Methods in Molecular Biology (2022) 2436: 39–53

DOI 10.1007/7651_2021_396

© Springer Science+Business Media, LLC 2021

Published online: 07 May 2021

Expansion of Human Pluripotent Stem Cells in Stirred Tank

Bioreactors

Marites T. Woon, Puspa R. Pandey, and Inbar Friedrich Ben-Nun

Abstract

Bioreactor technolology enables the expansion of mammalian cells, which can be translated to processes

compatible with Current Good Manufacturing Practice (cGMP) regulations. Cells are introduced to the

bioreactor vessel, wherein key parameters such as temperature, pH, and oxygen levels are tightly controlled

to facilitate growth over time. Here, we describe the microcarrier-based expansion of human pluripotent

stem cells in a 3 L stirred tank bioreactor.

Key words Bioreactor, Cell therapy, Human pluripotent stem cell expansion, Microcarrier, Scalable

1

Introduction

Stem cell technology has rapidly advanced since its development

and continues to revolutionize personalized medicine, drug devel-

opment and disease modeling [1]. Human pluripotent stem cells

(hPSCs) can differentiate, giving rise to clinically relevant cell types

such as cardiomyocytes [2] and neurons [3]. Holding remarkable

promise toward the development of curative therapies, consider-

able attention has been given in the large-scale expansion of these

invaluable cells [4–9].

Expansion of hPSCs required for cell-based therapies using

bioreactors addresses key limitations of conventional 2D culture

methods. The culture of these cells onto glass or plastic surfaces is a

process ubiquitious across academic and instustry-based labora-

tories. However, continued understanding of cell biology and

method improvements support the application of 3D culture meth-

ods instead [10]. 3D culture methods more accurately represent

the cellular microenvironment found in vivo, and cells cultured in

these systems respond accordingly [11]. Drug development studies

have demonstrated distinct differences in the responses of 2D vs 3D

cultured cells [12, 13], underscoring the need to recapitulate com-

plex cellular microenvironments to derive accurate responses

toward therapeutic candidates. Another critical limitation of 2D

culture systems is scalability. For example, approximately 1
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