Methods and compositions related to platelet releasate and platelet-rich fibrin

Abstract

To provide: a composition comprising human platelet releasate (hPR) as a xeno-free media supplement; and a method for preparing the supplement.SOLUTION: Provided is a method for preparing a platelet releasate, comprising the steps of: i) obtaining platelets from human blood, thereby obtaining platelet-rich-plasma (PRP); ii) adding CaCl2 to the PRP to a final concentration of greater than 25 mM, thereby generating a CaCl2 / PRP mixture; and, iii) agitating the CaCl2 / PRP mixture for less than 6 hours, thereby forming a clot and a releasate.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 802,623, filed February 7, 2019, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates generally to biotechnology, medical products, and the commercial manufacture of cell culture media supplements. Embodiments relate to compositions comprising cell culture media supplements and methods of preparing such supplements from platelet-rich plasma derived from animal blood. [Background technology]

[0003] background Mesenchymal stem cells (MSCs) have found widespread application in the treatment of trauma, wound care (bone and cartilage regeneration), myocardial infarction and autoimmune diseases. Furthermore, there is military significance in the use of MSCs, for example, in the treatment of wounded persons.

[0004] To support clinically relevant dosing in such a diverse range of clinical conditions, it is important to expand the cells on a large scale and under Good Manufacturing Practice (GMP) guidelines without compromising the uniqueness of MSCs. Conventional methods of stem cell expansion still face associated problems such as long expansion times and the use of animal-derived serum. The challenging process of large-scale cell expansion can be improved by the use of bioreactors and automation.

[0005] Common conventional techniques rely on the use of fetal bovine serum (FBS) as a supplement to growth media. FBS plays a vital role in actively promoting cell growth and proliferation. Although the risk of exposure to zoonotic diseases is small, regulatory efforts to scrutinize xenogeneic pathogens remain high. In addition, the risk of immune responses to animal-derived proteins requires strict regulation. To mitigate these issues, the FDA and other regulatory agencies have encouraged the use of xeno-free alternatives to FBS. However, the use of chemically defined serum-free media is not cost-effective for large-scale expansion proliferation.

[0006] Several reports on clinical grade cell expansion have shown the use of human platelet lysate (hPL) as a xeno-free supplement to growth media. hPL can sustain MSC growth without affecting their immunophenotype, but recent reports have shown rather inconsistent effects on MSC population doubling and immunosuppressive properties. Such inconsistencies may be due to differences in hPL production methods. In addition to this, supplementation with hPL induces gelation of the growth media, which results in a less favorable environment and expansion rates for conventional cell culture. In many cases, this issue is mitigated by the addition of heparin, a porcine-derived anticoagulant, but ultimately negates previous efforts to maintain xeno-free expansion conditions.

[0007] Currently, there is a need for more efficient protocols, methods and systems for commercial scale-up of hPL production, in which the need for heparin or other anticoagulants is alleviated. Additionally, there is a need for protocols that can be scaled up under Good Manufacturing Practice guidelines to provide commercial quantities of hPL. Such protocols should be cost-effective, be performed in a closed system to minimize the risk of contamination, provide a desirable composition rich in growth factors and cytokines, and the entire process should take a minimum amount of time from start to finish. Summary of the Invention

[0008] Disclosure Summary The present embodiments include, inter alia, compositions and methods relating to platelet releasates (hPR), particularly the large-scale production of hPR. The releasates according to the present disclosure are products or extracts obtained from cells engineered (e.g., degranulated) according to the methods of the present disclosure, and the releasates can be used as a supplement in cell culture media for culturing or expanding cells. hPR prevents gelling of the growth media, thereby reducing the need for heparin or other anticoagulants. Additionally, there are embodiments that include compositions and methods relating to platelet-rich fibrin, particularly the large-scale production of platelet-rich fibrin.

[0009] In some embodiments, the hPR is enriched with growth factors and contains less than about 0.05 mg / dL of fibrinogen. In some embodiments, the hPR contains FGF basic at a level of at least about 300 pg / ml. In some embodiments, the hPR contains SDF-1α at a level of about 50 pg / ml to about 20 pg / ml. In some embodiments, the hPR manufacturing process is carried out on an industrial scale and / or in a closed system that can result in up to 10 liters, 50 liters, 100 liters, or even hundreds of liters of hPR. Stem cells, such as mesenchymal stem cells, expanded in the presence of hPR have shown superior expansion rates compared to their commercially available counterparts.

[0010] Embodiments herein include compositions of platelet releasates, media supplements comprising platelet releasates, xeno-free serum supplements comprising platelet releasates, cell cultures expanded on platelet releasates, cell culture media comprising platelet releasates, sterilized platelet releasates, methods of sterilizing releasates, lyophilized releasates, therapeutic compositions, pharmaceutical formulations, methods of preparing platelet releasates, methods of manufacturing platelet releasates, methods of industrially producing platelet releasates, methods of commercially scaling up bone marrow derived mesenchymal stem cells, methods of expanding cells, methods of expanding cells on an industrial scale, methods of expanding cells for regenerative medicine, closed systems for producing platelet releasates, open systems for producing platelet releasates, methods of reducing process times for the production of platelet releasates, and methods of treating a subject using mesenchymal stem cells expanded on hPR.

[0011] Any one or more of the methods of the disclosure may include or exclude one or more of the following steps, may consist of one or more of the following steps, or may consist essentially of one or more of the following steps: obtaining a source or solution of whole blood, platelets or platelet rich plasma (PRP) or platelet concentrate, weighing, pooling, mixing, agitating, shaking, activating, agglutinating, centrifuging, freezing, thawing, weighing, filtering, double filtering, incubating, causing, concentrating, producing, clotting, gelling, separating, and packaging.

[0012] In some embodiments, the present disclosure relates to a method for preparing a platelet releasate, comprising: (i) obtaining platelets from human blood, thereby obtaining platelet-rich plasma (PRP); (ii) adding CaCl2 to the PRP to a final concentration of more than 25 mM; and (iii) stirring the CaCl2 / PRP mixture for less than 6 hours, thereby forming a clot and a releasate. In some embodiments of the method, the releasate comprises fibrinogen at a level of less than about 0.05 mg / dL. In some embodiments, the method further comprises concentrating by removing excess plasma prior to (ii).

[0013] In some embodiments of the disclosed methods, the releasant comprises FGF basicity at a level of at least about 300 pg / ml. In some embodiments, the releasant comprises FGF basicity at a level of about 300 pg / ml to about 550 pg / ml. In other embodiments, the releasant comprises FGF basicity at a level of about 350 pg / ml to about 520 pg / ml. In further embodiments, the releasant comprises FGF basicity at a level of about 400 pg / ml to about 500 pg / ml. In some embodiments, the releasant comprises FGF basicity at a level of about 450 pg / ml. In some embodiments, the emission is about 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495 , 500, 505, 509, 510, 515, 520, 525, 530, 535, 540, 545, or 550 pg / ml, at least about 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, or 550 pg / ml 0, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 509, 510, 515, 520, 525, 530, 535, 540, 545, or 550 pg / ml, or at most about 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 38 and / or 550 pg / ml, or any range derivable therein.

[0014] In some embodiments of the methods of the present disclosure, the releasant contains about 5.0 pg / ml to about 20 pg / ml of SDF-1α. In some embodiments, the releasant contains about 7.0 pg / ml to about 15 pg / ml of SDF-1α. In some embodiments, the releasant contains about 8.0 pg / ml to about 14 pg / ml of SDF-1α. In other embodiments, the releasant contains about 9.0 pg / ml to about 12.0 pg / ml of SDF-1α. In further embodiments, the release is at least about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 pg / ml or at least about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 pg / ml 0.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 pg / ml, or at most about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 pg / ml, or any range derivable therein.

[0015] In some embodiments of the method of the present disclosure, the final concentration of CaCl2 is greater than about 30 mM. In some embodiments, the final concentration of CaCl2 is about 25 mM to about 80 mM. In some embodiments, the final concentration of CaCl2 is about 30 mM to about 50 mM. In further embodiments, the final concentration of CaCl2 is about 40 mM to about 47 mM. In yet another embodiment, the final concentration of CaCl2 is about 45 mM. In some embodiments of the method of the present disclosure, the final concentration of CaCl2 is 80 mM. In some embodiments, the final concentration of CaCl2 is about 80 mM to about 90 mM.In some embodiments, the final concentration of CaCl2 is at least about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mM, at most about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mM, or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mM 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mM or more (or any value derivable therein). In some embodiments, the concentration will vary depending on the choice of calcium salt, as can be recognized by one of skill in the art. In some embodiments, the calcium salt is or includes calcium gluconate, calcium citrate, calcium phosphate, calcium chloride, calcium acetate, calcium carbonate, or other calcium salts.

[0016] In some embodiments, the method for preparing a platelet releasate takes from start to finish 4 hours or less, or 2.5 hours to 4 hours. In some embodiments, the duration of the method is at least 2, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 hours, at most 2, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 hours, or about 2, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 hours, or any range derivable therein. In some embodiments, the duration of the method is 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300 minutes, or about 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300 minutes, and any range derivable therein. The initiation of the method, in some embodiments, begins with testing the blood type of a blood sample, obtaining blood from a subject or biological sample, obtaining platelets from the biological sample, isolating platelets from the biological sample, thawing a sample of frozen platelets, or isolating plasma from the biological sample, and ends in some embodiments with agitating the mixture containing platelets to produce a releasate, centrifuging the releasate, filtering the releasate, storing the releasate, freezing the releasate, mixing the releasate with cell culture medium, or culturing cells with the releasate.In some embodiments, steps (i), (ii) and (iii) are understood to mean obtaining (e.g., isolating) human blood platelets, adding CaCl2 to the PRP, and stirring the CaCl2 / PRP mixture to form a clot, respectively. In some embodiments, step (i) further comprises concentrating the platelets by removing excess plasma. In some embodiments, the duration of steps (i), (ii) and (iii) is 2 hours to 6 hours (e.g., 3 hours to 4 hours). In some embodiments, the duration of steps (i), (ii), (iii) and (iv) is 2 hours to 6 hours (e.g., 3 hours to 4 hours).

[0017] In some embodiments of the disclosed methods, the CaCl2 / PRP mixture is stirred for less than about 4 hours. In some embodiments, the CaCl2 / PRP mixture is stirred for less than about 6 hours. In other embodiments, the CaCl2 / PRP mixture is stirred for less than about 180 minutes. In further embodiments, the CaCl2 / PRP mixture is stirred for about 30 minutes to about 150 minutes or 45 to 135 minutes. In yet other embodiments, the CaCl2 / PRP mixture is stirred for 60 minutes to 90 minutes. In some embodiments, the CaCl2 / PRP mixture comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 29, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 minutes, or at most 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 7, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 , 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 2 11, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 minutes, or 2 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 35 9, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 20 3, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 minutes, or any range derivable therein.

[0018] In some embodiments of the disclosed methods, the CaCl2 / PRP mixture is stirred at a constant revolutions per minute between 50 rpm and 500 rpm. In some embodiments of the disclosed methods, the CaCl2 / PRP mixture is stirred at a constant revolutions per minute between 150 rpm and 350 rpm.In some embodiments, the CaCl2 / PRP mixture is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 rpm or at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230 , 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 rpm, or approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 1 The mixture is stirred at an rpm of 60, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 rpm, or any value derivable therein.In some embodiments of the disclosed method comprising a batch size of about 50 L or about 100 L, the rpm is at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250 rpm, at most about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250 rpm. 5, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250 rpm or approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 The rotational speed may be 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250 rpm, or any value derivable therein.

[0019] The present disclosure relates to human platelet releasates derived from plasma (e.g., platelet-rich plasma). In some embodiments, the platelets are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days after donation, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days after donation, or any value derivable therein. "Day" means 24 hours or less, e.g., 3 days means 72 hours or less. In some embodiments, the releasants are from fresh platelets (i.e., within 24 hours of donation). In some embodiments, the platelets are expired. In some embodiments, expired platelets are at least 7 days after donation, at least 14 days after donation, at least 21 days after donation, at least 30 days after donation, or at most 7 days after donation, at most 14 days after donation, at most 21 days after donation, or at most 30 days after donation. In some embodiments, the platelets are stored at room temperature (e.g., 23° C. to 25° C.). In some embodiments, the platelets are cryopreserved platelets (e.g., 4° C. to 20° C.). In some embodiments, the platelets are frozen. In some embodiments, the platelets are frozen at about −55° C. to about −80° C., or any temperature within this range. In some embodiments, the plasma is further concentrated to obtain a platelet concentrate.In some embodiments, the platelet concentrate is at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 percent or more (or any range derivable therein) higher, or about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20 times higher (or any range derivable therein), or at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 percent or more (or any range derivable therein) higher. The plasma may have a concentration level of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20 times or more (or any range derivable therein) higher, or at most about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 percent or more (or any range derivable therein) higher, or at most about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20 times or more (or any range derivable therein) higher. In some embodiments, the plasma is not concentrated.

[0020] In some embodiments, the remnants are derived from mammalian platelet-rich plasma. In some embodiments, the platelets are derived from human platelet-rich plasma. In some embodiments, the platelets are derived from horse, dog, cow, chicken, cat, pig, rabbit, dolphin, sheep, mouse, rat, or monkey blood. In some embodiments, the platelets are derived from sports animals, farm animals, or pets.

[0021] In some embodiments, the disclosed method further comprises separating the clot from the releasate such that about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or less of the clot detectably remains in the separated releasate. In some embodiments, the clot is a fibrin clot. In some embodiments, the clot encapsulates cells and cellular debris from the blood.

[0022] In some embodiments, the method of the present disclosure further comprises filtering the discharge. In some embodiments, the discharge is filtered using a filter that is a 0.45 micron to 1.0 micron filter. In some embodiments, the filter is 0.45 micron, 0.50 micron, 0.55 micron, 0.60 micron, 0.65 micron, 0.70 micron, 0.75 micron, 0.80 micron, 0.85 micron, 0.90 micron, 0.95 micron, 1.0 micron, or any size derivable therein. In some embodiments, the filter is a 3 micron to 10 micron filter. In some embodiments, the filter comprises at least 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 microns, or at most 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 microns or any size derivable therein. In some embodiments, the filter is a 170 micron to 260 micron filter.In some embodiments, the filter is a 170 micron to 260 micron filter, hi some embodiments, the filter is at least 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260 microns, or at most 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260 microns, or any range derivable therein.

[0023] In some embodiments of the methods for preparing a platelet releasate, steps (i), (ii) and (iii) are performed in a closed bag. In some embodiments, steps (i), (ii) and (iii) are performed in a closed system. In some embodiments, steps (i), (ii) and (iii) are performed on an industrial scale in a closed system. In some embodiments of any of the methods presented herein, the methods further comprise a step (iv) of filtering the releasate. In some embodiments, steps (i), (ii), (iii) and (iv) are performed in a closed bag. In some embodiments, the filtering step is also part of the closed system. In some embodiments, some or all of steps (i), (ii), (iii) and (iv) are performed in a closed system. In some embodiments, some or all of steps (i), (ii), (iii) and (iv) are performed in an open system.

[0024] In some embodiments of the method for preparing a platelet releasate, the releasate is produced in a yield of about 0.2 liters to about 10 liters. In some embodiments, the releasate is produced in a yield of about 4.5 liters to about 10 liters. In some embodiments, the yield is at least 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 liters, at most 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 liters, or about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 liters, or any value derivable therein. In some embodiments of the method for preparing a platelet releasate, the releasate is produced at a yield of about 0.2 liters to about 100 liters. In some embodiments, the release is produced in a yield of about 50 liters to about 100 liters. In some embodiments, the yield is at least 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 liters, at most 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 6 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 liters or approximately 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0 ,7.5,8.0,8.5,9.0,9.5,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 liters, or any value derivable therein.

[0025] In some embodiments, the present disclosure relates to a releasant composition produced by any of the methods described herein. The amount of the releasant composition is about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 liters, at most about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0 , 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 liters, or at least about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 liters, or any value derivable therein.

[0026] In another aspect, the disclosure relates to a cell culture medium comprising the releasate produced by any of the methods described herein. In some embodiments, the cell culture medium does not contain added heparin or other anticoagulants.

[0027] A further aspect of the present disclosure relates to a method of culturing cells (e.g., stem cells), comprising expanding the cells on a cell culture medium comprising a releasate from human or mammalian platelet-rich plasma. In some embodiments, the releasate comprises fibrinogen at a level of less than about 0.05 mg / dL. In some embodiments, the releasate comprises FGF basic at a level of less than about 300 pg / ml. In some embodiments, the releasate comprises SDF-1α at a level of about 5.0 pg / ml to about 20 pg / ml. In some embodiments, the cell culture medium does not comprise added heparin. In some embodiments, the cell is a pluripotent stem cell (PSC), an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), a bone marrow derived mesenchymal stromal / stem cell (BM-MSC), an adipose derived mesenchymal stromal / stem cell (ADP-MSC), a T cell, a B cell, a natural killer cell, a dendritic cell, a peripheral blood derived mononuclear cell, a cancer cell cancer stem cell, a Chinese hamster ovary (CHO), a cord blood derived cell, a cord blood tissue derived cell, a placenta derived cell, a retinal cell, a neuronal cell, a fibroblast, an epithelial cell, an endothelial cell or a keratinocyte or a fibroblast, an osteoblast, an adipocyte, a chondrocyte, an endothelial cell, a cell of the immune system, a T cell, a B cell, a NK cell, an engineered cell (e.g., a chimeric antigen receptor (CAR) T cell), or a neuronal cell. In some embodiments, the release stimulates the release of a component from the cell. In some embodiments, the release stimulates the release of an exosome, an extracellular vesicle, a protein, a nucleic acid, or a combination thereof. In some embodiments, the releasant stimulates release of exosomes. In some embodiments, the method comprises recovering the released components from cells expanded on cell culture medium containing the releasant.

[0028] A further aspect of the disclosure relates to a method of treating a mammalian subject comprising administering to the mammalian subject a composition comprising a population of stem cells, the stem cells having been cultured with a releasant composition described herein. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the cells are pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) or any related lineage of differentiation. In some embodiments, the stem cells are bone marrow derived mesenchymal stem cells, bone marrow derived mesenchymal stromal / stem cells (BM-MSCs), adipose derived mesenchymal stromal / stem cells (ADP-MSCs), T cells, B cells, natural killer cells, dendritic cells, peripheral blood derived mononuclear cells. In some embodiments, the stem cells are autologous. In other embodiments, the stem cells are allogeneic. In some embodiments, the stem cells are xenogeneic. In some embodiments, the stem cells are engineered or manipulated cells.

[0029] In some embodiments, the population of stem cells comprises 2-100% of cells of a particular lineage of differentiation. In some embodiments, the method of culturing stem cells with the releasant composition of the present disclosure can be used to increase the percentage of a lineage to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 120%, 130%, 14 %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any value derivable therein.

[0030] A further aspect of the present disclosure relates to a method of treating a subject comprising administering to the subject a composition comprising stem cells, the stem cells having been cultured with releasate from human blood-derived platelets, the cells having been cultured with a releasate composition described herein.

[0031] A further aspect of the present disclosure is a method of treating a subject, comprising administering to the subject a composition comprising stem cells, the stem cells being cultured with release from human blood-derived platelets, the release comprising fibrinogen at a level of less than about 0.05 mg / dL. In some embodiments, the subject suffers from a bone disease, a bone defect, a bone injury, osteoporosis, osteoarthritis, or a spinal cord injury. In some embodiments, the subject suffers from a cartilage disease or a cartilage defect or injury. In some embodiments, the subject suffers from a bone, tendon, cartilage, or muscle injury. In some embodiments, the subject suffers from periodontal disease. In some embodiments, the subject suffers from an autoimmune disease. In some embodiments, the subject suffers from a myocardial infarction. In some embodiments, the subject suffers from or has suffered from graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), poly-trauma, systemic infection, or cancer. In some embodiments, the subject is a non-human animal subject.

[0032] Further aspects of the present disclosure relate to compositions comprising releasates from human or mammalian blood-derived platelets. In some embodiments, the releasates comprise fibrinogen at a level of less than about 0.05 mg / dL. In some embodiments, the releasates comprise FGF basic at a level of less than about 300 pg / ml. In some embodiments, the releasates comprise SDF-1α at a level of about 5.0 pg / ml to about 20 pg / ml. In some embodiments, the releasates comprise microvesicles from platelets. In some embodiments, the releasates comprise exosomes from platelets.

[0033] Some aspects of the present disclosure relate to formulations that include the releases described herein.The formulations can be used for therapeutic purposes, such as treating diseased or damaged tissues, including but not limited to bone, muscle, skin, nerve, tendon, connective tissue, ocular tissue, periodontal tissue, or cardiovascular tissue.In some embodiments, the formulations are used for therapeutic purposes, such as to treat or alleviate ocular conditions, including dry eye.In some embodiments, the formulations of the present disclosure can be artificial or synthetic tear formulations.

[0034] In some embodiments of the compositions, formulations and methods of the present disclosure, the releasant contains FGF basicity at a level of at least about 300 pg / ml. In some embodiments, the releasant contains FGF basicity at about 300 pg / ml to about 550 pg / ml, 350 pg / ml to 520 pg / ml, or about 400 pg / ml to about 500 pg / ml. In some embodiments, the releasant contains FGF basicity at about 450 pg / ml. In some embodiments, FGF basicity is at least 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 pg / ml or at most 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 pg / ml. 00, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 pg / ml, or about 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 pg / ml.

