Objective: to study the biological properties of macroporous cryostructurate from multicomponent concentrated collagen-containing solution (MCCS) as a promising matrix for the formation of cell- and tissue-engineered constructs. Materials and methods. A macroporous spongy carrier was obtained by cryostructuring of collagencontaining extract, prepared by acetic acid hydrolysis of chicken connective tissue (BIOMIR Service, Russian Federation). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (Sigma-Aldrich, USA) was used to make the cryostructurate water insoluble. The micromorphology of the sponge surface was studied using scanning electron microscopy. The cytotoxicity of the carrier was evaluated by reaction of the mouse NIH 3T3 fibroblast cell culture using automated microscope IncuCyte ZOOM (EssenBioscience, USA). Biocompatibility of the macroporous carrier was studied on cultures of human adipose tissue-derived mesenchymal stromal cells (AD-MSC), human hepatocellular carcinoma cell line HepG2 and human umbilical vein endothelial cell line EA.hy926 . The metabolic activity of cells was determined using PrestoBlue™ reagents (Invitrogen™, USA). Cell population development during long-term cultivation of the cell-engineered construct (CEC) was assessed by fluorescencelifetime imaging microscopy over the entire surface of the sample using a Leica Dmi8 inverted microscope with Leica Thunder software (Leica Microsystems, Germany). Results. Optical microscopy and scanning electron microscopy (SEM) showed the presence of pores of different sizes in the resulting biopolymer material: large pores with 237 ± 32 μm diameter, medium-sized pores with 169 ± 23 μm diameter, and small-sized pores with 70 ± 20 μm diameter; large and medium-sized pores were predominant. The studied media did not exhibit cytotoxicity. Cell adhesion and proliferation on the surface of the material and their penetration into the underlying layers during long-term cultivation were observed. The highest metabolic activity of the cells was observed for human AD-MSC on day 14, which corresponds to the normal dynamics of development of a population of cells of this type. The functional activity of HepG2 cells – albumin and urea production – was shown in the liver CEC model. Conclusion. The good adhesion and active proliferation that were shown for the three cell types indicate that the resulting biopolymer carrier is biocompatible, and that the spread of the cells into the inner volume of the sponge and active population of the sponge under prolonged culturing indicates that this material can be used to create cell- and tissue-engineered constructs.
Aim . To study the therapeutic potential of cryopreserved fetal liver cells seeded into macroporous alginategelatin scaffolds after implantation to omentum of rats with hepatic failure. Materials and methods. Hepatic failure was simulated by administration of 2-acetyl aminofl uorene followed partial hepatectomy. Macroporous alginate-gelatin scaffolds, seeded with allogenic cryopreserved fetal liver cells (FLCs) were implanted into rat omentum. To prevent from colonization of host cells scaffolds were coated with alginate gel shell. Serum transaminase activity, levels of albumin and bilirubin as markers of hepatic function were determined during 4 weeks after failure model formation and scaffold implantation. Morphology of liver and scaffolds after implantation were examined histologically. Results. Macroporous alginate-gelatin scaffolds after implantation to healthy rats were colonized by host cells. Additional formation of alginate gel shell around scaffolds prevented the colonization. Implantation of macroporous scaffolds seeded with cryopreserved rat FLCs and additionally coated with alginate gel shell into omentum of rats with hepatic failure resulted in signifi cant improvement of hepatospecifi c parameters of the blood serum and positive changes of liver morphology. The presence of cells with their extracellular matrix within the scaffolds was confi rmed after 4 weeks post implantation. Conclusion. The data above indicate that macroporous alginate-gelatin scaffolds coated with alginate gel shell are promising cell carriers for the development of bioengineered liver equivalents.
Abstract 2 H and 13 C NMR techniques were used to investigate the thermodynamic state of water molecules and of poly(vinyl alcohol) (PVA) chain segments in frozen 10% PVA solutions in 99.8% and 8.5% 2 H 2 O in the ‐66 to 3°C temperature range. The minimum amount of water not entering into the structure of ice is shown to correspond to a ratio of three water molecules to four PVA hydroxyl groups. The quantity of unfrozen water in the investigated systems was observed to change in the 5‐6°C range next to the melting point of pure water. The measurements show that, within the same temperature interval, the temperature decrease is accompanied by a sharp decrease in the 2 H spin‐lattice relaxation time T 1 , whereas 13 C T 1 and the width of the 13 C NMR signals increase up until the resolved PVA signals disappear. The correlation time of the motion of water deuterons and the PVA chain segments was calculated from the 2 H and 13 C T 1 data. The interrelation between the data obtained and the cryostructurization processes leading to the formation of gels in frozen aqueous PVA solutions is discussed.
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