Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

This study was designed to compare commercially available extender Bioxcell^® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 10⁶ spermatozoa/ml) in tris-citric egg yolk or Bioxcell^® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell^® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell^® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell^® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.     

The necessity of identity assessment of animal intestinal cell lines: A case report

Eight intestinal cell lines, established from different animal species were submitted to DSMZ (German Collection of Microorganisms and Cell Cultures) in order to analyze their species of origin and their microbial contamination. Species identity was determined by PCR targeting mitochondrial genes and hence confirmed by sequencing the amplified PCR products. For three cell lines (CIEB, CLAB, PSI-1) we confirmed the species identity, whereas the species of origin of the three other cell lines (B6, B10XI and IPEC) was not the expected one: B6 and B10XI cells, which were supposed to be of chicken origin were identified as porcine cells. IPEC, allegedly a sub clone of the well-known porcine intestinal cell line IPEC-J2, was of bovine instead of porcine origin. However, two further IPEC-clones, namely IPEC-1 and IPEC-J2, provided by another source were shown to be derived from the correct species (i.e. pig). Furthermore, six out of these eight cell lines turned out to be highly contaminated with mycoplasma. Alerted by this high incidence of infected and false specified cell lines, we feel obliged to inform all those working with animal intestinal cell lines and we strongly recommend verifying the species identity before using them. Also, the presence of mycoplasma should be tested when taking the cells in culture for the first time, and this mycoplasma control should be repeated at regular time intervals (e.g. every 4 weeks).     

The H-2KbtsA58 transgenic mouse: A new tool for the rapid generation of novel cell lines

The ability to generate expanded populations of individual cell types able to undergo normal differentiationin vitro andin vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periodsin vitro simply by growing them at 33°C in the presence of interferon, while still retaining the capacity to undergo normal differentiationin vivo andin vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2K^btsA58 transgenic mice and mutant mice of interest.     

Activation of Na/H exchanger 1 by neurotensin signaling in pancreatic cancer cell lines

Acidosis commonly observed in solid tumors like pancreatic cancer promotes genetic instability and selection of a more malignant phenotype of cancer cells. Overexpression or activation of integral membrane proteins mediating H⁺ efflux may contribute to extracellular acidification. Neurotensin (NT) induces intracellular alkalinization and stimulates interleukin-8 production in pancreatic cancer cells and, as demonstrated here, the stable NT analog Lys⁸-ψ-Lys⁹NT(8-13) enhances the amiloride-sensitive, Na⁺-dependent transmembrane H⁺ flux by a factor of 2.05 ± 0.28 and 2.69 ± 0.07 in BxPC-3 and PANC-1 pancreatic cancer cells, respectively, by phosphorylation of the Na⁺/H⁺ exchanger 1 (NHE1). Human genome-wide gene expression analysis was performed to detect effects of Lys⁸-ψ-Lys⁹NT(8-13) on BxPC-3 cells. Results indicated upregulation of genes involved in regulation of NHE1, hypoxic response and glycolysis in response to Lys⁸-ψ-Lys⁹NT(8-13) even under normoxic conditions. Therefore, our findings suggest that growth factors like NT may be implicated in the early progression of pancreatic cancer by localized acidification and induction of an aerobic glycolytic phenotype with higher metastatic potential in small cell aggregates.     

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler^® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.     

Growth of cancer cell lines under stem cell-like conditions has the potential to unveil therapeutic targets

Malignant tumors comprise a small proportion of cancer-initiating cells (CIC), capable of sustaining tumor formation and growth. CIC are the main potential target for anticancer therapy. However, the identification of molecular therapeutic targets in CIC isolated from primary tumors is an extremely difficult task. Here, we show that after years of passaging under differentiating conditions, glioblastoma, mammary carcinoma, and melanoma cell lines contained a fraction of cells capable of forming spheroids upon in vitro growth under stem cell-like conditions. We found an increased expression of surface markers associated with the stem cell phenotype and of oncogenes in cell lines and clones cultured as spheroids vs. adherent cultures. Also, spheroid-forming cells displayed increased tumorigenicity and an altered pattern of chemosensitivity. Interestingly, also from single retrovirally marked clones, it was possible to isolate cells able to grow as spheroids and associated with increased tumorigenicity. Our findings indicate that short-term selection and propagation of CIC as spheroid cultures from established cancer cell lines, coupled with gene expression profiling, represents a suitable tool to study and therapeutically target CIC: the notion of which genes have been down-regulated during growth under differentiating conditions will help find CIC-associated therapeutic targets.     