[0035] In some embodiments of the disclosed method or composition, the release comprises globulins, albumins, growth factors, cytokines, interleukins, interferons, chemokines, hormones, and glycoproteins such as fibronectin, vitronectin, laminin, or other compounds. In some embodiments, the release comprises microvesicles or extracellular vesicles. In some embodiments, the release comprises exosomes. In some embodiments, the release comprises TGFβ1, EGF, bFGF, PDGF-AA, PDGF-BB, PDGF-AB, SDF-1α, VEGF, or HGF. In some embodiments, the release comprises one or more of the proteins listed in Table 1. In some embodiments, the release does not comprise one or more of the proteins listed in Table 1. In some embodiments, the release includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, or 200 of the proteins listed in Table 1, or any value derivable therein. In some embodiments, the release does not include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, or 200 of the proteins listed in Table 1, or any value derivable therein. In some embodiments, the releasant comprises less than 0.01 pg / ml, 0.02 pg / ml, 0.03 pg / ml, 0.04 pg / ml, 0.05 pg / ml, 0.06 pg / ml, 0.07 pg / ml, 0.08 pg / ml, 0.09 pg / ml, 0.1 pg / ml, 0.2 pg / ml, 0.3 pg / ml, 0.4 pg / ml or 0.5 pg / ml of one or more of the proteins listed in Table 1. In some embodiments, the releasant comprises less than 0.1 pg / ml of one or more of the proteins listed in Table 1.

[0036] Some aspects of the present disclosure relate to methods of treating injured individuals using mesenchymal stem cells, endothelial cells, fibroblasts, cells of the immune system (e.g., T cells, B cells, NK cells or other modified cells), or neural cells expanded on hPR. The steps and embodiments discussed in this disclosure are contemplated as part of any of these methods.

[0037] A further aspect of the present disclosure relates to a method of promoting cell adhesion, cell differentiation or cell expansion in tissue culture comprising coating a tissue culture vessel with a composition comprising a releasate from human blood-derived platelets, the releasate comprising fibrinogen at a level of less than about 0.05 mg / dL, hi some embodiments, the vessel is a petri dish, flask or bioreactor.

[0038] A further aspect of the present disclosure relates to a method for preparing an osteobiological material, comprising adding a composition comprising release from human blood-derived platelets to said osteobiological material, wherein the release comprises fibrinogen at a level of less than 0.05 mg / dL. In some embodiments, the osteobiological material is an osteobiological graft material, a bone sponge or a bone putty. In some embodiments, the osteobiological material further comprises mammalian tissue, modified cells or engineered cells.

[0039] In some embodiments, the present disclosure relates to a method of preparing a clotting agent comprising adding to said clotting agent a composition comprising a releasate from human blood-derived platelets, wherein the releasate comprises fibrinogen at a level of less than about 0.05 mg / dL.

[0040] In some embodiments, the present disclosure relates to a method for preparing platelet-rich fibrin from mammalian blood-derived platelets, comprising: (i) obtaining (e.g., isolating) human blood platelets, thereby obtaining platelet-rich plasma (PRP); (ii) adding CaCl2 to the PRP to a final concentration of more than 25 mM; (iii) stirring the CaCl2 / PRP mixture for less than 6 hours, thereby forming a fibrin clot and a supernatant; (iv) adding an antifibrinolytic agent to prevent fibrinolysis; and (v) removing the supernatant to obtain platelet-rich fibrin. In some embodiments, the method further comprises concentrating by removing excess plasma. In some embodiments, the mammalian blood is human, horse, dog, cat, pig, cow, chicken, cat, pig, rabbit, dolphin, sheep, mouse, rat, monkey blood, sport animal blood, farm animal blood, or pet blood. In some embodiments, any process or method used to produce releasants may be used to produce platelet-rich fibrin. Such processes and methods may further include adding an antifibrinolytic agent to prevent fibrinolysis, and then removing the supernatant to obtain platelet-rich fibrin. In other aspects, the disclosure relates to a platelet-rich fibrin composition produced by the methods described herein. In some embodiments, the platelet-rich fibrin does not contain detectable levels of thrombin.

[0041] Aspects relate to kits that include a platelet releasate from mammalian blood-derived platelets. The kits may include additional components, such as cell culture media or other cell culture additives. In some embodiments, the kits include instructions for using the releasate as a cell culture media supplement.

[0042] It is contemplated that any method or composition or method step described herein can be implemented with respect to any other method or composition described herein, and that different embodiments can be combined. In certain embodiments, a method of generating platelet-rich fibrin includes one or more steps used to produce a releasate, or vice versa.

[0043] The use of one or more method steps or compositions may be used according to any of the methods described herein. Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of this disclosure also applies to other aspects of this disclosure, and vice versa. It is understood that the embodiments in the Examples section are embodiments applicable to all aspects of the technology described herein.

[0044] [The present invention 1001] (i) obtaining platelets from human blood, thereby obtaining platelet-rich plasma (PRP); (ii) adding CaCl to the PRP to a final concentration of greater than 25 mM, thereby generating a CaCl / PRP mixture; and (iii) stirring the CaCl2 / PRP mixture for less than 6 hours, thereby forming a clot and a release product. A method for preparing a platelet releasate comprising: [The present invention 1002] The method of claim 1001, wherein said release contains fibrinogen at a level of less than about 0.05 mg / dL. [The present invention 1003] The method of claim 1001, wherein said final concentration of CaCl2 is greater than about 30 mM. [The present invention 1004] The method of claim 1001, wherein the final concentration of CaCl2 is about 25 mM to about 80 mM. [The present invention 1005] The method of claim 1001, wherein the final concentration of CaCl2 is about 30 mM to about 50 mM. [The present invention 1006] The method of claim 1001, wherein the final concentration of CaCl2 is about 35 mM to about 50 mM. [The present invention 1007] The method of claim 1001, wherein the final concentration of CaCl2 is about 40 mM to about 47 mM. [The present invention 1008] The method of claim 1001, wherein the final concentration of CaCl2 is about 45 mM. [The present invention 1009] The method of claim 1001, wherein the final concentration of CaCl2 is about 80 mM. [The present invention 1010] Any of the methods of claims 1001 to 1009, wherein the CaCl2 / PRP mixture is stirred for less than 4 hours. [The present invention 1011] Any of the methods of claims 1001 to 1009, wherein the CaCl2 / PRP mixture is stirred for less than 180 minutes. [The present invention 1012] Any of the methods of claims 1001 to 1009, wherein the CaCl2 / PRP mixture is stirred for 30 minutes to 150 minutes. [The present invention 1013] Any of the methods of claims 1001 to 1009, wherein the CaCl2 / PRP mixture is stirred for 45 minutes to 135 minutes. [The present invention 1014] The method of any one of claims 1001 to 1009, wherein the CaCl2 / PRP mixture is stirred for 60 minutes to 90 minutes. [The present invention 1015] Any of the methods of claims 1001 to 1014, wherein the CaCl2 / PRP mixture is stirred at 50 rpm to 500 rpm. [The present invention 1016] Any of the methods of claims 1001 to 1014, wherein the CaCl2 / PRP mixture is stirred at 250 rpm. [The present invention 1017] The method of any one of claims 1001 to 1016, wherein the releasable product comprises a globulin, an albumin, a growth factor, a cytokine, an interleukin, an interferon, a chemokine, a glycoprotein, a fibronectin, a vitronectin, or a laminin. [The present invention 1018] The method according to any one of claims 1001 to 1016, wherein the releasable product comprises TGFβ1, TGFβ3, EGF, bFGF, PDGF-AA, PDGF-BB, PDGF-AB, SDF-1α, VEGF or HGF. [The present invention 1019] The method according to any one of claims 1001 to 1018, wherein said releasate comprises FGF basic at a level of at least 300 pg / ml. [The present invention 1020] The method according to any one of claims 1001 to 1018, wherein the releasant contains FGF basic at about 300 pg / ml to about 550 pg / ml. [The present invention 1021] The method according to any one of claims 1001 to 1018, wherein the releasant contains FGF basic at about 350 pg / ml to about 520 pg / ml. [The present invention 1022] The method according to any one of claims 1001 to 1018, wherein the releasant contains FGF basic at about 400 pg / ml to about 500 pg / ml. [The present invention 1023] The method of any of claims 1001 to 1019, wherein the releasant contains FGF basic at about 450 pg / ml. [The present invention 1024] The method according to any one of claims 1001 to 1023, wherein the releasant contains SDF-1α at about 5.0 pg / ml to about 20 pg / ml. [The present invention 1025] The method according to any one of claims 1001 to 1023, wherein the releasant contains SDF-1α at about 7.0 pg / ml to about 15 pg / ml. [The present invention 1026] The method according to any one of claims 1001 to 1023, wherein the releasant contains SDF-1α at about 8.0 pg / ml to about 14 pg / ml. [The present invention 1027] The method according to any one of claims 1001 to 1023, wherein the releasant contains SDF-1α at about 9.0 pg / ml to about 12.0 pg / ml. [The present invention 1028] The method of any of claims 1001 to 1027, wherein said platelets are derived from fresh platelets, from platelets kept at room temperature, or from platelets that have been previously frozen. [The present invention 1029] Any of the methods of claims 1001 to 1028, further comprising a step of separating the clot from the release. [The present invention 1030] The method of any one of claims 1001 to 1029, further comprising a step (iv) of filtering the release. [The present invention 1031] The method of claim 1030, wherein step (iv) comprises filtering the release using a filter having a pore size of between 0.45 microns and 1.0 microns. [The present invention 1032] The method of claim 1030, wherein step (iv) comprises filtering the release using a filter having a pore size of between 3.0 microns and 10 microns. [The present invention 1033] The method of claim 1030, wherein step (iv) comprises filtering the release using a filter having a diameter of between 170 microns and 260 microns. [The present invention 1034] The method of any one of claims 1001 to 1033, wherein steps (i), (ii) and (iii) are carried out in a closed bag. [The present invention 1035] The method of any one of claims 1031 to 1033, wherein steps (i), (ii), (iii) and (iv) are carried out in a closed bag. [The present invention 1036] The method according to any one of claims 1001 to 1024, wherein steps (i), (ii) and (iii) are carried out in a closed system. [The present invention 1037] The process according to any one of claims 1001 to 1024, wherein steps (i), (ii) and (iii) are carried out on an industrial scale in a closed system. [The present invention 1038] The process according to any one of claims 1031 to 1033, wherein steps (i), (ii), (iii) and (iv) are carried out on an industrial scale in a closed system. [The present invention 1039] The method of any one of claims 1001 to 1038, wherein the entire method is carried out in 4 hours or less. [The present invention 1040] The method according to any one of claims 1001 to 1027, wherein the duration of steps (i), (ii) and (iii) is 3 to 4 hours. [The present invention 1041] The method according to any one of claims 1001 to 1027, wherein the duration of steps (i), (ii), (iii) and (iv) is 3 hours to 4 hours. [The present invention 1042] The method of any one of claims 1001 to 1029, wherein the releasant is produced in a yield of about 0.2 liters to about 100 liters. [The present invention 1043] The method of any of claims 1001 to 1029, wherein the releasant is produced in a yield of about 4.5 liters to about 10 liters. [The present invention 1044] The method of any one of claims 1001 to 1029, wherein the releasant is produced in a yield of about 50 liters to about 100 liters. [The present invention 1045] The method of any of claims 1001 to 1044, further comprising, prior to (ii), concentrating platelets by removing excess plasma. [The present invention 1046] A releasable composition produced by any of the methods of the present inventions 1001 to 1045. [The present invention 1047] A cell culture medium comprising a releasate produced by any one of the methods of the present inventions 1001 to 1045. [The present invention 1048] 1047. A cell culture medium according to the present invention, which does not contain added heparin. [The present invention 1049] A method of culturing cells, comprising expanding the cells on a cell culture medium comprising a releasate derived from mammalian platelet-rich plasma. [The present invention 1050] The method of claim 1049, wherein said releasate contains fibrinogen at a level of less than about 0.05 mg / dL. [The present invention 1051] The method of any one of claims 1049 to 1050, wherein said releasate further comprises FGF basic at a level of at least about 300 pg / ml. [The present invention 1052] The method of any one of claims 1049 to 1050, wherein said releasate comprises FGF basic at about 300 pg / ml to about 550 pg / ml. [The present invention 1053] The method of any one of claims 1049 to 1050, wherein said releasate comprises FGF basic at about 350 pg / ml to about 520 pg / ml. [The present invention 1054] The method of any of claims 1049 to 1053, wherein the releasant contains FGF basic at about 450 pg / ml. [The present invention 1055] The method of any one of claims 1049 to 1053, wherein the releasant contains SDF-1 at about 5.0 pg / ml to about 20 pg / ml. [The present invention 1056] The method of any one of claims 1049 to 1053, wherein the releasant contains SDF-1 at about 7.0 pg / ml to about 15 pg / ml. [The present invention 1057] The method of any one of claims 1049 to 1053, wherein the releasant contains SDF-1 at about 8.0 pg / ml to about 14 pg / ml. [The present invention 1058] The method of any one of claims 1049 to 1053, wherein the releasant contains SDF-1 at about 9.0 pg / ml to about 12.0 pg / ml. [The present invention 1059] The method according to any one of claims 1049 to 1053, wherein said cell culture medium does not contain added heparin. [The present invention 1060] 4. The method of any of claims 1049 to 1053, wherein said cell is a pluripotent stem cell (PSC), an induced pluripotent stem cell (iPSC), a bone marrow derived mesenchymal stromal / stem cell (BM-MSC), an adipose derived mesenchymal stromal / stem cell (ADP-MSC), a T cell, a B cell, a natural killer cell, a dendritic cell, a peripheral blood derived mononuclear cell, a cancer cell cancer stem cell, a Chinese Hamster Ovary (CHO), a cord blood derived cell, a cord blood tissue derived cell, a placenta derived cell, a retinal cell, a neuronal cell, a fibroblast, an epithelial cell, an endothelial cell or a keratinocyte or a fibroblast, an osteoblast, an adipocyte, a chondrocyte, an endothelial cell, a cell of the immune system, a T cell, a B cell, a NK cell, a modified cell, or a neuronal cell. [The present invention 1061] The method of claim 1060, wherein said cells are mesenchymal stem cells. [The present invention 1062] The method of claim 1061, wherein said release stimulates differentiation of said cells into bone cells. [The present invention 1063] The method of any of claims 1049 to 1061, further comprising, prior to (ii), concentrating platelets by removing excess plasma. [The present invention 1064] The method of claim 1063, wherein said release stimulates differentiation of said cells into chondrocytes or adipocytes. [The present invention 1065] The method of any one of claims 1049 to 1061, wherein the release stimulates the release of a component from the cell. [The present invention 1066] The method of claim 1065, wherein the component comprises an exosome, an extracellular vesicle, a protein, a nucleic acid, or a combination thereof. [The present invention 1065] The method of any one of claims 1065 to 1066, further comprising the step of collecting said components. [The present invention 1066] A composition comprising a releasate from mammalian blood-derived platelets, said releasate comprising fibrinogen at a level of less than about 0.05 mg / dL. [The present invention 1067] The composition of claim 1066, wherein the release is derived from human blood. [The present invention 1068] The composition of any one of claims 1066 to 1067, which is a solution. [The present invention 1069] The composition of any one of claims 1066 to 1067, which is a dried or lyophilized powder. [The present invention 1070] Any of the compositions of claims 1066 to 1069, wherein the release contains FGF basic at a level of at least 300 pg / ml. [The present invention 1071] Any of the compositions of claims 1066 to 1069, wherein the release contains FGF basic at about 300 pg / ml to about 550 pg / ml. [The present invention 1002] Any of the compositions of claims 1066 to 1069, wherein the release contains FGF basic at about 350 pg / ml to about 520 pg / ml. [The present invention 1073] Any of the compositions of claims 1066 to 1069, wherein the release contains FGF basic at about 400 pg / ml to about 500 pg / ml. [The present invention 1074] Any of the compositions of claims 1066 to 1069, wherein the release contains FGF basic at about 450 pg / ml. [The present invention 1075] Any of the compositions of claims 1066 to 1074, wherein the release contains SDF-1 at about 5.0 pg / ml to about 20 pg / ml. [The present invention 1076] Any of the compositions of claims 1066 to 1074, wherein the release contains SDF-1 at about 7.0 pg / ml to about 15 pg / ml. [The present invention 1077] Any of the compositions of claims 1066 to 1074, wherein the release contains SDF-1 at about 8.0 pg / ml to about 14 pg / ml. [The present invention 1078] Any of the compositions of claims 1066 to 1074, wherein the release contains SDF-1 at about 9.0 pg / ml to about 12.0 pg / ml. [The present invention 1079] The composition of any of claims 1066 to 1074, which does not contain added heparin. [The present invention 1080] The composition of any of claims 1066 to 1079, wherein the release comprises one or more exosomes. [The present invention 1081] A therapeutic preparation comprising any one of the compositions of the present inventions 1066 to 1080. [The present invention 1082] A method of treating a mammalian subject, comprising administering to the subject a composition comprising a population of stem cells, the stem cells having been cultured with any one of the release compositions of the present inventions 1066 to 1080. [The present invention 1083] The method of claim 1082, wherein said releasate further comprises FGF basic at a level of at least about 300 pg / ml. [The present invention 1084] The method of claim 1082 or 1083, wherein the releasant contains SDF-1 at 5.0 pg / ml to 20 pg / ml. [The present invention 1085] The method of any one of claims 1082 to 1084, wherein the stem cells are mesenchymal stem cells. [The present invention 1086] The method of any one of claims 1082 to 1084, wherein the stem cells are bone marrow-derived mesenchymal stem cells. [The present invention 1087] Any of the methods of the present invention 1082 to 1084, wherein the stem cells are pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), bone marrow-derived mesenchymal stromal / stem cells (BM-MSCs), adipose-derived mesenchymal stromal / stem cells (ADP-MSCs), T cells, B cells, natural killer cells, dendritic cells, peripheral blood-derived mononuclear cells, cancer cells cancer stem cells, Chinese hamster ovary (CHO), umbilical cord blood-derived cells, umbilical cord blood tissue-derived cells, placenta-derived cells, retinal cells, neural cells, fibroblasts, epithelial cells, endothelial cells, or keratinocytes. [The present invention 1088] The method of any one of claims 1082 to 1084, wherein the population of stem cells comprises 2% to 100% of a single differentiated lineage. [The present invention 1089] 5. The method of any of claims 1082 to 1084, wherein said stem cells are autologous or allogeneic. [The present invention 1090] The method according to any one of claims 1082 to 1084, wherein said stem cells are modified or engineered cells. [The present invention 1091] 11. A method of treating a subject, comprising administering to the subject a composition comprising stem cells, the stem cells having been cultured with releasates from human blood-derived platelets. [The present invention 1092] The method of claim 1091, wherein said releasate contains fibrinogen at a level of less than about 0.05 mg / dL. [The present invention 1093] The method of any one of claims 1091 to 1092, further comprising FGF basic at a level of at least 300 pg / ml. [The present invention 1094] The method of any one of claims 1091 to 1093, wherein the releasant contains SDF-1 at 5.0 pg / ml to 20 pg / ml. [The present invention 1095] The method of claim 1091, wherein the subject is suffering from a bone disease, bone defect, bone injury, osteoporosis, osteoarthritis, or spinal cord injury. [The present invention 1096] The method of claim 1091, wherein the subject is suffering from a cartilage disease or a cartilage defect or a cartilage injury. [The present invention 1097] The method of claim 1091, wherein the subject is suffering from periodontal disease. [The present invention 1098] The method of claim 1091, wherein the subject is suffering from an autoimmune disease. [This invention 1099] The method of claim 1091, wherein the subject is suffering from a myocardial infarction. [The present invention 1100] The method of claim 1091, wherein the subject is suffering from graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), multiple trauma, systemic infection, or cancer. [The present invention 1101] (i) obtaining platelets from the blood of a mammal; (ii) adding CaCl2 to the PRP to a final concentration of greater than 25 mM; and (iii) agitating the CaCl2 / PRP mixture for less than 6 hours, thereby forming a clot and a releasate, the releasate comprising fibrinogen at a level of less than about 0.05 mg / dL. A method for preparing a platelet releasate comprising: [The present invention 1102] 1101. The method of claim 1101, wherein said mammalian blood is horse, cat, pig, dog, cow, chicken, cat, pig, rabbit, dolphin, sheep, mouse, rat, monkey blood, from sports animals, from farm animals, or from pets. [The present invention 1103] The method of any one of claims 1101 to 1102, further comprising, prior to (ii), concentrating said platelets by removing excess plasma. [The present invention 1104] A method for promoting cell adhesion, cell differentiation, or cell expansion in tissue culture, comprising the step of coating a tissue culture vessel with a composition comprising a releasate from mammalian blood-derived platelets, the releasate comprising fibrinogen at a level of less than 0.05 mg / dL. [The present invention 1105] The method of claim 1104, wherein the releasable material is derived from human blood-derived platelets. [The present invention 1106] The method of any one of claims 1104 to 1105, wherein the vessel is a petri dish, a flask, or a bioreactor. [The present invention 1107] 1. A method for preparing an osteobiological material, comprising the step of adding to the osteobiological material a composition comprising a release product from mammalian blood-derived platelets, the release product comprising fibrinogen at a level of less than 0.05 mg / dL. [The present invention 1108] The method of claim 1107, wherein the releasable material is derived from human blood-derived platelets. [The present invention 1109] The method of any one of claims 1107 to 1108, wherein said osteobiologic material is an osteobiologic graft material, a bone sponge, or a bone putty. [The present invention 1110] The method of any one of claims 1107 to 1108, wherein said osteobiologic material further comprises mammalian tissue, modified cells, or engineered cells. [The present invention 1111] A method for preparing a coagulant comprising adding to the coagulant a composition comprising a release product from mammalian blood-derived platelets, the release product comprising fibrinogen at a level of less than 0.05 mg / dL. [The present invention 1112] The method of claim 1111, wherein the releasable material is derived from human blood-derived platelets. [The present invention 1113] (i) obtaining platelets from the blood of a mammal, thereby obtaining platelet-rich plasma (PRP); (ii) adding CaCl to the PRP to a final concentration of greater than 25 mM, thereby obtaining a CaCl / PRP mixture; (iii) agitating the CaCl2 / PRP mixture for less than 6 hours, thereby forming a fibrin clot and a supernatant; (iv) adding an antifibrinolytic agent to prevent fibrinolysis; and (v) removing the supernatant to obtain the platelet-rich fibrin. 2. A method for preparing platelet-rich fibrin comprising: [The present invention 1114] 1113. The method of claim 1113, wherein said mammalian blood is human, horse, pig, cat, dog, cow, sheep, mouse, rat, monkey blood, from sports animals, from farm animals, or from pets. [The present invention 1115] The method of any one of claims 1113 to 1114, further comprising, prior to (ii), concentrating the platelets by removing excess plasma. [The present invention 1116] A platelet-rich fibrin composition produced by any of the methods of the present inventions 1113 to 1115. [The present invention 1117] 1116. The platelet-rich fibrin of the present invention, wherein said fibrin does not contain detectable levels of thrombin. [The present invention 1118] A kit comprising: (a) a platelet releasate from mammalian blood-derived platelets; and (b) instructions for using the releasate as a cell culture medium supplement. [The present invention 1119] The kit of claim 1118, further comprising a cell culture medium. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0045] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0046] [Figure 1A] 1A-1O show the amount and identity of various growth factors cytokines, interleukins, and interferons in exemplary releases of the present disclosure compared to plasma, platelet lysate, chemically defined medium, or FBS-supplemented medium. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 1G] See legend to Figure 1A. [Figure 1H] See legend to Figure 1A. [Figure 1I] See legend to Figure 1A. [Figure 1J] See legend to Figure 1A. [Figure 1K] See legend to Figure 1A. [Figure 1L] See legend to Figure 1A. [Figure 1M] See legend to Figure 1A. [Figure 1N] See legend to Figure 1A. [Figure 1O] See legend to Figure 1A. [Figure 1P] FIG. 1P shows a table correlating sample type with the sample ID used in FIGS. 2-6. "ACR" indicates releasate obtained from "Acrodose" pooled platelets. "APH" indicates releasate obtained from apheresis of a single individual. "Before" indicates sample before filtration. "After" indicates sample after filtration. "RT" indicates sample from platelets at room temperature. "RT" indicates sample from frozen platelets. [Diagram 2] 2 is a three-dimensional bar graph showing the values ​​of various growth factors and cytokines in an example release of the present disclosure. Each value represents a dilution factor of 2.0, i.e., each value is half the true concentration value. [Diagram 3] 3 is a three-dimensional bar graph showing the levels of various growth factors and cytokines in an example release of the present disclosure. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. [Figure 4] 4 is a three-dimensional bar graph showing the levels of various growth factors and cytokines in an example release of the present disclosure. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. [Diagram 5]5 is a three-dimensional bar graph showing the levels of various growth factors and cytokines in an example release of the present disclosure. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. [Figure 6] 6 is a three-dimensional bar graph showing the levels of various growth factors and cytokines in an example release of the present disclosure. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. [Figure 7] FIG. 7 shows a principal components analysis comparing exemplary releases of the present disclosure with plasma, platelet lysate, chemically defined medium, and FBS-supplemented medium. [Figure 8] 8 is a heat map comparing biomarkers from exemplary releases of the present disclosure with controls including plasma, a commercial platelet lysate, chemically defined medium, and medium supplemented with fetal bovine serum. [Figure 9A] 9 shows results obtained testing an exemplary method of preparing a platelet releasate according to the present disclosure using various concentrations of CaCl. Concentrations of CaCl ranging from 25 mM to 80 mM give a well-defined fibrin clot that encases the cellular debris. [Figure 9B] See legend to Figure 9A. [Figure 10] FIG. 10 shows the amount of fibrinogen in exemplary releasates of the present disclosure. "ACR" indicates releasate obtained from "Acrodose" pooled platelets. "APH" indicates releasate obtained from apheresis of a single individual. "Before" indicates sample before filtration. "After" indicates sample after filtration. "RT" indicates sample from room temperature platelets. "RT" indicates sample from frozen platelets. [Figure 11] 11A-11C show the effect of CaCl2 concentration in hPR on MSC doubling. Increasing CaCl2 concentrations are shown from left to right. [Figure 12A] 12A-12C show the effect of duration of platelet agitation on MSC doubling. Increasing agitation periods are shown from left to right, alternating between Acrodose and apheresis platelets. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13] Figure 13 shows the effect of hPR centrifugation duration on MSC doubling. ACR is Acrodose platelets and APH is apheresis obtained platelets. Increasing centrifugation duration is shown from left to right alternating between Acrodose and apheresis platelets. [Figure 14] Figure 14 shows the values ​​of various growth factors obtained in exemplary releasates of the present disclosure. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. Releases were from expired platelets at room temperature or from frozen expired platelets. [Figure 15] 15 shows the total protein, albumin and globulin values ​​obtained in exemplary releasates of the present disclosure, either from room temperature expired platelets or from frozen expired platelets. [Figure 16] Figures 16A-16C show comparative data between hPR and control of the present disclosure. Figure 16A shows total protein and growth factor values. Figure 16B shows the effect of hPR on MSC expansion. Figure 16C shows the luminescence intensity obtained for hPR and control. [Figure 17] Figure 17A shows a scheme according to one embodiment of the present disclosure for cGMP small scale manufacturing. Figure 17B shows an example of a bag for small scale yields of 30 ml volume. [Figure 18] FIG. 18 shows a scheme according to one embodiment of the present disclosure for cGMP small scale manufacturing. [Figure 19] FIG. 19 shows comparative data for total protein, globulins, and albumin between an exemplary human release according to the present disclosure and a commercial lysate. [Figure 20]20A-20B show comparative data for growth factors between an exemplary human release product according to the present disclosure and a commercially available lysate. Each value represents a dilution factor of 2.0, i.e., each value shown is half the true concentration value. [Figure 21] Figure 21 shows comparative data between an exemplary human release product according to the present disclosure and a commercially available lysate. Figure 21A shows MSC doubling after 5 days for cells grown in DMEM growth medium mixed with supplement. Figure 21B shows the luminescence intensity obtained for hPR and controls. Controls include commercially available platelet lysate, chemically defined medium, and medium supplemented with fetal bovine serum. The results show that at least twice the amount of commercially available lysate is required to produce a population doubling level (PDL) similar to that of the release product of the present disclosure. The results also show the superiority of the disclosed release product as a medium supplement for the expansion and growth of MSC. [Figure 22A] 22A-22F show heat maps comparing biomarkers from exemplary releasates (Releasate F1 and Releasate F2) of the present disclosure with controls. Controls include human serum (AB Serum), commercially available platelet lysates (Lysate A, Lysate B, Lysate C), and media supplemented with fetal bovine serum (FBS). These data show that the composition of the releasates is different from that of the platelet lysates. FIG. 22A shows a heat map of all biomarkers tested, grouped into five groups based on similarity. [Figure 22B] FIG. 22B shows a portion of the heatmap of biomarkers in group 1. [Figure 22C] FIG. 22C shows a portion of the heatmap of biomarkers in group 2. [Figure 22D] FIG. 22D shows a portion of the heatmap of biomarkers in group 3. [Figure 22E] FIG. 22E shows a portion of a heatmap of biomarkers in group 4. [Figure 22F] FIG. 22F shows a portion of a heatmap of biomarkers in group 5. [Figure 23]FIG. 23 shows population doubling levels of mesenchymal stem cells (MSCs) from three different donors cultured with various additives: fetal bovine serum (FBS), human AB plasma (AB serum), commercially available lysates (Lysate A, Lysate B, Lysate C) or exemplary releasates of the present disclosure (Releasate F1, Releasate F2). [Figure 24] FIG. 24 shows the results of protein concentration assays performed on cells expanded with various supplements: fetal bovine serum (FBS), human AB plasma (AB serum), commercially available lysates (Lysate A, Lysate B, Lysate C), exemplary releasates of the present disclosure (Releasate F1, Releasate F2), or pooled plasma. [Diagram 25] Figures 25A and 25B show scanning electron microscope (SEM) images of resting platelets (Figure 25A) and activated platelets (Figure 25B). [Figure 26] Figure 26A is a SEM image of an activated platelet showing local changes due to the activation process (CaCl2 addition, agitation), and Figure 26B is a transmission electron microscope (TEM) image showing a cross-section of an activated platelet demonstrating degranulation, where vesicles (e.g., alpha granules, dense granules, mitochondria, etc.) exit the cell and release their contents into the surrounding medium or remain intact. [Figure 27] Figure 27 shows a TEM image of a cross-section of an activated platelet, in which granules (e.g., alpha granules, dense granules, mitochondria, etc.) are observed to migrate toward the periphery of the cell and detach or be released as separate vesicles (arrows, top left and top right). Extracellular vesicles have been shown to either release their contents into the surrounding medium or remain intact (arrow, bottom left). [Figure 28] Figure 28 is a TEM image of a cross-section of an activated platelet showing degranulation, where granules are observed to migrate toward the periphery of the cell and detach or be released as separate vesicles. The vesicles are shown to vary in size (arrows, bottom left) and content. The cells are also shown to release factors into the surrounding medium (arrow, center left). [Figure 29]Figure 29 shows a TEM image of a cross-section of a platelet after degranulation, demonstrating that the cells are devoid of granules (e.g., alpha granules, dense granules, mitochondria, etc.) but remain intact (i.e., not lysed). Extracellular vesicles (dark strands) that are trapped with fibrin are also shown. [Diagram 30] Figure 30A shows an SEM image of platelets embedded or encapsulated in a fibrin matrix, and Figure 30B shows strands of fibrin polymer with platelets trapped within the mesh. [Diagram 31] FIG. 31 shows the results of bone marrow-derived MSC (BM-MSC) expansion and MSC marker analysis. [Diagram 32] Figures 32A-32G show microscopic images of BM-MSCs expanded in basal growth medium supplemented with one of various supplements, as indicated, to assess the adipogenic potential of BM-MSCs. [Diagram 33] Figures 33A-33G show microscopic images of BM-MSCs expanded in basal growth medium supplemented with one of various supplements, as indicated, to assess the chondrogenic potential of BM-MSCs. [Diagram 34] Figures 34A-34G show microscopic images of BM-MSCs expanded in basal growth medium supplemented with one of various supplements, as indicated, to assess the osteogenic potential of BM-MSCs. [Diagram 35] 35 shows the results of an immunomodulation experiment in which BM-MSCs were expanded in basal growth medium supplemented with one of various supplements as indicated and assessed for immunomodulation via synthesis of indoleamine 2,3-dioxygenase (IDO) in response to cytokine stimulation. Samples, from left to right for each condition, are FBS, lysate A, lysate C, releasate F1 and releasate F2. [Diagram 36] FIG. 36 shows the results of an immunomodulation experiment in which BM-MSCs were expanded in basal growth medium supplemented with one of various supplements, as indicated, and immunomodulation was assessed by co-culturing peripheral blood mononuclear cells (PBMCs) in the presence of BM-MSCs for 5 days. [Figure 37] FIG. 37 shows the results of an immunomodulation experiment in which BM-MSCs were expanded in basal growth medium supplemented with one of a variety of supplements, as indicated, and immunomodulation was assessed by co-culturing PBMCs in the presence of BM-MSCs for 5 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Description of exemplary embodiments I. Definition As used herein, the term "current good manufacturing practice (cGMP)" refers to a system of minimum guidelines that ensure that products are consistently manufactured and controlled according to quality standards. cGMP are designed to minimize risks associated with drug manufacturing that cannot be eliminated through end-product testing. These guidelines establish minimum requirements that manufacturers must meet to ensure that their products are consistently of high quality from batch to batch for their intended use.

