Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4⁺ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0×10⁷ cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-γ, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4⁺ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c×C57BL/6 F₁ mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-γ than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-γ-producing ability but rather derived from their poor responsiveness to IFN-γ.     

High-throughput microfluidic system for monitoring diffusion-based monolayer yeast cell culture over long time periods

We present a simple and high-throughput microfluidic system for diffusion-based monolayer yeast cell culture monitoring. Yeast cells are patterned into the micro-cavity array with a suitable height (4 μm) that keeps the cells fixed in monolayer during the cell division. Different sizes of cavities and different repeating times of injection were tested in order to realize as many single-cell/cavity as possible. Single-cell/cavity has been achieved in about 40% of 100 parallel cavities. As a demonstration, we apply this technology to investigate budding yeast and fission yeast cultures and show that it permits single-cell resolution over many cellular generations. Our results show that the technique provides an easy way to study the phenotype of single yeast cell cycle or cell-cell communication in high-throughput microfluidic system.     

Delayed spontaneous alternation in Purkinje cell degeneration mutant mice

Purkinje cell degeneration (pcd) mutant mice lose cerebellar Purkinje cells. It was found that pcd mice, contrary to normal mice, did not alternate spontaneously at any of 3 inter-trial intervals (15 s, 3 or 6 min). Deficits in spontaneous alternation are typical of animals with brain damage in areas important in spatial learning. Results are discussed in terms of a role for the cerebellum in spatially mediated behavior and behavioral inhibition.     

Optical imaging in microfluidic bioreactors enables oxygen monitoring for continuous cell culture

For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly(dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (> or =600 ps), high spatial resolution (1.4 microm), calibrated detection (0 to 300+/-8 microM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.     

An automatic monitoring system for epithelial cell height

This paper describes an automatic method to measure cell height (h) of epithelia grown as monolayers on transparent filter supports. Tissues are mounted in an Ussing-type chamber enabling solution exchange on both sides. The apical and basal side of the epithelial cells are marked with fluorescent beads. The image of the fluospheres is captured with a video camera and processed by a computer-based video imaging system. One basal reference bead in a gelatin layer on the filter support and up to three beads attached at the apical surface are used to monitor changes in cell height of three cells simultaneously. The focusing of the microbeads is done automatically by moving the objective with a piezoelectric device mounted on the nosepiece of the microscope. The algorithm for locating the bead is based on the changes in fluorescent light intensity emitted by the fluospheres. The method has an accuracy higher than 0.1 m and a time resolution as low as 6 s if measurements are restricted to one bead at the apical side. The method was tested on artificial model systems and used to measure volume changes in renal cultured epithelia (A6) after exposing the serosal surface to hypotonic solutions and replacing cell-impermeable sucrose by an organic compound (glycerol) with a smaller reflection coefficient. Serosal hypotonicity elicited a rapid volume increase followed by regulatory volume decrease, whereas the organic compound replacement caused a steady increase in cell volume.     

A microwell array system for stem cell culture

Directed embryonic stem (ES) cell differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. The optimized microwell array system is a potentially versatile tool for ES cell differentiation studies and high-throughput stem cell experimentation.     

Neuraminidase activity assays for monitoring MDCK cell culture derived influenza virus

