A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

Two new cell lines originated from the embryos of <Emphasis Type="Italic">Clostera anachoreta</Emphasis> (Lepidoptera: Notodontidae): characterization and susceptibility to baculoviruses

Two cell lines designated CAF-Clan I and CAF-Clan II have been established from embryos of Clostera anachoreta (Lepidoptera: Notodontidae) in TNM-FH medium containing 10% inactivated fetal bovine serum. CAF-Clan I consists of a mixture of three cell types: spherical cells, spindle-shaped cells, and giant cells. Most of the cultured cells formed a suspension in the medium and were subcultured more than 60 passages. CAF-Clan II mainly consists of spindle-shaped and spherical cells which attached to the culture surface and have undergone more than 40 passages. The cell population doubling time at 27°C of CAF-Clan I at passage 22 and CAF-Clan II at passage 24 was about 68.5 and 38.2 h, respectively. The chromosome number of both cell lines at passage 15 varied from 62 to 100 in the majority of cells, though a few cells exceeded 260 (n = 30). DNA amplification fingerprinting–polymerase chain reaction analysis confirmed that the origination of the two cell lines was C. anachoreta. The susceptibility of the cell lines to baculoviruses was tested. The results showed that CAF-Clan II was susceptible to infection of Autographa californica nucleopolyhedrovirus (AcMNPV) and Ecotropis oblique nucleopolyhedrovirus (EoNPV). Occlusion bodies (OBs) production was 129 ± 4 OBs/cell and 124 ± 15 OBs/cell for AcMNPV and EoNPV, respectively. CAF-Clan I was less susceptible to AcMNPV compared with CAF-Clan II, while non-permissive to EoNPV.     

Establishment and characterization of fibroblast cell lines from the skin of the Yangtze finless porpoise

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), as the sole freshwater subspecies of N. phocaenoides, is endemic to the Yangtze River and its adjacent lakes. Its population has declined significantly over recent decades. In this study, we established a skin-derived finite fibroblast cell line of the Yangtze finless porpoise, named YFP-SF1, using primary cell culture methods, and an immortalized cell line, T-YFP-SF1, through co-transfection (GFP and SV40 T antigens) techniques. YFP-SF1 proliferated continuously with a minimum population doubling time of 31 h and exhibited age-dependent changes in growth rate. T-YFP-SF1 cells exhibited fibroblast morphology and were characterized by a shorter doubling time, higher attachment efficiencies, colony formation at a low seeding density, and growth in low serum concentrations. Anchorage independence and foci formation in the cell monolayer were observed from passage 36. The chromosome number of YFP-SF1 and T-YFP-SF1 remained stable at 2n = 44 in the early passages, and the viability of thawed cells remained above 90% after cryopreservation in liquid nitrogen. Taken together, we have established fibroblast cell lines of Yangtze finless porpoise for the first time, which might assist as an in vitro model for this endangered mammal.     

家蚕胚胎细胞系BmE-SWU2的建立及其生物学特性研究

家蚕反转期胚胎组织经过一年多的原代培养,建立了BmE-SWU2细胞系。该细胞系细胞呈短梭形或圆形,细胞较小,属上皮型贴壁生长细胞系;细胞生长速度快,铺平率高于80%,繁殖力强,细胞群体倍增时间为51.8 h。BmE-SWU2细胞系的二倍体细胞（2n=56）比例达到89.76％,属二倍体细胞系,其染色体具有鳞翅目昆虫染色体的典型特征,中期染色体呈短杆状或颗粒状。BmE-SWU2细胞系对BmNPV高度敏感,TCID₅₀为1.581×10^-7。     

Establishment and characterization of a new <Emphasis Type="Italic">Aedes aegypti</Emphasis> (L.) (Diptera: Culicidae) cell line with special emphasis on virus susceptibility

A new cell line from the neonate larvae of Aedes aegypti (L) mosquito was established and characterized. The cell line at the 50th passage (P) level consisted of three prominent cell types, i.e., epithelial-like cells (92%), fibroblast-like cells (7%), and giant cells (∼1%). Karyological analysis showed diploid (2n = 6) number of chromosomes in >75% cells at P-50. The growth kinetics studied at 52nd passage level showed approximately tenfold increase in cell number over a 10-d study period. The species specificity studies using DNA amplification fingerprinting profile analysis using RAPD primers demonstrated 100% homology with the host profile showing the integrity of the cell line. Electron microscopy revealed the absence of mycoplasma or other adventitious agents. The cell line supported the multiplication of seven arboviruses, i.e., Chikungunya (CHIK), Japanese encephalitis, West Nile, dengue 2 (DEN-2), Chandipura, vesicular stomatitis, and Chittoor viruses. The cell line did not replicate Ganjam and Kaisodi viruses. CHIK virus yield in the new cell line was approximately 3log and 0.5log 50% tissue culture infective dose (TCID₅₀)/mL higher than Vero E6 and C6/36 cell lines, respectively. In the case of DEN-2 virus, it yielded 1log TCID₅₀/mL higher than Vero E6, but lesser than C6/36 cell line. Due to its high susceptibility to a broad spectrum of viruses, the new cell line may find application in virus isolation during epidemics and in antigen production.     

