infectious diseases such as HIV and, more recently, COVID-19.

However, in order to produce clinically relevant cell quantities for

existing autologous or allogeneic therapeutic approaches, repro-

ducible and controllable cultivation conditions are essential. In

this context, single-use bioreactors have become increasingly pop-

ular in recent years [6].

The fundamental principle behind single-use bioreactors is that

they make use of a plastic vessel or bag as a cultivation container,

instead of re-usable stainless-steel or glass. This gives these bio-

reactors a major advantage over their reusable counterparts, as they

can be put into operation immediately. The cultivation container is

purchased pre-assembled and sterile, eliminating the need for

cleaning and sterilization, while further reducing the risk of con-

tamination during the production process [7, 8]. Moreover, the

single-use cultivation containers, in which stirrers, spargers, and

measuring probes may be implemented, may then be decontami-

nated, and discarded directly following application, significantly

reducing the time between cultivations. This also reduces the vali-

dation work required for good manufacturing practice (GMP)

production, resulting in more runs realized per year and increased

process output. Earlier shortcomings of the technology, such as

leakage of the systems (with cubic meter range working volumes) or

the migration of leachables (bisphenol A or bis(2,4-di-tert-butyl-

phenyl) phosphate) in critical concentrations [9, 10] have also since

been brought under control through the use of improved materials

and the appropriate detection tests [11–14].

Oosterhuis [15] and Jossen et al. [16] describe the different

types of commercially available single-use bioreactors, which are

mainly stirred, wave-mixed, and orbitally shaken with a maximum

working volume of 6 m³, in their respective book chapters. Stirred

systems, however, are considered to be the best studied and most

commonly used of all the single-use bioreactor types, with such

systems successfully implemented for both the bench-top and pilot-

scale expansion of hMSCs.

1.1

Expansion of

hMSCs in Stirred

Single-Use Bioreactors

As hMSCs can be used for both autologous (patient-specific) and

allogeneic (off-the-shelf) cell therapies, the choice of approach has a

strong influence on the required production scale and thus on the

choice of the stirred single-use bioreactor system. Stirred, single-

use bioreactors in the benchtop range are usually sufficient for the

production of cell quantities required for autologous therapies. In

such systems, cells are generally expanded either as cell aggregates

(also known as spheroids) or on MCs. The use of spheroid-based

cultures means that the cells are in direct contact to one another

over a variety of cell junctions, thus enabling cell–cell interaction.

However, due to the heterogeneous nature of such spheroid-based

cultures, this method is more commonly used for the study of

complex 3D structures or for cell differentiation purposes in tissue

engineering, than for mass expansion. The main motivation for

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