Sticking Together - Clump Passaging for ES and iPS Cell Maintenance

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. 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

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?

Aggregates
Single Cells
Advantages
  • Allows for long-term expansion of human ES and iPS cells while maintaining a normal karyotype.
  • Right-size aggregates (50 - 200 µm) allow for good attachment to the matrix.
  • Ease of use and simple system to avoid unintended effects on the culture (no need for ROCK inhibitor3).
  • For cryopreservation, flow cytometry, clonal cell line generation, monolayer plating for transfection or differentiation, or any other endpoint analysis.
Disadvantages
  • Requires additional training if unfamiliar with the clump passaging technique.
  • Not optimal for setting up downstream applications such as specific differentiation protocols or endpoint analyses like flow cytometry.
  • Requires ROCK inhibitor to enhance cell survival (for first 24 hours only as longer exposure has shown changes in cellular metabolism4 and morphology5).
  • Requires more frequent genetic analysis to ensure that the karyotype is normal.

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.

Figure 1. Images of the H1 human ES cell line at a magnification of 40X (A) as a monolayer and (B) after 6 minutes at 15 - 25°C in GCDR.

Figure 2. Using a pipette, draw crosshatched lines in your monolayer culture.

Figure 3. Optimal aggregate size of 50 - 200 µm, shown at a magnification of 20X (A) and 40X (B).


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.

Figure 4. Human ES (H1) cells at a magnification of 40X (A) as a monolayer and (B) after 4 minutes in ReLeSR™ at 37°C.


STEMCELL Products

Product Catalog #
hPSC Genetic Analysis Kit 07550
Genomic DNA Purification Kit79020
STEMdiff™ Trilineage Differentiation Kit05230
mTeSR™185850
mTeSR™ Plus100-0276
TeSR™-E8™05990
TeSR™-AOF100-0401
Gentle Cell Dissociation Reagent (GCDR)100-0485
ReLeSR™05872, 100-0484
D-PBS (Without Ca++ and Mg++)37350
Falcon® Serological Pipettes, 5 mL38003
  1. Draper et al. (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22(1): 53–4.
  2. Buzzard et al. (2004) Karyotype of human ES cells during extended culture. Nat Biotechnol 22(4): 381–2.
  3. Beers et al. (2012) Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat Protoc7(11): 2029–40.
  4. Vernardis et al. (2017) Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor. Sci Rep 7: 42138.
  5. Närvä et al. (2017) A strong contractile actin fence and large adhesions direct human pluripotent colony morphology and adhesion. Stem Cell Reports 9(1):67–76.

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