Today, the therapeutic use of extracellular vesicles (EVs) typically involves human mesenchymal stromal cells (hMSCs). As more is learned, though, scientists are starting to think that hMSCs may be replaced by EVs interacting with the cell secretome. As such, they function like protocells but are less immunogenic, making them better candidates than cell therapy for certain applications, like fibrosis treatment.
Before that can happen, EVs need a scalable manufacturing system.
Adapting immortalized hMSCs to suspension culture “solves two main problems associated with EV manufacturing,” Qasim Rafiq, PhD, professor of bioprocess engineering and vice dean (health) in the faculty of engineering sciences, University College London (UCL), tells GEN. “Immortalization ensures consistency across batches, and the suspension method increases overall process productivity by manufacturing a higher amount of EVs on a volumetric basis.
“If proven clinically relevant,” Rafiq says, “this cell line and its respective EVs have the potential to completely change the fundamental approach in the vast majority of clinical trials relying on donor-derived, adherent hMSCs.”
The human telomerase reverse transcriptase (hTERT) immortalized human mesenchymal stromal cells (hMSCs) Rafiq and colleagues developed, called suspension hMSCs (S-hMSCs), “eliminate the need for microcarriers or other matrices to support adherent cell growth,” they report.
In this study, S-hMSCs doubled in about 55 hours. The cells retained approximately 90 percent of the CD73 and CD105 expression levels, and, “the CD90 receptor [was] downregulated during the suspension adaptation process.” Meanwhile, the transcripts coding for CD44, CD46, and CD47 were upregulated compared to those in adipose tissue-derived-hMSCs and hTERT-hMSCs.
The S-hMSCs generated EVs that averaged 150nm in size and bore the markers CD63, CD81, and TSG101. The negative marker, calnexin, was not expressed.
A 22-step transition
In the lab, the team gradually transitioned the cells from a static culture to a suspension culture. It took 22 steps, followed by 150 days of suspension.
“Perfusion mimic experiments led to a maximum cell yield reaching 8×106 cells/mL,” which the scientists call a “significant improvement over microcarrier-based studies using primary adherent hMSCs.”
While less than the yields of hollow-fiber or other bioreactor studies, this yield is five-fold greater than those of typical stirred-tank reactors. Thus, as Rafiq points out, “The suspension nature of the novel S-hMSCs allows for large-scale manufacturing in stirred tanks, making it feasible to produce several thousand liters.”
The scientists plan to increase cell and EV concentrations that, when combined with downstream process optimizations, will enable industrial-scale EV manufacturing. The group, he says, is “working with clinical partners now to demonstrate the clinical utility of the EVs.
“S-hMSCs represent the first step towards reducing the manufacturing costs associated with EV products,” Rafiq continues. “This cell line enables studies focused on bioprocess optimization, including pH, dissolved oxygen, and feeding strategies, to maximize EV production per batch.”
