Intracytoplasmic Sperm Injection (ICSI) - Eppendorf

TechnicalGuideIntracytoplasmic Sperm Injection(ICSI)Technical GuideEppendorf ® Cell Technology

Eppendorf Cell TechnologyIntracytoplasmic sperm injectionIntroductionThe number of infertile couples has increased inrecent years, and it is estimated that 15% to 20% ofall couples worldwide face infertility issues. Since itsintroduction into reproductive medicine in 1978, 1 in vitrofertilization has been considered a successful methodof treating tubal sterility, and after years of research it isestablishing itself as the preferred method of treatmentin the field of assisted reproduction. 2With increasingly intensive diagnoses of both partners,certain aspects of male subfertility are coming to theforefront; currently, almost 50% of infertility in couplescan be attributed to male subfertility. 3 Intracytoplasmicsperm injection (ICSI), one method of treating malesubfertility, has achieved a breakthrough 4 , and it isgaining in importance, raising hopes that these couplescan have children of their own.Preparation for ICSI involves obtaining an oocyte bymeans of follicle aspiration, removing the cumuluscells and corona radiata, and preparing the semen inaccordance with the Mini Swim-Up process 5 or otherpreparation procedures. 6 One sperm is then injectedinto the oocyte with a thin glass capillary (pipette).If fertilization occurs, the embryo is transferred intothe uterus 48 hours after microinjection.For the microinjection process described, Eppendorfoffers ideal tools for easing the workload: theTransferMan ® NK 2 micromanipulator (Fig. 1) isused for moving the holding capillary, and a secondTransferMan NK 2 is used for transferring the sperm;CellTram ® Air (Fig. 2) holds the oocyte; CellTram Oil(Fig. 3) transfers the sperm.In conjunction with an inverted microscope andHoffman Modulation Contrast ® or DIC (differentialinterference contrast) optics, microinjection canbe performed quickly and easily. With four easyto-understandbuttons, the TransferMan NK 2 issimple to operate, and for every TransferMan NK 2,two spatial coordinates are defined and stored.Fig. 2 CellTram AirFig. 1 TransferMan NK 2 micromanipulatorFig. 3 CellTram Oil

ProcedureInstrument Set-upThe capillary is easily moved in any direction (x, yand z) by means of a joystick. By simply pressing thejoystick button, it can repeatedly assume one of thepreprogrammed positions. A point in the focal plane ischosen as the oocyte holding side (Fig. 4, Position 1 H),where the sperm can be easily injected with the secondTransferMan NK 2 (Position 1 T). For Position 2, it isrecommended that the manipulators and capillariesuse a point outside of the drops in the overlay oil.Holding side (H)Position 2 HTransfer side (T)Position 2 TSperm Set-upMagnified 200x, a sperm cell is immobilized by quicklyusing a transfer capillary to press the tail of the spermcell against the bottom of the dish until it stops moving.The sperm cell is aspirated tail-first into the transfercapillary by rotating the knob of the CellTram Oil orCellTram vario.By pressing the joystick button, the transfer capillary,which now contains the sperm cell, moves into theoverlay oil. The petri dish is moved until an oocyte inone of the drops surrounding the sperm medium isvisible, then the oocyte is brought into focus. Theoocyte is held by the holding capillary (inner diameterapproximately 20 µm) of the second TransferMan NK2 and the simultaneous rotation of the knob of theCellTram Air in Position 1 H.Position 1 HPosition 1 TFig. 4 Schematic view of injection into oocyteThe transfer capillary and the holding capillary aredirected at the focal plane, and the positions arestored as Position 1 (T and H). Similarly, the positionsin the overlay oil are defined as Position 2 (T and H).The sperm and ova are put in several drops of oil ontothe petri dish. Pressing the joystick button lowers thetransfer capillary (inner diameter approximately 4 µmto 5 µm) to Position 1 T.•Injection into oocyteOocyte Set-upMagnified 100x, the oocyte is turned by the transfercapillary, which has been lowered again (Position 1 T)until the polar body comes to rest either at the12 o’clock or 6 o’clock position (the underpressureon the CellTram Air will vary slightly).

