FIELD OF THE INVENTION

This invention relates to a systems and methods for producing high levels of autologous IL-IRA cytokine as well as the means used therein, in particular in closed systems.

BACKGROUND

Therapeutically-effective proteins, such as autologous IL-IRA cytokine have been known for a long time. Cytokines such as interleukin play an important role in degenerative musculo-skeletal diseases, including osteoarthritis(OA), and a multitude of inflammatory disorders. Agents that inhibit the action of such cytokines have a high therapeutic potential in such diseases. There is a need for simple and rapid means and methods for the preparation of therapeutically active proteins such as the interleukin receptor antagonist (IRA).

US 2004156823 describes a method for inducing therapeutically-effective protein or protein mixture in a syringe (such as interleukin 1 receptor antagonist (IL-Ira)) from body fluid in a syringe in which the inner structures of the syringe are coated with inductors such as immunoglobulin incubated and the therapeutically-effective protein is formed in the body fluid.

In this method the body fluid is taken with a syringe directly from the subject.

The document presents 4 examples for inducing IL-Ira .

US 2003138910 provides methods and means for producing IL-IRa which serve as safe, cost-effective alternatives which can be carried out quickly for the use and for the production of conventional pharmaceutical products.

US 6623472 presents a method for inducing therapeutically-effective protein includes a syringe having inner structures coated with an inductor, disposing a therapeutically- effective protein therein with a body fluid and incubating the syringe and its contents. Thus there is a long unmet need to provide methods and means for producing high levels of interleukin receptor antagonists by a technique that can be carried out quickly for the use and production of pharmaceutical products.

SUMMARY OF INVENTION

There is provided in accordance with a preferred embodiment of the present invention a system for producing autologous IL-IRA cytokine comprising: (i)a blood collection tube, (ii)a tube cover, (iii) a portion of separation gel , (iv)an anticoagulant portion, (v)a plasma collection syringe comprising a sharp needle, (vi)a buffy coat collection syringe; and (vii)an incubation tube with cover; wherein the blood collection tube is a vacuum tube and the separation gel and the anticoagulant is in the vacuum tube.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation tube is adapted for incubation for sufficient time and temperature yielding high levels of the autologous IL-IRA cytokine.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further comprises a filter unit of 3- 50μιη.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation tube is selected from the group consisting of: a plastic tube, a glass tube, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the vacuum tube is adapted such that, when containing the whole blood, and centrifuged after treatment, yields separation fractions comprising, a first fraction of RBCs sediment, a second fraction of the gel , a third fraction comprising WBCs, platelets and), and fourth fraction of plasma solution.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further comprises a stopcock adapted as a turning plug which controls the flow of fluid from the plasma collection syringe to the blood collection tube. There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further comprises a stopcock adapted as a turning plug which controls the flow of fluid from the buffy coat collection syringe to the blood collection tube.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation tube comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the an anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant , oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the separation gel is at density of 1.08-1.09gr/cm ³ .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the centrifugation is being preformed for about about 10 to about 30 minutes.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the plasma is discarded up to 80% of plasma from the blood collection tube .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the plasma is discarded at least 50% of plasma from the blood collection tube .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the plasma is discarded about 80% of plasma from the blood collection tube .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein a higher potency of the IL-IRA is obtained when plasma portion containing IL-1RI molecules bound to IL-1RA, is discarded. There is provided in accordance with a preferred embodiment of the present invention a syringe system for producing autologous IL-1RA cytokine comprising: (i)a blood collection syringe, (ii)an anticoagulant portion, (iii) a syringe stopper, (iv) a sharp syringe needle, and (v) an incubation vessel with cover; wherein the blood collection syringe is adapted such that, when containing the whole blood ,and centrifuged after treatment, yields separation fractions comprising, a first fraction of RBCs sediment, a second fraction comprising WBCs, platelets and a third fraction of plasma solution.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the incubation vessel(30) containing WBCs and a portion of plasma, is incubated for sufficient time and temperature yielding high levels of the autologous IL-1RA cytokine.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein a portion of the RBCs is discarded resulting a fraction of WBCs as the bottom layer, and a fraction of plasma solution.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the an anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant , oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein portion of plasma transferred to the incubation vessel is between about 5% to about 100%

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the portion of plasma transferred to the incubation vessel is up to 5%.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein a higher potency of the IL-IRA is obtained in the incubation vessel when plasma solution containing IL- 1RI molecules bound to IL-IRA, is remained in the blood collection syringe. There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation vessel is selected from the group consisting of: plastic tube , glass tube, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation vessel comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the centrifugation is been preformed for about 10-30min .

There is provided in accordance with a preferred embodiment of the present invention a vessel system for producing autologous IL-1RA cytokine comprising: (i)a blood collection vessel, (ii)a vessel cover, (iii)an anticoagulant portion, (iv)a plasma and WBCs portion collection syringe comprising a sharp needle, (v)an incubation vessel with cover, and (vi)plasma portion collection syringe;

wherein the blood collection vessel includes an anticoagulant; further wherein the blood collection vessel is adapted such that, when containing the whole blood , placed in an upward position for sufficient time, yields separation fractions comprising a first fraction of WBCs sediment, a second fraction comprising WBCs , platelets and), and a third fraction of plasma solution; further wherein when the incubation vessel containing WBCs and plasma solution mixture centrifuged after treatment, yields separation fractions comprising a first fraction of WBCs sediment, and a second fraction of plasma solution.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the incubation vessel is incubated for sufficient time and temperature yielding high levels of the autologous IL-1RA cytokine.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the blood collection vessel is selected from the group consisting of: a syringe, a plastic tube ,a glass tube, a vacuum tube or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above,, wherein the incubation vessel is selected from the group consisting of: a plastic tube ,a glass tube, a syringe or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the fraction of RBCs sediment is formed using gravity force.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the tube in upward position is positioned for about 20-60minutes.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above,, further comprises a filter unit of 3- 50μιη.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above,, wherein the incubation tube comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the plasma is discarded up to 80% of plasma from the blood collection vessel.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the plasma is discarded at least 50% of plasma from the blood collection vessel.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the plasma is discarded about 80%> of plasma from the blood collection vessel.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein a higher potency of the IL-1RA is obtained when plasma portion containing IL-1RI molecules bound to IL-1RA, is discarded.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the centrifugation is being preformed for about 10-30 min .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation is being preformed at ambient temperature of 25-37 °c for 6-24 hours. There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation is being preformed with or without 5-6% C0 ₂ .

