Need For Cell Culture Research Articles

A population-epigenetic model to infer site-specific methylation rates from double-stranded DNA methylation patterns.

Cytosine methylation is an epigenetic mechanism in eukaryotes that is often associated with stable transcriptional silencing, such as in X-chromosome inactivation and genomic imprinting. Aberrant methylation patterns occur in several inherited human diseases and in many cancers. To understand how methylated and unmethylated states of cytosine residues are transmitted during DNA replication, we develop a population-epigenetic model of DNA methylation dynamics. The model is informed by our observation that de novo methylation can occur on the daughter strand while leaving the opposing cytosine unmethylated, as revealed by the patterns of methylation on the two complementary strands of individual DNA molecules. Under our model, we can infer site-specific rates of both maintenance and de novo methylation, values that determine the fidelity of methylation inheritance, from double-stranded methylation data. This approach can be used for populations of cells obtained from individuals without the need for cell culture. We use our method to infer cytosine methylation rates at several sites within the promoter of the human gene FMR1.

Comparative genomic hybridization in central and peripheral nervous system tumors of childhood and adolescence.

Brain tumors amount to less than 2% of all malignant neoplasms. However, they account for approximately 20% of all childhood cancers and are the leading cause of cancer mortality among children. Recently, enormous progress has been achieved in the field of pediatric neuro-oncology regarding the classification of children's brain tumors, as well as the understanding of the genetic events involved in their pathogenesis; thus leading to an emerging role of molecular diagnostic approaches using novel tools. Comparative genomic hybridization (CGH) is a technique that has revolutionized cytogenetic knowledge in the past decade. It permits the detection of chromosomal copy number changes without the need for cell culturing and gives a global overview of chromosomal gains and losses throughout the whole genome of a tumor. A survey of CGH-related publications on central and peripheral nervous system tumors in the pediatric and adolescent population revealed 884 cases. The CNS tumor groups most frequently examined by CGH were embryonal tumors (268 cases/30.3%) and ependymomas (241/27.2%), followed by astrocytic (163/18.4%), peripheral nerve (73/8.2%), choroid plexus tumors (56/6.3%), and craniopharyngiomas (38/4.3%). The most common CNS tumor entities were medulloblastomas (238/26.9%), classic ependymomas (160/18.1%), anaplastic ependymomas (70/7.9%), pleomorphic xanthoastrocytomas (53/6.0%), and pilocytic astrocytomas (50/5.6%). This article provides a short review of the CGH technique and its pitfalls, summarizes the current CGH-related data on pediatric brain tumors and muses on the future of CGH.

Comparative genomic hybridization: emphasis on use of formalin-fixed paraffin-embedded tissue

Comparative genomic hybridization (CGH) is a DNA-based technique that allows screening of the entire genome or individual chromosomes for the detection and location of chromosomal losses and gains. It accomplishes this without the need for cell culture and can therefore be employed on a wide range of routinely available specimens including fresh tissue and cell preparations, frozen tissue and formalin-fixed paraffin-embedded (FFPE) tissue. Therefore, large numbers of formalin-fixed tumours can be screened. This may then provide data regarding chromosomal gains and losses as both diagnostic tools and as markers of tumour progression. More specific investigation of the chromosomes can then be carried out by additional techniques such as fluorescent in situ hybridization, loss of heterozygosity analysis or DNA sequencing. CGH, however, is a difficult technique to perform and this is further confounded by the use of FFPE material. We describe some of the problems associated with using paraffin-embedded tissue as a source of DNA for use in CGH.

The role of comparative genomic hybridisation in prenatal diagnosis

The aims of this study were to assess the feasibility of using comparative genomic hybridisation instead of conventional cytogenetics in prenatal diagnosis and to determine the size of DNA loss that can be detected. Using comparative genomic hybridisation, six cases with standard aneuploidies were diagnosed correctly. This technique clearly identified a partial duplication of the long arm of chromosome 1 but was not capable of detecting the associated inversion. A small interstitial deletion on short arm of chromosome 10 also was detected precisely. Although the current comparative genomic hybridisation resolution is similar to the sensitivity of the highest resolution G banding, the latter is not a routine strategy in prenatal diagnosis. Comparative genomic hybridisation can allow full chromosome assessment equal to the highest resolution cytogenetic studies without the need for cell culture.

Comparative genomic hybridisation.

Comparative genomic hybridisation (CGH) is a technique that permits the detection of chromosomal copy number changes without the need for cell culturing. It provides a global overview of chromosomal gains...

