EP3752207A1

EP3752207A1 - Method for preparing an irradiated platelet lysate

Info

Publication number: EP3752207A1
Application number: EP19705334.1A
Authority: EP
Inventors: Bruno Delorme; Sabrina VIAU
Original assignee: Maco Pharma SAS
Current assignee: Maco Pharma SAS
Priority date: 2018-02-15
Filing date: 2019-02-12
Publication date: 2020-12-23
Also published as: FR3077823B1; FR3077823A1; US20210032590A1; WO2019158540A1

Abstract

The invention relates to a method for preparing an irradiated platelet lysate, comprising the following steps: provision of a starting platelet lysate comprising (i) platelet factors including growth factors and (ii) plasma proteins including coagulation factors and proteins other than the coagulation factors; and double irradiation of the starting platelet lysate, using UVC radiation and ionizing radiation, said double irradiation with UVC radiation and ionizing radiation being arranged to retain at least 75% of the total protein concentration of the starting platelet lysate, while reducing, by at least 40%, the concentration of at least one of the coagulation factors including fibrinogen, factor II, factor VII, factor IX, factor X and factor XI of the starting platelet lysate.

Description

Process for the preparation of an irradiated platelet ivsat

The invention relates to a method of preparing an irradiated platelet lysate and an irradiated platelet lysate obtained by such a method and a method of culturing cells using such an irradiated platelet lysate.

The invention applies to the field of blood platelet derivatives, and in particular to the field of cell culture for culturing cells for therapeutic use, more particularly mesenchymal stem cells.

In order to cultivate animal cells in vitro, RPMI (Roswell Park Memorial Institute), MEM (Modified Eagle Medium) or DMEM (Dulbecco Modified Eagle Medium) basic media consisting mainly of mineral salts, glucose, acids, are conventionally used. amino, vitamins and nitrogen bases. These basal media are usually supplemented extemporaneously with antibiotics to prevent bacterial contamination, L-glutamine - an unstable amino acid - and between 1.5 and 10% fetal calf serum as a nutritional supplement.

To use more efficient products and to avoid the use of xenogeneic products in cell cultures for therapeutic use, it has been proposed to replace fetal calf serum with human platelet lysate. The latter has the advantage of including a large amount of growth factors such as, for example, transforming growth factor beta (TGF-beta1, Transforming Growth Factor-betal), epidermal growth factor (EGF, Epidermal Growth Factor). , platelet derived growth factor AB (PDGF-AB, Platelet-Derived Growth Factor- AB), platelet-derived growth factor BB (PDGF-AB, Platelet-Derived Growth Factor-BB), growth factor 1 Insulin-Like Growth Factor-1 (IGF-1), Vascular Endothelial Growth Factor (VEGF, Vascular Endothelial Growth Factor) and Fibroblast Growth Factor 2 (FGF-2, Fibroblast Growth Factor 2), also called basic fibroblast growth factor (bFGF, Basic Fibroblast Growth Factor).

Thus, the following human or non-human animal cells have been cultured in the presence of platelet lysate: keratinocytes, renal epithelial cells, leukemia or solid tumor cell lines, as well as human primary cells such as adipocytes, amniotic fluid stem cells, bone marrow stromal cells, chondrocytes, corneal cells, endothelial cells, keratinocytes, mesenchymal stem cells, monocytes and osteoblasts (Pons, Miriam, et al., Fluman platelet lysate as validated replacement for animal serum to assess chemosensitivity. "ALTEX-Alternatives to animal experimentation (2018).

Platelet lysate is typically prepared from a platelet suspension in plasma or a plasma mixture and platelet additive solution, which is subjected to one or more freeze / thaw cycles to release the growth factors contained in the platelet lysate. platelets by cell lysis.

A disadvantage of such a platelet lysate is that it contains fibrinogen, from plasma, in an amount ranging from about 0.5 to 3 mg / ml, so that the addition of an anticoagulant to the basal medium is necessary to avoid coagulation of the basal medium (Burnouf T, Strunk D, Koh MB, et al .: Human platelet lysate: replacing bovine feast serum as a gold standard for human cell propagation? Biomaterials 2016; 76: 371-387) .

The most used anticoagulant is heparin. But commercially available heparins are generally of porcine origin, so that the culture medium, although devoid of fetal calf serum, still includes a xenogeneic product. In addition, at certain concentrations, heparin has a negative effect on cell proliferation (Viau, Sabrina, et al.) Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for expansion of human bone marrow mesenchymal stem cells. "PloS one 12.8 (2017): e0181406).

Several strategies have been proposed to eliminate the coagulation power of a platelet lysate:

A first method is described in WO 2013/003356, which consists in adding calcium chloride to the platelet lysate, inducing the conversion of fibrinogen to fibrin and the formation of a clot. The clot is then removed by centrifugation to obtain a lysate comprising less than 0.05 mg / ml of fibrinogen. This method, however, causes a loss of growth factors that would be trapped in the clot.

An indirect method is to remove plasma that contains fibrinogen from the starting platelet suspension, for example by performing several platelet wash cycles prior to preparing the platelet lysate. This strategy is for example described in document WO 2008/034803 and in document US 2012/0156306. The elimination of plasma, however, results in the elimination of other plasma proteins such as albumin, necessary for cell proliferation.

In WO 2017/162830, it is proposed to remove the plasma from the starting platelet suspensions and then heat the platelet lysate between 55 and 65 ° C for 20 to 40 minutes, so as to obtain an amount of fibrinogen less than 0.3 mg / ml. However, this method also reduces the amount of the other proteins present in the platelet lysate by at least 70%, which is undesirable for use in cell culture.

Laner-Plamberger et al. propose an original method of adding the non-defibrinogenic platelet lysate to the base medium to form a gel of fibrin, then destroy and remove this gel by vigorous stirring and centrifugation. The platelet lysate supplemented basal medium thus obtained is free of fibrinogen, but still contains the other plasma proteins (J Transi Med (2015) 13: 354). However, this method is complex to implement in the context of a standardized industrial production and controlled quality of platelet lysates.

