Creation of Embryoid Bodies from iPSCs using Complete KnockOut™ Serum Replacement Feeder-Free Medium

Introduction

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).

Embryoid Bodies are floating spherical colonies of iPSCs. They are setup in this manner when testing your cells for pluripotency. Allowing the colonies to grow as EBs in turn allows you to test the differentiation potential of the cells. EBs can be setup at a normally scheduled passage, but instead of plating the cells into fresh Geltrex-coated dishes, the cells are plated into non-tissue culture treated dishes to prevent attachment. bFGF is not added to EB medium.

Objective

The following protocols provide instructions for the differentiation of Induced Pluripotent Stem (iPS) cells using complete KnockOut™ Serum Replacement EB medium.

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Materials

  • Sterile Tissue Culture Hood
  • Incubator set at 37°C
  • Pipette-Aid
  • Water Bath set at 37°C
  • Sterile serological pipettes (5 mL, 10 mL)
  • 15 mL centrifuge tubes
  • 60 mm Tissue Culture treated dishes
  • 60 mm or 100 mm non tissue culture treated dishes
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Ordering Information

Sku Name Size Price Qty
12660012 KnockOut™ DMEM/F-12 500 mL USD 33.60
35050061 GlutaMAX™ Supplement 100 mL USD 30.40
10828028 KnockOut™ Serum Replacement 500 mL USD 277.00
21985023 2-Mercaptoethanol (55 mM) 50 mL USD 33.90
17105041 Dispase II, powder 5 g USD 536.00
14190144 DPBS, no calcium, no magnesium 500 mL USD 18.35

Protocols

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 complete KSR EB Medium

  1. To prepare 100 mL of Complete KnockOut™ Serum Replacement EB medium, aseptically combine the components listed in the table below.

    Component
    Stock Concentration
    Final Concentration
    Volume
    Knockout™ DMEM/F12 (Cat. no. 12660-012)
    -
    1X
    78 mL
    GlutaMAX™ -I (Cat. No. 35050-061)
    200 mM
    2 mM
    1 mL
    KnockOut™ SR (Cat. no. 10828-028)
    -
    20%
    20 mL
    Non-Essential Amino Acids (Cat. no. 11140-076)
    10 mM
    0.1 mM
    1

    Note: The KSR EB medium can be prepared and stored at 2-8°C for up to 2 weeks.

  2. 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.
  3. Aliquot appropriate volume (see Table 1) of KSR EB Medium needed for the day to a centrifuge tube warm to 37°C.

Passaging iPS cells from feeder-free conditions


  1. Observe the human iPSCs growing in complete KSR-Feeder Free medium under the microscope to confirm that the cells are 70–80% confluent and ready to be subcultured.

    Note: If colonies become too dense or too large, increased differentiation occurs.

  2. Cut out and remove any differentiated iPSC or hESC colonies prior to passaging the culture.
  3. Pre-warm the required volume of Dispase 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-EB medium 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 dish using a pipette, and rinse the cells twice with D-PBS.
  6. Gently add pre-warmed Dispase solution to the culture dish (e.g., 1 mL of Dispase solution per 60-mm culture dish). Swirl the culture dish to coat the entire cell surface.
  7. Incubate the culture dish at 37°C for 3 minutes.
  8. Remove the dish from the incubator, aspirate the Dispase solution, 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 15 mL centrifuge tube.
  10. Rinse the culture dish twice with DPBS, gently “spraying off” any cells that have not detached. Pool the rinse with the cells in the 15 mL 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 complete KSR-EB medium using a 5-mL serological pipette. Do not triturate. Note: it is critical at the step to gently resuspend the cells without using force to avoid damage.
  15. One 60 mm dish of cells can be transferred to one 60mm EB dish or one 100 mm EB dish. Place the cells onto a 60 mm or a 100 mm non tissue culture treated dish.
  16. Place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO2 in air.
  17. Change the medium on the EBs every other day by pulling off the entire volume of the dish and transferring it into a centrifuge tube. Keep the tube in the hood and allow the cells to settle to the bottom of the tube (about 5 minutes). Then, using a pipette, remove the supernatant from the tube and replace it with fresh, equilibrated KSR EB medium. Place the cells back onto the same dish.
  18. Continue to change the medium every other day. The EBs will grow in size over time.
  19. Alternatively, after 4 days, transfer the cells equally into 2 x 100mm tissue culture treated dishes to allow for attachment. The attachment of EBs will allow for further differentiation of the cells into various cell types.
  20. Keep the same schedule for fluid changes over time, however as the cells expand the volume per dish may need to be increased by 50% or 100%.
  21. Allow the cells to expand for time points at 14 days and 21 days, or even longer. The entire EB dish can be harvested for analysis.

Table 1 - Recommended Volumes

Component
35 mm Dish
60 mm Dish
100 mm Dish
Complete KSR EB Medium
-
5 mL
10 mL
Dispase
0.5 mL
1 mL
3 - 4 mL
DPBS for rinsing
2 mL
4 mL
10 mL


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Expected Results

Figure 1: Embryoid bodies at various time points (Day 3, 7, 9, and 16), all at 40 x magnification. Spheres gradually turn into masses of cells, and once transferred onto tissue culture treated dishes, they eventually attach and differentiate.

References

  • 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.

Appendix

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™ Serum Replacement EB medium using DMEM/F-12 including GlutaMAX™-I (Cat. no. 10565-018), aseptically combine the components listed in the table below.

Component
Stock Concentration
Final Concentration Volume
DMEM/F12 containing GlutaMAX™-I (Cat. no.
10565-018)
-
1X
79 mL
KnockOut™ SR (Cat. no. 10828-028)
-
20%
20 mL
Non-Essential Amino Acids (Cat. no. 11140-076)
10 mM
0.1 mM
1 mL

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

To prepare 100 mL of Complete KnockOut™ Serum Replacement EB medium using KnockOut™ DMEM (Cat. No. 10829-018), aseptically combine the components listed in the table below.

Component
Stock Concentration
Final Concentration
Volume
KnockOut™ DMEM (Cat. No. 10829-018)
-
1X
78 mL
GlutaMAX™ -I (Cat. No. 35050-061)
200 mM
2 mM
1 mL
KnockOut™ SR (Cat. no. 10828-028)-
20%
20 mL
Non-Essential Amino Acids (Cat. no. 11140-076)10 mM
0.1 mM
1 mL

Dissociation Enzymes/ Tools for Harvesting hESC/iPSC

Dissociation Enzyme /Tools
Application
Suggested concentration
StemPro® EZPassage™ tool (Cat. no. 23181-010)
Manual passaging
Sterile, disposable tool
StemPro® Accutase® (Cat no. A11105-01)
Monolayer of cells post passage, Dissociation into single cells
1X ready to use (1-2 minutes incubation at 37 C)
Dispase (Cat no. 17105-041)
Colony-like morphology post passage
2 mg/ml for 2-3 minutes incubation at 37 C
TrypLE™ Express (Cat no.12604-021)
Dissociation to single cells
1X ready to use
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