八字地老虎血球细胞系的建立

由八字地老虎Xestia c-nigrum血细胞建立了一株细胞系,命名为NEAU-Xc-960716H,原代培养90余天,现已传至70余代。细胞多为圆形,部分梭形,细胞群体倍增时间约为63 h。具有典型的鳞翅目昆虫染色体特征,数量多,形态为短杆状和球形。酯酶同工酶谱为5条主带,与同种昆虫（八字地老虎）的胚胎细胞系(NEAU-Xc-730E)酯酶图谱稍有不同,而与草地夜蛾细胞系(IPLB-SF-21)的酯酶图谱完全不同。该细胞系可以被八字地老虎核型多角体病毒XcNPV感染,但感染率较低。     

ESTABLISHMENT OF A CELL LINE FROM EMBRYONIC TISSUES OF THE FLESHFLY, SARCOPHAGA PEREGRINA (INSECTA: DIPTERA)

A continuous cell line was established from embryonic tissues of the fleshfly, Sarcophaga peregrine , and was designated as NIH-SaPe-4. The primary culture was initiated in October, 1977, and the cell line was passed 68 times during the following year. The cells were heterogeneous in morphology. Most cells were diploid and their chromosomes consisted of 4 metacentric, 6 sub-metacentric and 2 micro chromosomes. The population doubling time of the cell line was about 30 hr. The cells grew faster in Mitsuhashi-Maramorosch's medium than in Schneider's medium. The cells were either stored in the usual medium at 5°C for about 3 months, or in a medium containing 10% glycerol at –80°C for a longer period. Cell growth was suppressed by 20-hydroxy-ecdysone when at a greater concentration than 0.01 μg/ml, whereas insulin showed no effects on cell growth at a strength of 0.4 and 0.04 IU/ml.     

Factors affecting somatic embryogenesis in anther cultures of Chinese pink <Emphasis Type="Italic">(Dianthus chinensis</Emphasis> L<Emphasis Type="Italic">.)</Emphasis>

In this study, we aimed to maximize the rates of somatic embryogenesis achievable in anther cultures of Chinese pink (Dianthus chinensis L.) (2n = 2x = 30). The genotype of the donor plant was found to be a major factor in determining the success rate. Conditions imposed during anther culture (notably medium composition and light conditions) and pretreatments (namely, cold, heat, and mannitol incubations) were also found to influence somatic embryo induction. For example, the highest levels of embryogenic callus induction were achieved when the donor buds had been cold pretreated and the subsequent anther culture was maintained in darkness. Furthermore, there appeared to be an interaction of genotype with culture conditions. Thus, in cultures of the cultivar (cv.) ‘Carpet’, the highest rates of embryogenesis were obtained when the anthers had received a 5-d heat-shock, but such a thermal treatment did not generally produce a significant effect. Likewise, a 3-d mannitol pretreatment was optimal only for the cross-hybrid line ‘HC’. Assessment of the ploidy of the plants regenerated from the anther cultures revealed both diploid and tetraploid plants. Histological and cytological observations showed that all of these (both from n-pollen and 2n-pollen lines) derived from anther wall cells. Spontaneous chromosome doubling was inferred to have occurred during the embryogenic callus culture period.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

Development and characterization of a continuous cell line PSCF from Puntius sophore

A continuous Puntius sophore caudal-fin (PSCF) cell line of the pool barb Puntius sophore, an important freshwater food and ornamental fish of Asia and South East Asia, was developed from the caudal fin for the first time. The cell line was optimally maintained at 28° C in Leibovitz-15 (L-15) medium supplemented with 10% foetal bovine serum (FBS). The cytogenetic analysis revealed a diploid count of 50 chromosomes at 25th and 50th passage and 52 chromosomes at passage 70, 85 and 100. The viability of the PSCF cell lines was 75% after 6 months of storage in liquid nitrogen (-196° C). The most striking feature of the PSCF cells was its high increased growth ratio as evident from the population doubling time of 25 h at passage 100. The origin of the cell lines was confirmed by the amplification of 581 and 655 bp fragments of 16 S rRNA and cytochrome oxidase subunit I (COI) of mitochondrial DNA (mtDNA) genes, respectively. The PSCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the potential utility of the cells in gene expression studies.     

