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

Cryopreservation is essential for the long-term storage and banking of cartilage grafts. This paper reviews the developments on the cryopreservation of cartilage and transplantation of cryopreserved cartilage grafts during the past 10 years. It is stated that the current technologies for cryopreservation of cartilage grafts are not mature. Further systematic studies are necessary.
Animals ; Cartilage ; transplantation ; Cryopreservation ; Humans ; Tissue Preservation ; methods

OBJECTIVETo investigate the different parameters of the lyophilization procedures that affect the recovery of the rehydrated red blood cells (RBCs).

METHODSHuman RBCs loaded in tubes were cooled with 4 different modes and subjected to water bath at 25 degrees celsius;. The morphological changes of the RBCs were observed to assess the degree of vitrification, and the specimens were placed in the freeze-dryer with the temperature set up at 40, -50, -60, -70 and -80 degrees celsius;. The rates of temperature rise of the main and secondary drying in the lyophilization procedures were compared, and the water residue in the specimens was determined.

RESULTSThe protectant did not show ice crystal in the course of freezing and thawing. No significant difference was found in the recovery rate of the rehydrated RBCs freeze-dried at the minimum temperature of -70 degrees celsius; and -80 degrees celsius; (P > 0.05). The E procedure resulted in the maximum recovery of the RBCs (83.14% ± 9.55%) and Hb (85.33% ± 11.42%), showing significant differences from the other groups(P < 0.01 or 0.05). The recovery of the RBCs showed a positive correlation to the water residue in the samples.

CONCLUSIONFast cooling in liquid nitrogen and shelf precooling at -70 degrees celsius; with a moderate rate of temperature rise in lyophylization and a start dry temperature close to the shelf equilibrium temperature produce optimal freeze-drying result of human RBCs.

Erythrocytes ; cytology ; Freeze Drying ; Humans ; Tissue Preservation ; methods

In this brief review, some key issues related to cryopreservation of seeding cells, scaffolds, and engineered tissues are outlined. The importance of cryopreservation technology to the research and development of tissue engineered products is demonstrated. The biological or biochemical reaction rate must be reduced or completely shut off in order to preserve the tissue engineered products for a long period of time. Cryopreservation may be one of the possible approaches to the fulfillment of this requirement. Seeding cells are stored at low temperature. Tissue engineered scaffold products are usually lyophilized. Engineered tissues are preserved by vitreous cryopreservation technology.
Cell Count ; Cell Survival ; Cryopreservation ; methods ; Humans ; Tissue Engineering ; Tissue Preservation ; Tissue Survival

OBJECTIVETo investigate the effective method of preserving composite facial allograft so as to attenuate ischemic injury.

METHODSThe composite facial allografts were harvested from dog, perfused and preserved with 4 degrees C physiological sodium chloride and UW solution respectively. Immediately after the removal of the flap, after 12, 24, 48 h of preservation, MTT assay was used to determine the viability of several kinds of tissue, including skin, mucosa, muscle, bleed vessel, nerve and gland. The results of the two groups were compared in term of viability percentage. The pathology of several tissues were observed after 24 and 48 h of storage.

RESULTSThe viability percentage of every tissue conserved in UW solution for 48 hours was more than 75%. There was significant difference between physiological sodium chloride group and UW group (P < 0.05). Some changes, including Porous arrangement of fibers in connective tissue of skin and mucosa, hyalinization of tissue around the hair follicle and edema of cell in hair follicle, enlargement of space between muscle bundles and unclearness of boundary of acinus could be seen in physiological sodium chloride group while no significant change in UW group.

CONCLUSIONSUW solution could be considered as preservation solution for composite facial allograft.

Adenosine ; Allopurinol ; Animals ; Dogs ; Face ; Female ; Glutathione ; Insulin ; Male ; Organ Preservation Solutions ; Raffinose ; Tissue Preservation ; methods ; Transplantation, Homologous

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 summarize the experience of donor liver procurement and preparation in liver transplantation.

METHODSOne hundred and twenty-six cases of donor liver and kidney procurement and 105 cases of donor liver preparation from August, 2004 to December, 2006 were analyzed. The 105 donor liver grafts were all used for orthotopic liver transplantation.

RESULTSThe warm ischemia time of the graft ranged from 1 to 8.5 min with a mean of 4 min. The time of graft procurement ranged from 19 to 28 min (mean 22.5 min). Donor liver preparation lasted for 38 to 102 min in the 105 cases, with a mean of 51 min. The cold ischemia time of the donor liver was 5.5 to 13 h (mean 8 h). Anatomical variations were identified in 8 of the donor liver grafts.

