This protocol describes a 2D method for rapid, large-scale expansion of human pluripotent stem cells (hPSCs) as single cells in a 10-layer cell culture vessel, using mTeSR™ Plus, CloneR™2 and Corning® Matrigel®. Approximately 3 x 10⁹ viable hPSCs are generated in 10-11 days, when starting from a single well of a 6-well plate (~2 x 10⁶ cells) with one intermediate scale-up passage prior to expansion in a multi-layer cell factory.

Materials

• mTeSR™ Plus (STEMCELL Technologies, Catalog #100-0276)
• CloneR™2 (STEMCELL Technologies, Catalog #100-0691)
• TrypLE Express dissociation reagent (ThermoFisher, CAT#12605028)
• Optional: ACF Enzyme Inhibition Solution (STEMCELL Technologies, Catalog #05428)
• Sterile Ca⁺⁺/Mg⁺⁺free D-PBS (STEMCELL Technologies, Catalog #37350)
• hESC-Qualified Corning Matrigel® (Corning, 354277)
• T-175 tissue culture-treated flask (STEMCELL Technologies, Catalog #38073)
• Sterile serological pipettes
See More
• Sterile p20/200/1000 pipette tips
• 10-layer cell factory vessel (STEMCELL Technologies, Catalog #200-0557, Catalog #200-0556 or equivalent)
• T-25 vented tissue culture-treated flask (Corning, 430639)
• 15 mL and 50 mL Falcon® sterile conical tubes (STEMCELL Technologies, Catalog #100-0092, Catalog #100-0090)
• Sterile 2000 mL Bottle (Nalgene, Sigma-Aldrich Z364495) for cell factory seeding medium
• Sterile 1000 mL Bottle (Nalgene, Sigma-Aldrich Z364525) for cell factory harvest
See Less

Part I: Initial Harvest of ~2 Million hPSCs as Single Cells (Day 0)

1. Aspirate media from 1 well of a 6 well plate of hPSCs maintained in mTeSR™ Plus on Matrigel and rinse with 2 mL PBS, aspirate PBS
   Note: 1 well of a 6 well plate of hPSCs at a confluence of ≥ 60% will typically yield approximately 2 x 10⁶ viable cells. If harvesting at a lower confluence, more than 1 well may be required to yield 2 x 10⁶ viable cells.
2. Add 1 mL TrypLE Express and incubate at 37៰C for 5 minutes
3. Add equivalent volume of mTeSR™ Plus or ACF Enzyme Inhibition Solution to TrypLE to quench and pipette up and down ~1-2 times in the well(s) to dislodge single cells, and transfer whole volume to a 50 mL Falcon® tube
4. Rinse well with 2 mL PBS, transfer to same Falcon® tube to pool cells
5. Centrifuge at 300 x g for 5 minutes
6. Prepare 37 mL of seeding medium (10% CloneR™2 in mTeSR™ Plus): 3.7 mL of CloneR™2 + 33.3 mL of mTeSR™ Plus
7. Aspirate and resuspend in 1-2 mL of seeding medium
8. Aspirate Matrigel from the T-175 flask and fill with 35 mL of seeding medium
9. Seed 9000 cells/cm² into the T-175 flask
10. Evenly distribute cells by shaking the plate back and forth and incubate culture at 37°C for 4-5 days
    Note: A T-175 at 95% confluency will provide sufficient cells to seed a 10-layer cell factory
    Recommended seeding densities and expected yields are listed in Table 2.
11. Perform a complete medium change on day 1 after seeding
12. Perform medium changes on days 3 and 4, or optionally change medium daily

Part II: Harvest Intermediate Scale-Up in a T-175 Flask (Day 5)

1. On Day 5, or when cultures reach 90-95% confluence, aspirate media from T-175 flask
2. Rinse flask with 7 mL PBS and aspirate to remove PBS
3. Add 12 mL TrypLE Express and incubate at 37៰C for 5 minutes
4. Add equivalent volume of volume of mTeSR™ Plus or ACF Enzyme Inhibition Solution to TrypLE to quench and gently pipette up and down ~3X in flask to dislodge single cells, and transfer whole volume to a 50 mL Falcon® tube
5. Rinse flask with 7 mL PBS, transfer to same Falcon® tube to pool cells
6. Centrifuge at 300 x g for 5 minutes
7. Aspirate supernatant from pooled cell pellet and resuspend cells in small volume (20 mL) of seeding medium.
8. Count viable single cells with Nucleocounter A8 slide or hemocytometer with trypan blue
9. Calculate the number of cells required to achieve a seeding density of 9000 cells/cm² (or the optimal seeding density you have determined for your cell line of choice) for the cell factory and companion T-25 flask
   ex. 10-layer cell factory surface area = 6360 cm2, 9000 cells/cm² x 6360 cm² = 5.724 x 10⁷ cells required
   ex. T-25 surface area = 25 cm², 9000 cells/cm² x 25 cm² = 2.25x10⁵ cells required
10. Calculate the volume of Matrigel required to achieve 0.3-0.6% final concentration (v/v) for the cell factory and companion T25 flask
    ex. 10-layer cell factory seeding volume =1300 mL , 0.3% x 1300 mL = 3.9 mL Matrigel®
    ex. T-25 seeding volume = 5mL, 0.3% x 5 mL = 0.015 mL Matrigel®