There is provided in accordance with a preferred embodiment of the present invention a vessel system for producing autologous IL-IRA cytokine comprising: (i)a blood collection syringe comprising a sharp needle and a filter unit, (ii)a filtered blood sample tube, (iii) a tube cover, (iv) a plasma portion collection syringe with a sharp needle, and (v)an incubation vessel with a cover; wherein the blood collection syringe comprising a filter is adapted to be placed in a downward position containing the whole blood, such that the whole blood is passed through the filter yielding plasma platelets and RBC mixture(54), the filter adapted to retain a fraction of WBCs on or within the filter; further wherein when the a filtered blood sample tube centrifuged after treatment yields a separation fractions comprising a first fraction of RBCs sediment, a second fraction comprising, platelets and third fraction of plasma solution; further wherein when the filter comprising fraction of WBCs is washed with the plasma portion collection syringe comprising plasma yields a WBCs fraction.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the incubation vessel is incubated for sufficient time and temperature yielding high levels of the autologous IL-IRA cytokine.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the filtered blood sample tube is centrifuged for 10 to about 30 minutes.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the filtered blood sample tube is selected from the group consisting of: a plastic tube, a glass tube, a vacuum tube or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation vessel is selected from the group consisting of: a syringe, a plastic tube, a glass tube, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation is been preformed at ambient temperature of 25-37 °c for 6-24 hours. There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation is been preformed with or without 5-6% C0 ₂ .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation vessel comprises pretreated and washed borosilicate glass beads in a size of 0.5-5mm .

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the filter unit is selected from the group consisting of: a hydrophobic filter, hydrophilic filter or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the plasma is discarded up to 80% of plasma from the filtered blood sample tube.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the plasma is discarded at least 50%> of plasma from the filtered blood sample tube.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein the plasma is discarded about 80%> of plasma from the filtered blood sample tube.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further wherein a higher potency of the IL-IRA is obtained when plasma portion containing IL-1RI molecules bound to IL-1RA, is discarded.

There is provided in accordance with a preferred embodiment of the present invention a vessel system for producing autologous IL-IRA cytokine comprising: (i)an incubation vessel comprising washed and pretreated borosilicate glass beads for collection blood sample, (ii)a vessel cover; wherein the vessel comprising the washed and pretreated borosilicate glass beads and containing whole blood such that when incubated for sufficient time and temperature yield high levels of the autologous IL- IRA cytokine in the solution.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation vessel is selected from the group consisting of: a plastic tube, a glass tube, or any combination thereof. There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the washed and pretreated borosilicate glass beads are in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, further comprises an anticoagulant portion which is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above,, wherein the incubation is been preformed at ambient temperature of 25-37 °c for 6-24 hours.

There is further provided in accordance with a preferred embodiment of the present invention the system as defined above, wherein the incubation is been preformed with or without 5-6% C0 ₂ .

There is provided in accordance with a preferred embodiment of the present invention a method for producing of autologous IL-1RA cytokine in a closed system, the method comprising steps of: (a)obtaining a vacuum tube system for producing autologous IL-IRA cytokine comprising: (i)a blood collection vacuum tube, (ii)a tube cover, (iii)a portion of separation gel, (iv)an anticoagulant portion, (v)a plasma collection syringe comprising a sharp needle, (vi)a buffy coat collection syringe, and(vii)an incubation tube with a cover;

(b)placing the anticoagulant portion and the portion of separation gel in the vacuum tube,(c)filling the blood collection vacuum tube with blood sample, (d)separating by centrifugation there by obtaining separation fractions comprising, a first fraction of RBCs sediment, a second fraction of the gel , a third fraction comprising WBCs, platelets, growth factors and plasma solution fraction, (e)discarding a portion of plasma with the plasma collection syringe comprising a sharp needle, (f)extracting the remaining plasma and WBCs solution with the plasma collection syringe, (g)transferring the solution into the incubation tube; and (h)incubating the incubation tube; such that a high concentration of the autologous IL-IRA cytokine is obtained. There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation tube comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further comprises a filter unit of 3- 50μιη.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation tube is selected from the group consisting of: a plastic tube, a glass tube, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the separation gel is at density of 1.08-1.09gr/cm ³ .

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the discarding up to 80% of plasma from the blood collection vacuum tube.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the discarding at least 50% of plasma from the blood collection vacuum tube.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the discarding about 80%> of plasma from the blood collection vacuum tube.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further wherein a higher potency of the IL-IRA is obtained when plasma portion containing IL-1RI molecules bound to IL-1RA, is discarded.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant ,oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood. There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed with or without 5-6% C0 ₂ .

There is provided in accordance with a preferred embodiment of the present invention a method for producing autologous IL-IRA cytokine in a closed system, the method comprising the steps of: (a)obtaining a syringe system for producing autologous IL- lRA cytokine comprising: (i)a blood collection syringe, (ii) an anticoagulant portion, (iii)a syringe stopper, (iv)a sharp syringe needle; and (v)an incubation vessel with a cover;

(b)placing the anticoagulant portion in the blood collection syringe;(c)drawing whole blood with the blood collection syringe, (d)positioning the syringe in an downward position comprising a stopper, (e)separating the whole blood in the syringe by centrifugation yielding separation fractions comprising, a first fraction of RBCs sediment, a second fraction comprising WBCs, platelets and, and a third fraction of plasma solution, (e)discarding RBCs fraction from the syringe, (f)transferring the fraction of WBCs, platelets, growth factors and portion of plasma solution into incubation vessel; and (g)incubating the incubation vessel ;such that a high concentration of the autologous IL-IRA cytokine is obtained.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel contains pretreated and washed borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant ,oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours. There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed with or without 5-6% C0 ₂ .

