Large-Scale Expansion of hPSCs in 2D Monolayer Culture using mTeSR™ Plus
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.
Figure 1. Workflow Schematic for the expansion of hPSCs in a multi-layer cell factory in mTeSR™ Plus
Important Notes
- This protocol describes the steps required for expanding hPSCs in a 10-layer cell factory, if using a different format cell factory (2-layer, 5-layer etc.), volumes of reagents to use are listed in Table 1.
- This protocol recommends the direct pouring method for seeding, exchanging medium, and harvesting cells. To maintain sterility, it is critical to use aseptic technique and avoid making contact between open cell factory ports and media bottles when pouring.
- A 10-layer cell factory can not be viewed under a standard bright field microscope. To monitor cell health and confluence, it is recommended to seed a “companion flask”, such as a T-25 flask, at the same cell density per cm2, and fed at the same frequency and with the same volume of mTeSR™ Plus per cm2 as the cell factory.
- A seeding density of approximately 9000 cells/cm2 is recommended, however inherent biological variability of hESC and hiPSC lines may mean that seeding density optimization is required.
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
Part I: Initial Harvest of ~2 Million hPSCs as Single Cells (Day 0)
Before you begin: Coat a T-175 flask with Matrigel® as per standard pre-coating protocol. Alternatively, include matrigel in mTeSR™ Plus + CloneR™2 at step 8 at 0.3 - 0.6% as in Part III steps 4-6.
- 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.
- Add 1 mL TrypLE Express and incubate at 37៰C for 5 minutes
- 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
- Rinse well with 2 mL PBS, transfer to same Falcon® tube to pool cells
- Centrifuge at 300 x g for 5 minutes
- Prepare 37 mL of seeding medium (10% CloneR™2 in mTeSR™ Plus): 3.7 mL of CloneR™2 + 33.3 mL of mTeSR™ Plus
- Aspirate and resuspend in 1-2 mL of seeding medium
- Aspirate Matrigel from the T-175 flask and fill with 35 mL of seeding medium
- Seed 9000 cells/cm2 into the T-175 flask
- 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. - Perform a complete medium change on day 1 after seeding
- 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)
Before you begin: Thaw the required amount of hESC-Qualified Corning Matrigel® (Corning, 354277) to achieve a 0.3-0.6% final concentration on ice at 4°C, overnight. The day of harvest, in a sterile 2 L bottle, prepare 1330 mL seeding medium: 133 mL of CloneR™2 + 1197 mL of mTeSR™ Plus, and allow it to equilibrate to room temperature.
- On Day 5, or when cultures reach 90-95% confluence, aspirate media from T-175 flask
- Rinse flask with 7 mL PBS and aspirate to remove PBS
- Add 12 mL TrypLE Express and incubate at 37៰C for 5 minutes
- 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
- Rinse flask with 7 mL PBS, transfer to same Falcon® tube to pool cells
- Centrifuge at 300 x g for 5 minutes
- Aspirate supernatant from pooled cell pellet and resuspend cells in small volume (20 mL) of seeding medium.
- Count viable single cells with Nucleocounter A8 slide or hemocytometer with trypan blue
- 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 - 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)
- Pipette 5 mL of seeding medium into a sterile 15 mL conical centrifuge tube.
- 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.
- 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.
- Add appropriate volume of Matrigel® (calculated in part II step 10) to room temperature seeding medium + cells in both mixing containers
- Immediately mix by swirling and gentle inversion
- 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.
- Seed cell factory by pouring 1300 mL seeding suspension into an opened port
- 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. - 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)
- 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 - 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)
- Pour spent media into waste bottle as with feeds
- Rinse cell factory with 200 mL of PBS
a. Add PBS as with feeds, and distribute, then remove by pouring into waste bottle
- Add 200 mL of TrypLE Express OR 0.25% Trypsin-EDTA
- 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
- 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
- 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
- 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.
Table 1. Reagent volumes for different culture formats.
Table 2. Recommended seeding densities and typical cell yields for short-term, high-density single-cell passaging.
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.
Figure 3. hPSCs Harvested from Cell Factory Express Markers of the Undifferentiated State and Attach in Subsequent Passage with 100% Recovery After 24 Hours
hPSCs (SCTi003-A, WLS-1C, and H9) harvested on day 5 from cell factories were (A) 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 (WLS-1C and H9) ± SD. (B) Remaining hPSCs were subsequently seeded down in CloneR™2-supplemented mTeSR™Plus at 15,000 viable cells/well in Corning® Matrigel®-coated 96-well plates (n = 12 replicate wells). After 24 hours, wells were fixed with 4% paraformaldehyde, stained with Hoechst dye for cell quantification, and imaged on an ImageXpress® Micro 4 Microscope. Quantification of cell nuclei was done via a particle analyzing macro using ImageJ software, with the particle size threshold set to 300 pixels. Percent recovery was calculated as the number of Hoechst-stained nuclei divided by the number of viable cells seeded x 100%. Bars show mean ± SD, n = 10 biological replicates.
Figure 4. hPSCs Harvested from Cell Factory Frozen at -135°C in FreSR™-S Retain High Viability Upon Thaw and Attach Efficiently
hPSCs (SCTi003-A, WLS-1C, and H9) harvested as single cells from a cell factory were frozen down at 1 x 107 cells/vial in FreSR™-S at -135°C for ≥ 7 days. (A) Representative start, middle, and endpoint vials (spanning 2 hours of vialing time) were thawed into mTeSR™ Plus supplemented with CloneR™2 and assessed for viability. (B) 1 x 106 cells from each vial were seeded into mTeSR™ Plus supplemented with CloneR™2 per well of 6-well plate, in triplicate. After 24 hours, wells were harvested and % recovery was calculated as # viable cells harvested/# viable cells seeded x 100%. From the same thawed vials, 6-well plates were seeded in triplicate into mTeSR™ Plus supplemented with CloneR™2 at 9.6 x 104 cells per well. Wells were fed daily with mTeSR™ Plus and harvested at the end of the passage at day 5. Viable cell counts were performed and fold expansion was calculated as viable cells harvested divided by viable cells seeded. Bars are the average of the start, middle, and end vials ± SD (SCTi003-A n = 6, WLS-1C and H9 n = 3).
Figure 5. hPSCs Harvested from Cell Factory Are Capable of Differentiation to the Three Germ Lineages
H9 cells cultured for 5 days in a cell factory were dissociated by incubation with TrypLE™ Express Enzyme, and seeded as single cells into a 24-well plate for differentiation via the STEMdiff™ Mesoderm Induction Kit, the STEMdiff™ Definitive Endoderm Kit, and the STEMdiff™ SMADi Neural Induction Kit. (A) Early mesoderm cells show nuclear staining of Brachyury (red) and the absence of OCT4 (green) staining. (B) Definitive endoderm cells show nuclear staining of FOXA2 (red). (C) Neural progenitor cells showed nuclear staining of PAX6 (green) and SOX1 (red). All cells show nuclear staining by Hoechst dye (blue).
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