Robert Ian Freshney

University of Glasgow | UofG · Institute of Cancer Sciences

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and o...

•The WSU-CLL cell line was thought to come from chronic lymphocytic leukemia.•WSU-CLL is misidentified and is actually acute lymphoblastic leukemia cell line REH.•This makes WSU-CLL an unsuitable model for chronic lymphocytic leukemia.•Laboratories should check for cell line cross-contamination and test their stocks.•Adopting minimal reporting crit...

Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, includin...

This is the sixth edition of the leading text in the basic methodology of cell culture, worldwide. Rigorously revised, it features updates on specialized techniques in stem cell research and tissue engineering; updates on molecular hybridization, somatic cell fusion, hybridomas, and DNA transfer; new sections on vitrification and Organotypic Cultur...

No abstract available.

Estimates have been made of the absolute numbers of hepatogenic erythropoietic cells from 12.5 days post fertilization onwards in the mouse. All stages of maturation up to reticulocytes are present in the earliest samples but the least mature cells (proerythroblasts and basophilic erythroblasts) predominate; more mature cells (orthochromatic erythr...

In this chapter, we review the principles of cell culture and bioreactor design in the context of tissue engineering. In the first part, we describe the principles of cell and tissue culture, with emphasis on the initiation of culture, transformation, preservation, and validation of the cells and differentiation methods that utilize simple and well...

This book collects the most effective and cutting-edge methods and protocols for deriving and culturing human embryonic and adult stem cells-in one handy resource. This groundbreaking book follows the tradition of previous books in the Culture of Specialized Cells Series-each methods and protocols chapter is laid out exactly like the next, with ste...

Introduction Types of Cell Culture Isolation of Cells for Culture Subculture Cryopreservation Characterization and Validation Sources of Materials References

The disadvantages of serum are described, including variability, shelf life, availability, effect on down-stream processing, and potential for contamination. The development of selective media and the control of proliferation and differentiation are cites as further advantages, while the cost, availability, and multiplicity of media required, are c...

Epithelial cells are dealt with first in the chapter with invited protocols covered most of the common epithelial cell types, epidermis, cornea, breast, cervix, colon, liver, pancreas, lung, oral cavity, prostate and kidney. Mesenchymal cells are grouped together with invited protocols on fat cells, muscle, cartilage, bone, and endothelium. The thi...

Advice is given on the isolation and handling of human and animal tissue. The major methods of preparing a primary culture are described including primary explantation, enzymatic disaggregation in trypsin or collagenase, and mechanical disaggregation. Example protocols are provided for primary culture from mouse embryo fibroblasts, organ rudiments...

The effects of cell density on cell interaction are reviewed and different types of organotypic culture described and distinguished from organ culture. Organ culture uses undisaggregated tissue, while organotypic implies recombination of cells previously disaggregated and maintained as cell lines. Histotypic culture is defined as three-dimensional...

This chapter deals with methods for quantifying cell cultures, from cell counting hemocytometer and electronic) to biochemical estimations of DNA, protein and enzyme activity with sections on replicate sampling and data acquisition. There is an in depth assessment of methods for analysing cell proliferation, including analysis of growth curves, pla...

One of the most important issues in cell culture, contamination is discussed first by examining possible routes, including poor technique, environmental problems, contaminated equipment, particularly incubators, and importation of biopsies and cell lines. Monitoring for the main types of contamination, by visual observation or specific assays, is d...

The description of culture media starts with the physicochemical properties that are required, including control of CO 2 and bicarbonate concentration, pH, buffering, oxygen tension, osmolality, temperature, and viscosity. A description of the formulation of balanced salt solutions is followed by text and tables describing the composition of comple...

This chapter presents a number of techniques that are associated with cell culture though not necessarily culture techniques per se such as the protocols provided for autoradiography, in situ hybridization, FISH, cell hybridization, production of monoclonal antibodies, DNA transfer by lipofection and electroporation, and time-lapse video recording....