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel is selected from the group consisting of: plastic tube, glass tube, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the centrifugation is been preformed for about 10 to about 30 minutes .

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the transferring portion of plasma to the incubation vessel is up to 5%.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further wherein a higher potency of the IL-1RA is obtained when the plasma solution containing IL-1RI molecules bound to IL-1RA, is retained in the blood collection syringe.

There is provided in accordance with a preferred embodiment of the present invention a method for producing of autologous IL-IRA cytokine in a closed vessel system , the method comprising the steps of : (a)obtaining a vessel system for producing autologous IL-IRA cytokine comprising: (i)a blood collection vessel, (ii)a vessel cover, (iii)an anticoagulant portion, (iv)a plasma and WBCs portion collection syringe comprising a sharp needle, (v)an incubation vessel with a cover; and (vi)a plasma portion collection syringe;

(b)placing the anticoagulant in the blood collection vessel, (c)filling the blood collection vessel with blood, (d)placing the vessel in an upward position for sufficient time yielding separation fractions comprising a first fraction of WBCs sediment, and a second fraction comprising WBCs , platelets and and third fraction of plasma solution, (e)drawing the fractions of WBCs ,and plasma solution using a plasma portion collection syringe, (f)transferring the WBCs ,and plasma solution to the incubation vessel, (g)separating the incubation vessel by centrifugation, yielding separation fractions comprising a first fraction of WBCs sediment, and a second fraction of plasma solution, (h)discarding a portion of plasma with a buffy coat collection syringe ;and (i)incubating the incubation vessel; such that a high concentration of the autologous IL-IRA cytokine is obtained.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant ,oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed with or without 5-6% C0 ₂ .

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the discarding up to 80%> of plasma from the incubation vessel.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the discarding at least 50% of plasma from the incubation vessel.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the discarding about 80%> of plasma from the incubation vessel.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further wherein a higher potency of the IL-IRA is obtained when plasma portion containing IL-1RI molecules bound to IL-IRA, is discarded.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the blood collection vessel is selected from the group consisting of: a syringe, a plastic tube ,a glass tube, a vacuum tube or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel is selected from the group consisting of: a plastic tube ,a glass tube, a syringe or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the fraction of RBCs sediment is formed using gravity force.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the tube in upward position is positioned for about 20-60minutes.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further comprises a filter unit of 3- 50μιη.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the centrifugation is been preformed for about 10-30 min .

There is provided in accordance with a preferred embodiment of the present invention a method for producing of autologous IL-IRA cytokine in a closed vessel system , the method comprising the steps of : (a)obtaining a vessel system for producing autologous IL-IRA cytokine comprising: (i)a blood collection syringe comprising a sharp needle and a filter unit, with blood sample, (ii)a filtered blood sample tube, (iii)a plasma portion collection syringe with a sharp needle; and (iv)an incubation vessel with a cover;

(b)filling syringe with whole blood sample, (c)transferring content of the syringe through a filter unit to the filtered blood sample tube, (d)separating the filtered blood sample tube by centrifugation yielding a separation fractions comprising a first fraction of RBCs sediment, a second fraction comprising, platelets and third fraction of plasma solution, (e)drawing a portion of plasma using the plasma portion collection syringe, (f)flushing the filter containing WBCs with the obtained plasma in the a plasma portion collection syringe to the incubation vessel yielding a WBCs fraction(52);and (h)incubating the incubation vessel; such that a high concentration of the autologous IL-IRA cytokine is obtained. There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel contains pretreated and washed borosilicate glass beads in a size of 0.5-5mm.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the step of drawing up to 80% of plasma from the filtered blood sample tube.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the step of drawing at least 50% of plasma from the filtered blood sample tube.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the step of drawing about 80% of plasma from the filtered blood sample tube.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further wherein a higher potency of the IL-IRA is obtained when the plasma portion containing IL-1RI molecules that bind to IL-IRA, is discarded.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the filter unit is selected from the group consisting of: a hydrophobic filter, hydrophilic filter or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is been preformed with or without 5-6%> C0 ₂ .

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the filtered blood sample tube is centrifuged for 10 to about 30 minutes.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the filtered blood sample tube is selected from the group consisting of: a plastic tube, a glass tube, a vacuum tube or any combination thereof. There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel is selected from the group consisting of: a syringe, a plastic tube, a glass tube, or any combination thereof.

There is provided in accordance with a preferred embodiment of the present invention a method for producing of autologous IL-IRA cytokine in a closed vessel tube system, the method comprising the steps of: (a)obtaining a vessel system for producing autologous IL-IRA cytokine comprising: (i)an incubation vessel comprising washed and pretreated borosilicate glass beads for collection blood sample; (ii)a vessel cover; (b)filling the incubation vessel with whole blood sample; and

(c)incubating the incubation vessel; such that higher concentration of the autologous IL-IRA cytokine is formed .

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the borosilicate glass beads are pretreated and washed beads in a size of 0.5-5mm

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is being preformed at ambient temperature of 25-37 °c for 6-24 hours.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubating is being preformed with or without 5-6% CO.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, further comprises an anticoagulant portion which is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant, or any combination thereof.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the filtered blood sample tube is centrifuged for 10 to about 30 minutes. There is further provided in accordance with a preferred embodiment of the present invention the method as defined above, wherein the incubation vessel is selected from the group consisting of: a plastic tube, a glass tube, a vacuum tube or any combination thereof.