In WO 2016/193591, the platelet lysate is irradiated with ionizing radiation to be sterilized. Depending on the irradiation dose used, the irradiated platelet lysate may comprise less than 0.4 mg / ml of fibrinogen. According to the example described, a platelet lysate was prepared from platelet concentrates comprising platelets suspended in 30% plasma and 70% of a preservation solution, and then irradiated at a dose of 35 or 45 kGy. When the platelet lysate is added in an amount of 8% in the basal medium, the basal medium does not coagulate.

Further tests of gamma irradiation carried out by the applicant showed that the clotting power of the platelet lysate depended not only on the irradiation dose used, but also on the amount of protein in the platelet lysate and the amount of Platelet lysate added to basal medium.

Thus, a platelet lysate prepared from platelet concentrates comprising platelets suspended in 30% plasma, comprising between 20 and 30 g / l of total proteins, and irradiated at about 35 kGy according to the method described in document WO 2016/193591, coagulates at 15% and above in a MEM base medium lacking heparin. A platelet lysate prepared from platelet concentrates comprising platelets suspended in 100% plasma, comprising between 60 and 80 g / l of total proteins, and irradiated at 35 kGy according to the method described in document WO 2016/193591, coagulates as soon as % in a MEM base medium lacking heparin. To reduce the coagulation power of a platelet lysate, the Applicant has considered increasing the irradiation dose used up to 75 kGy to obtain a sterile and non-coagulable platelet lysate, even at high concentration in the basal medium. But at a very high dose of irradiation with gamma radiation, the plasma proteins of interest for cell growth are degraded.

There remains a need to obtain a platelet lysate with reduced clotting power in the presence of calcium, even when the platelet lysate is added at high concentration in a basal medium comprising calcium, while preserving the plasma proteins.

In addition, WO 2013/042095 and the article by S. Castiglia (Castiglia, Sara, et al., "Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions." Cytotherapy 16.6 (2014): 750-763) disclose platelet lysate obtained from buffy coat platelet concentrates that have been virally inactivated by UVA-type ultraviolet radiation in the presence of psoralen, a chemical intercalating agent for DNA. This viral inactivation technique, however, has the disadvantage of having to use a chemical agent which must then be removed from the treated product.

Another method of obtaining a platelet lysate free of pathogens is described in the article by S. Viau (Viau, Sabrina, et al., Pathogen reduction through additive-free short wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells. "PloS one 12.8 (2017): e0181406). In this article, platelet lysate is obtained from platelet concentrates that have been virally inactivated by UVC radiation in the absence of a chemical agent.

In these latter documents, platelet lysate prepared from virally inactivated platelet concentrates remains coagulable. The invention provides a method for preparing a platelet lysate with a reduced power of coagulation in the presence of calcium, while retaining the plasma proteins necessary in particular for cell proliferation.

Thus, according to a first aspect, the invention provides a process for preparing an irradiated platelet lysate comprising the following steps:

the provision of a platelet lysate in order to obtain a starting platelet lysate, said starting platelet lysate comprising, on the one hand, platelet factors including growth factors and, on the other hand, plasma proteins including coagulation factors and proteins other than coagulation factors,

the double irradiation of said starting platelet lysate, with UVC radiation of wavelength between 200 and 280 nm and with ionizing radiation having a wavelength of less than or equal to 100 nm, in order to obtain a platelet lysate irradiated by UVC radiation and ionizing radiation, said dual radiation by UVC radiation and ionizing radiation being arranged to retain at least 75% of the total protein concentration of said starting platelet lysate while reducing by at least 40% the concentration of at least one of said coagulation factors including fibrinogen, factor II, factor VII, factor IX, factor X and factor XI of the starting platelet lysate.

According to a second aspect, the invention relates to the irradiated platelet lysate obtained by the process according to the first aspect of the invention.

According to a third aspect, the invention provides a method for culturing cells, particularly animal cells and more particularly mesenchymal stem cells, comprising contacting said cells with a nutrient composition comprising a base medium and an irradiated platelet lysate. according to the second aspect of the invention.

Other objects and advantages will become apparent from the following description. Figures 1 to 6 show, respectively, the factor concentrations of IGF-1, TGF-beta1, bFGF, PDGF-AB, EGF and VEGF in three batches of UVC irradiated platelet lysate, as a function of irradiation dose. .

Figures 7 to 11 represent, respectively, the concentrations of factor II, factor VII, factor IX, factor X and factor XI coagulation factors, expressed as a percentage relative to normal human normal plasma, in three lots of irradiated platelet lysate. by UVC radiation, depending on the irradiation dose.

Figure 12 illustrates the amplification factors in the 7 ^th day of culture mesenchymal stem cells cultured in the presence of UVC radiation irradiated platelet lysate according to the radiation dose.

FIGS. 13 to 16 show, respectively, the growth factor concentrations bFGF, PDGF-AB, PDGF-BB and TGF-beta1, a non-irradiated platelet lysate (LP), a platelet lysate irradiated with UVC radiation at a dose of 1 J / cm ² and then irradiated by gamma radiation at a dose of 35 kGy (LP-UVC-G35) and a platelet lysate irradiated by UVC radiation at a dose of 1 J / cm ² and then irradiated with gamma radiation at a dose of 55 kGy (LP-UVC-G55).

Figures 17 and 18 show, respectively, the total protein concentrations and vitamin B12 concentrations, a non-irradiated platelet lysate (LP), a platelet lysate irradiated by UVC radiation at a dose of 1 J / cm ² then irradiated with gamma radiation at a dose of 35 kGy (LP-UVC-G35) and a platelet lysate irradiated by UVC radiation at a dose of 1 J / cm ² and then irradiated with gamma radiation at a dose of 55 kGy (LP -UVC-G55).

Figure 19 shows the amplification factors in the 7 ^th day of culture mesenchymal stem cells cultured in the presence of a non-irradiated platelet lysate (PL), a platelet lysate irradiated by UVC radiation at a dose of 1 J / cm ² then irradiated with gamma radiation at a dose of 35 kGy (LP-UVC-G35) and a platelet lysate irradiated by UVC radiation at a dose of 1 J / cm ² and then irradiated with gamma radiation at a dose of 55 kGy (LP-UVC-G55).

The invention relates to a method for preparing an irradiated platelet lysate for the purpose of obtaining a platelet lysate having a reduced coagulation power.

