WO2014053418A2 - Method for obtaining mesenchymal stem cells and use thereof - Google Patents

Abstract

The present invention relates to a method for obtaining mesenchymal stem cells (MSCs) from mammalian tissue or body fluid, comprising isolating said MSCs from tissue or body fluid and expanding said isolated MSCs in multiple cultivation passages, characterized in that the cell doubling log (CDL) per 24 hours of the MSCs is between 0.25 and 3.2 during expansion. In a further aspect, the current invention also relates to cell compositions obtained by the current method and to a container for storing such compositions.

Description

METHOD FOR OBTAINING MESENCHYMAL STEM CELLS AND USE THEREOF

TECHNICAL FIELD

The invention relates to a method for the isolation of mesenchymal stem cells, and the expansion thereof. The isolated stem cells can be used in regenerative therapies for e.g. joint, cartilage and tendon injuries, as well as to support and enhance the immune system. STATE OF THE ART

Stem cell therapy is a promising application in the relatively new field of regenerative medicine and surgery, including veterinary applications. Stem cells have the ability to differentiate into different cell types, can multiply massively, migrate spontaneously to damaged tissues, produce important factors for tissue repair and possess immu no-modulating properties. Various sources of mesenchymal stem cells (MSCs) have been described in humans, horses and other mammals; isolated mesenchymal stem cells were described originating primarily from bone marrow, adipose tissue, umbilical cord blood and umbilical cord matrix (Guest et al, 2008; Hoynowski et al, 2007; Koch et al, 2009; Radcliffe et al, 2010). However, the mesenchymal cells isolated through the currently known protocols exhibit a less than optimal ability to differentiate into cells for downstream applications. The obtained results are often also too variable to be readily and with confidence used. Therefore, there is a need for another, more standardized and better method of isolating mesenchymal stem cells. Furthermore, there is a need for an isolation method that allows allogenic application of the isolated mesenchymal stem cells.

A disadvantage of the use of autologous MSCs (from the individual itself) is that the, time-consuming (therapy comes sometimes too late), isolation is not always successful and the quality of MSCs varies between the different animals. A solution to this is thus the use of allogenic MSCs from selected donors. Use of allogenic MSCs is already reported for humans (Fang et al, 2007; Ringden et al, 2006) and horses (Carrade et al, 2011a; Carrade et al, 2011b). However, again such therapies are hampered by the quality of the isolated MSCs because of non- optimal isolation methods.

WO 2008/034740 discloses a method for the isolation and expansion of MSCs from peripheral blood of a mammal, by the addition of MCSF (macrophage colony stimulating factor) by which the cells are expanded and are then sorted. However, this method does not lead to a completely homogeneous population of MSCs, but to a mixture of hematopoietic, mesenchymal and pluripotent stem cells. WO 2009/040458, WO 2011/069121 and WO 2012076741 all disclose a method for isolating MSCs out of blood. Also Koch et a!., 2009, Martinello et a!., 2010, Koerner et a!., 2006 and Giovanni et a!., 2008 disclose methods for isolating MSCs. As mentioned however, the latter all are hampered for subsequent use, as the isolating methods are not optimal nor do they provide standardized results.

There is thus a need for a process to isolate mesenchymal stem cells in which a homogeneous population of MSCs is obtained, and for compositions comprising these homogeneous population of MSCs, to use in regenerative applications. By preference, use of the MSC is allogenic. Therefore, a rapid and simple process to obtain a MSC composition of high quality is necessary. It is well known that treatment with stem cells have the highest success rate when it is administered immediately after the inflammation phase caused by the damage (before infiltration of fibroblasts, and scar tissue formation) due to the ideal environment for cell growth at that time (Richardson et al, 2007).

With such a pure composition of MSCs, the theoretical applications are virtually unlimited and very accessible (especially when using allogenic MSCs).

SUMMARY OF THE INVENTION

The above invention relates to a method for the obtaining mesenchymal stem cells according to claim 1.

In a second aspect the invention relates to compositions according to claims 16 or 23 and a container for storing such compositions according to claim 31. In a final aspect, the invention relates to a method for the preparing such compositions prior to administration.

DESCRIPTION OF THE FIGURES Figure 1 shows an example of equine mesenchymal stem cells obtained according to an embodiment of the present invention, wherein said stem cells are positive for vimentin, fibronectin, and Ki67. Figure 2 shows an example of mesenchymal stem cells obtained from human blood, in which the stem cells are positive for vimentin, fibronectin, and Ki67.

Figure 3 is a schematic representation of the three-phase distribution of blood after centrifugation. Layer A is the plasma layer, Layer B the buffy coat, Layer C contains among others the erythrocytes.

Figure 4 show examples of compositions according to the present invention which are preserved in an embodiment of a container according to the current invention, preferably stored in a container, suitable for long-term storage at a minimum of - 80°C, and to administrate immediately after thawing.

Figure 5 shows the effect of mixing MSCs with different scaffolds (for example, based on hyaluronic acid or glycosaminoglycans) on the vitality of the mesenchymal stem cells.

Figure 6 shows a graphical representation of the significance of the inner diameter of a needle used to aspirate a composition according to the present invention, from a container.

