Gravisensitivity of endothelial cells: the role of cytoskeleton and adhesion molecules

The review addresses the effect of microgravity on the endothelial cells, an important mechanosensory element of the cardiovascular system that is known to undergo functional changes in space flight. The chalanges that arise in performing space flight experiments are presented, as well as approaches used to simulate microgravity effects in vitro. The role of cytoskeletal elements as the putative gravity sensors in the cells is demonstrated. The changes in the expression of adhesion molecules that may underlie the mechanisms of gravity sensing by endothelial cells are described. The possible reasons for the discrepancies between the results obtained, such as the differences between the cell lines and experimental design, the variation in time of cultivation, and the specific spaceflight related factors, are analyzed.     

小鼠骨髓源树突状细胞体外诱导培养及初步鉴定

用重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组小鼠白细胞介素4（rmIL-4）体外诱导小鼠骨髓细胞分化为树突状细胞,进行形态学变化观察,分析细胞表面分子,刺激T细胞增殖,探讨小鼠骨髓源树突状细胞（BMDC）体外诱导培养并进行初步鉴定。体外培养9d后BMDC可达80%以上,光镜下可见典型的树突状细胞形态。清楚表达成熟期主要表面标志物,可显著刺激同种异体混合淋巴细胞增殖。获得了较高纯度的BMDC,避免了使用传统磁珠分离方法所带来的成本高,操作复杂,产出率低的弊端,为研究BMDC功能以及运用开展下游实验提供材料。     

Dengue virus plaque formation on microplate cultures and its application to virus neutralization (38564).

Dengue virus plaque formation on BHK-21 cell microplate cultures was described. The clear plaques were visible usually 5 days after incubation in a CO-2 incubator at 37 degrees. The cells cultured in a 3-oz bottle were sufficient to prepare two microculture plates which were usually ready for use after 1-2 days of cultivation in the CO-2 incubator at 37 degrees. The overall procedures were easy and of economic advantage.     

Effect of magnetic nanoparticles Fe3O4 on viability, attachment and spreading of cells isolated from fetuses and new-born rats

This study shows that toxic effect of non-modified nanoparticles of Fe3O4 in vitro depends on metabolic and morphological condition of cells, derived from fetuses and newborn rats. During the cultivation of cells with magnetic nanoparticles, the new-born rats. During the cultivation of cells with magnetic nanoparticles, the latter bind to the cell surface and penetrate into the intracellular space. In this case, sorption of nanoparticles on the cell surface makes it difficult for cells to adhere to substrate, and the absorption by spread cells may prevent their proliferation. Magnetic nanoparticles are well absorbed by the upper layer of cellular aggregates. In this case, the cells of the inner layer remain intact. Consequently, the cell aggregates are able to respond to the constant magnetic field. These aggregates could potentially by used in cell transplantation for directed cell delivery.     

A new type porous carrier and its application to culture of suspension cells

A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate. The porous space of the carrier is an assembly of void spaces. The carrier was successfully applied to the cultivation of suspension animal cells. In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers. A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7×10⁷ cells per ml-carrier.     

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.     

Improvement in enzyme productivities from mold cultivations using the liquid-phase oxygen supply strategy

Cultivations of Aspergillus niger cells in which oxygen was provided through the liquid-phase oxygen supply strategy (which involves the need-based decomposition of H2O2 pulses to yield the necessary oxygen) were studied. Concentrations of H2O2 in the range of 1 to 5 mM were found to be suitable for use in cultivations. The growth rate constant of 0.17 h(-1) obtained in the H2O2-based cultivation, was comparable to that obtained in the cultivation with aeration; however, the maximum cell concentration in the H2O2-based cultivation was 124% of that obtained in cultivation with aeration. Maximum concentrations of catalase, protease and glucose oxidase obtained in the H2O2-based cultivation, were 240%, 172%, and 124% respectively, of those obtained in the cultivation with aeration. Further, the specific enzyme levels (units per gram cell) of catalase and protease obtained in the H2O2-based cultivation were 172% and 156% of those obtained in the cultivation with aeration, whereas, the specific glucose oxidase levels were comparable. In addition, the oxygen profiles inside the pellets ofA. niger during both the modes of oxygen supply were described using mathematical models. Studies to elucidate the mechanism of oxygen availability showed that the decomposition of the H2O2 supplied in the extracellular space occurred intracellularly, as well as extracellularly, to yield oxygen. Also, the proton motive force (PMF) was found to be involved in the process of oxygen availability from H2O2 to A. niger cells.     

