GeneDireX Newsletter 2024-Oct
Mesenchymal stem cells (MSCs) hold great therapeutic potential for cell-based tissue engineering and regenerative medicine due to their easy isolation, multipotency, immunomodulatory properties, and secretion of mediators that support tissue remodeling.1,2 MSCs, like every other cell, release extracellular vesicles (EVs), comprised of apoptotic bodies, microvesicles, and exosomes, for cell-to-cell communication via fusion with cell membranes.3,4
Exosomes are small, nanoscale extracellular vesicles derived from endosomes. They are spherical and have a lipid bi-layered single membrane, with a diameter ranging from 50–200 nm. Exosomes act as intercellular messengers carrying various components, including nucleic acids, proteins, lipids, and metabolites that reflect their cellular origin. These components sustain exosomes’ potential therapeutic functions that mimic those of the cells they come from and contribute to developing cell-free therapy. Additionally, exosomes could be engineered as smart carriers for delivering therapeutic agents. 2,5,6
GeneDireX, Inc. was established in the United States in 2008 and has been working in the field of biotechnology for over 10 years. Due to the expansion of our product portfolio and a shift in production focus, we are now located in Taiwan. One of our main development projects is cell culture media. Our cell culture media are carefully concocted to support the growth and maintenance of a wide variety of cell types. We provide a variety of basal media, specialty media, supplements, and custom formulations made with high-quality materials and manufacturing processes to meet the specific requirements of cell culture demands. Here, we provide two different cultural media, ADSC eXOrich and UCMSC eXOrich medium for the production of exosomes from human adipose-derived stem cells (ADSC) and umbilical cord-derived mesenchymal stem cells (UCMSC), respectively. This exosome production medium is a serum-free and ready-to-use medium. The composition is optimized for the exosomes (EVs) production.
For exosome purification, we recommend starting by preparing a conditioned medium after cell culture. The supernatant is collected from cultured cells, and large fragments of cell debris, waste products produced by cell metabolism, as well as impurities in the culture medium are removed through ultra-high-speed centrifugation or centrifugation filtration. Following this, exosomes can be concentrated using tangential flow filtration (TFF) or purified again through size exclusion chromatography (SEC). The diameter of the exosomes obtained through the above method is confirmed using nanoparticle tracking analysis (NTA), and the presence of common markers of exosomes is confirmed through protein identification.
Next, we will share our experience and results of using the ADSC eXOrich and UCMSC eXOrich medium in exosome production. We will use NTA analysis to confirm the diameter and concentration of particles and flow cytometry (FCM) combined with the cell membrane dye, FM4-64, to detect and analyze the exosome surface markers: CD9, CD63, and CD81. Generally, we recommend that MSCs be cultivated in dishes or flasks until 90% confluence under regular culture conditions, and then remove the original culture medium and wash cells with the PBS and eXOrich medium to remove the residual medium. The culture medium was then replaced with eXOrich medium only, and the cells were cultured for 3-4 days to collect the supernatant. We remove cell debris and large fragments or particles through centrifugation and filtration with using a 0.22um filter cup and concentrate exosomes using a filter cartridge, TFF-Easy (HansaBioMed).
Our results reveal that the most commonly found particle size (nm, denoted as mode) of exosomes secreted from ADSCs using alpha-MEM (Fig.1A) and IMDM (Fig.1B), and ADSC eXOrich medium (Fig.1C) is 118.9±3.1, 100.6±1.3, and 125.4±8.3, respectively. The concentration (particles/mL) of exosome (50-200 nm) secreted from ADSCs using alpha-MEM, IMDM, and ADSC eXOrich medium is 6.63×10⁸±5.35×10⁷, 7.37×10⁸±1.15×10⁷, and 2.11×10⁹±1.40×10⁸, respectively. The ADSC eXOrich medium could increase exosome production more than alpha-MEM or IMDM (Fig.1D).
Figure 1
Similar experimental data can be repeated using another culture medium, UCMSC eXOrich medium. The diameter of the mode of UCMSC-secreted exosomes using alpha-MEM is 118.4±2.9 nm, and the concentration of exosome (50-200 nm) is 2.22×10⁹±1.45×10⁸ particles/mL (Fig.2A). The diameter of the mode of UCMSC-secreted exosomes using UCMSC eXOrich medium is 114.2±2.3 nm and the concentration (50-200nm) is 4.99×10⁹±2.85×10⁸ particles/mL (Fig.2B), which is higher than alpha-MEM.