Figure 7 shows the effect of various concentrations of DMSO, when freezing examples of compositions according to an embodiment of the present invention, on the vitality of the cells in two different methods of thawing. Figure 8 shows a representation of mesenchymal stem cells isolated in accordance with an embodiment of the current invention.

Figure 9 shows a representation of mesenchymal stem cells isolated in accordance with an embodiment of the present invention, induced towards the formation of tenocytes.

Figure 10 A shows undifferentiated mesenchymal stem cells according to the present invention (left picture) which are differentiated to tenocytes (right picture) with clear fiber orientation. Differentiation was confirmed by expression of Collagen type I (Figure 10 B) and Smooth Muscle Actin (Figure 10 C). Figure 11 shows mesenchymai stem cells isolated according to an embodiment of the present invention, in which the cells were selected by diameter or cell size. By preference, cells were selected on the basis of the suspension diameter. Figure 12 A is an ultrasound scan of a patient with tendinitis, figure 12 B shows the evolution after 6 months of intensive, conservative therapy.

Figure 13 A is an ultrasound scan of a patient with tendinitis, figure 13 B shows the evolution, 29 days after treatment with a composition according to an embodiment of the present invention.

Figure 14 A is an ultrasound scan of a patient with chronic desmitis, figure 14 B shows the evolution, 35 days after treatment with a composition according to an embodiment of the present invention.

Figure 15 shows statistical results of patients (suffering from tendinitis or desmitis) treated with compositions according to embodiments of the present invention. Score 0 indicates 0% improvement 6 weeks after treatment and score 5 indicates 100% improvement ( 1 =20%, 2=40%, 3=60% and 4=80%).

Figure 16 shows immunocytochemistry on cytospins using Ki67 (A), collagen (Col) type II (B), vimentin (C) and p63 (D). Native mesenchymal stem ceils (MSCs) obtained according to the present invention were negative for p63 and positive for Ki67, Col II and vimentin, whereas chondrogenic induced MSCs according to the current invention were positive for p63, Col II and vimentin and slightly positive for Ki67. Arrows indicate a decreased signal for Ki67 in some chondrogenic induced MSCs. The relevant isotype controls were negative. Scale bar represents 25μηι. Figure 17 shows results of a flow cytometry experiment on MSCs obtained by an embodiment of the current invention. Flow cytometry confirmed a very low expression of major histocompatibility complex (MHC) class I and no expression of MHC class II on the native MSCs and chondrogenic induced MSCs. The light and dark grey histograms represent the relevant isotype control staining and marker antibody staining, respectively with the corresponding percentage of mean positive cells (gated as P2) ± SEM. Figure 18 shows representative images of peripheral blood (PB)-derived mesenchymal stem cells (MSCs) after staining for glycosaminoglycans with Safranin O in their undifferentiated state (A) and after chondrogenic induction of functional MSCs (B) and afunctional MSCs (C). Glycosaminoglycan production (black arrows) can be noticed only after induction of functional MSCs in (B). Scale bars represent 50μιη.

Figure 19 A and B shows an embodiment of the container according to the current invention, comprising an inner and outer compartment.

DETAILED DESCRIPTION

The invention relates to a process for the isolation of mesenchymal stem cells (MSCs), as well as a composition of mesenchymal stem cells obtained according to the present invention, and a method for its administration to a subject.

The method provides a relatively simple and rapid procedure to achieve a highly pure population of MSCs. This is often hampered by the fact that MSCs only represent a small percentage of the total amount of cells present in a tissue or fluid. For instance, if blood is used as a source for MSCs, only one MSG will present in the blood on a total population of IxlO⁶ leucocytes. Therefore, isolation and enrichment is necessary in order to come to a homogenous population. The resulting population of MSCs can be induced in a next step towards several specific cell types, or they can be used as such. The application of MSCs as such, or differentiated, are virtually unlimited. In the present invention, they will be used mainly in the treatment of lesions, as well as in the treatment of frequently occurring diseases or neurological disorders.

Unless defined otherwise, all terms used in the description of the invention, including technical and scientific terms, have the meaning as they are generally understood by a professional in the technical field of the invention. For a better evaluation of the description of the invention, the following terms are explained explicitly.

"A ", "an" and " the " refer in this document to both the singular and the plural, unless the context clearly implies otherwise. For example, " a segment " means one or more than one segment.

When "about" or "round" are used in this document additional to a measurable quantity, a parameter, a time period or time, etc., variations of +/- 20 % or less, preferably +/-10% or or less, more preferably +/-5% or less , still more preferably +/-!% or less , and even more preferably +/-0.1 % or less than the value cited and, to the extent that such variations can apply in the described invention. It should, however, be understood to mean that the value of the quantity in which the term "about" or "round" is used, itself is not disclosed specifically.

The terms "comprise", "comprising", "air", "include", "including", "law claiming", "content", "holding" are synonyms and are inclusive or open terms that indicate the presence of what follows and which do not preclude the presence or prevent other components, features, elements, members, steps, as known from, or described in the prior art.

Citing numerical intervals by endpoints includes all integers, fractions and/or real numbers between the endpoints, these endpoints included.

