Protocol Reprogramming Human Fibroblasts into ips Cells using the Stemgent VSVg Retrovirus Reprogramming Set: Human OKSM

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1 STEMGENT Page 1 VSVg Retrovirus Reprogramming Set: Human OKSM OVERVIEW The following protocol describes the reprogramming of BJ Human Fibroblasts (BJ cells) into induced pluripotent stem (ips) cells in a 6-well culture format. Viral transduction efficiency can vary and depends on factors such as the Multiplicity of Infection (MOI); the type, condition and passage number of the target cells to be transduced; the use of cationic polymers such as polybrene; and the growth medium requirements for the target cell type. If using cell types or conditions different from what is listed below, the protocol may need to be optimized. Product Description Cat. No. Format VSVg Retrovirus Reprogramming Set: Human OKSM set Product Components Size Storage VSVg Retrovirus hoct4 100 µl -70 C VSVg Retrovirus hklf4 100 µl -70 C VSVg Retrovirus hsox2 100 µl -70 C VSVg Retrovirus hc-myc 100 µl -70 C VSVg Retrovirus GFP 100 µl -70 C ADDITIONAL MATERIALS REQUIRED BJ Human Fibroblasts (early passage, p6) ( Cat. No ) EMEM FBS 1M HEPES DMEM 0.1% gelatin solution γ-irradiated Mouse Embryonic Fibroblasts (MEFs) (Globalstem) PBS 0.05% Trypsin/EDTA Pluriton Reprogramming Medium ( Cat. No ) Penicillin/Streptomycin (100X) 50 ml conical tubes 15 ml conical tubes 1.5 ml microcentrifuge tubes 6-well tissue culture plates 12-well tissue culture plates, logo, and all other trademarks are property of Inc Inc.

2 STEMGENT Page 2 VSVg Retrovirus Reprogramming Set: Human OKSM MATERIAL PREPARATION BJ Medium EMEM supplemented with 10% FBS 30 mm 1M HEPES Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Store BJ Medium at 4 C. MEF Medium DMEM supplemented with 10% FBS Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Store MEF Medium at 4 C. MEF Feeder Plates Add 1 ml of 0.1% gelatin solution to each well of a 6-well tissue culture plate and incubate overnight at 37 C and 5% CO 2. Aspirate the gelatin and plate γ-irradiated MEFs at a density of 2 x 10 5 cells per well in 2 ml of MEF Medium. Incubate overnight at 37 C and 5% CO 2. Pluriton Reprogramming Medium Pluriton Medium 1. Allow the 500 ml bottle of Pluriton Medium to thaw at 4 C for 2 days. 2. Aliquot the medium in 40 ml aliquots and store at -20 C for up to 3 months. Note: Avoid prolonged exposure of Pluriton Medium at 37 C. Pluriton Medium is stable for up to 2 weeks at 4 C. Avoid multiple freeze/thaw cycles. Pluriton Supplement 2500X 1. Thaw the 200 µl vial of Pluriton Supplement 2500X at 4 C. 2. Aliquot 4µl of the supplement into individual sterile microcentrifuge tubes. 3. Store Pluriton Supplement 2500X aliquots at -70 C for up to 3 months. Note: Avoid multiple freeze/thaw cycles. Pluriton Reprogramming Medium 10 ml Pluriton Medium 4 µl Pluriton Supplement 2500X 100 µl Penicillin/Streptomycin (100X) In a 50 ml conical tube, combine the Pluriton Medium, Pluriton Supplement, and Penicillin/Streptomycin just prior to use., logo, and all other trademarks are property of Inc Inc.

3 STEMGENT Page 3 VSVg Retrovirus Reprogramming Set: Human OKSM REPROGRAMMING TIMELINE DAY ACTIVITY MORPHOLOGY BJ Cell Preparation and Transduction ips Cell Colony Identification and Isolation -1 Plate BJ cells 0 Transduce BJ cells 1 Replace medium with fresh BJ Medium 4 Replace medium with fresh BJ Medium 5 Plate MEF feeder plates 6 Re-plate transduced cells onto MEF feeder plates 7-14 Incubate transduced cells in Pluriton Reprogramming Medium changing medium every 2 days 15+ Incubate transduced cells in Pluriton Reprogramming Medium changing medium every day. Emerging ips cell colonies can be monitored using StainAlive antibodies specific to cell surface pluripotency markers TRA-1-60 or TRA ips cell colonies typically begin to emerge around day 10. Generally, colonies can be picked between day 15 and 21. Pick ips cell colonies within a few days of proper morphology identification. As the overall cell confluence increases, they will become more difficult to isolate. REPROGRAMMING BJ CELLS Prepare Cells for Reprogramming If reprogramming BJ cells, follow the plating protocol listed below. If using another cell type, further optimization may be required. 1. For each well to be transduced, plate 8 x 10 4 BJ cells in 2 ml of BJ medium in a 6- well tissue culture plate. 2. Incubate overnight at 37 C and 5% CO 2. Transduce BJ Cells 3. Thaw one vial of each virus in a room temperature water bath. When completely thawed, place the tubes on ice. 4. An MOI of 10 is recommended when using the VSVg Retrovirus Reprogramming Set: Human OKSM to reprogram BJ cells. Calculate the amount of transduction units (TU) of each virus needed (See the specification sheet for lot specific virus titers):, logo, and all other trademarks are property of Inc Inc.

