Quick Protocols

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