Cord C. Uphoff

Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.

The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures.

P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine respiratory epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparin‐binding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages. Following purification of four recombinant proteins spanning P159 (F1P159, F2P159, F3P159 and F4P159), only F3P159 and F4P159 bound heparin in a dose‐dependent manner (Kd values 142.37 ± 22.01 nM; 75.37 ± 7.34 nM respectively). Scanning electron microscopic studies showed M. hyopneumoniae bound intimately to porcine kidney epithelial‐like cells (PK15 cells) but these processes were inhibited by excess heparin and F4P159. Similarly, latex beads coated with F2P159 and F4P159 adhered to and entered PK15 cells, but heparin, F2P159 and F4P159 was inhibitory. These findings indicate that P159 is a post‐translationally cleaved, glycosaminoglycan‐binding adhesin of M. hyopneumoniae.

Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines

SummaryMycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15–35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR delection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures

Detection of Mycoplasma Contaminations

Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The -specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma control and should be an established method in every cell culture laboratory. New cell lines as well as cell lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory and can also be used to determine the contaminating species.The described assay can be performed in less than 3 hours, including sample preparation, DNA extraction, PCR run, and analysis of the PCR products. Special emphasis is given to steps taken to avoid false-negative results due to the presence of inhibitors of the Taq polymerase in the crude samples and the interpretation of the results. The technique can also be adapted to the requirements of the pharmacopoeia.

Human Leukemia and Lymphoma Cell Lines as Models and Resources

Tumor cell lines are widely used as oncologic models and resources, forming, along with primary patient material and animal models, one of three major subjects for cancer investigation. With the advent of the Human Genome Project (HGP) and the ensuing provision of sequencing data and mapped clones, human cancer cell lines, notably those derived from leukemia-lymphoma (LL) have become increasingly productive tools for cancer gene ascertainment and characterization. Hence, the roles of putative novel cancer genes may be investigated using diverse panels of LL cell lines, both individually by PCR-based methods, and globally by transcriptional chip-profiling. Similar studies have also enabled the faithfulness with which cancer cell lines model their supposed in vivo counterparts to be quantified at last. Several recent transcriptional profiling studies indicate that of all tumor types well characterized human LL cell lines most accurately model the gene expression patterns of their corresponding primary tumors. Analysis using genomic arrays tells a similar story for the stability of chromosome rearrangements in LL cell lines. Well characterized LL cell lines also provide ideal tools for investigating the druggability of individual gene products, e.g. by measuring their transcript levels using q(uantitative)-PCR methods in cells subjected to treatments with small interfering (si)-RNAs. We provide a list of authentic, well characterized examples for prospective investigators, since many circulating cell lines have been cross-contaminated and describe DNA profiling methods which, together with classic and molecular cytogenetic analyses, inform authentication. We also review the problem of mycoplasma contamination and means for its eradication.

COMPARATIVE ANTIBIOTIC ERADICATION OF MYCOPLASMA INFECTIONS FROM CONTINUOUS CELL LINES

SummaryAccumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successfull alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1–3 wk) is a simple, inexpensive, and very practical approch for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66–85% of the cultures treated. Mycoplasma resistance was seen in 7–21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3–11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleased of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.

Biology of Monocyte-Specific Esterase

The monocyte-specific (carboxyl)esterase (MSE) is a marker enzyme which is well-known to hematologists as its detection is part of the traditii.mil cytochemical stainings of leukemia cells. There are a variety of synonyms for MSE among hematologists and biochemists. Biochemically. MSE is well-chemically, but should be discerned from other esterases with similar or identical substrate specificities and other features. Intensive analysis of normal and malignant hematopoietic cells and leukemia-lymphoma cell lines using isoelectric focusing established the specificity of this enzyme for monocytes and related cells, hence its designation as monocyte-specific esterase. Cloning of the gene led to its molecular characterization and provided new opportunities to examine MSE expression also at the RNA level which confirmed the monocyte /macrophage specificity. The availability of the gene sequences of various serine esterases and lipases which also hydrolyze ester bonds allowed for the identification of identical isolates from different tissues and the construction of an unrooted dendrogram based on sequence homologies of 22 enzymes The detailed regulation of the gene and the functional role of MSE hake remained largely unknown as of yet. However, DNA binding sites for various transcription factors have already been detected. Some evidence suggests involvement in physiological detoxification processes and in the immune defense against tumor cells. A more thorough understanding of the in vivo function of function of this truly unique enzyme should be helped by characterizing the signals anti signal transduction mechanisms which lead to MSE expression.

Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines.

We have identified new mutations in the p53 gene in 3/11 growth factor-independent and in 2/8 growth factor-dependent human acute myeloid leukemia (AML)-derived cell lines by single-strand conformational polymorphism (SSCP) and sequencing analysis. MEG-01 had a triplet deletion at codon 304; F-36P, NB-4 and MV4-11 showed point mutations at codon 344. F-36P had a second point mutation at codon 270 and NB-4 additionally at codon 319. M-MOK had a nucleotide substitution at codon 191. The frequency of p53 mutations in the cytokine-independent cell lines was comparable to that in the cytokine-dependent lines. These results suggest that loss of Wild type (wt) p53 is not the decisive event causing tumor cells to proliferate in vitro without externally added growth factors.

Expression of receptor tyrosine kinase HTK (hepatoma transmembrane kinase) and HTK ligand by human leukemia-lymphoma cell lines.

HTK (hepatoma transmembrane kinase) is a receptor tyrosine kinase belonging to the EPH subfamily of tyrosine kinases. Binding of its ligand (HTKL) results in tyrosine phosphorylation of HTK. In the present study, we analyzed the possible involvement of this ligand-receptor signalling system in hematopoiesis by examining the expression of both HTK and HTKL in a large and comprehensive panel of 70 continuous human leukemia-lymphoma cell lines. HTK and HTKL mRNA expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). HTK mRNA was detected in 68/70 cell lines; 58/70 cell lines were positive for HTKL mRNA expression; consequently, co-expression of both receptor and ligand was demonstrated in the majority of cell lines. Collectively, the wide-spread expression suggests a role for this ligand-receptor pair in hematopoietic development and/or function. Investigation of the details of signal transduction pathway that is activated by the HTK tyrosine kinase will help to define the exact biological function of the HTK-HTKL system.

ABL-BCR expression in BCR-ABL-positive human leukemia cell lines

Expression of normal ABL and BCR and of reciprocal fusion genes BCR-ABL and ABL-BCR was examined in a panel of 53 BCR-ABL-positive cell lines by RT-PCR to determine the influence of the various transcripts on leukemogenesis. Seventeen out of 18 lymphoid cell lines expressed ABL1a and/or ABL1b, whereas only 16 out of 35 myeloid cell lines expressed one or both normal ABL transcripts. Normal BCR was expressed in seven lymphoid cell lines; all cell lines from the m-bcr group (n = 9) were BCR-negative. Among the myeloid cell lines, 77% expressed the BCR gene. The M-bcr and m-bcr translocations were equally distributed among cell lines with lymphoid phenotype. The m-bcr translocation was not found in myeloid cell lines. b3-a2 constitutes the predominant form of fusion gene in myeloid cell lines with an incidence of about 68%. One myeloid cell line exhibited the mu-bcr variant. An ABL-BCR transcript of the 1a splice variant was not detected in any of the cell lines. ABL1b-BCR was expressed in all varieties of cell types and translocation forms: 56 and 66% in the lymphoid and myeloid cell lines, respectively; similar distributions were found for the fusion gene types: 67% among e1-a2, 73% among b2-a2, and 61% among b3-a2 translocations. Except for the lack of expression of normal BCR in m-bcr cell lines and of ABL1a-BCR expression in all cell lines, no consistent correlation of expression or lack of expression of BCR and ABL or of ABL-BCR reciprocal fusion genes could be found with cell lineages and translocation types. Further work is required to determine the exact role of the reciprocal fusion gene transcripts on the pathophysiological mechanisms of leukemogenesis.