Eucaryotic Cell Bank Technology Transfer to the ‘BioMoBiL’ Centre 29 / 10 / 2004 Konrad Kleszczyński Jolanta Grzenkowicz – Wydra

Where from ? ADVANCELL A biotech company that offers customized research services on in vitro models (Toxicology, Oncology, Dermatology, Aging diseases) Cell banking (156 cell lines) Growing and expansion of new cell lines and their distribution Cell lines quality control

Laboratory SOP Data Base Depositing Quality Control Essential equipment to cell laboratory: Flow Laminar Hood (Class II) CO2 Incubator Inverted Light Microscope Fridge and Freezers: - 20 ÷ -30OC - 80OC Water Cleaning System (e.g. Millipore) Centrifuges Other equipment

Laboratory SOP Data Base Depositing Quality Control Essential equipment for cell line depositing (Dewar’s containers): Liquid nitrogen container (3600 cryovials with the volume of 2 ml, conatainer capacity: 116 L, evaporation: 0,6 L / 24 hrs) Liquid nitrogen storage container (container capacity: 34 L, evaporation: 0,25 L / 24 hrs)

Laboratory SOP Data Base Depositing Quality Control Standard Operating Procedures An SOP is a set of instructions to complete a job safely, with no adverse impact on the environment and in a way that maximizes operational and production requirements. SOP performs: Depositing of samples Aparature: service, calibration, using Chemicals: taking-up confirmation, labelling, protocols All actions monitoring

Laboratory SOP Data Base Depositing Quality Control

Laboratory SOP Data Base Depositing Quality Control DataBase organizes and menages with depositing cell lines Access to the DataBase has just administrator who shows the place of the cryovial in the tank Worker’s access to DataBase is limited to fill in the questionaire that is present in Intranet All the actions are archived evereday

Laboratory SOP Data Base Depositing Quality Control

Laboratory SOP Data Base Depositing Quality Control Services that are offered by Cell Line Collection Cell line distribution Safe deposit (short- and longlasting deposit) Quality control Mycoplasma eradication Others

Laboratory SOP Data Base Depositing Quality Control Depositing Organization of Bank Cell Line Original Bank (OB) up to 5 cryovials Master Bank (MB) 10 - 15 cryovials Distribution Bank (DB) 15 - 30 cryovials 106 cells / ml

Laboratory SOP Data Base Depositing Quality Control Basic quality tests including: immunocytochemical test, test for presence of bacteria and mycoplasma detection Mycoplasma detection is rapid, simple and sensitive method that uses PCR amplification with a commercial kit Most of contaminations are caused by following mycoplasma species: M. arginini, M. fermentans, M. hyorhinis, M. orale, Acholeplasma laidlawii Contaminated growing culture is treated by various antibiotics as tetracyclin derivatives (minocyclin, BM-cyclin)

Laboratory SOP Data Base Depositing Quality Control C+ C- M 1 2 3 4 5 6 7 2% agarose gel electrophoresis of DNA amplification products C+ positive control C- negative control M DNA molecular weight marker 1-7 examined samples contaminated culture healthy culture with Mycoplasma DNA staining by Hoechst 33258

Laboratory SOP Data Base Depositing Quality Control