Feeder-Free Culture and Passaging of Human iPS Cells using Complete KnockOut™ Serum Replacement XenoFree Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells (1-3) with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.

GIBCO® media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout™ DMEM supplemented with KnockOut™ Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture (3-9). The launch of KnockOut™ SR XenoFree, based on the formulation of KnockOut™ SR but using human-derived or human recombinant proteins under cGMP conditions, allows the transition of pluripotent stem cell research to the clinic. This gold standard media system can now be used for feeder-free culture with the addition of KnockOut™ SR Growth Factor Cocktail.

Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the < conditions. Invitrogen has developed Knockout™ SR Growth Factor Cocktail to allow you to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout™ SR XenoFree.

Objective

The following protocols provide instructions for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut™ Serum Replacement XenoFree Feeder-Free medium (KSR XenoFree FF). Once adapted, instructions for continual maintenance are also provided.

• Sterile Tissue Culture Hood
• Incubator set at 37°C
• Pipette-Aid
• Water Bath set at 37°C
• Sterile serological pipettes (5 mL, 10 mL)
• Centrifuge
• 15 mL centrifuge tubes
• 60mm Tissue Culture treated dishes

Note: To maintain sterile culture conditions, all of the procedures in this protocol are carried out using sterile laboratory practices and conducted under a laminar flow hood.

Prior to starting, ensure that any media is equilibrated to 37°C and appropriately gassed.

Preparing CELLstart™-coated Culture Dishes

1. Dilute CELLstart™ (1 mL) 1:50 in Dulbecco’s Phosphate Buffered Saline (D-PBS) containing calcium and magnesium. Pipette the solution gently to mix. Do not vortex.
   
   Note: CELLstart™ dilutions greater than 1:50 may also work but should be optimized for individual cell lines
2. Cover the whole surface of each culture dish with the CELLstart™ solution (1 mL for a 35-mm dish, 1.5 mL for a 60-mm dish).
3. Incubate the dishes for 1–2 hours at 37°C.
4. Transfer each dish to a laminar flow hood and allow it to equilibrate to room temperate (about 1 hour) before use.
5. Note: You may store CELLstart™-coated culture dishes at 4°C for next-day use. Carefully wrap the dishes with Parafilm to prevent from drying.
6. Immediately before use, aspirate all CELLstart™ solution from the culture dishes. It is not necessary to rinse the vessels after removing CELLstart™.

Preparing Complete KnockOut™ SR Feeder-Free Medium

1. To prepare 1 mL of 10 μg/mL Basic FGF solution, aseptically mix the components listed below. Aliquot the solution and store at –20°C for up to 6 months..

Note: BSA can be substituted with HSA or Knockout™ SR at the same concentration. 

3. To prepare 100 mL of complete KnockOut™ SR XenoFree Feeder-Free (KSR XenoFree FF) medium asceptically combine the components listed in the table below.
   
   

   Component	Stock Concentration	Final Concentration	Volume
   Knockout™ DMEM/F12 (Cat. no. 12660-012)	-	1X	76.8 mL
   GlutaMAX™ -I (Cat. No. 35050-061)	200 mM	2 mM	1 mL
   KnockOut™ SR XenoFree (Cat. no. A1099202)	-	20%	20 mL
   KnockOut™ SR-GFC (Cat no. A1448601)	50X	1X	2 mL
   bFGF (Cat. no. PHG0024)	10 μg/mL	20 ng/mL	200 μL

   
   You may store the KSR XenoFree FF medium at 2-8°C for one week.
4. Just before pre-equilibrating the complete medium to temperature and gases, aseptically add the required volume of 2-mercaptoethanol (55 mM stock concentration) for a 0.1 mM final concentration. For example, to prepare 100 mL of KSR-FF medium add 182 μL of 55 mM 2-mercaptoethanol (1:550 dilution) Alternatively, the 2-mercaptoethanol may be added to the 1X completed medium and stored at 2–8°C for up to one week.