Figure 2
Furthermore, the expression of surface markers of exosome: CD9, CD63, and CD81, are analyzed using flow cytometry. The protocols have been modified according to the publication by Pospichalova V, et al. and described below.7 We utilize reference beads: NanoFCM™ Silica Nanospheres Cocktail #1, with diameters ranging from 68 to 155 nm, to set the voltages of photomultiplier tubes and the thresholds for light scattering (Fig.3A). The membrane‐specific, fluorescent amphiphilic styryl dye FM4‐64 is used to label exosomes because exosomes have lipid-bilayered single membrane structures. To obtain more accurate data, fluorescence minus one (FMO) controls were used to determine the cut-off point between background fluorescence and the fluorescence generated by positive populations within a sample of exosomes. The representative results of FCM analysis of ADSC-secreted exosome using ADSC eXOrich medium have been shown. FCM analysis revealed a raised population exhibited fluorescent signals after FM4-64 staining (Fig.3B). This population was gated to analyze the expression of CD9, CD63, and CD81. Here, we present the dot plot analysis results of CD63 versus CD9 (Fig.3C) and CD63 versus CD81 (Fig.3D).
Figure 3
According to FCM analysis data, we can verify that the exosomes produced by using ADSC eXOrich medium, express CD9, CD63, and CD81. To summarize the FCM analysis results, we have identified a population of particles exhibiting membrane structures with different diameters ranging from 68 to 155 nm at least. These particles contain CD9, CD63, and CD81, which are considered exosome markers.
In conclusion, combining NTA data with the FCM analysis results, our eXOrich medium is efficient and beneficial for producing exosomes from MSCs.
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References
- Hong P, Yang H, Wu Y, Li K, Tang Z. The functions and clinical application potential of exosomes derived from adipose mesenchymal stem cells: a comprehensive review. Stem Cell Res Ther. 2019;10(1):242. Published 2019 Aug 7. doi:10.1186/s13287-019-1358-y
- Zhang K, Cheng K. Stem cell-derived exosome versus stem cell therapy. Nat Rev Bioeng. Published online April 12, 2023. doi:10.1038/s44222-023-00064-2
- Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014;3:10.3402/jev.v3.24641. Published 2014 Aug 4. doi:10.3402/jev.v3.24641
- Ginini L, Billan S, Fridman E, Gil Z. Insight into Extracellular Vesicle-Cell Communication: From Cell Recognition to Intracellular Fate. Cells. 2022;11(9):1375. Published 2022 Apr 19. doi:10.3390/cells11091375
- Rezaie J, Feghhi M, Etemadi T. A review on exosomes application in clinical trials: perspective, questions, and challenges. Cell Commun Signal. 2022;20(1):145. Published 2022 Sep 19. doi:10.1186/s12964-022-00959-4
- Tan F, Li X, Wang Z, Li J, Shahzad K, Zheng J. Clinical applications of stem cell-derived exosomes. Signal Transduct Target Ther. 2024;9(1):17. Published 2024 Jan 12. doi:10.1038/s41392-023-01704-0
- Pospichalova V, Svoboda J, Dave Z, et al. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. J Extracell Vesicles. 2015;4:25530. Published 2015 Mar 31. doi:10.3402/jev.v4.25530
FAQ⎯Exosome production
Q1. How do we produce exosomes?
ANS: For exosome purification, we recommend starting by preparing a conditioned medium after cell culture. The supernatant is collected from cultured cells, and large fragments of cell debris, waste products produced by cell metabolism, as well as impurities in the culture medium are removed through ultra-high-speed centrifugation or centrifugation filtration. Following this, exosomes can be concentrated using tangential flow filtration (TFF) or purified again through size exclusion chromatography (SEC).
Q2. How do we enrich exosome production?
ANS: Exosomes are known as cell-secreted natural nanovesicles, and the low yield of exosome production from an MSC-conditioned medium under conventional culture conditions limits their applications. The optimal cell culture medium can enhance exosome secretion from MSCs. Therefore, we have developed two exosome enrichment media. ADSC eXOrich medium is for exosome secretion from adipose-derived stem cells and UCMSC eXOrich medium is for exosome secretion from umbilical cord mesenchymal stem cells.
Q3. Is the supplement needed?
ANS: In general, commonly used supplements like serum or human platelet lysate contain exosomes. Therefore, we do not recommend using supplements in the preparation of a conditioned medium for exosome isolation. When producing exosomes using ADSC eXOrich or UCMSC eXOrich medium, there is no need to add common nutritional supplements like serum or human platelet lysate. However, users can decide to add appropriate nutritional supplements based on their specific needs.
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