[0048] The term "cell culture" refers to the maintenance and propagation of cells in vitro. The cells can include stem and progenitor cells.

[0049] "Cell culture medium" is used for the maintenance of cells in in vitro culture. For some cell types, the medium may also be sufficient to support the expansion and growth of the cells in culture. The medium according to the present invention provides nutrients such as an energy source, amino acids and inorganic ions, as well as other compounds known to those skilled in the art.

[0050] The term "cell culture medium supplement" in the sense of the present disclosure refers to a medium additive that is added to a culture medium to stimulate the proliferation, differentiation and expansion of cells. Typically, the supplement contains one or more growth factors that are involved in stimulating proliferation and / or differentiation.

[0051] The term "cell expansion" is intended to mean the multiplication of cells, thereby resulting in an increase in cell number.

[0052] The term "growth factor" is intended to include proteins that stimulate cell proliferation by binding to specific receptors. Typically, growth factors act only on specific cell types that express their respective receptors.

[0053] "Subject", "individual" or "patient" or "injured person" are used interchangeably herein and refer to a vertebrate, such as a primate, mammal, or human. Mammals include, but are not limited to, horses, dogs, cats, pigs, cows, chickens, cats, pigs, rabbits, dolphins, sheep, mice, rats, monkeys, humans, farm animals, sport animals, and pets. Also intended to be included herein are any subjects involved in clinical research studies that do not show clinical signs of disease, or subjects used as controls.

[0054] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the described purpose. "Consisting essentially of," in the context of the biological compositions of the present disclosure, is intended to include all the recited active agents and excludes any additional active agents not recited, but does not exclude other components of the composition that are not active ingredients. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharma- ceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps for administering the compositions of the present invention or process steps for producing the compositions or achieving the intended results. Embodiments defined by each of these transitional phrases are within the scope of the present disclosure.

[0055] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination or to the exclusion of one another. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0056] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the art of cell biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0057] The term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes unspecified elements, steps, or ingredients. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term "comprising" can also be implemented in the context of the term "consisting of" or "consisting essentially of."

[0058] It is specifically contemplated that any limitations discussed with respect to one embodiment of the present invention may be applied to any other embodiment of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to manufacture or utilize any composition of the present invention. Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, such as in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legends.

[0059] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0060] The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0061] The term "mesenchymal stromal cells" refers to a subpopulation of fibroblasts or fibroblast-like non-hematopoietic cells that have the property of plastic adhesion and are capable of in vitro differentiation into cells of mesodermal origin. Mesenchymal stromal cells are derived from bone marrow, adipose tissue, umbilical cord (Wharton's gel), umbilical cord perivascular cells, umbilical cord blood, amniotic fluid, placenta, skin, dental pulp, breast milk and synovium. Mesenchymal stromal cells have clonogenic potential and can differentiate into several cells of mesodermal origin, such as adipocytes, osteoblasts, chondrocytes, skeletal muscle cells or visceral stromal cells. The term "mesenchymal stem cells" refers to the cultured (self-renewing) progeny of primary mesenchymal stromal cell populations. Mesenchymal stromal / stem cells (MSC) refer to mesenchymal stromal cells and / or mesenchymal stem cells.

[0062] The term "osteobiologic material" refers to any material capable of inducing and / or supporting existing or new bone growth. In some embodiments, the osteobiological material is a load-bearing material. The term also includes engineered materials (e.g., amniotic membrane- or placenta-derived materials, 3D printed constructs, etc.) that promote healing of fractures and bone defects.

[0063] Microcarriers are support matrices that support the growth of adherent cells. Microcarrier cell culture can be carried out in flasks or bioreactors (e.g., hollow fiber bioreactors, wave bioreactors, etc.).

[0064] A "releasate" according to the present disclosure is a product or extract obtained from cells when manipulated (e.g., degranulated) according to the protocols or methods of the present disclosure. The releasate describes a product obtained from cells in the absence of complete cell lysis. The releasate may be obtained from platelet cells (i.e., platelet releasate). The releasate may be used as a supplement in cell culture medium for culturing or expanding cells. The releasate may also be used in therapeutic applications, including bone biologics. The releasate may include all of the growth factors or compounds in Figures 1A-1O or Table 1. The releasate may exclude any one or any combination of the growth factors or compounds in Figures 1A-1O or Table 1. In some embodiments, the releasate includes microvesicles or exosomes from cells. In some embodiments, the releasate includes exosomes isolated or purified from cells after the manipulation (e.g., degranulation) procedure disclosed herein.

[0065] The terms "degranulate," "degranulated," "degranulating," and "degranulation" describe protocols or methods for cell manipulation disclosed herein whereby one or more vesicles or intracellular compartments of a cell 1) release their contents to the outside of the cell and / or 2) are removed or eliminated from the cell. Degranulation describes a process that does not include complete cell lysis.

[0066] As used herein, "treat," "treatment," or "therapy" refers to an approach or methodology used to obtain beneficial or desired clinical results, including the alleviation of symptoms, the reduction of inflammation, and the improvement of diseased or wounded tissues or organs.

[0067] The term "stem cell" refers to any cell that has the characteristics of being unspecialized, capable of renewal over long periods of time through cell division, and capable of being induced to become cells with specialized functions.

[0068] As used herein, the terms "comprising" or "comprising" are used in reference to compositions, methods, and each component thereof that is essential to the invention, but are still open to the inclusion of non-specified elements, whether essential or not.

[0069] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention. With respect to pharmaceutical compositions, the term "consisting essentially of" includes the recited active ingredients and excludes other active ingredients, but does not exclude any pharmaceutical excipients or other ingredients that are not therapeutically active.

[0070] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of an embodiment.

[0071] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will be apparent to those skilled in the art upon reading this disclosure, etc.

[0072] The word "and / or" means "and" or "or." By way of example, A, B and / or C includes A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination. In other words, "and / or" functions as an inclusive "or."

[0073] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the measuring or quantitating method.

[0074] II. Overview The inventors have developed a process capable of industrially producing platelet releasate (hPR), a xeno-free alternative to both FBS and platelet lysate. hPR prevents gelling of growth media and reduces the need for heparin or other anticoagulants. The disclosed process results in batches of hPR that are consistently and sufficiently rich in growth factors and other compounds beneficial to cell growth. In some embodiments, the hPR manufacturing process is carried out at an industrial scale in a closed system that can yield up to 10 liters, 50 liters, 100 liters, or hundreds of liters of hPR. In some embodiments, the yield is 4.5-10 L. In some embodiments, the yield is about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 liters, at least about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 liters, or at most about 2.0, 2.5, 3.0, 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 liters, or any value derivable therein. In some embodiments, the yield is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 liters, at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 liters, or at most about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, The volume of the hPR produced may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 liters or any value derivable therein. In some embodiments, for example, 10, 20, 30, 40, 50, or 100 liter batches of hPR are prepared in 10 L, 20 L, 30 L, 40 L, 50 L, or 100 L bags. In some embodiments, the yields can be pooled.

[0075] The release of the present disclosure may be used as a cell culture medium supplement for culturing, expanding or differentiating various cells, including stem cells and their associated lineages. In some embodiments, the release is used as a component of a storage medium for long-term storage of cells, such as cold storage or cryogenic storage. Exemplary cells include, but are not limited to, pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), bone marrow-derived mesenchymal stromal / stem cells (BM-MSCs), hematopoietic stem cells (HSCs), adipose-derived mesenchymal stromal / stem cells (ADP-MSCs), T cells, B cells, natural killer cells, dendritic cells, peripheral blood-derived mononuclear cells, cancer cells cancer stem cells, umbilical cord blood-derived cells, umbilical cord blood tissue-derived cells, placenta-derived cells, retinal cells, neural cells, fibroblasts, epithelial cells, endothelial cells or keratinocytes. In addition, the release of the present disclosure may be used to culture / expand Chinese hamster ovary (CHO) cells to produce antibodies, recombinant proteins and peptides. Other cells include fibroblasts, osteoblasts, adipocytes, chondrocyte endothelial cells, cells of the immune system, T cells, B cells, NK cells, modified / engineered cells and neural cells.

[0076] Salt activation Aspects of the disclosed method include activation of platelets using salts. Salts can act as clotting agents when added to platelets and can cause the formation of a fibrin clot that is gel-like and contains cellular debris. A variety of salts are contemplated and can be used in the disclosed method for preparing a release product, including, for example, calcium and magnesium salts. In some embodiments, calcium salts are used, such as calcium sulfate, calcium gluconate, calcium citrate, calcium phosphate, calcium chloride (CaCl2), calcium acetate, and / or calcium carbonate. In some embodiments, calcium chloride (CaCl2) is used. In some embodiments, magnesium salts are used, such as magnesium sulfate (MgSO4), magnesium gluconate, magnesium citrate, magnesium phosphate, magnesium chloride (MgCl2), magnesium acetate, and / or magnesium carbonate.

