At STEMCELL Technologies, our Research Scientists routinely passage human embryonic stem (ES) and induced pluripotent stem (iPS) cells as aggregates for maintenance. These established methods have been shown to allow the long-term expansion of many different cell lines while maintaining a normal karyotype expected of donor material. While it is possible to passage human ES and iPS cells as single cells, it has been demonstrated that this practice may place unwanted selective pressure on cell populations that could lead to genetic aberrations in the culture.^1,2 On the other hand, some researchers may prefer single-cell passaging for obtaining higher-density cultures or for compatibility with single-cell applications. For these researchers, we developed eTeSR™, a novel hPSC maintenance medium formulated specifically to maintain cell quality when passaging and maintaining hPSCs as single cells

This technical tip discusses the best practices of clump passaging, and when single-cell passaging is appropriate. Also included is a specialized technique for converting monolayer cultures into aggregate cultures using the enzyme-free dissociation reagents Gentle Cell Dissociation Reagent (GCDR; Catalog #100-0485) and ReLeSR™ (Catalog #05872, #100-0484).

When Should I Passage as Aggregates or Single Cells?

Clump Passaging Best Practices for Human ES and iPS Cell Maintenance

• No need for ROCK inhibitor.
• No antibiotics (aseptic technique is best and antibiotics may result in an adaptation of the cell line to grow well only when this is added to the medium).
• Aggregate size between 50 and 200 µm (optimize GCDR or ReLeSRTM incubation times to maintain aggregate size and avoid over-trituration of aggregates when harvesting cells).
• Leave the aggregates to settle for 24 hours prior to first medium change.
• Optimize your seeding density through aggregate counting and/or seeding different split ratios in order to passage cells at 50 - 75% confluency for scheduled passage day (usually around Day 7).
• Check your cell quality regularly through genetic analysis and functional assays (karyotyping or using hPSC Genetic Analysis Kit [Catalog #07550] or STEMdiff™ Trilineage Differentiation Kit [Catalog #05230]).

For complete instructions, refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1 (Document #10000005505), mTeSR™ Plus (Document #10000007757), TeSR™-E8™ (Document #10000005516), or TeSR™-AOF (Document #10000008160), available at www.stemcell.com or contact us to request a copy.

How to Transition from Monolayer into Aggregate Passaging

Using Gentle Cell Dissociation Reagent

The procedure below is for using Gentle Cell Dissociation Reagent (GCDR; Catalog #100-0485) to generate aggregates for passaging from a single-cell monolayer culture. Volumes given are for 6-well plates; if using alternative cultureware, adjust volumes according to surface area.

1. Aliquot sufficient TeSR™ medium (mTeSR™1, Catalog #85850; mTeSR™ Plus, Catalog #0100-0276; TeSR™-E8™, Catalog #05990; or TeSR™-AOF, Catalog #100-0401) and warm to room temperature (15 - 25°C). Do not warm the medium in a water bath.
2. Wash cells with 1 mL/well of D-PBS (Without Ca++ and Mg++; Catalog #37350) and aspirate.
3. Add 1 mL/well of GCDR and incubate at 15 - 25°C for 4 - 8 minutes.
   Note: Incubation time can vary depending on a number of factors including cell line and culture density. The culture should be checked under a microscope after 4 minutes and every minute following until the culture resembles the optimal image shown in Figure 1.
4. Carefully aspirate the GCDR, taking care not to shake/tap the plate.
5. Add 1 mL/well of TeSR™ medium.
6. Tilt the plate at approximately a 45° angle. Using a 1000 µL pipette tip, use the tip to gently score a crosshatched pattern in your monolayer culture in four directions: vertically, horizontally, and at 45° angles in both directions (Figure 2).
   Note: The scores should be approximately 0.5 cm apart and should start from the well wall and extend to the other. You should observe cell aggregates detaching as the plate is being scored. This is normal and indicates that the cells were incubated with GCDR for an appropriate time.
7. Still with the plate tilted, gently detach the remaining colonies by scraping with a cell scraper.
   Note: Take care to minimize the breakup of colonies.>
8. Transfer the detached cell aggregates to a 15 mL conical tube.
   Optional: Rinse the well with an additional 1 mL of TeSR™ medium to collect remaining cell aggregates.
9. Carefully pipette the cell aggregate mixture up and down to break up the aggregates as needed.
   
   Note: Refer to the appropriate TeSR™ medium Technical Manual for suggestions on how to break up cell aggregates cultured on different types of matrices. A uniform suspension of aggregates approximately 50 - 200 µm in size is optimal; do not create a single-cell suspension (Figure 3).
10. Refer to the appropriate TeSR™ medium Technical Manual for plating and passaging cells as aggregates.

Using ReLeSR™

The procedure below is for using ReLeSR™ (Catalog #05872, #100-0484) to generate aggregates for passaging from a single-cell monolayer culture. Volumes given are for 6-well plates; if using alternative cultureware, adjust volumes according to surface area.

1. Aliquot sufficient TeSR™ medium (mTeSR™1, Catalog #85850; mTeSR™ Plus, Catalog #0100-0276; TeSR™-E8™, Catalog #05990; or TeSR™-AOF, Catalog #100-0401) and warm to room temperature (15 - 25°C).
   Note: Do not warm the TeSR™ medium in a 37°C water bath.
2. Wash cells with 1 mL/well of D-PBS (Without Ca++ and Mg++; Catalog #37350) and aspirate.
3. Add 1 mL/well of ReLeSR™ and aspirate ReLeSR™ within 1 minute, so that colonies are exposed to a thin film of liquid.
4. Incubate at 37°C for 4 - 8 minutes.
   Note: Optimal dissociation time may vary depending on the cell line used. When passaging a cell line with ReLeSR™ for the first time, the optimal dissociation time should be determined by checking under the microscope after 4 minutes and every minute following until the culture resembles the optimal images shown in Figure 4.
5. Add 1 mL/well of TeSR™ medium.
6. Gently detach the colonies by scraping with a cell scraper.
7. Transfer the detached cell aggregates to a 15 mL conical tube using a 5 mL serological pipette (e.g. Catalog #38003).
   Note: A uniform suspension of aggregates approximately 50 - 200 µm in size is optimal; do not create a single-cell suspension (Figure 3).
8. Refer to the appropriate TeSR™ medium Technical Manual for plating and passaging cells as aggregates.

STEMCELL Products

This procedure has been optimized for use with human pluripotent stem cell (hPSC) maintenance reagents and multiple embryonic stem (ES) and induced pluripotent stem (iPS) cell lines. For upstream protocols and source materials, please see the mTeSR™ Plus Technical Manual and the Product Information Sheet for STEMCELL’s highly quality-controlled Healthy Control Human iPSC Line, Female, SCTi003-A.