A distinction is made between viability, metabolic toxicity, and cell survival assays and the implications of each for the design of assays. Protocols are provided for measuring viability by dye exclusion and dye uptake, and for the clonal growth assay of attached monolayer cells. Instructions are given on presenting and interpreting data. Metaboli...

The need for characterization is established, with particular emphasis on authenticating cell lines. Aspects of characterization include, identification of species, cell lineage or tissue of origin, and unique markers capable of identifying each individual cell line. As morphology is a major component of characterization, use of an inverted microsc...

Cloning is described as a method for isolating a specific cell type or genetic variant by diluting a cell inoculum to such an extent that the surviving cells grow as isolated colonies. Protocols are provided for dilution cloning of adherent cells and suspension cloning in agar or methylcellulose for non-adherent or transformed cells. As plating eff...

Reagents in common use are listed alphabetically with details of their preparation. These are reagents many of which will have appeared multiple times in the text, and include such items as phosphate-buffered saline, viability stain, EDTA, and trypsin. Details on storage are provided in most cases but sterilization is dealt with in Chapter 11. Keyw...

This chapter seeks to provide a background to aspects of basic cell biology which have specific relevance to cell culture, such as cell–cell adhesion, cell–matrix adhesion and signaling, cell proliferation, and differentiation. Endocrine, paracrine and autocrine signaling and the role of the cytoskeleton in matrix and cell contact signaling are als...

After providing a rationale for freezing cells for storage, the principles of cell freezing are discussed highlighting the optimum cell concentration, freezing medium, and cooling rate. The different types of liquid nitrogen cryofreezers are described with their relative merits and methods for preventing failure. Separate protocols are provided for...

The equipment requirements of a tissue culture laboratory are described, with some indication of the relative importance of each item, with items such a laminar flow cabinet, inverted microscope, CO 2 incubator, bench-top centrifuge, liquid nitrogen freezer, and water purification equipment being regarded as essential, while other items, such as an...

Various physical methods for separating different cell types are discussed from simple low technology methods, like density centrifugation, to high technology methods, like centrifugal elutriation and fluorescence-activated cell sorting (FACS). Particular emphasis is given to magnetic sorting, which is highly effective but not dependent on major ca...

Substrate materials are discussed and advice given on the choice of particular types of culture vessel. Issues governing selection of culture vessel are covered such as cell yield and size of vessel, suspension versus monolayer culture, venting, and sampling. Specialized systems are described, such as permeable supports (filter well inserts, hollow...

This chapter aims to provide a structured approach to dealing with problems of primary culture and serial subculture, including poor cell survival and proliferation, poor plating efficiencies, contamination (including cross-contamination), difficulties in recovering cells after cryopreservation, and determination of cell counts and viability. Preca...

This appendix provides a listing of most of the major suppliers of tissue culture reagents, media, culture vessels, and equipment, with full address, phone, fax, email and web site information in nearly all cases. Cell banks and scientific societies are also included within this list. Keywords: suppliers; resources; media; culture vessels; equipmen...

To avoid repetition, the sources of materials and equipment used in cell culture are not detailed in individual chapters but are collected together in this appendix. It lists, alphabetically, all the commercially available equipment and materials used in the protocols and supplements Appendix I, which deals with reagents that the users may wish to...

This chapter is intended for those who are involved in setting up a tissue culture laboratory, either by new build or refurbishment. It deals with the planning of the layout and services, and suggests possible floor plans for a small, medium, and large tissue culture laboratory. The required facilities are classified as the minimum requirements, de...

Elements of aseptic technique are described including control of the work area, aseptic manipulations, and care of laminar flow hoods and incubators. Advice is given on sterile pipetting and recommendations made against pouring. Protocols are provided for carrying out aseptic procedures in vertical laminar flow, or on the open bench, and a separate...

The chapter starts by considering the reasons for the lack of differentiation in most cultured cell lines, e.g., selection of immature cells and induction of cell proliferation. Stem cells and their plasticity are discussed, and the theory and practicalities of the induction of differentiation are presented. The major components of the inductive en...

