In this brief review, some key issues related to vitreous cryopreservation of living tissues (natural or engineered), including cells, embryos, tissues, organs, and engineered tissues, are outlined. The principle of vitreous cryopreservation for the biological activity and functionality is demonstrated. The procedures of cooling/ rewarming, composition and function of optimal cryoprotectants, and their effects on bioproducts are described. Vitrification could, therefore, prove to be a useful and effective method of bioproduct cryopreservation for a long period of time, particularly for organized tissues and organs. However, the toxicity of the cryoprotective agents and the devitrification occurring during the rewarming process need additional investigations. Several key areas of research on vitrification are also addressed.
Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Humans ; Organ Preservation ; methods ; Tissue Preservation ; methods

The purpose of this study was to establish a set of techniques for cryopreservation of platelets with dimethylsulphoxide (DMSO) to insure high quality of cryopreserved platelet. The methods were as following: (1) DMSO was filtered in stead of being sterilized before infusion into the bag with platelets. (2) The whole blood was centrifuged immediately after blood collection and the attached tube was tied on the top of the bucket. (3) The related centrifugal force was 480 x g, the accelerating and braking grades of the centrifuge for acceleration and deceleration were 9 and 4 respectively. (4) The flow rate of platelet rich plasma (PRP) could not be too high, 80 - 100 ml PRP should be harvested at 1 minute or so. The infusion rate of DMSO into the PRP was 1 ml/min. After the infusion of DMSO, the PRP bag must be put into the -80 degrees C ultra low freezer at once to make the product to be freezed as soon as possible. The cryopreserved platelet should be thawed in the cycling warm water at the temperature of 38 - 40 degrees C. (5) After thawing of platelet, the platelet, red blood cell and white blood cell were counted, and the bacteria culturing, tests for HBsAg, anti-HCV, anti-HIV, TP and ALT were carried out. The results showed that altogether 14 800 units of cryopreserved platelets were prepared including 80 units collected with blood cell separator, of which quality control was accomplished in 300 units. The manually collected platelet mean count >/= 2.4 x 10(10)/unit, while the apheresis platelet count >/= 2.5 x 10(11)/unit. The yield was over 70%. The contaminated red and white blood cells were Blood Platelets ; physiology ; Blood Preservation ; Cryopreservation ; Dimethyl Sulfoxide ; pharmacology ; Humans ; Platelet Transfusion ; Quality Control

Cell Differentiation ; drug effects ; Dimethyl Sulfoxide ; pharmacology ; HL-60 Cells ; Humans ; Proteomics

This study was aimed to search for effective cryoprotectants and freezing methods used in cord blood bank (CBB) for cryopreservation of cord blood hematopoietic stem cells. The non-programmed group using 8% final concentration of dimethyl sulfoxide (DMSO) and 5% final concentration hydroxyethyl starch (HES) (molecular weight 120,000) as protectants and group of conventional of programmed controller method using 10% DMSO only as cryoprotectant in cryopreservation of cord blood hematopoietic stem cells were compared. In each of the two groups, 15 cord blood units were used. In non-programmed group, cord blood units put in -80 degrees C refrigerator for 24 hours as a transitional step before deep-freezing in liquid nitrogen, when both of DMSO and HES had been added. The recoveries of the nuclear cells number, the yield of granulocyto-macrophage colony forming units (CFU-GM) and the cells viability in cord blood units before preservation and after thawing were tested for both methods. The results showed that no significant difference was found in above assays between two groups. The clinical application results also showed that hematopoietic engraftment rates after infusion were similar in both groups. It is concluded that the non-programmed method by -80 degrees C refrigerator as a transitional step and using the combined two protectants seems simple in operation and effective in clinical transplantation as well as the conventional programmed method.
Cryopreservation ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Hydroxyethyl Starch Derivatives ; pharmacology

