Abstract
Klinische Embryo Vitrifikation mit der Entwicklung von einzigartigen Vitrifikation Geräte im 21. Jahrhundert und mit der falschen Vorstellung entwickelt , dass extrem schnelle Abkühlung in einem "offenen" System (dh direkte LN 2 Kontakt) war eine Notwendigkeit Vitrifikation Erfolg zu optimieren. Das Dogma die Bedeutung der Kühlraten rund um führte zu unsicheren Praktiken vorbehaltlich der technischen Veränderung und zur Schaffung von Vitrifikation Geräte , die wichtige Qualitätskontrollfaktoren (zB einfache Bedienung, Wiederholbarkeit, Zuverlässigkeit, Kennzeichnung Sicherheit und Lagersicherheit) nicht berücksichtigt. Das Verständnis der Qualitätskontrolle Mängel anderer Geräte für die Entwicklung eines sicheren, reproduzierbaren erlaubt und zuverlässige uS-VTF Methode zur Minimierung richtet intra- und inter Techniker Variation. Ebenso wichtig ist, kombiniert es die Verfügbarkeit von zwei bestehenden FDA-konformen Geräten: 1) ein 0,3-ml-Ionomerharz Embryo Stroh mit internalisiert, zweifarbige, manipulations pDach Kennzeichnung mit wiederholbaren Dichtschweißung Potential; und 2) verkürzt, häufig verwendete, 300 & mgr; m ID sterile flexipettes, um direkt den Embryo zu laden (s), um eine hocheffiziente globale Vitrifikation Gerät zu erstellen. Wie andere aseptisch, geschlossen Vitrifikation Systeme (zB High Security Vitrifikation (HSV), Rapid-I und VitriSafe) effektiv in der Reproduktionsmedizin verwendet, microSecure Vitrifikation (uS-VTF) hat bewiesen , dass es eine hohe post-Erwärmung Überleben und Schwangerschaft erreichen können Ergebnisse mit seiner Aufmerksamkeit auf Einfachheit und geringere technische Variation. Obwohl die 0,3-ml Embryo Stroh eine interne hydrophobe Stecker enthalten, im Handel mit einem Standard-Samen Stroh ersetzt wurde besitzen Baumwoll Polyvinylpyrrolidon (PVP) Stecker, behielt er seine Ionomerharz Zusammensetzung Schweiß Abdichtung zu gewährleisten. Jedoch können die Wattestopfen fluid embryo Inhalte der flexipettes bei Kontakt Docht aus. Eine modifizierte uS-VTF Verfahren wurde angepasst, um eine zusätzliche innere Schweißnaht zu schließenversiegeln, bevor die Stecker auf dem Gerät Ladeseite. Der zusätzliche technische Schritt zum uS-VTF Verfahren hat seine erfolgreiche Anwendung nicht betroffen, da hohe Überlebensraten (> 95%) und die Schwangerschaftsraten heute fortsetzen.
Materials
| Name | Company | Catalog Number | Comments |
| Aluminum Cane | IVM | XC055 | |
| Ball bearings, 3/32" | VXB.com | KIT15977 | stainless steel |
| CBS semen/embryo straw, 0.3 mL | CryoBioSystems | 25292 | individual sterile |
| Color, ID rods, 30 mm | CryoBioSystems | 019021-26 | weighted |
| Culture tubes, 15 mL | Falcon | 352099 | Conical |
| Culture tubes, 10 mL | Falcon | 352057 | Snap-cap |
| Cryosleeves | Nalgene | 5016-001 | |
| Filter, 250 mL | Fisher Sci. | 09-740-2A | 0.22 μm |
| Flasks, Tissue Culture 50 mL | Falcon | 353014 | |
| Flexipettes, 300 μm ID | Cook Med. | K-FPIP-1300-10BS-5 | Sterile, 20/pack |
| Forcep, Large | Miltex | 6-30TC | |
| Forcep, Splinter - fine | Miltex | 17-305 | |
| Goblet | IVM | PA003 | |
| Heat Sealer, SYMS 1 | CryoBioSystems | 16399 | 110 V or 220 V with adapter |
| Hepes-buffered media | Life Global or | LGGH-100; 100 mL, or | stored at 2 - 8 ºC |
| Irvine Scientific | H-HTF; 90126; 100 mL | with non-essential AA's | |
| Labels, Cryo | GA International | CL-23T1 | Various colors |
| Liquid Nitrogen Tank, 40 L | MVE or Taylor Warton | various | liquid storage |
| LN2; Dewar flask, 0.5 L | Hampton Research | HR4-695 | Stainless steel |
| 6-well Custer Dishes | Biogenics | 015/020 | plasticware by case |
| Pipette Bulb, Micro Cap | Drummond | Fisher#13681451 | Hole on bulb apex |
| Petri Dishes, 35 mm | Falcon | 351006 | |
| Petri Dishes, 58 mm | Nunc | 150288 | |
| Petri Dishes, 100 mm | Falcon | 351029 | |
| Pipette Tips, ART long | Fisher Sci. | 02-707-80 | 10 - 100 μL |
| Pipet Aid | Drummond or Falcon | various | rechargeable |
| Pipetting Device, Stripper | Cooper Surgical | MXL3-STR | |
| Pipettes, Serological 1 mL | Falcon | 357521 | |
| Pipettes, Serological 2 mL | Falcon | 357507 | |
| Pipettes, Serological 5 mL | Falcon | 357543 | |
| Pipettes, Serological 10 mL | Falcon | 357551 | |
| Scissors, Surgical Mayo | Miltex | 5-SC-16 | |
| Stereomicroscope | Nikon, Olympus, Leica | various | |
| Sterile Gauze pads, 4" x 4" | Kendall Healthcare | 6939 | |
| Synthetic serum | Life Global, or | LGPS-20; 20 mL, or | stored at 2 - 8 ºC |
| Irvine Scientific | SS-99193; 12 x 10 mL | purchase low endotoxin lot | |
| Sucrose | Sigma Chemical Co. | #S9378 | Aliquot into 50 mL flasks, 1 year; 17.1 g/flask + Medium to 50 mL; makes a 1 M solution; Filter with 0.22 µm unit |
| Timer | Nalgene | 5016-001 | |
| Thawing Solution | Innovative Cryo Enterprises | BL-TS (≤1.0 M Sucrose) | T1, T2, T3, T4; tored at 2 - 8 ºC for ≤1 month after opening |
| Vitrification Solution*,** | Innovative Cryo Enterprises | BL-VS (≥7.9 M [Glycerol/EG]) | V1, V2, V3; stored at 2 - 8 ºC for ≤1 month after opening |
| * Non-permeating cryoprotective additives may include: sucrose, ficoll and sodium hyaluronate | |||
| ** Other commercial preparations are typically ethylene glycol (EG)/dimethyl sulfoxide (DMSO; 30% v/v; 4.8 M), but could be EG/propylene glycol (32% v/v; 5.2 M). Mixed solutions are typically used to reduce cryo-toxicity concerns of a high molar solution. Commercial solutions typically include an ES and VS solution. The formulation of commercial preparation is typically proprietary property. | |||
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