WO2018162740A2 - Cell culture supplements - Google Patents

Abstract

The present invention refers to: a combination of a) an heparin-free human platelet lysate devoid of serum and/or plasma components and b) an heparin-free human serum devoid of any platelet lysate components, or to an heparin-free platelet lysate devoid of serum and/or plasma components or to an heparin-free serum devoid of platelet lysate components, and to their use.

Description

CELL CULTURE SUPPLEMENTS

FIELD OF THE INVENTION

The present invention refers to the field of the cell culture medium supplements. In particular it refers to a combination of: a) an heparin-free human platelet lysate devoid of serum and/or plasma components and b) an heparin-free human serum devoid of any platelet lysate components, and to the single component a) and b), and to their use as cell culture supplements.

BACKGROUND ART

Human platelet derivatives (platelet lysate or platelet releasate) have been proposed as tissue culture supplements alternative to fetal or calf bovine serum. The adoption of an animal-free culture medium supplement is of particular relevance in establishing culture conditions for the isolation and expansion of cells intended for clinical applications. Thanks to the cell growth promoting activity of the platelet derived growth factors, platelet derivatives can support cell proliferation and differentiation. Platelet derivatives used as cell culture additives, are commonly provided in the form of platelet-lysate contained in a small amount of plasma. As known, plasma is the physiological fluid in vivo and cells come in contact with serum only during the injury healing or blood coagulation. However, the use of plasma in cell culture presents some problems. Citrate, a calcium chelator, is the standard plasma anticoagulant used in the process of plasma collection. When plasma is added to culture media, which usually contains calcium, fibrin clots may form. This can be prevented by adding thrombin (usually of animal origin) and calcium to plasma to induce clot formation and defibrination (Ayake S et al. J Transl Med. 2006; 4:40). To prevent coagulation and clot formation, another possibility is the addition of heparin (Kocaoemer A. et al Stem Cells 2007; 25:1270-8). Commercially available heparin is manufactured primarily from porcine sources, and although it is approved for human use, in some cases a hypersensitivity to heparin was reported (Huang Q. et al Anal Bioanal Chem 2012; 402: 1625-34; Bottio T. et al J Thorac Cardiovasc Surg. 2003; 126: 1 194-5). Being of animal origin, heparin represents a limit in the development of a totally xeno-free medium. Moreover, heparins are active factors that bind growth factors and may interfere with cell growth (Walker CP. et al. Br J Anaesth 2002; 88:848-63; Tiozzo R. et al. Thromb Res. 1991; 62:177-88). It was also shown that a relatively high concentration of heparin in culture media supplemented with human platelet lysate (PL) impaired adipo genie and osteogenic differentiation of mesenchymal stem cells (MSC). Other reports showed that heparin negatively affected proliferation and motility of vascular smooth muscle cells (Reilly CF et al. J Cell Physiol 1986; 129 (1): 1 1-9) and inhibited growth of osteoblasts and MSC under conventional culture conditions (Papathanasopoulos A et al. J Orthop Res 2011; 29(9): 1327-35). Others researchers showed that heparin also interfered with the functional capacity for migration and homing of BM- derived mononuclear cells used in cardiovascular repair (Seeger FH et al. Circ Res 2012; 11 1 :854- 62). To overcome the need of heparin addiction to the cell culture media containing platelet derivatives and at the same time to prevent the medium clotting after the PL addition, some options were proposed. A possible strategy is the derivation of a serum-converted platelet lysate from pooled Platelet Rich Plasma (PRP) obtained from buffy coats by a plasma-coagulation step in the presence of calcium chloride as shown by Henshaw MP et al. (Henshaw MP et al. Cytotherapy 2013; 15:1458-1468). These authors reported that this PL-serum was less efficient than the sister counterpart PL-plasma in supporting MSC proliferation although both lysates supported the tri- lineage cell differentiation potential. Moreover, it has to be considered the positive effect of the fibrinogen depleted lysate observed with regard to the immunosuppressive properties of MSC (Coplan IB et al. Biomaterials 2013; 34: 7840-7850). A second possibility is the production of a platelet lysate devoid of plasma obtained by repeated cycles of platelet washing with a saline solution prior their rupture and release of bioactive factors. This platelet lysate was shown to sustain cell proliferation, comparable to fetal bovine serum, in short term (1-7 days) cultures of renal epithelial cell lines, of both animal and human origin, grown in adhesion and in suspension cultures of human lymphoblastoid cell lines (Rauch C et al. Altex 28, 4/1 1). The mitogenic effect of platelet derived growth factors was visualized also by checking the activation of the ERK1/2 factors induced by the PL addition. However the sustainability of long term cell expansion in the presence of this PL as the only medium supplement was not investigated. The same group reported that commercially available Adipose-Derived Adult Stem Cell (ADSC), initially isolated in the presence of FCS, after 7 day culture in the presence of PL, maintained their differentiation potential toward the adipo-osteo and chondrogenic mesodermal lineages (Rauch C et al. J Adv Biotech and Bioeng 2014; 2: 39-4). No experimental evidence was given in those reports of a long term cell culture in the continuous presence of only platelet derived growth factors. Serum is a blood plasma from which coagulation factors have been removed. Traditionally, human serum is obtained by letting fresh whole blood, collected without any anticoagulant, to clot several hours at 4°C before high speed centrifugation. The first studies using MSC and human serum, obtained by conventional blood coagulation, as cell culture supplement, showed the efficient isolation and expansion of MSC while maintaining the osteo-adipogenic differentiation potential (osteogenic differentiation was higher in autologous serum rather than in FBS) (Stute N et al. Exp Hematol 2004; 32:1212-1225) and higher cell motility compared with FCS (Kobayashi T et al. J Bone Joint Surg Br 2005; 87: 1426-1433). In some cases serum was successfully derived by the clotting of umbilical cord whole blood (Shetty P et al. Cell Biol Int. 2007; 31(3): 293-8). Alternatively serum can be derived from blood plasma that has been treated with anticoagulants and from which blood cells, including red blood cells, white blood cells and platelets, were removed by centrifugation (platelet poor plasma) or by plasma directly collected by plasmapheresis. Precipitation of the coagulation factors is obtained by addition of calcium cations and/or thrombin treatment. When platelets are activated by thrombin, or other molecules they degranulate the alpha granules, resulting in the release of various kinds of growth factors. Therefore the growth factor content of serum may change depending on the presence of platelets in the source material and this may significantly change the biological effect of serum when used as supplement in a cell culture medium. Tanaka et al. described a most pronounced effect on human auricular chondrocytes when a serum derived from plasma including platelets was compared to a serum derived from a plasma depleted of platelets although no significant differences were observed on the cartilage matrix deposition by chondrocytes cultured under different serum conditions (Tanaka Y. Cell Biology International 2008, 32: 505-514). Several years ago, Rutherford et al. reported that exposure of quiescent cells to whole blood serum or platelet-poor plasma serum plus crude platelet factors preparations stimulated cell proliferation (Rutherford RB. Journal of Cell Biology 1976; 69:196-203). However, no investigations were made to distinguish between the role played by factors and molecules released by platelets and the ones present as serum components. Indeed, an increasing number of scientific publications report the use of platelet lysate as substitute of bovine serum in cell culture media. However the adopted protocols for the preparation of the platelet lysate always implicate the presence in the lysate of a certain percentage of plasma or serum proteins and factors. In the few examples where the lysate was obtained from washed platelets to remove the plasma components, the role played by the lysate on the induction and the sustainability of cell proliferation was tested in the presence of serum.

