The present invention refers to an autologous and allogenic fibrin carrier, method of obtaining an autologous and allogenic fibrin carrier of mammal cells and use thereof for transplantation of cultured cells, particularly human cells.

Fibrinogen is a blood plasma protein which is converted into fibrin being the end product of the blood coagulation process. Polymerization of fibrinogen into insoluble fibrin occurs in the presence of thrombin, calcium ions and XII factor . Resultant fibrin shows an ability to bind with multiple growth factors protecting them against denaturation and proteolysis. Therefore, fibrin can be used as a control and adjustment system for the release of growth factors, cytokines or other bioactive molecules influencing adhesion, proliferation, migration, differentiation and production of an extracellular matrix.

Hence, fibrinogen has been used for transplantations of cells. The main feature of fibrinogen is its plasticity which enables the prepared transplant to be fitted to the size of the defect and which results in good toleration in patients. High polymerization capacity from liquid into gel makes it possible to prepare any shape of the substrate for the suspension of cells and growth factors.

From the Polish patent document no. 54175 there is known a method for obtaining pure fibrinogen while fractionation of plasma, wherein the solid remain obtained by dissolving the sediment of Cohn fraction I at a temperature of + 30°C in an aqueous solution containing 0.9% of NaCl and 1% of glycose, used in the amount corresponding to 1/5 of the starting plasma volume during 1-2 minutes at + 30°C, is dissolved by incubation and centrifugation at 0°C by stirring at +30°C in an aqueous solution of a stabilizer containing 6.72 g of trisodium citrate dihydrate, 3.4 g of NaCl and 13 g of glucose acidified with concentrated hydrochloric acid up to pH = 6.3 in 1000 ml of the solution. The obtain solution is immediately frozen up to - 40°C and undergoes lyophilization. To dissolve the remain obtained from 400 ml to 500 ml of the starting plasma, 75 ml of the stabilizer's solution is used. Pure fibrinogen obtained after lyophilization is dissolved in apyrogenic distilled water prior to medical application. Furthermore, from the Polish patent application no. P-388247 there is known a method for obtaining fibrinogen, in particular for obtaining tissue glue, and a method of preparation thereof. According to the invention, a method for obtaining fibrinogen, particularly for obtaining tissue glue, comprising the donation of blood into a triple blood container, centrifugation of the collected blood, separation of plasma from the blood cells, and double freezing and thawing of plasma from the human blood consists in that the separated plasma is decanted to an empty transfer container, frozen at - 30°C, stored at a temperature below - 25°C for 12-24 hours, and then, from the freezer, it is transferred to a water bath and thawed at + 4°C for up to 2 hours. Then, plasma is refrozen again at - 30°C and stored at - 25°C for 12-24 hours. After this operation, the container with plasma is taken out of the freezer and connected with an empty transfer container, the drain is secured with the clamp and then immersed in a liquid medium at -i-4°C. The container with plasma with precipitate is centrifuged, and after centrifugation the container is placed in a manual press and supernatant is quickly separated from sediment, the clamp is released, the container with fibrinogen settlement is transferred to the freezer and kept at a temperature below -25°C.

The purpose and intention of the invention is to develop a fibrin carrier, a method of obtaining a more universal fibrin carrier for autologous and allogenic transplantations for all mammal cells, including chondrocytes, characterized by better usable and medical properties such as increased stability, increased growth and medicinal possibilities, making it possible to resign from additional cross-linking factors, such as xenogenic thrombin which may lead to allergic reaction and antifibrinolytic factors: tranexamic acid, aprotinin. Moreover, the purpose of the invention is to use a fibrin carrier for transplantation of mammal cells. It has turned out that introduction to fibrinogen of an additional blood-derived factor to fibrinogen did not lead to gel degradation but, contrarily, had a positive impact on the stabilization of the transplant and its quicker influence on the cells.

The required purpose is solved by an autologous and allogenic fibrin carrier characterized in that is a gel-like mixture of fibrinogen and platelet-rich plasma, mixed at a ratio 0.1 -2 to 0.1-2, favorably 1 : 1 of volume share, and calcium chloride in a 0.1%-99% solution, favorably a 10% solution at a ratio 0.1-2 to 0.1 -2, favorably 1 : 1 relative to the mixture of fibrinogen and platelet-rich plasma. An autologous and allogenic carrier is a gel-like mixture of fibrinogen and platelet-rich blood plasma combined at a ratio 0.1-2 to 0.1 to 2, favorably 1 :1 of volume share, and a solution of thrombin and calcium chloride at a ratio 1 : 1 relative to the mixture of fibrinogen and platelet-rich plasma at a ratio 0.1-2 to 0.1-2, favorably 1 :1. Fibrinogen is used either in cryoprecipitate taken from the blood donation of the recipient or the donor, or as ready-made commercial fibrinogen.