Development of new hCaM-Alexa Fluor biosensors for a wide range of ligands

Eight new fluorescent biosensors of human calmodulin (hCaM) using Alexa Fluor^® 350, 488, 532, and 555 dyes were constructed. These biosensors are thermodynamically stable, functional, and highly sensitive to ligands of the CaM. They resolve the problem of CaM ligands with similar spectroscopic properties to the intrinsic and extrinsic fluorophores of other biosensors previously reported. Additionally, they can be used in studies of protein-protein interaction through Förster resonance energy transfer (FRET). The variation in T_m (range 78.07–81.47 °C; 79.05 to WT) is no larger than two degrees in all cases in regards to CaM WT. The K_ds calculated with all biosensors for CPZ and BIMI (a new inhibitor of CaM) are in the range of 0.45–1.86 and 0.69–1.54 μm respectively. All biosensors retain their ability to activate Calcineurin about 70%. Structural models built “in silico” show their possible conformation taking the fluorophores in protein thus we can predict system stability. Finally, these new biosensors represent a biotechnological development applied to an analytical problem, which aims to determine accurately the affinity of inhibitors of CaM without possible interference, to be put forward as possible drugs related to CaM.     

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ⁻ mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ⁻ mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ⁻ mutant vaccine strain.     

Eradication of cross-contaminated cell lines: A call for action

This “white paper” was prepared and widely disseminated in an attempt to sound an alarm about the long-term existence of a grave, unresolved and growing problem that affects a significant portion of biomedical research, namely, the use of misidentified and cross-contaminated cell cultures. The “white paper” shows how bold action could bring about a profession-wide change in practice that would prevent further erosion. Misidentification and inter- and intra-specific cross-contamination of mammalian cell cultures used in research continues as a widespread problem despite an awareness that dates back more than 45 years. Awareness of the problem has led to a good understanding of the causes of cross-contamination and appropriate preventive measures. It has also led to the application of robust methods for the authentication of cell lines. Yet the problem continues unabated. Estimates of the incidence of research papers flawed by the use of misidentified and cross-contaminated cell cultures approximate 15–20%. The gravity of the situation calls for a strategy that would deliver a remedial message of authentication to virtually all cell culture researchers and also ensure compliance with the message. At the core of the strategy proposed herein is having cell line authentication as a condition for the award of research grants and for the publication of research findings.     

Metastases from metastases: comparative metastatic potential of human cancer cell lines originated from primary tumors or metastases in various tissues

Although metastases from original (primary) tumors are highly studied, metastases from metastatic sites (secondary tumors) are far less studied. Here, using data from metastasis map (MetMap) project reported in a recent study (Jin et al. in Nature 588(7837): 331–336. 10.1038/s41586-020-2969-2, 2020), we found that human cancer cell lines isolated from metastatic sites have higher potential to metastasize to another site in mice, compared to human cancer cell lines isolated from primary sites, for certain types of cancer including liver, lung and pancreas cancer. In contrast, for cancer types such as ovarian and skin cancer, human cancer cell lines originated from primary tumors have increased metastatic potential in mice, compared to human cancer cell lines originated from metastatic sites. This preliminary analysis points that the potential of metastases to further metastasize compared to that of primary tumors might be cancer type-dependent, and further research is needed to understand why certain cancer cell lines isolated from metastatic sites are more likely to spread to other organs.     