Platelet lysate refers to the product of platelet lysis, that is to say the product obtained after disintegration of the platelet cell membrane which leads to the release of the molecules (growth factors, cytokines) normally contained in the platelet lysate. inside the pads.

The lysis of the platelets is for example carried out by one or more freezing / thawing cycles, by the use of ultrasound or by a solvent / detergent treatment.

The method according to the invention firstly comprises the step of providing a platelet lysate to obtain a platelet lysate starting, said starting platelet lysate comprising on the one hand platelet factors including growth factors and plasma proteins including coagulation factors and proteins other than coagulation factors.

Platelet lysate is produced from platelets suspended in a liquid comprising plasma. Such a platelet suspension is, for example, a platelet concentrate or a mixture of platelet concentrates, a buffy coat, or a buffy coat, a platelet-rich plasma, or a platelet-rich plasma mixture. More particularly, the platelet suspension is a platelet concentrate obtained from apheresis or prepared from a blood donation or a mixture of platelet concentrates from apheresis or prepared from blood donations.

For example, the mixture comprises between 4 and 50 platelet concentrates, in particular between 5 and 30 platelet concentrates.

The preparation of a platelet lysate from a mixture of several platelet concentrates, in particular more than four platelet concentrates, is advantageous because it makes it possible to standardize the platelet lysate, that is to say to homogenize the concentration of its platelets. different components, including growth factor concentration (Viau S, Eap S, et al., A standardized and accurate clinical grade human platelet lysate for the effective expansion of human bone marrow mesenchymal stem cells.) May 2017, Volume 19, Issue 5 Supplement, Page S195).

Indeed, growth factor concentrations of a platelet lysate are dependent on the original platelet donor.

Platelet concentrates are either fresh, that is, qualified to be transfused to a patient, or expired, that is, stored for 5 days or more after preparation and can no longer be transfused to a patient. .

Such platelet concentrates include platelets suspended in a plasma-containing liquid medium.

For example, the liquid medium comprises only plasma. In another example, the liquid medium further comprises a platelet preservation solution, such as SSP + solution (Maco Pharma) or Intersol® (Fresenius Kabi). In a particular example, the liquid medium comprises from 20% to 100%, in particular 30% of plasma and from 0% to 80%, in particular 70% of platelet preservation solution.

Lysis of a platelet suspension comprising platelets in plasma provides a starting platelet lysate comprising on the one hand platelet factors normally contained within the platelets and on the other hand plasma components.

Plasma consists of 90% water, salts such as sodium, chlorine and calcium, lipids such as triglycerides and cholesterol, hormones, vitamins such as vitamin B12 and vitamin D and proteins such as albumin, immunoglobulins, coagulation factors including fibrinogen, antithrombin III involved in the coagulation chain, globulins, interleukins and interferons.

Thus, the starting platelet lysate to which the method of the invention is applied comprises in particular on the one hand platelet factors including growth factors and on the other hand plasma proteins including coagulation factors and proteins other than the factors coagulation.

These growth factors include TGF-beta1, EGF, PDGF-AB, IGF-1, VEGF and bFGF. Other growth factors found in the platelet lysate include connective tissue growth factor (CTGF) and stromal cell-derived factor 1 -alpha (SDF-1alpha, Stromal Cell- Derived Factor-1 alpha). These growth factors are said to be endogenous.

Endogenous substance is any substance produced by the platelets or included in the initial platelet suspension used to prepare the platelet lysate, as opposed to an exogenous substance introduced into the platelet lysate or initial platelet suspension. For example, a platelet lysate produced by freeze / thaw lysis of a platelet suspension comprises the following growth factor concentrations: TABLE 1

The starting platelet lysate further includes plasma proteins including coagulation factors and proteins other than coagulation factors.

Coagulation factors include fibrinogen, factor II, factor VII, factor IX, factor X and factor XI. Other coagulation factors are factor V and factor VIII. Other plasma proteins other than coagulation factors include albumin and antithrombin III, a protein involved in the coagulation chain.

The amount of total protein from the starting platelet lysate therefore depends on the percentage of plasma in the initial platelet suspension prior to platelet lysis.

For example, a starting platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 100% plasma comprises in particular the following components: Table 2:

In another example, a starting platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 30% plasma and 70% of a platelet preservation solution comprises in particular the following components:

Table 3:

The step of providing a platelet lysate is understood as providing a platelet lysate. That is, the method of the invention is carried out on a platelet lysate previously produced by lysing platelets of a platelet suspension.

After the step of providing a starting platelet lysate, the method according to the invention comprises the double irradiation of said starting platelet lysate by UVC radiation of wavelength between 200 and 280 nm and by ionizing radiation having a wavelength less than or equal to 100 nm on the other hand, in order to obtain a platelet lysate irradiated by UVC radiation and by ionizing radiation, said dual radiation by UVC radiation and by gamma radiation being arranged to conserve at least 75% the total protein concentration of said starting platelet lysate while reducing by at least 40% the concentration of at least one of said coagulation factors including fibrinogen, factor II, factor VII, factor IX, factor X and factor XI platelet lysate starting.

The double irradiation step is understood as a first irradiation followed by a second irradiation. In particular, the first irradiation is irradiation with UVC radiation and the second irradiation is irradiation with ionizing radiation. Alternatively, the first irradiation is irradiation with ionizing radiation and the second irradiation is irradiation with UVC radiation. The first and second irradiations are successive, that is to say carried out one after the other.

The step of irradiating said platelet lysate with UVC radiation of wavelength between 200 and 280 nm in order to obtain a platelet lysate irradiated by UVC radiation is especially designed to retain at least 75% of the total protein concentration of the starting platelet lysate while reducing by at least 20% the concentration of at least one of said coagulation factors including fibrinogen, factor II, factor VII, factor IX , factor X and factor XI of the starting platelet lysate.

UVC radiation refers to non-ionizing electromagnetic radiation, that is, radiation incapable of causing the ionization of atoms or molecules. The UVC radiation has a wavelength of between 200 and 280 nm, in particular 254 nm.

