Microenvironment design considerations for cellular scale studies

In vivo cellular microenvironments are not well-mimicked in present in vitro cell culture systems. Microtechnology, and microfluidics in particular, provides the tools to create in vivo-like cellular microenvironments in vitro. Features of in vitro cellular microenvironments are discussed and compared to macroscale cell culture environments; the concept of an effective culture volume (ECV) is introduced to facilitate the comparison. Current research using microtechnology to investigate in vitro cellular microenvironments is presented and areas where more research is needed in characterizing the in vitro microenvironment are outlined.     

Light source spectrum strongly influences the in vitro estimation of sun protection factor

Irradiation from an artificial solar simulator that matches the relative cumulative erythema effectiveness (RCEE) of sunlight is used for in vivo sun protection factor (SPF) testing, whereas irradiation that matches the spectrum of natural sunlight (NS) is generally used for in vitro SPF testing. This study was designed to clarify whether this difference is important. Eight sunscreens spread on artificial substrates and several optical filters as sunscreen mimics were used. Their in vitro SPF values were evaluated using RCEE-compliant and NS spectrum-matching light sources in calculation. The calculated in vitro SPF values obtained using NS light (SPF [NS]) were lower than those obtained using RCEE light (SPF [RCEE]). The in vitro SPF (RCEE) values showed a better correlation and better agreement with in vivo SPF values, as compared with the in vitro SPF (NS) values. A marked difference between in vitro SPF values obtained with the two light sources in calculation was found for sunscreens showing low transmittance in the ultraviolet B region. To obtain in vitro SPF values that correspond well to in vivo SPF values measured with currently accepted methodology, it is important to use an RCEE-compliant light source.     

Applications of high throughput microsomal stability assay in drug discovery

High throughput in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with high throughput assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between high throughput in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds in order to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. In this study the relationship between in vitro single time point high throughput microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies was examined using 306 real world drug discovery compounds. The results showed that in vitro Phase I microsomal stability t(1/2) is significantly correlated to in vivo clearance with a p-value<0.001. For compounds with low in vitro rat microsomal stability (t(1/2)<15 min), 87% showed high clearance in vivo (CL>25 mL/min/kg). This demonstrates that high throughput microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability (t(1/2)>15 min), no significant differentiation was observed between high and low clearance compounds. This is likely owing to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance. This finding supports the strategy used by medicinal chemists and drug discovery teams of applying the in vitro data to triage compounds for in vivo PK and efficacy studies and guide structural modification to improve metabolic stability. When in vitro and in vivo data are both available for a compound, potential in vivo clearance pathways can be diagnosed to guide further discovery studies.     

Calcium-Dependent Translocation of S100B Is Facilitated by Neurocalcin Delta

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells—especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.     

ANALYSIS OF THE BINDING OF PHYTOCHROME TO PARTICULATE FRACTIONS

Abstract— The binding of phytochrome to receptor sites in a particulate fraction of maize coleoptiles has been studied as a function of the level of far-red-absorbing phytochrome (P_fr) offered in vivo and in vitro. Evidence is presented that the binding is cooperative. The degree of cooperativity expressed by the Hill coefficient of the binding function is the same (1–6) both in vivo and in vitro , whereas the Hill coefficient of the state function in vivo is significantly higher (2-1). The highest Hill coefficient (3–5) was found for the in vitro binding function in squash hooks.     

Enantiomer ratio of MK-0767 in humans and nonclinical species

MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione-containing dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK-0767 contains a chiral center at the C-5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)-(R) to (-)-(S) enantiomers of MK-0767 were determined in plasma from humans (in vitro only) and nonclinical species used in the toxicological evaluation of rac-MK-0767, namely CD-1 mice, Sprague-Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (approximately 1.5-1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo. In mice, the ratio was higher in vitro (approximately 1) than in vivo (approximately 0.6), while in rabbits it was higher in vivo (approximately 1) than in vitro (approximately 0.5). These results suggested that differential binding of the MK-0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo, since in protein-free systems MK-0767 exists as the racemate.     

(Coixan polysaccharide)-graft-polyethylenimine folate for tumor-targeted gene delivery

CP-PEI-FA was prepared as an effective vector for in vitro and in vivo tumor-targeted gene delivery. The structures of the polymers were characterized, and their DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro/in vivo transfection were examined. The cytotoxicity of CP-PEI-FA was significantly lower than that of PEI 25 kDa and close to that of PEI 1200. The in vitro transfection of CP-PEI-FA was tested in C6 and HeLa cells (FR-positive cells) and A549 cells (FR-negative cells). CP-PEI-FA showed a high targeting specificity and good gene transfection efficiency in FR-positive cells. These results indicate that CP-PEI-FA is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA.     

