Standard Operating Procedure Immunology SOP Ref No SOP title Category SOP-IU008 Human PBMC isolation, freezing and thawing procedures Laboratory Version 1.1 Page 1 of 12
Isolation, freezing and thawing of Human PBMC MATERIALS (listed or equivalent) 1. R10 medium: - RPMI medium 1640 (GibcoBRL Cat# 21875) - 10% heat inactivated (56 C 45 min.) FBS (Defined, HyClone SH30070.03) - 1M HEPES buffer (GibcoBRL Cat# 15630), diluted 1:100-200mM L-glutamine (GibcoBRL Cat# 25030), diluted 1:100-100X penicillin-streptomicin solution (GibcoBRL Cat# 15140), diluted 1:100 2. Hank s Balanced Salt Solution (HBSS) w/o Ca ++, Mg ++, Cat.# 14175, Gibco BRL. 3. Accuspin System-Histopaque -1077 Cell Separation Tubes, Cat. # A7054, Sigma Diagnostics. Alternatively: sterile Leucosep 50ml tubes, cat#227290, Greiner. Shipped ambient but store at 2-8 C. 4. ACK Lysing Buffer, Cat# A10492, Gibco BRL. Store at room temperature. 5. Dimethyl Sulfoxide (DMSO), Cat#. D2650, Sigma. Store at room temperature 6. Sterile 2.0 ml polypropylene cryovials with screw cap and O-ring, Cat. #72.694.006, Sarstedt. 7. Freezing medium: 90% FBS, 10%DMSO. Store at 2-8 C, date of expiration, one month after date prepared 8. CTL Wash supplement cat#ctlw-010, CTL ltd 9. Benzonase 250U/µl #E8263-25ku, Sigma EQIPMENT (listed or equivalent) 1. Refrigerated centrifuge, w/swinging bucket rotor, able to hold at least four 50 ml tubes 2. -70 C freezer 3. Certified Biosafety hood 4. Set of micropipetters 5. Sterile individually wrapped 5 ml, 10 ml, 25 ml and 50 ml serological pipets (Costar) 6. Sterile polypropylene conical centrifuge tubes, 15 ml and 50 ml volumes (Fisher) 7. StrataCooler cell freezing box, Cat. #400005, Stratagene. 8. Electric Pipet-Aid Page 2 of 12
9. Sterile 2.0 ml polypropylene cryovials with screw cap and O-ring, Cat. #72.694.006, Sarstedt. The required precautions and procedures outlined for compliance with OSHA s Blood Borne Pathogen Standard should be followed when handling human blood products. All steps, other than centrifugations and cell counting, to be performed in a Biological Safety cabinet. Blood Sample Note: Whole blood should be transported and held at ambient temperature (18-22ºC) Part I Density Gradient Separation 1. Prior to use, bring Accuspin tubes to room temperature. Centrifuge tubes at 1000 x g for at least 30 seconds to bring separation medium level below frit/insert. 2. Label separation tubes with the subject number for the blood sample to be processed. Label one separation tube for each 10-15 ml of blood. 3. Use a sterile 25 ml pipet and add 10-15 ml of HBSS to each labeled separation tube. 4. Using a sterile 10 ml pipet, collect the blood from Vacutainer tubes and gently transfer into the HBSS in the separation tube with the matching subject number. Transfer a volume of blood, 10-15 ml, equal to that of the HBSS added to the tube. (Should be a 1:1 mixture of HBSS and blood). Note: alternatively, up to 20ml undiluted blood can be loaded directly to the Accuspin tube if reducing the tube number/subject is required 5. Centrifuge at 800 xg for 15 min at 20 C (± 2 C), with the centrifuge brake off. 6. Using a new 25 ml pipet for each subject, remove the upper plasma fraction down to within approximately 2 cm of the PBMC band (buffy coat or layer) located at the interface between the plasma fraction and the separation medium solution. 7. Using a new sterile 5 ml pipet, carefully and slowly pipet off the cells (buffy layer) at the interface above the frit of Accuspin. Be careful not to aspirate any more separation medium than necessary with the buffy layer. 8. Transfer the cells from one separation tube to one corresponding, pre-labeled sterile 50 ml tube. If more than one tube/subject has been generated do not pool all the cells into one 50 ml tube at this step. 9. Using a sterile 50 ml pipet, add HBSS to each tube containing collected cells to bring the volume up to 50 ml. Page 3 of 12
