the science of

exosomes

What is an Exosome?

Exosomes are Extracellular Vesicles (EVs), but not all EVs are exosomes. They contain  biologically active cargo (proteins, nucleic acids, and growth factors) which are enclosed in  a lipid bilayer membrane. Exosomes are one of the three main subtypes of EVs. The uniqueness of exosomes is that they can initiate metabolic reprogramming. They package biologically active cargo and deliver it to a neighboring cell and alter the recipient cell’s functions with its delivery.

They are measured in nanometers (nm) or billionths of a meter and have a size range of   30 - 150nm (by comparison a hair follicle is about 50 - 90 microns which is a thousand times larger than a nanometer). The other two subtypes of EVs are micro-vesicles and apoptotic bodies with a size range of 100nm - 1000nm and 50 - 5000nm respectively. They are also differentiated from exosomes based upon their biogenesis, release pathways, content, and function.

Exosomes are secreted by virtually every cell in the body and are cell specific. Each different type of tissue secretes an exosome specific to that type of tissue (heart, liver, lung, umbilical cord, placenta, etc.). It has since been found that exosomes participate in cell–cell communication and cell maintenance with their parent cells, thus playing a role in tissue repair and regeneration.

To be a “true” exosome the lipid bilayer membrane must be intact and its cargo must be biologically active. Scientists agree that the most important part of an exosome are the proteins because the proteins are the fuel that powers the change in a cell.

Given the structure of exosomes and their critical function to deliver biologically active cargo to neighboring cells, there are 5 vital components to creating a high quality exosome product!

Protein

Protein is the key to every metabolic function in the body. Metabolism is a chemical reaction in the body’s cells that changes food into energy. Our bodies need this energy to do everything from moving to thinking, growing and healing. Specific proteins in the body control the chemical reactions of metabolism.

Age is a key factor affecting metabolic rate and protein levels in the body. That is why we can heal and repair injured tissue much faster when we are younger. 

Our skin cells continuously proliferate, going through a process of growth, repair, and turnover throughout our lives. The fuel that gives the energy for this process to occur is protein. As we age the amount of protein available to enable this process diminishes. 

In addition, procedures such as microneedling and laser procedures have a significant impact on the protein in the skin. By applying an exosome product with a high concentration of active proteins after these procedures you can significantly affect the healing and regeneration process. It is therefore extremely important for a quality exosome product to contain a high concentration of protein to aid in the skin’s natural growth and repair process.

The Pro Xo™ line of exosome products provide the highest concentration of biologically active proteins of any product available on the market. 

Source

Pro Xo™ exosomes are derived from umbilical Cord Lining Stem Cells (CLSCs). These exosomes function exclusively to repair and regenerate not only mesenchymal tissue, such as bone, cartilage, and striated muscle, but also epithelial tissue, such as skin, cornea, and liver. 

The Umbilical Cord Lining Membrane (UCLM) comprises two layers: the amniotic (or epithelial) layer and the sub-amniotic (or mesenchymal) layer. Therefore, it is a rich source of two strains of stem cells: epithelial stem cells and mesenchymal stem cells. The UCLM is the best-known source for harvesting stem cells. The umbilical cord lining can yield 6 billion epithelial and 6 billion mesenchymal stem cells from a primary explant in one generation. In comparison, bone marrow produces a few million mesenchymal stem cells per bone marrow aspiration. 

CLSCs are immune privileged, resist rejection and are multipotent. Multipotent stem cells produce exosomes that are much broader reaching for all types of tissue repair and regeneration, most importantly the skin. Virtually all other sources of stem cells produce exosomes that are specific to the parent tissue, e.g. heart, liver, lungs, etc. Also important is the innate ability of CLSCs to promote anti-apoptotic, pro-angiogenic, and anti-fibrotic activities while creating and releasing  myriads of proteins. They also exhibit immuno-modulatory and anti-inflammatory effects.

Pro Xo™ exosomes come from umbilical cord lining tissue that was ethically sourced and donated for testing, which would have otherwise been discarded. They were then rigorously tested to create a master stem cell line that is free of any chromosomal aberrations, communicable diseases, bacteria or viruses. Only a select few were determined to be perfect for exosome production and included in the master stem cell line.

Conditioned Media

Conditioned media is the liquid component that the stem cells are suspended in while producing exosomes, but more importantly, it is the ‘DIET’ of these cells. The stem cells from which Pro Xo™ exosomes are derived are fed a diet of a specifically designed conditioned media that contains Amino Acids and Vitamins creating the perfect environment to properly nourish the stem cells as they secrete the exosomes. 

Creating this conditioned media took years of rigorous research and development. A multitude of formulations were tested before the perfect combination of amino acids and vitamins was discovered. 

