Physicians and scientists have been researching and developing clinical therapeutics using stem cells for the last half century. The clinical results of these stem cell treatments, many of which have no good solution within the framework of traditional medicine, have been remarkable. Pre-clinical and clinical research in this field has involved many different types of cells including embryonic stem cells, mesenchymal stem cells (MSCs), induced pluri-potential stem cells and other tissue-specific progenitor cells. Some of the most promising clinical results have been observed with therapies that employ mesenchymal stem cells, the youngest precursor cells of this connective tissue lineage, because of their innate ability to support the development of connective tissues such as skin, hair, bone, muscle and cartilage.
The evolution of cell-based therapy has seen a significant discovery, in which the beneficial clinical effects of cell therapy is not dependent on the stem cells themselves, but rather on the paracrine effect of their exosomes. Exosomes are nano-scale extracellular vesicles, extremely small membrane-enclosed vessels that contain proteins and RNA, which carry the inter-cellular, biologic message of the parent cell to target cells to influence their behavior. In the case of perinatal MSC exosomes, that message is the same message that supports the growth of a developing fetus. The natural function of these exosomes is to support development of the fetus, by reducing inflammation in utero, preventing rejection of the fetus by the maternal immune system, promoting vasculogenesis, stimulating cell growth and division and suppressing oncogenesis.
The scientific and medical community has realized over the past few years that there is tremendous therapeutic potential for the use of MSC exosomes to treat any number of autoimmune, inflammatory, degenerative, metabolic and malignant conditions, as well as traumatic injuries. Kimera Labs has realized this potential and has developed isolated MSC exosome products for use in clinical investigation. These products are unlike any other commercially available exosome products for many reasons. Kimera Labs products include sterile suspensions of isolated MSC exosomes derived from isolated, perinatal, mesenchymal stem cells.
In order to produce a purified, perinatal MSC exosome product, MSCs have to be selectively cultured to isolate only these cells. They subsequently produce exosomes, which are sterile-filtered from the conditioned media to remove any cells or other particulate matter of the same size as cells. Kimera exosomes then go through a proprietary, serial purification process that removes any particulate material and components of the culture medium leaving purified MSC exosomes, which are then suspended in sterile saline. Each lot of Kimera MSC exosomes begins anew with a new mesenchymal stem cell culture every 4 weeks.
Ensuring that the Kimera product is a pure MSC exosome requires characterization that extends way beyond protein and RNA assays and Nano Tracking Analysis (NTA) which indicates particle counts. These studies measure some relevant qualities of the biologic material that is assayed, but it does not confirm that the suspension is truly an MSC exosome. Identification of MSC exosomes requires advanced imaging modalities such as STORM super resolution fluorescence microscopy, which is capable of visualizing not only the exosome morphology, but more importantly the individual proteins that define the extracellular vesicle as an MSC exosome, such as CD 9, 63 and 81. By visualizing these specific proteins on these spheroid vesicles with this type of imaging, Kimera Labs is able to confirm the origin of these exosomes, and that they are in fact, MSC exosomes.
Realizing the profound potential therapeutic applications of MSC exosomes and seeing the remarkable progress Kimera Labs has made in exosome development, others have tried to manufacture, brand and market their own ‘MSC exosome’ products derived from adult bone marrow or placental tissue. Not understanding the science of exosomes, these exosome producers continue to promote the fallacy that they have superior MSC exosomes based on particle counts and inadequate protein assays. It is quite evident to the post-doctoral scientists at Kimera Labs and physicians well-versed in exosome technology, that these are not only inferior ‘exosome’ products, but also products that have a significant risk of contamination and potential for serious adverse effects.
Since Kimera Labs initiated the paradigm of characterizing its purified MSC exosome products with NTA particle counts (Fig. 1), others have followed that mode of characterizing their own products. Unfortunately, there may be a significant amount of particulate biologic material in these products that is somewhat similar in size to MSC exosomes. NTA erroneously counts these biologic particles as exosomes, yielding falsely elevated numbers. Kimera exosomes are derived from cell cultures, not tissue processing, so when the conditioned media is sterile-filtered there is little to no particulate material in the same size range as MSC exosomes, even before the serial purification process.
Multi-modality characterization of MSC exosomes intended for use in clinical research and future clinical applications ensures the purity and consistency of each production line. Confirmation of the quality of Kimera Labs exosomes utilizes advanced exosome characterization technologies such as ELISA protein analysis, high pressure liquid chromatography (HPLC), RNA sequencing, NanoSight Nano Tracking Analysis, Atomic Force Microscopy, Electron Microscopy and STORM super resolution fluorescence microscopy. Kimera Labs strict production and characterization requirements have been implemented to ensure consistent production of its exosome products including pharmaceutical-grade MSC exosomes.
Fig 2. Nano Tracking Analysis of ‘MSC exosomes’ indicating mutliple, inconsistent size distributions with multiple peaks between 50 and 600 nm, indicative of inconsistent samples and particulate debris, that does not represent MSC exosomes.
The most significant problem with the tissue-based products being marketed as ‘MSC exosomes’ is not its inferior quality, but rather its significant potential for causing adverse effects in potential clinical applications. Serious MSC exosome researchers have reported that even the presence of chromatin, which may adhere to the exosome membrane, has caused severe inflammatory responses in animal models. The type of particulate contamination reported on others’ NTA as ‘MSC exosomes’ may actually be other biologic material such as A and B blood-type antibodies, which could cause acute hemolytic reactions that can be life threatening.
Fig. 3. NTA (left) detects nano-particles based on light scattering. Super resolution fluorescence microscopy detects specific molecules, in this case CD 63 and 81 proteins, indicative of an MSC exosome.
In order to guarantee a high-quality, purified MSC exosome product, Kimera Labs uses STORM super resolution fluorescence microscopy to directly visualize the characteristic proteins found on an MSC exosome. This imaging modality confirms that the particles visualized on NTA of Kimera exosomes are MSC exosomes, not billions of particles of debris visualized on NTA and erroneously reported as MSC exosomes. It also confirms that the protein and RNA identified on ELISA or RNA sequencing assays are actually contained within the exosomes, not just floating in the suspension where they may be ultimately degraded by proteases and RNAses.