MULTILAYERED BLOOD PRODUCT

Abstract

A blood product (10), a method for preparing the blood product, a blood product obtainable by the method and a blood product preparing container means. The blood product comprises components from whole blood, especially fibrin, thrombocytes and leukocytes. The blood product (10) comprises a first layer (21), a second layer (22) and a third layer (23). The second layer (22) is adjacent to the first layer (21) and the third layer (23). The first layer (21) defines a first outer surface (24) of the blood product (10) and the third layer (23) defining a second outer surface (25) of the blood product (10). The first layer (21) comprises a majority of fibrin, the second layer (22) comprises a majority of thrombocytes and the third layer (23) comprises a majority of leukocytes.

Claims

1. A method to treat a wound, the method comprising: applying, to the wound, a self-supporting, three-layered blood product that comprises in consecutive layers: (a) a first layer including fibrin, (b) a second layer including thrombocytes, and (c) a third layer including leukocytes, wherein the blood product is configured for application to the wound so that the third layer is in direct contact with the wound.

2. The method of claim 1, wherein the third layer comprises leukocytes that are a majority by volume of the third layer.

3. The method of claim 1, wherein the three-layered blood product comprises in substantially parallel layers: fibrin in a majority by volume of a first layer, thrombocytes in a majority by volume of a second layer and leukocytes in a majority by volume of a third layer.

4. The method of claim 1, wherein the blood product conforms to continuous contours to which the blood product is applied.

5. The method of claim 1, wherein the blood product can withstand applied stress without rupturing.

6. The method of claim 1, further comprising removing, after a centrifugation, a blood product from a container as a single, self-supporting three-layered blood product of (a) fibrin, (b) thrombocytes, and (c) leukocytes, wherein the centrifugation is performed on whole blood with a centrifugal application force of at least 1000 times gravity so that the whole blood separates into (1) erythrocytes, (2) a blood product comprising fibrin, thrombocytes, and leukocytes, and (3) serum, and wherein the centrifugal application force is applied for a time that is sufficient to also separate the blood product into three layers of (a) the fibrin, (b) the thrombocytes, and (c) the leukocytes.

7. The method of claim 6, wherein the first material is selected from the group consisting of: polypropylene, polyethylene, polycarbonate, polyamide, acrylonitrile butadiene styrene, styrene, modified styrene, and polyurethane.

8. The method of claim 6, wherein the container has an inner surface that comprises a surface coating that lowers friction between the three-layered blood product and the container.

9. The method of claim 6, further comprising isolating the erythrocytes from the three-layered blood product during the coagulation.

10. The method of claim 6, further comprising a washing step where the three-layered blood product is washed so that if present, substantially all erythrocytes and serum attached to the three-layered blood product are detached.

11. The method of claim 6, further comprising adding a first substance selected from the group consisting of: fibroblasts, keratinocyte cells, and hyaluronic acid to the whole blood.

12. The method of claim 1, wherein the separating occurs concurrent with coagulation of the whole blood.

13. The method of claim 1, wherein the whole blood coagulates upon initiation of coagulation.

14. The method of claim 7, wherein the initiation of coagulation is by a first material that defines an inner surface of the container.

15. A method to induce healing is performed in three consecutive discrete layers so that the first layer protects the wound from environmental contamination, the second layer stimulates fibroblasts, and the third layer maintains sterility of the wound.