There is provided in accordance with a preferred embodiment of the present invention a portion of IL-IRA substantially free of IL1RI produced by a method of preparing autologous IL-IRA cytokine in a closed system, wherein the method comprises steps of obtaining a system selected from the group consisting of: a vessel system, a syringe system, or any combination thereof; further wherein a higher potency of the IL-IRA is obtained when the plasma containing IL-IRI molecules that bind to IL-IRA, is discarded.

BRIEF DESCRIPTION OF THE INVENTION

In order to understand the invention and to see how it may be implemented in practice, a few preferred embodiments will now be described, by way of non-limiting example only, with reference to be accompanying drawings, in which:

Fig. 1 shows a closed system for derivation of high levels of autologous IL-IRA cytokine in WBCs enriched plasma, in accordance with a preferred embodiment of the present invention; and,

Fig. 2 shows a closed syringe system for derivation of high levels of autologous IL- IRA cytokine, in accordance with a preferred embodiment of the present invention; and,

Fig. 3 shows a closed vessel system for derivation of high levels of autologous IL- IRA cytokine, in accordance with a preferred embodiment of the present invention; and,

Fig. 4 shows a closed vessel system for derivation of high levels of autologous IL- IRA cytokine, in accordance with a preferred embodiment of the present invention;

Fig. 5 shows a closed vessel system for derivation of high levels of autologous IL- IRA cytokine in a whole blood sample, in accordance with a preferred embodiment of the present invention; Fig 6 illustrates a flow chart of a method for producing of autologous IL-1RA cytokine in a closed system, in accordance with a preferred embodiment of the present invention;

Fig 7 illustrates a flow chart of a method for producing of autologous IL-1RA cytokine in a closed syringe system, in accordance with a preferred embodiment of the present invention;

Fig 8 illustrates a flow chart of a method for producing of autologous IL-1RA cytokine in a closed vessel system, in accordance with a preferred embodiment of the present invention;

Fig 9 illustrates a flow chart of a method for producing of autologous IL-1RA cytokine in a closed vessel system, in accordance with a preferred embodiment of the present invention; and

Fig 10 illustrates a flow chart of a method for producing of autologous IL-IRA cytokine in a closed vessel system, in accordance with a preferred embodiment of the present invention.

DETAILED DESCRIPTION

The following description is provided so as to enable any person skilled in the art to make use of the invention and sets forth the best modes contemplated by the inventor of carrying out this invention. Various modifications, however, will remain apparent to those skilled in the art, since the generic principles of the present invention have been defined specifically to provide methods and systems for producing high levels of autologous IL-IRA cytokine.

The term "Buffy coat collection syringe" is used herein to denote a second syringe which is used for collecting the fraction comprising WBCs platelets growth factor with or without the remaining plasma.

Reference is now made to fig. 1 which illustrates a closed system for deriving high levels of autologous IL-IRA cytokine in WBCs enriched plasma .

In accordance with the preferred embodiment of the present invention all the elements of the invention such as tubes, syringes etc are combined in a closed system. The closed system for producing autologous IL-IRA cytokine comprises : (a) a vacuum tube 1 for collecting a blood sample 5, (b) a cover for the tube 4, (c)a separation gel layer 2,(d)an anticoagulant 3,(e) (f) a plasma portion collection syringe 9 comprising a sharp needle 9a and an optional filter unit, (g) a buffy coat collection syringe 11, and (h) a tube for incubation 15 with a cover.

In some embodiments, test tubes are made of glass or MPA (modified poly Amide) or modified PET (Poly Ethylene Terephtalate). In some embodiments, the test tube has a layered structure such that the interior wall of the test tube comprises polypropylene. Test tubes can further be provided with a special cover. By way of non-limiting example the cover is made of butyl rubber or its halo derivative formulations at hardness between 40-60 Shore A. The hardness assures stable vacuum for at least the shelf life of the test tube which can be between 18-24 months.

The test tubes used can be of various sizes which depend of the required quantity of whole blood to be drawn from the treated subject. The test tubes have typically a size suitable for blood samples in the range of 4ml to 100ml.

The incubation tube is selected from the group consisting of: plastic tube , glass tube, or any combination thereof. The equipment consisting of a plastic hub, a hypodermic needle, and a vacuum tube. Under certain circumstances, a syringe may be used, often with a butterfly needle, which is a plastic catheter attached to a short needle. In the developing world, a needle and syringe are still the most common method of drawing blood.

In accordance with the preferred embodiment of the present invention ,fig. 1 shows the vacuum tube 1 consisting of a separation gel 2 layer at a density of 1.08- 1.09gr/cm and an anticoagulant layer 3. The anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant , oxalate based anticoagulant, or any type of anticoagulant designed for preventing clotting of blood.

A blood sample 100 is drawn from a subject to the blood collection tube which comprises the gel layer 2 and the anticoagulant layer 3. In order to perform a separation of whole blood 5 a centrifugation procedure 110 is performed at 300- 1500g for about 10 to about 30 minutes, resulting in four fractions. The first and heaviest fraction at the bottom of the tube comprises red blood cells (RBCs) layer 6 as a sediment layer, the second fraction comprises the gel layer 2 ,the third fraction, a buffy coat fraction, on the top of the gel, comprises: white blood cells (WBCs),platelets, and growth factors as a thin layer 7, and the last (fourth) fraction comprises plasma solution 8 as a supernatant layer.

The test tubes, by some embodiments, can contain an anticoagulant such as, but not limited to buffer citrate, modified ACD (citric/citrate dextrose), heparinate salts, EDTA salts, iodo acetate salts, oxalate salts, fluoride salts as water solutions or lyophilized material or wet or dry spray on inner wall and so forth.

