

Best practices for authenticating cell lines

Experiments using cell cultures are only valid to the extent that the cell culture is a true model system for the biological system being investigated. To assure that a cell line is and remains an appropriate biological model, its identity, purity, ploidy, and phenotype must be maintained. These characteristics comprise and determine the authenticity of a cell line. Routine monitoring of the cell line through microscopic examination of morphology can help to determine authenticity, as can the determination of phenotypic status. Assays designed to confirm cell identity and ploidy and freedom from cross-contaminating cell types may need to be performed at certain times, as such information may not be obtained through morphologic and phenotypic examinations alone. The best practices associated with establishing cell line authenticity are described in this article.     

Best practices for naming,receiving, and managing cells in culture

One of the first considerations in using an existing cell line or establishing a new a cell line is the detailed proactive planning of all phases of the cell line management. It is necessary to have a well-trained practitioner in best practices in cell culture who has experience in receiving a new cell line into the laboratory, the correct and appropriate use of a cell line name, the preparation of cell banks, microscopic observation of cells in culture, growth optimization, cell count, cell subcultivation, as well as detailed protocols on how to expand and store cells. Indeed, the practitioner should best manage all activities of cell culture by ensuring that the appropriate certified facilities, equipment, and validated supplies and reagents are in place.     

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.     

The epidermal stem cell: an overview

The epidermis, the intestinal epithelium and the bone marrow are constantly renewed. Once a terminally differentiated cell has fulfilled its function, it is eliminated. Thus, new differentiated cells need to be constantly produced by the proliferative compartment to ensure the function of the tissue. As this process continues throughout a lifetime, cells must exist with a large capacity for proliferation within each of these tissues. These cells must also be the depository of all the information necessary for suitable differentiation to occur. This cell population which is qualified as the stem population, has attracted, in recent years, considerable attention not only because of its role during development, but also because of its potential sensitivity to radiations and carcinogenesis and to antineoplastic drugs. The epidermis, which is a stratified and squamous epithelium, has appendages which developed from the primitive epidermis during embryonic life. These appendages are also renewed during the adult lifetime, as illustrated by hair growth. The epidermis proves to be a unique model with which the development and the renewal of a stratified epithelium can be studied.     

Membrane lipids and cell death: an overview

In this article we overview major aspects of membrane lipids in the complex area of cell death, comprising apoptosis and various forms of programmed cell death. We have focused here on glycerophospholipids, the major components of cellular membranes. In particular, we present a detailed appraisal of mitochondrial lipids that attract increasing interest in the field of cell death, while the knowledge of their re-modelling and traffic remains limited. It is hoped that this review will stimulate further studies by lipid experts to fully elucidate various aspects of membrane lipid homeostasis that are discussed here. These studies will undoubtedly reveal new and important connections with the established players of cell death and their action in promoting or blocking membrane alteration of mitochondria and other organelles. We conclude that the new dynamic era of cell death research will pave the way for a better understanding of the 'chemistry of apoptosis'.     

Optimization of porcine urothelial cell cultures: Best practices,recommendations, and threats

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.     

Asking nicely: Best practices for requesting data

Compiling disparate datasets into publicly available composite databases helps natural resource communities explore ecological trends and effectively manage across spatiotemporal scales. Though some studies have reported on the database construction phase, fewer have evaluated the data acquisition and distribution process. To facilitate future data sharing collaborations, Louisiana State University surveyed data providers and requestors to understand the characteristics of effective data requests and sharing. Data providers were largely U.S. natural resource agency personnel, and they reported that unclear data requests, privacy issues, and rigid timelines and formats were the greatest barriers toward providing data, but that they were motivated by improving science and collaboration. Data requestors identified challenges such as evolving needs, standardization issues, and insufficient resources (time and funding) as barriers to compiling data for these types of efforts. In a time of big data, open access, and collaboration, significant scientific advances can be made with effective requests and inclusion of data sets into larger and more powerful databases.     

An overview of viral and viral-like agents in cell culture systems

The first recognition of seriously contaminated biological products occurred almost 100 years ago. More recently, in the second half of this century, the potential for contamination by viral and viral-like agents became obvious when the SV-40 virus was identified as an endogenous agent in the cell cultures used to produce polio vaccines. The history of major contamination incidents is reviewed, and current issues are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Best practices for an insecticide-treated bed net distribution programme in sub-Saharan eastern Africa

Insecticide-treated bed nets are the preeminent malaria control means; though there is no consensus as to a best practice for large-scale insecticide-treated bed net distribution. In order to determine the paramount distribution method, this review assessed literature on recent insecticide treated bed net distribution programmes throughout sub-Saharan Eastern Africa. Inclusion criteria were that the study had taken place in sub-Saharan Eastern Africa, targeted malaria prevention and control, and occurred between 1996 and 2007. Forty-two studies were identified and reviewed. The results indicate that distribution frameworks varied greatly; and consequently so did outcomes of insecticide-treated bed net use. Studies revealed consistent inequities between urban and rural populations; which were most effectively alleviated through a free insecticide-treated bed net delivery and distribution framework. However, cost sharing through subsidies was shown to increase programme sustainability, which may lead to more long-term coverage. Thus, distribution should employ a catch up/keep up programme strategy. The catch-up programme rapidly scales up coverage, while the keep-up programme maintains coverage levels. Future directions for malaria should include progress toward distribution of long-lasting insecticide-treated nets.     

Estimating infection prevalence: Best practices and their theoretical underpinnings

Accurately estimating infection prevalence is fundamental to the study of population health, disease dynamics, and infection risk factors. Prevalence is estimated as the proportion of infected individuals (“individual‐based estimation”), but is also estimated as the proportion of samples in which evidence of infection is detected (“anonymous estimation”). The latter method is often used when researchers lack information on individual host identity, which can occur during noninvasive sampling of wild populations or when the individual that produced a fecal sample is unknown. The goal of this study was to investigate biases in individual‐based versus anonymous prevalence estimation theoretically and to test whether mathematically derived predictions are evident in a comparative dataset of gastrointestinal helminth infections in nonhuman primates. Using a mathematical model, we predict that anonymous estimates of prevalence will be lower than individual‐based estimates when (a) samples from infected individuals do not always contain evidence of infection and/or (b) when false negatives occur. The mathematical model further predicts that no difference in bias should exist between anonymous estimation and individual‐based estimation when one sample is collected from each individual. Using data on helminth parasites of primates, we find that anonymous estimates of prevalence are significantly and substantially (12.17%) lower than individual‐based estimates of prevalence. We also observed that individual‐based estimates of prevalence from studies employing single sampling are on average 6.4% higher than anonymous estimates, suggesting a bias toward sampling infected individuals. We recommend that researchers use individual‐based study designs with repeated sampling of individuals to obtain the most accurate estimate of infection prevalence. Moreover, to ensure accurate interpretation of their results and to allow for prevalence estimates to be compared among studies, it is essential that authors explicitly describe their sampling designs and prevalence calculations in publications.     

Towards an understanding of microtubule function and cell organization: an overview.

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.     

Calmodulin: an overview

Calmodulin is a 16,700-dalton Ca2+-binding protein ubiquitous in the eukaryotes. It has no intrinsic enzymatic activity, but it regulates a wide spectrum of enzymes that control many basic cellular processes, ranging from the metabolism of cyclic nucleotides, Ca2+, and glycogen to contractile activity and stimulus-secretion coupling. Mounting evidence now indicates that calmodulin is the major intracellular Ca2+ receptor that remained elusive despite three decades of extensive work by many investigators.