By retaining more than 75% of the total proteins of the starting platelet lysate, the UVC-irradiated platelet lysate can be used as a base supplement for cell culture. In particular, the albumin which represents more than 50% of the proteins in the plasma and which is a particularly important nutrient in the cell culture, is kept at least 80% relative to the starting platelet lysate.

In particular, at least 80%, and more particularly at least 90% of the total protein concentration in the UVC-irradiated platelet lysate is retained relative to the starting platelet lysate.

The UVC irradiated platelet lysate comprises a total protein concentration of from 20 to 80 mg / ml, depending on the plasma concentration of the initial platelet suspension.

For example, a platelet lysate produced from freeze / thaw lysis of platelet concentrates comprising platelets suspended in 100% plasma and then irradiated with UVC radiation comprises a total protein concentration ranging from about 55 mg / ml to about 80 mg. / ml. In another example, a platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 30% plasma and then irradiated with UVC radiation comprises a total protein concentration ranging from about 18 mg / ml to about 30 mg / ml.

In addition, by reducing by at least 20% the concentration of one of said coagulation factors including fibrinogen, factor II, factor VII, factor IX, factor X and factor XI, the coagulation power of the coagulation factor. Platelet lysate irradiated by UVC radiation is reduced.

That is, a base medium comprising calcium, for example about 0.2 g / l of calcium chloride will not coagulate in the presence of platelet lysate irradiated with UVC radiation or coagulate from a concentration platelet lysate irradiated by UVC radiation in the basal medium higher than that of a platelet lysate starting from which the basal medium coagulates.

For example, the alphaMEM basal medium coagulates in the presence of 5% or more of the starting platelet lysate, whereas this medium coagulates from 10% or more of platelet lysate irradiated by UVC radiation.

In particular, the UVC-irradiated platelet lysate is added in a range of from 2 to 25%, in particular in the range of 5 to 15%, and even more particularly in the range of 8 to 10% in a medium of based.

Since the UVC radiation-irradiated platelet lysate has a reduced coagulation power, its use as a base supplement for cell culture is possible, in certain concentrations, without the use of an anticoagulant such as heparin. In particular, irradiation with UVC radiation is arranged to reduce by at least 20% the concentration of each of the coagulation factors including fibrinogen, factor II, factor VII, factor IX, factor X and factor XI platelet lysate starting.

For example, platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 30% plasma and then irradiated with UVC radiation comprises the following plasma constituents.

Table 4:

In addition, the starting platelet lysate comprises in particular the endogenous growth factors TGF-beta1, EGF, PDGF-AB, IGF-1, VEGF and bFGF.

The irradiation with UVC radiation is in particular designed to retain at least 80% the concentration of one of the growth factors including IGF-1, PDGF-AB, EGF and VEGF of the starting platelet lysate, in particular to be able to use the platelet lysate irradiated as a complement to basal medium.

In particular, irradiation with UVC radiation is designed to retain at least 80% of the concentration of each of the growth factors including IGF-1, PDGF-AB, EGF and VEGF, in particular to be able to use the irradiated platelet lysate as a complement of basic medium.

For example, a platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 30% plasma and then irradiated with UVC radiation comprises the following growth factors. Table 5:

The irradiation with UVC radiation is in particular designed to retain at least 75%, particularly at least 80%, of the amplification factor of the mesenchymal stem cells cultured for 7 days in a base medium supplemented with starting platelet lysate.

According to a particular embodiment, irradiation with UVC radiation is carried out at a dose of between 0.01 to 2 J / cm ² , particularly between 0.5 J / cm ² and 1.5 J / cm ² , and more particularly at 1 J / cm ² .

A UVC radiation dose of less than 0.01 J / cm ² is not sufficient to degrade the coagulation factors present in the starting platelet lysate. A dose of UVC radiation greater than 2 J / cm ² damages the growth factors resulting in a significant loss of cell proliferation.

In particular, the starting platelet lysate is irradiated with UVC radiation in the liquid state.

For example, the starting platelet lysate in the liquid state is packaged in a UVC permeable container, such as a UVC permeable irradiation bag. A UVC permeable irradiation bag is in particular made of a material that does not have a maximum adsorption in the range from 200 to 280 nm. The irradiation bag is in particular made of ethylene vinyl acetate or polytetrafluoroethylene. The irradiation bag containing the starting platelet lysate is then placed in a UVC illumination apparatus. The bag is orbitally agitated during irradiation with UVC radiation, so as to homogeneously irradiate the entire platelet lysate.

Alternatively, the UVC radiation irradiation of the starting platelet lysate is performed under flow condition.

According to a particular embodiment, the method comprises, prior to irradiation with UVC radiation, a step of filtering said starting platelet lysate through a porosity filter of 0.65 μm or less, particularly 0.45 μm or less.

This filtration step makes it possible to eliminate any cell debris originating from the platelet lysis step and which could hinder the irradiation of the platelet lysate.

According to another particular embodiment, the method comprises, subsequent to irradiation with UVC radiation, a sterilizing filtration step of said platelet lysate irradiated by UVC radiation through a filter of porosity 0.45 μm or less, particularly 0.22. pm or less.

This filtration step through a sterilizing filter makes it possible to retain the bacteria having a size greater than 0.22 μm and, combined with irradiation with UVC radiation, makes it possible to obtain a platelet lysate having a reduced risk of bacterial and viral contamination. .

In order to further reduce the UVC-irradiated platelet lysate coagulation power, the method according to the invention comprises a step of irradiating the platelet lysate with ionizing radiation having a wavelength of less than or equal to 100 nm, which is particularly inferior. at 10 nm. Ionizing radiation having a wavelength of less than or equal to 100 nm comprises X-UV rays having a wavelength ranging from 10 nm to 100 nm, the X-rays having a wavelength ranging from 10 pm to 10 nm. nm and gamma rays having a wavelength of less than 10 μm.

A method of irradiation of a platelet lysate by ionizing radiation is described in particular in the patent application WO 2016/193591.

In such a method, prior to the ionizing radiation irradiation step, the method comprises a step of freezing said platelet lysate to irradiate the platelet lysate by ionizing radiation in the frozen state.

In particular, the freezing of the platelet lysate is carried out at a temperature between -10 ° C and -196 ° C, especially about -20 ° C or about -80 ° C.