10. Wash cells by centrifugation at 250 x g for 10 min at 20 C (± 2 C). Remove the supernatant and tap the bottom of tube to loosen the cell pellet. 11. Optional: pool cells from the same donor bleed. Resuspend the cell pellet in one tube with 10-20 ml of HBSS+5% HI-FBS (hereafter referred to as HF). Draw up resuspended cells in new sterile 25 ml pipet and transfer to next tube containing cell pellet from the same donor bleed. Use pipet to resuspend pellet in tube by mixing. Continue process until cells from all tubes for same donor bleed have been pooled. 12. Bring volume in tube up to 50 ml with HF. 13. Centrifuge at 250 x g for 10 min at 20 C (± 2 C). Remove the supernatant and tap the bottom of tube to loosen the cell pellet. 14. Add 6 ml of ACK Lysing buffer to each sample and resuspend cells (make certain that cells are resuspended well). Let sit for 5 min. 15. Bring volume of tube up to 50 ml with HF 16. Centrifuge at 250 x g for 10 min at 20 C (± 2 C). Remove the supernatant and tap the bottom of tube to loosen the cell pellet. Resuspend cells in 20 ml of HF and repeat wash (250 x g for 10 min at 20 C, ±2 C) 17. Finally resuspend cells in 10-20ml of R10 and take an aliquot for viable cell counting. 18. Calculate the total cell number in each sample using Hemacytometer, Guava, or other appropriate method. Part II Freezing NOTE: The freezing medium should be stored at 2-8 C until time of use 19. After counting the cell suspension, centrifuge at 250 x g for 10 min at 20 C (± 2 C). Note: While the cells are spinning, place the appropriate labels on the cryovials for each sample. Labels should include the correct subject code, study number, visit number, sample date, etc. 20. Remove the supernatant, tap the bottom of tube to loosen cell pellet, and then resuspend cells in volume of Freezing Medium resulting in an approximate cell concentration of 1 x 10 7 cells/ml. 21. Dispense 1ml cell suspension in freezing medium/vial 22. Place vials into StrataCooler and then place container in -70 C freezer. Page 4 of 12
Note: The complete process of cell resuspension in FM, aliquoting, vial placement into StrataCooler, and transfer of StrataCooler units into -70 C freezer should be performed in less than 30 min. 18. After 1-3 days at 70 C, vials should be transferred from StrataCooler units into standard freezer boxes. Transfer the box into a liquid N 2 container. Part III Thawing 19. Prepare complete Thawing medium (hereafter referred to as TM) by diluting 1:10 the CTL Wash supplement medium 10X stock solution in RPMI, and supplementing with L-glutamine 1:100 and Benzonase 50U/ml final. Prewarm TM and complete R10 medium in 37 o C (+/- 1 o C) waterbath 20. Label one 15 ml centrifuge tube per subject. 21. Remove PBMC vial(s) from liquid nitrogen and place on dry ice until ready to thaw. It is not recommended to thaw more than four vials at a time. 22. Thaw cells quickly with gentle agitation in 37 o C (+/- 1 o C) water bath. Stop with the visible ice chunk still in the vial. 22. Use a sterile 1 ml pipet to transfer each thawed cell suspension to the appropriately labeled 15 ml tube. 23. Rinse the vial(s) with 1ml TM, add it drop wise to the thawed cells 24. Use a 10 ml pipet to add TM to each tube drop wise in a 1-2 minutes of dispensing/mixing cycles diluting 5X the original volume (example: if 1 vial/subject has been thawed, add 4ml of TM); swirl gently tubes to mix to avoid shear forces. 25. Use a 10 ml pipet to add TM to each tube drop wise in a somewhat faster 30 seconds of dispensing/mixing cycles adding an equal volume as in step 24 (example: if 1 vial/subject has been thawed, add 4ml of TM); swirl gently tubes to mix to avoid shear forces. 26. Centrifuge at 18-20 o C at 250 x g for 10 minutes. 27. Aspirate the supernatant and tap bottom of tube to loosen cell pellet. 28. If ELISpot assay must be done use a 5 ml pipet to resuspend cells gently with 4 ml/thawed vial of R10 medium and transfer to one well/thawed vial of a 12-well plates for overnight resting at 37 C in 5% CO2 incubator. Cells are recovered and counted the next day for ELISpot set up. Page 5 of 12
