Directional Solidification Stage With Dynamically Variable Speeds: Assessment of Cell Viability After Interrupted Cooling

2011 ◽  
Author(s):  
Josh LaFountain ◽  
Ram V. Devireddy
Keyword(s):  
Cell Viability ◽  
Directional Solidification ◽  
Cooling Rate ◽  
Room Temperature ◽  
Adult Stem Cells ◽  
Membrane Integrity ◽  
Freezing Method ◽  
A Cell ◽  
Directional Freezing ◽  
Solidification Stage

A two-step directional freezing method was utilized to assess the membrane integrity in frozen/thawed adult stem cells (ASCs). Using a custom built directional solidification stage (DSS) we determined that cells cooled at two different rates (i.e., a slower rate between room temperature, 27 °C and −10 °C and a higher rate between −10 °C and −60 °C) experienced significantly higher loss of cell viability when compared with those cooled at a single and uniform slower cooling rate between 27 °C and −60 °C. Specifically, the cells cooled using the two-step freezing method exhibited a post-thaw cell viability of ∼17% while those that were cooled using the traditional method exhibited a cell viability of 49%. We also found that varying the cooling rate between 27 °C and −10 °C did not significantly affect the post-thaw viability of ASCs, suggesting that they are not susceptible to supra-zero cooling injury.

Directional freezing of spermatozoa and embryos

2014 ◽  
Vol 26 (1) ◽  
pp. 83 ◽  
Author(s):  
Amir Arav ◽  
Joseph Saragusty
Keyword(s):  
Cooling Rate ◽  
Drop Size ◽  
Mechanical Damage ◽  
Rapid Freezing ◽  
Wild Animals ◽  
Microscope Slide ◽  
Freezing Method ◽  
Wide Range ◽  
Sexed Semen ◽  
Directional Freezing

Directional freezing is based on a simple thermodynamic principle whereby the sample is moved through a predetermined temperature gradient at a velocity that determines the cooling rate. Directional freezing permits a precise and uniform cooling rate in small- and large-volume samples. It avoids supercooling and reduces mechanical damage caused by crystallisation. Directional solidification was used to date for slow and rapid freezing, as well as for vitrification of oocytes and embryos by means of the minimum drop size technique: small drops are placed on a microscope slide that is moved at high velocity from the hot base to the cold base. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The bovine sexed semen industry may benefit from the increased survival of spermatozoa after directional freezing.

scholarly journals 255 PRONUCLEUS FORMATION IN RAT ZONA-FREE OOCYTES CO-CULTURED WITH HOMOLOGOUS POST-THAW SPERMATOZOA

2005 ◽  
Vol 17 (2) ◽  
pp. 277
Author(s):  
Y. Seita ◽  
Y. Okuda ◽  
A. Takizawa ◽  
N. Hirahara ◽  
M. Koichi ◽  
...  
Keyword(s):  
Cooling Rate ◽  
Sperm Motility ◽  
Phase Contrast ◽  
Room Temperature ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Student’S T ◽  
Percentage Data ◽  
Student’S T Test

The aim of the present study was to develop an IVF system with frozen/thawed rat spermatozoa. We examined the effect of cooling rate to 5.0°C on post-thaw sperm motility and membrane integrity, and also investigated the ability of post-thaw spermatozoa to form pronuclei. Under room temperature, epididymal spermatozoa of Wistar rats were collected in 2.0 mL of egg yolk medium containing 8.0% (w/v) lactose monohydrate and 0.7% (v/v) Equex Stem. Samples were loaded into 0.25-mL straws and cooled to 5.0°C in the chamber of a programmed freezer. For cryopreservation, the samples were exposed to liquid nitrogen (LN) vapor for 10 min and then plunged into LN. Straws were thawed in a 37.0°C water bath for 10 s. Ovulated oocytes were collected and the zona pellucidae were removed with 0.1% pronase. One-hundred μL of thawed samples were put into a droplet of 400 μL R1ECM and pre-incubated for 1 h. R1ECM solution was added to the droplet to adjust to 0.5–1.5 × 106 sperm mL−1. The zona-free oocytes were then transferred into the droplet and co-cultured for 10 h. Oocytes were observed for pronuclei formation by means of an inverted phase contrast microscope. In Experiment I, the influence of sperm cooling rate to 5.0°C on sperm motility and membrane integrity was evaluated. Portions of samples were cooled at 54.0°C/min, 0.9°C/min, 0.5°C/min, and 0.3°C/min. The remainders were then frozen. The non-cooled samples were designated as controls. In Experiment II, we examined whether post-thaw spermatozoa have the ability to form pronuclei in vitro or not. All percentage data were arc-sine transformed and then analyzed by the Student's t-test. In Experiment I, the membrane integrity between the spermatozoa cooled at 0.5°C/min and the non-cooled spermatozoa was not different (38.1% vs. 37.2%; P > 0.05), but the integrity of these was higher than in spermatozoa cooled directly at 54.0°C/min (38.1% vs. 25.3%; P < 0.05). After culture for 1 h, the motility of spermatozoa cooled at 0.5°C/min was higher than that of those cooled at 54.0°C/min (61.3% vs. 53.3%; P < 0.05). At 2 h post-thaw the motility of spermatozoa cooled at 0.5°C/min was higher than that of spermatozoa cooled at 54.0°C/min and at 0.9°C/min (11.0% vs. 4.5%, 4.9%; P < 0.05). The membrane integrity of post-thaw spermatozoa cooled at 0.5°C/min was also higher compared to that of spermatozoa cooled at 54.0°C/min (22.5% vs. 8.4%; P < 0.01). In Experiment II, 28 (26.2%) of 107 oocytes had pronuclei when the post-thaw spermatozoa cooled at 0.5°C/min were used. The results indicated that the frozen/thawed spermatozoa cooled to 5.0°C at 0.5°C/min showed higher sperm motility and membrane integrity, and that spermatozoa can form pronuclei in homologous zona-free oocytes in vitro. Although in the rat sperm damage occurred during cooling to 5.0°C, and sperm motility and membrane integrity were also decreased by the cold shock, it is possible to decrease the damage by cooling slowly to 5.0°C at 0.5°C/min.