Alternatively, the platelet lysate is irradiated with ionizing radiation in a lyophilized state.

For irradiation with ionizing radiation in a frozen state, the platelet lysate is packaged in a container resistant to freezing and in particular in a freeze resistant bag. The freeze resistant material is especially ethylene vinyl acetate, polyethylene or a fluoropolymer such as fluorinated ethylene-propylene.

According to a particular advantageous embodiment, the irradiation with ionizing radiation is carried out after irradiation with UVC radiation, that is to say that the irradiation with ionizing radiation is performed on the platelet lysate irradiated by UVC radiation.

Even more advantageously, the irradiation with ionizing radiation is carried out after radiation by UVC radiation and subsequent to the sterilizing filtration of the platelet lysate irradiated by UVC radiation, that is to say on the platelet lysate irradiated by UVC radiation and then sterile filtered.

In this case, the irradiation step by ionizing radiation is carried out on the platelet lysate in its final packaging, in particular in a storage bag. The storage bag is for example made of a material resistant to freezing and radiation by ionizing radiation such as ethylene vinyl acetate.

Alternatively, irradiation with ionizing radiation is performed prior to irradiation with UVC radiation on the starting platelet lysate.

According to one embodiment, the ionizing radiation is gamma radiation having a wavelength of less than or equal to 10 μm.

Gamma radiation is an electromagnetic radiation composed of high energy photons of the order of 1.6 MeV. It is for example emitted by a source of cobalt 60.

The irradiation with ionizing radiation is arranged so as to keep at least 80% of the concentration of at least one of the endogenous growth factors selected from the group consisting of TGF-beta1 in the ionized radiation-irradiated lysate. , EGF, PDGF-AB, IGF-1 and VEGF of platelet lysate prior to irradiation with ionizing radiation.

In particular, the gamma radiation irradiation is arranged so as to preserve in the ionized radiation-irradiated lysate at least 80%, particularly at least 90%, and even more particularly 95% of the concentration of each of the growth factors. TGF-beta1, EGF, PDGF-AB, IGF-1 and VEGF, platelet lysate prior to irradiation with ionizing radiation. For example, irradiation with ionizing radiation is gamma irradiation carried out at an absorbed dose in the range of from 20 kGy to 75 kGy, especially from 35 kGy to 55 kGy.

The absorbed dose is the amount of energy imparted to the material per unit mass.

For example, the irradiation is carried out for a time in the range from 600 seconds to 1800 seconds, preferably from 900 seconds to 1200 seconds, and more preferably for 1075 seconds, with a source having an activity of 1 Mci (3, 7x1019 Bq).

According to the process of the invention, the platelet lysate undergoes a double irradiation by UVC radiation and by ionizing radiation, that is to say a first radiation by UVC radiation followed by a second irradiation by ionizing radiation, or a first irradiation with ionizing radiation followed by a second irradiation with UVC radiation.

Advantageously, irradiation with ionizing radiation and irradiation with UVC radiation are arranged together to conserve at least 75% of the total protein concentration of the starting platelet lysate.

Thus, the doubly irradiated platelet lysate retains its interest for use in cell culture or other application for which the proteins are of interest.

The conservation of most or all of the proteins of interest, as well as the conservation of the biochemical factors (factors equally important for cell growth) of such a doubly irradiated lysate makes it possible to compensate for the partial loss in certain growth factors induced by each of the two irradiations with UVC radiation and ionizing radiation. In addition, ionizing radiation irradiation and UVC irradiation are arranged together to reduce by at least 40% the concentration of at least one of the coagulation factors including factor II, factor VII, factor IX, factor X and factor XI of the starting platelet lysate.

In particular, ionizing radiation irradiation and UVC irradiation are arranged together to reduce by at least 40% the concentration of each of the coagulation factors including factor II, factor VII, factor IX and factor XI platelet lysate starting.

For example, a platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 30% plasma, irradiated with UVC radiation and then irradiated with ionizing radiation comprises the following components.

Table 6:

Thus, the coagulation power of the doubly irradiated platelet lysate is greatly reduced, so that it can be added at high concentration, i.e. at least 20% to a calcium-containing basal medium without coagulation. in the absence of anticoagulant such as heparin.

For example, irradiation with UVC radiation is carried out at a dose of between 0.01 to 2 J / cm ² , particularly between 0.5 J / cm ² and 1.5 J / cm ² , and more particularly at 1 J / cm ² , and irradiation with ionizing radiation is gamma irradiation performed at an absorbed dose in the range of 20 kGy to 60 kGy, particularly 35 kGy to 45 kGy. Since some growth factors are not impacted at the same level by irradiation with UVC radiation and irradiation with ionizing radiation, it is possible to modulate the amount of growth factors in a platelet lysate by modulating the respective parameters of each. both irradiations.

In addition, the irradiation by UVC radiation and gamma radiation irradiation having an antibacterial and antiviral effect, the method of the invention also makes it possible to obtain a highly viral and / or bacterial-proof product. .

In another aspect, the invention relates to an irradiated platelet lysate obtained by the process according to the first aspect of the invention.

The platelet lysate prepared according to the method of preparation of the invention has a particular profile of growth factors and proteins.

In particular, irradiation with UVC radiation impacts certain growth factors that are not or less impacted by irradiation with ionizing radiation. These growth factors include, in particular, EGF, TGF-beta1 and PDGF-BB factors.

For example, the UVC irradiated platelet lysate comprises an endogenous EGF growth factor concentration of less than 2800 μg / ml, and / or an endogenous TGF-beta 1 growth factor concentration of less than 70 000 μg / ml, especially lower. 40,000 ng / ml, and / or an endogenous growth factor PDGF-BB concentration of less than 12,000 μg / ml.

The same goes for vitamin B12 impacted by UVC radiation but not by ionizing radiation.

For example, the UVC-irradiated platelet lysate comprises a vitamin B12 concentration reduced by 10 to 30% relative to the lysate. platelet count. In particular, the concentration of vitamin B12 is in the range of 125 to 140 μg / ml.

Some growth factors or proteins are not or are little impacted by irradiation with UVC radiation and irradiation with ionizing radiation.