Adapting Human iPSCs to KSR XenoFree FF Medium

1. Culture the human iPSCs on human foreskin fibroblasts or MEF feeder cells until they are 70–80% confluent.
2. Pre-warm the required volume of TrypLE™ in a 37 °C water bath. Refer to Table 1 below for details on the volumes required.
3. Pre-equilibrate the required volume of KSR XenoFree FF in a 37°C water bath for15 min. Refer to Table 1 below for details on the volumes required.
4. Aspirate the medium from the culture dishes, and add an appropriate amount of TrypLE™ . Incubate the dishes at 37°C for 3-5 minutes.
5. Aspirate the TrypLE™ from each culture vessel and wash off the MEF feeder cells gently with D-PBS (2 to 3 times).
6. Add an appropriate amount of complete KSR XenoFree FF medium to each culture vessel. Use a cell scraper or a 5 mL pipette to gently scrape the cells off the surface of culture vessel.
   
   Note: see appendix for alternative adaptation protocols for hard to adapt iPS cell lines.
7. Collect the cell suspension from each culture dish into separate 15 mL conical tubes. Rinse each culture vessel with an appropriate amount of complete KSR XenoFree FF medium, and add the D-PBS rinse medium into the 15 mL conical tubes containing the cell suspension. Be cautious not to break the cell clumps into single cells
8. Centrifuge the 15 mL conical tubes at 200 × g for 5 minutes to pellet the iPSCs.
9. Aspirate the supernatant from the iPSC pellet. Resuspend the pellet in an appropriate amount of KSR XenoFree FF medium according to the split ratio (Table 1). Do not break the cell clumps to a smaller size, because the smaller clumps do not attach well to the surface.
   
   Note: We recommend a split ratio of 1:2 for the first 3 passages after the iPSCs have been passaged directly from the iPSC feeder-based culture medium to KSR XenoFree FF medium. Normally, a split ratio between 1:3 and 1:5 is appropriate, but passaging at 1:2 ensures the higher density of cells needed when adapting into a feeder-free culture.
10. Aspirate the CELLstart™ solution from the pre-coated culture vessel, and slowly add an appropriate amount of cell suspension to each culture vessel.
11. Move the culture dish back and forth and side to side several times to disperse the cells across the surface of the dish. Gently place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO₂ in air. Replace the spent medium with KSR XenoFree FF every day.

Passaging human iPS cells using KSR XenoFree FF

1. Observe the human iPSCs growing in complete KSR XenoFree FF under the microscope to confirm that the cells are 70–80% confluent and ready to be subcultured. Refer to Figure 1.
   
   Note: If colonies become too dense or too large, increased differentiation occurs.
2. Cut out and remove any differentiated iPSC colonies prior to passaging the culture.
3. Pre-warm the required volume of TrypLE™ in a 37 °C water bath. Refer to Table 1 below for details on the volumes required.
4. Pre-equilibrate the required volume of KSR XenoFree FF in a 37°C water bath for15 min. Refer to Table 1 below for details on the volumes required.
5. Aspirate the spent medium from the culture vessel using a pipette, and rinse the cells twice with D-PBS.
6. Gently add pre-warmed TrypLE™ to the culture vessel (e.g., 1 mL of TrypLE™ solution per 60-mm culture dish). Swirl the culture vessel to coat the entire cell surface.
7. Incubate the culture vessel at 37°C for 3 minutes.
8. Remove the vessel from the incubator, aspirate the TrypLE™, and gently wash the cells with D-PBS.
9. Gently scrape the cells off the surface of the culture dish using a cell scraper, and transfer the cells to a sterile 15mL centrifuge tube.
10. Rinse the culture dish twice with KSR XenoFree FF, gently “spraying off” any cells that have not detached. Pool the rinse medium with the cells in the 15mL tube.
11. Centrifuge the tube at 200 × g for 5 minutes at room temperature to pellet the cells.
12. Carefully aspirate the supernatant without disturbing the cell pellet and discard it.
13. Gently flick the tube to fully dislodge the cell pellet from the tube bottom.
14. Gently resuspend the cells in pre-equilibrated KSR XenoFree FF using a 5mL serological pipette. Do not triturate. Note: it is critical at the step to gently resuspend the cells without using force to avoid damage.
15. Transfer the cells to a fresh 60-mm CELLstart™-coated dish at the desired split ratio and move the culture dish back and forth and side to side several times to disperse the cells across its surface.
16. Place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO₂ in air.

