Vitrification is a promising approach for cryopreservation of adherent cells because it allows complete avoidance of ice formation. However, high cryoprotectant (CPA) concentrations are required to prevent freezing, and exposure to high CPA concentrations increases the risk of osmotic and toxic damage. Although cell membrane transport modeling can be used for rational design of CPA equilibration procedures, the necessary permeability data is extremely scarce for adherent cells. This study validates a method for in situ measurement of water and CPA permeability in adherent cells based on the fluorescence quenching of intracellular calcein. Permeability parameters for endothelial monolayers were measured during exposure to four common cryoprotectants (dimethyl sulfoxide, ethylene glycol, propylene glycol and glycerol) at temperatures of 4°C, 21°C and 37°C. Propylene glycol exhibited the highest permeability and glycerol the lowest. The data was fit using an Arrhenius model, yielding activation ...
Objective: To determine the permeability of unfertilized human oocytes to water and the cryoprotectant propane-1,2-diol over a range of temperatures and to use these data to predict osmotic responses under given conditions. Design: Laboratory-based study. Setting: Teaching hospital. Patient(s): Infertility patients donating unfertilized oocytes in excess of those required for treatment. Intervention(s): None. Main Outcome Measure(s): Water and cryoprotectant permeability were determined from measurements of oocyte volume excursions on exposure to 1.5 M propane-1,2-diol at 30°C, 24°C, and 10°C. Result(s): Permeability of human oocytes to water and cryoprotectant increased as temperature increased. The predicted response of oocytes, based on these data, closely matched the measured response of an oocyte on exposure to a widely used method for addition of cryoprotectant before freezing. Conclusion(s): Commonly used cryopreservation protocols involving slow cooling in the presence of ...
Cryoprotective Efficiency of Medium Combining Non-Penetrating and Penetrating Cryoprotectants When Freezing Erythrocyte Suspensions of Various Volumes
Microbial communities enriched from diverse environments have shown considerable promise for the targeted discovery of microorganisms and enzymes for bioconversion of lignocellulose to liquid fuels. While preservation of microbial communities is important for commercialization and research, few studies have examined storage conditions ideal for preservation. The goal of this study was to evaluate the impact of preservation method on composition of microbial communities enriched on switchgrass before and after storage. The enrichments were completed in a high-solid and aerobic environment at 55 °C. Community composition was examined for each enrichment to determine when a stable community was achieved. Preservation methods included cryopreservation with the cryoprotective agents DMSO and glycerol, and cryopreservation without cryoprotective agents. Revived communities were examined for their ability to decompose switchgrass under high-solid and thermophilic conditions. High-throughput 16S rRNA ...
Before freezing, embryos are first equilibrated in special solutions containing cryoprotecting agents. These agents protect the embryos from intracellular ice formation, which would be detrimental to their viability. The embryos are then placed in special straws, sealed and put in a machine with an integrated computer. This machine, the programmable freezer, lowers the temperature in a slow controlled manner until -196oC. There are several protocols of slow freezing, depending on the stage of the embryos and the type of cryoprotectant solutions used. The embryos are then plunged in liquid nitrogen and are stored until used.. Usually 1-3 embryos are placed in each straw. In this state, embryos may be preserved for a very long period of time. Embryos can be frozen at the 2PN stage (Day 1), cleavage stage (2-8 cells; Days 2-3), or blastocyst stage (Days 5-6 post oocyte retrieval).. Cryopreservation in liquid nitrogen at a temperature of -196oC does not require electric power. The only requirement ...
On Tue, 6 Jul 1999, jim wrote much, including: , Now to your point: A cryoprotectant which only surrounds and not infiltrates , the specimen can only be useful for the second method, since such a medium , contributes to the bulk of the specimen and does not lower the freezing point , of the specimen itself. Obviously this is true. I dont think even the makers of OCT compound and other such goo would claim a cryoprotective action. These materials serve to glue the frozen specimen to the cryostat chuck or bit of cork, and also provide some protection against sublimation of the ice (freeze drying) in stored frozen specimens. Gelatin (approx 2%) has a similar consistency and does the same job more cheaply. , To be effective during vitrification the cryoprotectant , must infiltrate the specimen. It must indeed. A.G.E.Pearse (Histochemistry Vol 1) makes the point that most of the cryoprotectives used with tissues - sucrose being the most popular one - penetrate only the extracellular spaces. He ...