In a first aspect, the invention relates to a method for obtaining or isolating of mesenchymal stem cells from tissue or body fluid of mammals. Said method comprises isolating the MSCs from tissue or body fluid and expanding said isolated MSCs in multiple cultivation passages. By preference, expansion will occur in at least 5 cultivation passages, preferably in at least 10 passages. By further preference, the expansion occurs in an expansion medium comprising a low glucose medium, up to 20% serum (e.g. fetal calf serum or fetal bovine serum) and antibiotics. Too high serum concentrations can lead to a kind of "adjustment phase" of the mesenchymal stem cells to the growth factors present in the serum, which can lead to a non -optimal division of the cells in the absence of serum. This can adversely affect the cells when they are used for regenerative purposes. By preference, said expansion medium does not comprise dexamethasone.

In a preferred embodiment of the method, the cell doubling log (CDL) per 24 hours of the MSCS will be between 0 (= cells did not double) and 3.2 during expansion, more by preference between 0.25 and 3.2, even more preferably between 0.5 and 2, most preferably between 1 and 1.6.

For the purpose of the current invention, the term "cell doubling log' (CDL) is to be understood as a measure for the doubling of a group of cells within a certain time period, in the present case preferably in 24 hours, and is to be calculated according to the following formula :

CDL = Log(Nf/Ni)/Log 2 ; whereby Nf is the final number of cells (here at time point= 24 hours) and whereby Ni is the initial number of cells at time point= 0.

The CDL is an important measure of the vitality of the MSCs, and of their functional ability, especially with regard to differentiation. CDL was previously described in Martinello et al ., 2010; Koch et al. , 2009 and Hoynowski et al. , 2007. It was found by the inventors of the current invention that the CDL of the cultivated cells during expansion should be between the proposed range in order to come to a stable and high quality population of MSCs. During the course of the expansion, the CDL should be monitored, and cells which show a deviated CDL, should be either taken out of culture or monitored even more closely. The CDL may be positively influenced by for instance variation in passage time and passage number; seeding density; by amending the media refreshing sequence or for instance by use of conditioned medium .

In another or a further preferred embodiment, said the cell doubling index (CDI) per 24 hours of cultivation of the MSCs should be between 1 and 3 during the expansion of the cells. For the purpose of the current invention, the term 'cell doubling index' or CDI should be defined as a numerical index which reflects the number of times a group of cells divided within a certain time period, in the current case 24 hours, and is to be calculated according to the following formula :

CDI = V(Nf/Ni);

whereby Nf is the final number of cells (here at time point= 24 hours) and whereby Ni is the initial number of cells at time point= 0.

Again, CDI is crucial for the vitality of the isolated cells and the quality, especially in view of any downstream applications.

Alternatively or additionally, the Cell Doubling or CD of the MSCs during the expansion should be between 0.5 and 4.5 during the expansion period and this within a period of 24 hours. For the purpose of the current invention, said Cell Doubling is to be understood as an indication whether or not a group of cells is able to double its amount within a certain period, in this particular case within 24 hours :

CD = (Nf/Ni)/2;

whereby Nf is the final number of cells (here at time point= 24 hours) and whereby Ni is the initial number of cells at time point= 0. In a further embodiment, the Population Doubling Time (PDT) between each passage during expansion (starting from the first passage) should be between 0.5 and 2.5, more preferably between 0.7 and 1.4, more preferably between 0.9 and 1.1. For the purpose of the current invention, the term Population Doubling Time (PDT) is to be calculated by the following formula :

PDT= ln(Nf/Ni)/ln2

whereby Nf is the final number of cells (here at time point= 24 hours) and whereby Ni is the initial number of cells at time point= 0.

Afunctional MSCs will lose their potency, which is translated in a deviation in CDL, GDI, CD, PDT or a deviation in combinations of the four parameters. Such deviation on its turn is a measure for lower differentiation capacity, different marker expression and overall low or unacceptable quality of the MSCs.

During the expansion and the various passages of the MSCs, the MSCs will start forming colonies. By preference, the percentage of colony forming units per number of seeded cells in a colony forming unit (CFU) assay will be between 50% and 250%; more preferably 50% and 150%; more preferably between 70% and 140%, more preferably between 80% and 120%. Again, afunctional MSCs will show a deviation in this percentage. A suitable CFU assay comprises the plating 10, 50 and 100 MSCs per 94 mm plate, whereby cells are fixed 8 days later at - 20°C for 10 minutes using ethanol. Crystal violet staining is subsequently used to visualize the CFUs macroscopicaliy whereby the total number of CFUs per plate are counted.

In a further embodiment, the MSCs will, after going through the expansion phase, be selected based on size/suspension diameter. For the purpose of the current invention, the term 'suspension diameter' should be understood as the largest diameter of a cell, measured in suspension. This is not to be confounded from the longitudinal or transverse diameter of cells in adhesion, whereby the diameter of a cell depends on the degree of extension of the cell on the substrate. As mentioned, the "suspension diameter' is the diameter of the cell in suspension, wherein in most cases the cell will have a spherical form.