While test tube may optionally include an anti-coagulant, in some procedures an anticoagulant may a priori not be included. For example, if the blood sample is withdrawn and maintained in cold conditions, an anti-coagulant may not be needed. In addition, in some procedures, the anti-coagulant may be mixed with the whole blood which was withdrawn from a subject, prior to inserting the whole blood into the tube. In some embodiments of the invention no gel will be used. Thus the components of this embodiment of the invention are vacuum tube 1 containing anticoagulant 3. This tube when contains whole blood sample is centrifuged at 300-1500g for about 10 to about 30 minutes, resulting in three fractions; namely the first and heaviest fraction at the bottom of the tube comprises red blood cells (RBCs) layer 6 as a sediment layer, the second fraction, a buffy coat fraction, on the top of the gel, comprises: white blood cells (WBCs),platelets, and growth factors as thin layer 7, and the third fraction comprises plasma solution 8 as a supernatant layer. In yet another embodiment of the invention no anticoagulant will be used, the blood collection tube containing separation gel layer and whole blood sample will be centrifuged, resulting in four fractions: the first and heaviest fraction at the bottom of the tube comprises red blood cells (RBCs) layer 6 as a sediment layer, the second fraction comprises the gel layer 2 ,the third fraction comprises a buffy coat fraction, on the top of the gel. The buffy coat fraction comprises: white blood cells (WBCs),platelets, and growth factors as thin layer 7, and the last fraction comprises plasma solution 8 as a supernatant layer. In both cases, (with or without gel), after centrifugation 110 a portion of plasma 8 is discarded 120 from the tube using a syringe 9 with a sharp needle 9a.

In some embodiments of the invention the amount of plasma 8 which extracted from the blood collection tube 1 is up to 80% of plasma. In another embodiment of the invention, the amount of plasma 8 which extracted from the tube 1 is at least 50% of plasma. In yet another embodiment of the invention the amount of plasma which extracted from the tube 1 is about 80% of plasma. Extraction of the plasma 120 and re-suspending of WBCs, platelets and growth factors is performed via repeated pumping of the plasma solution using a plasma collection syringe and an optional stopcock 10 as a valve or turning plug which controls the flow of fluid from a the plasma collection syringe to the blood collection tube .A higher potency of the IL-IRA is obtained when plasma portion containing IL-1RI molecules bound to IL-IRA, is discarded.

In another embodiment of the invention a two or three-way stopcock can be used to turn off the flow of one solution and turn on the flow of another.

In another embodiment of the invention, the plasma collection syringe 9 may also include a filter unit of 3-50μιη, attached to the long sharp needle 9a for piercing the stopper 10. The remaining amount of the top fraction comprising; plasma 8 ,and the fraction comprising WBCs and platelets 7 are drawn using a buffy coat collection syringe 11, attached to the hub of the long sharp needle or via attaching a syringe to another port of the stopcock 10 with an optional filter unit.

In another embodiment of the invention an optional stopcock 10 as a valve or turning plug controls the flow of fluid from the buffy coat collection syringe to the blood collection tube. In another embodiment of the invention a two or three-way stopcock can be used also to turn off the flow of one solution and turn on the flow of another. The plasma layer, and WBCs layer in the buffy coat collection syringe 11 are transferred to an incubation tube 15 closed with a cover 14. The incubation tube comprises washed and pretreated borosilicate glass beads 12 in size of 0.5-5mm. An incubation is carried out on the incubation tube at ambient temperature of 25-37°c with optional addition of C0 ₂ for 6-24 hours producing high levels of the autologous IL-IRA cytokine.

Reference is now made to fig. 2 which illustrates a closed syringe system for deriving high levels of autologous IL-IRA cytokine. The syringe system for producing autologous IL-IRA cytokine comprises: (a)a blood collection syringe 22,(b)an anticoagulant portion, (c)a syringe stopper 23,(d)a syringe sharp needle 21, and (e)an incubation vessel with a cover 30. The vessel is selected from a group consisting of: a syringe, a plastic tube, a glass tube, or any other suitable vessel.

Whole blood is drawn 200 from a subject using a blood collection syringe 22 having an anticoagulant layer 21. The anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant , oxalate based anticoagulant, or any type of anticoagulant designed for preventing clotting of blood. The syringe 22 closed 210 with a stopper 23, is centrifuged 220 in a downward position at 300-1500gr for aboutlO to about 30 minutes resulting in three fractions. The first and heaviest fraction comprises RBCs sediment 25 in the bottom of the syringe 22, upon it , the second fraction, buffy coat fraction comprises, WBCs, platelets and growth factors layer 26, and the third fraction comprises a plasma solution 27 as a supernatant layer. The RBCs layer 25 is discarded 230 from the blood collection syringe by removing the stopper 23 and pressing the plunger of the blood collection syringe. The remaining fractions comprise; a first fraction of WBCs sediment 26 and a second fraction of the plasma solution 27. The fraction of WBCs 26 and about 5% to about 100% of the plasma solution 27 are transferred to a vessel 240 suitable for incubation 30 with washed and pretreated borosilicate glass beads 29 of size 0.5 -5mm. An incubation of the vessel is carried out at ambient temperature of 25-37°c with optional addition of C0 ₂ for 6-24 hours producing high levels of the autologous IL-1RA cytokine.

In accordance with the preferred embodiment of the present invention, the incubation vessel is selected from the group consisting of: plastic tube , glass tube, or any combination thereof.

Reference is now made to fig. 3 which illustrates a closed vessel system for derivation of high levels of autologous IL-IRA cytokine via a gravitation method. Fig. 3 illustrates a vessel system for producing autologous IL-IRA cytokine comprising (i) a blood collection vessel 30, (ii) a vessel cover, (iii) an anticoagulant 31, (iv) a plasma and WBCs portion collection syringe comprising a sharp long needle, (v) an incubation vessel with a cover 39, and (v) p a plasma portion collection syringe.

In another embodiment of the invention The blood collection vessel is selected from a group consisting of: a plastic tube ,a glass tube, a vacuum tube or any other suitable vessel.