Fish cell lines: Establishment and characterization of three new cell lines from grass carp (Ctenopharyngodon idella)

Summary Three new cell lines were established from tissues of the grass carp,Ctenopharyngodon idella. Derived from the fin, snout, and swim bladder of two apparently healthy diploid fry, these cell lines have been designated GCF, GCS-2, and GCSB, respectively. The cells grew at temperatures between 24° and 36° C with optimal growth at 32° C and have been subcultured more than 50 times since their initiation in August 1986. Two of the lines remained diploid or pseudodiploid after 38 passages. The cells were tested for microbial contamination, and plating efficiencies were determined. The three cell lines were sensitive toRhabdovirus carpio (RVC), infectious hematopoietic necrosis virus (IHNV), golden shiner virus (GSV), chum salmon virus (CSV), and infectious pancreatic necrosis virus serotype VR299 IPNV). They were refractory to channel catfish virus (CCV), channel catfish reovirus (CRV), chinook salmon paramyxovirus (CSP), and an Ab serotype of IPNV. This work is a result of research sponsored by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under grant NA85AA-D-5G-095, and was undertaken while the first author was on leave from the Department of Fisheries, Huazhong Agricultural University, Wuhan, China. Salary support was provided by the People's Republic of China. Oregon Agricultural Experiment Station Technical Paper 8952.     

Cell surface oxygen consumption: A major contributor to cellular oxygen consumption in glycolytic cancer cell lines

Oxygen consumption for bioenergetic purposes has long been thought to be the prerogative of mitochondria. Nevertheless, mitochondrial gene knockout (ρ⁰) cells that are defective in mitochondrial respiration require oxygen for growth and consume oxygen at the cell surface via trans-plasma membrane electron transport (tPMET). This raises the possibility that cell surface oxygen consumption may support glycolytic energy metabolism by reoxidising cytosolic NADH to facilitate continued glycolysis. In this paper we determined the extent of cell surface oxygen consumption in a panel of 19 cancer cell lines. Non-mitochondrial (myxothiazol-resistant) oxygen consumption was demonstrated to consist of at least two components, cell surface oxygen consumption (inhibited by extracellular NADH) and basal oxygen consumption (insensitive to both myxothiazol and NADH). The extent of cell surface oxygen consumption varied considerably between parental cell lines from 1% to 80% of total oxygen consumption rates. In addition, cell surface oxygen consumption was found to be associated with low levels of superoxide production and to contribute significantly (up to 25%) to extracellular acidification in HL60ρ⁰ cells. In summary, cell surface oxygen consumption contributes significantly to total cellular oxygen consumption, not only in ρ⁰ cells but also in mitochondrially competent tumour cell lines with glycolytic metabolism.     

A functional role for Smad7 in sustaining colon cancer cell growth and survival

Initially identified as an inhibitor of transforming growth factor (TGF)-β mainly owing to its ability to bind TGF-β receptor type I and abrogate TGF-β-driven signaling, Smad7 can interact with additional intracellular proteins and regulate TGF-β-independent pathways, thus having a key role in the control of neoplastic processes in various organs. Genome-wide association studies have shown that common alleles of Smad7 influence the risk of colorectal cancer (CRC), even though the contribution of Smad7 in colon carcinogenesis is not fully understood. In this study, we assessed the expression and role of Smad7 in human and mouse models of sporadic CRC. We document a significant increase of Smad7 in human CRC relative to the surrounding nontumor tissues and show that silencing of Smad7 inhibits the growth of CRC cell lines both in vitro and in vivo after transplantation into immunodeficient mice. Knockdown of Smad7 results in enhanced phosphorylation of the cyclin-dependent kinase (CDK)2, accumulation of CRC cells in S phase and enhanced cell death. Smad7-deficient CRC cells have lower levels of CDC25A, a phosphatase that dephosphorylates CDK2, and hyperphosphorylated eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, knockdown of Smad7 associates with inactivation of eIF2α, lower CDC25A expression and diminished fraction of proliferating cells in human CRC explants, and reduces the number of intestinal tumors in Apc^min/+ mice. Altogether, these data support a role for Smad7 in sustaining colon tumorigenesis.     

Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines

Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.     

