Quick Protocols

PetakaG3 TMCell Culture CommonQuick Protocolswww.petaka.comlife in it!

PetakaG3 TMBasicsMaximum volume of media permittedin Petaka isMaximumvolume20 mlMedia injection and withdrawalshould always be done holdingPetaka in a VERTICAL POSITION(more will alter Petaka’s performance!)PetakaG3 TMReady to hostPetakas (RTHP)PREPARING “READY TOHOST” PETAKASHold the Petaka in a vertical position!1. Inject 0.5 ml of Antibiotic solution2. Pump 16 ml of MediumTotal16.5mlKeep 3.5 mlfree to addcells and serumlater3. Keep these Petakas refrigerated at4ºC in a vertical position in standsor in a plastic bag (zip bag)Dehydration and pH should becontrolled if RTHP are kept Inrefrigerator for months!SEEDING CELLS IN “READYTO HOST” PETAKASHolding Petaka a in vertical position1. Disinfect port with 90% Ethanol &a flame2. Warm the Petakas in an incubator,at 37ºC for 20 -30 min in aVERTICAL position3. Inject 1.5ml of serum (*)4. Inject up to 2ml of cell suspension5. Whilst holding the Petaka in avertical position, squeeze it untilthe level of media reaches the airexit & then release until the air inputstops. Then the inside/outsidepressure will be balanced6. Shake gently for 10-15 seconds7. Place in a stand & allow to incubatein a HORIZONTAL positionCulturing cells in mediasupplemented mediaPetakaG3 TM mlSEEDING THE CELLSHolding Petaka in a vertical position!1. Inject 1 ml of Serum for 5%concentration2. Inject 0.5 ml of Antibiotic solution3. Inject X ml of Cell suspension4. Inject (18.5 –X) ml of Medium20Total5. Whilst holding the Petaka in avertical position, squeeze it untilthe level of media reaches the airexit & then release until the air inputstops. Then the inside/outsidepressure will be balanced.6. Incubate Petaka according tothe Cell type (see INCUBATIONPOSITIONS)

PetakaG3 TMHarvestinganchored cellsADHERENT CELLS WITHTRYPSIN-EDTA1. Disinfect the port with 90% Ethanol& a flame2. Completely extract all the mediafrom the Petaka3.Inject 4 ml of 0.25% Tyrpsine-EDTAsolution4. Shake thoroughly5. Incubate at 37ºC for 3 min in ahorizontal position (both sides)6. Check Cell detachment under themicroscope7. WHEN CELLS ARE DETACHED!Tap the Petaka 2-3 times8. Pellet the cells using a Petakacentrifuge (see centrifugation)9. To pellet the cells in the bottomright hand corner of Petaka,position it with the port being theupper most corner of the device10. Introduce Petaka into thecentrifuge rotor pocket11. Set desired speed & time & begincentrifuge12. After centrifuge, the cell pellet willbe visible in the bottom right handcorner13. To extract the pellet, use a micropipette with a 1mm tip14. Transfer the cells to a conical tubePetakaG3 TMHarvesting freesuspended cellsNON ADHERENT CELLS1. Disinfect port with 90% Ethanol & aflame2. Tap the Petaka 2-3 times3. Pellet the cells using a Petakacentrifuge (see centrifugation)5. To pellet the cells in the bottom righthand corner of Petaka, position itwith the port being the upper mostcorner of the device6. Introduce Petaka into the centrifugerotor pocket7. Set desired speed & time & begincentrifuge8. After centrifuge, the cell pellet willbe visible in the bottom right handcorner9. To extract the pellet, use a micropipette with a 1mm tip10. Transfer the cells to a conical tubePetakaG3 TMKeeping cells in “invitro dormancy”(only adherent cells)PUTING THE CELLS IN“IN VITRO DORMANCY”1. After 75% confluent cell growth,remove the Petakas from the incubator2. Keep the Petakas at 20-25ºC in avertical position, away from directsunlight, or Infra-Red sources.3. Keep in a Polyspan Shield4. Periodically Check Cell shape underthe microscopeDO NOT CHANGETHE MEDIUM!In-dormancy survival time depends on:a) Cell typeb) Culture Mediac) Type of Buffer (in the media)d) Stability of the environmental variables

PetakaG3 TMRecovering cellsfrom “dormancy”RECOVERING CELLSFROM “DORMANCY”1. Disinfect port with 90% Ethanol & aflame2. Slowly withdraw the medium3. Inject the same volume of NEWmedium with the same additives4. Whilst holding the Petaka in a verticalposition, squeeze it until the level of media reaches the air exit & thenrelease until the air input stops.Then the inside/outside pressure willbe balanced.5. Incubate at 37ºC, in a vertical position,at least for 24 hours before cellSUBCULTUREPetakaG3 TMpH adjustment ofgrowing culturespH ADJUSTMENT WITHSODIUM BICARBONATE1. Disinfect port with 90% Ethanol & aflame2. Withdraw 1ml of Medium3. Whilst holding the Petaka in a verticalposition, squeeze it until the levelof media reaches the air exit & thenrelease until the air input stopsThen the inside/outside pressure will bebalanced4. Inject 1ml of 7.5% Sodium Bicarbonatesterile solution5. Shake gently for 10-15 seconds.6. Return the Petakas to the incubatorPetakaG3 TMMedia changeMEDIA CHANGE1. Disinfect port with 90% Ethanol & aflame2. Slowly withdraw all the mediumcompletely3. Inject the same volume of NEWmedium with the same additives4. Whilst holding the Petaka in a verticalposition, squeeze it until the levelof media reaches the air exit & thenrelease until the air input stops.Then the inside/outside pressure willbe balanced.5. Incubate at 37ºC, in a vertical position,at least for 24 hours before cellSUBCULTURE6. Return Petaka to the incubator