Summary of the invention

Present inventors wanted to distinguish between the role played by the platelet content and the serum/plasma components in inducting and sustaining proliferation of cells, derived from biopsies (primary cultures) or as cell lines, and unexpectedly observed that the two blood fractions had very well distinct and complementary roles. By applying blood fractionation procedures, the present inventors obtained two new "pure" blood fractions from large pools of donated blood, storable in a freeze-dried form and suitable to be used as cell culture medium supplements. In particular: (i) an heparin-free human platelet lysate without (or devoid of) serum and/or plasma components (herein defined as "virgin-Platelet Lysate", "v-PL" or "PL" or "Platelet Lysate" or "PL-free serum"); (ii) an heparin-free human serum without (or devoid of) Platelet Lysate components (herein defined as "Plasma-Serum", "Pl-s" or "HS or "Human Serum" or "Serum"). The present inventors unexpectedly found that, although the platelet released (PL in saline solution) was capable of recruiting quiescent cells, or even senescent cells, back in the cell cycle, by activating the cell proliferation machinery (ERK and AKT phosphorylation, Cyclin Dl induction, etc.) the Platele Lysate itself was unable to support cell proliferation unless the plasma or serum components were also present in the culture medium. Interesting, in cells that were constitutively stimulated, such as different cell lines of human or animal origin, or some cultures of cells derived from fetal tissues, the PL was not an absolute requirement of the culture medium and cell proliferation was observed by the simple addition of PL-free serum. In certain embodiments, the v-PL can be used as medium supplement to enhance cell yield, proliferation and number of duplications, in primaries cell cultures derived from different tissue biopsies or aspirates, and performed in standard culture media already containing conventional supplements, such as fetal bovine serum (FBS) or fetal calf serum (FCS). Moreover, inventors report that the v-PL, when used as the only medium supplement, promotes in tissue differentiated quiescent cells the activation of pathways triggering cell proliferation and the reprogramming of the same cells to progenitor cells. However, proliferation of these progenitors does not occur unless also the Pl-s is added as medium supplement. On the contrary the Pl-s, used as the only medium supplement, is not able to promote conversion to progenitors and proliferation of quiescent tissue differentiated cells, but it sustains cell proliferation in constitutively activated cell lines also in the absence of conventional medium supplements. In certain embodiments the two products can be combined in different ratios to allow the establishment of cell cultures starting from a biopsy or an aspirate and the in vitro expansion of different types of cells, including cells intended for cell therapy in humans, in complete absence of animal components. This consents to perform cell therapies with cells expanded in a safer conditions (absence of animal components in the medium). The use of the combined supplements allows to obtain in primary cell cultures, including but not limited to Mesenchymal Stem Cells (MSC) derived from bone marrow or adipose or cord blood and to articular chondrocytes from a cartilage biopsy, a number of cells otherwise not reachable with conventional culture medium supplements. This is particularly relevant in the case of elderly patients from which conventional culture medium supplements do not permit to obtain an adequate number of cells. Given that endothelial cells are the first cells in contact with PL after vessel injury, inventors treated HUVECs with PL for different times in presence or not of IL-la. The PL inhibited IL- 1 a-induced NF-κΒ activation already after 1 -hour treatment as well as after 16- hours treatment showing an anti-inflammatory activity at both the early and the late considered times. This finding is in agreement with the anti-inflammatory activity of PRP described in literature (Mazzocca, A. D., McCarthy, M. B. R., Intravia, J., Beitzel, K., Apostolakos, J., Cote, M. P., et al. (2013). An In Vitro Evaluation of the Anti-Inflammatory Effects of Platelet-Rich Plasma, Ketorolac, and Methylprednisolone. Arthrosc. J. Arthrosc. Relat. Surg. 29, 675-683) and provides a possible rationale to this effect. Inventors also observed that, at variance with the other so far investigated cell systems, PL did not significantly enhance the production of IL-6 and IL-8 induced by IL-la, nor induce a significant repression, although the trend was a decrease in the secretion of these two cytokines. The response of the different assayed primary cultures was quite variable as demonstrated by the standard deviation. Taken together, the data suggest that endothelial cells are protected by PL in the inflammatory milieu of the wound. A significant increase of PGE2 secretion in inflammatory conditions was detected by the inventors indicating that PL activated HUVECs possibly to contribute to resolution of tissue inflammation. The presented results provide an additional indication that PL exerts an early anti-inflammatory activity on HUVECs. Inventors also herein found that Platelet lysate enhanced the proliferation of HUVECs, without affecting their capability of forming tube-like structures on matrigel, and that resting confluent HUVECs were activated by PL that induced cell proliferation up to a cell concentration approximately double than the one of not PL stimulated, control cells. In agreement with these findings, proliferation-related pathways Akt and ERK were activated as well as the expression of the cell cycle activator Cyclin Dl enhanced. This an important finding because clot formation and platelet degranulation are the first tissue's response to an injury and, in particular, the multitude of platelet-released factors able to attract circulating cells and to restore the proliferation of resting resident cells physiologically leads to the tissue repair through the complex series of events typical of the wound healing process. Eventually, only after the effective repair of the injured tissue, normal blood perfusion resumes. Inventors demonstrated that resting cultured confluent HUVECs are activated and resume proliferation following a PL treatment suggesting that an activation of the resident cell population, possibly progenitors and differentiated cells, could occur also in vivo after an injury. Inventors also found out that platelet-released factors induced the stabilization of HIF-Ια and the phosphorylation of STAT3 in agreement with an involvement of PL in the activation of angiogenesis. In conclusion, inventors herein demonstrated a beneficial activity of PL treatment on HUVECs by the inhibition of inflammation, the enhancement of proliferation of cells, which retained the differentiation capability, the resumption of proliferation of quiescent cells and the activation of angiogenesis- related pathways, thus providing a rationale for the clinical use of PL in wound healing. Inventors also analyzed the response of human subcutaneous adipose tissue to platelet-derived factors under conditions mimicking as much as possible the wound micro environment in order to evaluate its contribute in supporting the repair process of skin lesions. For this purpose, inventors defined a clinically relevant model using Human Serum (HS), Platelet Lysate (PL or v-PL) and Interleukin- 1 a (IL-la) as effectors and primary human adipose-derived stromal cells (hASCs) and in toto human adipose tissue (hAT) cultured ex vivo as targets. HS (or Pl-s) was uncontaminated by platelet- derived factors, obtainable by the method herein discloses. Analogously, the PL (or v-PL) did not contain any plasmatic molecule since it has been prepared using platelets extensively washed with physiological saline before to have been disrupted. Consequently, such supplements allowed to study rigorously their separated and combined effects on adipose tissue. Considering that serum is the physiologic fluid present at the wound site following the coagulation process, inventors first evaluated the effect of HS on in vitro proliferation and differentiation of hASCs in comparison with FBS, normally used as gold standard for hASC culture. In particular, they found that HS sustained hASC proliferation with a rate significantly higher than FBS. However, no statistically significant difference was observed in the number of isolated CFU-fs between HS and FBS, demonstrating that the increased proliferation induced by HS was not related to a greater number of clonogenic progenitor cells. Moreover, they found that HS allowed adipogenic programme to carry on in hASCs by inducing the expression of two master regulators of adipogenesis PPARy2 and C/EBPa and the accumulation of lipid droplets within the cells while no induction was observed with FBS. Considering both the morphology and the amount of incorporated lipid droplets, the HS-induced adipogenesis showed to be qualitatively similar to adipogenesis obtained following a traditional differentiation protocol normally used for hASCs cultured in FBS (Scott, M. A., Nguyen, V. T., Levi, B., & James, A. W. (201 1). Current Methods of Adipogenic Differentiation of Mesenchymal Stem Cells. Stem Cells and Development, 20(10), 1793-1804). By promoting the osteogenic differentiation with a traditional protocol inventor obtained a strong deposition of mineralized matrix by HS committed hASCs even though lipid droplets were still revealed within cells by ORO staining. These findings suggest HS-expanded hASCs could represent a heterogeneous cell population constituted by progenitor cells at different stages of commitment. The coagulation process determines physiologically not only the conversion of plasma to serum but also the release of bioactive molecules from activated platelets by degranulation. For this reason, ivnentors studied the proliferative response of hASCs to platelet content. In particular, the presence of PL in the culture medium supplemented with HS significantly enhanced cell growth reaching at confluence state a higher cell density and determining spindle-like morphology and smaller dimensions in comparison with cells cultured in the absence of PL. PL determined in HS-expanded hASCs the early activation of proliferation-related Akt and ERK pathways, known to be involved in promoting cell and implicated in the induction of Cyclin Dl expression and in the repression of antiproliferative gene transcription, respectively. PL also induced the synthesis of Cyclin Dl after 4h treatment confirming the mitogenic role of PL in hASCs. Interestingly, inventors also observed that STAT3, which is involved in the cell-cycle progression, was activated by PL and the maximum level of the phosphorylated form was reached after 24h treatment. Taken together, these data suggest that quiescent progenitor cells could be possibly activated and induced to proliferate by platelet-derived factors also at the wound site. No significant variations in hASC growth were found between physiological and inflammatory conditions when PL was supplemented or not to culture medium containing HS thus suggesting IL-la was not involved in cell proliferation. Interestingly, inventors observed that hASCs treated with PL for 4 days showed a significant decrease of incorporated ORO in parallel with a significant decrease of ppary2 transcript level. Taken together, these data suggest that PL treatment would favour the presence of un-differentiated cells in cultures of committed hASCs. In particular, PL could possibly induce the de-differentiation of cells already committed in HS or induce the proliferation of un-committed cells. Moreover, inventors found that hASC expanded in presence of PL for 3 passages retained differentiation capability towards adipogenesis and osteogenesis. Considering a putative role of adipogenic lineage cells in the healing of damaged skin also in humans, they evaluated the activation of adipogenic progenitor cells following inflammation and induction by platelet lysate. In particular, they investigated the secretory and inflammatory response of HS-expanded hASCs to PL under both normal and inflammatory conditions. Inventors found the strong increase of pro-inflammatory cytokine IL-6 and IL-8 secretion from hASCs in presence of PL in inflammatory milieu respect to IL-la condition. Considering that the release of platelet-derived molecules is limited in time, inventors monitored the secretion of such cytokines up to 72h after the removal of PL stimulation finding the gradual decrease of their secretion with time. The production of COX-2, an enzyme involved in causing inflammation, was also temporarily induced in presence of IL-la by a burst of PL (24h). As a consequence of COX-2 increase, inventors revealed the synergistic induction of mPGES-1 by PL and IL-la even though mPGES-1 was already present at a remarkable basal level in the untreated cells. The mPGES-1 levels increased with time under all tested conditions, included the control condition. The activation of COX-2/mPGES-l pathway by PL under inflammatory condition led to a massive production of PGE2, a prostaglandin able to induce the functional switch of macrophages towards the alternatively activated M2 phenotype, involved in the dampening of inflammation. The strong but temporally transient increase of IL-6 and IL-8, the transient induction of COX-2 and the production of PGE2 following PL treatment under inflammatory condition suggest a possible contribution in vivo of subcutaneous adipose tissue first in establishing an inflammatory microenvironment and then in preparing the inflammation resolution during body's response to an injury. The ex vivo culture of in toto hAT showed that PL was able to induce the proliferation of small and elongated cells on the surface of hAT pieces under both physiological and inflammatory conditions, while the presence of such cells was limited in absence of PL. The presence of IL-la in the culture medium did not influence the cell proliferation. Moreover, PL induced cells to come out from hAT pieces, adhere to plastic and proliferate. Overall, these findings supported further the capability of platelet-derived factors of inducing in culture the cell growth not only of primary hASCs but also of in toto hATs and in in vivo subcutaneous adipose tissue. The methods of the invention allow to obtain two reagents (and not only cell culture supplements) to be considered as FCS substitute. The two separate reagents, in particular the Pl-s, allow to study cell "commitment" independently from the proliferation support by the serum. Serum alone is able to sustain the already committed cell proliferation (previously exposed to PL) independently from the necessity of the simultaneous presence of PL or to sustain the proliferation of constitutively activated cells which were not treated with PL, as in the case of cell lines.

Detailed description of the invention

It is therefore an object of the invention a combination of:

a) an heparin-free human platelet lysate devoid of serum and/or plasma components and

b) an heparin-free human serum devoid of any platelet lysate components.

The combination preferably comprises from 0.1% to 50% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components, more preferably from 0.5% to 40%, even more preferably from 0.9% to 30%, even more preferably from 1% to 5%, e.g. 0.9% or 4.5%. Other objects of the invention are an heparin-free platelet lysate devoid of serum and/or plasma components and an heparin-free serum devoid of platelet lysate components.

Preferably, said heparin-free human platelet lysate devoid of serum and/or plasma components and heparin-free human serum devoid of any platelet lysate components are produced as pool of several blood donors, preferably at least 3 for HS or 5 for PL.

A further object of the invention is a process for the preparation of the heparin-free platelet lysate devoid of serum and/or plasma components comprising:

a) obtaining by centrifugation a platelet rich blood fraction from an isolated buffy coat pool, said pool preferably comprising samples isolated from 2 to 500 subjects, more preferably from 100 to 250, even more preferably 5 to 10 or at least 25,

b) isolating by centrifugation the plasma free platelet concentrate from the obtained platelet rich blood fraction;

c) washing in saline solution, preferably isotonic, the isolated plasma free platelet concentrate;

d) subjecting to platelet lysis the washed plasma free platelet concentrate to obtain a platelet lysate devoid of serum and/or plasma components,

and optionally

e) lyophilizing the platelet lysate devoid of serum and/or plasma components.

In the process according to the invention the platelet rich blood fraction (or the platelet concentrate) may be leukocyte depleted prior platelet washing with saline solution or after platelet washing with saline solution. Preferably, the platelet concentrate (or the platelet rich blood fraction) can be leuko- depleted e.g. by filtration prior or after washing in saline solution. Preferably, in the process according to the invention after step c), the platelet concentrate is preferably resuspended in saline solution and the platelet concentration preferably adjusted to 10.0x10⁹ pit/ml. Preferably, in the process according to the invention the platelet lysis is obtained by three freeze -thawing cycles. In a preferred embodiment of the process of the invention, after platelet lysis a high speed centrifugation is performed to sediment platelet membranes and debris. The above centrifugation of step a) is preferably performed from 300 to 400 RCF (low speed), for 10 - 15 minutes. The above centrifugation of step b) is preferably performed from 2,000 to 2,300 RCF (high speed) for 20 - 30 minutes. The washing of step c) preferably comprises 3 washes. Preferably the saline solution is a physiological saline, more preferably 0.9% w/v NaCl solution. After step c), the platelet concentrate is preferably resuspended in saline solution and the platelet concentration preferably adjusted to from 2x10⁶ plt/ul to 12x10⁶ plt/ul, more preferably to lOxlO⁶ plt/ul. In the method of the invention, the platelet lysis may be obtained with any method known to the skilled man, including freeze - thawing, preferably in repeated cycles (e.g. 3), or sonication. After step d), a centrifugation is preferably performed, preferably at high speed, to sediment platelet membranes and debris. Before step e), the supernatant, comprising the cocktail of factors released by the platelets re-suspended in physiological saline (v-PL) may be collected and stored in aliquots at -20°C until use. Preferably, the platelet rich blood fraction is not leukocyte depleted and/or no anticoagulant is added and/or no additives are added to the washed isolated plasma free platelet concentrate in the above defined methods. Another object of the invention is a process for the preparation of the heparin-free serum devoid of platelet lysate components comprising:

a) obtaining by cryoprecipitation a cryo poor plasma from an isolated pool of plasma, said pool preferably comprising samples isolated from 2 to 100 subjects, preferably from at least 3 subjects, b) adding to the obtained cryo poor plasma calcium ions, preferably Calcium Gluconate or Calcium Chloride, and centrifugating to obtain heparin-free serum without platelet lysate components;

and optionally

c) lyophilizing the heparin-free serum without platelet lysate components.

Preferably before step a) whole blood units are centrifuged at high speed obtaining different phases: the plasma at the top, the BC layer (enriched in platelets and leukocytes) at the interface and the red blood cell fraction at the bottom. In step a) the plasma may be isolated by one subjects and then the obtained HS after step b) is pooled with others HS derived from plasma of other subjects. The starting plasma is preferably frozen. It was preferably thawed at 4°C to obtain a separation between the cryoprecipitate and the cryo-poor plasma (CPP). The concentration of the added calcium preferably corresponds to 2 mg/ml of CaCl₂ or to 5 mg / ml of Calcium Gluconate. The incubation time, or coagulation time, is preferably comprised within 2 hours or 6 hours at a temperature of 37°C. By adjusting calcium concentration and/or incubation temperature it is possible to obtain coagulation in a longer or shorter time. Following the coagulation step, the blood is preferably high speed centrifuged to separate the coagulum and the liquid phase (Pl-s or HS) recovered. The HS or v-PL may also be obtained starting from a single plasma unit or buffy coat sample, respectively, and then pooled with other HS or v-PL, respectively. HS may be divided into aliquots and lyophilized and stored at -20°C. The heparin-free platelet lysate or the heparin-free serum according to the invention are preferably obtained by the above methods. In the combination according to the invention, the heparin-free platelet lysate and/or the heparin-free serum are preferably obtained by the above method. The combination of the invention, or the heparin-free platelet lysate of the invention or the heparin-free serum of the invention are preferably lyophilized and/or frozen and/or freeze-dried and/or sterilized. Preferably the sterilization is performed before or after the freeze - drying step. Still preferably the sterilization is performed by gamma radiation or filtration. Another object of the invention is a cell culture medium supplement comprising or consisting of the above combination or the above heparin-free platelet lysate devoid of serum and/or plasma components, or the above heparin-free serum devoid of platelet lysate components. Said cell culture medium supplement preferably comprises from about 0.5% to about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and/or from about 5% to about 20% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components, more preferably it comprises about 1% or about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and/or about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components. Even more preferably said cell culture medium supplement comprises about 1% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components. Even more preferably said cell culture medium supplement comprises about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components. The above concentrations refer to the final concentrations of the cell culture medium supplement in the culture medium. It is a further object of the invention a process for the preparation of the cell or tissue culture medium supplement as above disclosed comprising mixing the heparin-free human platelet lysate devoid of serum and/or plasma components and the heparin-free human serum devoid of any platelet lysate components. Preferably said components are in liquid or powder form. Further object of the invention is the use of the heparin-free platelet lysate devoid of serum and/or plasma components as above defined as a cell culture medium supplement for:

