Microfluidics technology may be an effective method to solve some problems in cryopreservation. This review presents the research progress of microfluidics technology in the field of cell membrane transport properties, cryoprotectant addition and washout and the vitrification for cryopreservation of biological materials. Existing problems of microfluidics technology in the application of cryopreservation are summarized and future research directions are indicated as well.
Cell Membrane ; Cryopreservation ; Cryoprotective Agents ; Membrane Transport Proteins ; Microfluidics

The right choice of frozen protective agents and additives is an important factor to ensure the quality of frozen platelets. The immediate hemostatic function of frozen platelet in vivo is superior to liquid-stored platelets. In order to ensure the quality of frozen platelets better, it is important to understand the role of dimethylsulfoxide (DMSO) in the preservation of frozen platelets and its mechanism, and to understand the mechanism of DMSO enhancing the hemostatic function of frozen platelets and the effects of different factors on the frozen platelets. In this review, the long-term preservation of frozen platelets and its quality standards, the mechanism of DMSO effect on the molecular changes and inhibition of frozen platelets, different factors influencing preservation freezing platelets and the test of preservation effects, the application of frozen platelets to the military operation and disaster relief, the canine frozen platelet studies and so on are summarized.
Animals ; Blood Platelets ; Blood Preservation ; methods ; Cryopreservation ; Cryoprotective Agents ; Dimethyl Sulfoxide ; Dogs ; Humans

Vitrification is a promising alternative to tissue preservation, which will greatly avoid the ice-crystal formation and circumvent the hazardous effects associated with ice formation during the entire procedures. In this study, we evaluate the effect of vitreous cryopreservation of rabbit trachea by comparing the vitrification procedure with the conventional computer-programmed slow freezing approaches. Harvested tracheae were tailored and divided into groups and cryopreserved by vitrification and programmed freezing, respectively. The morphology and ultrastructure of the thawed tracheal fragments were examined by using HE staining, TUNEL test, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to assess the integrity of the tracheal fragments. The morphological studies demonstrated both cryopreservation procedure retain the interity of trachea, and that epithelium mucosae, cilia and cartilage cells were in good shape. Compared with slow freezing methods, vitrification was less detrimental to cartilage cells and had a higher survival rate of chondrocytes and coverage of epithelium and cilia. Therefore, vitrification method is a more satisfactory method to preserve trachea, the survival of chondrocytes in situ in cartilage tissue is adequate, and respiratory epithelium is soundly preserved.
Animals ; Cryopreservation ; methods ; Cryoprotective Agents ; chemistry ; Microscopy, Electron, Scanning ; Rabbits ; Tissue Preservation ; methods ; Trachea ; cytology ; ultrastructure

OBJECTIVETo investigate the effects of cryoprotectant with glycerol and the freezing-thawing procedure on the motility of

METHODSThe motion characteristics of human sperm from 18 selected specimens were assessed by computer-assisted human sperm. sperm analysis before and after adding hyper-osmolarity cryoprotectant with glycerol and the freeze-thaw procedure, and the data were evaluated in pairs.

RESULTSThe adding of cryoprotectant caused an increase in the proportion of rapid linear motile sperms (P < 0.05) and sperm velocities, including VCL, VSL and VAP (P < 0.005). But no changes were observed in the proportion of progressively motile sperms and sperm motility. Compared with the data from pre-frozen samples, velocities of post-thawed sperms and the percentage of motile sperms in each grade significantly declined (P < or = 0.01), so did ALH, while WOB, LIN and STR remained unchanged (P > 0.05). Significant differences in ALH, WOB, LIN and STR were observed only in comparison between the post-thawed and pre-treated samples.

CONCLUSIONThe number of Grade a sperms and sperm velocity increased after adding hyper-osmolarity cryoprotectant with glycerol. The sperm motile potential was impaired and even entirely destroyed in some cases by cryodamage. The influences on the sperm motion were differently induced in freezing-thawing procedures.

Cryopreservation ; Cryoprotective Agents ; pharmacology ; Freezing ; Glycerol ; pharmacology ; Humans ; Male ; Semen Preservation ; Sperm Motility ; drug effects

AIMTo evaluate two extenders and two cryoprotectant agents (CPA) for alpaca semen cryopreservation.

METHODSSemen samples were obtained from four adult alpacas (Lama pacos) and frozen using extender I (TRIS, citrate, egg yolk and glucose) or extender II (skim milk, egg yolk and fructose), each containing either glycerol (G) or ethylene glycol (EG) as CPA. Consequently, four groups were formed: 1) extender I-G; 2) extender I-EG; 3) extender II-G; and 4) extender II-EG. Semen was diluted in a two-step process: for cooling to 5 degrees (extenders without CPA), and for freezing (extenders with CPA). Viability and acrosome integrity were assessed using trypan blue and Giemsa stains.