Thus, the UVC irradiated platelet lysate and ionizing radiation include a PDGF-AB growth factor concentration in the range of 16,000 to 45,000 μg / ml.

Some growth factors or proteins are not affected by irradiation with UVC radiation, but are by irradiation with ionizing radiation.

For example, antithrombin III, a protein involved in the coagulation chain, is only weakly impacted by UVC irradiation, but is more strongly impacted by ionizing radiation.

Some growth factors are impacted by both UVC irradiation and ionizing radiation irradiation.

Thus, the UVC irradiated platelet lysate comprises an endogenous bFGF growth factor concentration of less than 140 μg / ml.

When the platelet lysate is further irradiated with ionizing radiation, the endogenous bFGF growth factor concentration is less than 90 μg / ml.

The platelet lysate irradiated by UVC radiation and ionizing radiation further comprises a fibrinogen concentration of less than 0.4 mg / ml.

Double irradiation with UVC radiation and ionizing radiation has no noticeable impact on the total protein concentration of the platelet lysate. Thus, the UV irradiated and irradiated ionizing platelet lysate comprises a total protein concentration of between 14 and 80 mg / ml, depending on the initial amount of plasma.

More particularly, the total protein concentration in a platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 100% plasma, irradiated with UVC radiation and with ionizing radiation comprises a total protein concentration ranging from about 55 mg / ml to about 80 mg / ml.

The total protein concentration in a platelet lysate produced from the freeze / thaw lysis of platelet concentrates comprising platelets suspended in 30% plasma, irradiated with UVC radiation and with ionizing radiation comprises a total protein concentration ranging from about 18 mg / ml to about 30 mg / ml.

According to a third aspect, the invention relates to a method for culturing cells, particularly animal cells, and more particularly mesenchymal stem cells, comprising contacting said cells with a nutrient composition comprising a base medium and a lysate platelet irradiated according to the second aspect of the invention.

The method applies for example to the culture of human or non-human animal cells, such as keratinocytes, epithelial cells, leukemic or solid tumor cell lines, adipocytes, amniotic fluid stem cells, stromal cells. bone marrow, chondrocytes, corneal cells, endothelial cells, mesenchymal stem cells, monocytes, osteoblasts and natural killer cells. For example, mesenchymal stem cells are human mesenchymal stem cells derived from bone marrow or umbilical cord blood.

According to a particular embodiment, the nutritional composition comprises from 2% to 25%, in particular from 5% to 15%, and even more particularly from 8% to 10% of irradiated platelet lysate according to the invention.

In particular, the irradiated platelet lysate is added extemporaneously in a preliminary manner to said base medium so as to form said nutritive composition.

As the irradiated platelet lysate has a reduced coagulation power, it is not necessary to add to the nutritional composition a heparin-type anticoagulant to prevent its coagulation and maintain it in a liquid state.

Thus, according to one embodiment of the method for culturing cells, particularly animal cells, the nutritional composition is in liquid form and free of anticoagulant.

Example 1: Platelet Lysate Irradiated by UVC Radiation

1 .1 Preparation of a platelet lysate

A lot of platelet lysate is prepared as described below.

Platelet concentrates (platelet concentrates) comprising 70% Intersol® Preservative and 30% Plasma were prepared from a mixture of five buffy coats and stored in storage pouches.

The storage bags were frozen at -80 ° C for about 24 hours before being thawed at 4 ° C for about 24 hours. The thawed storage bags are then centrifuged at a rate of 5000 g for 10 minutes so as to separate the supernatant comprising the platelet lysate from the sediment comprising cell debris.

The supernatant of each of the storage pockets is transferred to a mixing bag so as to obtain a mixture of platelet lysates (LP).

1.2. UVC irradiation

The mixture of platelet lysates is transferred, per volume of 500 ml, into irradiation pockets. Air and all bubbles are removed from the irradiation pockets.

The irradiation bags are then irradiated with a UVC illumination device (Macotronic UV, Maco Pharma, France), at different doses (0-3.2 J / cm ² ). The irradiation pockets are agitated at a speed of 1 10 rpm.

After irradiation with UVC radiation, the contents of the irradiation pockets are re-mixed in a transfer bag.

1 .3. Cytokine assays

The following tests, carried out on 3 batches (LP0, LP1, LP2), were conducted to characterize platelet lysates irradiated at different UVC doses:

- Assays using IGF-1 ELISA kits (ref DG100 / lot 341313) and TGF-Betal (ref DB100B / lot 340010),

- Assays using ELISA kits of bFGF (ref DFB50 / lot P104841), PDGF-AB (ref DHD00C / lot P101565), EGF (ref DE0000 / 1 and 339998), and VEGF (ref DVE00 / lot P100719)

The results, illustrated in FIG. 1, show that the concentration of IGF-1 in the platelet lysate is not affected by the UVC irradiation. The average of the 3 batches makes it possible to conclude that the PDGF-AB concentration is little or not impacted by the UVC irradiation (FIG. 4).

On the other hand, FIGS. 2,3, 5 and 6 show that the concentration of TGF-beta1, bFGF, EGF and VEGF decreases as the dose of UVC increases, starting from 0.8 J / cm ² .

Losses of EGF and VEGF are less important compared to losses of bFGF and TGF-beta1. 23% and 24% loss respectively for bFGF and TGF-beta1 at 0.8 J / cm ² , and up to 50% and 44% respectively at 1.6 J / cm ² .

1 .4. Plasma factor assays

Biochemical assays, performed on the same 3 lots, were conducted to characterize platelet lysates irradiated at different UVC doses.

The results of the plasma factor assays in the platelet lysate after UVC irradiation, shown in FIGS. 7 to 11, show that the UVCs have an effect on the factors II, VII, IX, X, and XI. As for TGF-beta1, the concentration of these plasma factors decreases as a function of the UVC dose delivered during the irradiation.

1 .5. Impact of UVC irradiation on LP efficiency (proliferation of CSMs)

Proliferation tests, performed on 3 batches, were conducted to characterize platelet lysates irradiated at different doses (Figure 12).

Mesenchymal stem cells (MSCs) were seeded in 6-well plates (triplicate for each condition) at 2500 cells / cm ² . All platelet lysates were used at 8% in alphaMEM medium. The experiment was performed twice.