16. The method of claim 15, wherein the healing is for an anastomosis procedure.

17. The method of claim 15, wherein the healing is to prevent or minimize post-surgical adherence.

18. The method of claim 15, wherein the healing is to seal a surgical site.

19. The method of claim 15, wherein the three discrete layers comprise, in consecutive layers: (a) a first layer including fibrin, (b) a second layer including thrombocytes, and (c) a third layer including leukocytes.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0077] The invention is explained in detail below with reference to the drawing, in which

[0078] FIG. 1 shows a cross sectional view of a first embodiment of the container means according to the invention,

[0079] FIG. 2 shows a cross sectional view of a second embodiment of the container means according to the invention,

[0080] FIG. 3 shows a cross sectional view of a third embodiment of the container means according to the invention,

[0081] FIG. 4 shows a cross sectional view of a fourth embodiment of the container means according to the invention,

[0082] FIG. 5 shows a cross sectional view of the first embodiment of the container means according to the invention,

[0083] FIG. 6 shows a cross sectional view of the second embodiment of the container means according to the invention,

[0084] FIG. 7 shows a cross sectional view of the third embodiment of the container means according to the invention,

[0085] FIG. 8 shows a cross sectional view of the fourth embodiment of the container means according to the invention,

[0086] FIG. 9 illustrates a cross sectional view of an embodiment of the blood product according to the invention, and

[0087] FIG. 10 shows a cross sectional view of an embodiment of the blood product according to the invention.

DETAILED DESCRIPTION

[0088] FIGS. 1 to 4 illustrates respectively a first, second, third and fourth embodiment of a container means 1. The container means 1 in the four embodiments comprises a cavity having an inner surface 3. The cavity being defined by a wall 4, a closed end 12 and an open end closeable by a lid 2. The container means 1 defines a volume of whole blood 5 and is closed to the surroundings via the lid 2 mounted on the open end of the container means 1. The container means 1 can be made of any material, and the material can be chosen, so that the material of the container means 1 due to contact with the whole blood 5 via the inner surface 3 activates the coagulation cascade of the whole blood 5, which is the case for the first embodiment in FIG. 1 and the third embodiment in FIG. 3. Alternatively, the material of the container means 1 can be chosen so that the material is inactive in relation to the whole bloods 5 coagulation cascade, which is the case for the second embodiment in FIG. 2 and the fourth embodiment in FIG. 3, where an object 7, made of a material that activates the bloods 5 coagulation cascade, instead is added to the whole blood 5, such that the object 7 is used to activate the whole bloods 5 coagulation cascade. The inner surface 3 of the container means 1 in the four embodiments can be coated and/or surface treated in order to lower the friction between the inner surface 3 and any components of the blood 5. Furthermore the material of the container means 1 can be chosen, so that a low friction between the inner surface 3 and any component of the blood 5 is obtained, e.g. by choosing a material with a low protein binding capacity. In the third and fourth embodiment shown in FIGS. 3 and 4 a compaction means 8, such as a filter, is placed in the container means 1, whereby the blood product 10 is compacted against the compaction means.

[0089] The compacting means 8 can be locked in the closed end 12 of the container means 1, where the erythrocytes are located, in the initial part of the process. At a later point in time the compacting means 8 can be released and provided that the density of the compacting means 8 is lower that the plasma, the compacting means will be forced to the top of the plasma. By removing the lid 2, the blood product 10 can be removed from the container means 1. The compaction means 8, or part of the compacting means, can be used to support the blood product 10 during transport from the container means 1.

[0090] The whole blood 5 in all four embodiments are subjected to a centrifugal force 6 acting downwards as illustrated, however the centrifugal force 6 is not limited to act in the shown direction. The centrifugal force 6 functions as a separation means, since the components of the whole blood 5 have different densities and thus will respond differently to the centrifugal force 6.

[0091] FIGS. 1 to 4 illustrates the four embodiments of the container means 1 at a point of time where the centrifugal force 6 just been applied, hence no separation of the whole blood 5 is visible, while FIGS. 5 to 8 respectively shows the first, second, third and fourth embodiment of the container means 1 at a point of time where the centrifugal force 6 has been acting sufficiently long and with sufficiently magnitude, hence the whole blood 5 has separated into serum 9, with the fibrin distributed through this serum part, platelets and leucocytes layer 10 and erythrocytes 11. At a later point in time the compacting means 8 can be released and provided that the density of the compacting means 8 is lower than that of the serum, the compacting means will be forced to the top of the serum and thereby releasing the fibrin from the wall and compacting the fibrin. By removing the lid 2, the blood product 10 can be removed from the container means 1. The compaction means 8, or part of the compacting means, can be used to support the blood product 10 during transport from the container means 1.