Three assay methods were investigated for monitoring the time-course of neuraminidase (NA) activity of tissue culture derived equine influenza A virus from large-scale microcarrier cultivation and several steps of downstream processing required for the production of inactivated vaccines. Measurements of neuraminidase activity by a thiobarbituric acid (TBA) and a fluorometric method using Amplex Red as a fluorogen (FL-AR) did not correlate with the increase of hemagglutinin (HA) during virus replication. Samples analysed by the TBA method showed unspecific interference from low molecular weight compounds (< 3 kDa) of cell growth medium and virus maintenance medium. Further investigations showed that this was probably caused by interfering reactions between reducing sugars and amino acids that can be overcome by dialysis of samples. On the other hand, the sensitivity of the FL-AR method was not sufficient for the required measuring range. However, a reliable and sensitive fluorometric assay method (FL-MU-NANA) was obtained using 4-methylumbelliferyl-alpha-d-N-acetylneuraminic acid (4-MU-NANA) as a substrate, which allowed the detection of neuraminidase activities as low as 0.09 mU/mL. In this assay, time-course of neuraminidase activities correlated well with increasing hemagglutinin activities during virus replication in a bioreactor. Analysis of samples from various downstream processing steps comprising of clarification, inactivation, ultrafiltration (UF) and size-exclusion chromatography for the purification of influenza virus showed that neuraminidase activity was preserved at comparatively high levels. Based on the hemagglutinin and neuraminidase activity of the clarified and inactivated virus harvest, the overall recovery after gel filtration was about 34.4% and 119.5%, respectively.     

Bovine ephemeral fever virus in cell culture and mice

Summary Light, immunofluorescent and electron microscopic observations were carried out sequentially on mice and VERO cell cultures infected with bovine ephemeral fever (BEF) virus. In early harvests from cell culture, 185×73 nm cone-shaped particles with nearly parallel sides predominated; these particles had all other features typical of the Rhabdoviruses (surface projections, envelope, axial channel, precisely coiled helical nucleocapsid with 35 cross-striations at a 4.8 nm interval). Identical particles were the most frequent form in mouse brain. Variation in shape toward more broadly based cone-shapes occurred late in infection and reflected anomalous morphogenesis or particle breakdown. T (truncated) particles were common at all stages of infection. It was concluded that the reported variation in length and shape of particles in various BEF virus isolates is not necessarily releated to strain variation but more likely to varying growth rates and T particle interference associated with varying degrees of adaptation to a host system. BEF viral morphogenesis took place primarily upon plasma membranes in vivo and in cell culture in association with small accumulations of intracytoplasmic matrix. Fusion of viral envelopes was observed, and an early syncytium formation occurred in infected cell cultures. Cytopathology was similarin vivo and in cell culture; a protracted stage of cell rounding was followed by extreme cytoplasmic vacuolation and condensation and then by lysis. Necrotic encephalomyelitis was severe in moribund mice but extraneural sites of viral propagation and damage were not found with any of the techniques employed. Since this strict neurotropism is most likelynot characteristic of naturally acquired BEF virus infection in nature, it was concluded that the mouse was an unsatisfactory host for the further study of pathogenesis.On leave from Center for Disease Control, Atlanta, Georgia, U.S.A.     

Pneumatic cell stretching system for cardiac differentiation and culture

This paper introduces a compact mechanical stimulation device suitable for applications to study cellular mechanobiology. The pneumatically controlled device provides equiaxial strain for cells on a coated polydimethylsiloxane (PDMS) membrane and enables real time observation of cells with an inverted microscope. This study presents the implementation and operation principles of the device and characterizes membrane stretching. Different coating materials are also analyzed on an unstretched membrane to optimize the cell attachment on PDMS. As a result, gelatin coating was selected for further experiments to demonstrate the function of the device and evaluate the effect of long-term cyclic equiaxial stretching on human pluripotent stem cells (hPSCs). Cardiac differentiation was induced with mouse visceral endoderm-like (END-2) cells, either on an unstretched membrane or with mechanical stretching. In conclusion, hPSCs grew well on the stretching platform and cardiac differentiation was induced. Thus, the platform provides a new possibility to study the effect of stretching on cellular properties including differentiation and stress induced cardiac diseases.     