a) inducing in-vitro the reprogramming to progenitor cells of differentiated cells, said cells being preferably mesenchymal cells and/or chondrocytes and/or keratinocytes and/or skin fibroblasts; b) enhancing in-vitro the number of obtainable primary cells, said cells being preferably derived from tissue biopsies or aspirates, and/or from bone marrow, adipose, cord blood, articular cartilage, bone, skin tissue,

c) rejuvenating in-vitro a culture of senescent cells, preferably of MSC (mesenchymal stem cells), said cell culture medium supplement preferably comprising from about 0.5% to about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components, more preferably comprising about 1% or about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components. The above concentrations refer to the final concentrations of the cell culture medium supplement in the culture medium. Another object of the invention is the use of the heparin-free serum devoid of platelet lysate components as above defined as a unique cell culture medium supplement to induce in-vitro cell growth, preferably of established cell lines, said cell culture medium supplement preferably comprising from about 5% to about 20% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components, more preferably comprising about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components. The above concentrations refer to the final concentrations of the cell culture medium supplement in the culture medium. A further object of the invention is the use of the combination as above defined as a cell culture supplement to enhance proliferation of cells in primary cell cultures, said cells being preferably adult stem cells, more preferably MSC, preferably derived from adipose, bone marrow and cord blood, said cell culture medium supplement preferably comprising from about 0.5% to about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and from about 5% to about 20% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components, more preferably comprising about 1% or about 5% (volume/volume) of the heparin- free human platelet lysate devoid of serum and/or plasma components and about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components. The above concentrations refer to the final concentrations of the cell culture medium supplement in the culture medium. Preferably, the cells cultured in the cell culture supplement as above defined are intended for cell therapy, preferably in humans. The heparin-free platelet lysate devoid of serum and/or plasma components according to the invention preferably comprises a not detectable concentration of fibrinogen, based on the method originally described by Clauss (InterMedical Sri, Villaricca (NA), Italy). The heparin-free serum devoid of platelet lysate components according to the invention preferably comprises not more than 0.5 ng PDGF-BB / ml. Preferably, the cell is cultured in a medium supplemented with 0.5 to 20 % of the cell culture medium supplement as defined above. Most preferably the cell is cultured in a medium supplemented with from 1% to 10% of the cell culture medium supplement as defined above. Even more preferably, the cell is cultured in a medium supplemented with 1 or 5 or 10 or 11 or 16 % of the cell culture medium supplement as defined above. E.g. when a medium is supplemented with 16% of the cell culture medium supplement according to the invention, the cell culture medium supplement may be a combination of the heparin-free platelet lysate devoid of serum and/or plasma components and the heparin-free serum devoid of platelet lysate components as above defined, respectively in the following percentages: 5% and 10%. Or e.g. when a medium is supplemented with 11 % of the cell culture medium supplement according to the invention, the cell culture medium supplement may be a combination of the heparin-free platelet lysate devoid of serum and/or plasma components and the heparin-free serum devoid of platelet lysate components as above defined, respectively in the following percentages: 1% and 10%. According to the invention, the cell is preferably selected from the group consisting of: a primary cell, a cell line, a cell obtained from a biopsy, an articular chondrocyte, a stem cell and an iPS cell. More preferably, the cell is a bone marrow mesenchymal stem cell or bone marrow stromal cell, preferably human (hBM-MSC), osteoblast, preferably human (hOB), skin fibroblast, preferably human (hSF), umbilical cord derived MSC, preferably human (hUC-MSC), articular chondrocytes, preferably human (hAC), adipose derived mesenchymal stem cell (AD-MSC) or adipose stromal cell, preferably human. Preferably the cell lines are: U-937, Hela, V-79 and HaCat. Human embryonic stem cells are excluded from the present invention. The term "cell culture medium supplement" also include the term "tissue culture medium supplement". The invention will be now illustrated by means of non-limiting examples referring to the following figures.

Fig. 1 Manufacturing process outline. Blood is donated for humanitarian reasons. An informed consent to the blood processing is obtained from all donors. For the preparation of the virgin-Platelet Lysate (v-PL), the starting material are discarded (for transfusional purposes) buffy coat (BC) units, from which it is derived a BC pool (up to 300 units). The Plasma-serum (Pl-s) is derived from several frozen plasma units. All the steps occured within a sterile closed system. The final v-PL and Pl-s solutions are divided into aliquots and lyophilized.

Fig. 2 (Table) Growth factor quantification. The platelet growth factors PDGF-BB and VEGF were quantified by an ELISA assay on the Pl-S and the v-PL.

Fig. 3 v-PL treatment induces proliferation of differentiated chondrocytes. Cells were obtained from digestion of articular cartilage biopsies of surgery discarded tissues after obtaining the patient informed consent to the biopsy processing. Recovered cells, cultured in standard conditions (adherent to plastic dishes), are de facto progenitors expressing type I collagen, with the capability to differentiate to mature cells expressing type II collagen and to organize a cartilage matrix, when transferred in suspension culture. Quiescent differentiated cells within the in vitro made cartilage, when exposed to PL, resumed proliferation (top panel on the right) following activation of the proliferation related pathways ERKs and AKT, and induction of cyclin Dl (top panel on the left), although the compact aggregates maintained the morphology of a cartilage rich in type II collagen and aggrecan. Moreover, in the cultures performed with PL elongated fusiform cells were present at the periphery of the aggregates, that expressed the nuclear protein PCNA, a typical marker of cell proliferation, (bottom panels). More specifically: Top panel on the left: Western blot analysis of proteins extracted from aggregates of cultured adult articular chondrocytes transferred in suspension culture in the absence of PL for 7 days and treated with 5% v-PL for different times (1 , 4, 8 and 24 h) using a-cyclin Dl , a- phospho Akt, a-phospho Erkl/2 antibodies. Actin was blotted as constitutively expressed protein control. After 1 hour v-PL treatment, an increase in the amount of cell proliferation related phospho Akt and Phospho Erkl/2 (Extracellular signal-regulated protein kinases 1 and 2) is observed. After 8 hours from treatment the Cyclin Dl protein expression is detected. This means that differentiated cells that do not proliferate when maintained in serum, once exposed to the PL mitogenic stimulus activate signal transduction pathways that promote cell growth in response to extracellular signals. Top panel on the right: To evaluate the effect of PL on proliferation in the cell aggregates, cell viability and proliferation was determined by MTT assay. Differentiated chondrocytes within the cell aggregates exposed to v-PL show an increase in cell proliferation while parallel control cells not supplemented with PL do not proliferate, (bottom panels) Bottom panels: The cartilage like tissue formed by chondrocytes maintained in suspension culture during 10 days, followed by another 7 day culture in the presence or absence of v-PL were paraffin embedded and sectioned. Sections were stained by immuno -histochemistry for Proliferating Cell Nuclear Antigen (PCNA), an index of cell proliferation.

Fig. 4 v-PL induces outgrowth from cartilage fragments of cells maintaining proliferation capability

Top panels on the right: Cartilage fragments of surgery discarded tissues were maintained in culture with medium containing 10% FCS ± 5% v-PL up to 12 months. The patient informed consent was always obtained before the biopsy processing. An induced cell proliferation, together with the release of chondroprogenitors, was observed in short and long term cultures of articular cartilage explants performed in the presence of platelet lysate. Only explants cultured with PL continued for more than one year to release a high number of proliferating cells as revealed by contrast phase microscopy image. The fact that PL treated cartilage biopsies continued to release cells for more than one year and at the same time presented an enhanced deposition of cartilage macromolecules, namely type II collagen and aggrecan, in the extracellular matrix indicates that PL induces the reprogramming of differentiated chondrocytes and promote the proliferation and release of chondroprogenitors that maintain the ability to turn into chondrocytes in a cartilage environment. Top panels on the left: Immunohisto chemical analysis performed on the paraffin embedded explants cultured for 3 weeks. The antibody against PCNA shows the presence of proliferating cells in the explants cultured with PL-s while the parallel explants not exposed to PL do not show positive cells. Graph at the bottom: Percentage of cell releasing explants in control medium (red line) and medium supplemented with PL (violet line) after different culture times (weeks).

Fig. 5 v-PL effect on cell proliferation - primary cultures. Adult tissue derived primary cells, AD-MSC (from adipo) and BM-MSC (from bone -marrow) or fetal tissue derived cells, UC-MSC (from umbilical cord) were initially selected and cultured in 10% FCS and then cultured in the presence of Pl-S and v-PL in different relative ratios. The patient or the woman informed consent was always obtained before processing of the samples. The cell proliferation rate was monitored by calculating their doubling number at different culture times. Pl-S alone is much less efficient in supporting proliferation than the combination of Pl-s+v-PL. This was especially evident in the case of BM-MSC where essentially no proliferation was observed with Pl-S alone.

Fig. 6 v-PL effect on cell proliferation - cell lines. The human cell lines U937, HeLa, HaCat and the hamster V79 cell line (from different sources. See materials and methods) were cultured in the presence of Pl-S and v-PL in different relative ratios. The cell proliferation rate was monitored by calculating their doubling number at different culture times. In this constitutively stimulated cell lines the supplement of v-PL was much less critical in supporting cell proliferation than in primary cells in agreement with its role in recruiting in the cell cycle quiescent and/or differentiated cells. Fig. 7 v-PL recruits to the cell cycle confluent resting cells . (graph) Confluent growth-arrested chondrocytes, obtained from cartilage biopsies and in vitro expanded as above, were maintained in 10% serum or treated with 5% v-PL. An MTT proliferation assay was performed in parallel on the cultures. Confluent cells treated with v-PL resumes proliferation whereas the parallel control culture does not. (bottom panel) Western blot analysis of proteins extracted from the cells treated with 5% PL probed with Cyclin Dl , phospho Akt, phospho Erkl/2 and Actin antibodies shows that proliferation pathways are activated by PL also in the absence of serum, although in this case cell proliferation did not occur (see graph). This indicates that v-PL commit quiescent cells to proliferation, but serum factors are required for the cells to proceed in the cycle.

Fig. 8 v-PL can rejuvenate a culture of senescent MSC. The positive effect of v-PL on resting cells was also verified on a primary culture of BM-MSC. A culture of bone-marrow derived MSC previously expanded in the presence of 10% FCS for about 10 population doublings was splitted in the different culture conditions: 1) 1% v-PL; 2) 10% Pl-s; 3) 10% Pl-s+1% v-PL. The patient informed consent was obtained before processing of the sample. After 3 weeks, one part of the cells of the culture in 10% Pl-s+1% v-PL was transferred in medium supplemented with Pl-s without v- PL. After additional passages, at a time that proliferation was arrested (senescent cells), part of the Pl-s culture was transferred again in medium supplemented with 10% Pl-s+1% v-PL (restoring in this way the mitogenic stimulus of the v-PL). As shown by the graph, 1% v-PL cannot support cell proliferation, but the addition of v-PL to senescent cells, maintained in the presence of Pl-s as the only supplement, rejuvenate the cells that resume proliferation

Fig. 9 Growth rate of cells cultured with the new supplements. The combined effect of Pl-s and v-PL on cell growth was tested on a primary culture of AD-MSC and compared to the control condition where cells were grown with the standard supplement FCS. The patient informed consent was obtained before processing of the sample.

The proliferation rate was monitored by the evaluation of the cumulative population doublings performed by the two parallel cultures.

Fig. 10 Modulation of NF-κΒ pathway in HUVECs treated with PL (v-PL) under physiological and inflammatory conditions. Sub-confluent HUVECs were treated for lh or 16h with complete medium supplemented with 5% PL (v-PL), or 100 U/mL IL-la, or 5% PL (v-PL) +100 U/mL IL-la or un-supplemented (control, CTR). Whole-cell extracts were analysed by ELISA-based TransAMTM NF-κΒ p65 kit. (A) Absorbance values of NF-κΒ activity after lh and 16h stimulation related to a representative experiment. (B), (C) Cell NF-κΒ activity after lh and 16h stimulation, respectively, expressed as fold increase over control following IL-la treatment and percentage value following PL (v-PL)+IL-l a treatment with respect to IL-la induced net increase (100%, after the subtraction of control value). For each condition, the average of 3 different experiments (mean ± SD) assayed in triplicate on different single-donor primary cultures are reported. For lh stimulation, * and ** symbols refer to p=0.0413 and p=0.0018, respectively. For 16h stimulation, * and ** symbols refer to p=0.0292 and p=0.0062, respectively.