[0092] FIG. 9 illustrates a schematic cross sectional view of the blood product 10 comprising a first layer 21, a second layer 22 and a third layer 23, where the second layer is adjacent to the first layer 21 and the third layer 23. The first layer 21 defines a first outer surface 24 of the blood product 10 and the third layer 23 defines a second outer surface of the blood product 10. The first layer 21 comprises a majority of fibrin, while the second layer 22 comprises a majority of thrombocytes and the third layer 23 comprises a majority of leukocytes. Thus the blood product 10 has a well defined multilayer structure with each layer having different compositions and therefore having different functionalities.

[0093] FIG. 10 shows a cross sectional view of a blood product obtained experimentally by using a test tube made of polypropylene as container means.

[0094] The invention has been described with reference to a preferred embodiment. However, the scope of the invention is not limited to the illustrated embodiment, and alterations, combinations and modifications can be carried out without deviating from the scope of the invention.

EXAMPLES

Example 1

[0095] 1. Take a plastic container (eg. a 2 ml microcentrifuge tube) and add a coagulation trigger, eg. 4 glass beads (diameter 2 mm) [0096] 2. Draw blood into the plastic container. [0097] 3. Mix the blood and coagulation activator end-over-end for 2 min. [0098] 4. Spin the container at 16200 g for 20 min. [0099] 5. After this the blood product will be formed in a layer between the red blood cells and the serum. [0100] 6. Remove the blood product using forceps.

[0101] In this method the needed spin time and spin speed will depend on the power of activation. E.g. if the coagulation activation is high, the cells must be separated before they are trapped in the fibrin network, this will require a high spin speed. Due to the high spin speed, spin time can be low.

Example 2

[0102] 1. Take a standard 50 ml centrifugation tube container and ad a coagulation trigger, eg. glass beads. [0103] 2. Draw blood into the centrifugation tube. [0104] 3. Mix the blood in the tube for 1 min. [0105] 4. Spin the tube at 3000 g for 20 min [0106] 5. Open the lid and loosen the fibrin (formed in the serum in the top part) from the wall (using a thin plastic stick or needle) [0107] 6. Spin the sample another 5 min at 3000 g. [0108] 7. After this the blood product will be formed in a layer between the red cell and serum. [0109] 8. Remove the blood product using forceps

Example 3

[0110] 1. Take a 20 ml container (inner diameter: 26 mm) prepared out of polyamide or polyurethane [0111] 2. Ad a lid and create a vacuum inside the container [0112] 3. Insert a needle in the patient [0113] 4. Connect the needle to the container without losing the vacuum [0114] 5. Draw blood into the plastic container by the help of the vacuum. [0115] 6. Spin the tube at 15000 g for 12 min [0116] 7. After this the blood product will be formed in a layer between the red cell and serum.

[0117] 8. Remove lid and remove the blood product with for forceps

Example 4

[0118] 1. Take a 20 ml container (inner diameter: 26 mm) prepared out of polyamide or polyurethane [0119] 2. Place a disk in the bottom of the container. The density of the disk shall be less than 1, preferable as low as possible. [0120] 3. Fixate the disk in the bottom of the container, eg compressing the wall of the container. [0121] 4. Add a lid and create a vacuum inside the container [0122] 5. Insert needle in the patient [0123] 6. Connect the needle to the container without losing the vacuum [0124] 7. Draw blood into a plastic container by the help of the vacuum. [0125] 8. Spin the tube at 3000 g for 8 min. At this stage the leucocytes will be on top of the red blood cells and the fibrin will be polymerized through the upper serum layer as the g force is low and not able to compress it on top of the platelets. [0126] 9. Release the disk in the bottom [0127] 10. Spin the tube at 3000 g for 2 min. This will cause the disk to move to the top and thereby collecting the leukocytes and platelets and compress the fibrin layer into one sheet. [0128] 11. Remove lid and remove the blood product, that is placed in top of the container