Figure 12 shows that both experiments generate the same proliferation pattern of CSMs in contact with the UVC irradiated platelet lysate.

On the one hand, it can be noted that the non-irradiated platelet lysate controls are quite similar. In addition, we observe a plateau of 0 J / cm ² at 1, 2 J / cm ² where the proliferation of CSMs does not seem to be impacted. Between 1, 2 J / cm ² and 1, 6 J / cm ² the proliferation of CSMs begins to decrease visibly.

These experiments determined that irradiation with UVC radiation at a dose of approximately 1 J / cm ² maintained a concentration of growth factors sufficient to ensure good cell proliferation, while visibly reducing the concentration of coagulation.

EXAMPLE 2 Industrial Production of Platelet Lysate Irradiated by UVC Radiation and Gamma Radiation

2.1 Preparation of platelet lysate

Several lots of platelet lysate (LP) were produced as described in Example 1 .1 above, from platelet concentrates comprising platelets suspended in 30% plasma and 70% of an additive solution.

2.2 UVC Radiation Irradiation

The mixture of platelet lysates is filtered through a 0.45 μm porosity filter before being irradiated with UVC radiation. The filtered platelet lysate mixture is transferred, per 500 ml volume, into irradiation pockets. Air and all bubbles are removed from the irradiation pockets.

The irradiation bags are then irradiated with a UVC illumination apparatus (Macotronic UV, Maco Pharma), at a dose of 1 J / cm ² . The irradiation pockets are agitated at a speed of 1 10 rpm.

After irradiation with UVC radiation, the contents of the irradiation pockets are re-mixed in a transfer bag and the mixture of UVC irradiated platelet lysates is filtered through a 0.2 μm porosity sterilizing filter to form a batch of platelet lysate irradiated with UVC (LP-UVC).

2.2 Gamma radiation irradiation

The UVC-irradiated platelet lysate mixture is then redistributed into 50 ml bags of ethylene vinyl acetate.

The 50 ml bags are frozen at -80 ° C and then irradiated with gamma radiation at an absorbed dose of 35 kGy or 55 kGy (LP-UVC-G35 and LP-UVC-G55). The same batch is used for irradiation at 35 kGy or 55 kGy.

2.3 Cytokine assay

In samples of LP, LP-UVC-G35 and LP-UVC-G55, the amount of bFGF (ref.SFB50 / lot P1 16487), PDGF-AB (ref. SHDOOC / lot P122623), PDGF-BB (ref.SBBOO / lot P1 16857) and TGF-betal (ref.SBI 00B / lot P1 19433).

In FIG. 13, the concentration of bFGF is impacted by the dual radiation by UVC radiation and by gamma radiation. LP-UVC-G35 loses 50% of its bFGF concentration and LP-UVC-G55 loses 61% of its concentration in bFGF. There is a dose effect of gamma irradiation on the loss of bFGF: the greater the dose of gamma irradiation applied, the greater the loss of bFGF.

In FIG. 14, the concentration of PDGF-AB is slightly impacted by the double radiation by UVC radiation and by gamma radiation: the LP-UVC-G35 loses 13% of its concentration of PDGF-AB, the LP-UVC-G55 loses 24% of its PDGF-AB concentration (Figure 14).

In FIG. 15, the concentration of PDGF-BB is more strongly impacted by the double irradiation by UVC radiation and by gamma radiation than the concentration of PDGF-AB: the LP-UVC G35 kGy loses 34% of its concentration of PDGF-BB LP-UVC-G55 loses 39% of its PDGF-BB concentration. The effect of the dose of gamma irradiation on the loss of PDGF-BB is not clearly demonstrated.

In FIG. 16, the concentration of TGF-beta1 is impacted by the double radiation by UVC radiation and by gamma radiation, with no effect of the gamma radiation dose: the LP-UVC-G35 loses 31% of its concentration of TGF-beta1 LP-UVC-G55 loses 34% of its concentration of TGF-beta1.

Based on these results, only the PDGF-AB concentration is weakly affected by dual irradiation with UVC radiation and gamma radiation. On the other hand, the concentration of bFGF decreases by about 50% for the doses studied.

2.4 Determination of proteins

The protein assay is performed using a BCA kit (UP40840 / C05KL03).

No noticeable effects of UVC and gamma irradiation were observed on the protein concentration (Figure 17). 2.5. Biochemical assays and coagulation factors

Biochemical analyzes were performed on the following 12 elements:

Among the 12 elements dosed, only vitamin B12 is impacted by the double irradiation by UVC radiation and by gamma radiation. The effect of the dose of gamma irradiation on the loss of vitamin B12 is not shown (Figure 18). According to Table 7 below, the concentrations of coagulation factors (Fil, FVII, FIX, FX, FXI) and Antithrombin III (ATM I), a protein involved in coagulation, are affected by the double irradiation with UVC radiation and by gamma radiation. A dose effect of gamma irradiation is observed on the loss of coagulation factors: the higher the dose of gamma irradiation applied, the greater the loss of coagulation factors.

Table 7: Percentages of difference in protein concentration relative to the starting LP

2.6. Gelification test

The assay was performed at different platelet lysate concentrations in alphaMEM basal medium without heparin: 2.5%, 5%, 8%, 10%, 15%, and 20%.

Table 8:

-: no gelation

+: gelled effect but almost liquid

++: gelled at 50%

+++: 100% gelled

For non-irradiated platelet lysate (LP), with a low percentage of platelet lysate (2.5%), there is no gelled effect. The results show that the higher the percentage of platelet lysate present in the basal medium, the greater the gelation of the basal medium.

At 5% LP, the basal medium already has a gelled effect, and at 8% platelet lysate, the basal medium is gelled up to 50%.

Basal medium containing LP-UVC begins to gel only at 10% LP-UVC concentrations, and at 15% LP-G35 concentrations.

At the highest percentages (15% and 20%), only basal media containing LP-UVC-G35 and LP-UVC-G55 remain ungelled.