... on the pregnancy rate of the vitrified-thawed Formosan sambar deer embryos. Hsin-Hung Lin, Chih-Hua Wang, Shann-Ren Kang, Chin-Hui Tseng, Mu-Jung Cheng, Wen-Lin Song, Ting-Chieh Kang, Shyh-Shyan Liu, and Perng-Chih Shen. The aim of this study was to investigate the effect of cryoprotectant components on the pregnancy rate of the vitrified-thawed Formosan Sambar deer blastocysts. The does were implanted with CIDR for 12 days and superovulated by intramuscular injection with eCG at day 10. The 4- to 8-cell stage embryos were collected by flushing the oviduct through midventral laparotomy at day 4 after mating. After collection, these embryos were subsequently cultured in the synthetic oviduct fluid solution (SOF) medium. Results showed that the blastocyst rate of cultured embryos was 29.6%. Thereafter, the blastocysts were vitrified with freeze medium containing 16.5% EG plus 16.5% DMSO (A) and 6.8 M EG (B), respectively. The pregnancy rate of vitrified ...
You might find some useful information in The Zebrafish Book, Chapter 7: http://zfin.org/zf_info/zfbook/cont.html#cont7 ======= Carrie R. Jones Zebrafish International Resource Center 5274 University of Oregon Eugene, OR 97403-5274 Phone: (541) 346-6028 ext. 22 Fax: (541) 346-6151 Email: cjones at zfin.org ======= May-Su YOU wrote: , I am Maysu You, now working at IMCB (Institute of Molecular and Cell , Biology) in Singapore. We are now working on sperm , squeezing,freezing and in vitro fertilization of Zebrafish. We got , around 90% of IVF successful rate when we used fresh sperm (from , both squeezed sperm and dissected testes) . So we moved to freezing , step. We tried two different cryoprotectants (methanol and BSMIS: , Buffered sperm motality inhibitor solution). After thawing, we , cannt get any IVF embryo. Therefore, we doubt about the freezing , procedure which we used at the moment. We also tried to observe the , sperm, but even with trypan blue, we are not sure wheather the , sperm ...
Cryoprotective solutions have been supplemented with macromolecules such as sucrose and human serum albumin (HSA). Several studies have shown that such molecules help to reduce physical damage and help to maintain osmotic pressure of the extracellular fluid (Shaw, et al. 2000). Besides its cryoprotective role, HSA facilitates gamete or embryo manipulation by preventing adsorption to the surface of petri dishes and pipettes through saturation of the potential binding sites. Also, the increased viscosity of the media, caused by the addition of HSA, promotes the ease of embryo handling and manipulation (Trounson and Gardner 2000 ...
Cryobiology: International Journal of Low Temperature Biology and Medicine is the official journal of the Society for Cryobiology.. It is published bi-monthly by Elsevier and contains research articles on all aspects of low temperature biology and medicine including:. ...
Dear Patsy, Your cryoprotectant solution sounds suspiciously like the solution we use to store vibratomed sections at -20C to actually keep them FROM freezing. It allows us to keep them very cold but without the damage of actually freezing them. The only difference is that we add glycerine to the solution. I suspect the ethylene glycol is the problem, I dont think it will allow the tissue to freeze. Try leaving it out of the mix and just make a 30% sucrose/0.1M phosphate buffered solution. Just my thoughts. Take care, Jo Dee Jo Dee Fish Research Technologist III Gladstone Institute of Cardiovascular Disease Telephone: (415) 734-2567 Fax: (415) 355-0824 E-mail: [email protected] Mailing address: The J. David Gladstone Institutes 1650 Owens Street San Francisco, CA 94158 -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Patsy Ruegg Sent: Friday, January 12, 2007 12:54 PM To: histonet Subject: ...
Cryobiology: International Journal of Low Temperature Biology and Medicine publishes research articles on all aspects of low temperature biology and...
Audrey U. Smith, circa 1960s Audrey U. Smith (1915-1981), the mother of Cryobiology, was born in India on 21 May 1915. She was educated Kings College, London (first-class B.Sc., 1935); Bedford College for Women (first-class B.Sc. in physiology, 1936); registered … Continue reading →. ...
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Excellent freezing of the specimens intended for examination in the electron microscope is one of the most important prerequisites for achieving reproducible results from the various subsequent cryopreparation methods.. The freezing method should produce microcrystalline or amorphous ice from the specimen water. To achieve this, the specimens must be frozen as quickly as possible at a freezing rate no lower than 10 000°C/s.. The conventional freezing methods in use are plunge freezing, jet spray and cold block (slamming) cryo fixation. However, due to the poor heat conductance of water, these methods can only satisfactorily freeze specimens measuring up to between 10 and 20µm.. Thicker specimens (such as tissue samples) could only be frozen in the past, if a cryoprotectant was added to lower the freezing point of the water in the specimen. The disadvantage of chemical cryoprotectants is that they often affect certain cell structures, causing different types of undesirable artefacts.. By using ...
On the strategy of reimbursing after the years premiums are paid, I also believe thats a crap shoot. It may not have been challenged yet under audit (Ive not heard of any cases), maybe because it hasnt been found (after all you have personal checks to vouch you paid personally, right?) If it gets caught AFTER one becomes disabled....that annual benefit will be a HUGE incentive for the IRS to attack that strategy. Thats a headache I dont want if my family is relying on ALL that tax free income ...