Establishment and characterization of a cell line from the mosquito Culex tritaeniorhynchus (Diptera: Culicidae)

We established a continuous cell line from the embryo of the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae), a known major vector of the Japanese encephalitis virus (family Flaviviridae, genus Flavivirus) in Asia. The cell line, designated NIID-CTR, was serially subcultured in VP-12 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS). It continued to grow for more than 60 passages over a 750-d period. The NIID-CTR cell line mainly comprised two morphologically distinct types of cells with adhesive properties: spindle-shaped and round cells. Most of the NIID-CTR cells at the 45th passage were diploid (2n = 6). The growth kinetics of the NIID-CTR cells was significantly affected by the FBS concentration in the medium. The population doubling time of the NIID-CTR cells was 20 h in the presence of 10 % FBS and 76 h in its absence. The DNA sequence of the mitochondrial cytochrome oxidase I gene confirmed that the NIID-CTR cell line was derived from C. tritaeniorhynchus. The cells were highly susceptible to Japanese encephalitis and Dengue viruses, thus providing a valuable tool for the study of mosquito-borne flaviviruses.     

Establishment of the AU-bek cell line and comparison with two other bovine cell lines

Summary Establishment of a new bovine cell line, AU-BEK, is reported. The cell line developed in a culture initiated from bovine embryonic kidneys by spontaneous cultural alteration to epithelioid cells that are indefinitely propagable. Epithelioid cells gradually increased to become the predominant cell. Whereas normal bovine cells have a diploid number of 60 chromosomes, of which only the two sex chromosomes are biarmed, AU-BEK cells at the 80th passage had a modal chromosome number of 84 and an average of 30 biarmed chromosomes per cell. AU-BEK cells are now in their 220th passage. Of the AU-BEK, MDBK, and CKT-1 bovine cell lines, the CKT-1 cell line had a karyotype closest to that of normal bovine cells. Their modal chromosome number was 57, and only three biarmed chromosomes were usually present. The bovine character of AU-BEK and CKT-1 cells was established by cytotoxic and viral susceptibility tests. Supported by the Alabama Agricultural Experiment Station. Publication No. 1115, School of Veterinary Medicine, Auburn University.     

Two new C3H mouse ascites tumor cell lines capable of proliferation in vivo and in suspension culture: Morphological,karyological, kinetic,and immunological properties

Summary The establishment of mouse tumor cell lines capable of proliferating in vivo and in suspension culture was undertaken. The MM-46 tumor line, initiated from primary mammary carcinoma arising in a C3H/He mouse, was maintained for over 100 generations in the peritoneal cavities of syngenic mice. At the 50th generation of the tumor suspension, cultures were initiated. The established cell lines, designated TMT-1 and TMT-2, were characterized in vitro and in vivo. The morphological finding indicated that TMT-1 and TMT-2 cells from mice closely resembled the MM-46 tumor cells. The oncogenic potential of the cultured cells was comparable to that of the original ascites tumor. The population doubling time of TMT-1 and TMT-2 cell lines was about 12 hr in mice, whereas the population doubling time of both cell lines lengthened to 20 hr in suspension culture. The increase of doubling time in culture was due to the prolongation of the G₁ period. The cell lines, TMT-1 and TMT-2, whether from culture or mice, possessed colony forming ability in soft agar medium. The colony forming ability of the cells decreased gradually through in vivo passages but it recovered upon recultivation of the cells from mouse to culture. Chromosome analysis and cytotoxicity test by anti-MM antiserum indicated that TMT-1 and TMT-2 cell lines closely resembled and had been derived from MM-46 tumor line. Therefore, it is possible to assay cell survival in vitro after in vivo experiments on these cells.     

In vitro cultivation of human renal cell cancer

Summary Two cell lines derived from primary human renal-cell cancers (RCC) have been established and characterized. Cell line 786-O has been in culture for longer than 1 year and has been subcultured more than 50 times. It has a doubling time of 45 hr and a hypertriploid karyotype and possesses a Y chromosome. Cell line 769-P also has been in culture for longer than 1 year. It has been subcultured 50 times and has a doubling time of 35 hr and a hypodiploid karyotype. Cells from both lines are epithelial, and they produce tumors in the cheek pouches of immunosuppressed hamsters. Neither cell line is contaminated withMycoplasma. Cells of the two lines can be distinguished from HeLa cells both by their karyotypes and by the mobility patterns of their isoenzymes of glucose-6-phosphate dehydrogenase. American Cancer Society Clinical Faculty Fellow, 1977–1980. Supported in part by Grants CA 13095-03 and CA 15551-03 from the National Cancer Institute.     

