Extracellular Vesicles (EVs)/Exosomes from MSCs are promising preclinical therapeutics. [1, 2, 3] RoosterBio’s goal is to make extracellular vesicles widely available for clinical trial efforts, accelerating the path to the clinic while reducing the friction to devise a scalable, fit-for-purpose biomanufacturing process. We now have developed robust upstream production systems [4] that can meet clinical demand, [5] coupled with downstream isolation methods [6] to purify and formulate MSC-EVs at great concentrations.

To fully harness their potential as a groundbreaking therapeutic modality, MSC-EVs need to integrate within a conventional Chemistry, Manufacturing, and Controls (CMC) paradigm, as regulatory agencies like the FDA require rigorous CMC sections for drug filings. [7] RoosterBio’s first step in this effort is to use current analytical tools to understand MSC-EV quality attributes and their variation across production processes. MSC-derived EVs are believed to retain many potentially therapeutic attributes from their MSC progenitors. However, variability in MSC function, which is influenced by factors such as tissue origin and donor-specific characteristics, [8, 9] can be reflected in the properties of the EVs they release. Therefore, a comprehensive understanding of the differences in EV quality attributes [10] arising from distinct tissue sources and individual donors is crucial to optimize the selection of starting materials for extracellular vesicle production, thereby ensuring consistency and efficacy in therapeutic applications.

Figure 1. A standardized RoosterBio process for upstream MSC-EV production.

Towards this end, we generated MSC-EVs in conditioned medium from a consistent but modifiable upstream workflow (Figure 1) with varied important parameters that must be considered by pioneering developers of MSC-EV therapies: tissue source, donor, production platform, and collection day (Table 1). We then used a panel of our most robust analytical assays for MSC-EVs (Table 2), all of which are available as part of our EV/Exosome analytical services program.

The Basics: Platforms & Particles

Extracellular particles accumulate from collection Day 2 to Day 5 (Figure 2A), as measured by nanoparticle tracking analysis (NTA). Therefore, a later collection day leads to greater particle number via MSC-EV harvests up until a maximum on Day 5. [4] This observation was consistent across all tested platforms. We additionally noticed a massive difference between production in 3D and 2D. When normalized to the cell number between these culture formats, this difference represents a >10-fold increase in particle production for 3D cultures versus 2D. An order of magnitude increase is truly transformative and further underscores that 3D platforms are key to MSC-EV therapeutic translation.

For tested 3D platforms, key upstream parameters such as microcarrier type, microcarrier concentration and seeding density remained constant. Therefore, a general similarity in particles per mL between scalable platforms indicates that the process is less sensitive to the platform of choice. This is good news, because it means that investigators can make practical decisions about 3D platforms while maintaining particle productivity. From here on, we used the stirred tank as the representative platform to generate additional results.

Although particle size also did not vary by platform used, we did find that particle size varied across different tissue sources (Figure 2B, 2C). Particles from umbilical cord (UC) MSCs are relatively larger (median 173 nm) than bone marrow (BM)-derived particles (median 158 nm), while adipose (AD)-derived particles are the smallest (median 143 nm). These results were our first clue that MSC source may impact extracellular vesicle properties.

Figure 2. (A) Bars represent mean and error bars ±SD for N=3 assay replicates. (B) Size distribution data are analyzed from N=3 measurements each. (C) Line represents the median, box represents the 25th-75th percentile, and whiskers represent the 5th-95th percentile. BM = bone marrow, UC = umbilical cord, AD = adipose tissue.

Sifting Through MSC Secretomes – Medium But Not Ordinary

This study generated conditioned medium from cells, which contains not only extracellular vesicles but also other cell-secreted biological content. The first step to understanding EV-specific content is to understand the total content in conditioned medium.

Protein content in samples ranged from 75-200 ug/mL for all tested conditions. Normalizing the extracellular particle number to the protein content as particles per protein reveals remarkable consistency across all samples (Figure 3A), including different tissue sources. This result suggests that both secreted particles and protein contents are highly correlated and conserved across production conditions. In contrast to protein results, the total RNA content did vary across tissue type. Furthermore, total RNA content did not increase substantially with collection day (Figure 3B), indicating that perhaps free RNA secreted by cells becomes degraded over time in culture.

The particles per protein metric is conventionally used to assess EV purity, since purification of EVs involves their separation from other proteins in the conditioned medium. As EVs are purified, the particles per protein increases significantly, [11] up to >1E11 P/mg. [12] Eventually, we would like to understand how biological content within EVs differs across tested parameters. Purifying EVs using downstream processing methods such as those established at RoosterBio would provide a better idea of EV-specific cargo compared to total biological content. An analytical comparison of purified EVs versus conditioned medium will be explored further in a future blog post.

Figure 3. (A) Bars represent mean and error bars ±SD for N=3 assay replicates. (B) Bars represent mean and error bars ±SD for N>2 assay replicates.

Figure 4. BM = bone marrow, UC = umbilical cord, AD = adipose tissue. ‘D’ indicates collection day, where D2 = Day 2, D5 = Day 5. The vertical axis represents protein size, where kDa = kilodaltons.