PetakaG3 TMFreezing cellsFirst follow the harvesting cellsprotocol up to the pellet formationand extraction.1. Disperse the cell pellet in FREEZINGMEDIUM at a final concentration:1,000,000 cells per ml of medium2. Transfer 1.5 ml of cell suspension intoeach Cryotube.3. Proceed freezing the Cryotubes(stepwise) up to -120ºC & store inLIQUID NITROGENIn order to remove the media whilstkeeping the cells within the Petakawe need to gather the cells in thebottom left hand corner, therefore forcentrifuging the air filter must be inthe uppermost corner of the Petaka4. Place the Petaka in the centrifuge, setspeed & time, then begin5. Remove Petaka from the centrifuge6. Pellet cells will have gathered in thebottom left hand corner7. Whilst holding the Petaka upright withthe pellet cells in the bottom corner,slowly remove the media, careful notto disturb the pellet cells8. Repeat centrifuge 2 more timesensuring the port is disinfected eachtimeThen to collect washed pellet cells,inject new medium, place the Petakain the centrifuge rotor pocket with theport being in the upper most corner.9. Set the centrifuge speed & time &begin10.Remove the Petaka from thecentrifuge11. Note the pellet cells in the bottomcorner under the port12. Remove the pellet cells using amicro pipette13. Transfer into a conical tubecontaining freezing media14. Transfer this into a cryotube15. Place in the freezer at least -80ºC orin liquid nitrogenPetakaG3 TMThawing frozen cells1. PREPARE “READY TO HOST”PETAKA WITH ONLY 15ml OF MEDIA2. Disinfect the port with 90% Ethanol& a flame3. Warm the RTHP in an incubator,at 37ºC for 10-20 minutes in aVERTICAL position.4. Inject 3ml of Serum (*)5. Remove the cells from the freezer6. Thaw the cryotube in a disinfectedwater bath at 38ºC for 1-3 minutes.7. Be sure the ice has melted8. Extract the cell suspension from thecryotube with a 1ml tip.9. Transfer the cell suspension into aPetaka(RTHP)10. Whilst holding the Petakain avertical position, squeeze it until thelevel of media reaches the air exit &then release until the air input stops.Then the inside/outside pressure willbe balanced.11.Shake gently for 10-15 seconds12. Incubate the Petaka in a horizontalposition according to the Cell type(see INCUBATION POSITIONS)PetakaG3 TMIn Situ monitoringadherent cells viabilityTRYPAN BLUE METHOD1. Disinfect port with 90% Ethanol & aflame2. Withdraw 1ml of Medium3. Inject 1ml of Trypan Blue 0.4%“sterile” solution4. Shake gently for 5-10 seconds5. Leave the Petaka in a vertical positionfor 10 minutes at room temperature6. Count proportion of “blue-stained”cells (dead cells) versus unstained perfield (living cells)7. Disinfect port with 90% Ethanol & aflame8. Slowly withdraw the medium9. Inject the same volume of NEWmedium10. Incubate in the required position

PetakaG3 TMShipping cells in PetakaPREPARING ADHERENTCELLS FOR SHIPMENT1. Withdraw the Petakas from theincubator when cells become around75% confluent2. Protect Petaka with Polyspan Shield3. Introduce Petaka/s in a mailerenvelope4. Ensure that during the trip Petakawill not be exposed to temperaturesbelow 10ºC or over 39ºC (optimaltemperature 20-22ºC)5. Optimal traveling time is up to 7-10days.Intercontinental delivery times havebeen successfully up to 14 to 21 days;however, depending on the cell type,final viability at destination could be aslow as 30-50%RECOVERING CELLS AFTERSHIPMENT1. Disinfect port with 90% Ethanol & aflame2. Slowly withdraw the medium3. Inject the same volume of NEWmedium4. Whilst holding the Petaka in a verticalposition, squeeze it until the levelof media reaches the air exit & thenrelease until the air input stops. Thenthe inside/outside pressure will bebalanced.5. Incubate at 37ºC, in vertical position,at least for 6-12 hours before cellSUBCULTUREPetakaG3 TMTissue cell isolationand primary culture1. Inside the laminar flow hood or inthe operating room, excise a tissuesample and place into a 10 cm tissueculture dish containing approximately10 ml of Hanks Balanced SaltSolution (HBSS).2. Inside the laminar flow hood, removeany fat and necrotic areas fromtissue sample. Maintain a sterileenvironment all times.3. In a minimal amount of HBSS,thoroughly mince the tissue sampleinto a fine slurry of 1mm3 or smallerparticles4. Transfer the minced tissue sampleinto a 50 ml conical tube and spindown at 2000 rpm for 3 minutes in arefrigerated centrifuge5. Fill the 50 ml conical containing thecollagenase digest up to 40 ml withHBSS and Spin at 3000 rpm for 5minutes. Discard the supernatant.6. Add warm DMEM with 10% FBSand 1% antibiotic, up to 10 Ml anddisperse the cell pellet.7. Suck up the mixture into a 10mlplastic pipette and eject it, repeatingboth operations 10X in order torelease free cells.8. Pass the mixture through a 100μmfilter (cell strainer) into a new 50 mlconical tube.9. Count cell concentration and seedtwo million cells in 1-5 ml suspensionper Petaka.10. Add media with 10% FBS and 2%Penicillin/Streptomycin solution upto 20 ml per Petaka.11. Incubate in the adequate position