Exosomes: Native vs Manufactured

Exosomes have, in a short period, become the focus of much attention in regenerative medicine. Of particular interest are exosomes that are derived from mesenchymal stromal cells. So what are exosomes? Exosomes are tiny, membrane-bound extracellular vesicles that have a unique generation pathway and are released from and taken up by most cells.(1) They are often so small that they are difficult to quantify by traditional methods. Therefore, either specialized equipment or ELISA assays are necessary for the quantification of exosome numbers in a product.

The contents of an exosome vary by the type and environment of the particular cell that produces it, but each exosome can typically carry a vast array of proteins, microRNA, mRNA, DNA, lipids, and peptides.(2) With this impressive cargo and its ability to transfer information, the use of exosomes as a potential cellular therapy is significant. However, there are challenges in the production and selection of exosomes.

Manufactured Exosomes

Large-scale production of exosomes is difficult and can alter certain cell behavior and characteristics.(2) In order to mass-produce exosomes, cells are plated on a tissue culture dish and grown. The cell culture media is removed and exosomes are isolated from the media. Exosomes manufactured in this manner have no specificity, the exosomes are being produced as a result of the environment they are in. The cells that are being grown in the artificial environment are problematic and the exosomes produced due to this artificial environment have the potential of having no therapeutic benefit.(3)

Exosomes from native tissue are more specific in their functionality and purpose

Wharton’s jelly of the umbilical cord and other birth tissues contain naturally produced exosomes. One function of the birth tissues is to provide a suitable environment for the developing fetus and prevent rejection of that fetus by the mother. Exosomes found in these tissues would contain cytokines, mRNA, miRNA, and DNA that would be specific to this function.

Exosomes are not just products of cell cultures but are abundantly present in our bodies. It is important to understand that the molecular mechanism involved in exosome production and distribution has not been fully understood and will take time to decipher.

1. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles and friends. J. Cell Biol. 200(4), 373–383 (2013).

2. Whitford W, Guterstam P. Exosome manufacturing status. Future Med Chem. 2019 May;11(10):1225-1236. doi: 10.4155/fmc-2018-0417. PMID: 31280675.

3. Brindley D, Moorthy K, Lee JH, Mason C, Kim HW, Wall I. Bioprocess forces and their impact on cell behavior: implications for bone regeneration therapy. J. Tissue Eng. 2011, 620247 (2011).

Keep It Cool

Cryopreservation is often been a topic of interest in science fiction where subjects are preserved and revived at a later time. As far-fetched as some of these scenarios may be, the concept of lowering the temperature of living tissues, such as cells, into a point of suspended animation is not only possible but has been the primary means for long-term preservation of tissues and cells.1

In order to maintain the viability of the donor tissue, it cannot be stored with simple cooling or freezing techniques for long periods of time due to the formation of ice crystals, osmotic shock, and membrane damage that occurs during the freezing and thawing process.2 Cells and tissues must be maintained at -136°C or colder to be optimally preserved with their viability intact for very long periods of time. There are a few cells and tissue types that can be stored at higher temperatures around -80°C such as amnion tissue, bone and cartilage. However, in order to preserve the naturally occurring factors found in Wharton’s Jelly, we align with scientific and research standards that specify the use of liquid nitrogen and cryoprotectants as an absolute necessity.3,4,5,6,7,8

Although the temperature in which human cell and tissue products are preserved is important, the methodology deployed in the freezing process is what truly determines the viability of the tissue’s characteristics. If cells or tissues are cooled down too slowly or quickly, various mechanical stresses, such as ice crystals or dehydration, in the cells or tissues can occur which will negatively affect clinical outcomes. The same goes for the use of proper cryoprotectant media, if you do not use the proper amount it can be ineffective or even harm the cells or tissue to be preserved.2,8

Our proprietary cryopreservation process was developed after years of experience using mammalian cells and tissues. The viability of our cryopreserved products has been verified in numerous in-house quality control checks, third-party laboratories, and by peers within the industry.