[0077] In some embodiments, the inventors have developed a method for preparing hPR from platelet-rich plasma based on the unexpected discovery that much higher salt concentrations, for example, on the order of 25 mM to 80 mM for CaCl2, result in an improved product. This discovery contradicts conventional recommendations and teachings in the art, resulting in a much more efficient process that can be completed in 3 to 4 hours from start to finish. Upon removal of the clot, the resulting liquid is a clear supernatant that is replete with growth factors, microparticles (e.g., exosomes), and other bioactive substances. The amount of CaCl added can result in a final concentration of CaCl of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 mM, or 100 mM, at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 mM, or 100 mM, or at most about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 mM, or 100 mM, or any other value derivable therein.

[0078] The source of platelets can be expired plasma-rich platelets or any suitable blood source. In some embodiments, the blood source is human. The source of platelets can be frozen or maintained at room temperature. The platelets can be frozen at -50°C or -80°C or any suitable temperature in between. The platelets can be frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours, or any range or value derivable therein. In some embodiments, the platelets are frozen for less than 24 hours. The platelets can be frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 100, 200, or 300 days, or for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 100, 200, or 300 days, or any range or value derivable therein.

[0079] Expired units of platelet rich plasma may be pooled with respect to ABO blood type. Blood count and / or infectious disease marker test results may be obtained and units may be pooled together accordingly. In some embodiments, type A plasma rich platelets are used. In some embodiments, type B plasma rich platelets are used. In some embodiments, type AB plasma rich platelets are used. In some embodiments, only type O plasma rich platelets are used. In some embodiments, platelets are 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 51 0, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 , 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 14 00, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790,1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, 2000, 2010, 2020, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140, 2 150, 2160, 2170, 2180, 2190, 2200, 2210, 2220, 2230, 2240, 2250, 2260, 2270, 2280, 2290, 2300, 2310, 2320, 2330, 2340, 2350, 2360, 2370, 2380, 2390, 2400, 2410, 2420, 2430, 2440, 2450, 2460, 2470, 2480, 2490, 25 00, 2510, 2520, 2530, 2540, 2550, 2560, 2570, 2580, 2590, 2600, 2610, 2620, 2630, 2640, 2650, 2660, 2670, 2680, 2690, 2700, 2710, 2720, 2730, 2740, 2750, 2760, 2770, 2780, 2790, 2800, 2900, 3000, 3100, 3200, 330 The pool may be up to a volume of 0, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milliliters (ml), deciliters (dl), or liters (l or L), including all values ​​and ranges therebetween.

[0080] In some embodiments of the disclosed methods, the platelets are concentrated prior to further processing. In some embodiments, the platelets are not concentrated prior to further processing. After adding salt (e.g., CaCl2) to the platelets, the mixture is agitated by any suitable method, such as, for example, using an orbital shaker to agitate the contents. Agitation can be performed at any suitable rpm. In some embodiments, the CaCl2 / PRP mixture is agitated at 50 rpm to 500 rpm. Agitation can be performed at any suitable rpm. In some embodiments, the CaCl2 / PRP mixture is agitated at 150 rpm to 350 rpm.In some embodiments, the CaCl2 / PRP mixture is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310 , 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 rpm, at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 23 5, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 rpm, or at most about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 15 5, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 rpm, or any value derivable therein.In some embodiments of the disclosed methods involving a batch size of 50 L or 100 L, the rpm is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 270, 275, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 8 0, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, or at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 11 The agitation may be 0, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, or any value derived therein. The agitation may include one or more mechanical forces. In some embodiments, the agitation is severe agitation and includes severe mechanical forces. In some embodiments, the agitation is gentle agitation and includes gentle mechanical forces.Mechanical forces that can be used for agitation include, for example, grinding (e.g., mortar / pestle, cryogrinder, glass homogenizer, bead homogenizer), beating with beads and / or spatulas, shearing (e.g., mixing; vortexing with glass, ceramic or steel beads; rotor / stator, dounce homogenizer homogenization), acoustic and ultrasonic treatment (e.g., bath and probe sonicators; infrasonic, sonic and ultrasonic wavelengths), French press / French pressure cell, optical methods (e.g., pyrolysis, cavitation), electromagnetic fields (e.g., microwave, shocking, electroporation), rocking, shaking (e.g., vortex shaker, platform shaker, orbital shaker, incubator shaker), stirring (e.g., magnetic stirring), fluidization, milling, freezing / heat treatment, and high pressure homogenization.

[0081] After addition of salt (e.g., CaCl2), the clotting time may be any suitable time and the mixture may be stirred. In some embodiments of the disclosed methods, the CaCl2 / PRP mixture is allowed to form a clot for 20 minutes to 4 hours. In some embodiments, the clotting time is 60 minutes to 90 minutes. The clotting time can be 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, 200 minutes, 205 minutes, 210 minutes, 215 minutes, 220 minutes, 225 minutes, 230 minutes, 235 minutes, 240 minutes, or any suitable amount in between. In some embodiments, the CaCl2 / PRP mixture is stirred for less than 180 minutes. In further embodiments, the CaCl2 / PRP mixture is stirred for 30 to 150 minutes or 45 to 135 minutes. In yet other embodiments, the CaCl2 / PRP mixture is stirred for 60 to 90 minutes. In some embodiments, some agents (such as kaolin) can accelerate clotting in 2 to 5 minutes.When such factors are used with the present method, the clotting times can be increased to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 225, 230, 235, 230, 235, 240, 245, 250, 250, 255 5, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 minutes, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 11 5, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 minutes, or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 minutes, or any range derivable therein.

[0082] After the clot is formed, the clot can be separated from the liquid material by any suitable method. In one embodiment, the clot is separated by centrifugation. In some cases, the mixture is centrifuged at a suitable temperature. For example, the mixture can be centrifuged at 4000 g at 4° C. In one embodiment, the clot is separated from the liquid material by decanting the liquid using an expressor or gravity separation techniques.about 500, 1000, 2000, 3000, 4000, 5000, about 6000, 7000, 8000 g, or more (or any range derivable therein), at least about 500, 1000, 2000, 3000, 4000, 5000, about 6000, 7000, 8000 g, or more (or any range derivable therein), or at most about 500, 1000, 2000, 3000, 4000, 5000, about 6000, 7000, 8000 g, or more (or any range derivable therein). , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes, and / or 1, 2, 3, 4, or more hours (or any range derivable therein), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes, and / or at least 1, 2, 3, 4 or more hours (or any range derivable therein), or at most 1, , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes, and / or for at most 1, 2, 3, 4, or more hours (or any range derivable therein).

[0083] In some embodiments, the method of the present disclosure further comprises filtering the exhaled material. Filtering the exhaled material reduces the amount of fibrinogen and other contaminants in the final product. Filtration can be performed in one or two passes. In some embodiments, the exhaled material is filtered using a filter that is between 0.45 microns and 1.0 microns. In some embodiments, the exhaled material is filtered using a filter that is between 0.45 microns and 0.65 microns. In some embodiments, the filter is between 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0 microns, or any size derivable therein. In some embodiments, the filter is a 3 micron to 10 micron filter.In some embodiments, the filter is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12. .2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 microns, at least 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6. 6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 microns or at most 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 microns, or any size derivable therein. In some embodiments, the filter is a 170 micron to 260 micron filter.In some embodiments, the filter is a 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260 micron filter, or any range derivable therein.

[0084] In some embodiments, the release is pooled after production. In some embodiments, the release is pooled after production. 20, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1 180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 166 0, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, 2000, 2010, 2020, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140,2150, 2160, 2170, 2180, 2190, 2200, 2210, 2220, 2230, 2240, 2250, 2260, 2270, 2280, 2290, 2300, 2310, 2320, 2330, 2340, 2350, 2360, 2370, 2380, 2390, 2400, 24 10, 2420, 2430, 2440, 2450, 2460, 2470, 2480, 2490, 2500, 2510, 2520, 2530, 2540, 2550, 2560, 2570, 2580, 2590, 2600, 2610, 2620, 2630, 2640, 2650, 2660, 2670 , 2680, 2690, 2700, 2710, 2720, 2730, 2740, 2750, 2760, 2770, 2780, 2790, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milliliters (ml), deciliters (dl), or liters (l or L), volumes, including all values ​​and ranges therebetween.

[0085] Growth factors and other bioactive compounds in the hPR The hPR products of the present disclosure retain multiple bioactive components, such as growth factors, cytokines and chemokines, in amounts and / or combinations such that stem cells, e.g., bone marrow derived mesenchymal stem cells, when expanded in the presence of hPR, have superior expansion rates compared to FBS, platelet lysate and chemically defined media. Growth factors, cytokines, chemokines, interferons or other components may be expressed as amphiregulin (AR) (colorectal cell-derived growth factor) (CRDGF), brain-derived neurotrophic factor (BDNF) (abrineurin), fibroblast growth factor 2 (FGF-2) (basic fibroblast growth factor) (bFGF) (heparin-binding growth factor 2) (HBGF-2), bone morphogenetic protein 4 (BMP-4) (bone morphogenetic protein 2B) (BMP-2B), bone morphogenetic protein 5 (BMP-5), bone morphogenetic protein 7 (BMP-7) (bone morphogenetic protein 1) (OP-1) (eptothemin alpha), beta-nerve growth factor (beta-NGF), pro-epidermal growth factor (EGF) [cleaved to become epidermal growth factor (urogastrone)], epidermal growth factor receptor (EC 2.7.10).1) (proto-oncogene c-ErbB-1) (receptor tyrosine protein kinase erbB-1), prokineticin-1 (endocrinoline-derived vascular endothelial growth factor) (EG-VEGF) (mambacaine), fibroblast growth factor 4 (FGF-4) (heparin-secreted transforming protein 1) (HST) (HST-1) (HSTF-1) (heparin-binding growth factor 4) (HBGF-4) (transforming protein KS3), fibroblast growth factor 7 (FGF-7) (heparin-binding growth factor 7) (HBGF-7) (keratinocyte growth factor), growth / differentiation factor 15 (G DF-15) (macrophage inhibitory cytokine 1) (MIC-1) (NSAID-activated gene 1 protein) (NAG-1) (NSAID-regulated gene 1 protein) (NRG-1) (placental TGF-beta) (placental bone morphogenetic protein) (prostate differentiation factor), glial cell line-derived neurotrophic factor (hGDNF) (astrocyte-derived trophic factor) (ATF), somatotropin (growth hormone) (GH) (GH-N) (growth hormone 1) (pituitary growth hormone), proheparin-binding EGF-like growth factor [cleaved and heparin-binding EGF-like growth factor (HB-E)] GF) (HBEGF) (diphtheria toxin receptor) (DT-R)], hepatocyte growth factor (hepatopoietin-A) (scatter factor) (SF) [cleaved to hepatocyte growth factor alpha chain; hepatocyte growth factor beta chain], insulin-like growth factor binding protein 1 (IBP-1) (IGF binding protein 1) (IGFBP-1) (placental protein 12) (PP12), insulin-like growth factor binding protein 2 (IBP-2) (IGF binding protein 2) (IGFBP-2), insulin-like growth factor binding protein 3 (IBP-3) (IGF binding protein protein 3 (IGFBP-3), insulin-like growth factor binding protein 4 (IBP-4) (IGF binding protein 4) (IGFBP-4), insulin-like growth factor binding protein 6 (IBP-6) (IGF binding protein 6) (IGFBP-6), insulin-like growth factor I (IGF-I) (mechanism of growth factor) (MGF) (somatomedin-C), insulin [cleaved to insulin B chain; insulin A chain], macrophage colony-stimulating factor 1 receptor (CSF-1 receptor) (CSF-1R) (M-CSF-R) (EC 2.7.10.1) (proto-oncogene c-Fms) (CD antigen CD115), tumor necrosis factor receptor superfamily member 16 (Gp80-LNGFR) (low affinity neurotrophin receptor p75NTR) (low affinity nerve growth factor receptor) (NGF receptor) (p75 ICD) (CD antigen CD271), neurotrophin-3 (NT-3) (HDNF) (nerve growth factor 2) (NGF-2) (neurotrophic factor), neurotrophin-4 (NT-4) (neurotrophin-5) (NT-5) (neurotrophic factor 4), tumor necrosis factor receptor superfamily member 11B (osteoclastogenesis inhibitory factor) (osteoprotegerin), platelet-derived growth factor subunit A (PDGF subunit A) (PDGF-1) (platelet-derived growth factor A chain) (platelet-derived growth factor alpha polypeptide), placenta growth factor (PlGF), Kit ligand (mast cell growth factor) (MGF) (stem cell factor) (SCF) (c-Kit ligand) [cleaved to soluble KIT ligand (sKITLG)], mast / stem cell growth factor receptor Kit (SCFR) (EC 2.7.10.1) (Piebald trait protein) (PBT) (proto-oncogene c-Kit) (tyrosine protein kinase Kit) (p145 c-kit) (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) (CD antigen CD117), pro-transforming growth factor alpha [cleaved to transforming growth factor alpha (TGF-alpha) (EGF-like TGF) (ETGF) (TGF type 1)], transforming growth factor beta-1 (TGF-beta-1) [cleaved to latency-associated peptide (LAP)], transforming growth factor beta-3 (TGF-beta-3) [cleaved to latency-associated peptide (LAP)], vascular endothelial growth factor A (VEGF-A) (vascular permeability factor) (VPF), vascular endothelial growth factor receptor 2 (VEGFR-2) (EC 2.7.10.1) (fetal liver kinase 1) (FLK-1) (kinase insert domain receptor) (KDR) (protein-tyrosine kinase receptor flk-1) (CD antigen CD309), vascular endothelial growth factor receptor 3 (VEGFR-3) (EC 2.7.10.1) (Fms-like tyrosine kinase 4) (FLT-4) (tyrosine protein kinase receptor FLT4), vascular endothelial growth factor D (VEGF-D) (c-Fos-induced growth factor) (FIGF). In some embodiments, it is specifically contemplated that any of these may be included in the yield, while in other embodiments, it is specifically contemplated that none of these may be included in the yield.

[0086] Other bioactive compounds include CC motif chemokine 21 (6Ckine) (β-chemokine exodus-2) (secondary lymphoid tissue chemokine) (SLC) (small inducible cytokine A21), tyrosine protein kinase receptor UFO (EC 2.7.10.1) (AXL oncogene), probetacellulin [cleaved to become β-cellulin (BTC)], CC motif chemokine 28 (mucosa-associated epithelial chemokine) (MEC) (protein CCK1) (small inducible cytokine A28), CC motif chemokine 27 (CC chemokine ILC) (cutaneous T cell-attracting chemokine) (CTACK) (ESkine) (IL-11 R-α-locus chemokine) (skinkine) (small inducible cytokine A27), CXC motif chemokine 16 (scavenger receptor for phosphatidylserine and oxidized low density lipoprotein) (SR-PSOX) (small inducible cytokine B16) (transmembrane chemokine CXCL16), CXC motif chemokine 5 (ENA-78(1-78)) (epithelial-derived neutrophil-activating protein 78) (neutrophil-activating peptide ENA-78) (small inducible cytokine B5) [cleaved to ENA-78(8-78); ENA-78(9-78)], CC motif chemokine 26 (CC chemokine IMAC) (eotaxin-3) (macrophage inflammatory protein 4-α) (MIP-4-α) (small inducible site kine A26) (thymic stromal chemokine-1) (TSC-1), CXC motif chemokine 6 (chemokine alpha 3) (CKA-3) (granulocyte chemotactic protein 2) (GCP-2) (small inducible cytokine B6) [cleaved to become small inducible cytokine B6, N-processed variant 1; small inducible cytokine B6, N-processed variant 2; small inducible cytokine B6, N-processed variant 3], growth regulatory alpha protein (CXC motif chemokine 1) (GRO-alpha(1-73)) (melanoma growth stimulatory activity) (MGSA) (neutrophil activating protein 3) (NAP-3) [cleaved to become GRO-alpha(4-73); GRO-alpha(5-73); GRO-alpha(6-73)];CXC motif chemokine 2 (growth regulatory protein beta) (Gro-β) (macrophage inflammatory protein 2-α) (MIP2-α) [cleaved to GRO-β(5-73) (GRO-β-T) (hematopoietic synergistic factor) (HSF) (SB-251353)]; CXC motif chemokine 3 (GRO-γ(1-73)) (growth regulatory protein gamma) (GRO-γ) (macrophage inflammatory protein 2-β) (MIP2-β) [cleaved to GRO-γ(5-73)]; CC motif chemokine 14 (chemokine CC-1 / CC-3) (HCC-1 / HCC-3) (HCC-1(1-74)) (NCC-2) (small inducible cytokine A14) [cleaved to HCC-1(3-74); HCC-1(4-74);HCC-1(9-74)], CC motif chemokine 16 (chemokine CC-4) (HCC-4) (chemokine LEC) (IL-10-inducible chemokine) (LCC-1) (liver expressed chemokine) (lymphocyte and monocyte chemoattractant) (LMC) (monotactin-1) (MTN-1) (NCC-4) (small inducible cytokine A16), interferon-9 (IL-9) (cytokine P40) (T cell growth factor P40), interleukin-17F (IL-17F) (cytokine ML-1), interleukin-18-binding protein (IL-18BP) (Tadekin-alpha), interferon lambda-2 (IFN-lambda-2) (cytokine Zcyto20) (Interleukin-28A) (IL-28A), Interferon lambda-1 (IFN-lambda-1) (Cytokine Zcyto21) (Interleukin-29) (IL-29), Interferon-31 (IL-31), C-X-C motif chemokine 10 (10 kDa interferon gamma-inducible protein) (gamma-IP10) (IP-10) (small inducible cytokine B10) [cleaved to become CXCL10(1-73)], CXC motif chemokine 11 (beta-R1) (H174) (interferon gamma-inducible protein 9) (IP-9) (interferon-inducible T cell alpha chemoattractant) (I-TAC) (small inducible cytokine B11), leukemia inhibitory factor (LIF) (differentiation stimulating factor) (Factor D) (melanoma-derived LPL inhibitor) (MLPLI) (Emfilermin), tumor necrosis factor ligand superfamily member 14 (herpes virus entry mediator ligand) (HVEM-L) (herpes virus entry mediator ligand) (CD antigen CD258) [cleaved to become tumor necrosis factor ligand superfamily member 14, membrane form;tumor necrosis factor ligand superfamily member 14, becomes soluble], lymphotactin (ATAC) (C motif chemokine 1) (cytokine SCM-1) (lymphotaxin) (SCM-1-α) (small inducible cytokine C1) (XC chemokine ligand 1), CC motif chemokine 8 (HC14) (monocyte chemoattractant protein 2) (monocyte chemotactic protein 2) (MCP-2) (small inducible cytokine A8) [cleaved to become MCP-2(6-76)], CC motif chemokine 7 (monocyte chemotactic protein 3) (monocyte chemotactic protein 3) (MCP-3) (NC28) (small inducible cytokine A7), CC motif chemokine 13 (CK-β-10) (monocyte chemotactic protein 4) (MCP-4) (NCC-1) (small inducible cytokine A13) [cleaved to CC motif chemokine 13, long chain; CC motif chemokine 13, medium chain; CC motif chemokine 13, short chain], CC motif chemokine 22 (CC chemokine STCP-1) (MDC(1-69)) (macrophage-derived chemokine) (small inducible cytokine A22) (stimulated T cell chemotactic protein 1) [cleaved to MDC(3-69); MDC(5-69); MDC(7-69)], macrophage migration inhibitory factor (MIF) (EC 5.3.2.1) (glycosylation inhibitor) (GIF) (L-dopachrome isomerase) (L-dopachrome tautomerase) (EC 5.3.3.12) (phenylpyruvate tautomerase), CC motif chemokine 20 (beta chemokine exodus-1) (CC chemokine LARC) (liver and activation-regulated chemokine) (macrophage inflammatory protein 3 alpha) (MIP-3-alpha) (small inducible cytokine A20) [cleaved, CCL20(1-67); CCL20(1-64);These include myeloid progenitor inhibitory factor (MPIF-1), also known as CCL20(2-70), CC motif chemokine 19 (β chemokine exodus-3) (CKβ-11) (Epstein-Barr virus-induced molecule 1 ligand chemokine) (EBI1 ligand chemokine) (ELC) (macrophage inflammatory protein 3β) (MIP-3-β) (small inducible cytokine A19), and myeloid progenitor inhibitory factor (MPIF-1), also known as CKβ8 and MIP-3. Alternative splicing of the MPIF-1 gene results in two mRNAs that encode the short (CKβ8) and long (CKβ8-1) isoforms of the chemokine. CC motif chemokine 23 (CK-β-8) (CKB-8) (macrophage inflammatory protein 3) (MIP-3) (myeloid progenitor inhibitory factor 1) (MPIF-1) (small inducible cytokine A23) [cleaved to CCL23(19-99); CCL23(22-99); CCL23(27-99); CCL23(30-99)], hepatocyte growth factor-like protein (macrophage stimulating protein) (MSP) [cleaved to hepatocyte growth factor-like protein α chain; hepatocyte growth factor-like protein β chain], platelet basic protein (PBP) (CXC motif chemokine 7) (leukocyte-derived growth factor) (LDGF) (macrophage-derived growth factor) (MDGF) (small inducible cytokine B7) [cleaved, connective tissue activating peptide III (CTAP-III) (LA-PF4) (low affinity platelet factor IV); TC-2; connective tissue activating peptide III (1-81) (CTAP-III(1-81)); beta thromboglobulin (β-TG); neutrophil-activating peptide 2 (74) (NAP-2(74)); neutrophil-activating peptide 2 (73) (NAP-2(73)); neutrophil-activating peptide 2 (NAP-2); TC-1; neutrophil-activating peptide 2 (1-66) (NAP-2(1-66));Neutrophil-activating peptide 2(1-63) (NAP-2(1-63))], osteopontin (bone sialoprotein 1) (nephropontin) (secreted phosphoprotein 1) (SPP-1) (urolithiasis protein) (uropontin), CC motif chemokine 18 (selective macrophage activation-associated CC chemokine 1) (AMAC-1) (CC chemokine PARC) (dendritic cell chemokine 1) (DC-CK1) (macrophage inflammatory protein 4) (MIP-4) (pulmonary and activation-regulated chemokine) (small inducible cytokine A18) [cleaved to become CCL18(1-68); CCL18(3-69); CCL18(4-69)], platelet factor 4 (PF-4) (CXC motif chief chemokine 4 (Iroplact) (oncostatin-A) [cleaved to platelet factor 4, truncated], stromal cell-derived factor 1 (SDF-1) (hSDF-1) (CXC motif chemokine 12) (intercrine reduced in hepatoma) (IRH) (hIRH) (pre-B cell growth stimulating factor) (PBSF) [cleaved to SDF-1-β(3-72);SDF-1α(3-67)], CC motif chemokine 17 (CC chemokine TARC) (small inducible cytokine A17) (thymus and activation-regulated chemokine), and CC motif chemokine 25 (chemokine TECK) (small inducible cytokine A25) (thymus-expressed chemokine), thymic stromal lymphopoietin. It is specifically contemplated that in some embodiments any of these may be included in the yield, while in other embodiments any of these may not be included in the yield.

