Establishment Of Continuous Cell Lines Research Articles

Optimized conditions for the long-term growth of primary cell cultures derived from the Asian citrus psyllid, Diaphorina citri (Liviidae: Hemiptera)

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a pest of significant importance to global citrus production, particularly as the vector of a phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) that causes the fatal citrus disease Huanglongbing or citrus greening. CLas is acquired as the psyllid feeds, replicates in ACP tissues, and persists throughout the life of the insect. The study of CLas has been hampered by the lack of a tractable in vitro culture system. As CLas replicates within psyllid tissues, we hypothesize that this bacterium also replicates in cultured ACP cells. In the current study, we evaluated a range of insect cell culture media, media combinations, and supplements for their ability to support the in vitro growth of ACP embryo-derived cells. Ninety-six primary cell cultures were initiated using approximately 12,000 dissected ACP eggs over a 12-month period. Of 19 media tested, 17 supported cell attachment, but only two media supported the long-term survival and growth of ACP embryonic cells over a period of more than 11months. Delineation of the optimal protocols and conditions for the maintenance of ACP primary cultures as described here provides a foundation for both establishment of continuous cell lines and testing for the replication of ACP-associated pathogens including CLas.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

海洋无脊椎动物胚胎细胞培养研究进展

胚胎细胞具有活跃的分裂增殖能力和多向分化潜能，是建立细胞系的最佳组织来源。但是由于微生物和原生动物污染以及体外培养条件下细胞不分裂等原因，海洋无脊椎动物的细胞培养研究目前仍停留在原代细胞培养和有限传代培养的水平上，尚无永生性细胞系的成功报道。本文综述了海洋无脊椎动物中5个门类包括多孔动物门、腔肠动物门、节肢动物门、软体动物门和棘皮动物门的胚胎细胞培养工作的研究进展，并对海洋无脊椎动物胚胎细胞培养的前景进行了展望。 Embryo cells with high potential in active proliferation and pluripotency are optimal cell sources for the establishment of continuous cell lines. However, due to the frequent occurrence of con-tamination by microorganisms and protozoans as well as the mitosis-arrestment of primary cul- ture cells, only primary cell cultures and limited subculture are successful in the cell culture of invertebrates, and no immortalized cell line has been established from invertebrates. This paper has reviewed the advances in the embryonic cell culture of marine invertebrates including Porifera, Coelenterata, Arthropoda, Mollusca and Echinodermata, and predicted its future outlook.

A METHOD FOR PROLONGED SURVIVAL OF PRIMARY CELL LINES

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.

Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA

Immortalization of human keratinocytes (HKc) by human papillomavirus type 16 (HPV16) is reproducible at a high frequency, is due directly to the presence of the viral sequences in the cells, and occurs independently from the genetic characteristics of the host cells. Ten human keratinocyte strains, each derived from a different individual, were transfected with pMHPV16d and selected with G418. Eight became established lines. Two strains, which failed to grow shortly after successful G418 selection, were negative for HPV16 DNA. No lines were established following transfection of the same HKc strains with vector sequences only. The immortalized lines maintained a constant number of copies of the viral genome integrated into the cellular DNA. Each line showed a unique integration pattern of HPV16 sequences into the cellular genome, but expressed similar patterns of viral messages. Sublines able to grow in the absence of growth factors (epidermal growth factor and bovine pituitary extract), and others which became resistant to differentiation stimuli (serum and calcium) were obtained by selection in growth factor-free medium and serum-supplemented medium, respectively. The establishment of continuous cell lines is a direct consequence of the presence of viral sequences; however, because none of these lines formed tumors in nude mice, additional events must be necessary for progression of malignancy. HPV16-immortalized human keratinocyte lines can be used to investigate and identify the viral factors involved with the modification of growth and differentiation control by HPV16.

Further Establishment of Continuous Cell Lines from Larval Fat Bodies of the Cabbage Armywarm, Mamestra brassicae (Lepidoptera : Noctuidae)

Further Establishment of Continuous Cell Lines from Larval Fat Bodies of the Cabbage Armywarm, Mamestra brassicae (Lepidoptera : Noctuidae)

Continuous cell lines with specific killer T-cell activity

THE establishment of continuous cell lines with effector T-cell activity is an obvious starting point in the isolation and characterisation of the T-cell receptor complex. Although nature provided the plasmacytomas which played a key part in elucidating B-cell immunoglobulin structure, there are no known examples of naturally occurring lymphomas with effector T-cell activity. Initial success with fusions between plasma cells and plasmacytomas encouraged extrapolation of this technique to T cells and thymic lymphomas1, but few promising results have so far been obtained. We have approached this problem by using the carcinogenic properties of mutagens to transform T lymphocytes responding in mixed lymphocyte culture (MLC), with the aim of isolating cell lines with killer T-cell activity. Here we report our success in the first step of isolating continuous, though antigen-dependent, killer T-cell lines.

Occurrence of Epstein-Barr virus in human leukocyte cultures.

Most human long-term lymphoid cell lines contain Epstein-Barr viral (EBV) DNA and virus-specific antigens. The available evidence suggests that EBV plays a role in the establishment of continuous cell lines of leukocytes derived from normal donors. These cell lines contain several copies of the whole EBV genome in a repressed state which can in some cases be activated by treatment with halogenated pyrimidines.

Established cell line (ICI 101) obtained from frozen human lymphoblastic leukaemia cells: Morphological and immunological comparison with the patient's fresh cells

We have established a cell line (ICI 101) from blood taken from a patient with lymphoblastic leukaemia and stored at − 70°C. A comparative morphological, virological and immunological study of this line and the fresh cells have been made. Virus particles, resembling murine leukaemia viruses, were detected in the fresh cells and found in samples from the cultures. The antigenic activity of the lymphoblasts, as demonstrated by immune adherence with the patient's serum, was present in the cultured cells. The antigenicity of the cultured cells was greater with the fresh cells (in immune adherence and fluorescence). This phenomenon is under study. This lymphoblastic line (ICI 101) which has retained its virus content and antigenic marker, is an excellent material for the study of human leukaemia. Finally, the isolation of this line from frozen cells shows that freezing of the original cells to − 70°C is not an obstacle to the establishment of continuous cell lines.