Part III: Seeding a 10-Layer Cell Factory (Day 5, continued)

1. Pipette 5 mL of seeding medium into a sterile 15 mL conical centrifuge tube.
2. Transfer the appropriate number of cells to seed the 10-layer cell factory into the 2 L bottle containing the remaining ~1300 mL of seeding medium.
3. Transfer the appropriate number of cells to seed the T-25 companion flask into the 15 mL conical centrifuge tube containing 5 mL of seeding medium.
4. Add appropriate volume of Matrigel® (calculated in part II step 10) to room temperature seeding medium + cells in both mixing containers
5. Immediately mix by swirling and gentle inversion
6. Seed vented T-25 flask with 5mL seeding suspension from 15 mL Falcon® tube, transport to incubator, and move in back and forth motion to distribute cells.
7. Seed cell factory by pouring 1300 mL seeding suspension into an opened port
8. Distribute suspension in cell factory:
   a. Tightly close both ports
   
   b. Place cell factory upright on its longest side, with ports facing towards you. Allow media to equilibrate to all layers
   
   c. Place cell factory upright on its shortest side, with ports facing towards you, and oriented to the top. Confirm media is evenly distributed between layers.
9. Carefully transport cell factory to incubator, with ports tilted slightly upwards, to prevent media from spilling between layers.

Part IV: Feeding (Days 6 - 9)

Feed on days 1, 3 and 4 post-seeding (optionally feed daily), monitoring companion flask to determine optimal day of harvest (≥90% confluent)

1. Feed cell factory by pouring spent media into waste bottle
   a. Remove both port caps
   
   b. Carefully lift cell factory, tilting towards one port to collect media, and pour out of port into waste bottle, being careful not to make contact between waste bottle and port
   
   c. Pour fresh media into one of the opened ports, likewise being careful not to make contact between media bottle and port
   
   d. Replace caps tightly
2. Distribute media as described above, and return to incubator
   Note: Feed companion flask on the same schedule and with proportional volume of media per cm² as cell factory (a 1300mL feed in a 10-layer cell factory is proportional to a 5mL feed in a T-25 flask)

Part V: Harvesting hPSCs from a 10-Layer Cell Factory (Day 10/11)

Harvest cell factory when companion flask confluence has reached 90-95% (day 5-6 post-seeding)

1. Pour spent media into waste bottle as with feeds
2. Rinse cell factory with 200 mL of PBS
   a. Add PBS as with feeds, and distribute, then remove by pouring into waste bottle
3. Add 200 mL of TrypLE Express OR 0.25% Trypsin-EDTA
4. Incubate at 37°C for 5-6 min
   a. Companion T-25 flask may be harvested in parallel and used as a proxy for observing the progress of the cell dissociation
5. Add 200 mL of ACF Enzyme Inhibition Solution or mTeSR™ Plus to flasks, replace caps and distribute as with feeds, then agitate by tilting cell factory and gently tapping while observing to confirm cells are detaching, and pour into sterile 1 L bottle
6. Rinse cell factory with 400 mL of sterile PBS, pour into same sterile collection bottle
   a. A 50 mL serological pipette may be used to triturate collected cell suspension to ensure any aggregates are dissociated to single cells
7. Distribute cell suspension into conical centrifuge tubes (50mL Falcon® or 225mL Falcon® PP) and centrifuge at 300 x g for 5 minutes to remove residual dissociation reagent. Resuspend and pool cells in mTeSR™ Plus, supplemented with 10% CloneR2, before performing cell counts and continuing with differentiation or freezing protocols.

Data Figures

Figure 2. Viable Cell Yield on Day 5 From Cell Factory in mTeSR™ Plus on Corning® Matrigel®

SCTi003-A, WLS-1C, and H9 hPSCs were seeded at 8000 - 11,500 cells/cm² in a suspension of CloneR™2 supplemented mTeSR™ Plus and 0.3% (v/v) Corning® Matrigel®, and maintained for 5 days with daily medium changes, with one skipped feed on day 2. On day 5, cultures were dissociated to single cells with TrypLE™ Express Enzyme and cell counts performed in triplicate using a NucleoCounter® NC-250™ to assess cell viability and yield. (A) hPSCs had viabilities greater than 95% (mean ± SD, n = 7) and (B) yielded, on average, 6.75 x 10⁵ viable cells/mL mTeSR™ Plus consumed (mean ± SD, n = 7), equivalent to 3.5 x 10⁹ viable cells in a 10-layer cell factory.