Platelet-rich plasma is used either as platelet-rich plasma obtained from the blood of the recipient or the donor, plasma obtained directly from the patient's blood using the apparatus, or ready-made platelet-rich plasma. Platelet-rich plasma contains from 4,6 x 10 ⁵ /μΐ to 2 x 10 ⁶ /μ1 of platelet cel ls.

A method of obtaining an autologous fibrin carrier comprising blood collection, separation of blood plasma from blood cells, freezing and thawing of blood plasma, characterized in that the blood donation collected from the blood donor is separated from the concentrate of blood cells, i.e. erythrocytes, leukocytes being removed, said separation having the form of centrifugation at + 20 to + 24°C for 5 to 15 minutes, with centrifugation force of 700 to 2000 x g; afterwards blood plasma is pushed through to an empty satellite container, wherein centrifugation takes place at + 20°C to +24°C for 5-15 minutes at a centrifugation force of 2100 to 5000 x g to separate platelet-rich plasma, which is stored in bags, favorably breathable swinging bags for maximum 7 days and favorably 3 days. The remaining platelet-poor plasma is used for preparation of fibrinogen in cryoprecipitate, by freezing blood plasma twice to the temperature of -30°C to -90°C and thawing blood plasma at about + 4°C, for example in a water bath, then it is centrifuged at about + 4°C for 5-20 minutes at centrifugation force of 1000 to 5000 x g, wherein supernatant is removed and resultant cryoprecipitate is mixed with platelet-rich plasma from the container, at a ratio from 0.1-2 to 0.1-2, favorably 1 :1, and with a calcium compound solution, favorably a 10% calcium chloride solution at a favorable ratio 1 :1 relative to the mixture of fibrinogen and platelet-rich plasma. Whereas the method of obtaining an allogenic fibrin carrier is characterized in that blood plasma without blood cells concentrate (erythrocytes, leukocytes), separated from the blood donation from at least two blood donors, where separation takes place by centrifugation at + 20°C to + 24°C for 5 to 15 minutes, at centrifugation force from 700 to 2000 x g, is pushed through to an empty satellite container, wherein centrifugation at + 20°C to + 24°C for 5-10 minutes, with centrifugation force from 750 to 2000 x g, separates platelet- rich plasma which is stored in bags, favorably breathable swinging bags, for maximum 7 days, and favorably 3 days. The remaining platelet-poor plasma is used for preparation of fibrinogen in cryoprecipitate by freezing blood plasma twice to the temperature - 30°C to - 90°C and thawing it at about + 4°C, for example in a water bath, then it is centrifuged for 5 to 15 minutes at centrifugation force from 1000 to 5000 x g, wherein supernatant is removed and resultant cryoprecipitate is mixed with platelet-rich plasma stored in a container at a ratio from 0.1-2 to 0.1-2, favorably 1 : 1, and with a calcium compound solution, favorably a 10% calcium chloride solution at a favorable ratio 1 :1 relative to the mixture of fibrinogen and platelet-rich plasma. Fibrinogen is prepared from another donor's plasma frozen at -30°C to -90°C and after a 16-week waiting period it is thawed at about + 4°C, for example in a water bath, then frozen again at -30°C to - 90°C and thawed at about + 4°C, then centrifuged at about + 4°C for 5 to 15 minutes at centrifugation force from 1000 to 5000 x g, wherein supernatant is removed, and resultant cryoprecipitate is mixed with platelet-rich plasma from the container at a ratio 0.1-2 to 0.1-2 , favorably 1 : 1, and with a calcium compound solution, favorably a 10% calcium chloride solution at a ratio from 0.1-2 to 0.1-2, favorably 1 :1 relative to the mixture of fibrinogen and platelet-rich plasma. Separation of plasma is finished at the level of 0.1 cm to 1 cm above the surface of blood cell concentrate: erythrocytes, leukocytes. Separation of platelet-rich plasma is finished when about 10 ml to 100 ml of blood plasma remains over its surface.

