Use of early passage fetal intestinal epithelial cells in semi-high-throughput screening assays: an approach to identify new innate immune system adjuvants

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries.     

Leukocyte-mediated cell dissemination and metastasis: findings from multiple types of human tumors

Our previous studies revealed that leukocyte infiltration could trigger human breast and prostate tumor invasion through focal disruptions of the tumor capsule, which selectively favors monoclonal proliferation of tumor progenitors or a biologically more aggressive cell clone overlying the focal disruptions. Our current study, involving multiple types of human tumors, further shows that leukocyte infiltration could also trigger tumor metastasis through the following pathways: [1] more leukocytes migrate to focally disrupted tumor capsules, which forms leukocyte aggregates surrounding newly formed tumor cell clusters, [2] the physical movement of leukocytes into proliferating tumor cells disrupts the intercellular junctions and cell-surface adhesion molecules, causing the disassociation of tumor cells from the tumor core, [3] leukocytes are conjoined with some of these tumor cells through plasma membrane fusion, creating tumor cell-leukocyte chimeras (TLCs), and [4] the leukocyte of TLCs impart migratory capacity to associated tumor cell partners, physically dragging them to different tissue sites. Our findings suggest a novel pathway for tumor cell dissemination from the primary sites and the subsequent journey to new sites. Our findings also provide a unique explanation for the cellular mechanism of leukocytes on tumor invasion and metastasis. If confirmed, our hypothesis and technical approach may significantly facilitate early detection and intervention of tumor invasion and metastasis.     

Characterization of a γ-glutamyl transpeptidase positive subpopulation of endothelial cells in a spontaneous tube-forming clone of rat cerebral resistance-vessel endothelium

A spontaneous tube-forming clone of rat cerebral resistance-vessel endothelium was characterized in long-term serial culture. In this study, a clone, RV-150 ECT, of cerebral resistance vessel endothelial cells in long-term culture has been shown to have a subpopulation of γ-GTP positive cells that are present in all cultures regardless of confluency status or tube-forming stages. In pre-confluent and confluent cultures, the γ-GTP positive cells are few in number, stain weakly, and are randomly distributed in the monolayers. In monolayer post-confluent cultures, γ-GTP positive cells increase in number, stain strongly, and begin to show signs of non-random distributions. In early post-confluent cultures that have become a mixture of monolayer and multilayer cells, there is a further increase in γ-GTP positive cells which begin to form distinct groupings. In mid post-confluent fultures, the multilayered areas of the culture have begun clustering to form clear multicellular aggregates. The γ-GTP positive cells at this stage are reduced in number and are predominately associated with the cell clusters. In late postconfluent cultures, the multicellular clusters develop clear cell cords between/among the clusters. At this stage the γ-GTP positive cells are associated exclusively with cell clustters. With cord development, the γ-GTP positive cells are associated with bothe clusters and cords, and are reduced in number apparently because of selective degeneration of these cells. The results of this study demonstrate that a phenotypically distinct subpopulation of endothelial cells exhibits characteristic features of the blood-brain barrier, namely γ-GTP. The ability of these cells to express this property in long-term serial culture suggests that this may represent a useful in vitro model to study the growth and differentiation of bloodbrain barrier vessels. © 1993 Wiley-Liss, Inc.     

Establishment and characterization of bovine mammary epithelial cell lines

Summary One bovine mammary epithelial cell clone, designated PS-BME-C1, and two bovine mammary epithelial cell lines, designated PS-BME-L6 and PS-BME-L7, were derived from mammary tissue of a pregnant (270 day) Holstein cow. The cells exhibit the distinctive morphologic characteristics of mammary epithelial cells and express the milk fat globule membrane protein, PAS-III. They form domes when cultured on plastic substrata and acinilike aggregates when cultured on a collagen matrix. These cells are capable of synthesizing and secretingα-lactalbumin andα-s₁-casein when cultured on a collagen matrix in the presence of insulin, cortisol, and prolactin. The cells have a near-normal diploid number and do not grow in suspension culture. When transplanted to the cleared mammary fat pads of female athymic nude mice, the cells readily proliferate forming noninvasive palpable spherical cellular masses within 8 wk after inoculation. The cells may become a useful tool to study the regulation of ruminant mammary epithelial cell growth and differentation. This work was supported by the Pennsylvania State University Experiment Station. The PS-BME cells are the property of The Pennsylvania Research Corporation. Scientists interested in obtaining the PS-BME clone or cell lines for their research may request them from the corresponding author.     

