Stem cell–based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed.Key points: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation• Single-use stirred bioreactor systems are preferred for the cultivation of both cell types• Future research should adapt and modify downstream processes to available single-use devices

The development of upstream bioprocesses necessitates small, instrumented bioreactors for investigating and optimizing production processes in a cost-effective manner. Due to advances in both the equipment and the materials used in additive manufacturing, 3D printing of customized bioreactors is now in the realm of possibilities. In this study, a small-scale 3D printed bioreactor suitable for mammalian and microbial cultivations was developed, featuring a working volume of 90 mL, inline pH and dissolved oxygen probes and a levitating magnetic stirrer. Aeration channels and a sampling port were printed directly into the vessel walls. Additionally, the vessel was equipped with a 3D printed customizable optical biomass-sensor. The bioreactor’s performance was evaluated through technical characterization and proof of concept cultivations, demonstrating that mixing time and oxygen mass transfer were sufficient for cultivating mammalian as well as microbial cells at high cell densities. Specifically, an Escherichia coli fed-batch cultivation achieved a maximum OD₆₀₀ of 204. Furthermore, a fed-batch cultivation of an IgG antibody-producing Chinese hamster ovary cell line reached a peak viable cell density of 10.2 × 10⁶ cells mL⁻¹ and a maximum product titer of 2.75 g L⁻¹. Using a three-parameter fit, the inline biomass signal could be correlated to the corresponding offline values with satisfactory accuracy, making it possible to monitor cell growth in real-time.

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies, efficient and scalable expansion procedures must be developed. For other adherent human cell types, the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study, a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings, indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( $${N}_{s1u}$$ N s 1 u ), thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics, computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner, improved process understanding allowed an expansion factor of ≈ 26 to be achieved, yielding more than 3 × 10 ⁹ cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability, identity, and differentiation potential throughout cultivation. Key points: • $${N}_{s1u}$$ N s 1 u  can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high‐resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However, achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L -scale, others, notably instrumented SU multiplate and fixed-bed bioreactors, remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods, operating ranges were identified for the Xpansion ^® 10 and Ascent™ 1 m ² bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application, almost 5 × 10 ⁹ viable cells could be produced within 5 days, achieving expansion factors of up to 35 without discernable impact on cell viability, identity, or differentiation potential. Key Points • Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion • Both the Xpansion ^® 10 multiplate and Ascent™ 1 m ² fixed-bed reactor accommodated the production of almost 5 × 10 ⁹ viable cells within 5 days • Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10 ⁻⁵ N cm ⁻² did not impair quality

Given the demands human induced pluripotent stem cell (hiPSC)-based therapeutics place on manufacturing, process intensification strategies which rapidly ensure the desired cell quality and quantity should be considered. Within the context of antibody and vaccine manufacturing, one-step inoculation has emerged as an effective strategy for intensifying the upstream process. This study therefore evaluated whether this approach could be applied to the expansion of hiPSCs in flasks under static and in microcarrier-operated stirred bioreactors under dynamic conditions. Our findings demonstrated that high density working cell banks containing hiPSCs at concentrations of up to 100 × 10⁶ cells mL⁻¹ in CryoStor^® CS10 did not impair cell growth and quality upon thawing. Furthermore, while cell distribution, growth, and viability were comparable to routinely passaged hiPSCs, those subjected to one-step inoculation and expansion on microcarriers under stirred conditions were characterized by improved attachment efficiency (≈50%) following the first day of cultivation. Accordingly, the process development outlined in this study establishes the foundation for the implementation of this intensified approach at L-scale.