Rat pleural mesothelial cells adapted to serum-free medium as a model for the study of growth factor effects

Mesothelial cells are the putative progenitors of mesotheliomas and cell lines have been used as tools to study the responses of these cells to various stimuli, including growth factors. The present study was undertaken to develop a rat mesothelial cell line capable of sustained growth under serum-free conditions with the object of avoiding the possible confounding effects of undefined serum components. Responses of mesothelial cells to epidermal growth factor were shown to differ under serum-free versus low-serum culture conditions. In contrast, a cell line, SFM1, adapted to growth in serum-free medium was characterized and found to exhibit responses to growth factors similar to the responses reported for human cell lines. This new line should prove to be a useful model for the study of these cells in vitro .     

Male diploid embryonic cell line ofDrosophila virilis

Summay A new established cell line 79f7Dv3g, ofDrosophila virilis consisting initially of male and female cells and represented now, after 6 yr of cultivation, only by male cells is described. The population doubling time is 36 h at 25° C. The cell culture is also able to grow in serum-free media for an indefinite time without special selection and has a population doubling time of 2 d.     

The differential effects of cadmium exposure on the growth and survival of primary and established cells from fish and mammals

The differential cytotoxic effects of cadmium on fish and mammalian epithelial cells in established and primary culture were assessed by looking at the reduction of the colony-forming ability and reduction in the extent of growth. The influence of medium composition on the toxicity of cadmium was also studied using serum-free and serum-containing media. The experiments using immortalized cell lines showed that mammalian cells were more sensitive than fish cells to cadmium. Both cell types were grown at the same serum concentration. However, using the normal primary system, human epithelial tissue explants showed less sensitivity to cadmium than did similar cultures from rainbow trout. It is likely that cellular mechanisms of cadmium resistance in the different cell types are responsible for these effects. As expected, cadmium proved to be more toxic when tested in serum-free medium. With fish skin primary cultures, reduction of cell numbers was observed at concentrations as low as 5 mol/L in serum-free medium compared to 100 mol/L in serum-containing medium. This was found to be due to the reduction in the activity of free cadmium ions, caused by the presence of serum in the medium. It is concluded that serum-free formulations are probably preferable when testing pollutants in vitro. The results highlight the importance of establishing species-specific pollution tests and standardizing variables.In summary, the results show species and culture media differences in cadmium toxicity and suggest that caution is required when extrapolating results for pollutant effects from one in vitro system to another.Abbreviations CE colony-forming efficiency - EPC epithelioma papulosum cyprini - KGM Clonetics Keratinocyte Growth Medium     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.     

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10⁷ cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

Vero细胞无血清培养技术的研究与应用

Vero细胞是世界卫生组织和我国生物制品规程认可的疫苗生产细胞系。随着对疫苗质量和安全性要求的不断提高,用无血清培养基取代含血清培养基培养Vero细胞已成为病毒疫苗生产的一个重要发展趋势。Vero细胞无血清培养的技术关键是研发或选择能支持细胞以贴附培养方式生长的无血清培养基。微载体培养是贴附依赖性细胞系规模化培养和病毒疫苗生产的有效技术途径。我们对Vero细胞无血清培养基的研发、Vero细胞无血清培养及病毒疫苗生产工艺做了讨论,对该领域存在的问题和发展策略进行了展望。     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Growth of kidney epithelial cells in hormone-supplemented,serum-free medium

Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors – insulin, transferrin, prostaglandin E₁, hydrocortisone and triiodothyronine – as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E₁ in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum-free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in the absence of fibroblast over-growth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

犬肾细胞MDCK无血清贴壁及单细胞悬浮培养

旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程,以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象,对2 656个可编码区进行分析,获得特异性靶点序列,并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性,筛选获得9个DNA序列,并设计了4对引物。经验证2对引物的特异性较好,其中引物SA3的基因组DNA检测限为13.7 fg/μL,菌体检测限为9.25×102 CFU/mL。结果验证     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

Interferon production by mammalian cells grown in a serum-free medium

Summary Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.     

Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts in coculture

Summary In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.     

The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein

UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.     

Coitinuous cell lines from embryonic tissues of ticks (Acari: Ixodidae)

Summary Six new cell lines were established in continous culture from embryonic tissues of ixodid ticks. Four were fromDermacentor variabilis and two fromD. parumapertus. The cells are mostly fibroblastic and diploid. Mosquito-borne viruses (Chikungunya, O'nyong, yellow fever, and St. Louis encephalitis) as well as tick-borne ones (Langat, Powassan, Colorado tick fever, Kemerovo, and Sawgrass) replicated in certain of these cell lines, but a nonvector-borne flavivirus, Modoc, did not. An undescribed virus fromD. occidentalis ticks, which could not be isolated in Vero cells or newborn mice, was readily isolated in theD. variabilis cell line.     

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions

Summary We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.