Ribosome induces transdifferentiation of A549 and H-111-TC cancer cell lines

Previously we reported that, lactic acid bacteria (LAB) can induce human dermal fibroblast (HDF) cells to form multipotent cell clusters which are able to transdifferentiate into three germ layer derived cell lineages. Later on, we confirmed that ribosome is responsible for the LAB-induced transdifferentiation and ribosomes from diverse organisms can mimic the LAB effect on HDF cells. In our present study we have shown that, upon incorporation of ribosomes, non-small cell lung cancer cell line A549 and gastric tubular adenocarcinoma cell line H-111-TC are transformed into spheroid like morphology those can be transdifferentiated into adipocytes and osteoblast. Our qPCR analysis has revealed that, during the formation of ribosome induced cancer cell spheroids, the expression of the cancer cell associated markers and cell cycle/proliferation markers were altered at different time point. Through our investigation, here we report a novel and a non-invasive approach for cancer cell reprogramming by incorporating ribosomes.     

DNA fingerprinting—a valuable new technique for the characterisation of cell lines

DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic “snap-shot” of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.     

HLA-A0201 positive pancreatic cell lines: new findings and discrepancies

Pancreatic cancer is being pursued as an immunotherapy target using antigen-specific vaccine approaches activating CD8(+) CTL and CD4(+) T-helper cells. CD8(+) CTL exert their anti-tumor effects in an HLA-restricted manner and only tumor cells carrying a matched HLA class I sub-type are targets for antigen-specific CTL. In the process of characterizing CD8(+) T cell responses against pancreatic cancer, we screened a number of human pancreatic tumor cell lines for HLA-A0201 positive (HLA-A2(+)) cell lines to be used in the evaluation of CTL function. This analysis revealed some new findings and discrepancies in the literature on the HLA sub-type of some commonly used pancreatic cell lines. We found that Capan-1 cells, originally reported to be HLA-A0201(+), actually only express HLA-A010101 and HLA-A300101 and were targets for HLA-A0201-restricted CTL only after transduction with an HLA-A0201-expressing lentivirus. Panc-1 cells were found to be HLA-A0201 positive, in agreement with published reports, while CF-Pac-1 cells were found to express both HLA-A020101 and HLA-A030101. We also found a normal human pancreatic ductal epithelial cell line, HPDE, to be HLA-A0201 positive. Our findings were verified with two different sequence-based typing methods, antibody staining followed by flow cytometry analysis, and functional analysis using an HLA-A0201-restricted peptide-specific T cell response.     

A method for prolonged survival of primary cell lines

Summary We have established a means for prolonged survival of primary cell cultures and establishment of continuous cell lines without genetic manipulations. Primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. Earlier attempts to circumvent this limitation have included transformation with oncogenes, spontaneous immortalization of primary cultures, and chemical carcinogenesis. We have found that addition of a complex of growth-promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. This same supplement allows the prolonged survival of primary cell cultures derived from adult rat ovaries. The rat ovary primary cultures consisted of mixed phenotypes, including epithelial, neuron-like, and mesenchymal cell types. Numerous cells stain positive for alkaline phosphatase in these cultures. Other primary cell lines were established from embryonic rat liver and from adult rat lungs, using the same supplement. The survival effect is reversible because cells degenerate when the supplement is removed. Therefore, the cell lines have neither acquired properties of a tumor cell line nor have they been immortalized by a virus infection. We expect that our approach will open the door to prolonged survival of other primary cell types.     

Cancer pagurus (Linnaeus, 1758) physiological responses to simulated live transport: Influence of temperature, air exposure and AQUI-S

Cancer pagurus is a commercially important crab, mostly appreciated in Southern European countries, including Portugal, being usually live transported from UK and France. Once in Portugal, crabs are redistributed across the country in small refrigerated vivier lorries in air or immersed conditions, while some are sent exposed to air to the Azores archipelago with mortality reaching 40-60%. In order to optimise transport conditions and survival, simulated live transport of immersed and air exposed crabs sedated or not with an anaesthetic, AQUI-S^®, was tested at different temperatures. It was found that crabs experienced stress during the experiment (with increased l-lactate, d-glucose and lowered pH), but with different magnitudes according to temperature, treatment and transport duration, resulting in 100% mortalities at 16 °C in immersed conditions and when exposed to air with AQUI-S^®. Results indicate that long duration transport in semi-dry conditions is viable at low temperatures (8 °C), while immersed transport is viable at 12 °C. AQUI-S^® was not an efficient solution at low temperatures in semi-dry conditions, but for short duration transport in immersed conditions at 16 °C it was the only treatment without mortality.