This very brief chapter provides a summary of major precautions and recommended practices regarding aseptic technique, avoidance of contamination, including cross-contamination, cell line acquisition and authentication, records keeping and standardization of procedures. Keywords: aseptic technique; contamination; authentication; records; standardiz...

A series of exercises is described, ranging from basic exercises including preparation of media, observation, feeding, and subculture, through advanced exercises including characterization, detection of mycoplasma, cryopreservation, primary culture, and cloning, to more specialized exercises, such as cell sorting, histotypic culture, and cytotoxici...

This appendix contains a list of tissue culture terms with their definitions (modified after Schaeffer, 1990) with additional definitions provided by the author.

This appendix lists other general and more specialized textbooks in the field of cell and tissue culture and, in addition, provides suitable background reading in cell and molecular biology. A number of journals are also listed which either focus on tissue culture technology, or contain articles, many of which employ tissue culture within their met...

Following some advice on terminology, finite and continuous cell lines are compared, and a table provided listing 62 misidentified or cross-contaminated cell lines in current use around the world. Dissociating procedures of differing degrees of severity are tabulated and protocols are provided for feeding and subculturing cell lines grown as monola...

The specific safety issues of a tissue culture laboratory are discussed with recommendations made for risk assessment, establishing safety procedures, and to accessing information on safety regulations. Laboratory safety is discussed under four headings, (1) general safety, including sharps, chemicals, gases, and liquid nitrogen, (2) fire risk aris...

The study of transformation has implications for cell line characterization, helps to identify genotypic and phenotypic changes relating to cancer, and provides technology which can be used to immortalize cell lines. The chapter begins with an analysis of transformation and a description of cellular changes associated with it. Among these is immort...

Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology. However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere in this volume, the technology, both simple and sophisticated, is available to remedy the problem...

This chapter covers some of the epithelial cell types not dealt with in the preceding chapters and reviews some of the common features used in isolation and propagation, including the use of collagenase digestion, serum-free selective media and matrix coating. Protocols are provided for corneal, thyroid, pancreatic and renal epithelial cells and ot...

The common conception of cytotoxicity is that the cell is killed by the cytotoxin and the assays employed tend to reflect this. There are, however, several distinct aspects of cytotoxicity, differing in cellular mechanisms, outcome, and, consequently, in the assay of their activity (Freshney, 1904). As requirements for in vitro assays become more d...

The role of the interleukin-6 (IL-6) group of cytokines in differentiation of two lung adenocarcinoma cell lines has been examined using induction of alkaline phosphatase and expression of surfactant protein A. Oncostatin M was the most active and potent for alkaline phosphatase in A549 cells, with IL-6 having similar activity but less potency. Nei...

Eight food-borne mycotoxins epidemiologically implicated in human disease were tested for their cytotoxic effects on human cells previously immortalised and transfected to introduce human cytochrome p450 (CYP 450) genes. Such cells retain many characteristics of normal cell growth and differentiation while simultaneously having the potential of eit...

This study focuses on the cytotoxic effects of fumonisin B1 (FB1) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two rela...

Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine int...

Two adherent cell lines MOG-H69V and MOG-H69VZ have been isolated from a continuous cell line, NCI-H69, derived from human small cell lung cancer by Carney et al, [1987]. They have been established and characterised morphologically, biochemically, and for growth characteristics in vitro Khan et al (19). In the present study both the parental and th...

The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of gro...

The adriamycin chemosensitivity and extent of gap junctional intercellular communication were assessed in a panel of seven human non-small cell lung cancer (NSCLC) cell lines. Communication was assessed by autoradiographic detection of transfer of 3H uridine nucleotides between coupled cells. The strength of coupling varied widely between the cell...

