Neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands.

Stem/progenitor cells are thought to have the potential in the treatment of severe neurodegenerative diseases. Recently, sympathoadrenal progenitors expressing specific markers of neural crest derivatives and capable to differentiate into neurons were discovered in adult bovine and human adrenal glands, but there was no reported data on cryopreservation of sympathoadrenal progenitors. The aim of the present study was to examine the neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands. Considering impact of various initial state of frozen biomaterial on cell recovery, we carried out a comparative estimation of cryopreservation outcome both for adrenal tissue fragments and isolated primary cells. The estimation consisted of determining cell yield, viability, ability to adhere, proliferate and differentiate in vitro. Cells isolated from the fresh adrenal glands were cultured until confluence. A formation of sympathoadrenal progenitors-embedded spherical cell colonies, whose cells are differentiated then into ßIII-tubulin-positive cells with neuron-like morphology, was observed on the monolayer. The colonies were well preserved after cryopreservation of cell culture with a cooling rate of 1 °C/min in the cryoprotectant media containing 5-15% of dimethylsulfoxide. Adrenal tissue fragments were cryopreserved in the presence of 10% dimethylsulfoxide at the cooling rates of 0.3; 1: 5; 40 and > 100 °C/min. Sympathoadrenal progenitors were recovered after cryopreservation with 0.3 °C/min cooling rate but not higher.

[Effect of magnetic nanoparticles Fe3O4 on viability, attachment and spreading of cells isolated from fetuses and new-born rats].

This study shows that toxic effect of non-modified nanoparticles of Fe3O4 in vitro depends on metabolic and morphological condition of cells, derived from fetuses and newborn rats. During the cultivation of cells with magnetic nanoparticles, the new-born rats. During the cultivation of cells with magnetic nanoparticles, the latter bind to the cell surface and penetrate into the intracellular space. In this case, sorption of nanoparticles on the cell surface makes it difficult for cells to adhere to substrate, and the absorption by spread cells may prevent their proliferation. Magnetic nanoparticles are well absorbed by the upper layer of cellular aggregates. In this case, the cells of the inner layer remain intact. Consequently, the cell aggregates are able to respond to the constant magnetic field. These aggregates could potentially by used in cell transplantation for directed cell delivery.

[Formation of spherical colonies as a property of stem cells].

The report deals with the ability of embryonic cells harvested from the nervous, hematopoietic and epithelial-muscular human tissues to form spheres during in vitro culturing. Judging from their own data and those reported elsewhere, the authors have hypothesized that formation of spherical colonies in vitro is a common feature of stem cells of various origin and varying degree of maturation. Probably, the formation of spheres provides for development of stem cells according to the locations and their inner temporal programs, which are specific and particular for every type of cells.

[Characteristics of human embryonic neuronal cells procured by non-enzyme method].

Isolation and culturing of human neuronal progenitor cells is of significant value for both fundamental research and therapeutic purposes. In this work, human embryonic neuronal cells were characterized as a heterogeneous population of progenitor cells with various differentiation potentials. During in vitro culturing the cells are capable of re-inoculating, proliferating, differentiating and migrating. While differentiating, these cells form neurons and glial cells. The present research demonstrates that depending upon the culturing conditions the embryonic neuronal cells may either form floating aggregates (incubation with embryonic serum), attach (incubation without serum), proliferate, or form neurospheres. Besides, peculiarities of aggregate differentiation during their incubation under various media are described.

[Isolation of two fractions of hepatocytes and characterization of their lipid composition]. / Vydelenie dvukh fraktsii gepatotsitov i kharakteristika ikh lipidnogo sostava.

Two fractions of hepatocytes were isolated from the rat liver by nonfermentative method. These fractions were different for mechanical stability to the action of perfusion factors. It has been shown that cells of these fractions were differently separated in the linear density gradient of sucrose. It was connected with structural and functional heterogeneity of hepatocytes in the liver. Results obtained allow us to confirm that hepatocytes in the liver form at least two classes of cells with different lipid content. The plasmatic membranes of hepatocytes with high content of lipids were inclined to damage in the process of liver perfusion as compared to cells with low lipid content.

Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion.

This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP(+)) indicates a predominantly mitochondrial localization of TPP(+) in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP(+), and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.

[Respiratory and detoxication activity of two fractions of hepatocytes isolated by a non-enzymatic method]. / Dykhatel'naia i detoksikatsionnaia aktivnost' dvukh fraktsii gepatotsitov, vydelennykh nefermentativnym metodom.

Slow centrifugation of isotonic sucrose medium containing the suspension of hepatocytes, obtained after disaggregation of liver, which has been preliminarily perfused by EDTA solution, results in the formation of two fractions of cells of different colour. Fraction 1 contains intact and metabolically active hepatocytes. Cells of fraction 2 have a damaged membrane, but they are capable of realizing biotransformation of xenobiotics in the presence of exogenous NADPH as well as maintaining efficiency of oxidative phosphorylation of mitochondria under the transfer to the medium simulating cytoplasm.