Directional Cooling of Adult Stem Cells

2006 ◽  
Author(s):  
R. Fuller ◽  
R. V. Devireddy
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Cell Membrane ◽  
Confidence Level ◽  
Adult Stem Cells ◽  
Fluorescent Dyes ◽  
Membrane Integrity ◽  
Ice Crystals ◽  
Cell Membrane Integrity ◽  
Solidification Stage

The effect of directional cooling on the immediate post thaw membrane integrity of adipose tissue derived adult stem cells (ASCs) was investigated using a directional solidification stage (DSS). ASCs were cooled at either 1, 5, 20 or 40 °C/min to an end temperature of −80°C in the presence and absence of a cryoprotective agent (dimethylsulfoxide, DMSO). After freezing to -80°C, the samples were thawed at 200°C/min and the ability of the frozen/thawed ASCs to exclude fluorescent dyes was assessed. ASCs frozen using the DSS in the presence of 0.85M (or 10% v/v) DMSO were found to have a higher post-thaw cell membrane integrity (confidence level of 99%) when compared with the ASCs frozen in its absence. Intriguingly, a comparison with corresponding data for ASCs that were frozen using a commercially available controlled rate freezer (CRF) suggests that the directionally cooled ASCs (both in the absence and presence of DMSO) exhibit a significantly lower post-thaw cell membrane integrity (confidence level of 95%). This lowering of post-thaw cell membrane integrity for ASCs frozen using the DSS is postulated to be related to the differences in the nature, and the associated damaging effects, of ice crystals formed in the DSS vs. the commercial freezer.

scholarly journals Biological Effects of XyloCore, a Glucose Sparing PD Solution, on Mesothelial Cells: Focus on Mesothelial-Mesenchymal Transition, Inflammation and Angiogenesis

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2282
Author(s):  
Valentina Masola ◽  
Mario Bonomini ◽  
Maurizio Onisto ◽  
Pietro Manuel Ferraro ◽  
Arduino Arduini ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Viability ◽  
Biological Effects ◽  
Membrane Integrity ◽  
Glucose Content ◽  
Mesenchymal Transition ◽  
Low Glucose ◽  
Endothelial To Mesenchymal Transition ◽  
Osmotic Agents

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.

scholarly journals Facile Green, Room-Temperature Synthesis of Gold Nanoparticles Using Combretum erythrophyllum Leaf Extract: Antibacterial and Cell Viability Studies against Normal and Cancerous Cells

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 893
Author(s):  
Olufunto T. Fanoro ◽  
Sundararajan Parani ◽  
Rodney Maluleke ◽  
Thabang C. Lebepe ◽  
Jose R. Varghese ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Cell Viability ◽  
Leaf Extract ◽  
Room Temperature ◽  
Klebsiella Oxytoca ◽  
Cost Effective ◽  
Average Diameter ◽  
Antibacterial Activities ◽  
Inhibition Concentration ◽  
First Time

We herein report a facile, green, cost-effective, plant-mediated synthesis of gold nanoparticles (AuNPs) for the first time using Combretum erythrophyllum (CE) plant leaves. The synthesis was conducted at room temperature using CE leaf extract serving as a reducing and capping agent. The as-synthesized AuNPs were found to be crystalline, well dispersed, and spherical in shape with an average diameter of 13.20 nm and an excellent stability of over 60 days. The AuNPs showed broad-spectrum antibacterial activities against both pathogenic Gram-positive (Staphylococcus epidermidis (ATCC14990), Staphylococcus aureus (ATCC 25923), Mycobacterium smegmatis (MC 215)) and Gram-negative bacteria (Proteus mirabilis (ATCC 7002), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13822), Klebsiella oxytoca (ATCC 8724)), with a minimum inhibition concentration of 62.5 µg/mL. In addition, the as-synthesized AuNPs were highly stable with exceptional cell viability towards normal cells (BHK- 21) and cancerous cancer cell lines (cervical and lung cancer).