旋毛虫肌幼虫细胞传代培养及超微结构观察

消化、分离观察旋毛虫(Trichinella spiralis)肌幼虫,获得肌幼虫细胞,用含10%胎牛血清的RPMI-1640培养液培养原代细胞,胰酶(含0.02?TA)消化法进行传代,透射电镜观察培养细胞超微结构,用多重PCR鉴定培养细胞。结果表明,在培养24～72h原代细胞开始贴壁,7～8d形成单层细胞,细胞间融合现象不明显,10～12d传一代。透射电镜显示旋毛虫细胞核为椭圆形,核膜、核仁清晰,核内染色质较丰富,胞浆含丰富的线粒体。细胞主要有两种类型:椭圆形和多角形,以椭圆形为主。多重PCR扩增培养细胞DNA,可见1条与旋毛虫肌幼虫DNA扩增产物相同的条带(173bp)。结果表明,旋毛虫肌幼虫细胞可在含10%胎牛血清的RPMI-1640培养液中传代培养。     

Circular RNAs Co-Precipitate with Extracellular Vesicles: A Possible Mechanism for circRNA Clearance

Backspliced circular RNAs (circRNAs) are prevalent in many eukaryotic systems and are spliced from thousands of different genes. Where examined, circRNAs are often highly stable and the mechanisms by which cells degrade and/or clear circRNAs from the cells are unknown. Here we investigated the possibility that cells can eliminate circRNAs into extracellular space, possibly within released vesicles such as exosomes and microvesicles. From three different cell lines and examining multiple circRNAs, we show that extracellular vesicle (EVs) preparations recovered from cell culture conditioned media contain established circRNAs. Moreover, these circRNAs are enriched over their linear counterparts within EV preparations when compared to the producing cells. This supports the idea that expulsion from cells into extracellular space, as by EVs release, can be a mechanism by which cells clear circRNAs. Moreover, since EVs can be taken up by other cells, excreted circRNAs could contribute to cell to cell communication.     

Cultivation of primary porcine hepatocytes in an OXY-HFB for use as a bioartificial liver device

Bioreactors being developed for bioartificial liver devices vary greatly in their construction. Until now, primary liver cells were cultivated either in sandwich configuration, as spheroids, or in special hollow fiber systems. Primary hepatocytes are demanding on their environment and have a high oxygen consumption. To get good results, optimal cultivation conditions are needed. The idea of the project was to investigate a new concept of an oxygenating hollow fiber bioreactor (OXY-HFB). The OXY-HFB should consist exclusively of oxygenating and internal heat exchange fibers to yield a simple and effective design. Primary liver cells were seeded on the surface of the fibers in the extrafiber space. Oxygen requirements and temperature control were supplied through the fibers. The culture medium was perfused through the extrafiber space and therefore brought into direct hepatocellular contact. The OXY-HFB concept offers different advantages. A high cell density of 2.5 x 10(7) cells/mL can be obtained. This results in a cell number of 2.5 x 10(9) liver cells per bioreactor. Furthermore, the OXY-HFB is easily handled because no incubator is required. To study the efficiency of this bioreactor technique, various parameters were investigated over a cultivation period of three weeks. These included urea synthesis, lactate formation, glucose elimination, albumin synthesis, oxygen level, and pH. Furthermore, the metabolites of diazepam were measured. The biochemical performance of the bioreactor remained stable over the investigated time period. These results demonstrate that porcine liver cells preserve their viability and primary metabolism in the OXY-HFB over the complete period of study.     

Bioprocess considerations for T-cell therapy: Investigating the impact of agitation,dissolved oxygen,and pH on T-cell expansion and differentiation

Adoptive T-cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single-use 24-well microbioreactor (micro-Matrix) was assessed for its use as a high-throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion-mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 10⁶ cells/ml. The distribution of CD8+ central memory T cells (T_CM), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T-cell manufacturing process.     

