Developing a Continuous Bioprocessing Approach to Stromal Cell Manufacture

Abstract

To this day, the concept of continuous bioprocessing has been applied mostly to the manufacture of molecular biologics such as proteins, growth factors, and secondary metabolites with biopharmaceutical uses. The present work now sets to explore the potential application of continuous bioprocess methods to source large numbers of human adherent cells with potential therapeutic value. To this purpose, we developed a smart multifunctional surface coating capable of controlling the attachment, proliferation, and subsequent self-detachment of human corneal stromal cells. This system allowed the maintenance of cell cultures under steady-state growth conditions, where self-detaching cells were continuously replenished by the proliferation of those remaining attached. This facilitated a closed, continuous bioprocessing platform with recovery of approximately 1% of the total adherent cells per hour, a yield rate that was maintained for 1 month. Moreover, both attached and self-detached cells were shown to retain their original phenotype. Together, these results represent the proof-of-concept for a new high-throughput, high-standard, and low-cost biomanufacturing strategy with multiple potentials and important downstream applications.

Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

“…Besides describing the step-by-step procedures to isolate, culture, and propagate human CSKs, the current article also highlighted the complexity of CSK culture protocol, compared to the SF culture, which can be propagated in a simple medium that contains only DMEM/F-12 and 5% FBS [ 12 , 39 , 40 , 41 ]. In addition, this article also highlighted the morphological and protein and gene expression differences between CSKs and SFs.…”

Bioprocess Design Considerations for Cultured Meat Production With a Focus on the Expansion Bioreactor

“…With lab-scale culture it is common practice to perform enzymatic dissociation using trypsin, however trypsin is animal-derived from cows or pigs and can be subject to batch-to-batch variation (Masters and Stacey, 2007). Other methods are available such as mechanical or shear-induced dissociation (Wang et al, 2018), and controllable biomaterials (Duffy et al, 2014;Miotto et al, 2017). Non-animal derived dissociation alternatives such as the recombinant enzyme TrypLE, derived from microbial sources, or an enzyme-free cell dissociation buffer such as Versene (EDTA) to promote aggregates could also be used (Masters and Stacey, 2007;Beers et al, 2012;Daniszewski et al, 2018).…”

Scale-Up Technologies for the Manufacture of Adherent Cells

“…Importantly, a continuous system has the potential to provide several advantages compared to current batch systems such as: reduction of footprint and resources, overall lower production costs, increase product quality, reproducibility and yield over time implementing a closed and automated system. For instance, a recent paper showed proof-of-concept data suggesting that an area of just 155 cm 2 (like a medium size tissue culture flask) can generate over 1 million of cells every 24 h ( 76 ). Thus, such a small area could generate ~100 million cells when working continuously for 3 months.…”

“…More than 30 years ago, the concept of continuous bioprocessing was introduced as an alternative to batch production; in general terms it means creating a continuous process of turning flowing raw material into intermediate or final products (73). A broad range of industries have adopted continuous processes, spanning chemical and oil refineries to food and life sciences (74)(75)(76). The reason for this implementation relates to the proven benefits continuous processing brings to reducing process cycle times, materials and energy used as well as waste production (77).…”