Pro Xo™ Premium exosomes are also suspended within the same conditioned media when packaged for use. That way they are always in the perfect environment until used on a patient.

Exosome Count

Having a “true” intact and biologically active exosome count is very important, but difficult to do since exosomes are extremely small. As a result, it is very expensive and time consuming to differentiate between EVs and ”true” exosomes for an accurate count.

Nanoparticle Tracking Analysis (NTA) has demonstrated an average of 177 billion EVs/mL of conditioned media. However, when applying the more sophisticated Fluorescent Nanoparticle Tracking Analysis (F-NTA) it was demonstrated that only an average 3.2 billion EVs/mL were membrane-enclosed particles and therefore exosomes.

Many companies simply rely on counting EVs of a certain size and claim they are all exosomes with no further analysis of the product. The Pro XoTM exosome count is determined using the most effective and sensitive methods to first isolate the exosomes and then analyze them to determine if they are intact and biologically active. This gives us the most enriched “true” exosome product available.

There are two critical factors in having the most accurate count of  “true” exosomes, the isolation and analysis methods used to determine the count. The isolation method(s) used are important to remove larger particles, but cannot differentiate between exosomes, micro-vesicles, apoptotic bodies and debris. 

Most exosome companies only use some form of ultracentrifugation or filtration to isolate the EVs in their products. While these methods are easy and cheap they can only separate EVs by size and cannot differentiate between exosomes, micro-vesicles, apoptotic bodies and debris. They can also cause considerable damage to the exosomes.

Pro Xo™ uses multiple isolation techniques including a specialized sequential filtration method whereby larger particles are filtered out first and sequentially smaller filters are used until the desired particle size is all that remains. We then employ several analyzation methods, described in the “testing” section to ensure we have the highest concentration of “true” exosome and protein-rich product available.

Testing

Pro Xo™ exosomes undergo the most rigorous testing and analyzation of any product on the market. It starts with the retrieval of the CLSCs from which our exosomes are derived and doesn’t end until we have the highest quality, protein rich, exosome products available.

Source & Conditioned Media Analysis

Karyotyping: Karyotyping is a test to examine chromosomes in a sample of cells to identify genetic abnormalities.

Our master stem cell line was karyotyped multiple times until senescence (the point at which cells stop dividing). This was done to detect any chromosomal abnormalities. Additional safety testing was completed to ensure there are no communicable diseases, bacteria or viruses. Only a select few tissues samples were determined to be perfect for exosome production and included in the master stem cell line.

Our master stem cell line contains no chromosomal abnormalities and is also free of any communicable diseases, endotoxins, bacteria, mycoplasma, Human Parvo B19, fungus, mold and other viruses.

Exosome & Protein Analysis

Nanoparticle Tracking Analysis (NTA): This uses laser light scattering based on the Brownian motion of particles to pinpoint particle size, but it also allows for the determination of their concentration. This is the first step used to isolate our exosomes.

Western Blot Testing: Western blotting is an important technique used in cell biology. By using a western blot, our scientists are able to identify specific proteins from a complex mixture of proteins extracted from the conditioned media. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody for visualization. 

The Western Blot test allows us to determine if the protein has been denatured or if it is still intact. Many methods of extracting exosomes can damage the proteins causing them to denature. A denatured protein will not stimulate change in a cell. Pro-Xo Aesthetics exosomes come from a proprietary extraction method that does not damage or denature the proteins. In addition, an independent study showed that when our exosomes were placed with skin fibroblasts, the fibroblasts were stimulated to produce more collagen and elastin.

Enzyme-Linked Immunosorbent Assay (ELISA ): ELISA is an assay technique in which an antibody against an antigen of interest is immobilized on the surface of a microplate and is used to isolate exosomes from a sample. It is designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. 

In the EV field, ELISA has been used to isolate exosomes from multiple different tissues and can even be quantitative when standards (of known exosome amounts) are used to create a calibration curve. The Pro-Xo Aesthetics exosome products have undergone ELISA testing and consistently measured 1mg of protein per 1ml of liquid. 

Florescent Nanoparticle Tracking Analysis (F-NTA): This is the final phase of testing and uses a special dye to help identify the intact and biologically active exosomes. The florescent component of this technology is important because it allows for the differentiation between EVs and “true” Exosomes. The other components such as micro-vesicles, apoptotic bodies and debris do not have the capabilities of exosomes.

It is the complete activated protein component that can stimulate a known response to the cell, whereas the incomplete components may have an incomplete response. Without protein, a cell does not have the ability to change.

Using multiple isolation methods consecutively and precise analyzation techniques we enrich the exosome content of the Pro Xo line of products. While this is costly and time-consuming it produces the highest quality and most effective products available.