In Vitro Multiplication and NMR Fingerprinting of Rare Veronica caucasica M. Bieb

Micropropagation of rare Veronica caucasica M. Bieb. was achieved by successful in vitro cultivation of mono-nodal segments on MS medium supplemented with 1.0 mg L^–1 6-benzylaminopurine (BA) and then transferring the regenerated plants on hormone free basal MS medium for root development. In vitro multiplicated plants were successively acclimated in a growth chamber and a greenhouse with 92% survival. The number of plastid pigments and the total phenolics content in in vitro cultivated and ex vitro adapted plants were unchanged, and no accumulation of reactive oxygen species (ROS) was detected by staining with 3-3′-diaminobenzidine (DAB) and 2′,7′-dichlorofluorescein diacetate (DCF-DA). Nuclear Magnetic Resonance (NMR) fingerprinting allowed for the identification of the major alterations in metabolome of V. caucasica plants during the process of ex situ conservation. Iridoid glucosides such as verproside, aucubin and catalpol were characteristic for in vitro cultivated plants, while in ex vitro acclimated plants phenolic acid–protocatechuic acid and caffeic acid appeared dominant. The successful initiation of in vitro and ex vitro cultures is an alternative biotechnological approach for the preservation of V. caucasica and would allow for further studies of the biosynthetic potential of the species and the selection of lines with a high content of pharmaceutically valuable molecules and nutraceuticals.     

In vitro angiogenesis assay for the study of cell-encapsulation therapy

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.     

氟康唑衍生物的设计合成及体外抗真菌活性研究

为了寻找活性更好的抗真菌化合物,基于已有的计算机辅助药物设计结果,保留氟康唑母体结构必需药效团,设计、合成了22个含对甲酚和胸腺嘧啶的氟康唑新衍生物.目标化合物的结构经1H NMR、元素分析和ESI-MS确证,初步体外抗真菌活性试验结果表明,化合物5l对7种真菌都表现出了较好的抗真菌活性(烟曲霉菌除外),化合物5a～5e,5g,5h,7a和7b对不同真菌表现出了一定的抗真菌活性.炔丙基取代氨基侧链结构的引入有利于提高该类目标化合物体外抗真菌活性,值得进一步深入研究.     

Cholesterylbutyrate solid lipid nanoparticles as a butyric acid prodrug

Cholesterylbutyrate (Chol-but) was chosen as a prodrug of butyric acid. Butyrate is not often used in vivo because its half-life is very short and therefore too large amounts of the drug would be necessary for its efficacy. In the last few years butyric acid's anti-inflammatory properties and its inhibitory activity towards histone deacetylases have been widely studied, mainly in vitro. Solid Lipid Nanoparticles (SLNs), whose lipid matrix is Chol-but, were prepared to evaluate the delivery system of Chol-but as a prodrug and to test its efficacy in vitro and in vivo. Chol-but SLNs were prepared using the microemulsion method; their average diameter is on the order of 100-150 nm and their shape is spherical. The antineoplastic effects of Chol-but SLNs were assessed in vitro on different cancer cell lines and in vivo on a rat intracerebral glioma model. The anti-inflammatory activity was evaluated on adhesion of polymorphonuclear cells to vascular endothelial cells. In the review we will present data on Chol-but SLNs in vitro and in vivo experiments, discussing the possible utilisation of nanoparticles for the delivery of prodrugs for neoplastic and chronic inflammatory diseases.     

Salinity stress enhances production of solasodine in Solanum nigrum L

Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs. The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied. The in vitro yield was compared with the yield from leaves of field grown plant. Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot. Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession. The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges. The data demonstrate that the solasodine production in cultures was growth dependent.     

DNA体外缩合的研究进展

DNA缩合不仅是自然界常见的生理现象,也是非病毒载体介导基因转染的关键步骤。了解DNA体外缩合的性质与特征,将有助于设计出高效的非病毒转基因载体。本文综述了近十几年来以多价阳离子为缩合剂诱发的DNA体外缩合的研究进展,着重介绍了缩合的机理、过程、影响缩合的因素以及应用于转基因的最新成果。     

Optimisation d’une sélection in vitro d’enzymes

Optimisation of an in vitro enzyme selection. Isolation of a catalyst for a given chemical reaction may be achieved by in vitro selection of enzymes from a protein library. Here, we investigate the polymerisation reaction on filamentous phage and the cross-linking of substrate on phage to optimise an in vitro selection for DNA polymerase activity. The efficiency of the optimised selection is measured by enrichment factors up to 3.8 × 10³, the highest described so far for an in vitro selection of proteins for catalysis. It should be useful for directed polymerase evolution towards novel catalytic activities. To cite this article: E. Orsi, J.-L. Jestin, C. R. Chimie 6 (2003).     

Use of Three‐Dimensional Arterial Models To Predict the In Vivo Behavior of Nanoparticles for Drug Delivery

Nanomaterials have been widely used for applications in biomedical fields and could become indispensable in the near future. However, since it is difficult to optimize in vivo biological behavior in a 3D environment by using a single cell in vitro, there have been many failures in animal models. In vitro prediction systems using 3D human‐tissue models reflecting the 3D location of cell types may be useful to better understand the biological characteristics of nanomaterials for optimization of their function. Herein we demonstrate the potential ability of 3D engineered human‐arterial models for in vitro prediction of the in vivo behavior of nanoparticles for drug delivery. These models enabled optimization of the composition and size of the nanoparticles for targeting and treatment efficacy for atherosclerosis. In vivo experiments with atherosclerotic mice suggested excellent biological characteristics and potential treatment effects of the nanoparticles optimized in vitro.     