[Exogenous NADPH restores the detoxication function of isolated hepatocytes lost in the process of freezing-thawing]. / Ekzogennyi NADPH vosstanavlivaet detoksikatsionnuiu funktsiiu izolirovannykh gepatotsitov, utrachennuiu v protsesse zamorazhivaniia-otogreva.

Freezing and thawing have been studied for their effect on the rat hepatocyte detoxication system. Freezing-thawing of hepatocytes in the medium without cryoprotectors is to impair functioning of the biotransformation system of biphenyl, a hydrophobic xenobiotic. It is found that inhibition of the first stage of xenobiotics biotransformation is a result of the loss of pyridine nucleotides and substrates necessary for NADP+ reduction.

[Role of albumin in the biotransformation of biphenyl by isolated rat hepatocytes]. / Rol' al'bumina v biotransformatsii bifenila izolirovannymi gepatotsitami krys.

The role of albumin in biotransformation of biphenyl, a lipophilic xenobiotic, by isolated rat hepatocytes has been studied. It is shown that in the absence of albumin biphenyl is quickly and almost completely bound by cells. The rate of formation of 4-hydroxybiphenyl, the reaction product, depends on the substrate concentration (in the range 8.5-70 microM) and conforms with the Mikhaelis-Menten equation. An increase in the biphenyl concentration in the incubating medium to 140 microM induces no changes in the rate of its biotransformation. Serum albumin, while binding biphenyl, also reduces its effective concentration in a cell, which prevents the cytochrome-P-450-dependent monooxygenase system of hepatocytes from the inhibiting effect of high concentrations of the xenobiotic.

[Isolation of rat hepatocytes by a nonenzymatic method: detoxifying and respiratory activity]. / Vydelenie gepatotsitov krys nefermentativnym metodom: detoksikatsionnaia i dykhatel'naia aktivnosti.

A procedure for isolation of rat hepatocytes involving perfusion by EDTA, mechanical disaggregation of the liver and separation of intact cells from damaged ones by low-velocity centrifugation in isotonic sucrose media is described. A layer of a darker colour formed after separation contains hepatocytes with native plasma membrane characterized by respiratory activity and rate of biotransformation being close to the values obtained with cells isolated with the application of collagenase.

Isolation of intact mitochondria and hepatocytes using vibration.

A method of isolation of rat liver cells and mitochondria suggesting tissue disaggregation by vibration has been proposed. Mitochondria obtained using vibration have better parameters of oxidative phosphorylation as compared to homogenization. Hepatocytes isolated by vibration had viability about 90%, as determined by trypan blue exclusion, and rates of respiration and xenobiotic metabolism comparable to that observed by the enzymatic method.

[Energy state of hepatocytes of fed rats isolated by the means of EDTA and vibration]. / Energeticheskoe sostoianie gepatotsitov sytykh krys, vydelennykh s pomoshch'iu EDTA i vibratsii.

The energy state of mitochondria in fed rat hepatocytes isolated by the use of non-enzymatic method including liver perfusion with an EDTA-containing solution with a further mild mechanical effect of tissue fragments by vibration has been studied. The isolation procedure used permits to obtain significant amounts of hepatocytes whose viability is not less than 80%. The endogenous respiration rate (10-15 nm O2/min.mln cells) is slightly stimulated by succinate. In the course of incubation in a balanced salt medium, the cells accumulate ATP and the lipophilic cation, tetraphenylphosphonium. Data from the inhibitory analysis testify to the fact that tetraphenylphosphonium accumulation reflects the membrane potential of intact cell mitochondria, which are in a metabolic state similar to state 3.

[Isolation of intact mitochondria from the rat liver using vibration]. / Vydelenie intaktnykh mitokhondrii iz pecheni krys s ispol'zovaniem vibratsii.

A method for isolating mitochondria from the rat liver is described. In this method the homogenization step is replaced by vibration with the frequency of 50 Hz realized by a simple device. Mitochondria isolated by vibration demonstrate higher indices of the oxidative phosphorylation with succinate and glutamate + malate used as substrates than those isolated by homogenization do. The method described permits decreasing considerably the isolation medium expenditure remaining the mitochondria yield per gram of the liver unchanged.

[A non-enzymatic method of preparing isolated hepatocytes: assessment of their morpho-functional activity]. / Nefermentativnyi metod polucheniia izolirovannykh gepatotsitov: otsenka ikh morfo-funktsional'noi aktivnosti.

A method for isolating hepatocytes using the two-step liver perfusion by chelate-containing solutions is described. The hepatocytes obtained are highly inactive for the trypan blue staining test, (not less than 90%), ATP content, the rate of endogenic respiration and its stimulation by succinate, a degree of xenobiotic oxidation.

[Functional characteristics of rat hepatocytes isolated by a nonenzymatic method and incubated in vitro]. / Nekotorye kharakteristiki sostoianiia gepatotsitov krys, izolirovannykh nefermentativnym sposobom i inkubiruemykh in vitro.

A study was made of the effect of normothermic incubation on the metabolic state of parenchymal liver cells, isolated by the nonenzymatic method. In the process of a 2 hour incubation in the nutritious sodium-containing medium, a high level of the energy supply system is maintained, determined by the contents of adenine nucleotides, the rate of endogenous respiration and by its stimulation by succinate.