A novel mechanism of autolysis in Helicobacter pylori: possible involvement of peptidergic substances

BACKGROUND: Helicobacter pylori survival in a hostile acidic environment is known to be caused by its production of urease, which is not released by known secretion pathways. It has been proposed that H. pylori cells undergo spontaneous autolysis during cultivation and that urease becomes surface-associated only concomitant with bacterial autolysis. The aim of this study was to elucidate mechanisms by which H. pylori cells undergo autolysis during cultivation. MATERIALS AND METHODS: Autolysis of H. pylori KZ109 cells was estimated by measuring the turbidity of the culture, by detection of cytoplasmic protein release into the culture supernatant and by scanning electron microscopic observation of H. pylori cells during cultivation. An autolysis-inducing factor (AIF) was partially purified from the culture supernatant by a partition method using ethyl acetate. RESULTS: Bacterial turbidity of KZ109 cells was drastically decreased after late-log phase accompanying release of urease and HspB into the extracellular space. Concomitantly, cell lytic activity was detected in the culture supernatant. Scanning electron microscopic observation suggested that partially purified AIF induced cell lysis. It was also shown that the AIF is different from other autolytic enzymes or substances so far reported. CONCLUSIONS: This study demonstrated the presence of the peptidergic autolytic substances in the culture supernatant of H. pylori KZ109 cells. The results of this study should be useful for further studies aimed at elucidation of the strategy of survival of H. pylori in the gastric environment and elucidation of the mechanisms of pathogenesis induced by H. pylori.     

Ultrastructure of Candida utilis in continuous culture with various concentrations of the carbon source introduced by different technics

The object of the work was to study changes in the ultrastructure of Candida utilis cells induced by increasing concentrations of glucose during chemostat cultivation and continuous cultivation with glucose pulse feeding. The results indicate that the cell structure and metabolism change vectorially with an increase of glucose concentration regardless of how glucose was added. An increase of the input glucose concentration is followed by an increase in the periplasmic space, the content of glycogen, the length and diameter of mitochondria, and the size of the nucleus. However, in the case of glucose pulse feeding, the above changes in the cell structure occur at a considerably lower input concentration as compared to the chemostat culture. Under these conditions, microtubuli are assembled in the cytoplasm in response to the glucose stimulus.     

Ultrastructure of the eye of a euphausiid crustacean

The compound eye of the Antarctic euphausiid Euphausia superba is a spherical clear zone eye. The dioptric system consists of a hexagonally-faceted cornea, two corneagenous cells, two crystalline cone cells which form the bipartite crystalline cone, and two accessory cone cells. The dioptric system of each ommatidium is separated from that of adjacent ommatidia by six distal pigment cells and a basement membrane. The proximal tip of the crystalline cone is cupped by the distal ends of the seven retinula cells whose nuclei are arranged in a staggered array slightly distal to the middle of the clear zone. In the distal half of the clear zone, each narrow retinula cell column is surrounded by large proximal extensions of the six distal pigment cells. The pigment cells narrow more proximally and terminate at the proximal basement membrane. A specialized axial channel complex extends from the crystalline cone through the clear zone, and is continuous with a conical refractive element which caps the distal end of the rhabdom. The rhabdom is fused, and made up of alternating highly birefringent layers of orthogonally-oriented microvilli. It is surrounded by a narrow extra-cellular space which is continuous with the distal refractive element and a second conical refractive element at the proximal end of the rhabdom.     

Mycology studies in space

Summary The postflight phase of the Apollo MEED mycology attempts to identify survival according to exposure to specific quantitative space flight factors, while the second phase of studies identifies qualitative change other than cell survival [57]. Initial changes incurred in space on a fungal cell can be monitored and further examined on return of the fungal species test system to Earth. The postflight studies present a better understanding of the space environmental influences on living cells and a more clear understanding of the fungal species under examination.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

EB病毒对脐带血细胞产生免疫球蛋白的影响

探讨EB病毒是否使培养的脐带血B细胞、CD5+B细胞产生免疫球蛋白及具有天然自身抗体性质的免疫球蛋白。无菌采集新生儿脐带血,RosetteSepTM法分离全B细胞,免疫磁珠分离CD5+B细胞,将分离得到的全B,CD5+B,CD5-B三组细胞分别用EB病毒、紫外线照射灭活的EB病毒(UVEBV)、TPA刺激,于培养的第10～30d,每间隔4d分别留取上清,ELISA法检测培养上清中的IgG,IgM及抗角蛋白自身抗体(AKautoAb)IgG,IgM,阳性对照为健康成人血清,阴性对照为未加刺激的培养细胞上清。EB病毒感染的3组细胞于培养的第7d发生转化,14d以后的培养上清中IgM,AKautoAbIgMOD值较对照组显著升高(p<0.01);UVEBV刺激细胞存活15d,其第14d的CD5+B细胞培养上清中IgMOD值高于对照组(p<0.05);TPA及未刺激的细胞存活7d,培养上清中未检测到免疫球蛋白。可见,EB病毒能使培养的脐带血B细胞、CD5+B细胞产生可能具有天然自身抗体性质的免疫球蛋白。     

Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation

O-glycosylation has been considered a limiting factor in protein secretion in filamentous fungi. Overexpression of the yeast DPM1 gene encoding dolichylphosphate mannose synthase (DPMS) in an Aspergillus nidulans mutant (BWB26A) deficient in O-glycosylation caused an increase in the number of secretory vesicles and changes in protein secretion. However, the secretory proteins, primarily O-mannosylated glucoamylase and N-glycosylated invertase, were mainly trapped in the periplasmic space. Different glycoforms of invertase were found insite the cells, in the periplasmic space and in the cultivation medium. Our data point to the importance of the cell wall as a barrier in protein secretion.     

In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique

Epithelial cells from tracheal mucosa offer significant potential as a cell source in development of tissue-engineered trachea. The purpose of this study was to investigate and optimize a suitable culture system for tracheal epithelial cells, including the methods of primary culture, passage, identification, and cryopreservation. Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique and were subjected to immunohistochemistry, immunofluorescence, and cryopreservation after purification. Epithelial cells reached confluency at 14–15 d. Immunohistochemical staining for cytokeratin showed brown yellow-positive cytoplasm and blue-counterstained nuclei, while immunofluorescence staining for cytokeratin showed green-positive cytoplasm and clear cell outline, indicating that the cultured cells had properties of epithelial cells. After recovery, epithelial cells exhibited high survival and viability. The results demonstrated that in vitro isolation and cultivation model was successfully established to provide high proliferative capacity, typical morphology and characteristics of tracheal epithelial cells from trachea mucosa by the use of the tissue explant technique.     

High-frequency interconversion of turbid and clear plaque strains of bacteriophage f1 and associated host cell death

Under normal cultivation conditions, a mixture of turbid and clear plaques is often apparent in cultures of bacterial cells infected with filamentous bacteriophages. Beginning with a culture of wild-type filamentous phage f1, which itself produces turbid plaques, a clear plaque strain (c1) was isolated. From c1, the turbid plaque strain t1 was isolated; from t1, the clear plaque strain c2 was isolated; and from c2, the turbid plaque strain t2 was isolated. Each of these strains was generated with a frequency of approximately 1 x 10(-4). Although filamentous phages have been thought not to induce host cell death, both turbid and clear plaque strains of f1 killed host bacteria. Plating of bacterial cells 1 h after infection revealed that colonies produced by cells infected with either wild-type f1 or strain c2 were smaller than those derived from uninfected cells, and that colony formation by infected cells was reduced by 15% and 38%, respectively. The time course of bacterial growth revealed that, at 4 h after infection, the number of CFU per milliliter of culture of cells infected with wild-type f1 or with strain c2 was reduced by 27% and 95%, respectively, compared with that for uninfected cells. Microculture analysis also revealed that the percentages of nondividing cells in f1 or c2 infected were 19% and 52%, respectively, 4 h after infection with wild-type f1 or with strain c2; no such cells were detected in cultures of uninfected cells. Negative staining and electron microscopy showed that 20% and 61% of cells infected with wild-type f1 or with strain c2 were dead 4 h postinfection. Finally, although the rates of DNA synthesis were similar for infected and uninfected cells, the rates of RNA and protein synthesis were markedly reduced in infected cells.     

Cell removal from fermentation broth by flocculation + sedimentation

Summary Cell separation by flocculation+sedimentation ofStreptoccocus equisimilis cultivation for hyaluronate lyase recovery, was investigated as a function of the pH of the fermentation broth, using three different cationic flocculants. The polyelectrolyte Superfloc N-100 appears to be the best of the three flocculants tested; after treatmen of pH 6.0 and 120 min free sedimentation, the cells are sedimented at 20% of the initial volume and 80% of the volume remained as a clear supernatant without loss of enzyme activity.