METHOD FOR PRODUCING A VIRUS-FREE PLATELET LYSATE MATERIAL

Abstract

The invention relates to a method for producing a material for preparing a substrate for cultivating living cells. The invention further relates to a platelet lysate material and its use.The solution of the problem underlying the invention is based on the combination of at least two different treatments with different virus inactivation procedures.Combining two or more procedures for inactivation of virus particles and/or virus components ensures that a wide range of viruses (e.g. enveloped and non-enveloped viruses) are inactivated so as to provide a safe product for clinical and pharmaceutical applications. Moreover, a platelet lysate material comprising blood platelet lysate is provided, wherein the material is a liquid or dry material that is essentially devoid of vims particles and/or virus components.

Claims

1. A method for producing a material for preparing a substrate for cultivating living cells, said method comprising: Providing a blood platelet lysate, and Exposing said platelet lysate to at least two different treatments, each treatment being capable of inactivating virus particles and/or virus components, so as to provide a virus-free or at least virus-reduced platelet lysate material.

2. The method according to claim 1, wherein the treatments comprise at least two different treatments selected from the group consisting of pasteurization, dry heat, vapor heat, irradiation with ultraviolet light, irradiation with gamma rays, irradiation with X-rays, electron-beam irradiation, nano-filtration, and solvent/detergent treatment.

3. The method according to claim 1, wherein said blood platelet lysate is liquid, said liquid platelet lysate being exposed to the treatments.

4. The method according to claim 1, wherein said blood platelet lysate is dried before the treatments, so as to provide a dry platelet lysate material, said dry platelet lysate material being exposed to the treatments.

5. The method according to claim 4, wherein the blood platelet lysate is dried by lyophilization.

6. The method according to claim 4, wherein the dry platelet lysate material is ground, so as to obtain a powder.

7. The method according to claim 4, wherein a liquid is added to the dry platelet lysate material, so as to obtain a reconstituted platelet lysate material.

8. The method according to claim 1, wherein the blood platelets are derived from thrombocyte concentrate and subsequently lysed by a freeze-thaw process.

9. A platelet lysate material comprising blood platelet lysate, said material being a liquid or dry material that is essentially devoid of virus particles and/or virus components.

10. The platelet lysate material according to claim 9 comprising 1-50% blood platelet lysate.

11. The platelet lysate material according to claim 9, wherein the blood platelets are derived from human blood.

12. The platelet lysate material according to claim 9, wherein said material is a powder.

13. Method of preparing a substrate for expanding living cells for clinical and/or therapeutic applications comprising: providing a platelet lysate that has been exposed to two different treatments that inactivate virus particles and/or virus components, wherein the substrate is virus-free or at least virus-reduced and is configured for expansion of the living cells for clinical and/or therapeutic applications.

14. The method according to claim 13, wherein said substrate is a liquid substrate or a three-dimensional, gel-like substrate.

15. The method according to claim 13, wherein the living cells are mesenchymal stem cells.

16. The method according to claim 3, wherein said blood platelet lysate is dried before the treatments, so as to provide a dry platelet lysate material, and said dry platelet lysate material is exposed to the treatments.

17. The platelet lysate material according to claim 9 comprising 1-40% blood platelet lysate.

18. The platelet lysate material according to claim 17 comprising 1-30% blood platelet lysate.

19. The platelet lysate material according to claim 18 comprising 1-20% blood platelet lysate.

20. The platelet lysate material according to claim 19 comprising 5-15% blood platelet lysate.

Description

DESCRIPTION OF EXEMPLARY AND PREFERRED EMBODIMENTS OF THE INVENTION

[0066] Mesenchymal stromal cells (MSCs) are a biologically important cell population that is able to support hematopoiesis, can differentiate along mesenchymal and nonmesenchymal lineages in vitro, is capable of suppressing alloresponses, and appears to be nonimmunogenic. These properties suggest emerging roles for mesenchymal stromal cells in cell therapy.

