Method:LymphoblastoidCellLinesfromFrozenWholeBlood

RosalieVeile

Purpose:

BloodSamplescanbestoredfrozenasabackupincaseanLCLisneededatalaterdate.

Timerequired:

15minutestofreeze1-4cryotubesplacingthemdirectlyintothe-135degreesCfreezer;oronehourtofreezethetubesusingtheCryomedfreezingchamber.Cellshavebeenshowntobemoreviableiftemperatureisloweredgraduallywiththefreezingchamber.

Procedure:

Freezingcells:
1. Pipette1.0mlofwholebloodinto2cryotubes(1.25ml).

2. Addtoeachcryotube100µldimethylsulfoxide(DMSO),or10%ofthevolumeofblood.

3. Immediatelybeginfreezingthewholebloodinthecryomedfreezingchamberuntilthechartdriveprinterreads-90degreesC.

4. Quicklytransferthefrozensampletolongtermstorageina-135degreesCfreezeroraliquidnitrogenstoragecontainer.Thesewholebloodsampleshavebeenshowntobeviableforaslongas5monthsbyG.Chenevix-Trench,etal.

Thawingcellsfortransformation:

1. WhenanLCLisneeded,thecellsarethawedrapidlyina37degreesCwaterbath:Placethecryotubesinabubblerack.Shaketheracktohelpthawthecells,usuallyfor1-2minutes.

2. Witha1mldisposablepipet,transferthesampletoa15mlconicalcentrifugetubefilledwith10mlofwashmedia.

3. Centrifugethecellsfor10minutesat1200rpm,nobrake,atroomtemperature.

4. Aspiratethewashmediatojustabovethecellpellet.Washthepelletagainwith10mlwashmediaandcentrifugeasinstep7.Repeatthewashatotalof4timesoruntiltheredcellcontaminationisminimal.Iftheredcellcontaminationisnoteliminated,severaldaysinculturewilldecreasetheamountofredcellssubstantially.

5. AspiratethewashmediaandresUSPendthecellpelletin300µlfilteredsupernatantfromaB95-8marmosetculturecontainingEpsteinBarrvirus.TransferthecellsuspensiontoaT-25cm2flaskandincubatefor2hoursat37degreesCwith5%CO2.Ifthereisaverysmallvolumeofcells,leavethecellsinthe15mlcentrifugetubefortheincubation.

6. Afterincubation,add800µlRPMI-1640containing20%fetalbovineserumand2XCyclosporinA.

7. Usinga5mldisposablepipet,plateoutcellsinserialdilutionina96-wellmicrotiterplate:Transferhalfofthecellsinthefirstwellintoasecondwell.Addenoughmediatofillthesecondwell.Thentakehalfofthiscell/mediamixtureandtransfertoathirdwell.Fillupthethirdwellwithmedia.Incubateat37degreesCwith5%CO2.

8. Feedthecellstwice-weeklybyremovinghalfoftheoldmediaandreplacingwithfreshmediauntiltransformedcoloniesareapparent(ususally2-3weeks).Thenewmediashouldcontain1XCSA.

9. Subculturecellstoa24-wellplatebeforetransferringtocultureflasks.Maintainthesubculturesongrowthmedia(noCSA).

Solutions:

• Growthmedia:(600ml)

   To 500 ml of sterile RPMI 1640 with 2 mM L-glutamine, add: 90.0 ml FBS 6.0 ml 200 mM (100X) L-glutamine 0.6 ml 50 mg/ml gentamicin reagent Note: Filter sterilize through a 0.22 µm filter and store up to 2 weeks at 4 degrees C.

• Washmedia:(1liter)

   To 1 liter of sterile RPMI 1640 with 2 mM L-glutamine, add: 10.0 ml 2.5 M (100X) hepes buffer 1.2 ml 50 mg/ml gentamicin reagent Note: Filter sterilize through a 0.22 µm filter and store up to 2 weeks at 4 degrees C.

• 2XCyclosporinAmedia:(1µg/ml)

   To 100 ml of growth media add 2 ml of 100X Cyclosporin A. 

• 100XCyclosporinA:(100ml)

   Dissolve 1 mg CSA in 0.1 ml ethanol, add 0.02 ml Tween 80 and mix well. While continually stirring, add 1 ml RPMI, drop by drop. Quanitate to a final volume of 100 ml with RPMI. Filter sterilize and store at 4 degrees C for up to 4 months.

References:

Chenevix-Trench,G.,etal,Am.J.Hum.Genet.46:635-636,1990.