Application-Oriented Bulk Cryopreservation of Human iPSCs in Cryo Bags Followed by Direct Inoculation in Scalable Suspension Bioreactors for Expansion and Neural Differentiation

Abstract

Stem cell-based therapies are promising tools for regenerative medicine and require bulk numbers of high-quality cells. Currently, cells are produced on demand and have a limited shelf-life as conventional cryopreservation is primarily designed for stock keeping. We present a study on bulk cryopreservation of the human iPSC lines UKKi011-A and BIONi010-C-41. By increasing cell concentration and volume, compared to conventional cryopreservation routines in cryo vials, one billion cells were frozen in 50 mL cryo bags. Upon thawing, the cells were immediately seeded in scalable suspension-based bioreactors for expansion to assess the stemness maintenance and for neural differentiation to assess their differentiation potential on the gene and protein levels. Both the conventional and bulk cryo approach show comparative results regarding viability and aggregation upon thawing and bioreactor inoculation. Reduced performance compared to the non-frozen control was compensated within 3 days regarding biomass yield. Stemness was maintained upon thawing in expansion. In neural differentiation, a delay of the neural marker expression on day 4 was compensated at day 9. We conclude that cryopreservation in cryo bags, using high cell concentrations and volumes, does not alter the cells’ fate and is a suitable technology to avoid pre-cultivation and enable time- and cost-efficient therapeutic approaches with bulk cell numbers.

Process Intensification in Human Pluripotent Stem Cell Expansion with Microcarriers

“…The hiPSCs, frozen at up to five-fold higher viable cell densities (VCDs) than described by other authors [11,16], were characterized under static cultivation conditions for attachment, growth, viability, morphology and quality following replating. The results of the study clearly indicated a freezing density dependent impact on initial attachment efficiency, especially for VCDs ≥ 5 × 10 6 cells mL −1 (Figure 2a).…”

“…This led to both fewer and more isolated colonies following the removal of RI, confirming the observations made regarding attachment efficiency. Considering that the impact of cryopreservation on the biological function of pluripotent stem cells at VCDs of up to 25 × 10 6 cells mL −1 is well documented [11,16,30], the observed initial disparities in attachment and growth were deemed unsurprising.…”

“…Recent studies with hiPSCs have suggested that non-uniform cell-cell aggregation or spheroid formation may lead to poor cell quality and impede efficient cultivation, especially when spheroid diameters exceed 400 µm [9,36,38]. To improve process homogeneity, Meiser et al [16] proposed using OSI, as this was shown to restrict cell aggregation within the first day of cultivation and led to more uniform spheroid size distributions. To observe whether similar effects could be observed when working with MCs, microscopic monitoring of the stirred cultivations was performed over a 5 d period (Figure A3).…”

“…Intensified production processes, which primarily serve to shorten process time and reduce the cost of manufacturing [13], have increasingly become the focus of antibody and vaccine manufacturers during the last 10 years. Within this context, one-step inoculation (OSI) has emerged as an effective intensification strategy, alongside the application of cells from high cell density [12,14,15] or large volume cell banks [14,16]. Also referred to as a frozen accelerated seed train [14], OSI describes the direct inoculation of the N-1 bioreactor Also referred to as a frozen accelerated seed train [14], OSI describes the direct inocula of the N-1 bioreactor using cryopreserved cells, thereby offering numerous advantag demonstrated by various working groups [12,[17][18][19][20].…”

“…These advantages includ reduction of expansion steps and the associated risk of contamination, an improvem in inoculum quality, and enhanced process reproducibility. When applied to expansion of hiPSCs as spheroids, Huang et al [11] confirmed that OSI could be use shorten the duration seed train without impacting cell quality under stirred condit Meiser et al [16] later observed that the OSI of hiPSCs led to a ≈15% reduction in aggregation and more uniform spheroid size after 24 h when using the orbitally sha CERO 3D bioreactor, allowing for 𝐸𝐹 of between 6-8 to be achieved within 3 d.…”