Direct detection of Campylobacter jejuni in human stool samples by real-time PCR

Our purpose was to establish a quick and accurate real-time PCR (rtPCR) method to detect Campylobacter jejuni directly from human diarrheal stool as an alternative to traditional culture methods. To determine the consistency of rtPCR and culture method, 256 clinical diarrheal stool samples and 50 normal stool samples from healthy individuals were examined, and the whole process was double-blinded. Our data showed that the sensitivity of rtPCR in pure cultures and stool was 102 CFU.mL-1 and 103 CFU.g-1, respectively. Of the 256 diarrheal samples, 10 specimens were successfully detected by both methods, whereas two specimens were PCR positive but culture negative. No positive results were found by these two methods in 50 normal specimens. Our data suggested that rtPCR was convenient in operation and time-saving (turnaround time 3.5-4 h), so it could be used for clinical diagnostic and epidemiological purposes.     

A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase.

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.     

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.     

Explant culture of gastrointestinal tissue: a review of methods and applications

The gastrointestinal (GI) tract is an important target organ for the toxicity of xenobiotics. The toxic effects of xenobiotics on this complex, heterogeneous structure have been difficult to model in vitro and have traditionally been assessed in vivo. The explant culture of GI tissue offers an alternative approach. Historically, the organotypic culture of the GI tract proved far more challenging than the culture of other tissues, and it was not until the late 1960s that Browning and Trier described the means by which intestinal tissues could be successfully cultured. This breakthrough provided a tool researchers could utilise, and adapt, to investigate topics such as the pathogenesis of inflammatory intestinal diseases, the effect of growth factors and cytokines on intestinal proliferation and differentiation, and the testing of novel xenobiotics for efficacy and safety. This review considers that intestinal explant culture shows much potential for the application of a relatively under-used procedure in future biomedical research. Furthermore, there appear to be many instances where the technique may provide experimental solutions where both cell culture and in vivo models have been unable to deliver conclusive and convincing findings.     

牛朊蛋白基因prnp敲除载体的构建及真核细胞转染

利用正负筛选策略(Positive-negative selection,PNS)对中靶细胞进行富集是提高体细胞基因打靶效率常用的策略之一。将动物的朊蛋白基因prnp敲除,使其不能表达朊蛋白(传染性海绵状脑病的致病蛋白),从而使其具有抵抗Prion病感染的能力。本研究采用正负筛选策略,构建了牛prnp基因的双等位基因敲除载体,经内切酶Sac Ⅱ线性化后,再通过电穿孔转染牛胎儿成纤维细胞,分别用600μg/mL G418、200nmol/mL Ganciclovir(GCV)进行正负药物筛选,最终获得了176个药物抗性细胞克隆,进一步采用PCR、测序、间接免疫荧光试验及Western blotting试验对细胞克隆进行鉴定,结果表明,其中的9个细胞克隆为中靶细胞,证明牛prnp基因被成功敲除。本研究为牛prnp的敲除提供了可行性依据,并为体细胞核移植生产敲除朊蛋白基因的转基因动物提供供体细胞。     

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.     

结核分枝杆菌H37Rv新基因Rv2742克隆表达及纯化

Rv2742是本课题组前期基于蛋白质基因组学策略从结核分枝杆菌Mycobacteriumtuberculosis H37Rv中发现、鉴定的遗漏注释基因。文中旨在建立结核分枝杆菌H37Rv漏注释蛋白Rv2742的可溶性诱导表达、纯化体系,为进一步探索Rv2742基因参与的生物学功能奠定基础。前期实验发现构建的pGEX-4T-2-Rv2742、pET-28a-Rv2742、pET-32a-Rv2742及pMAL-c2X-Rv2742原核表达载体均无法实现目的蛋白的诱导表达。但经密码子优化后,仅有pMAL-c2X-Rv2742载体能够实现目的蛋白的可溶性诱导表达。此外,通过比较不同宿主菌、温度及IPTG浓度对目的蛋白表达量的影响,发现目的蛋白在Rosetta (DE3)中,16℃及0.5mmol/LIPTG诱导条件下表达量最高。直链淀粉树脂(Amyloseresin)亲和层析柱纯化获得较纯的产物,经LC-MS/MS验证确认是Rv2742融合蛋白肽段序列。成功获得结核分枝杆菌H37Rv新基因Rv2742的重组蛋白,可进一步开展其潜在相互作用及免疫原性研究工作。     