[0087] Additional bioactive substances present in the releasate may include: inhibin βA chain (activin βA chain) (erythroid differentiation protein) (EDF), agouti-related protein, angiogenin (EC 3.1.27.-) (ribonuclease 5) (RNase 5), angiopoietin-1 (ANG-1), plasminogen (EC 3.4.21.7) [cleaved to plasmin heavy chain A; activation peptide; angiostatin; plasmin heavy chain A, short form; plasmin light chain B], cathepsin S (EC 3.4.22.27), tumor necrosis factor receptor superfamily member 5 (B cell surface antigen CD40) (Bp50) (CD40L receptor) (CDw40) (CD antigen CD40), teratocarcinoma-derived growth factor 1 (Cripto-1 growth factor) (CRGF) (epidermal growth factor-like crypt protein CR1), poly(A)-specific ribonuclease PARN (EC 3.1.13.4) (deadenylation nuclease) (polyadenylate-specific ribonuclease), Dickkopf-related protein 1 (Dickkopf-1) (Dkk-1) (hDkk-1) (SK), cadherin-1 (CAM 120 / 80) (epithelial cadherin) (E-cadherin) (uvomorulin) (CD antigen CD324) [cleaved to E-Cad / CTF1; E-Cad / CTF2; E-Cad / CTF3], epithelial cell adhesion molecule (Ep-CAM) (adenocarcinoma-associated antigen) (cell surface glycoprotein Trop-1) (epithelial cell surface antigen) (epithelial glycoprotein) (EGP) (epithelial glycoprotein 314) (EGP314) (hEGP314) (KS1 / 4 antigen) (KSA) (major gastrointestinal tumor-associated protein GA733-2) (tumor-associated calcium signal transducer 1) (CD antigen CD326), tumor necrosis factor ligand superfamily member 6 (apoptosis antigen ligand) (APTL) (CD95 ligand) (CD95-L) (Fas antigen ligand) (Fas ligand) (FasL) (CD antigen CD178) [cleaved, tumor necrosis factor ligand superfamily member 6, membrane form; tumor necrosis factor ligand superfamily member 6, soluble form (receptor-binding FasL ectodomain) (soluble Fas ligand) (sFasL); FasL form processed by ADAM10 (APL); FasL intracellular domain (FasL ICD) (FasL form processed by SPPL2A) (SPA)], low affinity immunoglobulin gamma Fc region receptor II-b (IgG Fc receptor II-b) (CDw32) (Fc-γRII-b) (Fc-γ-RIIb) (FcRII-b) (CD antigen CD32); low affinity immunoglobulin γ Fc region receptor II-c (IgGFc receptor II-c (CDw32) (Fc-γRII-c) (Fc-γ-RIIc) (FcRII-c) (CD antigen CD32), follistatin (FS) (activin-binding protein), galectin-7 (Gal-7) (HKL-14) (PI7) (p53-inducible gene 1 protein), intercellular adhesion molecule 2 (ICAM-2) (CD antigen CD102), interleukin-13 receptor subunit α-1 (IL -13 receptor subunit alpha-1) (IL-13R subunit alpha-1) (IL-13R-α-1) (IL-13RA1) (cancer / testis antigen 19) (CT19) (CD antigen CD213a1), interleukin-13 receptor subunit alpha-2 (IL-13 receptor subunit alpha-2) (IL-13R subunit alpha-2) (IL-13R-α-2) (IL-13RA2) (interleukin-13-binding protein (CD antigen CD213a2), interleukin-17B (IL-17B) (cytokine Zcyto7) (interleukin-20) (IL-20) (neuronal interleukin-17-related factor), interleukin-2 receptor subunit alpha (IL-2 receptor subunit alpha) (IL-2-RA) (IL-2R subunit alpha) (IL2-RA) (TAC antigen) (p55) (CD antigen CD25), interleukin-2 receptor subunit beta (IL-2 receptor subunit beta) (IL-2R subunit beta) (IL-2RB) (high affinity IL-2 receptor subunit beta) (p70-75) (p75) (CD antigen CD122), interleukin-23 subunit alpha (IL-23 subunit alpha) (IL-23-A) (interleukin-23 subunit p19) (IL-23p19), human TGF-β1 The cDNA encodes a 390 amino acid (aa) precursor containing a 29 amino acid signal peptide and a 361 amino acid proprotein, which is further cleaved into the N-terminal 249 amino acid latency-associated peptide (LAP) and the C-terminal 112 amino acid mature TGF-β-1, neural cell adhesion molecule (Nr-CAM) (neuronal cell surface protein Bravo) (hBravo) (NgCAM-related cell adhesion molecule) (Ng-CAM-related), plasminogen activator inhibitor 1 (PAI) (PAI-1) (endothelial plasminogen activator inhibitor) (SerpinE1), platelet-derived growth factor subunit A (PDGF subunit A) (PDGF-1) (platelet-derived growth factor A chain) (platelet-derived growth factor alpha polypeptide); platelet-derived growth factor subunit B (PDGF subunit B) (PDGF-2) (platelet-derived growth factor B chain) (platelet-derived growth factor beta polypeptide) (proto-oncogene c-Sis) (becaplermin), resistin (adipose tissue-specific secretory factor) (ADSF) (C / EBP-ε-regulated bone marrow-specific secretory cysteine-rich protein) (cysteine-rich secreted protein A12-α-like 2) (cysteine-rich secreted protein FIZZ3), stromal cell-derived factor 1 (SDF-1) (hSDF-1) (CXC motif chemokine 12) (intercrine reduced in hepatoma) (IRH) (hIRH) (pre-B cell growth-stimulating factor) (PBSF) [truncated to SDF-1-β(3-72); SDF-1-α(3-67)]; SDF-1α and SDF-1β are encoded by a single gene and arise by alternative splicing. The two proteins are identical except for four amino acid residues present in the carboxy terminus of SDF-1β that are absent in SDF-1α, interleukin-6 receptor subunit beta (IL-6 receptor subunit beta) (IL-6R subunit beta) (IL-6R-β) (IL-6RB) (CDw130) (interleukin-6 signal transducer) (membrane glycoprotein 130) (gp130) (oncostatin-M receptor subunit α) (CD antigen CD130), and sonic hedgehog protein (SHH) (HHG-1) [cleaved to form sonic hedgehog hedgehog protein N product; becomes sonic hedgehog protein C product], sialic acid-binding Ig-like lectin 5 (Siglec-5) (CD33 antigen-like 2) (obesity binding protein 2) (OB-BP2) (OB binding protein 2) (CD antigen CD170), interleukin-1 receptor-like 1 (protein ST2), transforming growth factor beta-2 (TGF-β-2) (BSC-1 cell proliferation inhibitor) (cetermin) (glioblastoma-derived T cell suppressor factor) (G-TSF) (polyergin) [cleaved to become latency-associated peptide (LAP)], angiopoietin-1 receptor (EC 2.7.10.1) (endothelial tyrosine kinase) (endothelial (Tunicainterna) endothelial cell kinase) (tyrosine kinase with Ig and EGF homology domain-2) (tyrosine protein kinase receptor TEK) (tyrosine protein kinase receptor TIE-2) (hTIE2) (p140 TEK) (CD antigen CD202b), thrombopoietin (C-mpl ligand) (ML) (megakaryocyte colony-stimulating factor) (megakaryocyte growth and differentiation factor) (MGDF) (myeloproliferative leukemia viral oncogene ligand), tumor necrosis factor receptor superfamily member 10D (decoy receptor 2) (DcR2) (TNF-related apoptosis-inducing ligand receptor 4) (TRAIL receptor 4) (TRAIL-R4) (TRAIL receptor with truncated death domain) (CD antigen CD264), triggering receptor expressed on myeloid cells 1 (TREM-1) (triggering receptor expressed on monocytes 1) (CD antigen CD354), vascular endothelial growth factor C (VEGF-C) (Flt4 ligand) (Flt4-L) (vascular endothelial growth factor-related protein) (VRP), and vascular endothelial growth factor receptor 1 (VEGFR-1) (EC 2.7.10.1) (Fms-like tyrosine kinase 1) (FLT-1) (tyrosine protein kinase FRT) (tyrosine protein kinase receptor FLT) (FLT) (vascular permeability factor receptor). In some embodiments, it is specifically contemplated that any of these may be included in the yield, while in other embodiments, it is specifically contemplated that any of these may not be included in the yield.

[0088] Further embodiments of releasants according to the present disclosure include one or more of the following bioactive compounds: C-X-C motif chemokine 13 (Angie) (B cell attractant chemokine 1) (BCA-1) (B lymphocyte chemoattractant) (C-X-C chemokine BLC) (small inducible cytokine B13), Eotaxin (C-C motif chemokine 11) (eosinophil chemotactic protein) (small inducible cytokine A11), C-C motif chemokine 24 (CK-β-6) (eosinophil chemotactic protein 2) (eotaxin-2) (myeloid progenitor inhibitory factor 2) (MPIF-2) (small inducible cytokine A24), granulocyte colony activator 1 (C-C motif chemokine BLC) (small inducible cytokine B13), eotaxin (C-C motif chemokine BLC) (small inducible cytokine B13), eotaxin (C-C motif chemokine BLC) (eosinophil chemotactic protein 2) (eotaxin-2) (myeloid progenitor inhibitory factor 2) (MPIF-2) (small inducible cytokine A24 ... B13), eotaxin (C-C motif chemokine BLC) (eosinophil chemotactic protein B13), eotaxin (C-C motif chemokine BLC) (eosinophil chemotactic protein B13), eotaxin (C-C motif chemokine BLC) (eosinophil chemotact Knee-stimulating factor (G-CSF) (pluripoietin) (filgrastim) (lenograstim), granulocyte-macrophage colony-stimulating factor (GM-CSF) (colony-stimulating factor) (CSF) (molgramostin) (sargramostim), CC motif chemokine 1 (small inducible cytokine A1) (T-lymphocyte-secreted protein I-309), intercellular adhesion molecule 1 (ICAM-1) (major group rhinovirus receptor) (CD antigen CD54), interferon gamma (IFN-γ) (immune interferon ron), interleukin-1α (IL-1α) (hematopoietin-1), interleukin-1β (IL-1β) (catabolin), interleukin-1 receptor antagonist protein (IL-1RN) (IL-1ra) (IRAP) (ICIL-1RA) (IL1 inhibitor) (anakinra), interleukin-2 (IL-2) (T cell growth factor) (TCGF) (aldesleukin), interleukin-4 (IL-4) (B cell stimulating factor 1) (BSF-1) (binetrakin) (lymphocyte leukocyte stimulating factor 1) (pitrocytopenic leukocyte antigen 1), interleukin-5 (IL-5) (B cell differentiation factor I) (eosinophil differentiation factor) (T cell exchange factor) (TRF) interleukin-6 (IL-6) (B cell stimulating factor 2) (BSF-2) (CTL differentiation factor) (CDF) (hybridoma growth factor) (interferon beta-2) (IFN-beta-2), interleukin-6 receptor subunit alpha (IL-6 receptor subunit alpha) (IL-6R subunit alpha) (IL-6R-alpha) (IL-6RA) (IL-6R 1) (membrane glycoprotein 80) (gp80) (CD antigen CD126), interleukin-7 (IL-7),Interleukin-8 (IL-8) (CXC motif chemokine 8) (chemokine (CXC motif) ligand 8) (emoctakin) (granulocyte chemotactic protein 1) (GCP-1) (monocyte-derived neutrophil chemotactic factor) (MDNCF) (monocyte-derived neutrophil activating peptide) (MONAP) (neutrophil activating protein 1) (NAP-1) (protein 3-10C) (T cell chemotactic factor) [cleaved and transformed into MDNCF-a (GCP / IL-8 protein IV) (IL8 / NAP1 type I); interleukin-8 ((Ala-IL-8)77) (GCP / IL-8 protein II) (IL-8(1-77)) (IL8 / NAP1 type II) (MDNCF-b); IL-8(5-77); IL-8(6-77) ((Ser-IL-8)72) (GCP / IL-8 protein I) (IL-8 / NAP1 type III) (lymphocyte-derived neutrophil-activating factor) (LYNAP) (MDNCF-c) (neutrophil-activating factor) (NAF); IL-8(7-77) (GCP / IL-8 protein V) (IL-8 / NAP1 type IV); IL-8(8-77) (GCP / IL-8 protein VI) (IL8 / NAP1 type V); IL-8(9-77) (GCP / IL-8 protein III) (IL8 / NAP1 type VI)], interleukin-10 (IL-10) (cytokine synthesis inhibitory factor) (CSIF), interleukin-11 (IL-11) (adipogenesis inhibitory factor) (AGIF) (oprelvekin), interleukin-12 subunit beta (IL-12B) (cytotoxic lymphocyte maturation factor 40 kDa subunit) (CLMF p40) (IL-12 subunit p40) (NK cell stimulatory factor chain 2) (NKSF2), interleukin-12 subunit alpha (IL-12A) (cytotoxic lymphocyte maturation factor 35 kDa subunit) (CLMF p35) (IL-12 subunit p35) (NK cell stimulatory factor chain 1) (NKSF1); interleukin-12 subunit beta (IL-12B) (cytotoxic lymphocyte maturation factor 40 kDa subunit) (CLMF p40) (IL-12 subunit p40) (NK cell stimulatory factor chain 2) (NKSF2), interleukin-13 (IL-13), interleukin-15 (IL-15), pro-interleukin-16 [cleaved,interleukin-16 (IL-16) (lymphocyte chemotactic factor) (LCF)], interleukin-17A (IL-17) (IL-17A) (cytotoxic T lymphocyte-associated antigen 8) (CTLA-8), CC motif chemokine 2 (HC11) (monocyte chemotactic protein 1) (monocyte chemotactic and activating factor) (MCAF) (monocyte chemotactic protein 1) (MCP-1) (monocyte secreted protein JE) (small inducible cytokine A2), macrophage colony-stimulating factor 1 (CSF-1 ) (M-CSF) (MCSF) (lanimostim) [cleaved to become processed macrophage colony-stimulating factor 1], C-X-C motif chemokine 9 (monokine induced by gamma interferon) (monokine induced by interferon gamma) (HuMIG) (MIG) (small inducible cytokine B9), CC motif chemokine 3 (G0 / G1 switch regulatory protein 19-1) (macrophage inflammatory protein 1-alpha) (MIP-1-alpha) (PAT 464.1) (SIS-β) (small inducible cytokine A3) (tonsillar lymphocyte LD78α protein) [cleaved to MIP-1-α(4-69) (LD78-α(4-69))], CC motif chemokine 4 (G-26 T lymphocyte secreted protein) (HC21) (lymphocyte activation gene 1 protein) (LAG-1) (MIP-1-β(1-69)) (macrophage inflammatory protein 1-β) (MIP-1-β) (PAT 744) (protein H400) (SIS-γ) (small inducible cytokine A4) (T cell activation protein 2) (ACT-2) [cleaved to MIP-1-β(3-69)], CC motif chemokine 15 (chemokine CC-2) (HCC-2) (leukotactin-1) (LKN-1) (MIP-1δ) (macrophage inflammatory protein 5) (MIP-5) (Mrp-2b) (NCC-3) (small inducible cytokine A15) [cleaved to CCL15(22-92); CCL15(25-92); CCL15(29-92)], platelet-derived growth factor subunit B (PDGF subunit B) (PDGF-2) (platelet-derived growth factor B chain) (platelet-derived growth factor β polypeptide) (proto-oncogene c-Sis) (becaplermin),CC motif chemokine 5 (EoCP) (eosinophil chemotactic cytokine) (SIS-δ) (small inducible cytokine A5) (T cell specific protein P228) (TCP228) (T cell specific protein RANTES) [cleaved to RANTES(3-68); RANTES(4-68)], metalloproteinase inhibitor 1 (erythroid enhancing activity) (EPA) (fibroblast collagenase inhibitor) (collagenase inhibitor of metalloproteinase 1) Tissue inhibitor of metalloproteinases 2 (TIMP-1), tumor necrosis factor (cachectin) (TNF-α) (tumor necrosis factor ligand superfamily member 2) (TNF-a) [cleaved, tumor necrosis factor, membrane form (N-terminal fragment) (NTF); intracellular domain 1 (ICD1); intracellular domain 2 (ICD2); C-domain 1; C-domain 2; tumor necrosis factor, becomes soluble form], lymphotoxin-α (LT-α) (TNF-β) (tumor necrosis factor ligand superfamily member 1), tumor necrosis factor receptor superfamily member 1A (tumor necrosis factor receptor 1) (TNF-R1) (tumor necrosis factor receptor type I) (TNF-RI) (TNFR-I) (p55) (p60) (CD antigen CD120a) [cleaved, tumor necrosis factor receptor superfamily member 1A, membrane form; tumor necrosis factor binding tag and tumor necrosis factor receptor superfamily member 1B (tumor necrosis factor receptor 2) (TNF-R2) (tumor necrosis factor receptor type II) (TNF-RII) (TNFR-II) (p75) (p80 TNF-α receptor) (CD antigen CD120b) (etanercept) [cleaved to tumor necrosis factor receptor superfamily member 1b, membrane form; tumor necrosis factor binding protein 2 (TBP-2) (TBPII)]. In some embodiments, it is specifically contemplated that any of these may be included in the yield, while in other embodiments, it is specifically contemplated that any of these may not be included in the yield.

