STANDARD OPERATING PROCEDURE

Transcription

1 STANDARD OPERATING PROCEDURE Culture of the Human Monocyte-Macrophage Cell Line; U937 SOP number: WP1/002 Protocol prepared by: Ewelina Hoffman & Abhinav Kumar Developed under NC3R project: NC/C013203/1

2 INTRODUCTION Procedure to culture human monocyte-macrophages (U937) in vitro. Cells were obtained from ATCC (Manassas, USA). Cell line description: Organism: Homo sapiens, human Tissue: Pleura/pleural effusion, lymphocyte, Myeloid Disease: histiocytic lymphoma Age: 37 years Gender: male Morphology: monocyte Growth Properties: suspension PROCEDURE Materials and reagents: Reagent Catalogue number Supplier 1. RPMI 1640 (with phenol red) R8758 Sigma Aldrich 2. Fetal Bovine Serum F7524 Sigma Aldrich 3. Penicillin/streptomycin P3781 Sigma Aldrich 4. L - glutamine G7513 Sigma Aldrich 5. RPMI 1640 (without phenol red) Life Technologies Preparing complete growth medium: RPMI-1640 supplemented with 10 % heat inactivated fetal bovine serum (FBS), 2mM L-glutamine and 100 U penicillin/ 0.1 mg/ml streptomycin. To prepare complete culture medium (CCM): Page 2 of 5

3 o RPMI ml o FBS 50 ml o 100 x concentrated penicillin/streptomycin 5 ml o 100 x concentrated L - glutamine 5 ml Cells subculturing: Subculturing, also referred to as passaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain. Cells should be maintained in T75 flasks between 1 x 10 5 to 4 x 10 5 viable cells/ml and sub-cultured to maintain this cell density in culture sustaining the cells in a logarithmic phase of growth. Cells should be cultured in a humidified incubator at 37 C with 5 % v/v CO 2. All reagents should be pre-warmed to 37 C for at least 30 min prior to use on cells. 1. Mark the new flasks with cell line, passage number, split ratio, date, and vial number if appropriate. 2. Gently pipette up and down along base of flask to remove adherent cells and ensure equally distributed cell suspension. 3. Remove a small sample to count cell number and record viability (if required). This will help determine how much volume of cells to remove and to ensure cells remain in optimum density. 4. Every 3-4 days or when medium becomes orange/yellow add 10 ml of fresh medium (maximum 50 ml in a T75 flask). 5. When cell density is > 4 x 10 5 cells/ml, remove one quarter of the cell suspension and transfer to new T75 flask containing 10 ml of fresh culture medium. For work package one work all experiments were conducted on cells between passages 4-25 from purchase. Page 3 of 5

4 APPENDIX 1 Thawing cells 1. Remove cells from liquid nitrogen cryostorage and thaw frozen cells rapidly (< 1 minute) in a 37 C water bath. Do not submerge to avoid decontamination. 2. Transfer cell suspension to a 15 ml falcon. Dilute the thawed cells 1 drop at a time using 10 ml prewarmed growth medium to avoid osmotic shock. Wash vial with CCM to remove residual cells. 3. Centrifuge cells at 200 x g for 5 min to remove DMSO. 4. Remove supernatant and re-suspend cells in 10 ml complete medium. 5. Plate thawed cells at high density (1 x 10 5 cells/ml) to optimise recovery e.g. T25 flask. 6. Incubate cells at 37 C, 5 % v/v CO 2 for 2 days to allow them to settle before changing medium. Note: Cryovials stored in liquid-phase present a risk of explosion when thawed. Page 4 of 5

5 APPENDIX 2 Freezing cells Freezing medium prepare 10 % v/v DMSO in CCM (9 ml CCM + 1 ml DMSO) Freeze at cell viability > 90 % and at low passage number. 1. Prepare freezing medium fresh. Use 10 % w/w DMSO in CCM. 2. Harvest cells by gently scraping. Gently pipette up and down along base of flask to remove adherent cells and ensure equally distributed cell suspension. 3. Remove cell suspension into a falcon tube and centrifuge at 200 x g for 5 min. Remove the old medium. 4. Re-suspend cells in 10 ml of CCM. 5. Determine the total number of cells and calculate percent viability using a haemocytometer and trypan blue exclusion assay. According to the desired viable cell density, calculate the required volume of freezing medium you will need. 6. Centrifuge cells at 200 x g for 5 min, remove the supernatant. 7. Re-suspend pellet in freezing medium at viable cell density 1x10 6 cells per cryovial. 8. Dispense aliquots into cryogenic storage vials (1 ml of cell suspension per cryovial). 9. Place the cryovials containing the cells in an isopropanol chamber (Mr Frosty) and store them at 80 C overnight. 10. Following day transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen. Page 5 of 5