Fig. 11 Pro-inflammatory cytokine secretion by HUVECs following PL stimulation under both physiological and inflammatory conditions. Sub-confluent HUVECs were treated for lh or 24h with complete medium supplemented with 5% PL (v-PL), or 100 U/mL IL-la, or 5% PL (v- PL)+100 U/mL IL-la, or without any supplement (control, CTR). At the end of the different stimulations, the supplemented media were removed and replaced with serum-free medium. After 24h incubation, the conditioned media were collected. Westem blot analysis of conditioned media for the secretion of IL-8 (A) and IL-6 (B). The densitometric analysis of western blots was performed on 3 or 4 independent single donor primary cultures (means ± SD) for lh or 24h treatments, respectively. The * symbol represents significant differences with p<0.0494. Under densitometric analysis histograms, representative westem blots are shown.

Fig. 12 PGE₂ secretion by HUVECs following PL (v-PL) stimulation under both normal and inflammatory conditions. Sub-confluent HUVECs were treated for 24h with complete medium supplemented with 5% PL (v-PL), or 100 U/mL IL-la, or 5% PL (v-PL) +100 U/mL IL-la, or without any supplement (control, CTR). Media were removed and replaced with serum-free medium for 24h incubation. The conditioned media were collected and analysed by Prostaglandin E2 ELISA kit. For each condition, PGE₂ production is expressed as fold change with respect to CTR. The average values and relative standard deviation values of 4 determinations performed in duplicate on 3 different single-donor primary cultures are presented. The * symbol represents a significant difference with p=0.0191.

Fig. 13 In vitro cultures of hASCs isolated and expanded in HS (Pl-s) or FBS. a) Cell proliferation monitored at different times by cell counting. The average values ± SD of 3 independent experiments performed in duplicate on different single-donor primary cultures are reported. The *** symbol corresponds to p=0.0006. Statistical analysis was performed using the unpaired t-Test. b) Cell viability evaluated at different times by MTT assay. The average values ± SD of 3 independent experiments performed each in triplicate are reported, c) Morphology of cells in FBS (left photo) and in HS (Pl-s) (right photo) one day after the seeding (day 0). Scale bar = 50 μηι. d) The CFE assay performed on hASCs immediately after their isolation using complete or control medium. Scans of dishes with methylene blue-stained colonies deriving from a representative experiment, e) Number of colonies per condition counted by naked eye. The average values ± SD are calculated referring to 5 independent experiments performed each in duplicate, f) 'Colony area percentage' and 'colony intensity percentage' calculated by ImageJ-plugin "ColonyArea" and referred to both tested condition. The average values ± SD are calculated referring to 5 independent experiments performed each in duplicate.

Fig. 14 Spontaneous adipogenic differentiation of hASCs cultured in HS (Pl-s) with respect to FBS.Representative ORO (Oil Red O) staining of hASCs cultured with HS (Pl-s) (a) or FBS (b, as control condition) with time. Scale bar = 50 μηι. The results were confirmed performing 3 independent experiments with different single-donor primary hASC cultures, c) Quantification of ORO incorporated in HS (Pl-s) expanded hASCs, normalized per cell number, at different times by spectrophotometric analysis. The average values ± SD of 3 independent experiments performed each in triplicate are reported. Real Time quantitative PCR analysis of ppary2 (d) and c/ebpa (e) transcript levels in HS (Pl-s) or FBS expanded hASCs at different times. The final results are expressed as means ± SD values considering 3 independent experiments assayed in triplicate. The *, ** and *** symbols refer to p<0.0422, p=0.0065 and p=0.0009, respectively. Statistical analysis was performed using the unpaired t-Test.

Fig. 15 Adipogenic and osteogenic differentiation of HS (Pl-s)-committed hASCs at P2 by using traditional differentiation protocols, a) Representative experiment assayed with ORO staining of adipogenesis-treated (ADIPO-INDUCED) and control (CTR) cells at the end of induction. The images on the right are magnification of the sections with dashed outline. Scale bar = 100 μηι. The results were confirmed performing 3 independent experiments with different single- donor primary hASC cultures, b) Quantification of ORO incorporated in adipogenesis-treated (INDUCED) and control (CTR) cells at the end of induction by spectrophotometric analysis. The average values ± SD of 3 independent experiments performed each in triplicate are reported, c) Representative experiment assayed with ARS (Alizarin Red S) staining of osteogenesis-treated (OSTEO-INDUCED) and control (CTR) cells at the end of induction. The lateral images with dashed outline are magnification of the treated and control cell layers. Scale bar = 100 μπι. The results were confirmed performing 3 independent experiments.

Fig. 16 Comparison of Pl-s and plasma as medium supplements. Human primary bone marrow- derived mesenchymal stem cells (BM-MSC) and human cell lines growing either in adhesion (HeLa) or in suspension (U-937) were cultured either with Pl-s or plasma as medium supplements. Cell proliferation was determined by direct cell counting. Number (n) of cell cultures used for the experiments is indicated on the graph for each cell type.

Examples

Materials and methods

Manufacturing process of virgin-Platelet Lysate (v-PL) and Plasma-serum (Pl-s)

Blood donated for humanitarian reasons is the starting material. An informed consent to the blood processing is obtained from all donors. An outline of the manufacturing process is reported in Fig. 1. All separation steps were within a sterile closed system. A high speed centrifugation of whole blood units separates different phases: the plasma at the top, the buffy coat (BC) layer (enriched in platelets and leukocytes) at the interface and the red blood cells fraction at the bottom. For the preparation of the virgin-Platelet Lysate (v-PL), from discarded BC units (for transfusional purposes) it was derived a BC pool (up to 300 units, preferably from 5 to 10 donors, more preferably at least 3 donors for HS (or Pl-s), or 5-10 donors or 25 donors for v-PL) which was centrifuged at low speed to obtain the Platelet Rich Plasma (PRP) at the top of the blood bag. PRP was high speed centrifuged to obtain a separation between an upper phase, the Platelet Poor Plasma (PPP) and a lower phase, the platelet concentrate. Recovered platelets were subjected to three washes in sterile saline solution, preferably a physiological saline as e.g. 0.9%w/v NaCl. After the third wash, the platelet concentrate was resuspended in saline solution and the platelet concentration adjusted to lOxlO⁶ plt/ul. The platelet concentrate underwent three freeze-thaw cycles. A high speed centrifugation was then performed to sediment platelet membranes and debris. The obtained supernatant, the plasma-free Platelet Lysate (v-PL) was divided into aliquots and preferably lyophilized.The Plasma-serum (Pl-s) was obtained from several frozen plasma units. Each plasma unit was slowly thawn at 4°C to obtain a separation between the cryoprecipitate and the cryo-poor plasma (CPP). The CPP was then subjected to a coagulation step at 37°C up to 6 hours after the addition of calcium choride (or calcium gluconate), preferably 2 mg/ml. Following the coagulation step, the blood bag was high speed centrifuged to separate the coagulum. The liquid phase, the Plasma-serum (Pl-s), was recovered, and the pool of several units was divided into aliquots and lyophilized.

Growth factor quantification by enzyme-linked immunosorbent assay (ELISA)

Elisa assay was performed for the quantification in the Pl-S and the v-PL of two platelet-derived growth factors, PDGF-BB and VEGF as representative indicators of a high and low concentrated platelet factor respectively. The assays were performed according to the manufacturer directives. Cell cultures (human Mesenchymal Stem Cells and human cell lines)

Primary cultures of Mesenchymal Stem Cells of different origin were established from human biopsies of discarded surgical samples of healthy donors. The patient/donor-informed consent was obtained prior sample processing. Human Bone -marrow derived Mesenchymal Stem Cells (BM- MSC) were obtained from bone-marrow and Adipose derived Mesenchymal Stem Cells (AD-MSC) were derived from liposuction waste according to published procedures (Muraglia A et al. Cytotherapy 2015; 17: 1793-1806). Umbilical Cord derived MSC (UC-MSC) were kindly provided by Dr. Introna M. (AO Papa Giovanni XXIII USS Center of Cell Therapy 'G. Lanzani' USC Hematology, Bergamo, Italy). Cells were isolated from cord blood tissue collected from pregnant women after either normal vaginal delivery or Cesarean sections. The UC processing was performed in accordance with the protocol for the isolation and expansion of UC-MSC as previously described (Capelli C. et al. Cytotherapy 2011; 13: 786-801).

Cell lines U-937, Hela and V-79 were purchased from the Interlab Cell Line Collection of the Biological Bank and Cell Factory of the IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro (Genoa, Italy). HaCat cell line was kindly provided by Dr. Pellegrini G. (Centre for Regenerative Medicine "Stefano Ferrari" Modena).

Primary and cell line cultures were grown in specific culture medium containing 100 IU/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine and, where indicated supplemented with PL-s and v-PL used alone or in different ratio combination. In some control experiments, medium was supplemented with 10% Fetal Calf Serum (FCS) instead of PL-s and v-PL. Cells were detached from the culture dish with 0.05% trypsin and 0.01% EDTA. Trypsin activity was neutralized with a trypsin soybean inhibitor solution (0.5 mg/ml in PBS).

Cell proliferation assay - cumulative doubling number calculation

For determining the doublings of BM-MSC, AD-MSC and UC-MSC in a long term culture, after the initial selection of the cells (passage 0) in FCS supplemented medium, at 70-80% confluence, cells were detached with trypsin/EDTA solution and replated at the density of 7 x 10⁴ cells for UC-MSC and 1 x 10⁵ cells for all the other cell types, in 60 mm 0 Petri dishes, in duplicate with medium supplemented with 10%Pl-s, 10%Pl-s+5% v-PL, 10%Pl-s+l%v-PL. Doublings were calculated by counting the number of cells at each passage until the end of the culture.

The human cell lines U-937 (pro-monocytic, suspension growth), HeLa (epithelial, adhesion growth), Hacat (keratynocytes, adhesion growth) and animal cell line V79 (hamster lung fibroblasts, adhesion growth) were cultured with the same supplements used for the primary cultures and their proliferative behaviour was monitored by counting the number of doubling number performed during culture time. At each passage, cells were plated at the following densities: U-937, 200,000 cells/ml in T25 flask; HeLa, 250,000 cells/plate 60 mm 0; HaCat, 250,000 cells/plate 60 mm 0; V79, 400,000 cells/plate 60 mm 0;

Human articular chondrocyte cultures

Femoral heads or femoral condyles were collected from patients undergoing hip or knee arthroplasty respectively, with their informed consent and the approval of the institutional Ethics Committee of the IRCCS AOU San Martino-IST National Cancer Research Institute (Genoa, Italy). The patients' age ranged from 61 to 81 year-old. All obtained samples were processed immediately after the surgery. After washing with sterile PBS, articular cartilage was isolated by using sterile blades, minced into small pieces before repeated cycles of digestion at 37 C in serum free F12 medium containing 0.25% (v/v) trypsin, 1 mg/ml hyaluronidase type II, 400 U/ml collagenase type I and 1,000 U/ml collagenase type II. Recovered chondrocytes were expanded as adherent cells (dedifferentiated chondrocytes) up to 6 passages in complete medium (Coon's F-12 medium supplemented with 2 mM L-glutamine, 1% penicillin / streptomycin, and 10% FCS) or complete medium supplemented with 5% v-PL. Culture media were changed twice a week. Cultures were performed at 37 °C and 5% C0₂.

Cell proliferation assay for articular chondrocytes - Crystal violet staining

After extensive washing with PBS, cells seeded in 96 multiwell plates were stained with 50 μΐ colorant solution [0.75% (g/ml) crystal violet (C3886, Sigma-Aldrich), 0.35% (g/ml) NaCl, 32.3%

(v/v) absolute ethanol, 8.64% (v/v) formaldehyde 37%] for 20 minutes at room temperature. Cells were then washed 5 times with water and dried by exposing the plate to air under a chemical hood.

To each well 100 μΐ eluent solution [50% (v/v) absolute ethanol and 1 % (v/v) acetic acid] were added and the absorbance at 595 nm measured within 10-30 minutes with a spectrophotometer AD 200 (Beckman Coulter, USA). Results were expressed as the average of at least three independent experiments.