Unlocking Extracellular Vesicle Cargoes for Protein & miRNA Signatures

Extracellular vesicles are conventionally defined by their nano size, a lipid membrane, and the presence of markers established by the broader field. Presence of the tetraspanins CD81, CD9, and CD63 are routinely used as EV surface markers, while TSG101 is an intraluminal EV marker. [13] CD73 is an MSC marker that we believe is a critical component of MSC-EV function. [14] Measuring the presence of these markers is, therefore, crucial to verify that the extracellular particles measured by NTA are likely MSC-EVs. To do this, we employ a capillary-based automated western blot (ProteinSimple Jess, Bio-Techne) to detect and verify the presence and size of each marker. In this study, all examined markers were detected, confirming the presence of MSC-EVs across all tested conditions. A sample set of results are presented in Figure 4, showing that marker presence appears consistently across different tissue sources and collection days.

To begin testing for internal extracellular vesicle content, we looked for specific microRNA sequences inside EVs by using a quantitative polymerase chain reaction (qPCR). This assay was developed to look for relevant MSC-EV microRNA species using an established reference microRNA (miR-16-5p). [15] For this assay, RNA external to EVs is degraded and EVs are isolated using ultracentrifugation. Therefore, results approximate the internal contents of EVs. All tested microRNA sequences were detected in all samples, and relative expression showed some differences in terms of both overall expression and expression across tissue types (Figure 5). These results and others highlight the importance of screening MSC-EV sources for microRNA content of interest, as microRNA contents in MSC-EVs likely vary depending on upstream conditions.

Figure 5. Bars represent mean and error bars ±SD for N=3 assay replicates. Data are normalized by the conventional ΔCt method. BM = bone marrow, UC = umbilical cord, AD = adipose tissue.

A Matter of Source: The Importance of Tissue & Donor

MSC therapeutic potential is greatly influenced by tissue source and donor, and so extracellular vesicles derived from MSCs can also show variability based on tissue sources and donors. Specifically, we observed differences in particle number and CD73 activity from different donors of bone marrow, umbilical cord and adipose derived MSCs. CD73 activity is a bioactivity/potency assay that measures the activity of MSC-EVs to convert ADP to adenosine. To learn more about our CD73 activity assay, please refer to our recently published blog. [14]

Figure 6. Bars represent mean and error bars ±SD for N=3 assay replicates. BM = bone marrow, UC = umbilical cord, AD = adipose tissue.

Donors from different tissue sources were selected to expand MSCs and collect EVs in stirred tank bioreactor systems (Table 1). CD73 activity was measured on Day 2 and Day 5 of EV collection and results showed that activity either remains stable or decreases with time (Figure 6A). This suggests that CD73 activity was least for EVs derived from adipose tissue (~1000 pmol/min per 1E9 particles) and remained stable over the collection phase. CD73 activity ranged from ~1000 to ~5000 pmol/min per 1E9 particles in EVs derived from umbilical cord. Finally, bone marrow-derived EVs showed CD73 activity in between EVs from adipose and umbilical cord. Because most variation in these results is explained by donor variation, we are hesitant to conclude that tissue source determines MSC-EV CD73 activity and thus recommend donor screening if CD73 activity is important for therapeutic applications. The relevance of changes in CD73 activity remains to be identified and is likely dependent on the intended EV therapeutic application. However, given that CD73 is a canonical MSC marker, it is reasonable to select CD73 activity as a specific functional attribute for MSC-EVs.

Similarly, we observed variation in extracellular particle number produced per cell across different tissue sources and donors (Figure 6B), ranging from 1×10⁴ to 4×10⁴ particles per cell, with bone marrow derived EVs the greatest on collection Day 5. Particle count is a major criterion when collecting EVs to understand where one can maximize productivity, and so these results help guide in selecting the tissue source and donor for EV generation depending on the criteria chosen and the relevant output. Although we focused on observed differences in these parameters, our platforms make it possible to test differences in other attributes between tissue sources or donors.

Acceleration Towards MSC-EV Characterization

Overall, this comprehensive study evaluated conditioned medium for MSC-EV quality attributes among different scalable production platforms using a standardized cell source and media system.

We found that extracellular particle number and size were generally consistent across platforms, demonstrating process robustness independent of platform used. Furthermore, extracellular particles per protein in conditioned medium was conserved across conditions, though RNA content varied and did not increase over time. Detection of tetraspanins, TSG101 and CD73 demonstrated the presence of MSC-EVs in all tested samples. We began to probe the internal contents of MSC-EVs by successfully measuring microRNA presence using qPCR.

Importantly, we found that some MSC-EV quality attributes varied by MSC source. This was first indicated by different EV size distributions from different MSC tissue sources. Testing different donors from the same tissue sources revealed donor-dependent differences in extracellular particle production. In a functional assay, MSC-EVs from different donors showed varying levels of CD73 enzymatic function. These results highlight the importance of screening donors for MSC-EV function of interest, as demonstrated in this study, these results can vary depending on the MSC donor used to generate MSC-EVs.

Ultimately, characterization of MSC-EVs in a CMC paradigm will be required for their translation as a therapeutic. RoosterBio continues to contribute to this effort by developing rigorous analytical methods that can fit into regulatory framework. The work presented in this blog uses these methods to help establish a foundation for selecting clinical manufacturing processes for extracellular vesicle production. Learn more by finding us at upcoming international meetings (including for ISCT and ISEV) where the work will be presented in more detail.

 

References

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11. Jadhav, S.H., Patel, G., Gupta, P. Dehgani, M., Staubach, S., Teshome, B., Jung, J., Lenzini, S., Garland, C., Boychyn, M., Rowley, J.A., Zakhem, E. Development of an End-to-End Scalable Purification Platform for Extracellular Vesicles. RoosterBio & Sartorius Poster Presented at ISCT 2023; Available from: https://tinyurl.com/yh28hk2y.
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