[0067] MSC were isolated from the caput femoris upon hip fracture after written consent using guidelines approved by the Ethic Committee of the University of Aachen. Bone fragments were flushed with PBS and mononuclear cells (MNC) subsequently isolated by a density gradient on Biocoll (Biochrom AG, Berlin, Germany). Cells were cultured in fibronectin coated cell culture dishes (Greiner, Kremsmunster, Austria) in Dulbecco's Modified Eagles Medium-Low Glucose (DMEM-LG; PAA, Pasching, Austria) with 2 mM L-glutamine (Sigma Aldrich, St. Louis, Mo., USA) and 100 U/mL penicilline/streptomycine (pen/strep; Lonza, Basel, Switzerland). Culture medium was supplemented with FCS (HyClone, Bonn, Germany) or HPL as indicated. Culture medium was always changed twice per week. When reaching 80% confluence cells were trypsinized, counted by in a Neubauer counting chamber (Brand, Wertheim, Germany) and replated at 10.sup.4 cells/cm.sup.2. Cumulative population doublings were calculated from the first passage onward.

[0068] Human platelet lysate (HPL) was generated from platelet units of healthy donors as described by Horn et al. (2010). In particular, HPL was generated by apheresis using the Trima Accel collection system (CaridianBCT, Garching, Germany). Such thrombocyte concentrates had a platelet content of 2.0 to 4.2×10.sup.11 platelets in 200 mL plasma supplemented with Acid-Citrate-Dextrose (ACD) (1:11 v/v). Platelet units were aliquoted, twice frozen at -80° C., re-thawed at 37° C. and centrifuged at 2600 g for 30 min at 4° C. to remove cell fragments and pooled in equal amounts. The supernatant was filtered through 0.22 μm GD/X PVDF filter device (Whatman, Dassel, Germany), optionally supplemented with 2 U/mL heparin to avoid gelatinization, and stored at −80° C.

[0069] Cells were cultured in cell culture dishes (Greiner, Kremsmunster, Austria) in Dulbecco's Modified Eagles Medium-Low Glucose (DMEM-LG; PAA) with 2 mM L-glutamine (Sigma Aldrich, St. Louis, Mo., USA) and 100 U/mL penicillin/streptomycin (pen/strep; Lonza, Basel, Switzerland). Culture medium/substrate was supplemented with HPL as indicated in the specification. Cells were cultured at 37° C. in a humidified atmosphere containing 5% carbon dioxide with medium changes twice per week.

[0070] Cell proliferation was evaluated via the Thiazolyl Blue Tetrazolium Bromide (MTT) assay. MSC were seeded on a 96-well plate (3,000 cells/well) in culture medium/substrate supplemented with 0, 1, 5, 10, or 20% of HPL as indicated in the specification. After 7 days of culture, cells were washed with PBS (PAA) and incubated with 1 mM MTT (Sigma-Aldrich) in PBS for 3.5 h. The excess solution was discarded and crystals were resolved in 4 mM HCl in isopropanol (both from Roth, Karlsruhe, Germany). Optical density (OD) was measured at wave length 590 nm with a Tecan Infinite 2000 plate reader (Tecan Trading, Männedorf, Switzerland).

EXAMPLE

[0071] Generation of Human Platelet Lysate:

[0072] Platelet units were kindly provided by the Institute for Transfusion Medicine, RWTH Aachen University Medical School after their expiry date (5 days after harvesting). Platelet units were generated by apheresis using the Trima Accel Collection System (CaridianBCT, Garching, Germany) and comprise 2.0-4.2×10.sup.11 platelets in 200 mL plasma supplemented with Acid-Citrate-Dextrose (ACD) (1:11 v/v). Aliquots of 45 mL were twice frozen at −80° C. and re-thawed at 37° C. and then centrifuged at 2,600×g for 30 minutes. The supernatant was filtered through 0.2 μm GD/X PVDF filters (Whatman, Dassel, Germany) and stored at −80° C. until use. Experiments of this study were performed with three HPL-pools each consisting of lysates of four donors but similar results were also observed using individual donor derived HPLs. HPLs consisted predominately of blood type A but a systematic analysis did not reveal any impact of the blood group.

