Development of a gastrointestinal tract microscale cell culture analog to predict drug transport

Microscale cell culture analogs (microCCAs) are used to study the metabolism and toxicity of a chemical or drug. These in vitro devices are physical replicas of physiologically based pharmacokinetic models that combine microfabrication and cell culture. The goal of this project is to add an independent GI tract microCCA to a multi-chamber chip microCCA representing the primary circulation. The GI tract microCCA consists of two chambers separated by a microporous membrane on which intestinal epithelial cells are cultured. Compounds of interest are pumped through the top chamber, allowing drug to be absorbed through the epithelial layer and circulated into the chip microCCA. The chip and GI tract microCCAs have been used to recreate the toxic effects of acetaminophen. Preliminary results have shown that the GI tract microCCA acts as a barrier to drugs entering the chip, mimicking in vivo function in this regard.     

Development of a microscale cell culture analog to probe naphthalene toxicity

Prediction of human response to drugs or chemicals is difficult as a result of the complexity of living organisms. We describe an in vitro model that can realistically and inexpensively study the adsorption, distribution, metabolism, elimination, and potential toxicity (ADMET) of chemicals. A microscale cell culture analog (microCCA) is a physical replica of the physiologically based pharmacokinetics (PBPK) model. Such a microfabricated device consists of a fluidic network of channels to mimic the circulatory system and chambers containing cultured mammalian cells representing key functions of animal "organ" systems. This paper describes the application of a two-cell system, four-chamber microCCA ("lung"-"liver"-"other tissue"-"fat") device for proof-of-concept study using naphthalene as a model toxicant. Naphthalene is converted into reactive metabolites (i.e., 1,2-naphthalenediol and 1,2-naphthoquinone) in the "liver" compartment, which then circulate to the "lung" depleting glutathione (GSH) in lung cells. Such microfabricated in vitro devices are potential human surrogates for testing chemicals and pharmaceutics for toxicity and efficacy.     

Rat kidney epithelial cell culture for metal toxicity studies

Summary Evaluation of the potential adverse human health effects of low-level chronic exposure to heavy metals is dependent on the basic knowledge of the cellular and molecular toxicology of these metals. The use of various cell culture systems has greatly facilitated our knowledge of the cellular effects. Inasmuch as most of the acute and chronic toxic effects of metals occur primarily on the renal proximal tubules, the development of a rat kidney epithelial cell culture has provided a unique system to study the uptake and mechanism of toxicity of metals and their intracellular binding ligands. In the presence ofd-valine, fibroblast growth was retarded and a primary epithelial monolayer culture was selectively grown from rat kidney cells. A distinct difference in the uptake of chemically similar divalent metals, such as Pb²⁺, Hg²⁺, Cd²⁺, and Zn²⁺, was observed in these cells. Both Pb²⁺ and Hg²⁺ were more avidly taken up by kidney cells than Cd²⁺ and Zn²⁺ salts and they also showed increased toxicity. On the other hand, the cellular uptake of Cd from cadmium-metallothionein (CdMT) was much less than from CdCl₂, but CdMT was about seven times more toxic than CdCl₂ when added to the renal cell culture. The cytotoxicity of CdCl₂ was decreased significantly with pretreatment of the cells with CdCl₂, although this had no effect on the toxicity of CdMT. The cellular toxicity of CdMT occurred probably during the process of its transport across the plasma membrane whereas that of CdCl₂ occurred after it had entered the cell. Thus rat kidney epithelial cells may be a useful tool to study the mechanism of renal toxicity of environmental chemicals and drugs. This work was funded by grants-in-aid of research from the Kidney Foundation of Canada.     

Anabolic-androgenic steroids: In cell culture

Summary Testosterone and related steroids at physiological concentrations positively stimulate in cell culture a number of reactions in a variety of tissues from different species of animals. Cells maintained in cell culture provide a means to study toxic effects in target organs and also the mechanism of action of these steroids.     

Application of cell culture toxicity tests to the development of implantable biosensors

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate, buffer, and a non-toxic sensor was readily obtained.     