Morphologic characterization of spontaneous nervous system tumors in mice and rats

Spontaneous rodent nervous system tumors, in comparison to those of man, are less well differentiated. Among the central nervous system (CNS) tumors, the "embryonic" forms (medulloblastoma, pineoblastoma) occur both in rodents and humans, whereas the human "adult" forms (gliomas, ependymomas, meningiomas) have fewer counterparts in rodents. In general, the incidence of spontaneous CNS tumors is higher in rats (>1%) than in mice (>0.001%). A characteristic rat CNS tumor is the granular cell tumor. Usually it is associated with the meninges, and most meningeal tumors in rats seem to be totally or at least partly composed of granular cells, which have eosinophilic granular cytoplasm, are periodic acid-Schiff reaction (PAS)-positive, and contain lysosomes. Such tumors are frequently found on the cerebellar surface or at the brain basis. Rat astrocytomas are diffuse, frequently multifocal, and they invade perivascular spaces and meninges. The neoplastic cells with round to oval nuclei and indistinct cytoplasm grow around preexisting neurons, producing satellitosis. In large tumors, there are necrotic areas surrounded by palisading cells. Extensive damage of brain tissue is associated with the presence of scavenger cells that react positively with histiocytic/macrophage markers. The neoplastic astrocytes do not stain positively for glial fibrillary acidic protein; they probably represent an immature phenotype. In contrast to neoplastic oligodendroglia, they bind the lectin RCA-1. Astrocytomas are frequently located in the brain stem, especially the basal ganglia. Rat oligodendroglial tumors are well circumscribed and frequently grow in the walls of brain ventricles. Their cells have water-clear cytoplasm and round, dark-staining nuclei. Atypical vascular endothelial proliferation occurs, especially at the tumor periphery. Occasionally in the oligodendrogliomas, primitive glial elements with large nuclei occur in the form of cell groups that form rows and circles. Primitive neuroectodermal tumors of rats, such as pineal tumors or medulloblastomas, appear to have features similar to those found in man. In mice, the meningeal tumors are mostly devoid of granular cells and the astrocytomas are similar to those occurring in rats, whereas spontaneous oligodendrogliomas are observed extremely rarely. Tumorlike lesions, such as lipomatous hamartomas or epidermoid cysts, are occasionally encountered in the mouse CNS. It is suggested that we classify rodent CNS lesions as "low grade" and "high grade" rather than as "benign" and "malignant." The size of CNS tumors is generally related to their malignancy. Tumors of the peripheral nervous system are schwannomas and neurofibromas or neurofibrosarcomas consisting of Schwann cells, fibroblasts, and perineural cells. Well-differentiated schwannomas are characterized by S-100 positivity and the presence of basement membrane. They show either Antoni A pattern with fusiform palisading cells or Antoni B pattern, which is sparsely cellular and has a clear matrix. The rat develops specific forms of schwannomas in the areas of the submandibular salivary gland, the external ear, the orbit, and the endocardium. Spontaneous ganglioneuromas occur in the rat adrenal medulla or thyroid gland. Compared to experimentally induced neoplasms, the spontaneous tumors of the rodent nervous system are poor and impractical models of human disease, although they may serve as general indicators of the carcinogenic potential of tested chemicals.     

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.     

生物型人工肝的在线快速细胞监测系统

目的 在不破坏生物反应器内细胞生存环境的条件下，在线快速了解反应器内肝细胞的生存状态、功能活性，以便及时采取相应的控制措施。方法 改变传统的大型分析仪器离线检测的方法，采用以单色光源、集成光电传感器为测量元件的单片机控制的专用监测系统，实现了对白蛋白、尿素的在线快速检测。结果 实验证明，该系统结构新颖简单，能够满足生物型人工肝系统的细胞功能活性检测任务。结论 该方案实际可行，为生物型人工肝的开发评价提供了及时的依据，从而使生物型人工肝脏的闭环控制实现成为可能。     