Precision capillaries for ICSIProduct features in commonTransferTip (ICSI)l Glass microcapillariesfor human sperm injectionand the containment ofl Sterilized by validatedgamma irradiationl NoncytotoxicFor sperm injection using theICSI techniquel 4 µm inner diameterl 7 µm outer diameterl Heat-formed spikehuman egg cellsl 1 mm outer diameter(continuous testing withmouse embryo test)eases penetrationl 35° tip angleVacuTipFor holding suspension cells(e.g., oocytes or blastocysts)l 15 µm inner diameterl 100 µm outer diameterl 35° tip anglel TransferTip-F,flexible flange, 0.4 mm longl VacuTipTo supply you with the ideal capillary for your personal work methods,we offer three types of TransferTips. They are differentiated by thelength and shape of the angled flange.l TransferTip-RP,rigid, parallel flange, 0.5 mm longl TransferTip-R,rigid flange, 1 mm long

Technical specificationsEppendorf TransferMan NK 2Control of axes:Operating range:Resolution:Drive:Max. working speed of instrument:X, Y, Z via central joystick; proportional kineticsX, Y, Z >20 mm eachX, Y, Z, approx. 40 nm per microstepDisc stepper motor, 40 nm per microstep7,500 µm/sWork radius Coarse: User-defined from 12,500 µm to 5 µm, in steps of 5 µmFine: approx. 0 µm to 1.5 µmCapillary exchange:Position storage:User profiles:Weight:Module unit, X, Y, Z complete:Control panel with power supply:Control panel dimensions (W x D x H):Power supply:Via programmable Home function3 independent positions can be stored and recalled for all 3 space coordinates4 independent profiles can be stored and recalled3.1 lb/1.4 kg6.0 lb/2.7 kg10.63 x 8.07 x 6.30 in/27.0 x 20.5 x 16.0 cm120 V–240 V/50 Hz–60 HzEppendorf CellTram microinjectorsCellTram Air CellTram Oil CellTram varioGeneration of pressure: Cylinder/piston system Cylinder/piston system Cylinder/piston systemair-filled, maintenance-free oil-filled, maintenance-free with gears, oil-filled,maintenance-freeDiameter of dial: 40 mm 42 mm/34 mm (coarse/fine)Volume changeper revolution: 88 µl 9.8 µl 9.6 µl/960 nl (coarse/fine)Total volume that can be set: 2.640 µl 980 µl 960 µlTotal piston stroke: 15 mm 50 mm(corresponds to 30 revolutions)(corresponds to 100 revolutions)Minimum volume that can be set:

Ordering informationDescription Applications Catalog No.TransferMan NK 2 920000011Prime Tech Piezo Impact Drill 920002307Instruments/accessoriesAdapters, for all commonly used microscopesavailable upon requestTwinTip-Holder, twin capillary holder For TransferMan NK 2 920007767Universal capillary holders For glass capillaries and grip heads 920007392CellTram Air, manual pneumatic pressure-control system For holding cells 920002021CellTram Oil, manual hydraulic pressure-control system For transferring cells or for manual microinjection 920002030CellTram vario, manual hydraulic microinjector,For transferring cells or for manual microinjectionwith gears 1:1 and 1:10 920002111ConsumablesVacuTip, sterile glass capillaries, 25 pcs. For holding large cells (e.g., oocytes) 930001015TransferTip-F (ICSI), sterile glass capillaries,For injecting sperm into an oocyte (ICSI)with flexible flange, 25 pcs. 930001031TransferTip-R (ICSI), sterile glass capillaries,For injecting sperm into an oocyte (ICSI)with rigid flange, 25 pcs. 930001066TransferTip-RP (ICSI), sterile glass capillaries,For injecting sperm into an oocyte (ICSI)with rigid, parallel flange, 25 pcs. 930001074For more information, contact your Eppendorf North America Cell Biology Sales Specialist at 800-645-3050.8301-C203E-07 © 2006 Eppendorf AG. Eppendorf ® , TransferMan ® , TransferTip ® , and CellTram ® are registered trademarks of Eppendorf AG. Hoffman ModulationContrast ® is a registered trademark of Modulation Optics, Inc. Product appearance, specifications, and/or prices are subject to change without notice.www.eppendorf.com • Email: info@eppendorf.com • Application hotline: 516-515-2258In the U.S.: Eppendorf North America 800-645-3050 • In Canada: Eppendorf Canada Ltd. 800-263-8715