Our commitment to quality and safety go hand in hand with our endeavor to protect and preserve the products that we develop.

1. Pegg D.E. “Principles of cryopreservation”. Methods Mol Biol. 2007;368:39–57.
2. Karlsson J.O., Toner M. “Long-term storage of tissues by cryopreservation: critical issues”. Biomaterials. 1996;17:243–256.
3. Miyahmoto, T., Ikeuchi, M., Noguchi, H., Hayashi, S., “Long-term Cryopreservation of Human and other Mammalian cells at -80C for 8 years” Cell Medicine Volume 10: 1-7, 2018
4. Hernández-Tapia LG, Fohlerová Z, Žídek J, Alvarez-Perez MA, Čelko L, Kaiser J, Montufar EB. “Effects of Cryopreservation on Cell Metabolic Activity and Function of Biofabricated Structures Laden with Osteoblasts”. Materials (Basel). 2020 Apr 22;13(8):1966.
5. Brockbank, K. G. M. “Essentials of cryobiology.” In Principles of Autologous, Allogeneic, and Cryopreserved Venous Transplantation. (Ed. K. G. M. Brockbank), RG Landes Company, Austin, TX (Medical Intelligence Unit Series) Springer-Verlag, 91-102, 1995.
6. Nishiyama, Y., Iwanami, A., Kohyama, J., Itakura, G., Kawabata, S., Sugai, K., Nishimura, S., Kashiwagi, R., Yasutake, K., Isoda, M., Matsumoto, M., Nakamura, M. and Okano, H. “Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and progenitor cells by a programmed freezer with a magnetic field.” Neuroscience Research Volume 107, June 2016, Pages 20-29
7. K. Imaizumi, N. Nishishita, M. Muramatsu, T. Yamamoto, C. Takenaka, S. Kawamata, K. Kobayashi, S. Nishikawa, T. Akuta “A simple and highly effective method for slow-freezing human pluripotent
8. Nover, Adam B et al. “Long-term storage and preservation of tissue engineered articular cartilage.” Journal of orthopaedic research : official publication of the Orthopaedic Research Society vol. 34,1 (2016): 141-8. doi:10.1002/jor.23034

Standards

The ethos of “do no harm” and then “do good” is a principle that we have ingrained into our minds and hearts. The safety of our clients is the most important aspect of our products and we achieve this by following quality standards and procedures that are higher than current industry standards.

All of our human cell and tissue products begin with the donor. We work closely with our vendors on a daily basis and have co-developed collection protocols to reduce the degradation of the tissue and minimize contamination. Along with the techniques involved with the collection of the tissue all donors are prescreened utilizing an extensive Donor Risk Assessment Interview (DRAI) that includes social, medical, travel and familial history.

After the donor has been cleared the collected tissue is transported to our laboratory and inspected. Each shipment of tissue is visually checked for abnormalities and then temperature tested to validate that proper cold transport had been conducted. Once the tissue has been received and cleared it is then prepped for processing. The tissue is cleaned utilizing aseptic techniques and transported into our ISO 7 Cleanroom where it undergoes our proprietary tissue processing without the use of digestive enzymes.

A total of 3 samples are taken from each processed tissue
• Sample A is quantitatively examined through our in-house flow cytometry
• Sample B is sent to a third-party laboratory that tests for sterility and bacterial endotoxins
• Sample C is analyzed in-house for Particulate testing

The remaining product is then sent into a vapor phase liquid nitrogen tank where they are held in quarantine.

In order for the products to be determined to be safe for release the test results from the samples, the donor’s bloodwork and medical history are reviewed by one of our MD medical reviewers. The process from the collection to distribution is tracked and recorded at every stage.

Our stringent quality control measures help to reduce not only variation within the product, but most importantly provides safety that healthcare providers can rely on. Only 70% of the donated tissues pass our rigorous quality inspection and standards. The source of our human cell and tissue products are abundant and readily available, but it’s our expertise in the process, the science and the data that sets us apart from the rest.

Better begins with Standards.