using this approach is to avoid implementing exogenous support

material, such as scaffolds or MCs, during the production process.

Furthermore, the absence of the exogenous support material means

that cells behave in a similar manner to native tissue [17, 18]. Spher-

oid formation takes place when cells self-attach and begin produc-

ing their own extracellular matrix (ECM), which not only acts as a

scaffold for the cells, but also mimics cell–cell and cell–matrix

signaling networks, enabling them to survive in suspension. As

various studies have shown, such 3D structures can lead to

increased secretion of growth and anti-inflammatory factors [19–

22]. Media composition also plays a crucial role in efficient spheroid

formation, as successful cell aggregation requires supplementation

of adhesion molecules (e.g., integrins, E-cadherin) to facilitate cell–

cell attachment. Bartosh et al. [19] showed that proliferation-

related genes are downregulated in hMSCs after aggregation.

Therefore, the maximum cell density in spheroid-based cultures is

limited to the specific spheroid size and number within a bioreactor

at the point of harvest. In addition, large spheroids are subject to

diffusion-related limitation (e.g., oxygen, nutrients), which is a

major drawback for high cell density cultures. Several studies have

shown that spheroids larger than 200–300 μm tend to induce

apoptosis or even unwanted spontaneous differentiation, due to

nutrient or oxygen limitation in the core of the spheroids [23–

27]. In this regard, the use of MCs presents an advantageous

alternative for the mass expansion of hMSCs.

Many different MCs, which are usually spherical in shape, have

been developed and tested over the years for the expansion of

hMSCs. These different MC types vary greatly with regard to size

(90–380 μm), core material (e.g., polystyrene, cellulose, dextran,

gelatine), and surface coating (e.g., collagen, laminin, fibronectin).

An overview of such commercially available MCs, including their

physical characteristics, may be found in several reviews [28–

30]. The core material and surface coating affect not only MC

sedimentation and cell growth, but also the stirrer speed required

to keep MCs in suspension and ensure adequate mass transfer. Rafiq

et al. [31] and Leber et al. [32] studied different MC types in

stirred single-use bioreactors (benchtop scale) for hMSCs under

predefined stirrer speeds (Njs ¼ Ns1). Both observed significant

differences in cell adhesion, cell growth, glucose consumption,

and metabolite formation depending on MC type. They found

that bone marrow-derived hMSCs grew best on collagen- and

recombinant protein-coated MCs, such as ProNectin^® F from

Solohill^® and Synthemax™II from Corning^®. This is not

surprising, as these MCs are coated with ECM proteins, which

contain the arginine-glycine-aspartate (RGD) sequence, known to

promote the attachment and growth of demanding cells. In gen-

eral, cell adhesion to MCs follows a Poisson distribution, with cell–

MC ratios of one, two, or three resulting in theoretical probabilities

Mesenchymal Stem Cell Expansion at Benchtop-Scale

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