4 STEMGENT Page 4 VSVg Retrovirus Reprogramming Set: Human OKSM Sample Calculation for transducing 8x10 4 cells at an MOI of 10 with a viral concentration of 2 x 10 7 TU/ml =40 5. Pre-chill a 50 ml conical tube on ice. 6. Carefully add the calculated volumes of each virus to the bottom of the pre-chilled 50 ml conical tube. 7. Aspirate the medium from the well of cells to be transduced. 8. For each well to be transduced, transfer 2 ml of room temperature BJ Medium to the virus suspension, mix briefly by pipetting and then transfer to the aspirated wells of the tissue culture plate. Note: The remaining undiluted virus can be refrozen once and stored at -70 C for up to 3 months. Avoid multiple freeze/thaw cycles. 9. Incubate overnight at 37 C and 5% CO After the overnight incubation, aspirate the medium and replace with 2 ml per well of fresh BJ Medium and then change medium every other day, until cells are ready to re-plate. 11. On day 5 post-transduction, plate inactivated MEFs onto 6-well tissue culture plates at a density of 2 x 10 5 cells per well. Re-plate Transduced Cells 12. On day 6 post-transduction, wash the well of transduced cells twice with 2 ml of PBS. 13. Aspirate the PBS and add 1 ml of 0.05% Trypsin/EDTA. 14. Incubate the plate for 2 to 3 minutes at 37 C and 5% CO 2 or until cells dissociate from the plate. 15. Add 2 ml of BJ Medium to neutralize the Trypsin/EDTA. 16. Pipette the medium across the surface of the well until the cells appear completely detached. 17. Transfer the cell suspension to a 15 ml conical tube. 18. Centrifuge for 5 minutes at 200 x g. 19. Aspirate the supernatant. 20. Re-suspend the cell pellet in 1 ml of Pluriton Reprogramming Medium. 21. Mix the cell solution gently in order to create a uniform suspension of single cells. 22. Count the total number of cells using a hemocytometer. Note: You may have up to 1 x 10 6 cells. This may be more than is required for re-plating. Excess transduced cells can be viably frozen or used for analysis. 23. In a 50 ml conical tube, transfer the volume of cell suspension required to provide the total number of desired cells for re-plating and then dilute with the appropriate, logo, and all other trademarks are property of Inc Inc.

5 STEMGENT Page 5 VSVg Retrovirus Reprogramming Set: Human OKSM amount of Pluriton Reprogramming Medium to bring the cell suspension to 5 x 10 3 cells/ml. Sample Calculation for One 6-well Plate: (6 wells) x (1 x 10 4 cells/well) = 6 x 10 4 cells resuspended in (2 ml of cell suspension per well) x (6 wells) = 12 ml total volume of Pluriton Medium 24. Aspirate the medium from a 6-well MEF Feeder Plate and wash each well with 2 ml of PBS. 25. Add 2 ml of cell suspension to each of the wells for a final cell density of 1 x 10 4 transduced cells per well. 26. Incubate at 37 C and 5% CO Replace the Pluriton Reprogramming Medium every other day until day 15 post transduction. 28. After day 15 post transduction, replace Pluriton Reprogramming Medium every day until all potential ips cell colonies have been picked for expansion and testing. 29. One day prior to colony isolation, plate MEFs onto 12-well tissue culture plates at a density of 1 x 10 5 cells per well. Single Colony Pick and Passage 30. Manually pick ips cell colonies to a single well of a 12-well MEF Feeder Plate. a. Using a sterile glass picking tool, gently separate the identified colony from surrounding cells. b. Using the glass picking tool, gently divide the colony into clusters that contain 30 to 40 cells. c. Using the glass picking tool, gently detach each colony cluster from the tissue culture well. d. Using a P200 pipette set at 100 μl, pipette the detached colony clusters out of the 6-well plate and into an individual well of the 12-well plate with 1 ml per well of Pluriton Reprogramming Medium. 31. Incubate the 12-well plate at 37 C and 5% CO The following day replace medium with fresh Pluriton Reprogramming Medium. Subsequently, colonies may be transitioned to another ips cell culture medium by changing the medium the next day (day 2 post-pick) with 50% Pluriton/50% medium of choice. Subsequent medium changes may then be made with 100% ips cell culture medium of choice. 33. Colonies can be subsequently passaged onto MEFs or a feeder-free matrix. Note: Pluriton Reprogramming Medium is a xeno-free medium and compatible with Stemedia Nutristem XF/FF Culture Medium. Nutristem is a trademark of Biological Industries., logo, and all other trademarks are property of Inc Inc.