用雙層毛細管儲存胚胎以避免冷凍過程病毒汙染

用雙層毛細管儲存胚胎以避免冷凍過程病毒汙染
缺點: 冷凍降溫較慢

http://humrep.oxfordjournals.org/content/20/2/492.full

Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws

1. V. Isachenko1,3, 
2. M. Montag1, 
3. E. Isachenko1, 
4. V. Zaeva2,
5. I. Krivokharchenko2, 
6. R. Shafei2 and 
7. H. van der Ven1

+Author Affiliations

1. ¹Department of Gynaecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany and ²Gynaecological Clinic ‘Ma-Ma’, Moscow, Russia

1. 3To whom correspondence should be addressed at: Department of Gynaecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Email: vovaisachenko@yahoo.com

• Received July 12, 2004.
• Revision received August 13, 2004.
• Accepted October 15, 2004.

 

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Abstract

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cooling of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronuclei stage oocytes (n=114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene glycol +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30–50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n=42) were rapidly cooled at a speed of 20 000°C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n=44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 200°C/min. For both groups, oocytes were thawed rapidly at a speed of 20 000°C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warming makes this process as efficient as conventional vitrification.

Figure 1.

Scheme and photographs of the container for vitrification using the ‘straw in straw’ mode: (1) 0.5 ml straw; (2) metal closing ball; (3) open-pulled straw; (4) vitrification medium with oocyte; (5) meniscus of vitrification medium.

Figure 3.

The same 2PN oocyte before vitrification (1), and after warming: for 5 min (2 and 3 at a different focus), 6 h (4), 18 h (5), 30 h (6), 42 h (7), 66 h (8), 78 h (9), 96 h (10) and 96 h, stained by Hoechst 33342 (11). Bar=30 μm.

Figure 2.

Development of oocytes after vitrification with direct cooling of open-pulled straws (OPS) in liquid nitrogen versus vitrification using cooling of OPS previously located in hermetically closed 0.5 ml straws. Superscripts a–b, c–d and e–f indicate significant differences (P < 0.05).