Example 5

[0129] 1. Take a 20 ml container (inner diameter: 26 mm) prepared out of polyamide or polyurethane [0130] 2. Place a disk in the bottom of the container. The density of the disk shall be less than 1, preferable as low as possible. [0131] 3. Fixate the disk in the bottom of the container, eg by compressing the wall of the container. [0132] 4. Create a vacuum inside the container and add a lid [0133] 5. Insert a needle in the patient [0134] 6. Connect the needle to the container without losing the vacuum [0135] 7. Draw blood into a plastic container by the help of the vacuum. [0136] 8. Spin the tube at 3700 g for 3 min. At this stage the leucocytes will be on top of the red cells and the fibrin will be polymerized through the upper serum layer as the g force is low and not able to compress it on top of the platelets. [0137] 9. Wait for 8 minutes. [0138] 10. Release the disk in the bottom [0139] 11. Spin the tube at 3000 g for 2 min. This will cause the disk to move to the top and thereby collecting the leukocytes and platelets and compress the fibrin layer into one sheet. [0140] 12. Remove lid and remove the blood product, that is placed in top of the container

[0141] As it appear from the above examples, the combination of coagulation activation, spin speed (g), spin time, rest time between spin can be varied within some limits.

[0142] These are illustrated on below tables:

TABLE-US-00001 Coagulation Coagulation Spin time after or activation speed 1. Spin time during 1 spin 2. Spin Low Low Long Long Yes, high Low Low Long Long Yes, can be low if fi- brin adhesion to con- tainer wall is mechan- ical released from wall or adheasion to the wall is low. Low High Short Long Yes, high High High Long Short No High High Short Short Yes, high

[0143] If processing with a disk:

TABLE-US-00002 Coagulation Coagulation Spin time after or 2. Spin were the activation speed 1. Spin time during 1 spin disk is released Low Low Long Long Low Low High Short Long Low High High Long Short Low High High Short Short Low

Example 6Optimization of Relative Centrifugation Force and Time

[0144] 1. Full blood was drawn into 6 ml EDTA tubes (Vacutainer, BD). [0145] 2. 20 ml container (inner diameter: 26mm) prepared out of polyamide or polyurethane were filled with 18 ml and spun at different RCFs for different times [0146] 3. Samples (300 l) were taken by a syringe at the bottom (approx 5 mm above the bottom) and top (approx 5 mm below the surface) after the above spin timesafterwards the spin was continued to obtain the next sample. Bottom samples were diluted 1:1 with D-PBS. [0147] 4. Samples were analyzed by automated cell counting (XE 2100, Sysmex Corporation) and cell numbers in the original sample calculated. Results are given in millions of cells per ml.

[0148] Millions of platelets per milliliter in the upper (top) and lower (bottom) part of blood centrifuged at the given relative centrifugation force (g) as a function of time (min):

TABLE-US-00003 Minutes 0 1 3 5 7 11 2000 g top 178 480 224 113 59 13 2000 g bottom 178 7 5 3 2 4 3000 g top 178 378 103 38 14 0 3000 g bottom 178 3 2 5 3 1 3700 g top 178 364 97 27 8 0 3700 g bottom 178 2 11 2 1 0

[0149] Millions of Leucocytes per milliliter in the upper (top) and lower (bottom) part of blood centrifuged at the given relative centrifugation force (g) as a function of time (min):

TABLE-US-00004 Minutes 0 1 3 5 7 11 2000 g top 7.43 0.28 0 0 0 0 2000 g bottom 7.43 0.89 0.02 0.13 0.26 0.12 3000 g top 7.43 0.19 0 0 0.01 0 3000 g bottom 7.43 0.07 0.04 0.11 0.02 0.02 3700 g top 7.43 0.05 0.03 0 0 0 3700 g bottom 7.43 0.03 0.59 0.07 0.02 0.01