By preference, MSCs of a cell size between 10 to 100 μηι are selected, more preferably between 15 and 80 μιη, more preferably between 25 and 50 μηη. In a further preferred embodiment, said selection occurs by a filtration step. The latter provides for a high population of single cells and avoids the presence of cell aggregates. Such cell aggregates may cause cell death during the preservation of the cells by freezing and may all have an impact on further downstream applications of the cells. For instance, cell aggregates may higher the risk of the occurrence of a capillary embolism when administered intravenously. In a further, more preferred embodiment, two or multiple filtration steps are used.

It is advisable to dilute the expanded MSCs prior to filtration, especially when intravenous application is envisioned. By preference, obtained MSCs are to be diluted to a cell concentration between O.OlxiO⁶ and lOxlO⁶ cells/ml prior to said selection, more by preference between O. lxlO⁶ and lxlO⁶ cells/ml, more preferably between O. lxlO⁶ and 0.5xl0⁶ cells/ml. By preference, dilution occurs in low glucose medium, such as DMEM LG (e.g. lg/ml).

Prior to expansion of the MSCs during the various passages, said MSCs are cultured in growth medium. The growth medium is by preference a low glucose growth medium supplemented with antibiotics, serum and dexamethasone. By preference, said MSCs are kept at least 2 weeks in growth medium. Surprisingly enough, it was found that the dexamethasone in the growth medium causes the stem cells to retain their specific characteristics and prevent them from differentiating. By preference, 1 % dexamethasone is used. By preference, said MSCs are seeded in said growth medium at a density of at least 100.000 cells/cm², more preferably of at least 2.5 x 10⁵ cells/cm².

The MSCs according to the present invention may originate from various sorts of tissues or body fluids, in particular from blood, bone marrow, fat tissue or amniotic fluid. By preference, the MSCs originate from blood, by preference peripheral blood. Blood appears to be an optimal source of MSCs. Blood is not only a non-invasive and painless source, but also simple and safe to collect and, consequently, easily accessible. Furthermore, MSCs isolated from blood are a promising therapeutic tool for certain degenerative or traumatic diseases in different animal species, because of their enormous plasticity and differentiation capacity (Giovannini et al, 2008; Koerner et al, 2006; Martinello et al, 2010; Zvaifler et al , 2000). The blood may originate from all mammals, especially horse, human, cat, dogs, rodents, etc. By preference, said origin of is equine. By preference, when using biood as an origin of MSCSs, the method comprises at least the following steps, prior to expansion of said MSCs: a) the collection of one or more blood samples from donors, in a sample bottle, coated with an anti-coagulant;

b) centrifuging said blood sample(s) thereby obtaining a 3-phase distribution phase consisting of plasma-phase, buffy coat, and erythrocytes-phase;

c) collecting said buffy coat and loading it on a density gradient;

d) collecting of the blood-inter-phase obtained from the density gradient of step c); and

e) isolating the mesenchymal stem cells from the blood-inter-phase by centrifugation.

In a step f) cells are subsequently plated at a concentration of at least 100.000 cells/cm², more preferably 2.5 x 10⁵/cm² mesenchymal stem cells and are kept in a low glucose growth medium supplemented with dexamethasone, antibiotics and serum .

By preference, 2.5 x 10 ''/cm ² cells, more preferably between 2.5 x 10⁵/cm² and 5 x lO'Vcm² cells are seeded in step f). This number is crucial to ultimately obtain a pure and viable population of MSCs at an acceptable concentration . The density in which the cells in step f) of the present method are seeded is essential, because seeding the cells too dense will lead to massive cell death during expansion and a non-homogenous population of mesenchymal stem cells. A too low cell density will however result in little or no colony formation of the MSCs, so that expansion is not or hardly possible, or it will take too much time, whereby in both cases the vitality of the cells is negatively influenced .

For the purpose of the current invention, the term 'anti- coagulant' should be understood as a composition that can prevent the coagulation of blood . Examples of anticoagulants used in the present invention include for instance EDTA or heparin .

The term "buffy coat' in this invention, is the fraction of non-coagulated blood, preferably obtained by means of a density gradient centrifugation, wherein the fraction is enriched with white blood cells and platelets. Figure 3 shows a schematic representation of a 3-phase distribution of a blood sample obtained by means of centrifugation. The buffy coat is the middle phase B, located between the plasma-phase A and the erythrocyte-phase C.

In particular, the buffy coat will be isolated from the other fractions and diluted by means of a suitable physiological buffer, such as for example, a phosphate, bicarbonate, or Tris buffer, preferably with a minimum ratio of 1 :2. This dilution factor is important, as lower dilution factors may lead to problems loading it on the density gradient from step c, due to a too heavy buffy coat fraction.

Preferably, the density gradient in step c) and d) of the present method is achieved by use of Percoll^®. More in particular, said Percoll^® will have a density between 1.08g/ml and 1.077g/ml.

By the term "blood-inter-phase" in the present invention that fraction of the blood is meant that is preferably obtained by means of a density gradient and is located between the bottom fraction, mainly consisting of erythrocytes and polymorph nuclear cells, and the upper fraction, mainly consisting of plasma polymorph nuclear cells. The blood-interphase is the source of blood mononuclear cells (BMCs) comprising monocytes, lymphocytes, and mesenchymal stem cells. In the present invention, the lymphocytes are washed away at 37 °C, while the monocytes die within 2 weeks in the absence of cytokines necessary to keep them alive. In this way, the MSCs are purified.