This study was aimed to investigate the protective effects of dimethylsulfoxide (DMSO) combined with trehalose on the cryopreserved platelets. The platelets were preserved at -80 degrees C. The experiments were divided into 5 groups: blank control group composed of apheresis platelet suspension; trehalose group composed of apheresis platelet suspension and 0.25 mol/L trehalose; DMSO group composed of apheresis platelet suspension and 5% DMSO; 5% combined group composed of apheresis platelet suspension, 5% DMSO and 0.25 mol/L trehalose; 2.5% combined group composed of apheresis platelet suspension, 2.5% DMSO and 0.25 mol/L trehalose. All the groups were thawed at 37 degrees C in a waterbath. The recovery rate of platelets and mean platelet volume (MPV) were assayed by using hemocytometer; the ultrastructural changes were examined by electron microscopy; the expressions of CD41, CD42b, CD61 and CD62p on platelets were detected by flow cytometry. The results indicated that single use of trehalose had no strong effect in increasing the recovery rate of platelets, but the morphology of platelets was close to normal. The DMSO showed significant effect in increasing the recovery rate of platelets and maintaining the intact property of platelets, however, the shape of platelets tended to sealing, and partial platelets still displayed heteromorphic changes. The combination of DMSO and trehalose revealed the protective effect on the external morphology and internal structure of platelets to be close to the normal homeostasis, and ensured an ideal recovery rate of the cryopreserved platelets and higher expression levels of CD41, CD42b, CD61 and CD62p in the same time. It is concluded that the combined use of DMSO and trehalose possesses the synergistic protective effect on the cryopreserved platelets, therefore, the combined use of both as the protective agent is hopeful to further raise the effectiveness of clinical infusion of the cryopreserved platelets.
Blood Platelets ; drug effects ; Blood Preservation ; methods ; Cryopreservation ; methods ; Dimethyl Sulfoxide ; pharmacology ; Humans ; Platelet Count ; Trehalose ; pharmacology

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Cryopreservation ; Dimethyl Sulfoxide ; pharmacology ; Dose-Response Relationship, Drug ; Hepatocytes ; physiology ; Swine

To study effect of vitrification state of protective solutions on recovery of red blood cells after lyophilization, four protective solutions composed of isotonic buffers containing 7% DMSO (v/v) and 20%, 30%, 40% or 50% polyvinylpyrrolidone (PVP) (w/v) were adopted. Vitrification state of protective solutions was examined first when white ice crystal appeared in any protective solution during freezing or thawing, if the used solution was not a vitrification solution. Red blood cells were lyophilized in MINILYO45 freeze-dryer after washing, mixing with protective solutions and prefreezing. After lyophilization, the samples were quickly rehydrated by 37 degrees C rehydration solution. The results showed that in vitrification and devitrification experiments, white ice crystal appeared in solution of 20% PVP + 7% DMSO and 30% PVP + 7% DMSO during freezing and thawing; vitrification appeared in solution of 40% PVP + 7% DMSO during freezing, but devitrification appeared during thawing; vitrification appeared in solution of 50% PVP + 7% DMSO during freezing and thawing. After rehydration, the recoveries of red blood cells and hemoglobin in 40% PVP + 7% DMSO group were (81.36 +/- 14.94)% and (77.54 +/- 12.86)%, which were significantly higher than that in 20% PVP + 7% DMSO, 30% PVP + 7% DMSO and 50% PVP + 7% DMSO groups (P < 0.01). The concentration of free hemoglobin in 40% PVP + 7% DMSO group was also significantly lower than that in other three groups (P < 0.01). With increase of PVP concentration in protective solutions, vitrification state and protective effect of these solutions also increased; when concentration of PVP in protective solution was 40% though it was not a vitrification solution, the effect of lyophilization was the best; but when concentration of PVP further increased to 50%, though it was a vitrification solution, the effect decreased. It is concluded that excessive vitrification state could not benefit lyophilization of red blood cells.
Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Dose-Response Relationship, Drug ; Erythrocytes ; cytology ; drug effects ; ultrastructure ; Freeze Drying ; methods ; Humans ; Microscopy, Electron ; Povidone ; pharmacology

