Effects of Cryoprotective Agents on the Bovine Articular Chondrocyte Viability

Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2 propanediol on the bovine articular chondrocyte viability were examined experimentally. The CPA was added at the concentrations of 0 6, 0 9, 1 2 and 1 5 mol/L and at 4 ℃ and 37 ℃ and removed at 37 ℃ in one step. CPA stepwise addition and removal at 0 6 and 1 2 mol/L and at 37 ℃ was also tested as an alternative protocol. Cell volume excursion during DMSO addition and removal was estimated and correlated well with cell survival rates. Solution makeup affects cell survival rate and a stepwise protocol can improve the cell survival rates significantly.

Protective effect of exogenous adenosine triphosphate on hypothermically preserved rat liver

AIM:To clarify the protective effect of exogenous adenosine triphosphate(ATP)on hypothermically preserved rat livers. METHODS:Establishment of continuous hypothermic machine perfusion model,detection of nucleotides in hepatocytes with HPLC,measurement of activities of LDH and AST in the perfusate,observation of histopathological changes in different experiment groups,and autoradiography were carried out to reveal the underlying mechanism of the protective effect of ATP. RESULTS:The intracellular levels of ATP and EC decreased rapidly after hypothermic preservation in control group,while a higher ATP and EC level,and a slower decreasing rate were observed when ATP-MgCl_2 was added to the perfusate (P<0.01).As compared with the control group,the activities of LDH and AST in the ATP-MgCl_2 group were lower(P<0.05). Furthermore,more severe hepatocyte damage and neutrophil infiltration were observed in the control group.Radioactive [α-^(32)P]ATP entered the hypothermically preserved rat hepatocytes. CONCLUSION:Exogenous ATP has a protective effect on rat livers during hypothermical preservation.However,Mg^(2+) is indispensable,addition of ATP alone produces no protective effect.The underlying mechanism may be that exogenous ATP enters the hypothermically preserved rat liver cells.

Effects of storage media,supplements and cryopreservation methods on quality of stem cells

Despite a vast amount of different methods, protocols and cryoprotective agents(CPA), stem cells are often frozen using standard protocols that have beenoptimized for use with cell lines, rather than with stem cells. Relatively fewcomparative studies have been performed to assess the effects of cryopreservationmethods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent forthe development of cryobiology and has been used universally for cryopreservation.However, the use of DMSO has been associated with in vitro and in vivotoxicity and has been shown to affect many cellular processes due to changes inDNA methylation and dysregulation of gene expression. Despite studies showingthat DMSO may affect cell characteristics, DMSO remains the CPA of choice, bothin a research setting and in the clinics. However, numerous alternatives to DMSOhave been shown to hold promise for use as a CPA and include albumin,trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, wewill discuss the use, advantages and disadvantages of these CPAs for cryopreservationof different types of stem cells, including hematopoietic stem cells,mesenchymal stromal/stem cells and induced pluripotent stem cells.

Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation

Background： L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods： The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F 1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion ofdimethyl sulfoxide （DMSO） and ethylene glycol （EG）, comparing with the control group （15% DMSO and 15% EG without L-proline）. The survival rate, 5-methylcytosine （5-mC） expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results： L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions： It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.

Effect of storage temperature and osmotic pre-treatment with alternative solutes on the shelf-life of gilthead seabream(Sparus aurata)fillets

The objective of the study was the kinetic modelling of the shelf-life of osmotically pre-treated fish during refrigerated and super-chilled storage.Fresh gilthead seabream(Sparus aurata)fillets were treated for 0-360 min at 15℃ in osmotic solutions 50:5 high dextrose equivalent maltodextrin:NaCl/100 g(HDM),40:10:5 HDM:trehalose:NaCl/100 g(HDM+treh)and 40:10:5 HDM:glucosamine:NaCl/100 g(HDM+gluc).Water loss,solid gain,salt content and water activity were monitored throughout treatment.Slices untreated and osmotically pre-treated for 45 min were aerobically packed and stored isothermally at 15,10,5,2.5,0,-1 and -3℃.Quality assessment was based on microbial growth(total viable count,Pseudomonas spp.,Brochothrix thermosphacta,Enterobacteriaceae spp.,H_(2)S-producing bacteria,lactic acid bacteria,yeasts and moulds),total volatile nitrogen(TVB-N),lipid oxidation(TBARs)and sensory scoring.Quality indices were kinetically modelled and temperature dependence of quality loss rates was modelled by Ratkowsky equation.Osmotic pre-treatment led to significant shelf-life extension of fillets,in terms of microbial growth,chemical changes and organoleptic deterioration.The pre-treatment with the alternative solutes led to depression of the freezing point(-1.8,-2.6,-3.2 and-3.5℃ for the untreated samples and the osmotically pre-treated with HDM,HDM t treh and HDM t gluc,respectively).TVB-N values were higher in untreated samples,followed by osmotically treated fillets,mainly at higher storage temperatures(i.e.10 and 15℃).Based on the mathematical models for sensory evaluation scoring,the shelf-life was 12,19,22 and 22 days at 0℃ for untreated and osmotically pre-treated with HDM,HDM t treh and HDM t gluc fish slices,respectively,while the respective values at -3℃ were 21,35,38 and 38 days.The alternative solutes had no significant effect on the quality and shelf-life of pre-treated fish fillet during storage at refrigerated conditions.

Cryopreservation of farm animal gametes and embryos:recent updates and progress

Cryopreservation has undergone tremendous advances and is widely used in animal production based on decades of study of cellular permeability, freezability and empirical generalization. Several improvement are particularly important: the cryopreservation protocol has been continuously re?ned over the years to achieve greater reproductive performance; cryoprotective agents are more effective and less toxic than previously; there has been signi?cant innovation in advanced cryopreservation systems and carriers. Despite this, there are still problems that urgently require practical solutions, such as remedies for cryodamage and encouraging the use of frozen–thawed porcine sperm in pig production.