CONCLUSIONSCold perfusion of the donor liver and repair of the hepatic artery are important procedures in donor liver procurement and preparation. Hemorrhage due to the donor graft should be prevented and the procedures should be performed in close cooperation with the recipient operation.

Adult ; Female ; Humans ; Liver Transplantation ; Male ; Middle Aged ; Organ Preservation ; methods ; Tissue Donors ; Tissue and Organ Procurement ; methods ; Young Adult

This study sought to find out a good way for the cryopreservation of tendon seeding cells so as to facilitate the preparation of tissue engineering tendons as products. The related questions are how different factors affect cell survival rate at the procedure of preservation and whether cryopreservation affects seeding cells' biological characters as well as collagen secretive function. The results of experiment indicate that DMSO is a more effective cryoprotectant in cryopreservation of tissue engineered tendon seeding cells. Blood serum nourishment is very important in cell culture, preservation and treatment. The same sustenance after cryopreservation increases cell survival rate. In the process of cryopreservation, the concentration of cells is important to cell survival rate; cell survival rate will decrease when it is less than 1.0 x 10(6)/ml. In the process of cryopreservation, the cooling speed is also important to cell survival rate, slow cooling method achieves higher cell survival rate than does the rapid cooling method. Cryopreservation by use of 10%DMSO+15%FCS+75%DMEM does not affect seeding cells' collagen secretive function greatly and does not affect seeding cells' growth curve, cell cycle and chromosome mode obviously. The prescription of 10%DMSO +15%FCS+75%DMEM is suited for the cryopreservation of tendon seeding cells.
Cell Count ; Cell Survival ; Cryopreservation ; methods ; Dimethyl Sulfoxide ; Humans ; Muscle, Skeletal ; cytology ; Tendons ; cytology ; Tissue Engineering ; Tissue Preservation ; methods

Human platelets have distinct characters when preserved by different methods. A efficient flow cytometric assay for different preserved platelets expression of CD62p and phosphatidylserine(PS) is in dire need. Efficient flow cytometric assay for CD62p and PS expressed by preserved platelets was established and the major conditions were optimized. The platelets need not to be washed to wipe off plasma and can be labelled diredtly during the sample preparation. It is efficient for flow cytometric analysis when fresh platelet riched plasma (FPRP) was set as negative control, thrombin actived FPRP, and liquid nitrogen treated FPRP were set as positive control respectively. Gly-Pro-Arg-Pro acetate salt (GPRP) was applied to prevent platelets aggregation and fibrin formation, stabilize platelets and minimize the artificial platelets activation. This is also the key to conquer difficulty of flow cytometric quantitive analysis when platelet, Ca(2+) and plasma coexist. This flow cytometric method is specially suitable for the multi-parameter assay including PS expression for cryopreserved platelets. Minimal sample manipulation, no fixation, and GPRP application resulted in minor artifacts and good sample stability. Results suggested, this flow cytometric assay for preserved platelets is simple and efficient. In addition, the author prepared four different methods treated platelets that can be easily distinguished through this flow cytometric assay. It not only makes sure the practicability of this flow cytometric assay, but also suggests the value of the treated platelets applied in preserved platelets flow cytometric ass
Blood Platelets ; metabolism ; Flow Cytometry ; methods ; Humans ; P-Selectin ; biosynthesis ; Phosphatidylserines ; analysis ; Reproducibility of Results ; Tissue Preservation

OBJECTIVETo establish a method for the germplasm preservation of R. glutinosa.

METHODPlantlets of different cultivars obtained by tip culture were inoculated into test tubes with MS medium supplemented with 0.2 mg.L-1 BA and 0.02 mg.L-1 NAA, and preserved at 4-6 degrees C in dark. At the same time, different media (A. distilled water + 10 g.L-1 agar; B. 1/2 MS + 5 g.L-1 agar; C. MS + 10 g.L-1 agar; D. 1/2 MS + 0.5 BA + 0.02 NAA + 10 g.L-1 agar; E. MS + 0.5 BA + 0.02 NAA + 10 g.L-1 agar) were set to conserve plantlets of "85-5" on the same condition.

RESULT4 months later, the death rate of "Xinggeda" was 73%, "Tucheng" 60%, "85-5" 33%, and "Beijing 1" 9%. All of the plantlets of "85-5" in different media kept alive. 10 months later, most of the preserved plantlets browned and wilted except those on medium A.

CONCLUSIONThe low temperature endurance of R. glutinosa is cultivar-dependent. Medium A can preserve "85-5" for more than 10 months in vitro.

Agar ; Culture Media ; Culture Techniques ; Plants, Medicinal ; growth & development ; Rehmannia ; growth & development ; Temperature ; Tissue Preservation ; methods