Single-Cell Passaging of 3D hPSC Suspension Cultures
This protocol describes a filter-free, single-cell passaging method for the short-term expansion of human pluripotent stem cells (hPSCs) in 3D suspension culture using TeSRâ„¢-AOF 3D and Gentle Cell Dissociation Reagent (GCDR). In combination with manual trituration, GCDR, a non-enzymatic dissociation reagent, enables the generation of single-cell suspensions while preserving key cell-surface proteins. In contrast, enzymatic dissociation reagents can disrupt cell-surface proteins, leading to reduced aggregation efficiency and limited culture expansion.
This filter-free, single-cell passaging method results in comparable culture expansion and cell viability to filter-based clump passaging, while preserving the expression of markers of the undifferentiated state and efficient specification to the three germ layers (Figures 1 and 2). Aggregate size distributions on culture Days 3 and 4 are consistent across both methods (Figure 3). Importantly, this method does not increase the risk of acquiring or propagating genetic abnormalities during short-term scale-up, as confirmed by SNP array analysis after five passages across four different hPSC lines.
This passaging method, designed for use up to five passages, yields healthy, high-quality hPSC aggregates. It is also compatible with automated culture platforms and short-term scale-up in bioreactors (Figure 4). For routine maintenance of hPSCs in 3D beyond five passages, we recommend filter-based clump passaging using GCDR in TeSRâ„¢-AOF 3D to reduce the risk of karyotypic abnormalities.
Materials
- TeSRâ„¢-AOF 3D (Catalog #100-0720)
- Gentle Cell Dissociation Reagent (Catalog #100-1077)
- Falcon® Conical Tubes, 50 mL (Catalog #100-0090)
- 37 µm Reversible Strainers, Large (Catalog #27250)
- Falcon® Serological Pipettes, 1 mL (Catalog #38001)
- Y-27632 (Catalog #72304)
Protocol
In the following passaging protocol, hPSC aggregates are dissociated to single cells using GCDR and resuspended in TeSRâ„¢-AOF 3D Seed Medium (with Y-27632). This protocol is for the dissociation of aggregates from an initial culture volume of 15 mL. For other culture volumes, refer to Table 1 for volumes of GCDR and TeSRâ„¢-AOF 3D Seed Medium (with Y-27632).
Table 1. Recommended Volumes of GCDR and TeSRâ„¢-AOF 3D Seed Medium When Dissociating Aggregates
- Aliquot and warm 5 mL of GCDR to 37°C.
- Bring TeSR™-AOF 3D Seed Medium to room temperature (15 - 25°C).
- Prepare enough TeSRâ„¢-AOF 3D Seed Medium (with Y-27632) to resuspend and seed all conditions. See Table 1 for recommended resuspension volumes.
- Image the cultures prior to passaging to assess aggregate morphology and size.
- Transfer aggregate suspension to a 50 mL conical tube.
Note: If desired, a 37 µm Reversible Strainer can be used to filter out non-aggregated single cells. Place the reversible strainer on a conical tube with the arrow pointing up and pass the entire culture volume through the strainer. Carefully flip the strainer onto a new conical tube and rinse the aggregates with the pre-warmed GCDR. Discard the strainer. Skip to step 8.
- Allow aggregates to settle at the bottom of the tube, and then aspirate the media.
- Add the pre-warmed GCDR to the conical tube.
- Incubate the tube in a 37°C water bath for 15 - 20 minutes. Gently flick the tube to resuspend the aggregates every 3 - 5 minutes.
Note: Optimal incubation time may vary depending on the cell line.
- Centrifuge the tube for 4 minutes at 100 x g to pellet the aggregates.
- Carefully aspirate the supernatant without disturbing the cell pellet.
- Flick the tube to resuspend the pellet and then add 1 mL of TeSRâ„¢-AOF 3D Seed Medium (with Y-27632).
- Use a P1000 pipette to gently triturate the cells 5 - 15 times (optimal number of triturations may vary depending on cell line, GCDR incubation time, and density of culture).
Note: If you are resuspending multiple pellets, perform steps 10 - 12 one tube at a time.
- Top up with the resuspension volume of TeSRâ„¢-AOF 3D Seed Medium (with Y-27632) as indicated in Table 1.
- Swirl the tube and take a sample for cell counts.
Note: Ensure cell density is in range for your desired cell counting method. If the cell suspension contains clumps, then a single cell counting method may not be accurate. If using the NC250 NucleoCounter® for an AO/DAPI count, ensure < 5% of cells make up aggregates composed of 5 or more cells. Cell viability should be > 80%.
- Seed the new vessel at the appropriate cell density in TeSR™-AOF 3D Seed Medium with 10 µM Y-27632.