Western blot analysis

Confluent cells on culture dishes or the cell aggregates in suspension in 15 ml falcon tubes in complete medium were washed with sterile PBS, maintained in serum free medium for 1 hour and treated with complete medium supplemented with 5% PL for different times. For HIF-1 alpha detection, after PL treatment, the cells were washed twice with cold PBS, then the dishes/falcon tubes, respectively, were put at -80°C for 10 minutes, then monolayer cells were scraped in cold Ripa buffer containing 50 mM Tris (pH7.5), 150 mM NaCl, 1% Deoxycholic acid, 1% Triton X- 100, 0.1% SDS, 0.2% NaN₃ and proteinase inhibitor cocktail (1 : 10, P2714, Sigma Aldrich) while for cells in suspension Ripa buffer was added directly to falcon tubes. Cells in RIPA buffer were transferred into cold 1.5 ml Eppendorf tubes and lyzed for 30 minutes on ice, centrifuged 10 minutes at 10,000 rpm, 4°C and the supernatant containing protein extract was collected and kept at -20° C. Protein concentration was quantified by Bradford assay [32]. 30-80 μg of proteins of each sample were loaded on the gel. Western blot analysis was performed according to Ulivi et al. [16].

Antibodies were a-Cyclin Dl (1 :250) and a- Actin (1 :200) from Santa Cruz Biotechnology), a- phospho Erkl/2 (1 : 1,000), a-Erkl/2 (1 : 1,000), a-phospho Akt (1 :1,000), a-Akt (1 :1 ,000), a-Histon H3 (1 :1 ,000) from Cell Signaling Technology), a-HIF-la (1 :500) from BD Bioscience. HRP conjugated anti mouse and anti rabbit antibodies (both 1 :5,000) from GE Healthcare, UK, peroxidase conjugated anti goat antibody (1 :10,000) from Jackson Immunoresearch (USA). ECL was purchased from GE Healthcare, UK.

Immunohistochemistry

Cells cultured on monolayer were fixed with 3.7% paraformaldehyde (PFA, Sigma) for 20 minutes at room temperature (RT) and analyzed by immunohistochemistry. Cell aggregates were washed 3 times with PBS, and either fixed with 3.7% PFA for 20 minutes at 4°C and embedded in paraffin [9] or fixed with 3.7% PFA for 3-4 hours on a lab shaker at 4° C and frozen. Both paraffin embedded and frozen samples were sectioned in slices of 5-6 μπι thickness. Slices were adhered on Superfrost Ultra Plus Slides (Thermo Scientific, Germany) coated with poly-L-lysine (Sigma Aldrich). Paraffin embedded slices were dewaxed to remove paraffin and processed for immunohistochemistry. Frozen section were pre -warmed for 1 hour at 37°C, immersed in boiling citrate buffer (pH 6) for antigen retrieval and cooled for 20 minutes at RT. Both types of sections were permeabilized with 0.2% Triton in PBS for 10 minutes, treated with 4% H₂0₂ for 30 minutes at RT to inhibit endogenous peroxidase activity, rinsed with PBS 3 times x 5 minutes, incubated with hyaluronidase type II (Sigma Aldrich) at concentration of 1 mg/ml in PBS (pH 6) for 30 minutes and washed with PBS. After incubation with 10% normal goat serum ( GS) in PBS for 1 hour at RT to inhibit nonspecific binding, the sections were incubated at 4° C for 16 hours with primary antibodies, washed 3 times with PBS and incubated for 1 hour at RT with Labeled polymer HRP anti-mouse (for a-collagen type I, a-collagen type II, a-collagen type II, a-collagen type X and a-Cyclin Dl) or Labeled polymer HRP anti-rabbit (for a-PCNA). Both Labeled polymer HRP anti-mouse and anti- rabbit were from Dako (Denmark). Then, sections were stained with 3 ,3'-Diaminobenzidine (DAB, Enzo Life Sciences, USA) for 3-5 minutes, counter-stained with Mayer's hematoxylin for 2 seconds, submersed in 0.1% NaHC0₃ for 1 minute, and finally mounted with Eukitt (O. Kindler GmbH, Germany). Images were acquired by a microscope Axiovert 200M (Carl Zeiss). Primary antibodies were: α-collagen type I (1 :500; SPl .D8-Developmental Studies Hybridoma Bank, University of Iowa, USA), α-collagen type II (1 :100; CIIC1 -Developmental Studies Hybridoma Bank, University of Iowa), α-collagen type X (1 :1 ,000; ab49945, Abeam, UK), a-aggrecan (1 :100; ab3778, Abeam), and a-PCNA (1 :200; PAB11790, Abnova, Taiwan), a-Cyclin Dl (1 :50; sc-8396, Santa Cruz Biotechnology). Primary antibodies were diluted in 5% NGS in PBS.

Statistic analysis All data are presented as means and standard error of the mean (SEM). Statistical analysis was performed according to unpaired t-test using online application of the Graphpad software (www.graphpad.com).

Materials

Medium 199 with Earle's Salts, foetal bovine serum (FBS), L-glutamine, penicillin G streptomycin sulphate and trypsin-EDTA were all obtained from Euroclone Life Sciences Division (Milan, Italy). 10 cm Petri dish, 96-well plate, 24-well plate were derived from Eppendorf S.r.l. (Milan, Italy). Recombinant human FGF-acidic (FGF-acidic), recombinant human FGF-basic (FGF -basic), animal- free recombinant human EGF and recombinant human interleukin-1 (IL-1 ) were purchased from Peprotech (London, UK). PHAREPA 25000 U.I./5 mL heparin sodium-salt was obtained from PharmaTex Italia (Milan, Italy). Hydrocortisone -water soluble, Bright-LineTM hemacytometer and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). Corning® Matrigel® Growth Factor Reduced Basement Membrane Matrix was acquired from Corning (Bedford, MA, USA). TransAMTM NF- κ B p65 kit was purchased from Active Motif (La Hulpe, Belgium). Prostaglandin E2 ELISA kit was from Cayman Chemical (Ann Arbor, MI, USA). NuPAGETM 4-12% Bis-Tris gels were from Invitrogen (Milano, Italy). AmershamTM ProtranTM 0.45 μ m NC, AmershamTM ECLTM western blotting detection reagents and AmershamTM hyperfilmTM ECL were obtained from GE Healthcare (Buckinghamshire, UK). Antibodies anti- interleukin-8 (IL-8), anti-interleukin-6 (IL-6), anti-cyclin Dl and anti-actin were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Antibodies anti-phospho-Akt, anti-Akt, anti- phospho-ERKi/2, anti-ERKi/2 and anti-phospho-STAT3 were acquired from Cell Signalling Technology (Danvers, MA, USA). Antibody anti-HIF-1 was obtained from BD Biosciences (San Jose, CA, USA).

HUVEC harvest and culture

Primary Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from "Centro di Risorse Biologiche" (CRB) of IRCCS Ospedale Policlinico San Martino (Genova, Italy) following the approval of this study by the institutional ethics committee. The CRB obtained the written informed consent by every umbilical cord donor. The HUVECs were CD31 - and CD106-positive (endothelial cell-specific markers) and CD90- and CD45-negative (fibroblast-specific and leukocyte-specific markers, respectively) as guaranteed by CRB. The cells were seeded at the density of 6.0xl0³ cells/cm² into gelatine-coated 10 cm Petri dishes and cultured in Medium 199 with Earle's Salts supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, 100 mg/L heparin, 10 μg/L FGF-acidic, 10 μg/L FGF -basic, 10 μg/L EGF, 1 mg/L hydrocortisone (complete culture medium). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Medium was changed 3 times per week and at 80% confluence cells were split 1 :2 by trypsinization with trypsin-EDTA. For the described experiments, HUVECs at passages from 3 to 6 were used. PL (v-PL) was supplemented to complete culture medium at a final concentration of 5% (v/v), approximately corresponding to the highest physiological concentration of platelets in the human blood, without adding heparin.

Proliferation assays

1) Crystal violet assay: HUVECs were seeded at the density of 2.5xl0³ cells/well on gelatine coated 96-well plate and incubated in complete culture medium for 24h to enable cell adhesion. The next day, medium was replaced with complete medium not supplemented (control cells) or supplemented with 5% PL (v-PL) (treated cells). The assay was performed in quintuplicate for each culture condition after 0, 2, 4 and 6 days of PL stimulation. At each time point, the culture media were removed and replaced with 50 μ L/well of 0.75% (w/v) crystal violet staining solution. After 20 min incubation, the staining solution was discarded and wells were extensively washed. When the plate was dry, 100 μ L/well of elution solution was added and dye absorbance was measured at 595 nm. The average of three independent experiments performed on different single-donor primary HUVEC cultures and the fold increase of the signal revealed after 6 days of PL (v-PL) treatment over control (mean ± SD) is shown.

2) Cell count: HUVECs were seeded on gelatine-coated 24-well plate and cultured in complete culture medium until reaching confluence. The medium was then replaced with complete medium supplemented (treated culture) or not supplemented (control culture) with 5% PL (v-PL). At 0, 3, 6, 10 days of PL (v-PL) stimulation, cell density was monitored in triplicate by cell counting using a Bright-Line™ Hemacytometer with an improved Neubauer chamber. Values are expressed as fold increase with respect to the value obtained at the time of the PL addition (first considered time point - day 0). Results are expressed as the average of three independent experiments performed on different single-donor primary HUVEC cultures (mean ± SD). Cell density ratio between PL-treated and control cells at 10th day of PL (v-PL) treatment is also reported. Ratio was separately calculated in 3 independent experiments and expressed as mean ± SD.

Tube formation assay Proliferating HUVECs were cultured in complete culture medium un-supplemented (control) or supplemented with 5% PL (v-PL) for a week. The cells were then trypsinized, re-suspended in serum-free medium (no supplements) and seeded at the density of 70,000 cells/well on matrigel- coated 24-well plate. Images were taken after 6h incubation at 37°C in a humidified atmosphere with 5% C02.

NF-KB activity assay

To evaluate the nuclear factor-κΒ ( F-κΒ) activity, TransAMTM NF-κΒ p65 kit was used. This ELISA-based kit is based on the binding of NF-κΒ active form to an oligonucleotide, containing NF-KB consensus decameric sequence 5'-GGGACTTTCC-3', immobilized on a 96-well plate. Subconfluent HUVECs were treated for lh or 16h with complete culture medium supplemented with 5% PL (v-PL), 100 U/mL IL-la, or 5% PL (v-PL) + 100 U/mL IL-la, or without any supplement (control). Media were removed and cells washed with PBS. Whole-cell extracts were prepared following manufacturer's instructions. The NF-κΒ active form contained in the whole-cell extracts specifically binds to the consensus sequence and the primary antibody recognizes an epitope on subunit p65, which is accessible only when NF-κΒ is activated and bound to its target site. An HRP-conjugated secondary antibody provides a colorimetric signal quantified by spectrophotometry. Specificity of the assay was checked by adding soluble wild-type and mutated consensus oligonucleotides acting as competitors for NF-κΒ binding. For the reported representative experiment, results are expressed as the absorbance values measured in the presence of the mutated oligonucleotide minus those measured in the presence of the wild-type oligonucleotide. This assay was performed in triplicate on 3 independent experiments corresponding to different single-donor primary HUVEC cultures. For each stimulation time, the over the control fold increase of NF-KB activity induced by IL-la stimulation and the percentage value of NF-κΒ activity induced by PL (v- PL)+IL-la treatment with respect to IL-la net increase are reported (means ± SD).

PGE₂ quantification

To quantify the PGE₂ production, Prostaglandin E2 ELISA kit was used. This assay is based on the competition between PGE₂ present in the sample and a PGE₂ tracer for a limited amount of anti- PGE₂ monoclonal antibody. Sub-confluent HUVECs were treated for 24h with complete culture medium supplemented with 5% PL (v-PL), 100 U/mL IL-la, or 5% PL (v-PL) + 100 U/mL IL-la, or without any supplement (control). Cells were then extensively washed with PBS for removing residual factors and then incubated in serum- free medium for 24h. The different conditioned media were collected and assayed following manufacturer's instructions. Results are expressed as fold change referring to control. This assay was performed in duplicate on 3 independent experiments corresponding to different single-donor primary HUVEC cultures.

Statistical analysis

All data are presented as means and standard deviations based on independent experiments performed on three different primary HUVEC cultures, each of them derived from a single donor. The statistical analysis was performed using the unpaired t-Test for NF-κΒ activity and proliferation assays or using the ordinary one-way ANOVA for IL-8, IL-6, Cyclin Dl, HIF-Ια and phospho- STAT-3 densitometric analysis and PGE2 quantification. If ANOVA detected statistically significant differences within the data set, Tukey's or Dunnett's multiple comparison tests were used to calculate the significant differences for IL-8 and IL-6 densitometric analysis and PGE2 quantification or for Cyclin Dl, HIF-Ια and phospho-STAT-3 densitometric analysis, respectively. All tests were run setting a confidence interval of 95% (p<0,05).

