Cell Culture and Single Cell Passaging of Human Pluripotent Stem Cells Without the Need for ROCK Inhibitor

Human PSCs are typically propagated as colonies and passaged by clump passaging techniques (i.e., cutting colonies into small clumps or clusters for passaging) which is labor intensive and adds to variability in cell seeding³. Alternatively, culture of dissociated hPSCs as a single cell suspension requires the utilization of small molecules such as Rho-associated protein kinase (ROCK) inhibitors (e.g., Y-27632 or thiazovivin) to prevent dissociation-induced apoptosis⁴. The use of completely dissociated single cell suspension for passaging generates a monolayer culture that has advantages of higher culture scalability, rapid expansion, and high efficiency⁵. However, culture conditions which involve single cell dissociation of hPSCs have been shown to cause genomic alterations in hPSCs during long-term cultures^6,7.

Recently, a recombinant isoform of human Laminin, namely Laminin-521, has been shown to support long-term single cell culture without the need for ROCK inhibitors^8-12. This work has been performed using surfaces freshly-coated with rLaminin-521. However, manual coating procedures are labor-intensive and can introduce variability into the culture environment.

Here we present Corning PureCoat rLaminin-521 cultureware, a sterile pre-coated, ready-to-use, animal-free surface manufactured under cGMP conditions that enables stem cell scientists to efficiently scale-up their cell production in xeno-free culture environments.

PureCoat rLaminin-521 cultureware supported long-term culture, and single cell passaging of hiPSCs in xeno-free NutriStemTM XF/FF culture medium. The hiPSCs remained undifferentiated as demonstrated by the expression of OCT-3/4 and SSEA-4 markers. Cells maintained pluripotency through 10 passages and successfully differentiated into cells from the three germ lineages. Human iPSCs possessed a normal karyotype after 10 passages on PureCoat rLaminin-521 cultureware as a single cell suspension without the use of ROCK inhibitor. PureCoat rLaminin-521 cultureware removes manufacturing bottlenecks associated with self-coating protocols with respect to time, labor, and coating variability, and provides a robust and scalable platform for hPSC culture.

Materials and Methods

Human induced pluripotent stem cell (hiPSC) culture and passaging on Corning Matrigel matrix

Human episomal iPSC line (Thermo Fisher Scientific) was used 
for this study and was maintained in feeder-free conditions on Corning Matrigel hESC-qualified matrix (Corning Cat. No. 354277) in mTeSR™1 medium (STEMCELL Technologies) according to manufacturers’ instructions. To passage cells on Matrigel matrix, culture medium was aspirated and cells were washed once with DPBS (2 mL/well; without calcium and magnesium). Cell dissociation buffer (1 mL/well; Enzyme-free, PBS; Thermo Fisher Scientific) was added to the wells and incubated for 3 to 5 minutes at room temperature. The separation of the cells within the colonies was observed under the microscope. The cell dissociation buffer was carefully aspirated from the wells, and the cells were dislodged from the plate as small cell clumps/clusters by forcefully adding fresh medium to the well. The small cell clumps/clusters were seeded onto a freshly-coated Matrigel matrix plate at
 a ratio of 1:10 to 1:14 in mTeSR1 medium (2 mL/well) and incubated at 37°C and 5% CO2 in a humidified incubator. The medium was changed daily and the cells were passaged once a week. The hiPSCs were kept in continuous culture under these conditions until needed for the experiments.

Long-term single cell hiPSC culture on Corning® PureCoatTM rLaminin-521 cultureware

NutriStem XF/FF culture medium (1 mL/well, Stemgent) was added to each well of PureCoat rLaminin-521 cultureware (6-well plate, Corning Cat. No. 356290). These plates were equilibrated in a humidified incubator at 37°C for 30 minutes to 1 hour until the cells were ready to be seeded.

Human iPSCs growing on Corning Matrigel® matrix (or rLaminin-521) coated surfaces were washed once with DPBS
(2 mL/well). StemPro® Accutase® cell dissociation reagent
(1 mL/well, Thermo Fisher Scientific) was added to the wells
 and the cells were incubated at 37°C and 5% CO2 in a humidified incubator for 3 to 4 minutes. The plate was gently tapped
 on the side to further detach the cells from the surface. The cells were gently pipetted up and down 6 to 10 times to achieve a single-cell suspension. Fresh medium (3 to 4 mL/well) was added to the cell suspension to dilute the Accutase, and the cell suspension was centrifuged at 100 × g for 4 minutes. The supernatant was removed and the cells were resuspended in culture medium. The cells were counted using a Vi-CELL™ Cell Viability Analyzer (Beckman Coulter). The hiPSCs were seeded at a density of 50,000 cells/cm² on to the pre-equilibrated rLaminin-521 plates. A complete medium change was performed 48 hours after cell seeding and cells were fed daily until they were ready to be passaged. Cell morphology was observed daily using an EVOS® XL Cell Imaging System (Thermo Fisher Scientific). Cells were passaged every 4 to 5 days when the cell confluence was more than 80%.

Undifferentiated marker expression using flow cytometry analysis

Cells were dissociated from rLaminin-521 plates with Accutase as described above and samples were prepared for FACS according to the manufacturer’s instructions for the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences). The cells were analyzed with a BD FACSCalibur™ flow cytometer (BD Biosciences) and data were analyzed with the BD CellQuest™ Pro software (BD Biosciences).

Tri-lineage differentiation

To assess whether the hiPSCs maintained pluripotency, Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems) was used according to the protocols provided. The cells were plated onto Corning Matrigel matrix-coated 24-well plates and differentiated into the three lineages separately. The three germ layers were stained with antibodies for Otx2 (ectoderm marker), brachyury (mesoderm marker), and SOX17 (endoderm marker). The cells were counterstained with the corresponding secondary antibody (Anti-goat IgG-NL557, R&D Systems) and nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific). The stained differentiated cells were imaged using a fluorescence microscope (Olympus IX70).

Karyotype analysis

The hiPSCs were dissociated with Accutase as described above and seeded on Corning Matrigel matrix-coated T-25 flasks with ROCK inhibitor Y27632 (5 μM, Sigma). Live cell samples were submitted for karyotyping by G-banding analysis (Cell Line Genetics).

Results and Discussion

Human iPSCs were cultured on Corning PureCoat rLaminin-521 cultureware in NutriStem XF/FF culture medium for 10 passages. Throughout this study, the hiPSCs exhibited the typical undifferentiated hPSC morphology as characterized by the presence of small cells and high nucleus to cytoplasm ratio (Figure 1A). The cells maintained a high viability (average of 88.9% ±3.4) and cell expansion rates were consistent between passages as evidenced by the fold expansion rate at each passage (Figure 1B).