In a further embodiment of the current invention, the isolation of the mesenchymal stem cells from the blood-inter-phase is preferably done by means of centrifugation of the blood-inter-phase (after isolation of the inter-phase), after which the cell pellet is washed once at least with a suitable buffer such as a phosphate buffer. Following a minimum period of 2 weeks ( 14 days), preferably 3 weeks (21 days) it will be clear if mesenchymal stem cell colonies are visible in the culture bottles. In a subsequent step g) at least 133stem cells/cm² expansion are transferred to medium containing low glucose, serum and antibiotics for the purpose of expanding the MSCs. As mentioned, this medium will include a maximum of 20 % serum (such as FBS or FCS).

Preferably, the expansion of the mesenchymal stem cells will occur in minimal five cell passages. This way, a sufficient amount cells can be obtained . Preferably, the cells are split at 70 to 80 % confluency. The mesenchymal stem cells can be maintained up to 50 passages in culture. After this the risk of loss in vitality, senescence or mutation formation occurs. Examples 1 and 2 describe protocols according to the present invention for the isolation of mesenchymal stem cells from the blood of e.g . horse and human.

After the expansion stage the mesenchymal stem cells are ready to use for various downstream purposes, such as regenerative therapies. However, preferably, the obtained MSCs or derivatives thereof (induced or differentiated cells) are frozen and stored at a temperature of at least -80 °C, optionally in liquid nitrogen containers. An example of a method for the freezing and storage of the MSCs is set forth in Example 3. A crucial factor in the freezing of the MSCs is the composition of the cryogenic medium, in particular, the concentration of DMSO. DMSO prevents ice crystal formation in the medium d uring the freezing process, but may be toxic to the cells in hig h concentrations. In a preferred form, the concentration of DMSO comprises up to 20%, more preferably, the DMSO concentration in the cryogen comprises 10%. The cryogenic medium further comprises low- glucose medium such as low- glucose DMEM . Figure 7 shows the influence of the DMSO concentration on the vitality of the stem cells, or compositions according to the present invention during defrosting . A percentage of 10% DMSO showed the best results. Freezing in accordance with the process of the present invention results in a minimal preservation time of the cells at -80°C for at least 6 months.

In a second aspect, the current invention relates to a composition comprising mesenchymal stem cells. By preference, said composition obtained by the method according to the present invention preferably comprise of 90% mesenchymal stem cells. More preferably, it will comprise of at least 95% mesenchymal stem cells, more preferably of at least 99%, most preferably 100%.

In a preferred embodiment, said composition comprises at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90% of single cells and whereby said single cells have a suspension diameter of between 10 μηη and 100 μηι . As previously mentioned , the d iameter of the cells as well as their single-cell nature is crucial for any downstream application and for the vitality of the cells.

The nature of the cells can be ascertained by means of markers specific for mesenchymal stem cells. Preferably, markers are selected from the group consisting of CD29, CD44, CD90, CD105, vimentin, fibronectin, collagen type II, Ki67 and CK18 or any combination thereof. Preferably, said MSCs are positive for vimentin (Figure 16), fibronectin, Ki67 (Figure 16), or any combination thereof. By means of the latter, the purity of the obtained MSCs can be analyzed, and the percentage of MSCs determined. Figures 1 and 2 show mesenchymal stem cells are obtained according to an embodiment of the present invention, respectively, isolated from horse (Figure 1), and human blood (Figure 2). Both stem cell populations are positive for vimentin, fibronectin and Ki67.

By preference, said MSCs will have a low or undetectable level of MHC I, MHC II and/or p63 protein (Figure 16) expression. For the sake of the current invention, said low level should be understood as less than 25%, more preferably less than 15% of the total cells expressing said MHCI, MHC II or p63. Especially the absence of MHC I and MHC II are hereby important for further downstream applications, as this will result in a very low immunogenicity of the cells, which is no doubt a huge advantage in terms of downstream applications. Figure 17 shows results of a flow cytometry experiment on MSCs obtained by an embodiment of the current invention. Flow cytometry confirmed a very low expression of major histocompatibility complex (MHC) class I and no expression of MHC class II on the native MSCs and chondrogenic induced MSCs.

By preference, the composition is formulated for intravenous, intra-articular, intramuscular or intra-lesional, intra-arterial, topical, subconjunctival administration to mammals or through regional perfusion. These modes of administration will depend heavily on the desired application of stem cells and/or their differentiated form.

If desired, the obtained mesenchymal stem cells can be induced or differentiated towards adult cells. Induction and differentiation is preferably done by the addition of specific growth factors and/or other differentiation - inducing factors to the medium of the cells. The nature of these factors will crucially depend on the differentation and the desired adult cell type. In a preferred embodiment, the MSCs according to the present invention can differentiate into tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, or fibroblasts. Figures 9 and 10 show MSCs according to an embodiment of the present invention which were induced and differentiated into tenocytes. The nature of the differentiated cells was morphologically confirmed by the observation of the typical fiber structure (Fig. 10 A), and also via the expression of specific markers such as smooth muscle actin (Figure 10 C) and collagen type I (Figure 10 B).