体外抗原诱导小鼠T细胞对NK细胞功能的抑制及其可能分子机制研究

目的:为了探讨抗原诱导T细胞抑制对NK细胞功能的抑制及其可能机制.方法:在体外,分别利用抗原、抗原 抗CD80抗体、刀豆蛋白A、抗CD3抗体先行诱导小鼠T细胞,并于诱导后24、48、72、96小时分别引入NK细胞,通过51Cr释放试验动态观察NK细胞的功能状况,激光共聚焦显微镜观察T细胞抑制NK细胞功能的作用模式,同时用RT-PCR方法检测经诱导的T细胞Bc1与Bc2表达情况.结果:①各组在24、48、72小时相点,上清液对NK功能均无显著影响,而到96小时,抗原激活组与抗原 抗CD80抗体诱导组两组上清液对NK细胞的杀伤能力表现出促进作用,与其它各时相点相比有统计学意义(P＜0.05),且后者低于前者,二者相比也具统计学意义(P＜0.05).②刀豆蛋白A与抗CD3抗体刺激组T细胞从T细胞接受刺激24小时起至96小时,均表现了抑制NK细胞功能.抗原 抗CD80抗体耐受诱导组与抗原激活组T细胞则是在诱导后48小时方表现出对NK细胞的抑制,与其它两组相比差异具有统计学意义(P＜0.05).③只有同种抗原刺激48小时的细胞培养物可检出Bc1、Bc2.其他时相、其他细胞刺激剂作用下均未能检出Bc1,Bc2的表达.结论:经诱导的T细胞能以直接接触的方式抑制NK细胞功能,但不同诱导剂活化的T细胞引起NK细胞抑制的机制可能不同.小鼠HLA-G的类似物-Bc1与Bc2的表达与抗原信号有关,抗原诱导的小鼠T细胞可能通过表达Bc1与Bc2来抑制NK细胞的功能,从而参与移植耐受的形成,这一结论提示这类非经典MHC Ⅰ类分子有可能成为移植耐受的检测标志.     

Genetic control of susceptibility to spontaneous testicular germ cell tumors in mice

Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men. TGCT is a polygenic trait and genes that control susceptibility for TGCT development have not yet been identified. The 129/Sv inbred strain of mice is an important experimental model to study the genetics and development of TGCTs. We review several novel approaches that were developed to study the susceptibility of TGCTs in the 129/Sv mouse model and its application in humans. These approaches showed that several spontaneous and engineered mutations interact with 129/Sv-derived susceptibility genes to enhance or suppress susceptibility; two of these mutations (Ter and Trp53) revealed novel linkages for susceptibility genes in sensitized polygenic trait analysis. Linkage analysis with a chromosome substitution strains suggests that as many as 100 genes control susceptibility. Bilateral TGCTs result from the coincidental occurrence of unilateral tumors. These results highlight the important contributions that this mouse model can make to studies of TGCT susceptibility in humans.     

Non-invasive flow cytometric monitoring of pHi in cell culture processes using EGFP

We have described a new method for monitoring of cell culture processes using green fluorescent protein (GFP) fluorescent intensity. GFP has been used as a non-invasive fluorescent reporter for various cellular processes. In this study, enhanced (EGFP) was found to be a very sensitive indicator of pHi in in vitro cell culture, and responded rapidly to extracellular pH (pHe) changes. EGFP transfected cells were evaluated for pHi changes by flow cytometry, by measuring EGFP fluorescent intensity, and compared to that of the pH-sensitive fluoroprobe, SNARF. EGFP intensity was found to reflect pHi values of cells at different pHe in the presence of nigericin and was affected by the addition of HCl and NaOH. Significant changes in pHi were detected at different stages of batch culture and when using different cell density and media composition. The EGFP assay can be used to minimise the perturbation of cells and processes under study, thus leading to accurate information about the physiological state of single cells in a population. The results establish the application of EGFP as a non-invasive indicator of pHi for monitoring of mammalian cell culture processes.     