The next day, gently replace the spent medium with KSR XenoFree FF to remove cell debris. Replace the spent medium everyday thereafter. Observe the iPS cells daily and passage them as needed (approximately every 4–5 days). Passaging is recommended when the cells reach 70-80% confluence. For iPS cell cryopreservation and thawing, refer to our protocol titled “Cryopreserving and Recovering Human iPSCs in Complete KnockOut™ Serum Replacement XenoFree with or without feeders”.

Figure 1: The phase contrast image below shows iPSCs grown on CELLstart™-coated culture dishes using complete KnockOut™ Serum Replacement XenoFree Feeder-Free medium. The iPSCs exhibit morphology similar to hESCs, characterized by large nuclei and scant cytoplasm. (40x magnification)



Table 1 - Recommended volumes

• Takahashi, K. et al. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676.
• Takahashi, K. et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872.
• Yu, J. et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318 :1917–1920.
• Maherali, N. et al. (2008) Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell 3:595–605.
• Li, W. et al. (2009) Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4:16–19.
• Liao, J. et al. (2009) Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4:11–15.
• Dimos, J.T. et al. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221.
• Aasen, T. et al. (2008) Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnli 26:1276–1284.
• Park, I.H. et al. (2008) Generation of human-induced pluripotent stem cells. Nat Protoc 3:1180–1186.

Knockout DMEM/F12 and GlutaMAX™ can be substituted with the following alternatives:

1.  DMEM/F-12 containing GlutaMAX™-I (Cat. no. 10565-018)

To prepare 100 mL of complete KnockOut™ SR XenoFree Feeder-Free (KSR XenoFree FF) medium using DMEM/F-12 containing GlutaMAX™-I (Cat. no. 10565-018), aseptically combine the components listed in the table below.

2.  Knockout™ DMEM (Cat. No. 10829-018) and GlutaMAX™-I (Cat. No. 35050-061)

To prepare 100 mL of complete KnockOut™ SR XenoFree Feeder-Free (KSR XenoFree FF) medium using Knockout DMEM (Cat. No. 10829-018) aseptically combine the components listed in the table below.

Alternative bFGF pack sizes

1. Coat plates with CELLstart™ as described above
2. Passage 1: 75% MEF-conditioned medium + 25% KSR XenoFree FF
3. Passage 2: 50% MEF-conditioned medium + 50% KSR XenoFree FF
4. Passage 3: 25% MEF-conditioned medium + 75% KSR XenoFree FF
5. Passage 4 & thereafter: 100% KSR XenoFree FF
   
   Note: If iPS cell lines are extremely problematic, a further level of caution can be taken by maintaining a culture in each prior passage medium while starting the next level of adaptation. For example, when passaging the 25/75 MEF-conditioned medium / KSR XenoFree FF culture (as described above), iPS cells can be passaged into both 100% KSR XenoFree FF AND 25/75 medium. If the 100% culture does poorly, adaptation can be resumed using the backup 25/75 culture.

Direct and partial sequential strategy:

1. Coat plates with CELLstart™ as described above
2. Split cells into three plates (Plate 1, 2 & 3)
3. At the first passage, seed Plate 1 directly into KSR XenoFree FF and Plates 2 & 3 into the MEF-conditioned medium / KSR XenoFree FF culture.
4. On the next day, fluid-change Plate 2 with KSR XenoFree FF that day, and every day thereafter.
5. For Plate 3 at the second passage, try plating these cells directly into KSR XenoFree FF at a 1:2 split ratio (as above).