[0089] Additional bioactive agents that may be present in the release or composition of the present disclosure include one or more of the following: tumor necrosis factor receptor superfamily member 9 (4-1BB ligand receptor) (CDw137) (T cell antigen 4-1BB homolog) (T cell antigen ILA) (CD antigen CD137), CD166 antigen (activated leukocyte adhesion molecule) (CD antigen CD166), T lymphocyte activation antigen CD80 (activating B7-1 antigen) (BB1) (CTLA-4 counter-receptor B7.1) (B7) (CD antigen CD80), tumor necrosis factor receptor superfamily member 17 (B cell maturation protein) (CD antigen CD269), monocyte differentiation antigen CD14 (myeloid cell-specific leucine-rich glycoprotein) (CD antigen CD14) [cleaved, monocyte differentiation antigen CD14]. CD14, urinary form; monocyte differentiation antigen CD14, becomes membrane-bound], tumor necrosis factor receptor superfamily member 8 (CD30L receptor) (Ki-1 antigen) (lymphocyte activation antigen CD30) (CD antigen CD30), CD40 ligand (CD40-L) (T-cell antigen Gp39) (TNF-related activation protein) (TRAP) (tumor necrosis factor ligand superfamily member 5) (CD antigen CD154) [cleaved to become CD40 ligand, membrane form; CD40 ligand, soluble form], carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein 1) (BGP-1) (CD antigen CD66a), tumor necrosis factor receptor superfamily member 21 (cell death receptor 6) (CD antigen CD358), tyrosine-protein kinase receptor TYRO3 (EC 2.7.10.1) (tyrosine protein kinase BYK) (tyrosine protein kinase DTK) (tyrosine protein kinase RSE) (tyrosine protein kinase SKY) (tyrosine protein kinase TIF), endoglin (CD antigen CD105), receptor tyrosine protein kinase erbB-3 (EC 2.7.10.1) (proto-oncogene-like protein c-ErbB-3) (tyrosine kinase-type cell surface receptor HER3), E-selectin (CD62 antigen-like family member E) (endothelial leukocyte adhesion molecule 1) (ELAM-1) (leukocyte-endothelial cell adhesion molecule 2) (LECAM2) (CD antigen CD62E),Tumor necrosis factor receptor superfamily member 6 (Apo-1 antigen) (apoptosis-mediating surface antigen FAS) (FASLG receptor) (CD antigen CD95), Fms-related tyrosine kinase 3 ligand (Flt3 ligand) (Flt3L) (SL cytokine), tumor necrosis factor receptor superfamily member 18 (activation-inducible TNFR family receptor) (glucocorticoid-inducible TNFR-related protein) (CD antigen CD357), tumor necrosis factor receptor superfamily member 14 (herpes virus entry mediator A) (herpes viral entry mediator A) (HveA) (tumor necrosis factor receptor-like 2) (TR2) (CD antigen CD270), intercellular adhesion molecule 3 (ICAM-3) (CDw50) (ICAM-R) (CD antigen CD50), contactin-2 (axonal glycoprotein TAG-1) (axonin-1) (transient axonal glycoprotein 1) (TAX-1), interleukin-1 receptor type 1 (IL-1R-1) (IL-1RT-1) (IL-1RT1) (CD121 antigen-like family member A) (interleukin-1 receptor alpha) (IL-1R-α) (interleukin-1 receptor Receptor type I) (p80) (CD antigen CD121a) [cleaved to interleukin-1 receptor type 1, membrane type (mIL-1R1) (mIL-1RI); interleukin-1 receptor type 1, soluble type (sIL-1R1) (sIL-1RI)], cytokine receptor common subunit gamma (interleukin-2 receptor subunit gamma) (IL-2 receptor subunit gamma) (IL-2R subunit gamma) (IL-2RG) (γC) (p64) (CD antigen CD132), interleukin-10 receptor subunit beta (IL-10 receptor subunit beta) (I L-10R subunit beta) (IL-10RB) (cytokine receptor class II member 4) (cytokine receptor family 2 member 4) (CRF2-4) (interleukin-10 receptor subunit 2) (IL-10R subunit 2) (IL-10R2) (CD antigen CDw210b), interleukin-17 receptor A (IL-17 receptor A) (IL-17RA) (CDw217) (CD antigen CD217), interleukin-21 receptor (IL-21 receptor) (IL-21R) (novel interleukin receptor) (CD antigen CD360),Lysosomal membrane protein 2 (85 kDa lysosomal membrane sialoglycoprotein) (LGP85) (CD36 antigen-like 2) (lysosomal membrane protein II) (LIMP II) (scavenger receptor class B member 2) (CD antigen CD36), neutrophil gelatinase-associated lipocalin (NGAL) (25 kDa α-2-microglobulin-related subunit of MMP-9) (lipocalin-2) (oncogene 24p3) (siderocalin LCN2) (p25), L-selectin (CD62 antigen-like family member L) (leukocyte adhesion molecule 1) (LAM-1) (leukocyte surface antigen Leu-8) (leukocyte-endothelial cell adhesion molecule 1) (LECAM1) (lymph node homing receptor) (TQ1) (gp90-MEL) (CD antigen CD62L), lymphatic endothelial hyaluronan receptor 1 (LYVE-1) (cell surface Face retention sequence-binding protein 1 (CRSBP-1) (extracellular link domain-containing protein 1) (hyaluronan receptor), MHC class I polypeptide-related sequence A (MIC-A), MHC class I polypeptide-related sequence B (MIC-B), pro-neuregulin-1, membrane-bound isoform (pro-NRG1) [cleaved and transformed into neuregulin-1 (acetylcholine receptor-induced activity) (ARIA) (breast cancer cell differentiation factor p45) (glial cell growth factor) (heregulin) (HRG) (Neu differentiation factor) (sensory and motor neuron-derived factor)], platelet-derived growth factor receptor beta (PDGF-R-β) (PDGFR-β) (EC 2.7.10.1) (beta platelet-derived growth factor receptor) (beta platelet-derived growth factor receptor) (CD140 antigen-like family member B) (platelet-derived growth factor receptor 1) (PDGFR-1) (CD antigen CD140b), platelet endothelial cell adhesion molecule (PECAM-1) (EndoCAM) (GPIIA') (PECA1) (CD antigen CD31), MAPK / MAK / MRK overlapping kinase (EC 2.7.11.22) (MOK protein kinase) (renal tumor antigen 1) (RAGE-1), hepatitis A virus cell receptor 1 (HAVcr-1) (kidney injury molecule 1) (KIM-1) (T cell immunoglobulin and mucin domain-containing protein 1) (TIMD-1) (T cell immunoglobulin mucin receptor 1) (TIM) (TIM-1) (T cell membrane protein 1),Tumor necrosis factor receptor superfamily member 10C (antagonist decoy receptor for TRAIL / Apo-2L) (decoy TRAIL receptor without death domain) (decoy receptor 1) (DcR1) (lymphocyte inhibitor of TRAIL) (TNF-related apoptosis-inducing ligand receptor 3) (TRAIL receptor 3) (TRAIL-R3) (TRAIL receptor without intracellular domain) (CD antigen CD263), elafin (elastase specific inhibitor) (ESI) (peptidase inhibitor 3) (PI-3) (protease inhibitor WAP3) (skin-derived anti-carp proteinase) (SKALP) (WAP 4 disulfide core domain protein 14), urokinase plasminogen activator surface receptor (U-PAR) (uPAR) (monocyte activation antigen Mo3) (CD antigen CD87), vascular cell adhesion protein 1 (V-CAM) 1) (VCAM-1) (INCAM-100) (CD antigen CD106), and tumor necrosis factor receptor superfamily member 27 (X-linked ectodysplasin-A2 receptor) (EDA-A2 receptor). In some embodiments, it is specifically contemplated that any of these may be included in the yield, while in other embodiments, it is specifically contemplated that any of these may not be included in the yield.

[0090] In the compositions disclosed herein, either the bioactive compound or the growth factor is present in an amount of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 460, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1020, 1040, 1060, 1080, 1090, 1100, 1110, 1120, 1140, 1160, 1200 , 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 2000, 3000, 4000, 5000 pg / ml or more (or , 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540), , 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 2000, 3000, 4000, 5000 pg / ml or more (or any range derivable therein), or at most about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 84 The bioactive compounds may have concentrations of 0, 860, 880, 900, 920, 940, 960, 980, 1000, 2000, 3000, 4000, 5000 pg / ml, or more (or any range derivable therein), or none of the bioactive compounds or growth factors may have detectable concentrations in any of the compositions discussed herein or before or after any of the steps of the methods discussed herein. In certain embodiments, any one or more of the bioactive compounds or growth factors are specifically excluded in the compositions discussed herein.

[0091] In one example, Figures 1A-10 show the amount and identity of various growth factors cytokines, interleukins, and interferons in exemplary releasates of the present disclosure compared to plasma, platelet lysate, chemically defined medium, or FBS-supplemented medium. In a further example, Table 1 shows the amount and identity of various growth factors cytokines, interleukins, and interferons in exemplary releasates of the present disclosure (Releasate F1 and Releasate F2).

[0092] Basic fibroblast growth factor (bFGF), also referred to as FGF-β or FGF basic, is a growth factor and signaling protein encoded by the FGF2 gene. In some embodiments, the platelet releasate comprises FGF basicity at a level of at least about 100 pg / ml. In some embodiments, the platelet releasate comprises FGF basicity at a level of at least about 200 pg / ml. In some embodiments, the platelet releasate comprises FGF basicity at a level of at least about 300 pg / ml. In some embodiments of the methods of the present disclosure, the releasate comprises FGF basicity at a level of at least about 300 pg / ml. In some embodiments, the releasate comprises FGF basicity at a level of about 100 pg / ml to about 800 pg / ml. In some embodiments, the releasate comprises FGF basicity at a level of about 300 pg / ml to about 550 pg / ml. In other embodiments, the releasate comprises FGF basicity at a level of about 350 pg / ml to about 520 pg / ml. In further embodiments, the releasant contains FGF basicity at a level of about 400 pg / ml to about 500 pg / ml, hi some embodiments, the releasant contains FGF basicity at a level of about 450 pg / ml.The emissions are approximately 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560 , 570, 580, 590, 600, 700, or 800 pg / ml or any value or range derivable therein, at least about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420 , 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, or 800 pg / ml, or any value or range derivable therein, or at most about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 , 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, or 800 pg / ml, or any value or range derivable therein. In some embodiments, it is specifically contemplated that the composition (e.g., releasate) comprises FGF basicity. In other embodiments, it is specifically contemplated that the composition does not comprise FGF basicity or does not comprise a detectable or biologically relevant amount of FGF basicity (e.g., less than 0.1 pg / ml).

[0093] In some embodiments of the methods of the present disclosure, the releasant contains SDF-1α at about 2.0 pg / ml to about 50 pg / ml. In some embodiments of the methods of the present disclosure, the releasant contains SDF-1α at about 4.0 pg / ml to about 30 pg / ml. In some embodiments of the methods of the present disclosure, the releasant contains SDF-1α at about 5.0 pg / ml to about 20 pg / ml. In some embodiments, the releasant contains SDF-1α at about 7.0 pg / ml to about 15 pg / ml. In some embodiments, the releasant contains SDF-1α at about 8.0 pg / ml to about 14 pg / ml. In other embodiments, the releasant contains SDF-1α at about 9.0 pg / ml to about 12.0 pg / ml. In further embodiments, the emissions are 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8. 0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, or 50 pg / ml of SDF-1α, or any value or range derivable therein, and at least 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 ...7, 5.8, 5.9, 5.1, 5. 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pg / ml of SDF-1α, or any value or range derivable therein, or at most 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pg / ml of SDF-1 alpha, or any value or range derivable therein. In some embodiments, it is specifically contemplated that the composition (e.g., releasate) comprises SDF1-alpha. In other embodiments, it is specifically contemplated that the compositions do not contain SDF-1α or do not contain detectable or biologically relevant amounts of SDF-1α (e.g., less than 0.1 pg / ml).

[0094] In some embodiments of the disclosed methods and compositions, the release comprises microvesicles or exosomes from the cells. The microvesicles or exosomes may comprise growth factors or other proteins from the cells. The microvesicles or exosomes may vary in size and composition. In some embodiments, the microvesicles or exosomes comprise one or more of growth factors, cytokines, chemokines, DNA, RNA, microRNA (miRNA), and other nucleic acids. In some embodiments, it is specifically contemplated that the release comprises microvesicles. In other embodiments, it is specifically contemplated that the composition does not comprise microvesicles or does not comprise a detectable or biologically relevant amount of microvesicles. In some embodiments, it is specifically contemplated that the release comprises exosomes. In other embodiments, it is specifically contemplated that the composition does not comprise exosomes or does not comprise a detectable or biologically relevant amount of exosomes.

[0095] It is contemplated herein that any data presented may include a standard deviation of about 2% to about 10%. This standard deviation is believed to apply across all data from high throughput growth factors to cytokine and chemokine analysis. In some embodiments, one or more standard deviation values ​​are applied in assessing acceptable or expected amounts of cytokines or biomarkers in a composition. In some embodiments, 1, 2, 3, 4, 5, 6 or more standard deviation values ​​are applied.

[0096] In general, the disclosure provides methods for large-scale manufacturing of platelet releasate (hPR) from expired units of platelets using cGMP. In some embodiments, the methods produce hPR that contain negligible amounts of fibrinogen. In some embodiments, the hPR contains less than about 0.05 mg / dL of fibrinogen. In some embodiments, the hPR contains any amount of fibrinogen between about 0.00 and 0.05 mg / dL. The fibrinogen is removed by conversion to a fibrin clot that also traps other cellular debris, resulting in a clear supernatant that is hPR.

[0097] hPR according to the present disclosure may retain other plasma-derived components such as globulins and albumin, as shown in FIG.

[0098] In certain embodiments, the releasate composition of the present disclosure is added to other components, nutrients, or media to form a cell culture or cell storage medium. The releasate can be added to an existing cell culture medium, such as Minimum Essential Medium (MEM) or Dulbecco's Modified Eagle Medium (DMEM), and used for cell culture. The cell culture medium according to the present disclosure is formulated to provide nutrients (e.g., growth factors, etc.) necessary for the growth or maintenance of cells, including stem cells and / or progenitor cells, such as mesenchymal stem cells. Such cell culture media, in some embodiments, also do not include added heparin and coagulated materials.

[0099] Closed system The method of preparing hPR according to the present disclosure can be carried out in a closed system that ensures a contaminant-free final product. The closed system can include, for example, a pre-filtration module of whole blood or platelet-rich plasma, a platelet retention module, a mycoplasma and virus retention module, and the like. The modules can be connected through module connections for the addition and removal of by-products. In some embodiments, bags such as those used in blood storage or bioreactors are used. Such bags can be connected to other bags and filtration systems by tubing. The connections are made, for example, using a sterile welder and a tube sealer. The closed system can reduce the need for clean rooms. If the cells collected for transfusion are not exposed to the surrounding atmosphere, the contents of the bag (i.e., the cells) remain sterile. By extension, all unit operations and downstream processes performed in this manner (i.e., by sterile welding one bag to another bag or filter) will maintain the sterility of the contents.

[0100] While some embodiments involve the use of closed systems, in some embodiments the release is prepared in an open system using flasks, tubes, cell culture reservoirs, bioreactors, etc., which may be within a biosafety cabinet, isolator or clean room.

[0101] Therapeutic Uses / Formulations / Combinations In some embodiments, the hPR composition of the present disclosure can be used as a therapeutic substance.Some aspects of the present disclosure relate to a formulation that includes the release described herein.The formulation can be used for therapeutic purposes, such as treating damaged, wounded or diseased tissue, including but not limited to bone, muscle, skin, nerve, tendon, connective tissue, eye, periodontal tissue or cardiovascular tissue.hPR can be used as an improved substitute for dissolution in clinical applications.

[0102] The compositions of the present disclosure may be formulated in any suitable manner for medical treatment, including, but not limited to, liquids, gels, powders, ointments, aerosols, sprays, etc. The compositions described herein may be delivered to tissue by any suitable means, including, but not limited to, surgical implantation, injection, topical application, wound dressings, etc.

[0103] In some embodiments, hPR is combined with any known osteobiologics and coagulation agents. The former promote healing mechanisms through the presence of growth factors, cytokines and chemokines, while the latter may have applications in trauma, field care, etc. In some embodiments, hPR (alone or in combination with other agents) can be used to coat tissue culture plastics, microcarriers, etc. to promote cell attachment, differentiation and expansion). In some embodiments, the hPR of the present disclosure is combined with biomaterials, mammalian tissues, modified or engineered cells and tissues.

[0104] In some embodiments, the present disclosure provides a method of treating a subject, comprising administering to the subject a composition comprising stem cells, the stem cells being cultured with releasates from human blood-derived platelets, the releasates comprising fibrinogen at a level of less than about 0.05 mg / dL, less than about 0.05 mg / dL. In some embodiments, the subject suffers from a bone disease, bone defect, bone injury, osteoporosis, osteoarthritis, or spinal cord injury. In some embodiments, the subject suffers from a cartilage disease or cartilage defect or injury. In some embodiments, the subject suffers from bone, tendon, cartilage, or muscle injury. In some embodiments, the subject suffers from periodontal disease. In some embodiments, the subject suffers from an autoimmune disease. In some embodiments, the subject suffers from a myocardial infarction. In certain embodiments, the releasates are allogeneic to the target patient, and in other embodiments, the releasates are autogenic or xenogenic to the target patient.

[0105] In certain embodiments, the actual dosage of the composition administered to a subject or patient can be determined by physical and physiological factors such as the size of the damaged or wounded tissue, the severity of the condition, the type of condition being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration.

[0106] Platelet-rich fibrin In addition to the release products of the present disclosure, which may be derived from fresh or expired platelet-rich plasma or platelet concentrate, another useful product, fibrin clots, may be used for therapeutic purposes. The solid or gelatinous clot material is rich in growth factors and contains sufficient amounts and clotting factors to become a coagulable structure for medical use.

[0107] In some aspects, the present disclosure relates to a method for preparing platelet-rich fibrin from fibrinogen mammalian blood-derived platelet concentrate, comprising: (i) obtaining human blood platelets, thereby obtaining platelet-rich plasma (PRP); (ii) adding CaCl2 to PRP to a final concentration of more than 25 mM; (iii) stirring the CaCl2 / PRP mixture for less than 6 hours, thereby forming a fibrin clot and a supernatant; (iv) adding an antifibrinolytic agent to prevent fibrinolysis; and (v) removing the supernatant to obtain platelet-rich fibrin. In some embodiments, the method further comprises concentrating by removing excess plasma. In some embodiments, the mammalian blood is human, horse, dog, cat, pig, cow, chicken, cat, pig, rabbit, dolphin, sheep, mouse, rat, monkey blood, from sports animals, from livestock, or from pets. Any one or more steps discussed herein in the context of hPR can be implemented in the method for generating platelet-rich fibrin. In certain embodiments, the releasant generating method further comprises isolating or separating the obtained clot. In certain embodiments, the method comprises one or both of the following steps: adding an antifibrinolytic agent to the separated clot to prevent fibrinolysis; and removing the supernatant to obtain platelet-rich fibrin. Antifibrinolytic agents include, but are not limited to, aprotinin, tranexamic acid, aminomethylbenzoic acid, or aminocaproic acid. Serpins are also antifibrinolytic, examples of which include aprotinin, alpha 1 antitrypsin, C1 inhibitor, and camostat. In some embodiments, the concentration of antifibrinolytic agent added or its final concentration plus the separated clot is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 89, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7. 0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10 .5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 μM or mM (or any range derivable therein), at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5 .2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15. 5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 μM or mM (or any range derivable therein), or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0. 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3. 2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6. 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9. 4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 μM or mM (or any range derivable therein). In some embodiments, the concentration of aprotinin ranges from about 1.0 μM to about 10 mM; tranexamic acid ranges from about 1 μM to about 100 mM; aminocaproic acid ranges from about 0.01 mM to about 50 mM.

[0108] The therapeutic or medical uses of fibrin clots are known to those skilled in the art. The therapeutic or medical uses of fibrin clots span many different fields, including orthopedics, sports medicine, regenerative dentistry, cosmetic, plastic and maxillofacial surgery. Such uses include, but are not limited to, use as or with biological adhesives, biological hydrogels, topical preparations for skin care and / or hemostatic agents to prevent blood loss at sites of vascular injury. Growth factors and cytokines in fibrin clots can play a role, for example, in wound healing, new or rapid vascularization of healing tissue, bone regeneration or soft tissue maturation.

[0109] The following examples are included to demonstrate certain embodiments of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure. The examples should not be construed as limiting in any way. The contents of all cited references (including references cited throughout this application, issued patents, published patent applications) are expressly incorporated herein by reference. In the event that the definition of a term in a document incorporated herein by reference conflicts with that used herein, the definition used herein shall apply. EXAMPLES

[0110] III. Working Examples Example 1 - Method for preparing human platelet releasate hPR is manufactured in a closed system using expired units of platelets from adult blood. Immediately after expiration, the units of platelets are fractionated to isolate platelet-rich plasma (PRP). After leukoreduction, platelets were pelleted by centrifugation at 4000 g for 10 min. Platelets were pooled into batches based on the results of ABO typing and infectious disease testing. The pool of PRP was agitated to release growth factors. This step was followed by another round of centrifugation at 4000 g for 15 min to separate cell debris and clotting factors. The supernatant, which is hPR, was collected in aliquots and stored at -80°C. The batches of hPR were characterized by measuring (i) total protein, (ii) growth factor concentration, and (iii) growth kinetics of MSCs, as shown in Figure 19, Figures 20A and 20B, and Figures 21A and 21B.

[0111] The following is an example of a protocol for preparing a human platelet releasate according to the present disclosure. 1. A unit of (platelet rich plasma) PRP was weighed out. 2. The following parameters or test results were recorded for each unit used: ABO type, blood counts, and infectious disease markers (IDM). 3. Although only type O (Rh+ and Rh-) PRP has been used as an example, any ABO blood type may be used. 4. Expired units were pooled to a final volume of 250ml per bag. 5. Each PRP unit was connected to two additional empty transfer bags. 6. The PRP units were balanced by placing them in a centrifuge set at 4000g at 4°C. 7. After centrifugation for 15 minutes, the units were gently transferred to a G4 / G5 expressor system. 8. The supernatant was expressed, i.e. the plasma was placed in an empty bag. 9. Plasma bags were stored at -80°C until further use. 10. A second bag was used to store concentrated PRP (50ml or more) for further processing. 11. Using a deep freeze, the bags were frozen to -55°C. 12. The bags were incubated in a -80°C freezer overnight. 13. The unit was incubated up to this step to achieve the required production volume. 14. Using a water bath set at 37°C, the bag was thawed for 30 minutes. 15. Platelets were activated by injecting calcium chloride at a final concentration of 40 mM using a sterile welded connection. 16. The contents of the bag were agitated by placing the unit in an orbital shaker apparatus at 38-40°C and 250 rpm for 90 minutes. 17. After the mixing step, the concentrated PRP units were balanced in a centrifuge set at 4000 g at 4°C. 18. The units were centrifuged for 15 minutes. 19. Using a G4 / G5, the supernatant from all bags (50 ml) was transferred to a 1 litre bag. 20. PRP units were pooled in 1 liter bags up to a maximum volume of 500 ml. 21. The units were then incubated in a 1 L bag containing 500 ml of Unfiltered Human Platelet Releasate (UN-hPR) at 4° C. until further use. 22. From this step onwards, all manipulations were carried out in a biosafety cabinet (BSC). 23. The bags were transferred to a Biosafety Cabinet (BSC) containing a vacuum filtration system (Stericup) for three stage filtration. 24. 500 ml of UN-hPR was transferred to a 0.45 micron Stericup. 25. After filtration, the Stericup system bottle was collected and its contents transferred to a 0.22 micron Stericup for a second filtration. 26. After filtration, the contents in the 0.22 micron Stericup system transfer bottle were collected and transferred to a 0.10 micron Stericup for a third filtration. 27. After filtration, the 0.10 micron Stericup system transfer bottle was retrieved and a 25 ml aliquot of Filtered Human Platelet Releasate (Fil-hPR) was transferred into a 30 ml storage bottle. 28. The storage bottles (20 bottles of 500ml each) were transferred to a freezer set at -20°C or below -80°C.

