**3.1.1 Open Pulled Straw (OPS)**

Open Pulled Straw (OPS) have been designed to guarantee a ultra rapid freezing without ice crystals formation. The system, ideated by G. Vajta in 1998, is based on the hypothesis that decreasing the standard straw diameter, the volume of solution to vitrify is reduced too, raising the cooling rate. This method is so characterized by a very high cooling and warming rates (over 20,000◦/min) and a short contact with concentrated cryoprotective additives (less than 30 sec over -180◦). This approach reduces the possibility of chilling injury and toxic and osmotic damage. Several OPS have been developed reducing the diameter of standard straws of a half, increasing the cooling rate by 10 times and reducing by 30% the concentration of CPA and the time of exposition. Common OPS are standard 0.25 mL straws with one extremity pulled and thinned by heating. This solution increases the superficies/volume rate and hastens the cooling rate of the 2 *μ*L drop set to contain the embryo. The Open Pulled Straw produced by *MTG* are made of PVC: with a length of 93 mm, straws can have an inner tip diameter approximately of 0.65 mm for Standard OPS and of 0.3 mm for super fine OPS. Before plunging the thin straw into liquid nitrogen, embryos are treated with highly concentrated cryoprotectant (CPA) solutions of ethylene glycol (EG) and dimethyl-sulfoxide (DMSO), in variable percentage.

## **3.1.2 Cryoloop**

Cryoloop is generally applied to investigate the contribution given by cortical areas to network interactions and cerebral functions.

The Cryoloop is manufactured from straight 23 gauge hypodermic stainless steel tubing, having external and internal diameters respectively of 0.635 mm and 0.33 mm. Methanol, drawn from a external reservoir, is pumped in a Teflon tube directly in the Cryoloop that is in contact with the brain. Before reaching the Cryoloop, tubes containing Methanol are coiled and immersed in a bath of methanol and dry-ice pellets. The mixture cools the flowing methanol at a temperature of -75◦. A microthemocouple, connected to a digital thermometer allows to monitor the temperature of the Cryoloop.

The use of Cryoloop device in human oocytes vitrification is under investigation. Experiments are now focused on animal oocytes and blastocytes cryopreservation. Cryoloops used for vitrification consist of a nylon loop of 10 or 20 micron diameters mounted on a stainless steel pipe inserted into the lid of a cryovial (Fig. 7). One of the main producers is the Hampton Research Corporation.

For vitrification, blastocytes are placed on a cryoloop that has been coated with a thin film of cryoprotectant solution. Blastocytes on the cryoloop are placed into the cryovial, which is submerged and filled with liquid nitrogen and the vial is sealed. Studies demonstrate that both mouse and human blastocystes can be successfully vitrified by suspension on a nylon loop and immersing directly into nitrogen. Mouse oocytes cryopreservation has provided successful results, but this method has not been applied to human oocytes. Tests on rabbit oocytes showed a good survival rate approximately of 80% for four different protocols.

#### **3.1.3 Cryo-electron microscopic (CryoEM)**

The Cryo-electron microscopic technique involves freezing biological samples in order to view the samples with the lowest distortion and the fewest possible artifacts.

8 Will-be-set-by-IN-TECH

Open Pulled Straw (OPS) have been designed to guarantee a ultra rapid freezing without ice crystals formation. The system, ideated by G. Vajta in 1998, is based on the hypothesis that decreasing the standard straw diameter, the volume of solution to vitrify is reduced too, raising the cooling rate. This method is so characterized by a very high cooling and warming rates (over 20,000◦/min) and a short contact with concentrated cryoprotective additives (less than 30 sec over -180◦). This approach reduces the possibility of chilling injury and toxic and osmotic damage. Several OPS have been developed reducing the diameter of standard straws of a half, increasing the cooling rate by 10 times and reducing by 30% the concentration of CPA and the time of exposition. Common OPS are standard 0.25 mL straws with one extremity pulled and thinned by heating. This solution increases the superficies/volume rate and hastens the cooling rate of the 2 *μ*L drop set to contain the embryo. The Open Pulled Straw produced by *MTG* are made of PVC: with a length of 93 mm, straws can have an inner tip diameter approximately of 0.65 mm for Standard OPS and of 0.3 mm for super fine OPS. Before plunging the thin straw into liquid nitrogen, embryos are treated with highly concentrated cryoprotectant (CPA) solutions of ethylene glycol (EG) and dimethyl-sulfoxide

Cryoloop is generally applied to investigate the contribution given by cortical areas to

The Cryoloop is manufactured from straight 23 gauge hypodermic stainless steel tubing, having external and internal diameters respectively of 0.635 mm and 0.33 mm. Methanol, drawn from a external reservoir, is pumped in a Teflon tube directly in the Cryoloop that is in contact with the brain. Before reaching the Cryoloop, tubes containing Methanol are coiled and immersed in a bath of methanol and dry-ice pellets. The mixture cools the flowing methanol at a temperature of -75◦. A microthemocouple, connected to a digital thermometer

The use of Cryoloop device in human oocytes vitrification is under investigation. Experiments are now focused on animal oocytes and blastocytes cryopreservation. Cryoloops used for vitrification consist of a nylon loop of 10 or 20 micron diameters mounted on a stainless steel pipe inserted into the lid of a cryovial (Fig. 7). One of the main producers is the Hampton

For vitrification, blastocytes are placed on a cryoloop that has been coated with a thin film of cryoprotectant solution. Blastocytes on the cryoloop are placed into the cryovial, which is submerged and filled with liquid nitrogen and the vial is sealed. Studies demonstrate that both mouse and human blastocystes can be successfully vitrified by suspension on a nylon loop and immersing directly into nitrogen. Mouse oocytes cryopreservation has provided successful results, but this method has not been applied to human oocytes. Tests on rabbit oocytes showed a good survival rate approximately of 80% for four different protocols.

The Cryo-electron microscopic technique involves freezing biological samples in order to view

the samples with the lowest distortion and the fewest possible artifacts.

**3.1 Open supports**

**3.1.1 Open Pulled Straw (OPS)**

(DMSO), in variable percentage.

network interactions and cerebral functions.

allows to monitor the temperature of the Cryoloop.

**3.1.3 Cryo-electron microscopic (CryoEM)**

**3.1.2 Cryoloop**

Research Corporation.

Fig. 7. CryoLoop Properly Installed in a CrystalCap Copper Magnetic - *Hampton Research Corporation*

Biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing (about 3,000◦/min), usually in ethane slush close to liquid nitrogen temperature. Specimens, maintained at liquid nitrogen temperature or colder, are contained into the high-vacuum of the electron microscope column. The frozen sample grid is then kept at liquid nitrogen temperature in the electron microscope and digital micrographs are collected with a camera. Images obtained from the cryo-electron microscopy are usually very noisy and have very low contrast. It is necessary to smooth the noise as well as enhance the contrast.