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Hexamethylene bisacetamide (HMBA), sodium butyrate (NaBt), and cyclic AMP (cAMP) have been shown to induce differentiation, which may regulate tumour growth differently from conventional cytotoxic drugs. It was the intention in the present study to determine whether alterations could be induced in the phenotype of small cell lung cancer (SCLC) cell...

Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to deter...

Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth ra...

Three sublines have been derived from the parental line Mv1Lu by transfection with normal and mutated Ha-ras, and myc oncogenes, and subsequent cloning. All the oncogenes have increased the growth rate of the cell in vitro, increased their plating efficiency in monolayer and suspension, and reduced their serum dependence. Growth in vivo as xenograf...

Mink lung epithelial cells were transfected with c-myc and activated H-ras genes. The transfected sublines formed colonies in soft agar and were tumorigenic when injected subcutaneously into athymic nude mice. DNA synthesis was measured in each of the cell lines by 3H-thymidine incorporation and in the parent line there was dose related stimulation...

The development of pleiotropic drug resistance (PDR) in vivo in solid tumour models suggests that a similar process may occur in the clinic. A subline of the Ridgway osteogenic sarcoma (ROS)--a murine subcutaneously-growing solid tumour--with moderate resistance (1.5 fold) to actinomycin D was selected by repeated suboptimal treatment with this dru...

Clinical applications consequent upon elucidation of the role of oncogenes in contributing to the malignant cellular phenotype have mainly focussed on refinement of existing prognostic models for patient survival. Current research on breast cancer has shown a relationship between amplification of the c-erbB-2 or Her-2/neu oncogenes and disease prog...

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradren...

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline > adrenaline > salbutamol < noradrenaline, which is consistent with the pr...

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The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to...

The glucocorticoid hormones methyl prednisolone and dexamethasone were shown to be cytostatic, but not cytotoxic, at high cell densities for early passage and continuous cell lines from human glioma at 0.25 microM and above, in the presence or absence of serum. In the absence of serum both steroids at 2.5 nM increased the saturation density close t...

The calcium antagonist verapamil has been used to overcome multidrug resistance in a number of ani mal and human tumour models. In vivo data derived from human solid tumours is, however, limited. We have investigated the ability of verapamil to increase sensitivity to etoposide (VP16), adriamycin (ADR) and vincristine (VC) in the human non-small ce...

The sensitivity of 7 human non-small cell lung cancer cell lines to each of 7 cytotoxic drugs was determined. None of the cell lines used in these experiments had been previously exposed to cytotoxic drugs in vitro. A pattern of cross-resistance (P less than 0.05) between the drugs adriamycin (ADR), vincristine (VC) and etoposide (VP16) was noted s...

The effects of a non-ionic polyoxyethylated lauryl ether surfactant (Brij 30) on monolayer uptake and spheroid penetration of adriamycin have been studied. Co-incubation of adriamycin with Brij 30 increases intracellular adriamycin levels by 2-3-fold. Although, in the concentrations used, Brij 30 alone is not cytotoxic, adriamycin and Brij 30 mixtu...

Using growth delay and clonogenic cell survival as end points, we have shown that the 3-dimensional structure of human lung tumour spheroids confers a degree of resistance to the anthracyclines adriamycin and 4'-deoxydoxorubicin, relative to cells grown as monolayer. 4'-deoxydoxorubicin induces a longer growth delay and greater clonogenic cell kill...

4'-Deoxydoxorubicin (4'-deoxy) is a new adriamycin analogue with a similar spectrum of antitumour activity but is significantly more lipophilic than the parent compound. We report the kinetics and uptake of the two drugs by human non-small cell lung tumour cells in monolayer culture and the relationship between intracellular drug levels and cytotox...

Glucocorticoids are cytostatic for human glioma grown at a high cell density in cell culture. The effect is not cytotoxic, appears to involve a modification of the cell surface, and has been detected with methyl prednisolone, dexamethasone, and beta-methasone. Glucocorticoids were also found to reduce malignancy-associated properties (plasminogen a...

Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 μM, respectively. The Ka apparent value for dopamine was increased by the D-l antagonis...