[0112] Figures 2-6 and 10-20 show quantitative comparative analysis of various growth factors, cytokines, interferons, total proteins, globulins, albumin and other components in hPR batches obtained using the above protocol. The data unexpectedly show that the amount of growth factors and cytokines was higher in hPR produced by the present method compared to controls containing commercial human platelet lysate (hPL), chemically defined medium (CDM) and fetal bovine serum (FBS). These results also show that the proliferation rate of MSCs is greater in basal medium supplemented with hPR compared to FBS, CDM and hPL.

[0113] A variation of the protocol according to the present disclosure involves filtering the emissions using a 0.45 micron to 0.65 micron filter. Recent data suggests that it may be possible to filter the emissions as part of a closed system.

[0114] Example 2 - Scaled-up method for preparing human platelet releasates The inventors have developed a process for producing consistent lots of hPR that does not require the addition of anticoagulants to the growth medium. The yield of human platelet releasate using the protocol outlined in Example 1, or modifications thereof, is: Scaling up When used in combination with hPR, up to 10 L of hPR can be achieved in less than 4 hours. The effect of hPR in a large-scale cell expansion system was also tested. Large-scale production of hPR can produce values ​​as high as 24 L every 4 hours.

[0115] Example 3 - CaCl on fibrin clot formation 2 Concentration effect The inventors tested various concentrations of CaCl2 on clot formation when using the protocol of Example 1. The concentrations tested include 10 mM, 20 mM, 40 mM, 80 mM, 100 mM and 200 mM. Unexpectedly, low concentrations below 10 mM (close to the prior art recommendations) did not result in a clear fibrin clot encapsulating the cells. Similarly, higher concentrations of CaCl2, i.e., above 80 mM, did not result in a clear fibrin clot and clear supernatant (releasate). The optimal CaCl2 concentration ranged from 25 mM to 80 mM. At such concentrations, the fibrin clot is a clear gel containing all the cell debris leaving the supernatant as a clear liquid. The data are shown in Figure 9.

[0116] Example 4 - CaCl in hPR on MSC doubling 2 Concentration Effects We studied the effect of 10 mM, 20 mM, 40 mM, 80 mM, 100 mM and 200 mM CaCl2 in hPR on the population doubling level of bone marrow derived mesenchymal stem cells. For this experiment, expired Acrodose or apheresis platelets stored at room temperature were used. The control was cells cultured in medium containing 10% commercial xeno-free substitute instead of FBS. Cell titer Glo luminescence assay was performed on cell cultures at day 5 and day 7. The data are shown in Figure 11. The concentrations that resulted in the highest PDL levels were 20 mM and 40 mM CaCl2.

[0117] Example 5 - Effect of agitation duration on fibrin clot formation We studied the effect of platelet agitation duration on the population doubling level of bone marrow-derived mesenchymal stem cells. For this experiment, expired Acrodose or apheresis platelets stored at room temperature were used. Controls were cells cultured in medium containing 10% commercial xeno-free substitute instead of FBS. At the time points of PDL3 and PDL7, a cell titer Glo luminescence assay was performed. The data are shown in Figure 12. Agitation for 60-90 min was shown to be sufficient, with periods up to 180 min giving similar results.

[0118] Example 6 - Effect of hPR centrifugation duration on MSC doubling We also tested the effect of hPR centrifugation duration on the population doubling level of bone marrow-derived mesenchymal stem cells. For this experiment, we used expired Acrodose or apheresis platelets stored at room temperature. The control was cells cultured in medium containing 10% commercial alternative to FBS. At the time of PDL3, we performed a cell titer Glo luminescence assay. The data is shown in Figure 13. The results show that centrifugation for 15 minutes was a sufficient duration to generate hPR that gave sufficient population doubling levels.

[0119] Example 7 - Effect of using a refill period on MSC doubling Human bone marrow-derived MSCs were cultured in α-MEM supplemented with commercially available human platelet lysate and various human platelet releasate preparations at concentrations of 0.5%, 1% and 2.5%, 5% and 10%. Cell culture supplements were typically used in the range of 1%-20% suspended in cell culture medium. Cells were seeded in 24-well tissue culture plates at a seeding density of 5,000 cells / well. Cell doublings were monitored over a 5-day period and assessed using a CellTiter Glo luminescent assay. The results shown in Figure 21A and Figure 21B indicate that the proliferation rate of MSCs is superior in medium supplemented with hPR compared to FBS, CDM and hPL. In some instances, the amount of commercially available lysate used to reach the same PDL levels obtained by hPR was more than doubled.

[0120] Example 8 - Production or preparation of human platelet-rich fibrin (hPRF) The following is an example of a protocol for preparing human platelet-rich fibrin according to the present disclosure. 1. A unit of (platelet rich plasma) PRP was weighed out. 2. The following parameters or test results were recorded for each unit used: ABO type, blood counts and infectious disease markers (IDM). 3. Only type O (Rh+ and Rh-) PRP has been used as an example, but any other ABO type can be used. 4. Expired units were pooled to a final volume of 250ml per bag. 5. Each PRP unit was connected to two additional empty transfer bags. 6. The PRP units were balanced by placing them in a centrifuge set at 4000g at 4°C. 7. After centrifugation for 15 minutes, the units were gently transferred to a G4 / G5 expressor system. 8. Squeeze out the supernatant, i.e., plasma into the empty bag. 9. Plasma bags were stored at -80°C until further use. 10. A second bag was used to store concentrated PRP (50ml or more) for further processing. 11. Using a deep freeze, the bags were frozen to -55°C. 12. The bags were incubated in a -80°C freezer overnight. 13. The unit was incubated up to this step to achieve the required production volume. 14. Using a water bath set at 37°C, the bag was thawed for 30 minutes. 15. Platelets were activated by injecting calcium chloride at a final concentration of 40 mM using a sterile welded connection. 16. The contents of the bag were agitated by clamping the unit on an orbital shaker apparatus at 38°C-40°C, 250 rpm for 90 minutes. 17. After the mixing step, the concentrated PRP units were balanced in a centrifuge set at 4000 g at 4°C. 18. The units were centrifuged for 15 minutes. 19. Using the G4 / G5, discard the supernatant. 20. Transfer the sediment to a homogenizer. 21. To prevent fibrinolysis, add an appropriate concentration of an antifibrinolytic agent (e.g., aprotinin or tranexamic acid or aminocaproic acid). 22. Transfer the platelet-rich fibrin to a sterile container (e.g., 5 cc capacity) and store at 4°C.

[0121] Example 9 - Preparation of Platelet Releasate Preparation Preparation of an exemplary release (Release F1) The following is an example of a protocol for preparing a human platelet releasate (Releasate F1) according to the present disclosure. 1. A unit of (platelet rich plasma) PRP was weighed out. 2. The following parameters or test results were recorded for each unit: ABO type, blood counts and infectious disease markers (IDM). 3. Although only type O (Rh+ and Rh-) PRP was used in this example, any ABO blood type can be used. 4. PRP units were adjusted to a final volume of 250ml per bag. 5. Using a sterile tube welder, each 250 mL pre-weighed PRP unit was connected to an empty 300 mL transfer bag. 6. The PRP units were balanced by placing them in a centrifuge set at 4000g at 4°C. 7. After centrifugation for 15 minutes, the units were gently transferred into an extractor system, e.g. Compomat G5 by Fresenius Kabi. 8. The supernatant, i.e., plasma, was transferred to an empty transfer bag, leaving 50 mL of plasma with the platelets. 9. The transfer bag containing 200 mL of transferred plasma was stored at -80°C for further use or disposal. 10. The bag containing 50 ml of plasma, i.e. platelets with concentrated PRP, was used for further processing. 11. The bags containing the concentrated PRP were first frozen to -55°C to -80°C using a quick-cool freezer. 12. The bag containing the concentrated PRP was then transferred to a -80°C freezer for storage. NOTE: Concentrated PRP units were collected, processed, and stored at -80 °C until the essential manufacturing volume was reached. This storage facilitates accumulation of concentrated PRP units for large-scale manufacturing. 13. The bag containing the concentrated PRP was removed from the -80°C freezer and thawed for 30 minutes using a water bath set at 37°C. NOTE: If PRP units are available in abundance at step 1 for large-scale manufacturing, steps 11, 12 and 13 may be omitted. 14. Platelets were activated by injection of sterile calcium chloride solution at a final concentration of 40 mM using a sterile welded connection. 15. The contents of the bag were agitated by placing the unit in an orbital shaker apparatus at 38°C-40°C, 250 rpm for 90 minutes. 16. After the mixing step, the calcium chloride treated concentrated PRP units were balanced in a centrifuge set at 4000 g at 4°C. 17. The units were centrifuged for 15 minutes. 18. 170 micron to 260 micron filter system. For example, a new transfer bag system was assembled by sterile welding / connecting a 1 L transfer bag to a Fenwal y type blood component recipient set containing a 170 micron to 260 micron filter. This transfer bag assembly is used in the next step to collect and pool the human platelet releasate. 19. Using the extractor system, the supernatant in all bags (called human platelet releasate) was transferred into a collection bag system. 20. With 50 mL of human platelet release per unit, 10 units were pooled in a 1 liter transfer bag of the collection bag system to a maximum volume of 500 mL. 21. The pooled units were then stored overnight at -80°C in 1 L bags containing 500 ml of human platelet releasate. 22. After overnight treatment at -80°C, the human platelet releasates were thawed in a 37°C water bath for 30 minutes. 23. The thawed human platelet releasate was connected to the leukoreduction system using a sterile tube welder. The filtered human platelet releasate was collected in the attached 1 L transfer bag. 24. Additionally, sterile filtration of the human platelet releasate was performed using a 0.65um in-line filter (eg, Macopharma in-line filter) attached to the 1 L transfer bag containing the product. 25. After filtration, 25 ml aliquots of the filtered human platelet releasate were transferred into 30 ml storage bottles. 26. The storage bottles (20 bottles of 500ml each) were transferred to a freezer set at or below -80°C.

[0122] Preparation of an exemplary release (release F2) The following is an example of a protocol for preparing a human platelet releasate (Releasate F2) according to the present disclosure. This exemplary protocol does not include a step of centrifuging the platelet-rich plasma. 1. A unit of (platelet rich plasma) PRP was weighed out. 2. The following parameters or test results were recorded for each unit: ABO type, blood counts and infectious disease markers (IDM). 3. Although only type O (Rh+ and Rh-) PRP was used in this example, any ABO blood type can be used. 4. PRP units were adjusted to a final volume of 250ml per bag. 5. The bag containing the PRP was first frozen to -55°C to -80°C using a quick-cool freezer. 6. The bag containing the PRP was then transferred to a -80°C freezer for storage. NOTE: PRP units were collected, processed, and stored at -80 °C until the essential manufacturing volume was reached. This storage facilitates accumulation of PRP units for large-scale manufacturing. 7. The bag containing the PRP was removed from the -80°C freezer and thawed for 30 minutes using a water bath set at 37°C. NOTE: If PRP units are available in abundance at step 1 for large-scale manufacturing, steps 5, 6 and 7 may be omitted. 8. Platelets were activated by injection of sterile calcium chloride solution at a final concentration of 40 mM using a sterile welded connection. 9. The contents of the bag were agitated by placing the unit in an orbital shaker apparatus at 38°C-40°C, 250 rpm for 90 minutes. 10. After the mixing step, the calcium chloride treated PRP units were balanced in a centrifuge set at 4000 g at 4°C. 11. The units were centrifuged for 15 minutes. 12. 170 micron to 260 micron filter system. A new transfer bag system was assembled by sterile welding / connecting a 1 L transfer bag to a Fenwal y type blood component recipient set containing a 170 micron to 260 micron filter. This transfer bag assembly is used in the next step to collect and pool the human platelet releasate. 13. Using the extractor system, the supernatant in all bags (called human platelet releasate) was transferred into a collection bag system. 14. With 250 mL of human platelet release per unit, 4 units were pooled into a 2 liter transfer bag in the collection bag system to a maximum volume of 1000 mL. 15. The pooled units were then stored overnight at -80°C in 2L bags containing 1000ml of human platelet releasate. 16. After overnight treatment at -80°C, the human platelet releasates were thawed in a 37°C water bath for 30 minutes. 17. The thawed human platelet releasate was connected to the leukoreduction system using a sterile tube welder. The filtered human platelet releasate was collected in the attached 2 L transfer bag. 18. Additionally, sterile filtration of the human platelet releasate was performed using a 0.65um in-line filter (eg, Macopharma in-line filter) attached to the 2L transfer bag containing the product. 19. After filtration, 50 ml aliquots of filtered human platelet releasate were transferred into 60 ml storage bottles. 20. The storage bottles (20 bottles of 1000ml each) were transferred to a freezer set at -80°C or below.

[0123] Example 10 - Differential biomarker expression in platelet releasates Antibody array analysis was performed on various compositions: releasate F1 and releasate F2, human AB serum, FBS, and three commercially available platelet lysates (lysates A, B, and C). For antibody array analysis, all samples were processed as a service by Raybiotech (Norcross, GA) using the Quantibody Human Cytokine Antibody Array 4000. This quantitative array gives the concentrations of 200 human growth factors, cytokines, chemokines and other factors simultaneously (see Table 1). All experiments were performed according to the recommended manufacturer's instructions provided in the antibody array package insert. Briefly, after a 30-minute incubation with blocking buffer, 100 μL of two-fold diluted samples were added to each well of the glass slide. After the defined incubation period and extensive washing, biotin-labeled detection antibodies and detection antibody cocktails were added for 1-2 hours (at room temperature) and then washed. Streptavidin conjugated with a Cy3 equivalent dye was then added and incubated for 1 hour at room temperature. Slides were thoroughly washed and then scanned using a microarray scanner. Each protein had a standard curve that included measurements from known dilutions of purified standard proteins. Each slide array contained a positive control sample that was used for normalization purposes. Measurements were based on the fluorescence intensity of the labeled antibody bound to each spot and calculated from the average of four spots per antibody. Slides were measured and analyzed using Raybiotech software and normalized to the positive control. Final results were expressed as picograms of protein / ml extract.

[0124] The results of the antibody array analysis are shown in Table 1 and Figures 22A-F and demonstrate differential biomarker expression in the releasates compared to the other samples. Figure 22A shows the levels of all proteins in all samples, separated into five groups (1-5) as indicated. Figures 22B-22F show the protein levels for each of the five groups shown in Figure 22A. Values ​​marked as 0.0 in Table 1 were below the limit of detection (LOD). Values ​​in italics in Table 1 were above the maximum standard (MAX) of the assay.

[0125]

Table 1

[0126] Example 11 - MSC doubling time with platelet release Bone marrow-derived MSCs (BM-MSCs) were seeded in wells of a 24-well plate at a density of 5,000 cells / well. Growth medium consisting of DMEM (Gibco) with 1x Glutamax (Gibco) supplemented with either 5% lysate, 10% FBS, 10% AB serum, or 5% release formulation was added to the wells. Commercially available lysate (5% supplement concentration) and FBS and AB serum (10% supplement concentration) served as controls. Each condition, including controls, was run in triplicate. Cells were expanded for 5 days under standard cell culture conditions, i.e., using a humidified 37°C, 5% CO2 / 95% air environment. On day 5, cell proliferation was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Briefly, the medium was removed and cells were washed three times in sterile PBS. 200ul of the prepared CellTiter-Glo reagent was added to each well and then incubated at 37°C for 2 minutes with agitation. Upon removal from the incubator, the well plate was left at room temperature for 10 minutes to allow the luminescence signal to equilibrate. The entire contents of each well were then transferred to a well of a luminometer-compatible 96-well plate. The luminescence of each well was measured using a Tecan Spark plate reader (Morrisville, NC). The number of cells per well was determined using a standard curve of relative light units (RLU) for known concentrations of cells run in parallel with the experimental samples. The population doubling level (PDL) was calculated from the cell count using the following formula: PDL=3.322(LogA-LogB), where A is the number of cells (per well) at the end of the growth period and B represents the initial number of cells seeded in each well (5000 in this experiment).

[0127] The results of the MSC doubling experiment are shown in FIG.

[0128] Example 12 - Quantification of Protein in Release Formulations Total protein assays were performed for various additives: FBS, human AB plasma (AB serum), one of three commercially available lysates (Lysate A, Lysate B, or Lysate C), one of two platelet releasate preparations (Releasate F1, Releasate F2), or pooled plasma. Total protein assays were performed by testing samples (i.e., FBS, lysate, releasate, etc.) with a Total Protein Reagent Kit (Listing No. 7D73) using an Architect C8000 system following the instructions provided in the package insert. To quantify the concentration of fibrinogen in the samples, a Fibrinogen Assay Kit from Diazyme (Reference No. DZ768A-K) was used following the assay procedure set forth in the package insert. The concentrations of albumin and globulin in the test samples were quantified using Sebia's Capillary Protein (E) 6 kit by capillary electrophoresis using the CAPILLARYS system. The results are shown in FIG. 24.

[0129] Example 13 - Electron microscopy of platelets Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed on resting and activated platelets. For SEM, platelets from different stages of production of the releasant formulation were fixed using PBS buffer containing 4% formaldehyde and 1% glutaraldehyde. After fixation, platelets were treated with osmium tetroxide and washed using sodium cacodylate buffer, followed by a series of washes with 20%, 40%, 60%, 80%, and 100% ethanol to dehydrate the platelets. The cells were then sputter-coated with gold-palladium alloy, stored under vacuum for 24 hours, and imaged using a JEOL JSM-6610LV. Transmission electron microscopy (TEM) was performed by fixing platelets with PBS buffer containing 4% formaldehyde and 1% glutaraldehyde. After fixation, platelets were treated with osmium tetroxide and washed with sodium cacodylate buffer, followed by a series of washes with 20%, 40%, 60%, 80%, and 100% ethanol to dehydrate the platelets. The cells were then treated with a 1:1 ratio mixture of propylene oxide and epoxy resin. After 24 h of incubation under vacuum, the cells were embedded in 100% epoxy resin and baked in an oven for hardening. The hardened blocks containing the cells were sectioned at 90 nm thickness using an ultramicrotome equipped with a diamond cutter and imaged using a JOEL 1230. The degranulation process was observed, involving the release of vesicles and intracellular material from the platelets to the external environment. Figures 25-30 show the results of these electron microscopy experiments. In particular, Figure 29 shows platelets after degranulation, showing that the platelets lack granules but remain intact (i.e., not lysed).

[0130] Example 14 - CD marker expression on BM-MSCs expanded with release Bone marrow-derived MSCs (BM-MSCs) were expanded in basal growth medium supplemented with 10% FBS, 10% AB-serum, 5% commercial lysate (lysate A, lysate B, or lysate C), 5% release F1, or 5% release F2. To clearly demonstrate the effect of the release formulation on the differentiation potency of MSCs, the MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. After expansion, the cells were characterized for MSC-associated CD marker expression. The results are shown in Figure 31. CD marker expression of BM-MSCs expanded in release formulations (F1 and F2) met the standardized criteria for characterization of MSCs proposed by the International Society for Cellular Therapy (ISCT).

[0131] Example 15 - Assessment of adipogenic potential BM-MSCs were expanded in basal growth medium supplemented with 10% FBS, 10% AB serum, 5% commercial lysate (lysate A, lysate B, or lysate C), 5% release F1 or 5% release F2. After expansion, the pluripotency of the cells was evaluated using differentiation medium. BM-MSCs expanded in release formulations (F1 and F2) bound to tissue culture-treated plastic showed pluripotency and met the standardized criteria for the characterization of MSCs proposed by the International Society for Cellular Therapy (ISCT). To clearly demonstrate the effect of release formulations on the differentiation potency of MSCs, MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. The results of this experiment are shown in Figure 32A-G and suggest that release F1 is preferred in stimulating adipogenesis compared to release F2. The releasate preparations (F1 and F2) can also be used to assess the differentiation potential and / or suitability of the donor's cells prior to expansion (i.e., donor selection) or after cell expansion (i.e., lot or batch evaluation, a.k.a., release criteria). In this context, suitability testing refers to the ability of the donor's MSCs to differentiate into adipocytes.

[0132] Example 16 - Evaluation of cartilage formation ability BM-MSCs were expanded in basal growth medium supplemented with 10% FBS, 10% AB serum, 5% commercial lysate (lysate A, lysate B, or lysate C), 5% release F1 or 5% release F2. After expansion, the pluripotency of the cells was evaluated using differentiation medium. BM-MSCs expanded in release formulations (F1 and F2) bound to tissue culture-treated plastic showed pluripotency and fulfilled the standardized criteria for the characterization of MSCs proposed by the International Society for Cellular Therapy (ISCT). To clearly demonstrate the effect of release formulations on the differentiation potency of MSCs, MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. The results of this experiment are shown in Figure 33A-G and suggest that release F1 is preferred in stimulating chondrogenesis compared to release F2. The release formulations (F1 and F2) can also be used to assess the differentiation potential and / or suitability of the donor's cells prior to expansion (i.e., donor selection) or after cell expansion (i.e., lot or batch evaluation, a.k.a., release criteria). In this context, suitability testing refers to the ability of the donor's MSCs to differentiate into chondrocytes.

