Closed Vitrification System as A Platform for Cryopreservation of Tissue Engineered Constructs.

BACKGROUND: Cryopreservation of mesenchymal stromal cells (MSCs) and MSCs-based tissue engineered constructs (TECs) is a promising strategy for regenerative medicine. OBJECTIVE: To examine vitrification system consisting of multicomponent vitreous solution, closed type container, human adult MSCs and two-step exposure procedure as a platform for cryopreservation of MSCs-based TECs. MATERIALS AND METHODS: Vitrification properties of solutions were studied by visual analysis and calorimetry. Viability (trypan blue, MTT-test), metabolic activity (Alamar Blue assay) and adhesion of cells were assessed both after exposure with vitreous solutions and following rapid cooling-thawing in standard cryovials. RESULTS: The feasibility of the vitrification system was tested on MSCs suspensions (S-MSCs) and alginate encapsulated MSCs (AE-MSCs). The minimal concentrations of cryoprotectants, which allowed avoiding ice formation during rapid cooling and rewarming comprised 10 % for dimethylsulfoxide, 20 % for ethylene glycol, 20 % for 1.2-propanediol and 0.5 M sucrose. To achieve viability and metabolic activity rates of AE-MSCs comparable to S-MSCs after vitrification the extension of the exposure time within the same vitreous solution was sufficient. After vitrification both S-MSCs and AE-MSCs retained the capacity to osteogenic and adipogenic differentiation. CONCLUSION: Data demonstrate that this vitrification system can be used as a platform for development of effective protocols for cryopreservation of MSCs-based TECs.

Some Methods for Substantiating Diagnostic Decisions Made Using Machine Learning Algorithms.

Various classification algorithms used in the diagnosis of breast cancer based on microwave radiometry data are considered. In particular, their principles of operation and the possibility of substantiating diagnoses using numerical data are discussed. A substantiation algorithm based on decision trees and a naive Bayesian classifier is presented. Examples of substantiation are given for breast cancer.

Mitochondria-targeted plastoquinone derivative SkQ(1) decreases ischemia-reperfusion injury during liver hypothermic storage for transplantation.

The ability of the mitochondria-targeted plastoquinone derivative 10-(6'-plastoquinonyl)decyl triphenylphosphonium (SkQ(1)) to decrease ischemia-reperfusion injury in isolated liver during hypothermic storage (HS) was studied. Rat liver was stored for 24 h at 4°C without or in the presence of 1 µM SkQ(1) with following reperfusion for 60 min at 37°C. The presence in the storage medium of SkQ(1) significantly decreased spontaneous production of reactive oxygen species and intensity of lipid peroxidation in the liver during HS and reperfusion. The GSH level after HS in solution with SkQ(1) was reliably higher, but reperfusion leveled this effect. At all stages of experiment the presence of SkQ(1) did not prevent the decrease of antioxidant enzyme activities such as catalase, GSH peroxidase, GSH reductase, and glucose-6-phosphate dehydrogenase. The addition of SkQ(1) to the storage medium improved energetic function of the liver, as was revealed in increased respiratory control index of mitochondria and ATP level. SkQ(1) exhibited positive effect on the liver secretory function and morphology after HS as revealed in enhanced bile flow rate during reperfusion and partial recovery of organ architectonics and state of liver sinusoids and hepatocytes. The data point to promising application of mitochondria-targeted antioxidants for correction of the ischemia-reperfusion injury of isolated liver during long-term cold storage before transplantation.

Coupling of gelatin to inner surfaces of pore walls in spongy alginate-based scaffolds facilitates the adhesion, growth and differentiation of human bone marrow mesenchymal stromal cells.

We have developed a novel wide-pore scaffold for cell 3D culturing, based on the technology of freeze-drying of Ca-alginate and gelatin. Two different preparation methodologies were compared: (i) freeze-drying of Na-alginate + gelatin mixed solution followed by the incubation of dried polymer in saturated ethanolic solution of CaCl2; (ii) freeze-drying of the Na-alginate solution followed by the chemical "activation" of polysaccharide core with divinylsulfone with subsequent gelatin covalent attachment to the inner surfaces of pore walls. The scaffolds produced using the first approach did not provide adhesion and proliferation of human bone marrow mesenchymal stromal cells (MSCs). Conversely, the second approach allowed to obtain scaffolds with a high adherence ability for the cells. When cultured within the latter type of scaffold, MSCs proliferated and were able to differentiate into adipogenic, osteogenic and chondrogenic cell lineages, in response to specific induction stimuli. The results indicate that Ca-alginate wide-pore scaffolds with covalently attached gelatin could be useful for stem cell-based bone, cartilage and adipose tissue engineering.

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.

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.

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.

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.

The reduction of Alamar Blue by peripheral blood lymphocytes and isolated mitochondria.

Alamar Blue is a widely used nontoxic indicator of cell proliferative activity, which penetrates quickly through the biological membranes and can be easily reduced by intracellular enzymes. Accumulation of reduced fluorescent form of Alamar Blue during short-term culture of human peripheral blood lymphocytes may be used as a cell viability test since it was prevented by disruption of plasma membrane by digitonin. The inhibition of Alamar Blue reduction by NaN3 indicates that its metabolism is associated with mitochondrial activity. A compaative study of Alamar Blue reduction and oxygen consumption on isolated rat liver mitochondria shows, that the Alamar Blue reduction is not associated with the activity of specific complex of respiratory chain and it seems to be an integral indicator of oxidation-reduction activity of respiratory chain components.

The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates.

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.

Dynamics of the changes in homogenate respiratory activity after rat whole liver storage at 4 degrees C.

Homogenate respiratory activity was studied after different storage terms of the whole rat liver at 4 degrees C in sucrose-based solution and following normothermic reperfusion. Preservation of homogenate respiratory activity in all metabolic states after normothermic reperfusion of the control liver (60 min, 4 degrees C) is shown. Further storage (6 and 24 hrs) of isolated liver under the mentioned above conditions strengthens the substrate respiration of homogenate both after storage and after normothermic reperfusion. At the same time oxidative phosphorylation does not practically change. No change was noted in respiratory activity in the states 3, 4ADP and 3DNP after 24 hrs of liver storage in respect of a previous term. Following normothermic liver reperfusion contributes to a statistically true reduction of mentioned parameters of respiration, that correlates with a decrease in the degree of respiration and phosphorylation coupled of the studied system.