Overview: Evaluation of metabolism-based drug toxicity in drug development

Drug metabolism can be a key determinant of drug toxicity. A nontoxic parent drug may be biotransformed by drug metabolizing enzymes to toxic metabolites (metabolic activation). Conversely, a toxic drug may be biotransformed to nontoxic metabolites (detoxification). The approaches to evaluate metabolism-based drug toxicity include the identification of toxic metabolites and the evaluation of toxicity in metabolically competent and metabolically compromised systems. A clear understanding of the role of drug metabolism in toxicity can aid the identification of risk factors that may potentiate drug toxicity, and may provide key information for the development of safe drugs.     

Incorporation of 3T3-L1 cells to mimic bioaccumulation in a microscale cell culture analog device for toxicity studies

Deficiencies in the early ADMET (absorption, distribution, metabolism, elimination, and toxicity) information on drug candidates extract a significant economic penalty on pharmaceutical firms. We have developed a microscale cell culture analog (microCCA) device that can potentially provide better, faster, and more efficient prediction of human and animal responses to a wide range of chemicals. The system described in this paper is a simple four-chamber microCCA ("lung"-"liver"-"fat"-"other tissue") designed on the basis of a physiologically based pharmacokinetics (PBPK) model of a rat. Cultures of L2, HepG2/C3A, and differentiated 3T3-L1 adipocytes were selected to mimic the key functions of the lung, liver, and fat compartments, respectively. Here, we have demonstrated the application of the microCCA system to study bioaccumulation, distribution, and toxicity of selected compounds. Results from the bioaccumulation study reveal that hydrophobic compounds such as fluoranthene preferentially accumulated in the fat chamber. Only a small amount of fluoranthene was observed in the liver and lung chambers. In addition, the presence of the differentiated 3T3-L1 adipocytes in the microCCA device significantly reduced naphthalene and naphthoquinone-induced glutathione (GSH) depletion. These findings suggest the potential utilization of the microCCA system to assess ADMET characteristics of the compound of interest prior to animal or human trials.     

小鼠睾丸支持细胞体外培养特性

支持细胞是睾丸的重要组成部分,其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持细胞,并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究,发现支持细胞的贴壁时间主要集中在接种后2～4h;当培养液中氨根离子浓度高于2.3mmol/L,渗透压高于326mosm/kg,pH≤6.8时支持细胞生长进入衰亡期;在氨基酸代谢方面,发现培养过程中丙氨酸和谷氨酸浓度迅速增加,缬氨酸、亮氨酸和异亮氨酸浓度略有降低,丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养及工艺优化奠定了基础。     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Summary The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible). This work was submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Environmental Science, Institute of Environmental Sciences.     

An in vitro approach to the study of target organ toxicity of drugs and chemicals

Summary A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.     

实时无标记细胞分析系统(RTCA)在药物心肌毒性检测和生物活性研究中的应用

实时无标记细胞分析系统(Real time xCELLigence analysis system,RTCA)是一种新型细胞检测技术,能够连续监测、记录及分析细胞活动产生的各种信息,在药物研究中的心肌毒性评估和细胞生物活性考察方面都可以发挥重要作用.文中首先对RTCA的原理与特点进行了介绍,然后分别对RTCA在心肌毒性...     

Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract

AIMS: The study of two human strains of Lactobacillus to be used as probiotics in the gastrointestinal tract. METHODS AND RESULTS: The Lactobacillus acidophilus UO 001 and Lact. gasseri UO 002, were resistant to the gastrointestinal conditions (pH 2 and 3, presence of pepsin, pancreatin or bile salts), the resistance was enhanced in the presence of skimmed milk. Additionally, adhered to Caco-2 cells through glycoproteins in Lact. gasseri and carbohydrates in the case of Lact. acidophilus. These strains are able to inhibit the growth of certain enteropathogens: Salmonella, Listeria and Campylobacter without interfering with the normal microbiota of the gastrointestinal tract, as stated by using the mixed culture and the spot agar test. Finally, strongly adherent Lact. gasseri were found to inhibit the attachment of Escherichia coli O111 to intestinal Caco-2 cells under the condition of exclusion. CONCLUSIONS: These results indicate that the two strains of Lactobacillus from human origin present important properties for survival in, and colonization of, the gastrointestinal tract, that give them potential probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Two strains of Lactobacillus isolated from human vagina of healthy premenopausal women could be promising candidates to be used in the preparation of probiotic products and for their use as health-promoting bacteria.     