[0133] Example 17 - Evaluation of bone formation ability BM-MSCs were expanded in basal growth medium supplemented with 10% FBS, 10% AB serum, 5% commercial lysate (lysate A, lysate B, or lysate C), 5% release F1 or 5% release F2. After expansion, the pluripotency of the cells was evaluated using differentiation medium. BM-MSCs expanded in release formulations (F1 and F2) bound to tissue culture-treated plastic showed pluripotency and met the standardized criteria for the characterization of MSCs proposed by the International Society for Cellular Therapy (ISCT). To clearly demonstrate the effect of release formulations on the differentiation potency of MSCs, MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. The results of this experiment are shown in Figure 34A-G and suggest that release F2 is preferred in stimulating bone formation compared to release F1. The release formulations (F1 and F2) can also be used to assess the differentiation potential and / or suitability of the donor's cells prior to expansion (i.e., donor selection) or after cell expansion (i.e., lot or batch evaluation, a.k.a., release criteria). In this context, suitability testing refers to the ability of the donor's MSCs to differentiate into bone cells.

[0134] Example 18 - Evaluation of the immunomodulatory potential of the release IDO synthesis assay BM-MSCs were expanded in basal growth medium supplemented with 10% FBS, 10% AB serum, 5% commercial lysate (lysate A, lysate B, or lysate C), 5% release F1, or 5% release F2. To clearly demonstrate the effect of the release formulation on MSC differentiation potency, MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. After expansion, cells were evaluated for their immunomodulatory potential by synthesis of indoleamine 2,3-dioxygenase (IDO) in response to stimulation from the cytokines interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNFα). An IDO-specific inhibitor was used as a control. Cells expanded in the release formulation showed increased production of IDO as shown in Figure 35, and increased IDO response leads to increased kynurenine production, and increased kynurenine production was quantified in this assay. IDO acts on tryptophan present in the basal growth medium to form kynurenine (KYN), and therefore an increase in kynurenine levels is proportional to an increase in IDO synthesis in cells. The release preparations (F1 and F2) can also be used to evaluate / test the differentiation potential and / or suitability of the donor's cells before expansion (i.e., donor selection) or after cell expansion (i.e., lot or batch evaluation, a.k.a., release criteria). In this context, suitability / differentiation potential testing refers to the ability of the donor's MSCs to modulate the immune response.

[0135] PBMC culture assay BM-MSCs were expanded in basal growth medium supplemented with 5% release F1 or 5% release F2. To clearly demonstrate the effect of the release formulation on the differentiation potency of MSCs, MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. After expansion, cells were evaluated for immunomodulatory potential by co-culturing peripheral blood mononuclear cells (PBMCs) for 5 days in the presence of BM-MSCs. Phytohemagglutinin (PHA) was used to induce the expansion of PBMCs. MSCs expanded in release formulations (F1 and F2) exhibited immunomodulatory properties. As shown in Figure 36, BM-MSCs moderated the proliferation of PBMCs. Release formulations (F1 and F2) can also be used to evaluate / test the differentiation potential and / or suitability of a donor's cells before expansion (i.e., donor selection) or after cell expansion (i.e., lot or batch evaluation, a.k.a. release criteria). In this context, compatibility / differentiation potential testing refers to the ability of the donor's MSCs to modulate the immune response by moderating the proliferation of PBMCs.

[0136] Treg stimulation assay BM-MSCs were expanded in basal growth medium supplemented with 10% FBS, 10% AB serum, 5% commercial lysate (lysate A or lysate C), 5% release F1 or 5% release F2. To clearly demonstrate the effect of the release formulation on the differentiation potency of MSCs, the MSCs used across the different conditions were obtained from the same donor and maintained in the same PDL for all conditions. After expansion, the cells were evaluated for immunomodulatory potential by co-culture of PBMCs with BM-MSCs. Regulatory T cells (T-reg) were measured. Phytohemagglutinin (PHA) was used to induce expansion of PBMCs. Interleukin-1 (IL2) favors selective upregulation of regulatory T cells. As shown in Figure 37, BM-MSCs expanded in release formulations (F1 and F2) showed superior immunomodulatory properties. As shown in Figure 37, MSCs expanded in release formulations do not attenuate the expansion of T-regs, a desirable property in immunomodulation. The releasate preparations (F1 and F2) can also be used to assess / test the differentiation potential and / or compatibility of the donor's cells before expansion (i.e. donor selection) or after cell expansion (i.e. lot or batch evaluation, a.k.a. release criteria). In this context, compatibility / differentiation potential testing refers to the ability of the donor's MSCs to modulate the immune response by moderating the proliferation of PBMCs without inhibiting the proliferation of T-regs.

[0137] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that the factors described herein can be substituted with certain factors that are chemically and physiologically related, while still achieving the same or similar results. All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0138] References The references cited in this application, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025076468000010.tif172166TIFF2025076468000011.tif216166TIFF2025076468000012.tif216166TIFF2025076468000013.tif136166

Claims

1. (i) obtaining platelets from human blood, thereby obtaining platelet-rich plasma (PRP); (ii) adding CaCl to the PRP to a final concentration of greater than 25 mM 2 Addition of CaCl 2 generating a PRP mixture; and (iii) the CaCl 2 / Stirring the PRP mixture for less than 6 hours, thereby forming a clot and release product. A method for preparing a platelet releasate comprising:

2. 10. The method of claim 1, wherein the releasant contains fibrinogen at a level of less than about 0.05 mg / dL.

3. CaCl 2 2. The method of claim 1, wherein the final concentration of is greater than about 30 mM.

4. CaCl 2 2. The method of claim 1, wherein said final concentration of is from about 25 mM to about 80 mM.

5. CaCl 2 2. The method of claim 1, wherein said final concentration of is about 30 mM to about 50 mM.

6. CaCl 2 2. The method of claim 1, wherein said final concentration of is about 35 mM to about 50 mM.

7. CaCl 2 2. The method of claim 1, wherein said final concentration of is about 40 mM to about 47 mM.

8. CaCl 2 2. The method of claim 1, wherein the final concentration of is about 45 mM.

9. CaCl 2 2. The method of claim 1, wherein the final concentration of is about 80 mM.

10. CaCl 2 The method of any one of claims 1 to 9, wherein the / PRP mixture is stirred for less than 4 hours.

11. CaCl 2 The method of any one of claims 1 to 9, wherein the / PRP mixture is stirred for less than 180 minutes.

12. CaCl 2 The method of any one of claims 1 to 9, wherein the / PRP mixture is stirred for 30 minutes to 150 minutes.

13. CaCl 2 The method of any one of claims 1 to 9, wherein the / PRP mixture is stirred for 45 minutes to 135 minutes.

14. CaCl 2 The method of any one of claims 1 to 9, wherein the / PRP mixture is stirred for 60 minutes to 90 minutes.

15. CaCl 2 The method of any one of claims 1 to 14, wherein the / PRP mixture is stirred at 50 rpm to 500 rpm.

16. CaCl 2 The method of any one of claims 1 to 14, wherein the / PRP mixture is stirred at 250 rpm.

17. 17. The method of any one of claims 1 to 16, wherein the releasable product comprises a globulin, an albumin, a growth factor, a cytokine, an interleukin, an interferon, a chemokine, a glycoprotein, a fibronectin, a vitronectin or a laminin.

18. 17. The method of any one of claims 1 to 16, wherein the releasant comprises TGFβ1, TGFβ3, EGF, bFGF, PDGF-AA, PDGF-BB, PDGF-AB, SDF-1α, VEGF or HGF.

19. The method of any one of claims 1 to 18, wherein the releasant comprises FGF basic at a level of at least 300 pg / ml.

20. The method of any one of claims 1 to 18, wherein the releasant comprises FGF basic at about 300 pg / ml to about 550 pg / ml.

21. The method of any one of claims 1 to 18, wherein the releasant comprises FGF basic at about 350 pg / ml to about 520 pg / ml.

22. The method of any one of claims 1 to 18, wherein the releasant comprises FGF basic at about 400 pg / ml to about 500 pg / ml.

23. The method of any one of claims 1 to 19, wherein the releasant comprises FGF basic at about 450 pg / ml.

24. The method of any one of claims 1 to 23, wherein the releasant comprises SDF-1α at about 5.0 pg / ml to about 20 pg / ml.

25. The method of any one of claims 1 to 23, wherein the releasant comprises SDF-1α at about 7.0 pg / ml to about 15 pg / ml.

26. The method of any one of claims 1 to 23, wherein the releasant comprises SDF-1α at about 8.0 pg / ml to about 14 pg / ml.

27. The method of any one of claims 1 to 23, wherein the releasant comprises SDF-1α at about 9.0 pg / ml to about 12.0 pg / ml.

28. 28. The method of any one of claims 1 to 27, wherein the platelets are derived from fresh platelets, from platelets kept at room temperature, or from previously frozen platelets.

29. 29. The method of any one of claims 1 to 28, further comprising the step of separating the clot from the releasate.

30. 30. The method of any one of claims 1 to 29, further comprising the step (iv) of filtering the release.

31. 31. The method of claim 30, wherein step (iv) comprises filtering the release using a filter that is between 0.45 microns and 1.0 microns.

32. 31. The method of claim 30, wherein step (iv) comprises filtering the release using a filter that is between 3.0 microns and 10 microns.

33. 31. The method of claim 30, wherein step (iv) comprises filtering the release using a filter that is between 170 microns and 260 microns.

34. 34. The method of any one of claims 1 to 33, wherein steps (i), (ii) and (iii) are carried out in a closed bag.

35. 34. The method of any one of claims 31 to 33, wherein steps (i), (ii), (iii) and (iv) are carried out in a closed bag.

36. 25. The method of any one of claims 1 to 24, wherein steps (i), (ii) and (iii) are carried out in a closed system.

37. 25. The process of any one of claims 1 to 24, wherein steps (i), (ii) and (iii) are carried out in a closed system on an industrial scale.

38. 34. The process of any one of claims 31 to 33, wherein steps (i), (ii), (iii) and (iv) are carried out in a closed system on an industrial scale.

39. 39. The method of any one of claims 1 to 38, wherein the entire method is carried out in 4 hours or less.

40. 28. The method of any one of claims 1 to 27, wherein the duration of steps (i), (ii) and (iii) is 3 to 4 hours.

41. 28. The method of any one of claims 1 to 27, wherein the duration of steps (i), (ii), (iii) and (iv) is between 3 hours and 4 hours.

42. 30. The method of any one of claims 1 to 29, wherein the releasate is produced in a yield of about 0.2 liters to about 100 liters.

43. The method of any one of claims 1 to 29, wherein the releasate is produced in a yield of about 4.5 liters to about 10 liters.

44. The method of any one of claims 1 to 29, wherein the recharge is produced in a yield of about 50 liters to about 100 liters.

45. 45. The method of any one of claims 1 to 44, further comprising, prior to (ii), concentrating the platelets by removing excess plasma.

46. 46. ​​A releasant composition produced by the method of any one of claims 1 to 45.

47. 46. ​​A cell culture medium comprising a releasate produced by the method of any one of claims 1 to 45.

48. 48. The cell culture medium of claim 47, which does not contain added heparin.

49. A method of culturing cells, comprising expanding the cells on a cell culture medium comprising a releasate derived from mammalian platelet-rich plasma.

50. 50. The method of claim 49, wherein the releasate comprises fibrinogen at a level of less than about 0.05 mg / dL.

51. 51. The method of claim 49 or 50, wherein the releasate further comprises FGF basic at a level of at least about 300 pg / ml.

52. 51. The method of claim 49 or 50, wherein the releasant comprises FGF basic at about 300 pg / ml to about 550 pg / ml.

53. 51. The method of claim 49 or 50, wherein the releasant comprises FGF basic at about 350 pg / ml to about 520 pg / ml.

54. The method of any one of claims 49 to 53, wherein the releasate comprises FGF basic at about 450 pg / ml.

55. The method of any one of claims 49 to 53, wherein the releasant comprises SDF-1 at about 5.0 pg / ml to about 20 pg / ml.

56. The method of any one of claims 49 to 53, wherein the releasant comprises SDF-1 at about 7.0 pg / ml to about 15 pg / ml.

57. The method of any one of claims 49 to 53, wherein the releasant comprises SDF-1 at about 8.0 pg / ml to about 14 pg / ml.

58. The method of any one of claims 49 to 53, wherein the releasant comprises SDF-1 at about 9.0 pg / ml to about 12.0 pg / ml.

59. 54. The method of any one of claims 49 to 53, wherein the cell culture medium does not contain added heparin.

60. 54. The method of any one of claims 49-53, wherein the cell is a pluripotent stem cell (PSC), an induced pluripotent stem cell (iPSC), a bone marrow derived mesenchymal stromal / stem cell (BM-MSC), an adipose derived mesenchymal stromal / stem cell (ADP-MSC), a T cell, a B cell, a natural killer cell, a dendritic cell, a peripheral blood derived mononuclear cell, a cancer cell cancer stem cell, a Chinese Hamster Ovary (CHO), a cord blood derived cell, a cord blood tissue derived cell, a placenta derived cell, a retinal cell, a neuronal cell, a fibroblast, an epithelial cell, an endothelial cell or a keratinocyte or a fibroblast, an osteoblast, an adipocyte, a chondrocyte, an endothelial cell, a cell of the immune system, a T cell, a B cell, a NK cell, an engineered cell, or a neuronal cell.

61. 61. The method of claim 60, wherein the cells are mesenchymal stem cells.

62. 62. The method of claim 61, wherein said release stimulates differentiation of said cells into bone cells.

63. The method of any one of claims 49 to 61, further comprising, prior to (ii), concentrating the platelets by removing excess plasma.

64. 64. The method of claim 63, wherein said release stimulates differentiation of said cells into chondrocytes or adipocytes.

65. The method of any one of claims 49 to 61, wherein the release stimulates the release of a component from the cell.

66. 66. The method of claim 65, wherein the components comprise exosomes, extracellular vesicles, proteins, nucleic acids, or combinations thereof.

65. 67. The method of claim 65 or 66, further comprising collecting the component.

66. A composition comprising a releasate from mammalian blood-derived platelets, said releasate comprising fibrinogen at a level of less than about 0.05 mg / dL.

67. 67. The composition of claim 66, wherein the release is from human blood.

68. 68. The composition of claim 66 or 67, which is a solution.

69. 68. The composition of claim 66 or 67, which is a dried or lyophilized powder.

70. The composition of any one of claims 66 to 69, wherein the release comprises FGF basic at a level of at least 300 pg / ml.

71. The composition of any one of claims 66 to 69, wherein the release comprises FGF basic at about 300 pg / ml to about 550 pg / ml.

2. The composition of any one of claims 66 to 69, wherein the release comprises FGF basic at about 350 pg / ml to about 520 pg / ml.

73. The composition of any one of claims 66 to 69, wherein the release comprises FGF basic at about 400 pg / ml to about 500 pg / ml.

74. The composition of any one of claims 66 to 69, wherein the release comprises FGF basic at about 450 pg / ml.

75. The composition of any one of claims 66 to 74, wherein the release comprises SDF-1 at about 5.0 pg / ml to about 20 pg / ml.

76. The composition of any one of claims 66 to 74, wherein the releasant comprises SDF-1 at about 7.0 pg / ml to about 15 pg / ml.

77. The composition of any one of claims 66 to 74, wherein the releasant comprises SDF-1 at about 8.0 pg / ml to about 14 pg / ml.

78. The composition of any one of claims 66 to 74, wherein the release comprises SDF-1 at about 9.0 pg / ml to about 12.0 pg / ml.

79. The composition of any one of claims 66-74, which does not contain added heparin.

80. The composition of any one of claims 66-79, wherein said released product comprises one or more exosomes.

81. A therapeutic formulation comprising the composition of any one of claims 66 to 80.

82. 81. A method of treating a mammalian subject, comprising administering to the subject a composition comprising a population of stem cells, the stem cells having been cultured with a releasable composition according to any one of claims 66 to 80.

83. 83. The method of claim 82, wherein the releasate further comprises FGF basic at a level of at least about 300 pg / ml.

84. The method of claim 82 or 83, wherein the releasant comprises SDF-1 at 5.0 pg / ml to 20 pg / ml.

85. The method of any one of claims 82 to 84, wherein the stem cells are mesenchymal stem cells.

86. The method of any one of claims 82 to 84, wherein said stem cells are bone marrow-derived mesenchymal stem cells.

87. 85. The method of any one of claims 82-84, wherein said stem cell is a pluripotent stem cell (PSC), an induced pluripotent stem cell (iPSC), a bone marrow derived mesenchymal stromal / stem cell (BM-MSC), an adipose derived mesenchymal stromal / stem cell (ADP-MSC), a T cell, a B cell, a natural killer cell, a dendritic cell, a peripheral blood derived mononuclear cell, a cancer cell cancer stem cell, a Chinese hamster ovary (CHO), a cord blood derived cell, a cord blood tissue derived cell, a placenta derived cell, a retinal cell, a neuronal cell, a fibroblast, an epithelial cell, an endothelial cell, or a keratinocyte.

88. The method of any one of claims 82-84, wherein the population of stem cells comprises between 2% and 100% of one differentiated lineage.

89. The method of any one of claims 82 to 84, wherein said stem cells are autologous or allogeneic.

90. The method of any one of claims 82 to 84, wherein the stem cells are modified or engineered cells.

91. 11. A method of treating a subject, comprising administering to the subject a composition comprising stem cells, the stem cells having been cultured with releasates from human blood-derived platelets.

92. 92. The method of claim 91, wherein the releasate comprises fibrinogen at a level of less than about 0.05 mg / dL.

93. 93. The method of claim 91 or 92, further comprising FGF basic at a level of at least 300 pg / ml.

94. The method of any one of claims 91 to 93, wherein the releasant comprises SDF-1 at 5.0 pg / ml to 20 pg / ml.

95. 92. The method of claim 91, wherein the subject is suffering from a bone disease, bone defect, bone injury, osteoporosis, osteoarthritis, or spinal cord injury.

96. 92. The method of claim 91, wherein the subject is suffering from a cartilage disease or a cartilage defect or injury.

97. 92. The method of claim 91, wherein the subject is suffering from periodontal disease.

98. 92. The method of claim 91, wherein the subject is suffering from an autoimmune disease.

99. 92. The method of claim 91, wherein the subject has suffered a myocardial infarction.

100. 92. The method of claim 91, wherein the subject is suffering from graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), multiple trauma, systemic infection, or cancer.

101. (i) obtaining platelets from mammalian blood; (ii) Add CaCl to PRP to a final concentration of >25 mM 2 adding; and (iii) the CaCl 2 agitating the PRP mixture for less than 6 hours, thereby forming a clot and a releasate, the releasate containing fibrinogen at a level of less than about 0.05 mg / dL. A method for preparing a platelet releasate comprising:

102. 102. The method of claim 101, wherein the mammalian blood is horse, cat, pig, dog, cow, chicken, cat, pig, rabbit, dolphin, sheep, mouse, rat, monkey blood, from sports animals, from farm animals, or from pets.

103. 103. The method of claim 101 or 102, further comprising, prior to (ii), concentrating the platelets by removing excess plasma.

104. A method for promoting cell adhesion, cell differentiation, or cell expansion in tissue culture, comprising the step of coating a tissue culture vessel with a composition comprising a releasate from mammalian blood-derived platelets, the releasate comprising fibrinogen at a level of less than 0.05 mg / dL.

105. 105. The method of claim 104, wherein the releasable material is derived from human blood-derived platelets.

106. 106. The method of claim 104 or 105, wherein the container is a Petri dish, a flask, or a bioreactor.

107. 1. A method for preparing an osteobiological material, comprising the step of adding to the osteobiological material a composition comprising a release product from mammalian blood-derived platelets, the release product comprising fibrinogen at a level of less than 0.05 mg / dL.

108. 108. The method of claim 107, wherein the releasable material is derived from human blood-derived platelets.

109. 109. The method of claim 107 or 108, wherein the osteobiologic material is an osteobiologic graft material, a bone sponge, or a bone putty.

110. 109. The method of claim 107 or 108, wherein the osteobiologic material further comprises mammalian tissue, modified cells, or engineered cells.

111. A method for preparing a coagulant comprising adding to the coagulant a composition comprising a release product from mammalian blood-derived platelets, the release product comprising fibrinogen at a level of less than 0.05 mg / dL.

112. 112. The method of claim 111, wherein the releasable material is derived from human blood-derived platelets.

113. (i) obtaining platelets from the blood of a mammal, thereby obtaining platelet-rich plasma (PRP); (ii) CaCl to a final concentration of >25 mM 2 is added to the PRP, thereby 2 / Obtaining a PRP mixture; (iii) the CaCl 2 agitating the PRP mixture for less than 6 hours, thereby forming a fibrin clot and a supernatant; (iv) adding an antifibrinolytic agent to prevent fibrinolysis; and (v) removing the supernatant to obtain the platelet-rich fibrin.

2. A method for preparing platelet-rich fibrin comprising:

114. 114. The method of claim 113, wherein said mammalian blood is human, horse, pig, cat, dog, cow, sheep, mouse, rat, monkey blood, from a sports animal, from a farm animal, or from a pet.

115. 115. The method of claim 113 or 114, further comprising, prior to (ii), concentrating the platelets by removing excess plasma.

116. 116. A platelet-rich fibrin composition produced by the method of any one of claims 113-115.

117. 117. The platelet-rich fibrin of claim 116, wherein said fibrin does not contain detectable levels of thrombin.

118. (a) Platelet releasates from mammalian blood-derived platelets; and (b) cells. and instructions for using the releasate as a culture medium supplement.

119. 119. The kit of claim 118, further comprising cell culture medium.

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