Overview of Cell Models: From Organs Cultured in a Petri Dish to Organs-on-Chips

In this review, we tried to elucidate the origin and development of different animal and human cell culture methodologies used to evaluate the effects of various factors and substances in vitro. Organ cultures and conventional two-dimensional cultures of dissociated cells of various types, such as primary, tumor, induced pluripotent, stem, etc., have their advantages and drawbacks but usually do not represent accurate models for studying biological processes that take place in living organisms. Nowadays, high-throughput cell assays on the basis of various methods of signal detection (optical utilizing colorimetric, luminescent and fluorescent methods of detection, and electrochemical) are widely used at early stages of drug development for selection of the most active compounds and evaluation of their cytotoxic effects. The use of animals as models for drug testing is being criticized because of the lack of correlation between the results obtained in studies on them and on humans, and also because of the high cost and ethical issues. Therefore, much effort is put to create models based on human cells. This is how cultures emerged that utilize a three-dimensional network to simulate the architecture of tissues in vivo, and then so-called organs-on-chips—microfluidic microfabricated devices combining several types of cells—that replicate physical and chemical parameters of the microenvironment of cells in living organisms. In summary, experimental cell models have come a long way from the whole organs cultivated in a growth medium to almost complete reconstruction of organs in vitro based on the cutting-edge engineering approach with the use of different cell types. This currently enables one to replicate complex biological processes and study the influence of different substances and factors on them more successfully.     

Perinatal development of mammillotegmental connections in rats

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and after incubation in a fixative fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other conducting systems of the mammillary bodies. On days 14-15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.     

Oxidation of luminol with peroxidase from royal palm leaves

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Palm tree peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring

Three amperometric enzyme electrodes have been constructed by adsorbing anionic royal palm tree peroxidase (RPTP), anionic sweet potato peroxidase (SPP), or cationic horseradish peroxidase (HRP-C) on spectroscopic graphite electrodes. The resulting H(2)O(2)-sensitive biosensors were characterized both in a flow injection system and in batch mode to evaluate their main bioelectrochemical parameters, such as pH dependency, I(max), K(M)(app), detection limit, linear range, operational and storage stability. The obtained results showed a distinctly different behavior for the plant peroxidase electrodes, demonstrating uniquely superior characteristics of the RPTP-based sensors. The broader linear range observed for the RPTP-based biosensor is explained by a high stability of this enzyme in presence of H(2)O(2). The higher storage and operational stability of RPTP-based biosensor as well as its capability to measure hydrogen peroxide under acidic conditions connect with an extremely high thermal and pH-stability of RPTP.     

Cyclometalated ruthenium(II) complexes as efficient redox mediators in peroxidase catalysis

Cyclometalated ruthenium(II) complexes, [Ru(II)(C~N)(N~N)(2)]PF(6) [HC~N=2-phenylpyridine (Hphpy) or 2-(4'-tolyl)pyridine; N~N=2,2'-bipyridine, 1,10-phenanthroline, or 4,4'-dimethyl-2,2'-bipyridine], are rapidly oxidized by H(2)O(2) catalyzed by plant peroxidases to the corresponding Ru(III) species. The commercial isoenzyme C of horseradish peroxidase (HRP-C) and two recently purified peroxidases from sweet potato (SPP) and royal palm tree (RPTP) have been used. The most favorable conditions for the oxidation have been evaluated by varying the pH, buffer, and H(2)O(2) concentrations and the apparent second-order rate constants ( k(app)) have been measured. All the complexes studied are oxidized by HRP-C at similar rates and the rate constants k(app) are identical to those known for the best substrates of HRP-C (10(6)-10(7) M(-1) s(-1)). Both cationic (HRP-C) and anionic (SPP and RPTP) peroxidases show similar catalytic efficiency in the oxidation of the Ru(II) complexes. The mediating capacity of the complexes has been evaluated using the SPP-catalyzed co-oxidation of [Ru(II)(phpy)(bpy)(2)]PF(6) and catechol as a poor peroxidase substrate as an example. The rate of enzyme-catalyzed oxidation of catechol increases more than 10000-fold in the presence of the ruthenium complex. A simple routine for calculating the rate constant k(c) for the oxidation of catechol by the Ru(III) complex generated enzymatically from [Ru(II)(phpy)(bpy)(2)](+) is proposed. It is based on the accepted mechanism of peroxidase catalysis and involves spectrophotometric measurements of the limiting Ru(II) concentration at different concentrations of catechol. The calculated k(c) value of 0.75 M(-1) s(-1) shows that the cyclometalated Ru(II) complexes are efficient mediators in peroxidase catalysis.     

Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase.

Anionic sweet potato peroxidase (SPP; Ipomoea batatas) was shown to efficiently catalyse luminol oxidation by hydrogen peroxide, forming a long-term chemiluminescence (CL) signal. Like other anionic plant peroxidases, SPP is able to catalyse this enzymatic reaction efficiently in the absence of any enhancer. Maximum intensity produced in SPP-catalysed oxidation of luminol was detected at pH 7.8-7.9 to be lower than that characteristic of other peroxidases (8.4-8.6). Varying the concentrations of luminol, hydrogen peroxide and Tris buffer in the reaction medium, we determined favourable conditions for SPP catalysis (100 mmol/L Tris-HCl buffer, pH 7.8, containing 5 mmol/L hydrogen peroxide and 8 mmol/L luminol). The SPP detection limit in luminol oxidation was 1.0 x 10(-14) mol/L. High sensitivity in combination with the long-term CL signal and high stability is indicative of good promise for the application of SPP in CL enzyme immunoassay.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.