As a consequence, and in a third aspect of the current invention, the invention also relates to a cell composition obtained by differentiation of the MSCs composition according to any of the previous embodiments, whereby the cells of said cell composition are tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, keratinocytes, neurons or fibroblast.

In a further embodiment, the composition or cell composition of the current invention may be mixed with components selected from the group consisting of platelet- rich plasma (PRP), hyaluronic acid or glycosaminoglycans. The latter are known to have additional beneficial functions during downstream applications of the compositions according to the current invention. In one embodiment, e.g. when said (cell) composition is used for joint or tendon pathologies, such composition can be used with components/carriers selected from the group of platelet- rich plasma (PRP), hyaluronic acid, compositions based on hyaluronic acid, glycosaminoglycans, or compositions based on glycosaminoglycans. Mixing of the composition with such carrier substances may in some cases be desirable to increase the effectiveness of the composition or create a synergistic effect. PRP, for example, a substance rich in growth factors, stimulate the stem cells after implantation. Preferably, both the stem cells and PRP are harvested from the same donors for compatibility reasons. Carrier substances can also be used to counteract gravity: stem cells follow the law of gravity and therefore have difficulties reaching higher lesions without a carrier in which they can migrate. In addition, the carrier substances themselves also have beneficial effects on the pathological environment in which they contribute to the tissue repair itself and also provide a good stem cell niche to help differentiation of the cells in this area.

Examples of hyaluronic acid, glycosaminoglycans or compositions on this basis include OSTENIL®, OSTENIL® +, Adant® and Adequan® (see Figure 5). Figure 5 shows a schematic representation of the effect of dilution of the composition with hyaluronic acid - glycosaminoglycan components on the vitality of the composition according to the present invention. Arthramid® or R- Gel® are toxic and must be avoided to use in combination with MSCs.

Preferably, the cells from the composition are isolated from the blood of human, cat, dog or horse. By preference, the compositions according to the current invention are equine derived. In a particular aspect of the current invention, any embodiment of the above- mentioned compositions may be used for allogenic administration to a subject. Allogenic use allows a better control of the quality of the MSCs, as different donors may be screened, and the optimal donors may be selected. In view of preparing functional MSCs, the latter is indispensable. This is in contrast to autologous use of MSCs, as in this case, quality of the cells cannot be ensured.

For instance when blood MSCs were isolated, blood from a donor was used who was later also recipient of his isolated mesenchymal stem cells. In another embodiment, blood is used from donors in which the donor is preferably of the same family, gender or race as the recipient of the mesenchymal stem cells isolated from the blood of donors. In general, these donors will be tested on common current transmittable diseases or pathologies, in order to avoid the risk of horizontal transmission of these pathologies or diseases through the stem cells. Preferably, the donor animals are kept in quarantine. When using donor horses they can be, for example tested for the following pathologies: equine infectious anemia (EIA), equine rhinopneumonia (EHV-1, EHV-4), equine viral arteritis (EVA), West Nile virus (WNV), African Horse Sickness (AHS), Dourine (Trypanosoma), piroplasmosis, glanders (malleus, glanders), equine influenza A, Borreliosis (Borrelia burgdorferi, Lyme disease).

By preference, the current invention has its application in the veterinary field.

The compositions according to the current invention may be used for the following downstream applications:

- treatment of trauma is selected from the group of skin trauma, cartilage trauma, tendon traumas, traumas of ligaments, traumas of bones, traumas of mucus membranes, cysts or fractures; - treatment of neurological and neurodegenerative diseases selected from the group of Cushing's syndrome, difficulty of breathing or paresis of the extremities;

- treatment of acute or chronic inflammatory disease states selected from the group of laminitis, periostitis, gastritis, osteoarthritis, inflammation caused by viral, bacterial, parasitic or mycotic agents in mammals;

- treatment of hypersensitivity reactions such as insect hypersensitivity (summer eczema for example), drug hypersensitivity, hypersensitivity to dust and other types of hypersensitivity;

- treatment of hypersensitivity reactions

- treatment of stomach dilatation and torsion-complexes;

- treatment of infertility in mares or precocity in foals.

As mentioned, the compositions according to the current invention will by preference be frozen in order to allow long-time storage of the compositions. The current invention therefor also relates to a container for preserving a composition, by preference at low temperature, such as temperatures below -20°C.

In a preferred embodiment, said container comprises an outer compartment and an inner compartment, whereby the inner compartment is suitable for receiving said composition or cell composition according to the current invention . By preference, the inner compartment is removable from said outer compartment. Both the outer and inner compartment are closeable by a cap. In a preferred embodiment of the current invention, the ratio between the maximal outer diameter Dl of the inner compartment and the maximal inner diameter D2 of the outer compartment will be in the range of between 0.5 and 0.95, more preferably between 0.7 and 0.9.

In a further embodiment, the ratio between the maximal length of the outer compartment I I and the maximal length L2 of the inner compartment provided with cap is between 1 and 1.5, more preferably between 1 and 1.3, even more preferably between 1 and 1.2. By preference, I I comprises the inner length, whereas L2 comprises the outer length of the respective compartments.