We have investigated the mechanism of resistance to adriamycin (ADR) of 3 human glioma cell lines in culture. The cell lines had different inherent sensitivities to ADR. Verapamil increased the ADR sensitivities of the 2 most resistant cell lines (G-UVW and G-CCM) by up to 5-fold. This effect was not seen in a sensitive cell line (G-MCF). Although...

Clinical usage of glucocorticoid hormones in the reduction of intra-cerebral pressure in cases of glioma has suggested that they may also have a cytostatic effect (Lieberman et al., 1977). Investiga-tion of the effect of glucocorticoids in vitro demon-strated that although they were cytostatic at high cell densities that they are also capable of in...

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Cells cultured from anaplastic astrocytoma (Kernohan and Sayre, grades III and IV) will proliferate on confluent monolayers of normal glia, while cells cultured from normal brain will not. The growth of a cell line containing a high proportion of well-differentiated glioma cells (G-CCM) was partially inhibited, though not as much as normal glia, wh...

The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to...

There is now clear evidence that cells cultured from human and animal tumours can be induced to differentiate in vitro by recognised hormones, regulatory peptides, polar solvents and cytotoxic drugs. Examples can be found from several different types of tumour with the bulk of the data deriving from neuroblastoma and myeloid leukaemia. There is no...

The phenotypic expression of cells derived from human anaplastic astrocytomas, rat glioma, normal human adult and foetal brain tissue have been examined for differentiated and malignancy-associated properties. Glial fibrillary acidic protein (GFAP), high affinity glutamate and gamma-amino butyric acid (GABA) uptake and glutamine synthetase were use...

In vitro studies have shown that glucocorticoids have a cytostatic effect on glioma cells at high cell densities but enhance cell survival and proliferation at low cell densities. The cytostatic effect is not cytotoxic and may be mediated via a membrane modification altering cell-cell interaction. Cell interaction is also implicated in differentiat...

In vitro studies have shown that glucocorticoids have a cytostatic effect on glioma cells at high cell densities but enhance cell survival and proliferation at low cell densities. The cytostatic effect is not cytotoxic and may be mediated via a membrane modification altering cell-cell interaction. Cell interaction is also implicated in differentiat...

A method has been developed for measuring the drug sensitivity of human gliomas in short-term culture, using scintillation counting or autofluorography. Cell cultures prepared from malignant astrocytomas were treated with anticancer drugs whilst in exponential growth in microtitration plates. After drug treatment and a recovery period, residual via...

The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to...

Survival and proliferation of cell cultures from human anaplastic astrocytomas were shown to be enhanced by glucocorticoids with an optimal concentration of approximately 2.5 x 10(-5)M (10 micrograms/ml). The stimulation of proliferation was only observed in a clonal growth assay and was reversed as the size of individual colonies reached approxima...

An autofluorographic record of drug titrations in microtitration plates has been obtained using cultures labelled with [35s] methionine after treatment with cytostatic drugs. By adding scintillation fluid directly to each culture well of the microtitration plate, and then centrifuging the evaporate the toluene and leave a flat even film of fluor, i...

Cultures of human astrocytoma have been derived by collagenase digestion and are presumed, from their aneuploid karyotypes, to be predominantly neoplastic. Early passage cultures in proliferative phase have been cloned in the presence of dexamethasone and betamethasone, both commonly used in management of patients with brain tumours. These steroids...

An attempt has been made to construct an assay potentially suitable for use with primary cultures of human tumours to measure the survival of exponentially growing monolayer cultures after exposure to anti-neoplastic drugs. Cell survival was assessed using their protein synthetic capacity after removal of drugs. HeLa cells were employed to avoid th...

Mammary cancer directed and nonspecific immunoassays were made in 3 groups of female patients. One group had primary mammary cancer treated by mastectomy and postoperative radiotherapy plus an autograft of irradiated tumor (AIT) 40-66 mth previously. A second age matched group had mammary cancer comparable to the first group in clinical presentatio...