Scalable passaging of adherent human pluripotent stem cells

Abstract

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive, result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs, we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate, formulated as a hypertonic solution, gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium, retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together, this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology, high levels (>80%) of pluripotency markers OCT4, SSEA-4, TRA-1-60 andTRA-1-81, a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers, both in vivo and in vitro.

Figure 1. Subculture of WA09 hESCs with hypertonic citrate solution supports detachment of colonies as multicellular aggregates.

Confluent WA09 hESC cultures maintained in mTeSR™1 on Matrigel™ were dissociated in a 1 mM sodium citrate solution at increasing osmolalities. Using image analysis software, six images of detached cells from each of three independent cultures (n = 3) were taken after 10 or 20 minutes of treatment with the citrate solutions. These images were quantified to assess the size of the multicellular colony aggregates produced. Error bars indicate standard error of the mean. Representative brightfield images of detached hESC multicellular colony aggregates, after 20 minutes of treatment, are shown below each osmolality. Using the isotonic osmolality (270 mOsmol/kg) as a reference, all other osmolalities exhibited statistically fewer single cells and aggregates less than four cells (P<0.05); scale bar: 100 µm.

Figure 2. Comparison of hypertonic citrate solution with EDTA in the detachment of hPSCs as multicellular aggregates.

(A) Percentage of WA09 cells recovered from multi-layer flasks using either the Hypertonic Citrate Solution (1 mM; 570 mOsmols/kg) or EDTA (0.5 mM); P>0.05. (B) Size quantification and brightfield images of the hESC aggregates obtained after a 20 minute treatment with hypertonic citrate or EDTA show the cell aggregates collected using the hypertonic citrate solution were larger and contained fewer single cells and very small aggregates (P<0.05). Error bars indicate standard error of the mean. All conditions, n = 3; scale bar: 200 µm.

Figure 3. Comparison of WA09 cell post-detachment viabilities using conventional methods and the optimized hypertonic citrate solution.

Using the hypertonic citrate solution as a reference, all other conventional detachment methods exhibited lower cell viability (P<0.05). Error bars indicate standard error of the mean. All conditions, n = 3.

Figure 4. Post-detachment cell viability impacts the rate of cell expansion.

(A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, Dispase or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10⁵ viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10⁵ and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.

Figure 5. WA09 hESCs subcultured for over 25 passages using hypertonic citrate retain their stem cell characteristics.

(A) Immunodetection of Oct4, Sox2 and Nanog antigens (green); SSEA-4, Tra-1-60 or Tra-1-81 antigens (red). Individual cell nuclei are visualized using DAPI (blue). Scale bar: 200 µM. (B) Flow cytometric analysis performed on cells cultured in either StemPro® or mTeSR™1 using antibodies that detect Oct4, SSEA-4, Tra-1-60, and Tra-1-81 antigens. Cells expressing each pluripotent antigen, detected using a specific antibody are illustrated in red. The isotype control used to detect non-specific binding is shown in gray. (C) Immunohistochemistry performed on embryoid bodies differentiated for 7 days in suspension culture and 7 days on gelatin-coated plates. Antibodies detecting Beta-III-Tubulin (TUJ1), Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown (green). Nuclear staining is shown using DAPI (blue). Scale bar: 200 µM. (D) Tissue sections of teratomas produced from undifferentiated hESCs contain cells from the indicated germ layers. Sections are shown counterstained with Hematoxylin and Eosin. Scale bar: 200 µm.