Development of high throughput optical sensor array for on-line pH monitoring in micro-scale cell culture environment

On-line pH detection of cell culture environment is necessary in a bioprocess or tissue engineering. Devices by means of electrochemical mechanisms for this purpose have been reported to be less suitable compared with optical-based sensing principles. More recently, some non-invasive optical sensing systems have been proposed for online pH monitoring of cell culture environment. However, these devices are not for multi-target pH monitoring purpose, and are large in scale and thus not appropriate for the pH monitoring at a micro scale such as in microbioreactor or microfluidic-based cell culture platform. To tackle these issues, an optical fiber sensor array for on-line pH monitoring was proposed using microfluidic technology. The working principle is based on the optical absorption of phenol red normally contained in culture medium. Different from other device of the similar working principle, the proposed device requires less liquid volume (less than 0.8 μl), is non-invasive, and particularly can be configured as an array for high throughput pH monitoring. The present device has been optimized for the shape of detection chamber in a microfluidic chip with the aid of computational fluid dynamics (CFD) simulation, to avoid flow dead zone and thus to reduce the response time of detection. Both simulation and experimental results revealed that the design of oval detection chamber (axis, 1.5 and 2.0 mm) can considerably reduce the response time. Preliminary test has proved that the optical pH detection device is able to detect pH with average detection sensitivity of 0.83 V/pH in the pH range of 6.8–7.8, which is normally experienced in mammalian cell culture.     

Dynamic rotational seeding and cell culture system for vascular tube formation

Optimization of cell seeding and culturing is an important step for the successful tissue engineering of vascular conduits. We evaluated the effectiveness of using a hybridization oven for rotational seeding and culturing of ovine vascular myofibroblasts onto biodegradable polymer scaffolds suitable for replacement of small- and large-diameter blood vessels. Large tubes (12 mm internal diameter and 60 mm length, n = 4) and small tubes (5 mm internal diameter and 20 mm length, n = 4) were made from a combination of polyglycolic acid/poly-4-hydroxybutyrate and coated with collagen solution. Tubes were then placed in culture vessels containing a vascular myofibroblast suspension (10(6) cells/cm(2)) and rotated at 5 rpm in a hybridization oven at 37 degrees C. Light and scanning electron microscopy analyses were performed after 5, 7, and 10 days. Myofibroblasts had formed confluent layers over the outer and inner surfaces of both large and small tubular scaffolds by day 5. Cells had aligned in the direction of flow by day 7. Multiple spindle-shaped cells were observed infiltrating the polymer mesh. Cell density increased between day 5 and day 10. All conduits maintained their tubular shape throughout the experiment. We conclude that dynamic rotational seeding and culturing in a hybridization oven is an easy, effective, and reliable method to deliver and culture vascular myofibroblasts onto tubular polymer scaffolds.     

Features of chromosomal abnormalities in spontaneous abortion cell culture failures detected by interphase FISH analysis

Cytogenetic analysis of reproductive wastage is an important stage in understanding the genetic background of early embryogenesis. The results of conventional cytogenetic studies of spontaneous abortions depend on tissue culturing and are associated with a significant cell culture failure rate. We performed interphase dual-colour FISH analysis to detect chromosomal abnormalities in noncultured cells from two different tissues-cytotrophoblast and extraembryonic mesoderm-of 60 first-trimester spontaneous abortions from which cells had failed to grow in culture. An original algorithm was proposed to optimize the interphase karyotype screening with a panel of centromere-specific DNA probes for all human chromosomes. The overall rate of numerical chromosomal abnormalities in these cells was 53%. Both typical and rare forms of karyotype imbalance were found. The observation of six cases (19%) of monosomy 7, 15, 21 and 22 in mosaic form, with a predominant normal cell line, was the most unexpected finding. Cell lines with monosomies 21 and 22 were found both in cytotrophoblast and mesoderm, while cells with monosomy 7 and 15 were confined to the cytotrophoblast. The tissue-specific compartmentalization of cell lines with autosomal monosomies provides evidence that the aneuploidy of different human chromosomes may arise during different stages of intrauterine development. The effect of aneuploidy on selection may differ, however, depending on the specific chromosome. The abortions also revealed a high frequency of intratissue chromosomal mosaicism (94%), in comparison with that detected by conventional cytogenetic analysis (29%; P<0.001). Confined placental mosaicism was found in 25% of the embryos. The results of molecular cytogenetic analysis of these cell culture failures illustrate that the diversity and phenotypic effects of chromosomal abnormalities during the early stages of human development are underestimated.