Materials

Type I collagenase was purchased from Worthington (Lakewood, NJ, USA). The MEM Alpha Medium (IX) GlutaMAX™ without nucleosides (aMEM) was from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Foetal bovine serum (FBS), penicillin G-streptomycin sulphate solution and normal goat serum were from Euroclone Life Sciences Division (Milan, Italy). Dimethyl sulphoxide (DMSO) was obtained from Panreac (Barcelona, Spain). Human interleukin- 1 alpha (IL-la) was from PeproTech (London, UK). Bright-Line™ Hemacytometer with an improved Neubauer chamber, crystal violet powder, thiazolyl blue tetrazolium bromide (MTT), insulin from bovine pancreas, dexamethasone, L(+)-ascorbic acid sodium salt, β- glycerophosphatedisodium salt hydrate, Oil Red O (ORO), Alizarin Red S (ARS), inhibitor cocktail and poly-L-lysine solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formaldehyde 37% weight solution was from Chem-Lab (Zedelgem, Belgium). Methylene blue hydrate powder was purchased from Honeywell Riedel-de Haen® (Seelze, Germany). The RNeasy® Plus Micro Kit and Omniscript® RT Kit were from Qiagen (Milan, Italy). Power SYBR® Green PCR Master Mix and 7500 Fast Real-Time PCR System were from Applied Biosystems® Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). The NuPAGETM4-12% Bis- Tris gels and streptavidin-peroxidase of HistoMouseTM-SP kit were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). AmershamTM ProtranTM 0.45 μηι NC, secondaryanti- rabbit and anti-mouse horseradish peroxidase-linked immunoglobulins G (IgGs), AmershamTM ECLTM western blotting detection reagents and AmershamTMHyperfilmTM ECL were obtained from GE Healthcare (Buckinghamshire, UK). Primary antibodies raised against interleukin-8 (IL-8), interleukin-6 (IL-6), Cyclin Dl and Actin were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Primary antibodies raised against phospho-Akt, Akt, phospho-ERKl/2, ERK1/2 and phospho-STAT3 were acquired from Cell Signalling Technology (Danvers, MA, USA). Primary antibodies raised against cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase- 1 (mPGES-1) and Prostaglandin E2 ELISA kit were from Cayman Chemical (Ann Arbor, MI, USA). Secondary anti-goat peroxidase-conjugated immunoglobulins G (IgGs) were from Jackson ImmunoResearch (Bar Harbor, MA, USA). Primary antibody raised against PCNA was from Abnova (Taipei City, Taiwan). Secondary biotinylated immunoglobulins G (IgGs) and 3,3'- diaminobenzidine (DAB) were from Dako (Carpinteria, CA, USA).

Primary hASC cultures

Primary human adipose-derived stromal cell (hASC) cultures were obtained from subcutaneous adipose tissue liposuction wastes derived from healthy female donors. All donors provided the written informed consent. Protocol and procedures were approved by the Local Ethical Committee. The hASCs were isolated following the protocol reported by Estes, Diekman, Gimble, & Guilak,

2010, with some modifications. Briefly, liposuction aspirate was extensively washed with phosphate-buffered saline (PBS, composed by 136.9 mM NaCl, 2.7 mM KC1, 8.0 mM Na2HP04, 1.5 mM KH2P04, with pH 7.4) (1 :1 volume). After that, the fat sample was digested with 0.1% type I collagenase in PBS (1 :1 volume) at 37°C for lh shaking the mix by hand every 5-10 minutes. The digested sample was centrifuged at 290g for 5 minutes at room temperature and, at the end of centrifugation, shacked vigorously to ensure all the stromal cells could be properly released and separated from the remaining tissue. After a further centrifugation, the upper mature adipocyte and middle collagenase/PBS layers were aspirated off leaving the pellet, which were composed by the stromal vascular fraction (SVF). The pellet was re-suspended in aMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate for neutralizing the residual collagenase. The SVF was centrifuged, re-suspended in PBS and splitted in two half. After a final centrifugation, one half was re-suspended with a volume of aMEM supplemented with 10% (v/v) HS (Pl-s), 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate (complete medium) corresponding to half of initial volume of lipoaspirate, while the other half with an identical volume of aMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate (control medium). The SVF cells were plated at an equivalent to about 0,04 mL of liposuction aspirate per cm² for every condition. Adhering hASC cultures were incubated at 37°C in a humidified atmosphere with 5% CO2 and media were changed 3 times per week. At 80% confluence, cells were harvested by trypsinization and frozen in liquid nitrogen using aMEM supplemented with 50% (v/v) HS (Pl-s) or FBS, 10% (v/v) DMSO, 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate for long-term storage (passage 0, P0). After to be thawed, cells were expanded for one passage (PI) and used at passage 2 (P2) for all the described experiments, except for CFE assay and differentiation assays of PL-expanded cells. In particular, for differentiation assays of PL (v-PL)-expanded cells, HS (Pl-s)-isolated hASCs were expanded for one passage with complete medium and for further 3 passages with complete medium supplemented with 5% PL (v- PL) and used at passage 5 (P5).

Proliferation assays

I. Cell counting: HS (Pl-s)- and FBS-isolated hASCs at P2 were plated at 4157 cells/cm2/well in 6- well plates using the complete or control culture medium according to their isolation condition. They were incubated for 24h to enable cell adhesion to wells and, the next day (day 0), medium was replaced with complete medium, un-supplemented or supplemented with 5% PL (v-PL), or control medium. For each culture condition, the cell number was measured in two different wells, after 0, 2, 4, 6 and 8 days of stimulation, using a Bright-Line™ Hemacytometer with an improved Neubauer chamber. The final result was expressed as the average of 3 independent experiments performed on different single-donor primary hASC cultures ± SD value.

II. Crystal violet staining assay: HS (Pl-s)-isolated hASCs were plated at 2.5x103 cells/well in 96- well plate and incubated in complete medium for 24h. The next day (day 0), medium was replaced with complete medium un-supplemented or supplemented with 5% PL (v-PL), 100 U/mL IL-la, or 5% PL (v-PL) + 100 U/mL IL-la. Crystal violet staining assay was performed in quintuplicate for each culture condition after 0, 1 , 2, 3 and 4 days of the different stimulations. At every time point, the culture media were removed and replaced with 50 μΕΛνεΙΙ of 0.75% (w/v) crystal violet staining solution (prepared using 0.375 g crystal violet powder, 0.175 g NaCl, 16.15 mL absolute ethanol and 4.32 mL formaldehyde 37% weight solution and bringing to 50 mL with distilled water). After 20-minute incubation, the staining solution was discarded and wells were extensively washed with distilled water. When the plates were dry, 100 μίΛνεΙΙ of elution solution (composed by 50% v/v absolute ethanol and 1% v/v acetic acid in PBS) was added. For each well, the optical density (OD)was measured at 595 nm. As final result, it was reported the n-fold increase of OD respect to day 0, for each culture condition, expressed as the average of 3 independent experiments performed on different single-donor primary hASC cultures ± SD value. In toto hAT cultures

In toto human adipose tissue (hAT) samples were obtained from resection material of abdominoplasty on healthy female donors. All donors provided the written informed consent. The inner portion of subcutaneous fat was minced into pieces with the diameter of 3-4 mm, paying attention to avoid visible blood vessels. Such pieces were cultured in complete culture medium supplemented with 5% PL (v-PL), 100 U/mL IL-la, or 5% PL (v-PL) + 100 U/mL IL-la, or without any supplement, for 14 days. In toto hAT cultures were incubated at 37°C in a humidified atmosphere with 5% C02 and media were changed 3 times per week.

Adipogenic and osteogenic differentiation

To induce adipogenic and osteogenic differentiation, HS-isolated hASCs at P2 or PL (v-PL)- expanded hASCs at P5 were plated at 8314 cells/cm²/well in 24-well plates and were expanded for at least 4 days in complete medium. After this expansion step, one half of sub-confluent cell wells was treated with specific induction media for adipogenic and osteogenic differentiation for 14 days and the other half was cultured with complete medium as control. Adipogenic differentiation medium was composed by complete medium supplemented with 6 ng/mL insulin and 10^"7 M dexamethasone (Scott, Nguyen, Levi, & James, 2011). Osteogenic differentiation medium was composed by complete medium supplemented with 50 μg/mL ascorbic acid, 10 mM β- glycerophosphate and 10^"7M dexamethasone (Muraglia et al., 2017). The media were changed 3 times per week. At day 14, the presence of intracellular lipid droplets was detected in all the cell cultures induced to adipogenesis and in half of those induced to osteogenesis, with their relative control cultures, by Oil Red O (ORO) staining. Cell layers were fixed with a formol-calcium solution (comprised of 10 mL formaldehyde 37% weight solution, 1 g calcium chloride, 90 mL distilled water) for 10 min at room temperature. They were extensively washed with distilled water and stained with 0,5% (w/v) ORO in propan-2-ol, diluted 3:2 in distilled water, for 20 min at room temperature. The dye excess was washed away with distilled water and photos of stained cell layers were taken by Hamamatsu Color Chilled 3CCD Camera C5810. For adipogenic induction experiment, incorporated ORO was extracted by incubating stained cell layers in 100% propan-2-ol for 10 min at room temperature with mild agitation. For control and induction conditions, the optical density (OD) was measured at 530 nm in triplicate and expressed as the average of 3 independent experiments performed on different single-donor primary hASC cultures ± SD value. Analogously, the amount of incorporated ORO was also evaluated in hASCs plated at P2 at 4157 cells/cm2/well in 24-well plates and cultured in complete medium, un-supplemented or supplemented with 5% PL, or in control medium for 0, 4 and 8 days, without any induction. The final result is expressed as means ± SD values considering at least 3 independent experiments assayed in triplicate on different single-donor primary cultures. At the end of osteogenic induction, the matrix mineralization in the induced cultures was valuated by Alizarin Red S (ARS) staining. Cell layers were fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at room temperature. They were washed with distilled water and stained with 2% (w/v) ARS in distilled water at pH 4.2 for 10 min at room temperature. After a washing step with absolute ethanol and others with distilled water, the stained wells were allowed to air dry and finally scanned by Epson Perfection V330 Photo Scanner. The osteogenic differentiation was assayed in duplicate on 3 different single-donor primary cultures.

PGE2 quantification

For the quantification of PGE2 in conditioned media, Prostaglandin E2 ELISA kit was used. This assay is based on the competition between PGE2 present in the sample and a PGE2- acetylcholinesterase (AChE) conjugate (PGE2-tracer) for a limited amount of anti-PGE2 monoclonal antibody. By adding the substrate of AChE, a colorimetric reaction occurs and therelative absorbance is measured spectrophotometrically at 405 nm. The PGE2 concentration relative to the sample is calculated using a standard curve. The hASC 24h,72h-conditioned media were prepared in the same way reported for western blot analysis and assayed following manufacturer's instructions. For each culture condition, the PGE2 concentration is reported as average value of 3 independent experiments performed in duplicate on different single-donor primary cultures ± SD.

Statistical analysis

All data are presented as means and standard deviations based on independent experiments performed on at least three different primary hASC cultures, each of them derived from a single donor. The statistical analysis was performed using the unpaired t-Test for cell counting assay, real time quantitative PCR analysis, CFE assay and ORO quantification or using the ordinary one-way ANOVA for IL-8, IL-6 and Cyclin Dl densitometric analysis, PGE2 quantification and crystal violet assay. If ANOVA detected statistically significant differences within the data set, Tukey's multiple comparison test was used to calculate the significant differences for IL-8 and IL-6 densitometric analyses, PGE2 quantification and crystal violet assay. Dunnett's multiple comparison test was used for Cyclin Dl densitometric analysis. All tests were run setting a confidence interval of 95% (p<0,05).

RESULTS Production of virgin-Platelet Lysate (v-PL) and Plasma-serum (Pl-s)

v-PL and Pl-s were produced according to the protocol described in materials and methods and outlined in Fig. 1. Concentrations of PDGF-BB and VEGF determined by ELISA in the two preparations are reported in Table of Fig. 2. Freeze-dried aliquots were stored at -20° C for subsequent use as cell culture supplements.

In cartilage permissive culture conditions, PL (v-PL) stimulates proliferation of differentiated chondrocytes within a cartilage specific matrix.

