Page 469 - IJB-9-2

International Journal of Bioprinting Bioprinting of exosomes

are imposed on biological culture fluids to separate EXOs, nanoparticle tracking analysis, visualization of particles by
remains the gold standard for isolation and concentration electron microscopy to determine the size and structure,
of EXOs [23,90] . However, this technique is not amenable for quantification of total protein content via bicinchoninic
scaled-up manufacturing processes, as it leads to EXO acid assay, analysis of proteome by mass spectrometry,
aggregation and is often characterized by low EXO yield and identification of specific positive and negative
and purity [23,90] . In contrast, tangential flow filtration (TFF), exosomal surface markers using immunoblotting and flow
which involves the use of a permeable membrane filter and cytometry techniques [14,90] . In addition, engineered EXOs
tangential fluid flow to separate and purify biomolecules need to be assessed quantitatively and qualitatively to
of specific sizes, shows potential for large-scale EXO characterize exogenously loaded therapeutic cargo via mass
production, as it has demonstrated consistent production spectrometry, and their potency must be evaluated using
between batches and an improvement in quality, but relevant functional assays . Furthermore, other exosomal
[14]
more importantly, it is 100-fold more efficient in isolating components that originate from producer cells, such as
EXOs compared to standard UC [91-93] . Size-exclusion nucleic acids, proteins, and lipids, should be thoroughly
chromatography (SEC) is another frequently employed investigated to understand and prevent unwarranted issues
technique used for EXO purification. It addresses associated with immunogenicity, genotoxicity, and/or
limitations associated with UC, including the elimination carcinogenicity . The standardization of these methods
[90]
of protein or cell debris contamination and the prevention is paramount for establishing safety and efficacy profiles
of EXO aggregation. It is also a viable option for large-scale that are critical for the successful clinical development
EXO separation and purification . While UC, TFF, and and translation of produced EXOs. Hence, the quality
[90]
SEC isolate and purify EXOs based on size and/or density, control and acceptance criteria used to assess the quality
these techniques do not inherently possess the ability to and consistency of EXO production should be inherently
purify specific EXO subpopulations or engineered EXOs based on identity, purity, safety, and efficacy of therapeutic
loaded with therapeutic factors . Hence, immune capture EXOs .
[90]
[23]
approaches, such as affinity chromatography that is Finally, there is also a lack of understanding on
suitable for up-scaled production of EXOs, can be availed the impact of storage conditions on EXO stability and
for purification of EXO subpopulations or engineered bioactivity . This understanding is imperative to discern
[83]
EXOs [94,95] . However, it should be highlighted that the the effects of storage-mediated changes on EXO size,
elution of intact EXOs is a challenge for chromatography- number, cargo profiles, cellular uptake behavior, and
based purification methods. No single EXO isolation/ bioactivity because these attributes inherently define the
purification step has been proven efficient; hence, a therapeutic attributes of produced EXOs. As storage at
combination of different isolation/purification techniques 4°C affects the biological activity and protein content of
has been employed . For instance, a combination of TFF EXOs , the current consensus for EXO storage appears
[90]
[98]
and bind-elute SEC protocols has been demonstrated to be to be -80°C [14,23,99] . However, as different EXO types or
more effective in purifying EXOs from C2C12 myoblast subpopulations may demand different storage conditions,
cultures compared to a single-step purification technique . it becomes crucial to optimize the storage conditions for
[96]
Along similar lines, the TFF-SEC combination method each EXO-based therapeutic . Furthermore, factors such
[23]
was shown to be efficient in the isolation of EXOs from as the constituents and pH of storage buffer, number of
urine . More notably, clinical trials employing therapeutic freeze-thaw cycles, and storage container material also
[97]
EXOs routinely utilize a combination of TFF with UC play crucial roles, as they may alter the characteristics of
purification protocols . Collectively, these observations therapeutic EXOs [100] . Recent studies have demonstrated
[23]
suggest that careful consideration is required for choosing that the addition of cryoprotectants in EXO storage
the ideal combination of protocols for the optimal isolation formulations, such as the Food and Drug Administration
and purification of various therapeutic EXOs. (FDA)-approved excipient trehalose, significantly
Furthermore, there is a dearth of high-throughput improves the stability of EXOs [101,102] . Another promising
methodologies to accurately assess EXOs quantitatively approach is the lyophilization of EXOs, which has been
and qualitatively, so as to determine their purity, dosage, shown to increase the stability and the shelf life of freeze-
[99,103]
and potency. Nevertheless, rapid advances are being made dried EXOs .
with the advent of novel technologies that could accelerate From the regulatory point of view, therapeutic EXOs
the clinical translation of exosome-based therapeutics. need to be manufactured in a current good manufacturing
Conventional characterization studies that evaluate practice (cGMP) facility in accordance with the adhered
the quality of produced EXOs typically include the regulations for manufacturing traditional biologics, such
determination of particle quantity and concentration using as recombinant proteins and antibodies . One important
[90]

Volume 9 Issue 2 (2023) 461 https://doi.org/10.18063/ijb.690