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

Mammalian cells are extensively used for production of biopharmaceuticals. Most cells used in industry have infinite proliferative capacity, which provides a high number of cells and corresponding productivity. However, infinite cells will continue to multiply even after cell density reaches sufficient levels. This excess proliferation aggravates the culture environment and induces low productivity. Therefore, after cell density reaches sufficient levels, downregulation of proliferation would prevent such aggravation and extend the culture period and improve productivity. To realize such suitable proliferation, we aimed to establish a novel cell line whose proliferation was spontaneously downregulated after reaching a sufficient population level. Mutagenesis using high-energy beam irradiation was used. CHO-DP12 cells were irradiated with 2.5 Gy X-rays and screened with hydroxyurea and 5-fluorouracil to eliminate any cells multiplying after confluence and to concentrate desired mutants. One clone was established and named CHO-M1. Cell cycle analysis indicated that CHO-M1 cells had a similar cell cycle profile in the exponential growth phase, but cells rapidly accumulated in G1 phase just before confluence and did not progress through the cell cycle. This suggested that until confluence, proliferation of CHO-M1 was similar to parental CHO, but after confluence, it was inhibited and under G1 arrest. The specific antibody production rate of CHO-M1 was kept high, even after confluence, while that of parental CHO was drastically decreased in stationary phase. These results suggest that the desired cell line was successfully established and that high-energy beam irradiation could be an efficient mutagenic technique for breeding industrial cells.     

Attachment and growth of human embryonic stem cells on microcarriers

The use of human embryonic stem cells (hESCs) for cell-based therapies will require large quantities of genetically stable pluripotent cells and their differentiated progeny. Traditional hESC propagation entails adherent culture and is sensitive to enzymatic dissociation. These constraints hamper modifying method from 2-dimensional flat-bed culture, which is expensive and impractical for bulk cell production. Large-scale culture for clinical use will require innovations such as suspension culture for bioprocessing. Here we describe the attachment and growth kinetics of both murine embryonic stem cells (mESCs) and hESCs on trimethyl ammonium-coated polystyrene microcarriers for feeder-free, 3-dimensional suspension culture. mESCs adhered and expanded according to standard growth kinetics. For hESC studies, we tested aggregate (collagenase-dissociated) and single-cell (TrypLE-dissociated) culture. Cells attached rapidly to beads followed by proliferation. Single-cell cultures expanded 3-fold over approximately 5 days, slightly exceeding that of hESC aggregates. Importantly, single-cell cultures were maintained through 6 passages with a 14-fold increase in cell number while still expressing the undifferentiated markers Oct-4 and Tra 1-81. Finally, hESCs retained their capacity to differentiate towards pancreatic, neuronal, and cardiomyocyte lineages. Our studies provide proof-of-principle of suspension-based expansion of hESCs on microcarriers, as a novel, economical and practical feeder-free means of bulk hESC production.     

Microengineering neocartilage scaffolds

Advances in micropatterning methodologies have made it possible to create structures with precise architecture on the surface of cell culture substrata. We applied these techniques to fabricate microfeatures (15-65 microm wide; 40 microm deep) on the surface of a flexible, biocompatible polysaccharide gel. The micropatterned polymer gels were subsequently applied as scaffolds for chondrocyte culture and proved effective in maintaining key aspects of the chondrogenic phenotype. These were rounded cell morphology and a positive and statistically significant (p < 0.0001) immunofluorescence assay for the production of type II collagen throughout the maximum culture time of 10 days after cell seeding. Further, cells housed within individual surface features were observed to proliferate, while serial application of chondrocytes resulted in the formation of cellular aggregates. These methods represent a novel approach to the problem of engineering reparative cartilage in vitro.     

Promising system for selecting healthy in vitro-fertilized embryos in cattle

Conventionally, in vitro-fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors--(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage--pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos.     

Metabolic regulation of manganese superoxide dismutase expression via essential amino acid deprivation

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging.     

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.     

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.     

Monolayer culture of cells originating from apreimplantation bovine embryo

Summary The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm² had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged Expansion to a 25-cm² flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1∶2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes. Published as publication no. 1891 College of Veterinary Medicine, Auburn University, Alabama 36849. This work was funded in part by an Auburn University Faculty Research Grant-in-aid. Preliminary results of the study were presented in abstract form at the 1987 Annual Conference of the International Embryo Transfer Society.