Example 7Growth Factor Release as a Response to Chronic Wound Fluid

[0150] 1. The blood product was generated by the method given in example 1. [0151] 2. The blood products was cut in half and placed in 100 l PBS1% BSA or 100 l chronic wound fluid (collected over 24 hrs from a venous leg ulcer) and incubated at 37 degrees celcius. [0152] 3. After given time points (1, 2, 3.5, 7, 14, 22 and 29 hours) the samples were spun at 16000 g for 10 min, and the supernatant transferred to a new tube, added 1/10 th (81 l sample+9 l PI) Protease Inhibitor (Complete, Roche) and frozen at 80 degree celcius. [0153] 4. Platelet derived growth factorAB levels in were determined by using an ELI-SA kit (DuoSet ELISA cat. No. DY222, R&D systems) as described by the manufacturer. PDGF-AB concentrations in the original samples were calculated.

[0154] Platelet derived growth factor AB (PDGF-AB) release from blood product (ng/ml blood product) as a function of time (hrs).

TABLE-US-00005 Time (hours) 1 2 4 7 22 29 Chronic Wound fluid 0 0 0 0 0 0 (control) Blood product in PBS 153 172 160 174 206 233 Blood product in chronic 426 490 602 497 234 275 wound fluid

Example 8

[0155] 1. The blood product was generated by the method given in example 1. [0156] 2. Two blood products were incubated in 1 ml DMEM (PAA, Germany) at 37C and 5% CO2 for 48 hrs. [0157] 3. In parallel 2 blood products were incubated in 1 ml DMEM (PAA, Germany) including lipopolysaccharide ((10 ng/ml) (LPS derived from Escherichia coli; L2654; Sigma-Aldrich) at 37 C and 5% CO2 for 48 hrs. [0158] 4. After 48 hrs the media were transferred to microcentrifuge tubes and spun 20 min at 16200g. The supernatants were frozen at 80 C until analysis. [0159] 5. Proteome profiler Arrays ARY007 and ARY005 (both R&D systems) were performed as described by R&D systems, except that buffer 4 (ARY007) were used in both kits. 420 ul supernatant were diluted in 500 ul buffer 4 and 580 ul buffer 5 as described by R&D systems. [0160] 6. Reactivity with arrays were detected using chemiluminescent HRP substrate (Immobilon Western, Millipore, US). [0161] 7. Light emission were captured using a Fluorchem 3000 system (Alpha Innotech, US). Mean pixel density were extracted by the Flourchem software (Alpha Innotech, US).

TABLE-US-00006 Blood product + Blood product Lipopolysaccharide Substance detected (Mean pixel density) (Mean pixel density) CXCL8 (IL-8) 2605 404 CXCL10 (IP-10) 305.5 48.5 MMP-8 518 214 IL-1ra 1825.5 1117.5 IL-16 171 105 MMP-9 2663.5 1729.5 Angiopoietin-1 676.5 537.5 PDGF-AB/PDGF-BB 851.5 704.5 PDGF-AA 1357 1159.5 CXCL16 300 256.5 TIMP-1 2507.5 2170.5 Endostatin 187 175.5 Angiogenin 689 650.5 MIF 1989 1880.5 IGFBP-2 1942 1883.5 IGFBP-3 444 468 EGF 612.5 670.5 IGFBP-1 291.5 401 sICAM-1 635.5 894.5 PAI-1 1940 2897.5 CCL5 (RANTES) 4104 6151.5 CD26 458.5 828.5 VEGF 246.5 891.5 CXCL1 (GRO-alpha) 336 2236 CCL4 (MIP-1-beta) 7 117.5 CCL2 (MCP-1) 6.5 334 G-CSF 7.5 429.5 IL-1 Beta 7 600 IL-6 8 2094.5