In some embodiment of the invention a blood sample 32 is drawn 300 from a subject to the blood collection vessel 30, closed with a cover 32 and positioned in an upward position 310 for 20-60 min. Gravity causes the precipitation of RBCs, thereby resulting in three fractions. The first fraction in the bottom of the vessel comprises: a sediment of RBCs layer 33 , the second fraction comprises WBCs , platelets and growth factors 34 on top of the RBCs layer and the third fraction comprises a plasma solution 35 as a supernatant layer. The WBCs layer and the plasma layer are transferred 320 to an incubation vessel 39 via a plasma and WBCs portion collection syringe. Centrifugation 340 of the incubation vessel 39 comprising washed and pretreated borosilicate glass beads 29 of size 0.5 -5mm is preformed at 200- 15 OOg for aboutlO to about 30 minutes, resulting in three fractions. The first fraction in the bottom of the vessel comprises the glass beads 37,upon it, the second fraction, a buffy coat fraction, comprising WBCs , platelets , growth factors 34, and the third fraction comprises the plasma solution 35.

In yet another embodiment of the invention, after centrifugation 340 a portion of plasma 35 is discarded from the incubation tube using a plasma collection syringe with a sharp needle.

In some embodiments of the invention the amount of plasma 8 which extracted from the blood collection tube 1 is up to 80% of plasma. In another embodiment of the invention, the amount of plasma 8 which extracted from the tube 1 is at least 50% of plasma. The incubation vessel 39 containing the glass beads 37, and a fraction of a buffy coat fraction, comprising WBCs , platelets , growth factors 34, is incubated at 25-37°c with optional addition of C0 ₂ for 6-24h, to produce high levels of the autologous IL-1RA cytokine.

In another embodiment of the invention, the buffy coat fraction comprising: WBCs, platelets and growth factors and the plasma fraction. Those fractions are transferred from the blood collection vessel to the a centrifugation vessel via a plasma and WBCs portion collection syringe . Centrifugation at 200-1500g for aboutlO to about 30 minutes of the centrifugation vessel resulting in two separate fractions comprising: first fraction of WBCs and second fraction of plasma.

A portion of the plasma is discarded using a plasma collection syringe and the WBCs and the remaining plasma are transferred to an incubation vessel which comprises washed and pretreated borosilicate glass beads in size of 0.5-5mm. In another embodiment of the invention the amount of plasma which extracted from the tube is up to 80%) of plasma.

In another embodiment of the invention the amount of plasma which extracted from the tube is at least 50% of plasma. In another embodiment of the invention the amount of plasma which extracted from the tube is about 80% of plasma.

In accordance of the invention, the incubation vessel is incubated at 25-37°c with an optional addition of C0 ₂ for 6-24h, to produce high levels of the autologous IL-1RA cytokine . In another embodiment of the invention the incubation vessel is selected from a group consisting of: a plastic tube ,a glass tube, or any other suitable vessel.

Reference is now made to fig. 4 which illustrates a closed system for derivation of high levels of autologous IL-IRA cytokine via filtration. Fig. 4 shows a vessel system for producing autologous IL-IRA cytokine comprising: (a)a filtered blood sample tube 50; (b) a tube cover, (c)a blood collection syringe 55 comprising a sharp needle 55a and a filter unit 55b ,(d) an incubation vessel with a cover 58 , and (e)a plasma portion collection syringe 57 with a sharp needle 57a.

In another embodiment of the invention, the tube is selected from a group consisting of: a plastic tube, a glass tube, a vacuum tube or any other suitable vessel.

In accordance with the preferred embodiment of the present invention, the whole blood sample is drawn 500 from a subject, using a blood collection syringe 55. The whole blood sample is passed 510 through a special filter 55b such as hydrophilic filter or hydrophobic filter. The filter includes multi-sized pores which captures nucleated cells as WBCs, while letting the RBCs and the plasma flow through to a filtered blood sample tube . The filtered blood sample tube 50 is centrifuged 520 at 300-1500g for about 10 to about 30 minutes to obtain cell free plasma, resulting in three fraction. The first and heaviest fraction at the bottom of the vessel comprises RBCs sediment 51, the second fraction comprises platelets 52, and the third fraction comprises plasma solution 53 as a supernatant layer .The plasma layer is drawn 530 by a plasma portion collection syringe 57. The next step is performing a flushing 550 by attaching the filter 540 consisting of the captured WBCs fraction 55b to the plasma portion collection syringe 57 consisting the plasma 57a. The flushing obtains and release the WBCs to an incubation vessel with a cover 58 which contains washed and pretreated borosilicate glass beads 59 in size of 0.5 -5mm. The incubation vessel containing the WBCs fraction 58 is then incubated at 25-37°c with optional addition of C0 ₂ for 6-24h, to produce high levels of the autologous IL-IRA cytokine .

In another embodiment of the invention the incubation vessel is selected from a group consisting of: a plastic tube, a glass tube, or any other suitable vessel

Reference is now made to fig. 5 which illustrates a closed system for derivation of high levels of autologous IL-IRA cytokine in a whole blood sample.

Fig. 5 shows an incubation vessel 60 with a cover 62 for producing autologous IL- IRA cytokine. The vessel comprises washed and pretreated borosilicate glass beads 61 in size of 0.5 -5mm and may also comprises an anticoagulant layer. The incubation vessel is selected from a group consisting of: a syringe, a plastic tube, a glass tube, a vacuum tube or any other suitable vessel. The anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant, or any type of anticoagulant designed for preventing clotting of blood. A whole blood sample 64 is drawn to the incubation vessel 60 and incubated at 25-37°c with optional addition of C0 ₂ for 6-24hours, to produce high levels of the autologous IL-IRA cytokine.

