玻璃化冷凍基本概念

玻璃化冷凍須解決3大問題
chemical toxicity 化學毒性
osmotic stress 滲透壓
nitrogen (LN) contamination 液態氮汙染

提高升溫降溫速度可
下降chemical濃度解決化學毒性
下降osmotic stress


最易受傷害chilling injury之溫度為25度下降至0度(dangerous zone)

---載具必以最快速度放入LN2

2-cell 胚胎最易受冷凍過程傷害

SPS封閉式載具最大缺點為升溫降溫速度比開放式較慢

http://humrep.oxfordjournals.org/content/24/4/797.full

Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush

1. Saar Yavin1,2, 
2. Adaya Aroyo1,2, 
3. Zvi Roth2 and 
4. Amir Arav1,3

+Author Affiliations

1. ¹Institute of Animal Science, Volcani Centre, PO Box 6, Bet Dagan 50250, Israel
2. ²Department of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

1. 3Correspondence address. E-mail: arav@agri.huji.ac.il

• Received January 8, 2007.
• Revision received May 21, 2008.
• Accepted June 20, 2008.

 

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Abstract

BACKGROUND Vitrification is becoming the method of choice for embryo cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of liquid nitrogen (LN) contamination.

METHODS An innovative vitrification method that combines LN slush and sealed pulled straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with LN.

RESULTS Ultrarapid cooling of murine embryos at 32 200°C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 ± 3.5%, 106/196) than cooling at 1700°C/min in 0.25 ml straws (10 ± 2.1%, 21/197) (P < 0.05). Embryos at the 2-cell stage cryopreserved in 75% vitrification solution (VS) (100% VS contains ∼5 M ethylene glycol, 0.6 M trehalose and 6% w/v bovine serum albumin) in SPS formed blastocysts at a higher rate (79 ± 3.6%, 99/126) than cryopreservation in 100% VS (31 ± 6.5%, 16/51), however, this was not significantly different from the fresh control group (88 ± 4.6%, 43/49). Early stage embryos at the 2 pronuclei- and 4–8-cell stage formed blastocysts at rates of 68 ± 4.5 and 60 ± 3.7%, respectively, after vitrification in 87.5% VS.

CONCLUSIONS This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

Figure 1

Schematic illustrating sealed pulled straws (SPS) loaded with embryos.

VS, vitrification solution.

Figure 2

Cooling rate measurements: SPS plunged into liquid nitrogen (LN) slush (dotted line), super open pulled straws plunged into LN (broken line), SPS plunged into LN (broken line with dotts) and 0.25 ml straw plunged into LN (continuous line).

(a) between 25 and −140°C and (b) through the ‘dangerous zone’ 25 to 0°C.