Cell freeze-drying can be divided into the freezing and drying processes. Mechanical damage caused by ice crystals and damage from solute during freezing shall not be ignored and lyoprotectants are commonly used to reduce those damages on cells. In order to study the mechanism of lyoprotectants to protect cells and determine an optimal lyoprotectant formula, the thermophysical properties and percentage of unfrozen water of different lyoprotectants in freezing were investigated with differential scanning calorimeter (DSC). The survival rate indicated by trypan blue exclusion test and cell-attachment rate after 24 h using different lyoprotectants to freeze hepatoma Hep-G cells were measured after cell cryopreservation. The results show that 40% (W/V) PVP + 10% (V/V) glycerol + 15% (V/V) fetal bovine serum + 20% (W/V) trehalose formula of lyoprotectant demonstrate the best effect in protecting cells during freezing, for cell-attachment rate after 24 h is 44.56% ± 2.73%. In conclusion, the formula of lyoprotectant mentioned above can effectively protect cells.
Calorimetry, Differential Scanning ; Cryopreservation ; Cryoprotective Agents ; chemistry ; Freeze Drying ; Freezing ; Hep G2 Cells ; Humans ; Trehalose ; chemistry

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

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

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.
Cryopreservation ; Cryoprotective Agents/chemistry ; *Flow Cytometry/standards ; Lymphocyte Subsets/*cytology ; Quality Control ; Reagent Kits, Diagnostic ; Time Factors

OBJECTIVESeveral cryoprotectants were employed to study the protective effect on the freeze-drying process of the oleanolic acid-loaded nanosuspensions (OLA-LS) in order to select the optimum formulation.

METHODThe protective effect was evaluated by measuring the mean particle size of samples before and after freeze-drying process.

RESULTSucrose with the concentration of 10% was selected as the optimum cryoprotectant. The average size of excellent sample was 236.3 nm (versus 211.2 nm of fresh one), and a much higher polydispersity index of 0.242 (versus 0.180).

CONCLUSIONThe optimum lyophilized powder could be obtained with suitable type of cryoprotectant with appropriate concentration and proper freeze-drying conditions.

Cryoprotective Agents ; chemistry ; pharmacology ; Drug Stability ; Freeze Drying ; methods ; Microscopy, Electron, Transmission ; Nanoparticles ; chemistry ; ultrastructure ; Oleanolic Acid ; chemistry ; Particle Size ; Solubility ; drug effects ; Sucrose ; chemistry ; pharmacology ; Suspensions

BACKGROUND: The red blood cell (RBC) deformability test is a useful method for measuring the ability of RBCs to adapt their shape to the flow conditions. Using this test, several investigators have shown the relationship between RBC deformability and numerous clinical conditions. For the quality control (QC) of RBC deformability test, we evaluated whether frozen-thawed-deglycerolized RBCs can be used as QC materials. METHODS: Packed RBCs were frozen with 40% (wt/vol) glycerol and stored at -80degrees C for 3 months. For 10 different frozen RBC panels, RBCs were thawed, deglycerolized and stored at 4degrees C for 4 weeks. Using microfluidic ektacytometer, we measured RBC deformability of the thawed RBCs. The stability of thawed RBCs was tested once a day for 28 days of storage time and was analyzed by simple regression analysis. The precision of the test using thawed RBCs was analyzed for 7 days of storage time by calculation of CV values of intra-assay (10 measurements/assay) and between-day measurements. RESULTS: Frozen-thawed-deglycerolized RBCs were stable for 1 week. Within-run and between-day precisions of the RBC deformability test during 7 days of storage of thawed RBCs were 1.4-2.9%, and 1.9-2.8%, respectively. CONCLUSIONS: Frozen-thawed-deglycerolized RBCs used in RBC deformability test showed satisfactory within-run and between-run precisions and stability for one week after thawing, and may be used as QC materials for this test.
Blood Preservation ; Cryopreservation ; Cryoprotective Agents/chemistry/isolation & purification ; *Erythrocyte Deformability ; Erythrocytes/immunology/physiology ; Glycerol/chemistry/isolation & purification ; Hematologic Tests/standards ; Humans ; Quality Control

Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast's own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 degrees C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.
Animals ; Antifreeze Proteins ; biosynthesis ; Blotting, Western ; Cold Temperature ; Coleoptera ; Cryoprotective Agents ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Freezing ; Insect Proteins ; biosynthesis ; Mice ; Pichia ; metabolism ; Sf9 Cells

OBJECTIVETo establish cryopreservation system of shoot-tips from Fritillaria anhuiensis.

