In our recent work in different bioreactors up to 2.5 L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, NJS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at NJS and detachment in-situ in 15 mL ambr™ bioreactors, 100 mL spinner flasks and 250 mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.
Abstract BACKGROUND Traditional large‐scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface‐to‐volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two‐phase system for expansion and non‐enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein‐rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non‐enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION The prospect of a temporary microcarrier that can be used to expand cells and then ‘disappear’ for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality.
Lab-grown meat, also known as cultivated meat, is an emerging novel food with the potential to address some of the current global challenges, while providing a sustainable food option. It is real meat with the same structure, composition, and taste, but it doesn't require animal slaughter and is produced in a controlled manner. Due to its complex structure with multiple cell types, the production of cultivated meat is challenging. Commercial availability and affordability of these novel food products will be determined by the designed manufacturing processes. This chapter discusses the different elements required to produce cultivated meat such as suitable cell sources, growth media, scaffolds and biomaterials. Different manufacturing routes and emerging technologies such as cell encapsulation in hydrogels or novel bioreactor designs with applicability in the production of cultivated meat will also be identified and discussed. The engineering and biological challenges will be discussed in the context of lessons to be learnt from other areas such as advanced therapeutics manufacturing.
Abstract As more and more cell and gene therapies are being developed and with the increasing number of regulatory approvals being obtained, there is an emerging and pressing need for industrial translation. Process efficiency, associated cost drivers and regulatory requirements are issues that need to be addressed before industrialisation of cell and gene therapies can be established. Automation has the potential to address these issues and pave the way towards commercialisation and mass production as it has been the case for ‘classical’ production industries. This review provides an insight into how automation can help address the manufacturing issues arising from the development of large-scale manufacturing processes for modern cell and gene therapy. The existing automated technologies with applicability in cell and gene therapy manufacturing are summarized and evaluated here.
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