Application of a simple culture of Plasmodium berghei for assessment of antiparasitic activity.

and 1992. Application of a simple culture of Plasmodium berghei for assessment of antiparasitic activity. International Journal for Parasitology 22: 1137–1142. Mouse erythrocytes infected with Plasmodium berghei were incubated for a short period in microplate wells. The parasites changed morphologically from the immature ring form to mature schizonts, and free merozoites were released. However, reinvasion of the erythrocytes appeared not to be possible in this system. This intraerythrocytic one-step growth of the parasite could be determined quantitatively by counting incorporation of 'H-hypoxanthine. The incorporation was markedly decreased by addition of certain antiparasitic agents to the culture. The sensitivity of this growth inhibition test was comparable to or higher than the mouse protection test. The results suggested the practical utility of this simple assay in screening antimalarial activity.     

Pressure-driven perfusion culture microchamber array for a parallel drug cytotoxicity assay

This article reports a pressure-driven perfusion culture chip developed for parallel drug cytotoxicity assay. The device is composed of an 8 x 5 array of cell culture microchambers with independent perfusion microchannels. It is equipped with a simple interface for convenient access by a micropipette and connection to an external pressure source, which enables easy operation without special training. The unique microchamber structure was carefully designed with consideration of hydrodynamic parameters and was fabricated out of a polydimethylsiloxane by using multilayer photolithography and replica molding. The microchamber structure enables uniform cell loading and perfusion culture without cross-contamination between neighboring microchambers. A parallel cytotoxicity assay was successfully carried out in the 8 x 5 microchamber array to analyze the cytotoxic effects of seven anticancer drugs. The pressure-driven perfusion culture chip, with its simple interface and well-designed microfluidic network, will likely become an advantageous platform for future high-throughput drug screening by microchip.     

Characterization of monolayer and organ cultures of cloned and enriched lymphohematopoietic stromal cell populations

The role of hematopoietic microenvironments in the regulation of maturation and differentiation of hematopoietic cells, although heavily debated, remains uncertain. Several investigators have suggested that the adherent “stromal” cell populations, which grow as colonies in cultures of lymphomyeloid tissues, include the cells involved in such regulatory processes. Grossly, the colonies described by several investigators appear similar morphologically, and the cells giving rise to them have been variously termed (1) fibroblast colony forming cells (FCFC), (2) plaque forming units-culture (PFU-C), (3) macrophage colonies, and (4) marrow stromal cells. FCFC have been reported to re-establish their parent microenvironment when transplanted in an allogeneic system. In this study, cloned and enriched cell populations obtained from such colonies in cultures of murine lymphomyeloid tissues have been characterized by their growth in culture and using morphological, histochemical, and electron microscopic techniques. The results demonstrated that, although the initial stromal colonies appeared to be identical, the constituent cell types varied considerably. Some colonies were comprised primarily of macrophages, while others appeared to contain predominantly fibroblasts; two additional cell types that established colonies have not yet been satisfactorily identified. These results demonstrate the heterogeneity of lymphomyeloid stromal colonies. There is a need for caution in the analysis of experiments in which uncharacterized stromal cell colonies are transplanted or employed as supporting monolayers in culture systems in experiments designed to evaluate the origins and functions of lymphohematopoietic stroma.     

Comparison of nuclear polyhedrosis virus replication in five lepidopteran cell lines

Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides.     

In vitro culture of ovaries of a viviparous gall midge

Summary Ovaries of the viviparous pedogenetic gall midgeHeteropeza pygmaea can be cultured in hemolymph obtained from X-ray-sterilized larvae of the same species. In this culture medium, formation of follicles is essentially the same as in vivo, and sometimes female larvae develop from these follicles. The ovaries of such larvae, in their turn, have been cultured in vitro to produce larvae. In this way, in vitro development from oogonium to larva has been maintained for several generations. When using hemolymph obtained from larvae grown under different conditions, the in vitro cultured ovaries produce a second type of egg which probably is male-determined. Ovarian development in vitro has been studied with differential interference contrast optics and time-lapse cinemicrography. This work was supported by the Swiss National Science Foundation Grant No. 3.2010.73.