Minute 4200 rpm spin
EVs mediate a series of cellular functions such as the transport of materials and intercellular communication 1. EVs are highly specialized messenger molecules, which can deliver biological signals. For these reasons, the potential of extracellular vesicles in therapeutic application in regenerative medicine is drawing increasing interest.
I. Platelet-Rich Plasma
Platelet-rich plasma (PRP) is a preparation for therapeutic purposes that is increasingly accepted for various musculoskeletal disorders, due to its theoretical potential to repair tissues with poor-healing capacity 1. PRP therapy uses injections of a patient’s own concentrated platelets. In a PRP preparation, platelets are concentrated by centrifugation from a peripheral blood draw resulting in three fractions: 1) Red blood cells (RBCs) 2) Platelet-rich plasma (PRP) and 3) Platelet-poor plasma (PPP). According to the FDA, PRP is considered a blood product. Per the FDA guidance “Regulatory Considerations for Human Cells, Tissues, and Cellular and Tissue-Based Products: Minimal Manipulation and Homologous Use” 2, July 2020, Section V.A, pg. 22:
for example, platelet rich plasma (PRP, blood taken from an individual and given back to the same individual as platelet rich plasma) is not an HCT/P under 21 CFR Part 1271 because it is a blood product.
II. SuperShot® PRPis a PRP preparation that includes an additional centrifugation step whereby the low-density lipid rich plasma fraction is precipitated out of the Platelet-poor Plasma fraction, following initial centrifugation, and incorporated into the PRP. In a standard PRP procedure, the PRP fraction is injected, leaving many biological molecules such as extracellular vesicles (EVs), discarded with the PPP. EVs mediate a series of cellular functions such as the transport of materials and intercellular communication 1. EVs are highly specialized messenger molecules, which can deliver biological signals. For these reasons, the potential of extracellular vesicles in therapeutic application in regenerative medicine is drawing increasing interest 2. The SuperShot® PRP process increases the concentration of EVs in PRP when compared to a standard PRP process.
SuperShot® Solution: SuperShot is packaged as 2mL solution in a 10x concentrate of: 23.3% Poly(ethylene glycol) 6000 (PEG)/10% Dextran 500 (Dex). It is diluted 10x in PPP to a working concentration of 2.3% PEG/1% Dex.
SuperShot is manufactured in a 503b facility (Compound Preferred, 1125 Hollipark Dr., Idaho Falls, ID 83401).
SuperShot® is terminally sterilized during manufacture.
When added to PPP, SuperShot® enables separation of low-density plasma-born molecules upon low-speed centrifugation. PEG and dextran together result in aqueous polymer two phase system, which is required for the purification of biological materials. The PEG/Dex solution results in the precipitation of small low-density molecules, including extracellular vesicles (50-500nm in diameter). PEG precipitation is simple, fast, and scalable; does not deform EVs 3.
The SuperShot® PRP Process: First a PRP centrifugation process is performed. A small portion of the PPP is aseptically removed and combined with SuperShot® 10x solution. The PPP including SuperShot® is then centrifuged at 4200 RPM for 1 min. The result is a pellet of low-density biomolecules including EVs. The supernatant, which now consists of the depleted PPP and PEG/Dex is removed and discarded, leaving behind the EF pellet, which is then resuspended into PRP.
Low density vesicles are isolated from platelet poor plasma with SuperShot®. Distinct types of these extracellular vesicles are isolated and added to PRP.
FDA’s regulatory authority over Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/P) is defined in 21 CFR 1271. 21 CFR 1271.3(d) defines what is and is not included under the definition of HCT/P: (d) Human cells, tissues, or cellular or tissue-based products (HCT/Ps) means articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient. Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under parts 607 and 207 of this chapter, respectively.