In conclusion, the doubly irradiated platelet lysate-containing basal medium does not gel, regardless of the percentage of this doubly irradiated platelet lysate used (up to 20% high doses), even without the addition of heparin. Thus, double irradiation significantly degrades the coagulation power of a platelet lysate. 2.7. Proliferation tests

The cells used are human primary mesenchymal stem (MSC) stem cells derived from bone marrow from two different donors (M065 and M068). The experiment was performed blind by two experimenters.

On the first day, 6-well plates are seeded at 2500 cells / cm ² in triplicate. Human CSMs are at P3.

The basic medium used is the alphaMEM medium. The platelet lysates are those of Table 8, at 8% in the basal medium.

We change the environment every 3 days. After 7 days of cell culture, the cells are counted with ViCell.

Only a small impact of UVC irradiation and gamma irradiation on the efficiency of platelet lysate was observed: 4% loss of cell proliferation caused by UVC and gamma radiation at 35 kGy (Figure 19) .

In contrast, at 55 kGy, gamma irradiation has a greater impact on platelet lysate efficiency. Thus, the cumulative effect of UVC irradiation and gamma irradiation at 55 kGy results in an 18% loss of cell proliferation compared to LP control. However, it can be noted that the doubly irradiated LP at 55 kGy remains more effective than the 10% SVF + bFGF conditions at 1 ng / ml (results not shown).

The absence of an impact on the cell proliferation of the double irradiation of the platelet lysate by UVC and gamma radiation at 35 kGy could be explained by the fact that, on the one hand, the cytokines of interest and, on the other hand, the different biochemical factors important for cell growth (vitamins, ...), do not are not or poorly impacted. The relative loss of some of these factors (still present even if for some in smaller quantities) could explain the lack of effect on cell proliferation. Moreover, at gamma irradiation of 55 kGy, all cytokines being more impacted than at 35 kGy, this would no longer allow optimal proliferation of cells, which could explain the negative effect of double irradiation. UVC and gamma radiation at 55 kGy on cell proliferation (-18%).

Claims

A process for preparing an irradiated platelet lysate comprising the steps of:

the double irradiation of said starting platelet lysate by UVC radiation of wavelength between 200 and 280 nm and by ionizing radiation having a wavelength of less than or equal to 100 nm, in order to obtain a platelet lysate irradiated by UVC radiation and ionizing radiation, said dual radiation by UVC radiation and ionizing radiation being arranged to retain at least 75% of the total protein concentration of said starting platelet lysate while reducing by at least 40% the concentration of at least one of said coagulation factors including fibrinogen, factor II, factor VII, factor IX, factor X and factor XI of the starting platelet lysate.

2. Method according to claim 1, characterized in that the double radiation by ionizing radiation and by UVC radiation is arranged to reduce by at least 40% the concentration of each of the coagulation factors including factor II, factor VII, factor IX, factor X and factor XI of the starting platelet lysate.

3. Method according to one of claims 1 or 2, characterized in that irradiation UVC radiation is arranged to retain at least 75% of the total protein concentration of said platelet lysate starting, while reducing at least 20% the concentration of at least one, in particular each, of the coagulation factors including fibrinogen, factor II, factor IX, factor X and factor XI of the starting platelet lysate.

4. Method according to any one of claims 1 to 3, characterized in that the starting platelet lysate comprises at least the endogenous growth factors TGF-beta1, EGF, PDGF-AB, IGF-1, VEGF and bFGF, and in that the irradiation with UVC radiation is arranged to retain at least 80% of the concentration of at least one of the growth factors including IGF-1, PDGF-AB, EGF and VEGF, and in particular at least 80% of each of the growth factors including IGF-1, PDGF-AB, EGF and VEGF in the starting platelet lysate.

5. Method according to any one of claims 1 to 4, characterized in that irradiation UVC radiation is carried out at a dose of between 0.01 to 2 J / cm ² , particularly between 0.5 J / cm ² and 1.5 J / cm ² , and more particularly at 1 J / cm ² .

6. Method according to any one of claims 1 to 5, characterized in that the starting platelet lysate is irradiated by UVC radiation in the liquid state.

7. Method according to any one of claims 1 to 6, characterized in that it comprises, prior to irradiation with UVC radiation, a filtration step of said starting platelet lysate through a porosity filter 0, 65 μm or less, particularly 0.45 μm or less.

8. Method according to one of claims 1 to 7, characterized in that it comprises, subsequent to irradiation with UVC radiation, a sterilizing filtration step of said platelet lysate irradiated by UVC radiation through a porosity filter 0.45 μm or less, particularly 0.22 μm or less.

9. Method according to any one of claims 1 to 8, characterized in that it comprises, prior to the irradiation step by ionizing radiation, a step of freezing said platelet lysate to irradiate the platelet lysate by ionizing radiation in the frozen state.

10. Method according to any one of claims 1 to 9, characterized in that the irradiation by ionizing radiation is performed after radiation by UVC radiation.

A method according to any one of claims 1 to 10, characterized in that the ionizing radiation is gamma radiation having a wavelength of less than or equal to 10 μm.

12. Process according to any one of claims 1 to 1 1, characterized in that the irradiation with ionizing radiation is carried out at an absorbed dose in the range from 20 kGy to 75kGy, in particular from 35 kGy to 55 kGy.

13. Irradiated platelet lysate obtained by the method according to any one of claims 1 to 12, characterized in that it comprises a total protein concentration of between 18 and 80 mg / ml, a fibrinogen concentration of less than 0.4. mg / ml, and an endogenous bFGF growth factor concentration of less than 90 μg / ml.

The irradiated platelet lysate according to claim 13, characterized in that it comprises an endogenous growth factor TGF-beta1 concentration of less than 70,000 μg / ml, an endogenous EGF growth factor concentration of less than 2,800 μg / ml. and an endogenous growth factor PDGF-BB concentration of less than 12,000 μg / ml.

15. Platelet lysate according to one of claims 13 or 14, characterized in that it comprises a vitamin B12 concentration of between 120 and 140 μg / ml.

A method for culturing animal cells, particularly mesenchymal stem cells, comprising contacting said cells with a nutrient composition comprising a basal medium and an irradiated platelet lysate according to any one of claims 13 to 15.

17. The method of claim 16, characterized in that the nutrient composition is in liquid form and free of anticoagulant.