Furthermore, the essence of the invention is a specific use of an analogous and allogenic fibrin carrier. Use of an autologous fibrin carrier for obtaining a gel-like transplant, that is a carrier formed by mixing fibrinogen and platelet-rich plasma obtained from the recipient's blood, favorably from a single donation, or - ready- made fibrinogen of a proper phenotype and platelet-rich plasma taken directly from a blood donation from the patient-recipient using the apparatus in the operating room, wherein in the process of obtaining the carrier, and prior to the addition of calcium chloride solution (0.1% to 99%) at a ratio 0.1-2 to 0.1-2, favorably 1 :1 of volume share relative to the mixture of fibrinogen and platelet-rich plasma, 5,000 to 100 million of the recipient's cells or allogenic and xenogenic cells are added, which are cultured in a known way and, on the transplantation date, undergo processing which comprises trypsinization, washing in a serum-containing medium, counting and lifetime assessment. Use of an autologous fibrin carrier for obtaining a gel-like transplant, that is a carrier formed by mixing, in a special mold, fibrinogen and platelet-rich plasma obtained from the recipient's blood, favorably from a single donation, or ready-made fibrinogen characterized by proper phenotype and platelet-rich plasma taken directly from a blood donation using the operating room's apparatus from the patient- recipient, wherein during the process of obtaining the carrier, before a solution of calcium chloride (0.1-99%) is added at a ratio 0.1 to 0.1-2, favorably 1 : 1 of volume share to the mixture of fibrinogen and platelet-rich plasma - 5000 to 100 million cells obtained from the recipient, separated from the surrounding tissue by mechanical or enzymatic means, are added.

Use of an allogenic fibrin carrier for obtaining a gel-like transplant, that is a carrier formed by mixing fibrinogen and platelet-rich plasma obtained from the donor's blood from at least two donations, or fibrinogen from one donor and platelet-rich plasma from another donor, or ready-made fibrinogen and ready-made platelet-rich plasma, wherein, during the process of obtaining a carrier and prior to the addition of a calcium chloride solution (0.1 % - 99%) at a ratio 0.1-2 to 0.1-2, favorably 1 : 1 of volume share relative to the mixture of fibrinogen and platelet-rich plasma - 5000 to 100 million recipient's cells or allogenic and xenogenic cells are added, cultured in a known way and processed on the transplantation date by trypsinization, washing in a serum-containing medium, counting and vitality assessment.

Use of an allogenic fibrin carrier for obtaining a gel-like transplant, i.e. a fibrin carrier obtained by mixing fibrinogen and platelet-rich plasma from the donor's blood, from at least two donations, or fibrinogen from one donor and platelet-rich plasma from another, or ready-made fibrinogen and ready-made platelet-rich plasma, wherein during the process of obtaining a carrier, and prior to the addition of a calcium chloride solution (0.1-99%) at a ratio 0.1 -2 to 0.1 - 2, favorably 1 : 1 of volume share relative to the mixture of fibrinogen and platelet-rich plasma -5000 to 100 million cells are added, obtained from the recipient and separated from the surrounding tissue by mechanical or enzymatic means.

A fibrin carrier - according to the invention - enriched with platelet-rich plasma is a stronger biological scaffold characterized by synergy-based growth properties resulting from the stimulation of platelet-depending growth factors. Consequently, tissue regeneration and wound healing processes are faster. Furthermore, an improved fibrin carrier can be applied widely for autologous and allogenic transplantations and for all kinds of cells, both human and animal ones. It can also be used externally, in the form of a gel, spray etc.

A method of obtaining a fibrin carrier is characterized by an ease of preparation, a possibility to utilize blood ingredients fully - for example from a single donation, and by relatively low production costs.

A favorable characteristic of the inventions is also a possibility to resign from a necessity to use animal-origin thrombin, which may lead to allergic reactions but also to a possible infection with an animal-origin disease.

Example 1. An autologous fibrin carrier contains:

Cryoprecipitate of fibrinogen with a concentration of autologous fibrinogen of 65 g/1 - 9 ml

autologous platelet-rich plasma with 0,70 x lO ^{1 1} platelet cells - 9 ml

10% calcium chloride solution -18 ml

Example 2. A allogenic fibrin carrier contains:

Cryoprecipitate of fibrinogen with a concentration of allogenic fibrinogen of 80 g/1 - 9 ml

allogenic platelet-rich plasma with 0,70 x 10 ^{1 1} platelet cells - 50 ml

calcium salt solution converted into Ca ⁺² ions - 9 ml

Example 3. 450 ml, that is 1 blood donation taken from the patient-recipient 3 days before the transplantation to a container with CPD preservative fluid is centrifuged at + 22°C for 8 minutes at centrifugation force 1400 x g to separate about 280 ml of blood cell concentrate: erythrocytes and leukocytes of RBC concentrate, from 200 ml of platelet-rich plasma, which is then pressed through a press to an empty satellite container.