In an early study from inventors' laboratory, they demonstrated that dedifferentiated growth plate cartilage cells, transferred in suspension culture in agarose coated dishes in the presence of ascorbic acid, re-differentiated and formed a tissue with morphological and structural properties of cartilage [Zerega B et al. J Bone Miner Res. 1999 Aug; 14(8): 1281-9]. Similarly, to obtain cell aggregates organized as cartilage-like tissue, human articular cartilage cells expanded in adhesion were transferred to suspension at a concentration of lxl0⁵/ml in different Falcon tubes containing 5 ml medium in the presence of ascorbic acid for 10 days. Cultures were split and half of the cell aggregates were maintained in the same culture medium, whereas the medium of the other half was supplemented with 5% PL. Cell proliferation was determined by MTT in both control and PL- supplemented aggregates after additional 4 and 7 days culture. Control aggregates did not show any cell proliferation, whereas a cell proliferation was observed in PL-supplemented aggregates (Fig. 3 top right panel). In agreement with this finding, in the PL supplemented aggregates, a maximum of AKT and ERK1,2 activation, monitored by western blot, was observed already after 1 hour from the addition of PL, whereas a peak of cyclin Dl expression was reached after 8 hours (Fig. 3 top left panel). The occurrence of a cell proliferation in the PL-supplemented aggregates was confirmed by immunohistochemistry by looking at the expression of the proliferating cell nuclear antigen PCNA. In the PL-supplemented aggregates, but not in the control aggregates, PCNA was detected not only in peripheral elongated cells (Fig. 3 bottom panels) but also in chondrocytes within the aggregate. In the same aggregates, the presence of type I, type II and type X collagens and aggrecan was investigated in serial sections. In control and PL-supplemented aggregates the presence of a large amount of type II collagen and aggrecan was observed, while some type I collagen was detected only at the periphery of the PL supplemented aggregates (not shown). No type X collagen was observed in all aggregates.

PL (v-PL) induces cell proliferation and the release of chondroprogenitors in cultured cartilage explants Since inventors' final aim was to establish biological basis for a possible therapeutic application of PL on damaged cartilage, inventors performed static cultures of human articular cartilage small fragments in the absence (CTR) or in the presence of PL. Culture conditions allowed the adherence of the cartilage chips to the surface of plastic dishes. After 3 weeks of culture, the tissue chips were collected, frozen embedded and analyzed by immunohistochemistry with a- PCNA antibodies (Fig. 4 top left panels). In contrast to the cartilage fragments cultured in the absence of PL, in the cartilage fragments cultured in the presence of PL, a PCNA positivity was observed in many elongated cells at the border of the sample. In the same sample PCNA positivity was observed also in some cells within the cartilage matrix. Collagen staining showed an increase of the type II collagen expression compared to controls (not shown). It was reported that human articular cartilage, in spite of being cultured with chemo tactic stimuli, has limited cell outgrowth potential [Zingler C et al. Osteoarthr Cartil. 2016; 24(1):124-128]. Inventors performed static cultures of human articular cartilage small fragments, as above, in condition allowing their adherence to the surface of the Petri dish, either in complete medium or in complete medium supplemented with 5% PL (12 wells per condition; 2 fragments each well). Cartilage chips and outgrown cells were observed with a bright field microscope (Fig. 4 top right panels). Few cells were released from the control chips, whereas a large number of cells with an elongated morphology migrated out of the PL treated chips. After 2 months 100% of the PL treated chips had released a large number of cells whereas only 50% of the control chips had released few cells (Fig. 4 graph of bottom panel). The PL treated cartilage chips were removed and the cells remained adherent to the dish were trypsin treated and further expanded in culture as adherent cells without the need of PL supplement in the culture medium, whereas cells released in the absence of PL were few and unable to proliferate (not shown). Cells obtained from the PL treated chips and expanded in the absence of PL expressed type I collagen. However, they maintained the memory of the original phenotype and, when transferred to a suspension culture reverted to the synthesis of type II collagen, aggrecan and no type X collagen resulting in aggregates resembling real articular cartilage.

v-PL effect on cell proliferation: primary cultures vs cell lines

Primary cultures obtained from adult derived tissues, AD-MSC (from adipose) and BM-MSC (from bone -marrow) or fetal tissue derived cells, UC-MSC (from umbilical cord) were initially selected and cultured in 10% FCS and at pO (passage 0) cultured in the medium supplemented with Pl-S and v-PL in different relative ratios (10% Pl-s vs 10% Pl-s+1 % v-Pl or 10% Pl-s+5% v-PL) for about 30 days. Cell proliferation was monitored by evaluating the cumulative population doubling number at different times of the relative culture. In general, cells cultured with Pl-s alone showed a low or no proliferation rate in comparison to cells grown with the combination of Pl-s and v-PL In particular BM-MSC essentially did not proliferate in the presence of Pl-s alone (only about 2 cell doublings in 30 months) while underwent 10.5 and 17 cell doublings in 10% Pl-s+1 % v-PL and 10% Pl-s+5% v- PL culture medium respectively. AD-MSC and UC-MSC performed about 10 doublings in 10% Pl- s and about 25 and 20 doublings in the presence of Pl-s and v-PL respectively. For both AD-MSC and UC-MSC no major differences were observed between the condition 10% Pl-s+1% v-PL and 10% Pl-s+5% v-PL.

The blood-derived supplements used in the same combinations of the primary cultures, were also tested with human, U937, HeLa, HaCat and animal V-79 cell lines. Also in this case the cell proliferation rate was monitored by calculating the cell doubling number at different times in culture. v-PL was not a requirement to support cell proliferation. Indeed, Pl-s alone was able to support cell growth as well as the combination of Pl-s and v-PL in different percentages both for suspension (U-937) and adhesion (HeLa and HaCat) human cell growth. Moreover, the human additives were also able to sustain the survival and proliferation of animal derived cells (V-79, adhesion cell growth).

v-PL promotes re-entry in the cell cycle of resting cells

Confluent growth-arrested dedifferentiated human articular chondrocytes were maintained in the original culture medium supplemented with 10% FCS or additionally supplemented with 5% v-PL. A crystal violet proliferation assay was performed in parallel on both cultures (Fig. 4 upper panel). Confluent cells treated with v-PL resumed proliferation, whereas the control culture maintained in FCS only remained quiescent and did not proliferate.

A western blot analysis of proteins extracted from control cells and cells treated with 5% v-PL for different times (1 , 4, and 8 hours) was performed using α -cyclin D l , - phospho Akt, -phospho Erkl/2 antibodies (Fig. 5). Actin was blotted as an internal control. After 1 h from the v-PL treatment inventors observed an increase in the amount of phospho Akt and Phospho Erkl/2 (Extracellular signal-regulated protein kinases 1 and 2). After 8h the activation of these proteins was almost completely over whereas an expression of the Cyclin D l protein was detected in the v-PL treated cells.

Interestingly, the western blot analysis also showed that these proliferation pathways were activated by PL not only in cells resuming proliferation, but also in cells treated with v-PL in the absence of serum, although in this case cell proliferation did not occur (Fig. 5 upper panel). This confirmed inventors' previous observation that confluent quiescent cells that did not proliferate when maintained in serum, once exposed to the PL mitogenic stimulus were able to activate signal transduction pathways that promote cell growth in response to extracellular signals. However, the presence of serum proteins is an absolute requirement for cell proliferation to occur.

v-PL effect on the inflammatory, proliferative and angiogenesis-related response of primary human umbilical vein endothelial cells (HUVEC)

Since endothelial cells are the first cell population responding to platelet-derived factors, the effect exerted by PL on the inflammatory, proliferative and angiogenesis-related response of primary human umbilical vein endothelial cells (HUVEC) was also analyzed.

It was found that PL exerted a protective effect on HUVECs in inflammatory milieu by inhibiting IL- l a-activated NF-κΒ pathway and by inducing the secretion of PGE2, described as a pro- resolving factor towards macrophages in the wound microenvironment.

PL down-regulates NF-κΒ pathway in an inflammatory milieu

Inventors evaluated the activation of NF-κΒ pathway, a key player in the inflammatory phase response (Lawrence, T. (2009). The Nuclear Factor NF- B Pathway in Inflammation. Cold Spring Harb. Perspect. Biol. 1), in sub-confluent HUVECs treated for lh or 16h with complete culture medium supplemented with 5% PL, or 100 U/mL IL-la, or 5% PL+100 U/mL IL-la or un- supplemented (control, CTR). Figure 10, Panel A, reports the NF-κΒ activity in a representative experiment where cells were exposed to different culture conditions. Panels B shows the over the control fold increase of NF-κΒ activity induced by IL-la stimulation for 1 hour and the percentage reduction in the IL- l a-induced NF-κΒ activity when the culture was supplemented also with PL. Panel C shows the fold increase and the percentage reduction after 16h stimulation of the cells. Values are reported as average values ± SD values of 3 independent experiments. The NF-KB activity increase was statistically significant with respect to control after both lh and 16h cell stimulation with IL-la (p=0.0413 and p=0.0062, respectively, Figure 10B,C), and statistically significantly decreased after both lh and 16h stimulation in cells treated with PL+IL-la with respect to the IL-la treated cells (p=0.0018 and p=0.0292, respectively, Figure 10B,C). These results indicate an anti-inflammatory activity of PL on HUVECs both at early and late times. Considering the negative regulation of NF-κΒ pathway by PL in an inflammatory milieu, inventors evaluated the production of two pro-inflammatory cytokines, IL-8 and IL-6, following lh and 24h stimulations with PL under both physiological and inflammatory conditions. By western blot analysis of conditioned media, inventors did not observe a significant variation of pro-inflammatory cytokine secretion induced by IL-la in PL+IL-la treated cells. The reported densitometric analyses refer to the average of 3 or 4 independent experiments performed on different primary HUVEC cultures for lh or 24h stimulations, respectively (Figure 11 A,B).

PL increases PGE2 secretion by HUVEC in an inflammatory milieu

A quantitation of PGE2 released by HUVECs in the presence of PL was performed in order to detect a possible protective activity in an inflammatory environment. Results reported in Figure 12 show a significant increase of PGE2 secretion in PL+ILla treated cells with respect to the control (p=0.0191) indicating that PL-activated HUVECs could contribute to resolution of tissue inflammation also by a paracrine mechanism.

Moreover, v-PL enhanced the proliferation of HUVECs, without affecting their capability of forming tube-like structures on Matrigel, and activated resting quiescent cells to re-enter cell cycle and proliferate reaching a cell density significantly higher than the un-stimulated cells. In agreement with these findings, proliferation-related pathways Akt and ERKs were activated as well as the expression of the cell-cycle activator Cyclin Dl was enhanced demonstrating that quiescent cells, at a stage of basic metabolism, were activated by PL and resumed proliferation possibly contributing to vessel restoration. Finally, PL induced in HUVECs the stabilization of HIF-Ια and the phosphorylation of STAT3, which are involved in the angiogenesis, suggesting the possible in vivo contribution of PL to new vessel formation by activation of resident progenitor cells located in the vessel walls.

V-PL can rejuvenate a culture of senescent MSC

The positive effect of v-PL on resting cells was also verified on a primary culture of BM-MSC. Cells previously isolated and expanded in 10% FCS, after about 10 population doublings, were splitted and transferred to different culture conditions: 1) 1% v-PL; 2) 10% Pl-s; 3) 10% Pl-s+1% v- PL. After 5 passages in the new culture conditions (corresponding to about 7.5 doublings), to half of the cells in 10% Pl-s+1% v-PL the v-PL was removed, leaving the cells in 10% Pl-s only and the culture continued. After about 7 additional passages, the culture was again splitted. Half of the culture was maintained in 10% Pl-s while the other half was transferred to 10% Pl-s+1% v-PL (restoring in this way the mitogenic stimulus of the v-PL) (Fig.6). The 1% v-PL alone did not support cell viability and proliferation (yellow asterisk line). Also the 10% Pl-s alone culture condition was not permissive for the cell growth (red square line) while the combination of the two supplements allowed a good proliferation rate (blu diamond line). When the mitogenic stimulus of v-PL was removed from the culture, after an adaptation step of some passages to the new culture condition, cells entered a quiescent status (violet circle line). However, when v-PL was again provided to this senescent culture, cells resumed proliferation (green triangle line).

Pl-s versus Plasma

A comparison of the efficacy of Pl-s and plasma in sustaining cell proliferation was performed taking advantage of primary cultures of BM-MSC and different cell lines growing either in adhesion (HeLa) or in suspension (U-937) (Figure 16). Plasma- and plasma-derived serum were equally able to sustain cell proliferation. However, especially for BM-MSC and HeLa, both cell types growing in adhesion, the Pl-s was slightly more efficient than the plasma from which it was derived.

Differentiation Potential of Cells Cultured with the New Supplements

Maintenance of the differentiation potential by the cells expanded in the presence of the new medium supplements was confirmed. Specific in vitro assays routinely performed in the lab were adopted. In particular, osteogenic differentiation was tested for BM-MSC and AD-MSC and chondrogenic differentiation for dedifferentiated articular chondrocytes. To note that, in experiments where inventors compared differentiation of MSC expanded in the presence of the new culture medium supplements and in the standard culture condition, i.e., with FCS as medium additive, inventors observed a higher extent of osteogenic differentiation in cells cultured with 10% Pl-s + l%v-PL and a possible shortening of the time before the onset of the osteogenic differentiation. In the stimulated culture condition, it is clearly evident a calcium-enriched matrix and a type II collagen positivity for the MSC and the chondrocytes, respectively.

Growth rate of cells cultured with the new supplements compared to the growth rate of cells cultured in the presence of the fetal bovine supplement.