Assessment of Genetic Changes in Hepatocellular Carcinoma by Comparative Genomic Hybridization Analysis: Relationship to Disease Stage, Tumor Size, and Cirrhosis

Hepatocellular carcinoma (HCC) is a common and highly malignant tumor that is prevalent in Southeast Asia. Although the etiological factors associated are now well recognized, the interactions between individual factors and the molecular mechanisms by which they lead to cancer remain unclear. Cytogenetic analysis on HCC has been limited because of poor hepatocyte growth in vitro. The recently developed technique of comparative genomic hybridization (CGH), however, permits screening of the entire genome without the need of cell culture. CGH was applied to the study of genomic aberrations in 67 surgically resected samples of HCC, 3 of adenomatous hyperplasia (AH), and 12 of nontumorous cirrhotic liver surrounding the tumors. All samples were from patients of a racially and etiologically homogeneous population in Southern China, where chronic hepatitis B virus infection is the main etiological factor. CGH analysis of the HCC samples revealed frequent copy number gain of 1q (48/67 cases, 72%), 8q (32/67 cases, 48%), 17q (20/67 cases, 30%), and 20q (25/67 cases, 37%) and common losses on 4q (29/67 cases, 43%), 8p (25/67 cases, 37%), 13q (25/67 cases, 37%), and 16q (20/67 cases, 30%). Our finding of a high incidence of 1q gain strongly suggested this aberration was associated with the development of HCC. Genomic abnormalities were detected in 1 of the 3 AH specimens but absent in all 12 cirrhotic tissues surrounding the tumor. Clinical staging classified 3/67 HCC cases as T1, 53 cases as T2, and 11 cases as T3. No significant difference in the pattern of genomic imbalances was detected between stages T2 and T3. A significant copy number loss of 4q11-q23 was, however, identified in those tumors larger than 3 cm in diameter. Of particular interest was the identification of 8q copy number gain in all 12 cases of HCC that arose in a noncirrhotic liver, compared with only 20/55 cases in HCC arising in a cirrhotic liver. We suggest that 8q over-representation is likely associated with a growth advantage and proliferative stimulation that have encouraged malignant changes in the noncirrhotic human liver.

Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis

In clinical settings, chromosome studies are usually performed on solid tissue other than solid tumors for one of two reasons: the tissue biopsy is the only tissue available from the patient, or tissues other than the standard peripheral blood lymphocytes must be examined because of suspected mosaicism. The Basic Protocol, Alternate Protocol 1, and Alternate Protocol 2 describe a method for culturing tissue samples to be used for preparation of metaphase chromosomes and subsequent examination or for biochemical or DNA analysis. The Basic Protocol describes mechanical disruption of the specimen to provide free access of the cells to the culture surface, whereas Alternate Protocol 1 describes a more efficient enzymatic disruption procedure. Support Protocol 1 and Support Protocol 2 describe methods of collecting tissue samples. Alternate Protocol 2, which can sometimes be applied to placental tissue, involves use of mitotically active chorionic villus tissue to prepare metaphase chromosome spreads directly without the need for cell culture.

Cultivation of anchorage-dependent mammalian cells and production of various metabolites

Cell culture is an important tool for the production of various biologically active products such as pharmaceuticals, viral vaccines, viral insecticides, monoclonal antibodies, polypeptides and growth factors. These products are produced by normal, transformed and genetically engineered cells. The majority of these cells are anchorage dependent and require a solid surface to attach to in order to proliferate, metabolize nutrients and produce biomolecules. The need for cell culture was intensified with the enhanced development of mammalian genetic engineering, which raised a new demand for advanced cell culture technologies. Intensive studies have been undertaken by scientists to develop new technologies for large-scale cell culture. A major effort was invested to develop cell culture systems with high ratios of surface area to volume. At present there is a wide variety of systems, each with its own advantages and limitations. In this short review, the various large-scale cell culture systems are described. Cell culture technology in general, and in particular the substrates to which the cells adhere, play important roles in the product formation of a given macromolecule; therefore the system should be chosen very carefully and adapted to the biological process. The production process of tissue plasminogen activator from normal human fibroblasts and a new cell carrier, “Fibra-cel”, which is made of non-woven polyester fabric, are presented as examples in order to demonstrate the state of the art in this field.

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens

The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

Skeletal muscle cell populations. Separation and partial characterization of fibroblast-like cells from embryonic tissue using density centrifugation.

Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll density centrifugation. Isolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle- and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flattened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.

Rapid diagnosis of chlamydial infections with the MicroTrak direct test.

A direct test on clinical specimens, using fluorescein-labeled monoclonal antibodies, for Chlamydia trachomatis (MicroTrak [Syva Co.]) was evaluated for the rapid diagnosis of chlamydial infections. Asymptomatic females attending pregnancy or planned parenthood clinics were tested by the direct test and by a cell culture method. Of 401 paired, endocervical specimens, 398 (99.3%) gave identical direct and culture results. The overall sensitivity of the direct test was 96.3% (26 of 27), and the specificity was 99.5% (372 of 374) as compared with that of culture. More than four-fifths of the direct smears were read within ca. 2 min. In this study population, the performance of the direct specimen test was comparable to that of cell culture methods. Rapid turnaround time and elimination of the need for cell culture make the direct test a practical method for the specific diagnosis of chlamydial infections.