Reference is now made to fig. 6 which illustrates a flow chart of the method for producing of autologous IL-IRA cytokine in a closed system, the method comprising steps of: (a)obtaining a vacuum tube system for producing autologous IL-IRA cytokine 600 comprising: (i)a blood collection vacuum tube; (ii)a tube cover; (iii)a portion of separation gel;

(iv)an anticoagulant portion; (v)a plasma collection syringe comprising a sharp needle; (vi) an portion collection syringe; and (vii) an incubation tube with a cover; (b)placing the anticoagulant portion and the portion of separation gel in the vacuum tube 610, (c)filling the blood collection vacuum tube with blood sample 620, (d) separating by centrifugation yielding four separation fractions. The first fraction comprises RBCs sediment, the second fraction comprising the gel, a third fraction comprises WBCs, platelets and, and fourth fraction comprises plasma solution 630,(e)discarding a portion of plasma with the plasma collection syringe comprising a sharp needle 640 , (f) extracting the remaining plasma and WBCs solution with the plasma collection syringe 650, (g)transferring the solution into incubation tube 660, and (h)incubating the incubation tube; such that a high concentration of the autologous IL-IRA cytokine is obtained 670.

In another embodiment of the invention the method as described above, wherein the incubation tube comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm. In another embodiment of the invention the method as described above, further comprises a filter unit of 3-50μιη.

In another embodiment of the invention the method as described above, wherein the incubation tube is selected from the group consisting of: a plastic tube, a glass tube, or any combination thereof. In another embodiment of the invention the method as described above, wherein the separation gel is at density of 1.08-1.09gr/cm .

In another embodiment of the invention the method as described above, wherein the discarding up to 80% of plasma from the blood collection vacuum tube. In another embodiment of the invention the method as described above, wherein the discarding at least 50% of plasma from the blood collection vacuum tube.

In another embodiment of the invention the method as described above, wherein the discarding about 80% of plasma from the blood collection vacuum tube.

In another embodiment of the invention the method as described above, wherein the anticoagulant is selected from the group consisting of: a citrate based anticoagulant,

EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant, or any combination thereof. In another embodiment of the invention the method as described above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

In another embodiment of the invention, the method as described above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours. In another embodiment of the invention the method as described above, wherein the incubating is being preformed with or without 5-6%> C0 ₂ .

Reference is now made to fig. 7 which illustrates a flow chart of the method for producing autologous IL-IRA cytokine in a closed system, the method comprising the steps of : (a)obtaining a syringe system for producing autologous IL-IRA cytokine 700 comprising:

(i)a blood collection syringe; (ii)an anticoagulant portion; (iii)a syringe stopper; (iv)a sharp syringe needle; and, (v)an incubation vessel with a cover;

(b)placing the anticoagulant portion and the portion of separation gel in the blood collection syringe 710;(c)drawing whole blood with the blood collection syringe 720,

(d) positioning the syringe in an downward position comprising a stopper 730,

(e) separating the whole blood in the syringe by centrifugation yielding separation fractions comprising, a first fraction of RBCs sediment, a second fraction comprising WBCs, platelets and a third fraction of plasma solution 740, (f)discarding RBCs fraction from the syringe 750, (g)transferring the remained solution of WBCs, platelets and growth factors into incubation vessel 760, and (h)incubating the incubation vessel such that a high concentration of the autologous IL-IRA cytokine is obtained 770.

In another embodiment of the invention the method as described above, wherein the anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant, or any combination thereof. In another embodiment of the invention the method as described above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

In another embodiment of the invention the method as described above, wherein the incubation vessel contains pretreated and washed borosilicate glass beads in a size of 0.5-5mm

In another embodiment of the invention the method as described above, wherein the incubating is being preformed at ambient temperature of 25-37 °c for 6-24 hours. In another embodiment of the invention the method as described above, wherein the incubating is been preformed with or without 5-6% C0 ₂ .

In another embodiment of the invention the method as described above, wherein the incubation vessel is selected from the group consisting of: plastic tube, glass tube, or any combination thereof.

In another embodiment of the invention the method as described above, wherein the centrifugation is been preformed for about about 10 to about 30 minutes .

Reference is now made to fig 8 which illustrates a flow chart of the method for producing of autologous IL-IRA cytokine in a closed vessel system, the method comprising the steps of : (a)obtaining a vessel system for producing autologous IL- lRA cytokine 800 comprising:

(i)a blood collection vessel, (ii)a vessel cover, (iii)an anticoagulant, (iv)a plasma portion collection syringe comprising a sharp needle, (v)an incubation vessel with a cover; and (vi)a buffy coat collection syringe.

(b)placing the anticoagulant in the blood collection vessel 810, (c)filling the blood collection vessel with blood 820, (d)placing the vessel in an upward position for sufficient time yielding separation fractions comprising a first fraction of RBC sediment, and a second fraction comprising WBCs , platelets and a third fraction of plasma solution 830,(e)drawing the fractions of WBCs ,and plasma solution using a plasma portion collection syringe 840, (f)transferring the WBCs ,and plasma solution to the incubation vessel 850,(g)separating the incubation vessel by centrifugation, yielding separation fractions comprising a first fraction of WBCs sediment, and a second fraction of plasma solution 860, (h)discarding a portion of plasma with a buffy coat collection syringe 870 , (i)incubating the incubation vessel such that a high concentration of the autologous IL-IRA cytokine is obtained 880. In another embodiment of the invention the method as described above, wherein the incubation vessel comprises washed and pretreated borosilicate glass beads in a size of 0.5-5mm.

In another embodiment of the invention the method as described above, wherein the anticoagulant is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant ,oxalate based anticoagulant, or any combination thereof.

In another embodiment of the invention the method as described above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood. In another embodiment of the invention the method as described above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours.

In another embodiment of the invention the method as described above, wherein the incubating is being preformed with or without 5-6% C0 ₂ .

In another embodiment of the invention the method as described above, wherein the discarding up to 80% of plasma from the incubation vessel.

In another embodiment of the invention the method as described above, wherein the discarding at least 50% of plasma from the incubation vessel.

In another embodiment of the invention the method as described above, wherein the discarding about 80%> of plasma from the incubation vessel.

In another embodiment of the invention the method as described above, wherein the blood collection vessel is selected from the group consisting of: a syringe, a plastic tube ,a glass tube, a vacuum tube or any combination thereof.