Inventors compared the proliferation rate of cells derived from a primary culture AD-MSC maintained in a medium containing the standard animal-based supplement 10% FCS with the one of the same cells maintained in a medium with the new additive in the ratio 10% Pl-s+1% v-PL. Cultures were monitored for about 30 days and the population doublings were calculated. Cells grown with 10% FCS performed a significant lower number of doublings in comparison to the parallel culture maintained with 10% Pl-s+l%v-PL (6 vs 25 doublings respectively).

HS (Pl-s) sustains hASC proliferation more efficiently than FBS

Primary hASCs were isolated in presence of HS (Pl-s), which was devoid of platelet-derived factors, and characterized respect to cells isolated with FBS. In particular, the cells were isolated using a culture medium supplemented with 10% HS (Pl-s) or 10% FBS (complete or control medium, respectively) and were frozen in liquid nitrogen at P0 for long-term storage. After thawing, (92.8±8.1)% and (88.6±9.6)% of viable cells were obtained with complete and control medium, respectively, by trypan blue exclusion test. The proliferation of HS (Pl-s)- and FBS-expanded hASCs was monitored at P2 for 8 days by cell counting. The HS (Pl-s)-expanded hASCs had a higher proliferation rate respect to FBS-expanded cells, showing a significant (5.75±1.35)-fold increase of cell density after 8 culture days (Figure 13a, p=0.0006 for 10%FBS vs. 10%HS (Pl-s) at day 8). In parallel experiments, inventors evaluated

cell viability in the same conditions used for cell counting by MTT assay, obtaining trends similar to those relative to cell proliferation (Figure 13). This finding indicates that hASCs were metabolically active independently from the cell density. Cell morphology was different depending on culture conditions (Figure 13c). In agreement with the reported cell density, HS (Pl-s)-expanded hASCs were smaller than FBS-expanded cells, which showed a spreading and flatter shape. Although HS sustained cell proliferation more efficiently than FBS, inventors did not find a significant difference in the number of colony-forming unit-fibroblasts (CFU-fs) between complete and control medium by CFE assay (Figure 13d,e). For the quantitative analysis of CFE assay, inventors used the ImageJ- plugin "Colony Area", which calculates the percentage of dish area covered by colonies (colony area percentage) and the intensity- weighted area percentage (colony intensity percentage). Both the output parameters increased with the complete medium respect to the control one but these differences were not significant (Figure 13f).

HS (Pl-s) induces spontaneous adipogenic differentiation in hASCs

During hASC culture in presence of HS (Pl-s), inventors observed spherical deposits within the cells increasing in number and dimension with time while they were absent in hASCs cultured with FBS. The ORO staining of hASCs at P2 cultured with 10% HS (Pl-s) for 0, 4 and 8 days showed ORO positive cells with red-stained lipid deposits increasing with time while FBS-expanded hASCs were totally negative (Figure 14a,b). Quantitation of the amount of incorporated ORO, normalized per cell number, showed a trend of increase with time in agreement with the observation of the progressive increase of deposits (Figure 14c), suggesting the spontaneous differentiation of ASCs towards adipogenesis. Analysis of total RNA extracted from hASCs cultured with HS (Pl-s) and FBS for 0, 4 and 8 days showed that transcript levels of ppary2 and c/ebpa, two master regulators of adipogenesis, gradually increased with time in HS (Pl-s)-expanded hASCs at variance with FBS- expanded cells in which they remained at a basal level with time (Figure 14d,e). At 8th day, ppary2 and c/ebpa expression reached a level significantly higher in HS (Pl-s)-cultured cells with respect to FBS-cultured ones (p=0.0422 and p=0.0318 for 10%HS (Pl-s) vs. 10%FBS, respectively).

Committed HS (Pl-s)-expanded hASCs differentiate towards adipogenesis and osteogenesis

From observing HS (Pl-s)-expanded hASC cultures after ORO staining, inventors realized that cell layers contained some regions with ORO-negative cells also at high cell density after 8 culture days. For this reason, inventors induced committed HS (Pl-s)-expanded hASCs to differentiate into the adipogenic lineage in order to investigate qualitatively the capability of ORO-negative cells to differentiate towards adipogenesis. Sub-confluent hASCs at P2 were treated with complete medium supplemented with 6 ng/mL insulin and 10^"7 M dexamethasone or un-supplemented (control) for 14 days obtaining in induced cultures ORO-positive cells with intracellular lipid droplets morphologically similar to those recognised in control cultures at the end of induction (Figure 15a). The amount of incorporated ORO in the treated cells did not significantly vary between induced and control cultures although a trend of increase was shown (Figure 15b). However, inventors still found several ORO-negative cells in induced cell layers. To verify the presence of immature or less committed cells in HS (Pl-s)-expanded hASC cultures, inventors induced the osteogenic differentiation in sub-confluent HS (Pl-s)-expanded hASCs containing lipid droplets at P2 by using complete medium supplemented with 50 μg/mL ascorbic acid, 10 mM β- glycerophosphate and 10^~7 M dexamethasone or un-supplemented (control) for 14 days. At the end of induction, ARS staining revealed a great amount of calcium-rich deposits in the extracellular matrix of induced cells but not in the control cultures (Figure 15c). In a parallel set of experiments, inventors still found ORO- stained lipid droplets within induced ASCs. Surprisingly, these droplets were qualitatively very similar to those found in the control cultures. Interestingly, when chondrogenic differentiation was induced on HS (Pl-s)-expanded hASCs at P2 by pellet-culture method using serum-free medium supplemented with 6.25 μg/mL insulin, 6.25 μg/mL human apotransferrin, 1.25 μg/mL linoleic acid, 5.35 μg/mL bovine serum albumin, 1 mM sodium pyruvate, 50 μg/mL ascorbic acid, 10^"7 M dexamethasone and 10 ng/mL TGF-βΙ for 21 days, poor metachromatic toluidine blue staining and no collagen type II production were detected in the extracellular matrix of pellets by histological analysis.

Discussion

Despite the fact that platelet-derived mitogens were crucial to promote the reentry in the cell cycle (commitment) of confluent cells of early passages and even of late passages of primary cultures, v- PL used as single component did not sustain viability and proliferation of either cell lines or primary cell cultures. On the contrary Pl-s was ineffective in quiescent resting cells but supported proliferation of the same cells after v-PL treatment and of cells constitutive ly stimulated as in the case of established cell lines. In agreement with these findings, the combination of the two components was highly effective in supporting proliferation of primary cell cultures at a higher level than FBS in control cultures, whereas the Pl-s added to the medium as a single component sustained the growth of several human cell lines in adhesion or in suspension at a growth rate comparable to the one of FBS control cultures. When inventors determined the efficiency of Pl-s and the plasma from which it was derived with regard to the ability to sustain cell proliferation in cultures of both primary cells and cell lines, they observed a comparable activity in the two medium supplements. However, a slight proliferation advantage was observed in cultures supplemented with the Pl-s. This was especially true for cells growing adherent to the Petri dish compared to cells growing in suspension. This finding suggests that, during the coagulation process, an activation of proteins and factors favoring adherence of proliferating cells is occurring, possibly through enzymatic proteolysis. The use of the two combined products allowed to establish cell cultures from tissue biopsies or aspirates in complete absence of animal components and to in vitro expand different types of cells, intended for cell therapy in humans, maintaining their differentiation potential and without introducing karyotype alterations. These cells include, but are not limited to, MSC derived from bone marrow, adipose or cord blood, and articular chondrocytes. To note is that this not only could consent to perform cell therapies with cells expanded in a safer condition (absence of animal components in the medium) but, in some cases, could allow to isolate and expand transplantable cells otherwise not achievable with conventional culture medium supplements. This is particularly relevant in case of elderly patients from which conventional culture medium supplements often do not permit to obtain an adequate number of cells.

Claims

1. A combination of:

a) an heparin-free human platelet lysate devoid of serum and/or plasma components and

b) an heparin-free human serum devoid of any platelet lysate components.

2. The combination according to claim 1 , comprising from 0.1% to 50% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components.

3. An heparin-free platelet lysate devoid of serum and/or plasma components.

4. An heparin-free serum devoid of platelet lysate components.

5. A process for the preparation of the heparin-free platelet lysate devoid of serum and/or plasma components comprising:

a) obtaining by centrifugation a platelet rich blood fraction from an isolated buffy coat pool, said pool preferably comprising samples isolated from 2 to 500 subjects, more preferably from 100 to 250,

b) isolating by centrifugation the plasma free platelet concentrate from the obtained platelet rich blood fraction;

c) washing in saline solution, preferably isotonic, the isolated plasma free platelet concentrate; d) subjecting to platelet lysis the washed plasma free platelet concentrate to obtain a platelet lysate devoid of serum and/or plasma components,

and optionally

e) lyophilizing the platelet lysate devoid of serum and/or plasma components.

6. The process according to claim 5 wherein the platelet rich blood fraction is leukocyte depleted prior platelet washing with saline solution.

7. The process according to claim 5 wherein the platelet rich blood fraction is leukocyte depleted after platelet washing with saline solution.

8. The process according to claims 5 or 6 or 7 wherein after step c), the platelet concentrate is preferably resuspended in saline solution and the platelet concentration preferably adjusted to from lO.OxlO⁹ pit/ml.

9. The process according to any one of claims 5-8 wherein the platelet lysis is obtained by three freeze -thawing cycles.

10. The process according to any one of claims 5-9 wherein after platelet lysis a high speed centrifugation is performed to sediment platelet membranes and debris.

11. A process for the preparation of the heparin-free serum devoid of platelet lysate components comprising:

a) obtaining by cryoprecipitation a cryo poor plasma from an isolated pool of plasma, said pool preferably comprising samples isolated from 2 to 100 subjects,

b) adding to the obtained cryo poor plasma calcium ions, preferably Calcium Gluconate or Calcium Chloride, and centrifugating to obtain heparin-free serum without platelet lysate components;

and optionally

c) lyophilizing the heparin-free serum without platelet lysate components.

12. The process according to claim 11 wherein in step b) CaCl₂ 2 mg/ml is added.

13. The heparin-free platelet lysate of claim 3, being obtained by the method according to any one of claims 5-10.

14. The heparin-free serum of claim 4, being obtained by the method according to claim 11 or 12.

15. The combination according to claim 1 or 2, wherein the heparin-free platelet lysate is obtained by the method according to any one of claims 5-10, and/or the heparin-free serum is obtained by the method according to claim 11 or 12.

16. The combination of claim 1 or 2 or 15, or the heparin-free platelet lysate of claim 3 or 13 or the heparin-free serum of claim 4 or 14, being lyophilized and/or frozen and/or freeze-dried and/or sterilized.

17. A cell culture medium supplement comprising or consisting of the combination of claim 1 or 2 or 15 or 16, or the heparin-free platelet lysate devoid of serum and/or plasma components of claim 3 or 13 or 16, or the heparin-free serum devoid of platelet lysate components of claim 4 or 14 or 16.

18. The cell culture medium supplement of claim 17 comprising from about 0.5% to about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and/or from about 5% to about 20% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components, preferably comprising about 1% or about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and/or about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components.

19. Use of the heparin-free platelet lysate devoid of serum and/or plasma components as defined in any one of claims 3, 13 or 16 as a cell culture medium supplement for:

a) inducing in-vitro the reprogramming to progenitor cells of differentiated cells, said cells being preferably mesenchymal cells and/or chondrocytes and/or keratinocytes and/or skin fibroblasts; b) enhancing in-vitro the number of obtainable primary cells, said cells being preferably derived from tissue biopsies or aspirates, and/or from bone marrow, adipose, cord blood, articular cartilage, bone, skin tissue,

c) rejuvenating in-vitro a culture of senescent cells, preferably of MSC (mesenchymal stem cells), said cell culture medium supplement preferably comprising from about 0.5% to about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components

more preferably comprising about 1% or about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components.

20. Use of the heparin-free serum devoid of platelet lysate components as defined in claims 4 or 14 or 16 as a unique cell culture medium supplement to induce in-vitro cell growth, preferably of established cell lines

said cell culture medium supplement preferably comprising from about 5% to about 20% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components, more preferably comprising about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components.

21. Use of the combination according to claim 1 or 2 or 15 or 16 as a cell culture supplement to enhance proliferation of cells in primary cell cultures, said cells being preferably adult stem cells, more preferably MSC, preferably derived from adipose, bone marrow and cord blood said cell culture medium supplement preferably comprising from about 0.5% to about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and from about 5% to about 20% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components,

more preferably comprising about 1% or about 5% (volume/volume) of the heparin-free human platelet lysate devoid of serum and/or plasma components and about 10% (volume/volume) of the heparin-free human serum devoid of any platelet lysate components.

22. The use according to any one of claims 19-21 wherein the cells cultured in the cell culture supplement are intended for cell therapy, preferably in humans.