Establishment and characterization of an embryonic cell line from Gampsocleis gratiosa (Orthoptera: Tettigoniidae)

The first continuous cell line from the embryo of Gampsocleis gratiosa (Orthoptera: Tettigoniidae), designated as RIRI-GG1, was established. This cell line was serially subcultured in modified Grace medium. The cells were grown adherent to a culture flask and had spindle-like and polygonal shapes. The chromosome number ranged from 26 to 79 at the 50th passage, and 68% of cells had a diploid chromosome number. The growth rate was determined at the 53rd passage, and the population doubling time was calculated to be 122.1 h. The rDNA internal transcribed spacer and the mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-GG1 cell line was derived from G. gratiosa. This cell line had no apparent susceptibility to Autographa californica nucleopolyhedrovirus and Bombyx mori nucleopolyhedrovirus.     

A new insect cell line from the longicorn beetle Plagionotus christophi (Coleoptera: Cerambycidae)

We have established a continuous cell line from the fat body tissue of the longicorn beetle Plagionotus christophi. The cells have been serially subcultured in MGM450 medium, and the line has been designated as PC-1. The cells were grown in suspension and comprise largely flattened spindle- or oval-shaped cells morphologically related to blood cells of longicorn beetles. The chromosome number ranged from 19 to 36, with a mode of 20 (diploid). The growth curve was determined at the 100th passage, and the population doubling time was calculated to be 3.79 d. Isozyme analysis of malic enzyme, phosphoglucose isomerase, and phosphoglucomutase revealed that PC-1 cells were enzymatically distinct from coleopteran XP-1, AnCu-35, dipteran AeAl-2, and lepidopteran GaMe-LF1 cell lines.     

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.     

Establishment and cryopreservation of a cell line derived from caudal fin of endangered catfish Clarias dussumieri Valenciennes, 1840

We describe a new cell line, Clarias dussumieri fin (ClDuF), from the caudal fin of C. dussumieri using the explant technique followed by cryopreservation. The cryopreserved CiDuF cells were validated for quality and other characteristics. They showed typical epithelial morphology in vitro and epithelial cells outgrew their fibroblast cells after the fifth passage. ClDuF cells had a characteristic sigmoid curve with population doubling in 24 h. Immunotyping of the ClDuF cells against cytokeratin suggested the epithelial lineage. Chromosome analysis showed normal diploid (2n = 50) numbers and the cells did not contain any contamination, including Mycoplasma and other microbes. Partial sequencing of fragments of mitochondrial 16s rRNA and COI genes of ClDuF confirmed that the cell line was initiated from C. dussumieri. Cells at the 10th and 25th passages had more than 80% and 70% viability in the culture, respectively, after 6 months of storage at LN₂. These ClDuF cells were morphologically identical to the cells before freezing and the genetic resource of C. dussumieri was preserved. The species-specific cells can serve as a valuable source for virus isolation, conservation and cloning of somatic cells.     

Activation of metastatic potential in african green monkey kidney cell lines by prolongedin vitro culture

Summary Studies on the tumorigenicity of Vero kidney cells ofCercopithecus aethiops monkey origin were extended to various passage levels of BSC-1 aneuploid cells and to low passage CV-1 diploid cells (derived also fromC. aethiops monkey kidney). It was found that BSC-1 cells—like Vero cells—showed increased tumorigenicity with increasing passage level in antitymocyte globulins (ATG) treated newborn rats and in nude mice. Cells passaged over 250 times in cultures formed invasive adenocarcinomas in newborn rats. Their malignant tumor growth was further demonstrated around the 500 passage level when tumor metastases were detected in the lungs of four of the 14 inoculated rats. Vero cells induced such lung metastases in rats already at passage 227. CV-1 diploid cells at low passage level produced small nodules of epithelioid cells in newborn rats at 6th day after inoculation that had disappeared by the 21st day, and caused no local invasion nor lung metastasis. In vitro tumorigenicity tests on BSC-1 and CV-1 cells, using chick embryo skin, human muscle and colony formation in agarose, confirmed the animal test results. The results of this study indicate that BSC-1 and Vero cell lines at low and high passage levels may prove to be useful tools to study the molecular basis of malignancy. Editor's Statement In this paper Contreras et al. document the increased malignancy of commonly-used monkey cell lines upon long-term culture. These observations have implications for the use of these cell lines in studies of cancer cell biology, as well as the use of these lines for the production of biologicals. David W. Barnes