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Deriving Clinically-Relevant Numbers of Human iPSCs From Optimized Bioreactor Culture

Review of “Optimized serial expansion of human induced pluripotent stem cells using low‐density inoculation to generate clinically relevant quantities in vertical‐wheel bioreactors” from STEM CELLS Translational Medicine by Stuart P. Atkinson

The generation and differentiation of clinically relevant numbers of homogenous pluripotent stem cells in bioreactors can reduce costs, improve monitoring and control, and create an environment that promotes higher cell densities [1] when compared to traditional monolayer culture [2-4]. However, researchers led by Michael S. Kallos (University of Calgary, Alberta, Canada) noted a significant lack of systematic optimization when it came to the bioreactor-mediated expansion of human induced pluripotent stem cells (hiPSCs). In their new STEM CELLS Translational Medicine article [5], Borys et al. now describe an optimized protocol employing a vertical‐wheel bioreactor that achieved the rapid generation of clinically-relevant numbers of high-quality hiPSCs that can be safely employed for various therapeutic purposes.

The authors optimized inoculation and agitation as well as oxygen and nutrient availability to promote the efficient and safe expansion of preformed suspended aggregates formed by seeding single hiPSCs into non-adherent suspension wells, where cell‐cell adhesion and secretion of endogenous extracellular matrix stimulates attachment and growth [6]. Additionally, the process employed defined, serum‐free medium and a single‐use, low‐shear, vertical‐wheel bioreactor that they hoped would provide the perfect environment for the safe and rapid culture of high‐quality hiPSCs. Specifically, the team employed a novel vertical‐wheel bioreactor system that combines radial and axial flow components to produce uniform distributions of hydrodynamic forces and lower shear stress compared with traditional horizontal‐blade based bioreactors [7, 8].

Encouragingly, following the definition of optimized parameters, which included the use of 21% O2, the authors established a 32-fold expansion of a small starting number of hiPSCs in only six days with only a single 50% medium replacement on day four; furthermore, the team then demonstrated the ability to replicate their protocol over four serial passages (in 28 days) to generate clinically-relevant numbers of hiPSCs. Importantly, the resultant hiPSCs maintained genomic stability at the chromosomal level, their pluripotent nature (as evidenced by the expression of pluripotent markers such as SSEA‐4, TRA‐1‐60, Oct‐4, and Nanog), and the functional ability to form teratomas in vivo, suggesting their overall high-quality status. 

Overall, the authors describe an economical and reproducible means to rapidly generate clinically-relevant numbers of high-quality hiPSCs and hence foster the translation of production to clinical and manufacturing settings and the development of advanced hiPSC-based therapeutics.

For more on the implementation of optimized bioreactors to generate clinically-relevant numbers of hiPSCs, stay tuned to the Stem Cells Portal!

References

  1. Rodrigues CAV, Fernandes TG, Diogo MM, et al., Stem cell cultivation in bioreactors. Biotechnology Advances 2011;29:815-829.
  2. Ludwig TE, Levenstein ME, Jones JM, et al., Derivation of human embryonic stem cells in defined conditions. Nature Biotechnology 2006;24:185-187.
  3. Reubinoff BE, Pera MF, Fong C-Y, et al., Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology 2000;18:399-404.
  4. Richards M, Fong C-Y, Chan W-K, et al., Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nature Biotechnology 2002;20:933-936.
  5. Borys BS, So T, Colter J, et al., Optimized serial expansion of human induced pluripotent stem cells using low-density inoculation to generate clinically relevant quantities in vertical-wheel bioreactors. STEM CELLS Translational Medicine 2020;9:1036-1052.
  6. Sart S, Bejoy J, and Li Y, Characterization of 3D pluripotent stem cell aggregates and the impact of their properties on bioprocessing. Process Biochemistry 2017;59:276-288.
  7. Croughan MS, Giroux D, Fang D, et al., Chapter 5 - Novel Single-Use Bioreactors for Scale-Up of Anchorage-Dependent Cell Manufacturing for Cell Therapies, in Stem Cell Manufacturing, J.M.S. Cabral, et al., Editors. 2016, Elsevier: Boston. p. 105-139.
  8. Rodrigues CAV, Silva TP, Nogueira DES, et al., Scalable culture of human induced pluripotent cells on microcarriers under xeno-free conditions using single-use vertical-wheel™ bioreactors. Journal of Chemical Technology & Biotechnology 2018;93:3597-3606.