RESULTSWhen compared, the motility after thawing was higher (P >0.05) in groups II-EG (20.0% +/- 6.7%) and II-G (15.3 % +/- 4.1% ) than that in groups I-G (4.0 % +/- 1.1%) and I-EG (1.0 % +/- 1.4%). Viable spermatozoa with intact acrosomes in groups II-EG (18.7 % +/- 2.9%) and II-G (12.7 % +/- 5.9%) were higher than that in groups I-G (5.7% +/- 1.5%) and I-EG (4.0% +/- 1.0%).

CONCLUSIONThe skim milk- and egg yolk-based extenders containing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders.

Animals ; Camelids, New World ; Cryoprotective Agents ; administration & dosage ; Freezing ; Male ; Semen ; Semen Preservation ; Sperm Motility

AIMTo establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender.

METHODSSemen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity.

RESULTSThe post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity.

CONCLUSIONCynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Animals ; Cryopreservation ; Cryoprotective Agents ; Glycerol ; chemistry ; Macaca fascicularis ; Male ; Semen Preservation ; Spermatozoa ; cytology

This study was purposed to evaluate the effect of different lyophilizing protectants including human albumin, glucan, polyvinyl pyrrolidone and glycerine on lyophilized trehalose-loading red blood cells (RBC), then to screen the optimal lyophilizing protectant. The RBC were incubated in 800 mmol/L concentration of trehalose solution at 37°C for 7 hours, and washed 3 times with PBS solution to obtain the trehalose-loading RBC. The trehalose-loading RBC in control group were directly lyophilized without lyophilizing protectants, the trehalose-loading RBC in the experimental group were mixed with Lyophilizing protectants. The samples of 2 groups were kept at room temperature for 30 minutes, pre-frozen at -80°C for 24 hours, then lyophilized in freeze-dryer for 24 hours. Finally the samples were quickly rehydrated by 6% HES at 37°C. The recovery rate and hemolysis rate of hemoglobin were detected by using cyanohemoglobin detection kit. The water content of unhydrated samples were detected at the same time. The results showed that when the moisture content of sample was 3% - 5%, the recovery rate of hemoglobin in control group was 33.57 ± 2.89%, and that in experimental group was 51.15 ± 1.98%, there was statistically significant difference between the control and experimental group (P < 0.05). When the different concentration of dextran solution was chosen as protectants, the recovery rate of hemoglobin of lyophilized RBC was obviously lower. The higher concentration of dextran, the better the recovery rate. The recovery rate of hemoglobin was 22.15 ± 4.12% when the concentration of dextran was 36%, there were statistically significant difference between the two groups (P < 0.05). When the different concentration of polyvinyl pyrrolidone (PVP) solutions was chosen as protectants, especially the concentration below 40%, the recovery rate of hemoglobin of lyophilized RBC was significantly belower than the control group, there was statistically significant difference between the two groups (P < 0.05). When 10% glycerol was used as protectants, the recovery rate of hemoglobin was 3.93 ± 1.80%. There was also statistically significant difference between the two groups (P < 0.05). It is concluded that human serum albumin shows an important protective effect on the lyophilization of the trehalose-loading red blood cells. The dextran and PVP at the concentration lower than 40% can decrease the protective effect of trehalose in cells. Glycerol can not be chosen as protectant for lyophilized trehalose-loading red blood cells.
Blood Preservation ; methods ; Cryoprotective Agents ; pharmacology ; Erythrocytes ; drug effects ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4-12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients' conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.
Cryopreservation ; methods ; Cryoprotective Agents ; adverse effects ; pharmacology ; Humans ; Organ Preservation ; methods

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

Lyophilization is the best preservation of platelets at present. Lyophilized platelets could be stored in circumstance temperature, small space and be transported with portability, but lyophilizing may damage platelet membrane, denature protein and induce platelet activation. The chain of events including freezing and drying that lead to activation are related to the phase transition of platelet membrane lipid. Application of protectants in accordance with damage mechanism of lyophilization could alleviate the damnification. Human platelets can be preserved using lyophilization successfully in the presence of trehalose in laboratory presently, that retains normal functions and activities after rehydration. These findings suggest that the application of lyophilized platelets in transfusion medicine will be carried out in future. In this paper damage effect of lyophilization on platelets, application of cryoprotectants in the platelet lyophilization and experimental studies on platelet cryopreservation were reviewed.
Blood Platelets ; physiology ; Blood Preservation ; Cryoprotective Agents ; pharmacology ; Freeze Drying ; Humans