By use of the container according to the current invention, a temperature- controlled freezing protocol, normally used for freezing of cell compositions, was no longer needed . The optimal dimensions allow the container with regard to inner and outer compartment allow a decreased freezing rate. It is believed that the latter is achieved by the volume of air present between the inner and outer compartment, which act as a sort of buffer against a freezing rate that is too fast. The inventors have carefully optimized the dimensions container of the current invention in order to achieve an optimal freezing rate. It was found that outside the ranges of diameter and length, non-optimal freezing was achieved, with either a freezing rate that was too fast or too slow, both leading to damaged cells.

As mentioned, both compartments may be provided with a cap to close off the respective compartments from outside contaminants or air. In a further preferred embodiment, the cap of the outer compartment is engaged with the cap of the inner compartment. The latter has as an advantage that this allows easy handling of the container, and that the inner compartment is easily taken out of the outer compartment, through the engaging caps. Moreover, an ideal and homogenous air buffer is established around the cell-containing inner tube by engaging the inner tube to the outer tube. By preference, one of the caps, or both caps are provided with an internal screw thread. In a further embodiment, said cap of the inner compartment may be provided with a pierceable septum. Such pierceable septum consists by preference of a flexible material such as rubber.

Preferably, both container and reservoir are made of materials suitable for cryogenic application. Figure 4 and 19 illustrates an embodiment of the container according to the present invention.

Figure 19 shows an embodiment of the container according to the current invention. The container (1) is provided with two compartments, an outer compartment (2) and an inner compartment (3), whereby in a closed position of the container 1, the inner compartment (3) is embedded in the outer compartment 2. Both the outer and inner compartment are closeable by a cap (4, 5). By preference, both caps (4, 5) are provided with a screw thread (6, 7).

The inner compartment (3) will have the following parameters (see figure 19A) : - length L2: the maximal length of the inner compartment when engaged with cap (5), including the length of the outer part of the cap (5).

- length 12 : the maximal length of the inner compartment without being engaged with the cap (5);

- D2 : the maximal outer diameter of the inner compartment.

The outer compartment (2) will have the following parameters:

- length LI : the maximal length of the outer compartment when engaged with cap (4), including the length of the outer part of the cap (4). - length I I : the maximal length of the outer compartment without being engaged with the cap (4);

- Di : the maximal inner diameter of the outer compartment. As seen on Figure 19B, the container and the compartments are thus designed that in closed configuration, the cap (4) of the outer compartment (2) will engage with the cap ( 5) of the inner compartment (3). By preference, engagement occurs by the screw thread mechanism (6) of the cap (4), which will be able to engage with the upper side of the cap ( 5) of the inner compartment (3). In a possible embodiment, and for the latter purpose, the cap (5) of the inner compartment will be provided with a recess ( not visible on the figures) which is able to engage with said screw thread (6). In another embodiment, said screw thread (6) will be open at the bottom, or provided with a recess or opening , for engagement with the cap (5) of the inner compartment (3).

By means of this engagement principle and upon opening the container, the inner compartment (3) will spontaneously be withdrawn from the outer compartment (2). The latter is a great advantage in terms of handling time and ease of handling .

In a final aspect, the current invention describes a method for preparing a composition or cell composition according to the current invention, prior to administering it to a subject, wherein the subject is a mammal and whereby said administration may be intravenous, intra-articular, intramuscular or intra-lesional, intra-arterial, topical, subconjunctival or through regional perfusion, comprising the steps of:

a) thawing said container comprising the composition, frozen at at least -80°C, whereby said thawing is carried out at a temperature between 20°C and 37°C, preferably between 25°C and 37°C, and in a time span of maximal 20 minutes, more preferably maximal 5 minutes;

b) aspirating the sample from the container by means of a need le with an inner diameter of at least 0.3mm, preferably at least 0.35mm ; and

c) optionally mixing of the composition with components selected from the group consisting of platelet-rich plasma ( PRP), hyaluronic acid or glycosaminoglycans.

In a final step d ), the composition is administered to a subject, whereby said subject is a mammal, preferably a dog, cat, horse, monkey or human . The cell diameter of the injection needle is crucial in this respect, in order to avoid damage to the cells. Figure 6 shows the essential effect of the inner diameter of the needle used for aspiration of the composition. A 23G needle (inner diameter 0 : 33 mm) had a significantly more positive effect on the vitality of the cells present in the composition than a 25G needle (inner diameter 0: 26 mm).

Defrosting the composition can be accomplished by thawing in a hot water bath or in the palm of one's hand or by any other method within the temperature limits (Figure 7).

Preferably, the composition is administered within 2 minutes after thawing, in order to safeguard the vitality of the composition.

In what follows, the invention is described on the basis of non-limiting examples which illustrate the invention, and are not intended to limit the scope of the invention.