27. If flow cytometry-based assays must be done, use a 5 ml pipet to resuspend cells gently with 4 ml of R10 medium, leave the cell suspension in the 15ml tube and keep tubes in a CO2 incubator for 2-4 hours before counting viable cells and set up the ICS assay. Appendix A: Preparation of Complete RPMI Medium R10 Reagents (all provided as sterile solutions by manufacturer) 1. RPMI medium 1640 2. Fetal bovine serum 3. 1M HEPES Buffer 4. 200 mm L-glutamine 5. 100X penicillin-streptomycin solution Procedure (to be performed in a biosafety cabinet using aseptic technique) 1. Thaw one 500 ml bottle of fetal bovine serum (FBS) by placing in a 4 o C refrigerator overnight (2 o C - 8 o C acceptable). Label bottle with date of thaw. 2. Place bottle of thawed FBS in a 56 o C water bath and heat-inactivate for 45 minutes, swirling bottle every 15 minutes to mix. When complete, label bottle with date of inactivation and store at 4 o C for up to one month. Alternatively, prepare 50ml aliquots recording on tubes the FBS lot number and thawing, heat inactivation and freezing dates, store aliquots frozen at - 20 C for one year. 3. To one 500 ml bottle of RPMI medium 1640, add the following reagents using aseptic technique: a. 50 ml of heat-inactivated FBS c. 5 ml of 200 mm L-glutamine b. 5 ml of 1M HEPES buffer d. 5 ml of 100X penicillin-streptomycin 4. Mix well and label bottle with expiration date (two months from date of preparation) and lot number. Assign a lot number as follows: R10 - MMDDYY where M = month, D = day and Y = year using the date the medium was prepared. For example, lot number R10-070498 for medium prepared on July 4, 1998. 5. In Appendix B, record date upon which complete RPMI medium was prepared, record the assigned lot number and expiration date for the complete RPMI medium, record date that serum was thawed and heat-inactivated and record lot numbers and expiration dates of each reagent used to prepare complete RPMI medium. 6. Store the complete RPMI medium at 4 o C and discard after expiration date. Page 6 of 12
Appendix B: Batch Record for Preparation of Complete RPMI Medium R10 (make copies and use one new sheet each time medium is prepared) Complete RPMI medium information: date prepared lot number (R10-MMDDYY) Expiration date Fetal bovine serum information: date heat-inactivated, aliquotted, frozen date 50ml aliquot thawed All reagent information: Reagent Vendor Lot # Exp. Date RPMI 1640 GibcoBRL fetal bovine serum HyClone HEPES GibcoBRL L-glutamine GibcoBRL penicillin-streptomycin GibcoBRL SIGNATURE DATE Page 7 of 12
Appendix C: Batch Record of Samples frozen (make copies and use one new sheet for each assay set-up) Mark with not applicable (n.a.) when appropriate Sample information Bleed and PBMC date Freezing date Operator Sample key sample code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 subject identification clinical site Sample Timepoint PBMC yield #of frozen vials # cells/vial SIGNATURE DATE Page 8 of 12
Appendix D: Batch Record of Samples thawed (make copies and use one new sheet for each assay set-up) Mark with not applicable (n.a.) when appropriate Sample information Bleed and PBMC date Thawing date Freezing date Operator Sample key sample code subject identification clinical site Sample Timepoint #of thawed vials Cells yield (specify if same day or after O/N rest) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SIGNATURE DATE Page 9 of 12
Appendix E: HBSS + 5% FBS (HF) Batch Sheet HBSS + 5% FBS (HF) Batch Sheet Store at 4 C/Use at room temperature Assigned lot # (HF-MMDDYY): Expiration date (1 month): Prepared by: Date prepared: Item Source Catalog # Exp. Date Lot # ml added FBS HBSS FBS heat-inactivation date SIGNATURE DATE Page 10 of 12
Appendix F: Freezing Medium (FM) Batch Sheet Freezing Medium (FM) Batch Sheet Store and use at 4 C Assigned lot # (FM-MMDDYY): Expiration date (1 month): Prepared by: Date prepared: Item Source Catalog # Exp. Date Lot # ml added FBS DMSO FBS heat-inactivation date SIGNATURE DATE Page 11 of 12
Appendix G: Thawing medium (TM) Batch Sheet Thawing Medium (TM) Batch Sheet Prepare fresh/use prewarmed at 37 C Assigned lot # (TM-MMDDYY): Expiration date (3 days if protected from light): Prepared by: Date prepared: Item Source Catalog # Exp. Date Lot # ml or µl Wash supplement RPMI 1640 L-glutamine Benzonase CTL ltd GibcoBRL GibcoBRL added SIGNATURE DATE Page 12 of 12