Plasma separation process is finished when about 1.0 cm of plasma is remain above the surface RBC concentrate. RBC concentrate is removed. Plasma undergoes centrifugation at about +22°C for 9 minutes at centrifugation force of 5000 x g. After centrifugation, platelet-rich plasma is separated from blood cells concentrate in the amount of about 50 ml, over which 0.5 cm of plasma floats on the surface, and then it is kept in a breathable swinging container for 3 days. The remaining 150 ml of platelet-poor plasma is then used for preparation of fibrinogen in cryoprecipitate, which takes 2 days and consists in freezing blood plasma to -60°C and keeping it in a container in a freezer for about 14 hours, and then thawing it at +4°C in a water bath. Then it is again frozen at -72°C and kept in a container in a freezer for 12 hours and then thawed at +4°C. After thawing plasma is centrifuged at +4°C for 9 minutes at centrifugation force of 5000 x g. After centrifugation and removal of supernatant, 5-10 ml of cryoprecipitate is obtained. 1 ml of platelet-rich plasma is added to 1 ml of cryoprecipitate and mixed. Then, 2 ml of a 10% calcium chloride solution is added to obtain 4 ml of an autologous fibrin carrier suspension.

Example 4. Out of 1 unit of frozen plasma, about 270 ml after a 16-week storage period, having its origin in a single donation of an honorary donor, allogenic fibrinogen is prepared in the form of cryoprecipitate by freezing at -55°C, storage in a container in a freezer for 15 hours, thawing at about +4°C and repeating the process. Thawed plasma is centrifuged at +4°C for 9 minutes at centrifugation force of 1000 x g. After centrifugation, supernatant is removed and cryoprecipitate is obtained in the amount of 5 ml to 7 ml. Independently, about 3 days before the transplantation from a single donation, 450 ml of an honorary donor's blood is separated by centrifugation at +22°C for 8 hours at centrifugation force of 1400 x g giving away about 280 ml of RBC concentrate depleted of leukocytes, from 195 ml of blood plasma with platelets, and pressed through a press to an empty satellite container. To avoid introduction of RBC concentrate cells to the container, plasma separation process is stopped when about 1.0 cm of blood plasma floats over the surface of RBC concentrate. The resultant plasma with platelets is centrifuged at +22°C at centrifugation force of 2300 x g for 15 minutes. After centrifugation, about 50 ml of platelet-rich plasma is separated from platelet-poor plasma which is removed. Then, the obtained platelet-rich plasma with 60 mm of plasma floating on its surface is kept swinging for 3 days in a container. 1 ml of platelet-rich plasma concentrate is added to 1 ml of cryoprecipitate and mixed. Then, 2 ml of a 10% calcium chloride solution is added to the resultant suspension to obtain 4 ml of an allogenic fibrin carrier.

Example 5. 30 million recipient's cells in a special mold, cultured in a known method and trypsinized, washed in a serum-containing medium, counted and after vitality assessment are added to 1,8 ml of the suspension obtained by combination of 0.9 ml of FBG cryoprecipitate of FBG concentration of 50 g/1 with 0.9 ml of platelet- rich plasma concentrate with 0.80 x 10 ⁶ platelets/μΐ obtained from a single recipient's donation. Then, 1.8 ml of a 10% aqueous solution of calcium chloride is added. The mold is clamped on both sides so that the thickness of the gel-like transplant with an autologous fibrin carrier is 1 cm.

Example 6. As in Example 5, 35 million cells obtained from a piece of the recipient's tissue and separated mechanically from the surrounding tissue are added to the suspension of cryoprecipitate and plasma. Then, 6 ml of a 10% aqueous solution of calcium chloride is added to obtain a gel-like transplant containing an autologous fibrin carrier.

Example 7. 30 million cells in a special mold, obtained from a piece of the recipient's tissue after mechanical separation from the surrounding tissue are added to 5 ml of the suspension obtained by combination of 2.5 ml of cryoprecipitate with fibrinogen concentration of 57 g/1 and 2.5 ml of platelet-rich plasma concentrate with platelet cells concentration in plasma of 0.65 x 10 ⁶ platelets/μΐ from two donations of two different donors. Then, 5 ml of a 10% aqueous solution of calcium chloride is added. The mold is clamped from both sides so that the thickness of the obtained gel, referred to as fibrograft, is 1 cm.