The study was purposed to develop a novel cryopreserved agent (CPA) for platelets, to investigate the morphology of cryopreserved platelets in different CPA and the CD62P expression on membrane of platelets after stimulating by thrombin, as well as to compare the effect of adding UDP-Gal on preserved efficiency of preservation solutions. A novel cryopreserved agent consisting of 2% DMSO, thrombosol and UDP-Gal was developed on basis of using higher concentration of DMSO. The morphology of chilled platelets was observed by transmission electron microscope and compared with fresh platelets. The expression of CD62P on the membrane of platelets was detected at 0, l, 3 months. The results indicated that the significant effect of cryopreservation on morphology of platelets was found according to percentages of round, dendritic and irregular shapes of cryopreserved platelets. The protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal were better than that of 5% DMSO. Compared with fresh platelets, the expression of CD62P on platelet membrane decreased obviously after cryopreservation, but not observed difference at preservation for 1 month and 3 months, as well as among 3 kinds of different CPA. It is concluded that the protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal on morphology of platelets are similar, but better than that 5% DMSO. The reaction of cryopreserved platelets to thrombin decreases, while the significant difference is not found among these 3 kinds of CPA. The addition of UDP-Gal to cryopreserved agents not show the protective effect on platelets.
Blood Platelets ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Humans ; P-Selectin ; biosynthesis ; genetics ; Uridine Diphosphate Galactose ; pharmacology

Even when DNA sequencing of purified DNA template failed under the optimal condition, it can be generally contributed to high GC content. GC-rich region of template causes a secondary structure to produce shorter readable sequence. To solve this problem, the sequencing reaction was modified by using dimethyl sulfoxide (DMSO). It was found that 5% (v/v) of DMSO in the reaction mixture recovers sequencing signal intensity with reduced frequency of ambiguous bases. When DMSO was added to sequencing reaction of DNA template with normal GC content, it did not show any adverse effect. Sequencing accuracy and unambiguous base frequency were significantly improved at concentration of 2% to 5% (v/v) DMSO in GC-rich DNA template. DMSO has been empirically introduced to enhance the efficiency of PCR in GC-rich templates. However, the underlying mechanism of improved cycle sequencing by DMSO is unknown. Thus, cycle sequencing reaction was remodified with other additives such as N-methyl imidazole, N-methyl2-pyrrolidone, N-methyl-2-pyridone and glycerol, possessing the similar chemical properties as DMSO. Most of methyl nitrogen ring-containing chemicals did not improve sequencing accuracy, whereas only glycerol mimicked the positive effect of DMSO by the same extent. In the present study, we suggest that the treatment of DMSO improve cycle sequencing by the alteration of structural conformation of GC-rich DNA template.
Base Composition ; DNA/chemistry* ; Dimethyl Sulfoxide/pharmacology* ; Plasmids/genetics ; Polymerase Chain Reaction/methods* ; Sequence Analysis, DNA* ; Solvents/pharmacology ; Solvents/chemistry ; Templates

Spinal dorsal horn neurons play an important role in the processing of sensory information and are also targets of modulation by both endogenous and exogenous drugs. Propofol is an intravenous anesthetic and whether it has direct modulatory actions on sensory neuronal responses of the spinal cord dorsal horn has not been well studied. In the present study, a single dose (0.5 micromol) of propofol dissolved in dimethyl sulfoxide (DMSO) was directly applied onto the dorsal surface of the spinal cord and its effect was evaluated in 25 wide-dynamic-range (WDR) neurons and 10 low-threshold mechanoreceptive (LTM) neurons by using extracellular single unit recording technique in sodium pentobarbital anesthetized rats. Compared with the DMSO treatment, propofol produced a significant inhibition of WDR neuronal activity evoked by both noxious heat (45, 47, 49 or 53 degrees C, 15 s) and mechanical (pinch, 10 s) stimuli applied to their cutaneous receptive fields (cRF) on the ipsilateral hind paw skin. To investigate whether propofol exerts a modulatory effect on non-nociceptive afferent-mediated activity, the responses of WDR or LTM neurons to non-noxious brush and pressure were also evaluated. The non-noxious mechanically-evoked responses of both WDR and LTM neurons were significantly suppressed by propofol. The present results indicate that propofol has direct actions on the dorsal horn neurons of the spinal cord in rats. However, since both non-nociceptive and nociceptive afferent-mediated activity can be suppressed, the spinal effects of propofol are not likely to be specifically associated with anti-nociception.
Anesthetics, Intravenous ; Animals ; Dimethyl Sulfoxide ; Electrophysiology ; Male ; Mechanoreceptors ; drug effects ; Nociceptors ; physiology ; Posterior Horn Cells ; physiology ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; physiology