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 TeSR™-AOF, Y-27632 and CellAdhere™ Laminin-521. Approximately 1.6 x 10⁹ viable hPSCs are generated in 11 - 12 days, when starting from a 6-well plate (~12 x 10⁶ cells) with one intermediate scale-up passage prior to expansion in a multi-layer cell factory.

Materials

• TeSR™-AOF (STEMCELL Technologies, Catalog #100-0401)
• CellAdhere™ Laminin-521 (STEMCELL Technologies, Catalog #200-0117)
• Dulbecco’s Phosphate Buffered Saline, with Ca++, Mg++ (ThermoFisher, Catalog #14040141)
• Y-27632 (STEMCELL Technologies, Catalog #72304)
• TrypLE Express dissociation reagent (ThermoFisher, Catalog #12605028)
• Optional: ACF Enzyme Inhibition Solution (STEMCELL Technologies, Catalog #05428)
• Sterile Ca⁺⁺/Mg⁺⁺free D-PBS (STEMCELL Technologies, Catalog #37350)
• T-225 vented tissue culture-treated flask (Corning, 431082)
• Sterile serological pipettes
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• 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
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Part I: Initial Harvest of 6-well plate of hPSCs as Single Cells (Day 0)

1. Aspirate media from a 6 well plate of hPSCs maintained in TeSR™-AOF on CellAdhere™ Laminin-521 and rinse with 2 mL PBS per well, 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 wells may be required.
2. Add 1 mL TrypLE Express per well and incubate at 37៰C for 8 - 10 minutes
3. Add equivalent volume of TeSR™-AOF 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 with 2 mL PBS per well, transfer to same Falcon® tube to pool cells
5. Centrifuge at 300 x g for 5 minutes
6. Prepare 47 mL of seeding medium (10 µM ROCK inhibitor Y-27632 in TeSR™-AOF): 94 µL of 5 mM Y-27632 + 46.906 mL of TeSR™-AOF
7. Aspirate supernatant from tube, and resuspend cells in 1 - 2 mL of seeding medium
8. Aspirate CellAdhere™ Laminin-521 from the T-225 flask and fill with 45 mL of seeding medium
9. Seed 11,000 cells/cm² into the T-225 flask
10. Evenly distribute cells by shaking the plate back and forth and incubate culture at 37°C for 5 - 6 days
11. Evenly distribute cells by shaking the plate back and forth and incubate culture at 37°C for 4-5 days
    Note: A T-225 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.
12. Perform a complete medium change on day 1 after seeding
13. Perform medium changes on days 3 and 4, or optionally change medium daily

Part II: Harvest Intermediate Scale Up in a T-225 Flask (Day 6)

1. On Day 5, or when cultures reach 90 - 95% confluence, aspirate media from T-225 flask
2. Rinse flask with 10 mL PBS and aspirate to remove PBS
3. Add 22 mL TrypLE Express and incubate at 37៰C for 8 - 10 minutes
4. Add equivalent volume of TeSR™-AOF 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 two 50 mL Falcon® tubes
5. Rinse flask with 10 mL PBS, transfer to the same Falcon® tubes to pool cells
6. Centrifuge at 300 x g for 5 minutes.
7. Aspirate supernatant from the two 50 mL Falcon® tubes and add 12.5 mL of seeding medium to each one. Resuspended cells and pool them into one tube.
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 11,000 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, 11,000 cells/cm² x 6360 cm² = 7.00 x 10⁷ cells required
   ex. T-25 surface area = 25 cm², 11,000 cells/cm² x 25 cm² = 2.75x10⁵ cells required

Part III: Seeding a 10-Layer Cell Factory (Day 6, 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. 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.
5. Mix the cell factory seeding suspension in the 2 L bottle by swirling and gentle inversion.
6. Seed cell factory by pouring 1300 mL seeding suspension into an opened port
7. 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.
8. Carefully transport the cell factory to the incubator, with ports tilted slightly upwards, to prevent media from spilling between layers.

Part IV: Feeding (Days 7 - 10/11)

Feed on days 1, 3 and 4/5 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 5 mL feed in a T-25 flask)

Part V: Harvesting hPSCs from a 10-Layer Cell Factory (Day 11/12)

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 8 - 10 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 TeSR™-AOF 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 (50 mL Falcon® or 225 mL Falcon® PP) and centrifuge at 300 x g for 5 minutes to remove residual dissociation reagent. Resuspend and pool cells in TeSR™-AOF, supplemented with 10 µM ROCK inhibitor Y-27632, before performing cell counts and continuing with differentiation or freezing protocols.

Data

Figure 2. Viable Cell Yield on Day 5 From Cell Factory in TeSR™-AOF on CellAdhere™ Laminin-521

SCTi003-A, WLS-1C, and H9 hPSCs were seeded at 8500 - 12,500 cells/cm² in a suspension of 10 µM ROCK inhibitor Y-27632 in TeSR™-AOF on CellAdhere™ Laminin-521, 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 93% (mean ± SD, n = 6) and (B) yielded, on average, 3.1 x 10⁵ viable cells/mL TeSR™-AOF consumed (mean ± SD, n = 6), equivalent to 1.6 x 10⁹ viable cells in a 10-layer cell factory.

Markers of the Undifferentiated State

Figure 3. hPSCs Harvested from Cell Factory Expressed Markers of the Undifferentiated State

hPSCs (SCTi003-A, WLS-1C, and H9) harvested on day 5 from cell factories were assessed by flow cytometry for expression of OCT-4 and TRA-1-60 (n = 3). Both markers were expressed on greater than 90% of cells. Bars are the mean of 2 technical replicates ± SD.