Modern Trends in the In Vitro Production and Use of Callus,Suspension Cells and Root Cultures of Medicinal Plants

This paper studies modern methods of producing and using callus, suspension cells and root cultures of medicinal plants in vitro. A new solution for natural product production is the use of an alternative source of renewable, environmentally friendly raw materials: callus, suspension and root cultures of higher plants in vitro. The possibility of using hairy root cultures as producers of various biologically active substances is studied. It is proven that the application of the genetic engineering achievements that combine in vitro tissue culture and molecular biology methods was groundbreaking in terms of the intensification of the extraction process of compounds significant for the medical industry. It is established that of all the callus processing methods, suspension and root cultures in vitro, the Agrobacterium method is the most widely used in practice. The use of agrobacteria has advantages over the biolistic method since it increases the proportion of stable transformation events, can deliver large DNA segments and does not require special ballistic devices. As a result of the research, the most effective strains of agrobacteria are identified.     

In Vitro Confirmation of Siramesine as a Novel Antifungal Agent with In Silico Lead Proposals of Structurally Related Antifungals

The limited number of medicinal products available to treat of fungal infections makes control of fungal pathogens problematic, especially since the number of fungal resistance incidents increases. Given the high costs and slow development of new antifungal treatment options, repurposing of already known compounds is one of the proposed strategies. The objective of this study was to perform in vitro experimental tests of already identified lead compounds in our previous in silico drug repurposing study, which had been conducted on the known Drugbank database using a seven-step procedure which includes machine learning and molecular docking. This study identifies siramesine as a novel antifungal agent. This novel indication was confirmed through in vitro testing using several yeast species and one mold. The results showed susceptibility of Candida species to siramesine with MIC at concentration 12.5 µg/mL, whereas other candidates had no antifungal activity. Siramesine was also effective against in vitro biofilm formation and already formed biofilm was reduced following 24 h treatment with a MBEC range of 50–62.5 µg/mL. Siramesine is involved in modulation of ergosterol biosynthesis in vitro, which indicates it is a potential target for its antifungal activity. This implicates the possibility of siramesine repurposing, especially since there are already published data about nontoxicity. Following our in vitro results, we provide additional in depth in silico analysis of siramesine and compounds structurally similar to siramesine, providing an extended lead set for further preclinical and clinical investigation, which is needed to clearly define molecular targets and to elucidate its in vivo effectiveness as well.     

Identification of Toxic 2,4-Decadienal in Oxidized, Low-Density Lipoprotein by Solid-Phase Microextraction

The oxidation of low-density lipoprotein (LDL) in vitro was studied by a combination of solid-phase microextraction and GC/MS. 2-trans,4-cis-2,4-Decadienal, which is strongly toxic in vitro, was detected as the early oxidation product. This compound is degraded further to hexanal and (by cyclization of 4-hydroxy-2-nonenal) to 2-pentylfuran.     

Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization

Efforts to improve the in vitro embryo production process in pigs have included modifying culture medium and number of spermatozoa inseminated in order to reduce the incidence of polyspermy. Polyspermy is a pathological condition which results in aberrant embryonic development. The microchannels are designed to more closely mimic the function of the oviduct and create a flow pattern of spermatozoa past the oocytes similar to the pattern in the oviduct. In vitro fertilization of porcine oocytes in the microchannels has produced a higher incidence of monospermic penetration (p<0.05) as compared to the oocytes fertilized in the traditional microdrop system with comparable penetration and male pronucleus formation rates. Additionally, cleavage rates of the embryos as well as development to the blastocyst stage are similar. Here we demonstrate that the biomimetic microchannel in vitro fertilization system can reduce polyspermy and, therefore, increase the number of potentially viable embryos without reducing the overall in vitro production efficiency.     

Modulation of Triterpene Saponin Production: In Vitro Cultures,Elicitation, and Metabolic Engineering

Saponins are secondary metabolites that are widely distributed in the plant kingdom and are often the active components in medicinal herbs. Hence, saponins have a potential for the pharmaceutical industry as antibacterial, virucidal, anti-inflammatory, and anti-leishmanial drugs. However, their commercial application is often hindered because of practical problems, such as low and variable yields and limited availability of natural resources. In vitro cultures provide an alternative to avoid problems associated with field production; they offer a system in which plants are clonally propagated and yield is not affected by environmental changes. Additionally, treatment of in vitro cultures with elicitors such as methyl jasmonate may increase the production of saponins up to six times. In vitro cultures are amenable to metabolic engineering by targeting specific genes to enhance saponin production or drive production towards one specific class of saponins. Hitherto, this approach is not yet fully explored because only a limited number of saponin biosynthesis genes are identified. In this paper, we review recent studies on in vitro cultures of saponin-producing plants. The effect of elicitation on saponin production and saponin biosynthesis genes is discussed. Finally, recent research efforts on metabolic engineering of saponins will also be presented.