METHODTaking vitrification as system of cryopreservation, the shoot tips with length 2-3 mm were precultured in MS medium enriched with 0.4 mol x L(-1) sucrose for 3 d. They were treated for 20 min with 60% PVS2 at 25 degrees C, and then subjected to ice-cooled vitrification solution for 60 min and transferred to 2 mL cryotubes with fresh vitrification solution (PVS2) and plunged into liquid nitrogen. After rapid thawing in 40 degrees C water bath for 1 min, shoot-tips were expelled into MS medium containing 1.2 mol x L(-1) sucrose for 20 min. Further recovery and growth took place on regeneration medium in the dark at 25 degrees C for 2 weeks, and then with light/dark cycle of 12/12 h. The genetic integrity of cryopreserved shoot-tips was identified through products of PCR with arbitrary primers.

RESULT AND CONCLUSIONThe highest survival rates of shoot-tips reached 79.9% by vitrification, and the regeneration rates were 52.3%. No changes were found between treated materials and untreated materials in genomic DNA.

Cryopreservation ; methods ; Cryoprotective Agents ; chemistry ; Fritillaria ; genetics ; metabolism ; Genomic Instability ; genetics ; Plant Shoots ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Preservation, Biological ; Survival Analysis ; Vitrification

OBJECTIVETo investigate the effect of Astragalus Extractum on canine isolated kidney during hypothermia perfusion and preservation.

METHODSIsolated kidneys in the control group were hypothermia perfused and preserved using conventional hypertonic adenine citrate solution (HC-A), and for those in the experimental group, using HC-A plus Astragalus extract instead. The changes of renal tissue construction were observed with light microscopy and electron microscopy. Moreover, the kidney transplantation model of dog was established to determine the changes of biochemical parameters before and after transplantation. Data were analysed synthetically.

RESULTSThe ultrastructural injury in preserved kidney of the experimental group was significantly milder than that in the control group. Parameters determined in the early stage of transplantation showed that the blood creatinine level was significantly lower and the endogenous creatinine clearing value was higher in the experimental group than that in the control group, the difference was significant (P < 0.05).

CONCLUSIONWhen Astragalus extractum is used in preserving kidney with hypothermia perfusion, it shows definite protective effect on the ischemic reperfusion injured kidney.

Animals ; Astragalus Plant ; chemistry ; Cryoprotective Agents ; pharmacology ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hypothermia, Induced ; In Vitro Techniques ; Kidney ; Kidney Transplantation ; Male ; Reperfusion Injury ; prevention & control ; Transplantation, Autologous

In order to acquire freezing model of the cryopretective solution (NaCl-propylene glycol-water ternary system) for platelet, the melting points (T(f)) of this cryopretective solutions with different solute concentration and different ratio of PG mass to NaCl mass were measured by using a differential scanning calorimeter (DSC), and these experimental data were fitting by computer. An empirical equation was derived which characterized the Tf as a function of the solute concentration and the ratio of PG mass to NaCl mass inside this solution. It was concluded that the equilibrium freezing model for human platelets in this system could be used to instruct platelet cryopreserving techniques.
Blood Platelets ; drug effects ; Cryopreservation ; methods ; Cryoprotective Agents ; chemistry ; pharmacology ; Humans ; Models, Chemical ; Organ Preservation Solutions ; chemistry ; pharmacology ; Propylene Glycol ; chemistry ; pharmacology ; Sodium Chloride ; chemistry ; pharmacology ; Temperature ; Water ; chemistry ; pharmacology