[0073] Culture Medium and HPL-Gel:

[0074] Culture medium consisted of Dulbecco's Modified Eagles Medium-Low Glucose (DMEM-LG; PAA, Pasching, Austria) with 2 mM L-glutamine (Sigma Aldrich, St. Louis, Mo., USA) and 100 U/mL penicillin/streptomycin (pen/strep; Lonza, Basel, Switzerland) and HPL (10% if not indicated otherwise). Cells were cultured on tissue culture plastic (TCP; Greiner, Kremsmünster, Austria). For gel preparation, HPL containing cell culture medium without heparin was filled in cell culture plates. Due to the calcium comprised in the medium the coagulation cascade was initiated and the HPL-medium gelatinized within one hour at 37° C. That is, the blood platelet lysate, either pure or diluted, spontaneously gelatinizes at approximately 37° C. without the necessity to add any coagulation-inducing agent such as, for example, thrombin and/or calcium gluconate. The resulting HPL-gel was used as cell culture substrate which was seeded with the same amount of MNC. For comparison, gels consisting of 80% collagen G (3 mg/mL collagen type I/III in 12 mM HCl; Biochrome, Berlin, Germany) were used as described before. Cells were cultured at 37° C. in 6 well plates or T75 tissue culture flasks (Nunc Thermo Fisher Scientific, Langenselbold, Germany).

[0075] Isolation of Human Mesenchymal Stromal Cells from Bone Marrow:

[0076] MSC were isolated from mononuclear cells (MNC) by plastic adherence. In brief, bone fragments from the caput femoris of patients undergoing femoral head prosthesis were flushed with phosphate-buffered saline (PBS). Subsequently, MNC were isolated by Ficoll density gradient centrifugation (Biocoll Separating Solution, density 1.077 g/l, Biochrom AG, Berlin, Germany) and washed twice with PBS. At least 5×10.sup.5 MNC were then resuspended in culture medium either on TCP, HPL-gel or collagen-gels in 6-well plates. Cells were cultured at 37° C. in a humidified atmosphere with 5% CO.sub.2. The first medium exchange was performed after 48 hours to remove non-adherent cells. Thereafter, half-medium changes were performed twice per week and MSC were further passaged as described below.

[0077] Lyophilization:

[0078] Freezing is done by placing the gel in a freeze-drying flask and rotating the flask in a shell freezer which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger scale, freezing can be accomplished using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in temperature (annealing). The freezing temperatures are between −50 C and −80 C.

[0079] During the primary drying phase, the pressure is lowered (to the range of a few millibars), and enough heat is supplied to the frozen gel for the ice to sublime. In this initial drying phase, about 95% of the water in the material is sublimated. This phase may be slow because, if too much heat is added, the material's structure could be altered. In this phase, pressure is controlled through the application of partial vacuum. The vacuum speeds up the sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plates provide a surface for the water vapour to re-solidify on. Condenser temperatures are typically below −50 C (−60 F).

[0080] The secondary drying phase aims to remove unfrozen water molecules, since the ice was already removed in the primary drying phase. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0° C., to break any physico-chemical interactions that have formed between the water molecules and the frozen material. The pressure can also be lowered in this stage to encourage desorption (for example, in the range of microbars, or fractions of a pascal).

[0081] After the freeze-drying process is complete, the vacuum is broken with an inert gas, such as nitrogen, before the material is sealed. At the end of the operation, the final residual water content in the product is extremely low, around 1% to 4%.

Non-Patent Literature

[0082] Horn, P.; Bokermann, G.; Cholewa, D.; Bork, S.; Walenda, T.; Koch, C.; Drescher, W.; Hutschenreuther, G.; Zenke, M.; Ho, A.; Wagner, W.: Comparison of Individual Platelet Lysates for Isolation of Human Mesenchymal Stromal Cells. Cytotherapy 12: 888-898; 2010.