In another embodiment of the invention the method as described above, wherein the incubation vessel is selected from the group consisting of: a plastic tube ,a glass tube, a syringe or any combination thereof. In another embodiment of the invention the method as described above, wherein the fraction of RBCs sediment is formed using gravity force. In another embodiment of the invention the method as described above, wherein the tube in upward position is positioned for about 20-60minutes.

In another embodiment of the invention the method as described above, further comprises a filter unit of 3-50μηι.Ιη another embodiment of the invention the method as described above, wherein the centrifugation is being preformed for about about 10 to about 30 minutes. Reference is now made to fig. 9 which illustrates a flow chart of the method for producing of autologous IL-1RA cytokine in a closed vessel system , the method comprising the steps of : (a) obtaining a vessel system for producing autologous IL- 1RA cytokine 900 comprising: (i)a blood collection syringe comprising a sharp needle and a filter unit, with blood sample; anticoagulant (ii)a filtered blood sample tube; (iii)a plasma portion collection syringe with a sharp needle; and (iv)an incubation vessel with a cover;

(b)filling syringe with whole blood sample 910, (c)transferring content of the syringe through a filter unit to the filtered blood sample tube 920, (d)separating the filtered blood sample tube by centrifugation yielding a separation fractions comprising a first fraction of RBCs sediment, a second fraction comprising, platelets and third fraction of plasma solution 930, (e)drawing a portion of plasma using the plasma portion collection syringe 940, (f)flushing the filter containing WBCs with the obtained plasma in the a plasma portion collection syringe to the incubation vessel yielding a WBCs fraction 950, (g)incubating the incubation vessel; such that a high concentration of the autologous IL-1RA cytokine is obtained 960.

In another embodiment of the invention the method as described above,wherein the incubation vessel contains pretreated and washed borosilicate glass beads in a size of 0.5-5mm.

In another embodiment of the invention the method as described above, wherein the step of drawing up to 80% of plasma from the filtered blood sample tube.

In another embodiment of the invention the method as described above, wherein the step of drawing at least 50% of plasma from the filtered blood sample tube.

In another embodiment of the invention the method as described above, wherein the step of drawing about 80%> of plasma from the filtered blood sample tube.

In another embodiment of the invention the method as described above, wherein the filter unit is selected from the group consisting of: a hydrophobic filter , hydrophilic filter or any combination thereof.

In another embodiment of the invention the method as described above, wherein the incubating is being preformed at ambient temperature of 25-37 °c for 6-24 hours. In another embodiment of the invention the method as described above, wherein the incubating is being preformed with or without 5-6%> C0 ₂ .

In another embodiment of the invention the method as described above, wherein the filtered blood sample tube is centrifuged for about 10 to about 30 minutes. In another embodiment of the invention the method as described above, wherein the filtered blood sample tube is selected from the group consisting of: a plastic tube,a glass tube, a vacuum tube or any combination thereof.

In another embodiment of the invention the method as described above, wherein the incubation vessel is selected from the group consisting of: a syringe, a plastic tube, a glass tube, or any combination thereof.

Reference is now made to fig. 10 which illustrates a flow chart of the method for producing of autologous IL-1RA cytokine in a closed vessel tube system, the method comprising the steps of: (a)obtaining a vessel system for producing autologous IL- 1RA cytokine 1000 comprising: (i)an incubation vessel comprising washed and pretreated borosilicate glass beads for collection blood sample;(ii)a vessel cover; (b)filling the incubation vessel with whole blood sample 1010, and (c) incubating the incubation vessel such that higher concentration of the autologous IL-1RA cytokine is formed 1020 .

In another embodiment of the invention the method as described above, wherein the borosilicate glass beads are pretreated and washed beads in a size of 0.5-5mm

In another embodiment of the invention the method as described above, wherein the incubating is been preformed at ambient temperature of 25-37 °c for 6-24 hours.

In another embodiment of the invention the method as described above, wherein the incubating is been preformed with or without 5-6% C0 ₂ .

In another embodiment of the invention the method as described above, further comprises an anticoagulant which is selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant, or any combination thereof.

In another embodiment of the invention the method as described above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

In another embodiment of the invention the method as described above, wherein the filtered blood sample tube is centrifuged for about 10 to about 30 minutes.

In another embodiment of the invention the method as described above, wherein the incubation vessel is selected from the group consisting of: a plastic tube, a glass tube, a vacuum tube or any combination thereof.

It is known in the literature ("Differential binding of human interleukin-1 (nonreceptor antagonist to natural and recombinant soluble and cellular IL-1 type I receptors", Morten Svenson,Susanne Nedergaard,Peter M. H. Heegaard,Teri D.

Whisenand3, William P. Arend3,Klaus Bendtzen. Eur. J. Immunol. 1995.25: 2842- 2850) that IL-1RI molecules (soluble proteins found in the plasma), which

specifically bind to the IL-IRA reduce the efficacy of the IL-lRA.The present invention provides means and methods for discarding a part of the plasma containing IL-IRI - bound IL-IRA, the remaining IL-IRA preparation is of greater potency. It is herein acknowledged that the present invention provides means and methods for not only producing high concentrations and amounts of IL-IRA., but the IL-IRA so producd is of high efficacy.

This is a soluble protein found in the plasma and we believe that if we reduce its concentration by discarding part of the plasma, the IL-IRA will be more potent than in the whole blood.

In another embodiment of the invention a portion of IL-IRA substantially free of IL- 1RI produced by a method of preparing autologous IL-IRA cytokine in a closed system, wherein the method comprises steps of obtaining a system selected from the group consisting of: a vessel system, a syringe system, or any combination thereof; further wherein a higher potency of the IL-IRA is obtained when the plasma containing IL-IRI molecules bound to IL-IRA, is discarded.

In the foregoing description, embodiments of the invention, including preferred embodiments, have been presented for the purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments were chosen and described to provide the best illustration of the principals of the invention and its practical application, and to enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth they are fairly, legally, and equitably entitled.