STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 101 MOD: 2 nd Issue Page: 1 of 6 Procedure Type: General Laboratory Procedure

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1 THE UNIVERSITY OF NEWCASTLE DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 101 MOD: 2 nd Issue Page: 1 of 6 Procedure Type: General Laboratory Procedure Title: Procedure for subculturing human, murine and GM cell lines 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED Subculturing human, murine, and GMO cell lines HAZARDS 1. Refer to GLP 102 Working with human, murine and GMO cell lines for a risk assessment covering general aspects of this work. RISK ASSESSMENT 1. Refer to GLP 102 Working with human, murine and GMO cell lines for a risk assessment covering general aspects of this work. RISK CONTROL 1. Refer to GLP 102 Working with human, murine and GMO cell lines for a risk assessment covering general aspects of this work. WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Petrina Guthrie Kathryn Skelding Leonie Ashman DATE 30 th January nd December rd December 2008 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 6 Title: Procedure for subculturing human, murine and GM cell lines I have read and understood the Risk Assessment for GLP Procedure for subculturing human, murine and GM cell I agree to abide by the Risk Controls outlined in this Assessment. Name Signature Date

3 Page: 3 of 6 2. Purpose: 2.1 To outline the procedure for splitting human, murine, and GMO adherent and suspension cell 3. Equipment: 3.1 As outlined in GLP 102 Working with human, murine and GMO cell lines and GLP 079 Trypan blue cell count and GLP 103 Trypsinisation of adherent cell 4. Materials: 4.1 As outlined in GLP 102 Working with human, murine and GMO cell lines and GLP 079 Trypan blue cell count and GLP 103 Trypsinisation of adherent cell ul pipette tips - sterile 5 Reagents: 5.1 As outlined in GLP 102 Working with human, murine and GMO cell lines and GLP 079 Trypan blue cell count and GLP 103 Trypsinisation of adherent cell 6 Set Up: 6.1 Ensure that you have read and understood the Safety Precautions (Section 7 of this SOP) prior to commencing this procedure. 6.2 Adherent cells should be subcultured when near confluency. 6.3 Non-adherent cell lines should be subcultured when 8-10 x 10 5 /ml or as per notes for individual cell 6.4 Do not let cells overgrow. 6.5 Check under the microscope before beginning and as appropriate do cell count/viability determination as per GLP 079 Trypan blue cell count. 6.6 Media, PBS, and trypsin (if required) should be warmed to 37 C in the 37 C waterbath before beginning. 6.7 Set up Biosafety cabinet as outlined in GLP Safety Precautions: 7.1 Good laboratory techniques are to be used at all times.

4 Page: 4 of Any material that has been in contact with human, murine, or GMO cell lines is treated as potentially infectious. 7.3 All work with human, murine, or GMO cells must be performed in Class II biosafety cabinet. 7.4 Wear gloves and lab gown at all times. 7.5 Always decontaminate waste as soon as possible. 7.6 Ensure that all flasks are immediately labeled with the name of the cell line, the date of storage, passage number and initials. 8 Methods: 8.1 Remove the flask of cells from the incubator and check under the microscope to check whether the culture is near confluent and ready to split. 8.2 Subculture (adherent cells) or do cell count (non-adherent cells) 8.3 The following work must be carried out aseptically in a Class II biosafety cabinet NB All items must be sprayed with 70% ethanol prior to entering or reentering the biosafety cabinet. All items must be sprayed with 70% ethanol upon removal from the biosafety cabinet. 8.4 Adherent cell lines Refer to GLP 103 Trypsinisation of Adherent Cell Lines for the method to be used to disperse adherent cell lines in solution prior to splitting To remove trypsin from trypsinised adherent cells: Transfer cells/media to 50ml falcon tubes Spin down the cells at x g at room temperature for 5 mins to pellet the cells Remove the supernatant from the tube using a sterile glass pipette or mixing cannula attached to the vacuum pump and trap. Spray the vacuum pump plastic tubing with 70% ethanol both before and after use Resuspend pellet by flicking the bottom of the tube with your finger to disperse cells Add 10mls of Medium to the tube using a pipet boy and a 10ml serological pipette and gently resuspend the cells by pipetting up and down. For cells that are harder to resuspend try using a transfer pipette. 8.5 Suspension cell lines

5 Page: 5 of Place flask/s in the hood, pipette cells up and down using a pipette boy and 10ml serological pipette Some suspension cell lines tend to colonize the flask so ensure that the bottom of the flask is sprayed when pipetted up and down to loosen any cells that may have adhered. In addition tap the flask with the palm of your hand to loosen any particularly adhered cells and ensure the cells are evenly dispersed. Both adherent and Suspension cell lines: 8.6 Perform cell count GLP 079 Trypan blue cell count. 8.7 Dilute into fresh medium to give appropriate density. 8.8 Once cells are dispersed in solution, whether adherent cell lines or suspension cell lines, the following method should be followed. 8.9 Seed a new flask (T25, T75, T180) with a dilution of cells, the amount will depend on the size of the flask used and the cell line used. Alternatively, perform a cell count (refer to GLP 079 Trypan blue cell count) and refer to individual cell line data sheet for optimal seeding concentration and volume Add approximately 13ml media to a medium T75 flask and 26ml media to a large T180 flask Label all flasks/plates/dishes with name, date, passage number and dilution/ concentration and place in the 37 o C 5% CO 2 Incubator For suspension cell lines add ml media to the flask depending on the size of the flask and dilution of cells reseeded. 9 Maintenance: 9.1 Refer to individual cell line data sheet for details on cell maintenance. 9.2 Adherent cultures will need to be subcultured again when near confluent, however the concentration at which subculturing occurs is specific to each cell line refer to individual cell line data sheet. 9.3 Check cells under microscope to assess confluence. 9.4 Limit cell-lines to 30 passages (each time the cells are subcultured = 1 passage), as genetic drift may occur in long-term culture. 9.5 Do not subculture cell lines at high ratios, many cell lines will die if cultured at too low density. 10 Shutdown:

6 Page: 6 of Waste disposal: Refer to GLP 102 Working with human, murine and GMO cell 10.2 PC2 biosafety cabinet clean up: Refer to GLP 102 Working with human, murine and GMO cell 11 Review Date: 11.1 This procedure should be reviewed by: 2 nd December Change History: 12.1 Issue Number: 1 st Issue Date Issued: 22 nd February Issue Number: 2 nd Issue Date Issued: 3 rd December 2008 Reason for Change: Review and refinement of procedure following contamination problems.