scholarly journals Zinc, Zinc Transporters, and Cadmium Cytotoxicity in a Cell Culture Model of Human Urothelium

Toxics ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 94
Author(s):  
Soisungwan Satarug ◽  
Scott H. Garrett ◽  
Seema Somji ◽  
Mary Ann Sens ◽  
Donald A. Sens
Keyword(s):  
Dna Methylation ◽  
Cell Culture ◽  
Cell Viability ◽  
Zinc Transporters ◽  
Cell Culture Model ◽  
Induced Expression ◽  
Glutathione Biosynthesis ◽  
Culture Model ◽  
A Cell ◽  
Cd Exposure

We explored the potential role of zinc (Zn) and zinc transporters in protection against cytotoxicity of cadmium (Cd) in a cell culture model of human urothelium, named UROtsa. We used real-time qRT-PCR to quantify transcript levels of 19 Zn transporters of the Zrt-/Irt-like protein (ZIP) and ZnT gene families that were expressed in UROtsa cells and were altered by Cd exposure. Cd as low as 0.1 µM induced expression of ZnT1, known to mediate efflux of Zn and Cd. Loss of cell viability by 57% was seen 24 h after exposure to 2.5 µM Cd. Exposure to 2.5 µM Cd together with 10–50 µM Zn prevented loss of cell viability by 66%. Pretreatment of the UROtsa cells with an inhibitor of glutathione biosynthesis (buthionine sulfoximine) diminished ZnT1 induction by Cd with a resultant increase in sensitivity to Cd cytotoxicity. Conversely, pretreatment of UROtsa cells with an inhibitor of DNA methylation, 5-aza-2’-deoxycytidine (aza-dC) did not change the extent of ZnT1 induction by Cd. The induced expression of ZnT1 that remained impervious in cells treated with aza-dC coincided with resistance to Cd cytotoxicity. Therefore, expression of ZnT1 efflux transporter and Cd toxicity in UROtsa cells could be modulated, in part, by DNA methylation and glutathione biosynthesis. Induced expression of ZnT1 may be a viable mechanistic approach to mitigating cytotoxicity of Cd.

Mechanical properties of NiAl-Mo composites produced by specially controlled directional solidification

2012 ◽  
Vol 1516 ◽  
pp. 255-260 ◽  
Author(s):  
G. Zhang ◽  
L. Hu ◽  
W. Hu ◽  
G. Gottstein ◽  
S. Bogner ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Fracture Toughness ◽  
Directional Solidification ◽  
Creep Resistance ◽  
Room Temperature ◽  
Growth Direction ◽  
Nominal Composition ◽  
Potential Candidate ◽  
In Situ Composites

ABSTRACTMo fiber reinforced NiAl in-situ composites with a nominal composition Ni-43.8Al-9.5Mo (at.%) were produced by specially controlled directional solidification (DS) using a laboratory-scale Bridgman furnace equipped with a liquid metal cooling (LMC) device. In these composites, single crystalline Mo fibers were precipitated out through eutectic reaction and aligned parallel to the growth direction of the ingot. Mechanical properties, i.e. the creep resistance at high temperatures (HT, between 900 °C and 1200 °C) and the fracture toughness at room temperature (RT) of in-situ NiAl-Mo composites, were characterized by tensile creep (along the growth direction) and flexure (four-point bending, vertical to the growth direction) tests, respectively. In the current study, a steady creep rate of 10-6s-1 at 1100 °C under an initial applied tensile stress of 150MPa was measured. The flexure tests sustained a fracture toughness of 14.5 MPa·m1/2at room temperature. Compared to binary NiAl and other NiAl alloys, these properties showed a remarkably improvement in creep resistance at HT and fracture toughness at RT that makes this composite a potential candidate material for structural application at the temperatures above 1000 °C. The mechanisms responsible for the improvement of the mechanical properties in NiAl-Mo in-situ composites were discussed based on the investigation results.

An Ultrafast Vitrification Method for Cell Cryopreservation

2017 ◽  
Vol 140 (1) ◽  
Author(s):  
Fengmin Su ◽  
Nannan Zhao ◽  
Yangbo Deng ◽  
Hongbin Ma
Keyword(s):  
Thin Film ◽  
Cooling Rate ◽  
Experimental Results ◽  
Low Concentration ◽  
Temperature Range ◽  
Thin Film Evaporation ◽  
Film Evaporation ◽  
A Cell ◽  
Cryoprotective Solution ◽  
Ultrafast Cooling

Ultrafast cooling is the key to successful cell vitrification cryopreservation of lower concentration cryoprotective solution. This research develops a cell cryopreservation methodology which utilizes thin film evaporation and achieves vitrification of relatively low concentration cryoprotectant with an ultrafast cooling rate. Experimental results show that the average cooling rate of dimethylsulfoxide (DMSO) cryoprotective solution reaches 150,000 °C/min in a temperature range from 10 °C to −180 °C. The ultrafast cooling rate can remarkably improve the vitrification tendencies of the cryoprotective solution. This methodology opens the possibility for more successful cell vitrification cryopreservation.

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