Cryoprotective Effect of Pectin Tanacetan from Tanacetum vulgare L.

We researched the ability of tanacetan pectin from inflorescences of common tansy Tanacetum vulgare L. to change the osmolarity and freezing point of water in solutions of cryoprotectants: glycerol-3.5%, dimethyl sulfoxide (DMSO)-10%, dimethylacetamide-10% (DMAC), and 1.2-propanediol (1.2-PD)-10%, as well as the effect of solutions of tanacetan (0.2%, 0.4%) on the kinetics of crystallization processes and the nature of crystal formation. We used a combination of protector and pectin that we tested earlier, which provided effective protection for human leukocytes and platelets, as well as bovine spermatozoa, at temperatures below freezing (-20°C and -80°C). It has been established that tanacetan slows down the process of water freezing in glycerol, but not in DMSO, DMAC, and 1.2-PD, promotes deeper supercooling of the medium, and affects the morphological structure of ice. The addition of pectin to the cryosolution increases the activity of the main cryoprotectant glycerol even at its low concentrations. The combination of glycerol and tanacetan can be effective in freezing biological materials, which is confirmed by the preservation of leukocytes at -20°C and -80°C for 7 days, platelets at -80°C for 30 days, and spermatozoa at -80°C within 1 day. A comprehensive analysis of the chemical, physicochemical, and cryoprotective properties of tanacetan indicates the prospect of using pectin in the cryopreservation of biological objects at temperatures of electric freezers.

Apple Pectin as a New Component for Cryopreservation of Nucleated Cells.

This study explored the ability of apple pectin AU-701 to change the freezing point of water in cryoprotectant solutions with different penetrating abilities using glycerol, dimethyl sulfoxide (DMSO), 1,2-propanediol (1,2-PD), dimethyl acetamide (DMAC), hydroxyethyl starch (HES), hexamethylenebistetraoxyethylurea (or substance A-378), and in biological fluid (human venous blood). An effective interaction was used to protect human blood leukocytes at ultrafreezer temperature (-80°C). Apple pectin affects the freezing temperature of water in different ways and it depends on the medium in which it is dissolved, as it either slows down the freezing process (in glycerol) or accelerates it (venous blood). The addition of apple pectin to the cryosolution increases the activity of the base cryoprotector (glycerol) even at low concentrations. Therefore, the combination of these substances can be effective in freezing biological substances, which is proved by indicators of safety of leukocytes during the freezing process at low temperature (-80°C) for 14 days.

Effect of Pectins on Water Crystallization Pattern and Integrity of Cells During Freezing.

The ability of various pectin polysaccharides to modify the morphological structure of ice during the phase transitions from water to ice was studied. Pectins were isolated from Sosnowsky's hogweed Heracleum sosnowskyi Manden (heracleuman-N6HS), tansy Tanacetum vulgare L. (tanacetan-N7TVF), and Rauwolfia serpentina Benth callus (rauwolfian-N8RS). Pectins were isolated by multistep extraction. The effect of pectins was assessed using osmometry, thermographic analysis, and cryomicroscopy. A concentrate of leukocytes was used as the sample for the subsequent freezing step. The condition of the leukocyte membrane, and lysosomal and phagocytic activity after a freezing-warming process were assessed. Osmotic concentrations of the pectin polysaccharide solutions were found to be very low. The 0.4 wt % N7TVF solution had the highest osmotic concentration as well as freezing point; however, the duration of its crystallization plateau was lower than that of the 0.4 wt % and 0.2 wt % N6HS solutions. All studied polysaccharide solutions demonstrated a high linear rate of ice crystal growth. There were statistically significant differences between the melting rates for the 0.2% solutions of the pectins, N6HS and N7TVF, N6HS and N8RS, as well as between concentrations for the pectin N7TVF and between concentrations for the pectin N8RS. The data on the integrity of cells that are frozen in a medium containing polysaccharides may indicate a cryoprotective effect of the N7TVF and N8RS pectins, that is, tanacetan from tansy and rauwolfian from rauwolfia. The most effective modifier among the substances, which were studied by us, was the N7TVF pectin polysaccharide (tanacetan from tansy).

Use of Inert Gases for the Preservation of Nuclear Blood Cells

Abstract The subject of the study was the stability of human white blood cell membranes subject to noble gases (xenon ad krypton, 0.6 mPa) clathrate cryoanabiosis (‒80°C). A unique portable stainless steel low pressure container with a compartment for flexible plastic container was designed to ensure that the cells are saturated with gases. The samples were warmed after 1 and 30 days in a water bath (+38°C) for 35-50 sec, while the container was being tilted (2-3 times per second), until the temperature of the biological object reached +3±1°C. It was demonstrated that after 30 days of clathrate anabiosis (-80°C) over 95% (of the original number) of leukocytes remain viable, and cell membranes of 54.5±3.4% of them is resistant to trypan blue; granulocyte survival rate is 73.5±2.7%, original lipid peroxidation rate and antioxidant activity are retained. Biological object cryopreservation in noble gases environment is a promising trend in biology and medicine.

The Cryoprotectant Effect of Polysaccharides from Plants and Microalgae on Human White Blood Cells.

The use of carbohydrates as cryoprotectants is increasing. In this study the effects of incorporating polysaccharides extracted from plants and microalgae originating in northern Russia, into cryoprotectant solutions used to preserve human white blood cells were investigated. Cells in the presence of the polysaccharides were cooled to either -40°C or -80°C, using a two-step cooling process. The morphological and functional indicators of the cryopreserved leukocytes were assessed by light microscopy. When combined with glycerol, the pectin-polysaccharides Lemnan from common duckweed (Lemna minor L.) and Comaruman from marsh cinquefoil (Comarum palustre L), were capable of lowering the freezing point of the cryoprotectant solution and helped to preserve the integrity of the human white blood cell membranes at temperatures below zero. In addition, the increase in phagocytic activity of neutrophils was confirmed. In the context of the contemporary search for effective cell cryoprotectants, the results of this research demonstrate that the cryopreservation of biospecimens in a polysaccharide environment is a promising trend in applied medicine, which can be considered an alternative to traditional cryogenic nitrogen techniques.