PROTOCOL. Generating NuFF-Conditioned Pluriton Medium. Required Materials. Page 1

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1 Page 1 Supportive feeder cells are extremely important to the overall health of cells undergoing reprogramming, as well as successful growth and proliferation of the resulting or established human induced pluripotent stem (ips) cell cultures. The quality of fibroblasts used for generating conditioned medium can ultimately impact the overall success of an experiment. NuFF-Conditioned Pluriton Medium is routinely used in reprogramming experiments to supplement a culture with an expired co-culture feeder layer or when reprogramming in feeder-free conditions, such as Matrigel or defined matrices. To generate NuFF-Conditioned Pluriton Medium, Pluriton Medium must be incubated with inactivated newborn human foreskin fibroblasts (NuFF cells) for a 24 hour period in order to become conditioned prior to culturing target cells or established human ips cells. NuFF cells used in this process must be mitotically inactivated, by either gamma-irradiation or mitomycin-c treatment, both of which block further cell proliferation without inhibiting cellular metabolism. Use only reprogramming-qualified or validated NuFF cells that have been previously tested to support pluripotent cell cultures and/or reprogramming experiments. After thawing, inactivated NuFF cells will sufficiently condition medium for up to 7 to 10 days. If more conditioned medium is required than can be collected following this protocol, increase the number of NuFF vials thawed and T150 flasks cultured. Do not extend the number of collection days. NuFF-Conditioned Pluriton Medium can be made prior to an experiment and frozen at -20⁰C until use (up to 3 months). Required Materials Product Description Cat. No. Format Storage Stemgent Reprogramming-Qualified NuFF Cells GSC-3006G 4 x 10 6 cells/vial Liquid Nitrogen Stemgent bfgf, human recombinant μg -70 C Pluriton Reprogramming Medium ml -20 C

2 Page 2 Day 1 Plate NuFF Feeder Cells Approximately 4 x 10 6 mitotically inactivated NuFF cells should be plated in one T150 flask to generate NuFF-Conditioned Pluriton Medium. 1. Thaw one vial of inactivated NuFF cells in a 37 C waterbath until only a small ice crystal remains. 2. Transfer the contents of the vial of NuFF cells directly to a 15 ml conical tube. 3. Gradually add 5 ml of NuFF culture medium to the cells while gently agitating the contents of the vial. 4. Centrifuge the cells for 4 minutes at 200 x g. 5. Aspirate the supernatant and resuspend the cell pellet in 6 ml of NuFF culture medium. 6. Count the cells in solution and calculate the live cell density. 7. Add 25 ml of NuFF culture medium to the T150 flask. 8. Add the 6 ml of NuFF cell suspension (4 x 10 6 cells), to the medium in the flask. 9. Evenly distribute the NuFF cell suspension in the flask. 10. Incubate the cells overnight at 37 C and 5% CO 2. Day 2 Exchange Medium After allowing the inactivated NuFF cells to attach overnight, the culture can be washed and prepared to generate NuFF-Conditioned Plurition Medium. 1. Supplement 35 ml of Pluriton Medium with 35 µl of bfgf (at 4 µg/ml stock concentration) to a final bfgf concentration of 4 ng/ml. 2. Aspirate the NuFF culture medium from the T150 tissue culture flask. 3. Add 12 ml of PBS to the cells to wash. Aspirate the PBS. 4. Add 35 ml of Pluriton Medium supplemented with bfgf to the T150 flask. 5. Incubate the cells overnight at 37 C and 5% CO 2.

3 Page 3 Day 3 to Day 8 Collect and Freeze NuFF-Conditioned Pluriton Medium After each 24 hour incubation, the medium in the T150 flask can be collected as NuFF- Conditioned Pluriton Medium. Each day the conditioned medium should be collected and replaced with fresh Pluriton Medium supplemented with bfgf to a final concentration of 4 ng/ml. Each daily collection of 35 ml of NuFF-Conditioned Pluriton Medium should be frozen and stored at -20 C until the media can be pooled together on Day 9. Repeat the collection and exchange of medium daily through Day 9 (7 days of medium collection total). 1. With a 10 ml pipet, transfer 35 ml of NuFF-Conditioned Pluriton Medium from the T150 flask to a sterile 50 ml conical tube. 2. Freeze the NuFF-Conditioned Pluriton Medium at -20 C. 3. Add 35 ml of Pluriton Medium supplemented with bfgf to the cells in the T150 flask. 4. Incubate the cells overnight at 37 C and 5% CO 2. Day 9 Collect, Pool, and Re-Aliquot NuFF-Conditioned Pluriton Medium 1. Thaw all aliquots of previously-collected NuFF-Conditioned Pluriton Medium at 4 C. 2. Collect the final 35 ml of NuFF-Conditioned Pluriton Medium from the NuFF cells in the T150 flask. 3. Pool all collected and thawed NuFF-Conditioned Pluriton Medium and filter using a 0.22 µm pore size, low protein-binding filter. 4. Dispense filtered NuFF-Conditioned Pluriton Medium into 40 ml aliquots and re-freeze at -20 C until use. Day 9+ Using NuFF-Conditioned Pluriton Medium 1. Thaw a 40 ml aliquot of NuFF-Conditioned Pluriton Medium 4 C. 2. Transfer the appropriate volume of NuFF-Conditioned Pluriton Medium to a sterile conical tube. NOTE: The remaining 30 ml of NuFF-Conditioned Pluriton Medium from this aliquot can be stored at 4 C and used over the next 2 to 5 days. 3. If using low oxygen culture conditions (5% O 2 ), equilbrate the NuFF-Conditioned Pluriton Medium for in a sterile 15 ml conical tube with a vented cap incubated at 37 C, 5% CO 2 and 5% O 2 for a minimum of 2 hours to overnight. 4. Just prior to use, thaw one aliquot of Pluriton Supplement (2500X) and add the appropriate volume to the equilibrated NuFF-Conditioned Pluriton Medium to generate NuFF- Conditioned Pluriton Reprogramming Medium. 5. Exchange medium and culture cells as directed in the reprogramming or culture protocol.

4 Page 4 Recipes NuFF Culture Medium 45 ml DMEM 5 ml FBS 0.5 ml GlutaMAX-1 1. Filter-sterlize using a 0.22 µm pore size, low protein-binding filter. 2. Store NuFF culture medium at 4 C for up to 2 weeks. bfgf Solution (4 ug/ml) 50 µg Stemfactor bfgf, human recombinant 12.5 ml 0.1% BSA in PBS 1. Briefly centrifuge the lyophilized vial of bfgf. 2. Use 500 µl of 0.1% BSA in PBS to reconstitute the lyophilized bfgf in the vial. 3. Transfer the reconstituted bfgf to a 15 ml conical tube. 4. Add 12 ml of 0.1% BSA in PBS to yield a 4 µg/ml working solution. 5. Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. 6. Aliquot into single-use volumes in sterile, low protein-binding microcentrifuge tubes. 7. Store bfgf aliquots at -20 C for up to 6 months. NuFF-Conditioned Pluriton Reprogramming Medium 10 ml NuFF-Conditioned Pluriton Medium (equilibrated) 4 µl Pluriton Supplement (2500X) 1. Thaw an aliquot of Pluriton Supplement on ice. 2. Transfer 10 ml of pre-equilibrated NuFF-Conditioned Pluriton Medium to a 15 ml conical tube. 3. Add 4 µl of Pluriton Supplement to a final concentration of 1X just prior to use.

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