Figures
Figure 1. hPSC Aggregates Passaged As Single Cells Maintain Equivalent Expansion to Clump-Passaged hPSCs over Five Consecutive Passages
Human pluripotent stem cells (hPSCs) from four cell lines were cultured as aggregates in TeSRâ„¢-AOF 3D in non-Tissue Culture (TC)-treated six-well plates on an orbital shaker. The aggregates were passaged every 3 - 4 days by dissociation to single cells or clumps with GCDR. Single-cell passaged cultures maintained equivalent expansion compared to clump-passaged cultures over five passages. Points represent the average cumulative viable cells of three technical replicates for each cell line (n = 4 cell lines), and error bars represent the standard deviation of the replicates.
Figure 2. hPSCs Maintained in TeSRâ„¢-AOF 3D and Passaged As Single Cells Highly Express Markers of the Undifferentiated State and Differentiate Efficiently to the Three Germ Layers
(A) Human pluripotent stem cells (hPSCs) from four cell lines were maintained as aggregates in TeSRâ„¢-AOF 3D and passaged as single cells with GCDR for five passages. All cell lines highly expressed markers of the undifferentiated state, with > 80% staining positive for OCT4 and TRA-1-60, as determined by flow cytometry. Bars represent the average percent positive cells of three technical replicates for each cell line (n = 4 cell lines), and error bars represent the standard deviation of the replicates. (B) Two hPSC lines were maintained as aggregates in TeSRâ„¢-AOF 3D and passaged as single cells with GCDR for five passages before being differentiated toward the three germ layers using STEMdiffâ„¢ Trilineage Ectoderm Medium, STEMdiffâ„¢ Trilineage Mesoderm Medium, or STEMdiffâ„¢ Definitive Endoderm Kit. Both hPSC lines demonstrated efficient differentiation to all three lineages (> 80% double positive marker expression). Bars represent the mean of three technical replicates for each cell line (n = 2 cell lines) and error bars represent the standard deviation.
Figure 3. hPSCs Passaged As Single Cells in TeSRâ„¢-AOF 3D Reach Equivalent Aggregate Size Distribution to Clump-Passaged hPSCs by Day 3 of Culture
(A) The human induced pluripotent stem cell (hiPSC) line SCTi003-A was cultured as aggregates in TeSRâ„¢-AOF 3D in non-TC-treated six-well plates on an orbital shaker. Wells were imaged from Day 1 - 4 on a benchtop microscope. hPSC aggregates that were seeded as single cells were smaller than clump-seeded aggregates on Day 1, but caught up in size by Day 3. hPSCs from both passaging conditions show characteristic hPSC aggregate morphology. (B) Aggregate images were analyzed to compare the size distributions of the single-cell and clump-passaged cultures. Each point represents the diameter of one aggregate from a total of three replicate wells, and the bars represent the median aggregate diameter at each time point. By Day 3 of culture, the median aggregate diameter, and overall size distribution, is comparable between the single cell and clump-seeded cultures.
Figure 4. Single-Cell Passaging Enables Efficient Scale-Up of hPSCs in Suspension Culture
Two hPSC lines were cultured as aggregates in TeSRâ„¢-AOF 3D and passaged as single cells. Both cell lines maintained expected expansion (daily fold expansion > 1.4) when scaling up in recommended culture vessels. Each dot represents one technical replicate and the bars represent the mean daily fold expansion of the technical replicates and cell lines combined.
Troubleshooting
The resulting single-cell suspension should look comparable to the microscope images shown in Figure 5. See Table 2 for troubleshooting steps if your resulting single-cell suspension differs.
Figure 5. hPSC Aggregates Can Be Dissociated to Single Cells with GCDR
The hiPSC line SCTi004-A was cultured as aggregates in TeSRâ„¢-AOF 3D and then dissociated to single cells with a 15 minute incubation in GCDR followed by gentle trituration with a pipette. Microscope images were taken of single cells seeded into non-TC-treated six-well plates in TeSRâ„¢-AOF 3D at a density of 5 x 10â´ viable cells/mL. This dissociation method consistently results in cell viability > 90% and < 5% of cells in aggregates with five or more cells. Optimal GCDR incubation time and number of triturations may vary by cell line.
Table 2. Common Problems and Solutions for 3D hPSC Single-Cell Passaging
- Ensure input hPSC culture is of high quality
- Increase GCDR incubation time to 20 or 25 minutes
- Increase the number of triturations with the P1000 pipette
- Ensure input hPSC culture is of high quality
- Increase GCDR incubation time to 20 or 25 minutes
- Decrease the number of triturations with the P1000 pipette and/or triturate slower
- Ensure input hPSC culture is of high quality
- Ensure cell viability is > 90%
- Ensure Y-27632 is present in the Seed Medium
- Ensure the culture volume and agitation rate is correct
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