A. Yu. Petrenko

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

INTRODUCTION Human fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me(2)SO concentration during cryopreservation of HFL hematopoietic cell preparations. METHODS Human fetal liver (HFL) cells of 8-12 weeks of gestation were cryopreserved with a cooling rate of 1 degrees C/min down to -80 degrees C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me(2)SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose. RESULTS The addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me(2)SO/0.3M sucrose mixture was comparable to cryopreservation in 5% Me(2)SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34(+) cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells. CONCLUSION The inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me(2)SO, replacing serum and increasing the efficiency of cryopreservation.

Comparison of the methods for seeding human bone marrow mesenchymal stem cells to macroporous alginate cryogel carriers

We performed a comparative study of the localization, distribution, metabolic activity, and surface properties of human bone marrow mesenchymal stromal cells after static and perfusion seeding to macroporous alginate cryogels. A simple perfusion system for mesenchymal stromal cell seeding to macroporous alginate cryogel sponges proposed in this study resulted in rapid and uniform distribution of cells within the whole volume of the scaffold preserving functional and morphological properties of the cells.

A mechanism of latent cryoinjury and reparation of mitochondria

Abstract Changes in the systems of oxidative phosphorylation and ion transport of rat liver mitochondria have been studied during storage or incubation after freeze-thawing. It has been found that two different processes take place in frozen-thawed mitochondria, one of them tends toward resealing membrane defects and is accompanied by a partial reparation of function; and another one leads to a decrease of the membrane potential, release of K+ and Ca2+ from the matrix, accumulation of lipid peroxides, and uncoupling of oxidative phosphorylation (latent cryoinjury). The latent cryoinjury appears as a result of oxidation of endogenous pyridine nucleotides under conditions of high permeability of the inner membrane and low membrane potential, thus causing activation of the membrane lipid peroxidation and enzyme hydrolysis, leakage of cations, and deenergization of mitochondria. Inhibition of the latent cryoinjury favors the restoration of mitochondrial function after freezethawing

Growth and adipogenic differentiation of mesenchymal stromal bone marrow cells during culturing in 3D macroporous agarose cryogel sponges

We studied the possibility of population of macroporous agarose cryogel sponges by mesenchymal stromal bone marrow cells with their subsequent adipogenic differentiation. After 7-day culturing of mesenchymal stromal cells in agarose cryogel, the level of cell proliferation was 35%. After 3-week culturing in a medium inducing adipogenesis we observed accumulation of intracellular neutral lipids positively stained with Oil Red O. These findings can be used for the development of bioengineering constructions of the adipose tissue on the basis of spongy carriers.

Phenotypical properties and ability to multilineage differentiation of adipose tissue stromal cells during subculturing

Morphological and immunophenotypical properties of human adult adipose tissue stromal cells (ATSC) at cultivation passage 0 and 4 as well as their ability to induced in vitro differentiation into adipogenic and osteogenic directions were studied in this work. It was shown that primary cultures of ATSC were characterized by the presence of the lower number of cells expressing mesenchymal markers (CD73, CD105) than the cells of the 4th passage, but contained endothelial progenitor cells expressing CD34 and capable to form capillary-like structures within extracellular matrix. Both cell populations could equally differentiate into adipogenic and osteogenic lineages.

Hepatoprotective Effect of Fetal Tissue Cytosol and Its Thermostable Fraction in Rats with Carbon Tetrachloride-Induced Hepatitis

Pretreatment of rats with cytosol of fetal tissue or its thermostable fraction prevented death of animals from CCl4 intoxication, decreased serum transaminase activities and level of TBA-reactive products, and normalized the prooxidant/antioxidant balance in the liver. The effect of cytosol was more pronounced than that of its thermostable fraction.

Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived from human fetal liver

The study of the differentiation potential of multipotent stromal progenitor cells (PC) in embryogenesis is a crucial issue for understanding their biology and role in the tissue regeneration of an adult organism. In this study, in monolayer culture, osteogenic and adipogenic potencies of fibroblast-like PCs derived from human fetal liver of 8–11 gestation weeks were investigated before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells includes immature stromal fibroblast-like PCs, which were able to induce osteogenic and adipogenic differentiation. The short-term exposure of recently isolated human fetal liver cells to cryoprotectant DMSO led to alterations in the properties of fibroblast-like PCs. Under subculture conditions, an increase in the number of fibroblast-like PCs capable of inducing osteogenic differentiation in vitro was discovered. It is necessary to take this established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs into consideration when developing cryopreservation methods for stem cells.

Effect of transplantation of human fetal tissues on prooxidant-antioxidant equilibrium in the liver and blood rats after partial hepatectomy in rats.

We studied the effect of transplantation of fetal liver cells and postnuclear cytoplasmic fraction from human fetal soft tissues on the prooxidant-antioxidant equilibrium in the liver and blood of rats after partial hepatectomy. The preparations increased antioxidant activity and decreased the intensity of lipid peroxidation, which probably contributes to their therapeutic effects.