EXAMPLES Example 1 : Example Protocol for the isolation of mesenchymal stem cells from the blood of horses

- Take five samples peripheral blood of horses and Collect it in EDTA tubes (5 x 10 ml)

- Transport the blood at 4 °C

- Centrifuge the tubes for 20 minutes at room temperature (10 accel and decel 10)

- Collect the buffy coat into a sterile 15 ml tube and dilute the cells 1 :2 with PBS at room temperature

- Transfer the solution in the same amount of Percoll (between 1 : 08 and 1.77 g/ml) at room temperature

- Centrifuge for 15 minutes at room temperature (without brake: 10 accel and decel 2)

- Collect the interphase

- Wash the interphase 3x with PBS by centrifugation for 8 minutes at room temperature - Resuspend the obtained pellet in growth medium with dexamethasone and count the cells

- Seed 20 to 40 x 10⁶ BMCs per T75 flask (seed 4 flasks)

- Renew growth medium within 48 hours after seeding in order to remove floating lymphocytes and other cells (avoids extensive cell death)

- Change the growth medium 2 times per week

- Trypsinize the cells at 70-80% confluency

- Seed 0.5 x 106 cells per T75 flask of the first passage in expansion medium

- Split cells at 70-80% confluence until passage 5 (P5)

Figure 1 shows mesenchymal stem cells isolated according to the protocol of Example 1, wherein the cells are positive for the markers: vimentin, fibronectin, and Ki67.

Example 2 : Example Protocol for the isolation of mesenchymal stem cells from the blood of humans, cats or dogs

The protocol of Example 1 can be used without problems in blood samples from other mammals, such as human, dog or cat. Figure 2 shows mesenchymal stem cells are isolated from human blood, positive for the markers: vimentin, fibronectin, and Ki67.

Example 3 : Example protocol for cryo-preservation of MSCs

- Trypsin 0.25% -EDTA 0.02% [50ml] :

• 10% Trypsin (stock solution = 2.5%) [5ml]

· 1% Versene (EDTA) [500μΙ]

• Dilute with PBS lx [44.5ml]

(or commercially available Tryp-EDTA 0.25%)

a. Remove the media

b. Add 0.25% trypsin-EDTA to the cells;

c. Incubate for up to 10 minutes at 37°C (check that all cells came loose); d. Add the same amount of warm medium (37°C, containing FBS) or pure FBS in order to block the trypsin activity; e. Centrifuge the solution at 300G for 8 minutes at room temperature (10 accel and decel 10);

f. Remove the supernatant;

g. Add 10ml lx PBS and resuspend the cells, to wash away the remaining medium, centrifuge at 300G for 8 minutes at room temperature (10 accel & decal

10);

h. Remove the supernatant;

i. Resuspend the cells in an appropriate quantity of medium,

j Add 1ml cells/cryotube;

k. Place the cryotubes overnight in a plastic container with isopropanol (KT) at -80°C.

I. Transfer the labeled tubes in a box at -80°C.

- General principle: [lml/cryotube]

• MSCs + DMEM low glucose [0.9ml]

• + 10% DMSO [ΙΟΟμΙ]

Example 4: Example of composition of media used

- Growth Medium [50ml] :

• Low glucose DMEM [39ml]

20% FBS [10ml]

· 1% low dexamethasone (stock = 10-9 M) [500μΙ]

• 1% Antibiotics - antifungals (penicillin/streptomycin/amphotericin B) [500μΙ]

- Expansion medium [500ml] :

• Low glucose DMEM [395ml]

20% FBS [100ml]

• 1% Antibiotics - antimycotics [5ml] Example 5

Examples of compositions of the present invention which may be marketed and used for downstream applications are the follwing : a. A composition of non-differentiated mesenchymal stem cells (see Figure 8) isolated from mammals, preferably for use in orthopedic lesions and pathologies such as osteoarthritis, cartilage damage, cyst structures. Preferably, such composition is administered intra-articularly.

b. A composition of mesenchymal stem cells induced towards chondrocytes, preferably for use in cases of severe cartilage injuries (Figure 18). c. A composition of mesenchymal stem cells induced towards tenocytes. Preferably, such composition is used in applications of tendon lesions, lesions of the ligaments and other tendopathies (see figure 9 and 10). d. A composition of non-differentiated mesenchymal stem cells, with a diameter of less than 40 μιτι (see Figure 11), preferably for use in treatment of Musculoskeletal pathologies, endocrine pathologies or for regional perfusion. The composition is preferably administered intravenously.

e. A composition according to point d, wherein said composition is stimulated so that an increased capacity for migration to pathological sites in the body.

Example 6 : Treatment of tendinitis in horses with a composition comprising mesenchymal stem cells induced towards tenocytes

Ten horses with severe tendinitis of the superficial digital flexor tendon and 15 horses with chronic desmitis of the musculus interosseous medius were treated with a composition according to the present invention, including mesenchymal stem cells are induced (pre-differentiation) towards tenocytes in combination with PRP as a carrier substance. Administration of the composition occurred intra-lesional under ultrasound guidance into the injured tendon. 80% of the treated horses showed improvement after only 6 weeks, where normally only after at least 6 months of intensive, conventional conservative therapy similar results are obtained .

Figure 12 A is an example of an ultrasound scan of a patient with tend initis, which was treated using conventional conservative therapy. Recovery was partially observed after 6 months of therapy (Figure 12 B).

Figure 13 A is an example of an ultrasound scan of a patient with tendinitis, treated with a composition of tenocytes according to the present invention . Figure 13 B shows the evolution, 29 days after treatment. A remarkable improvement was visible.

Figure 14 A is an ultrasound scan of a patient with chronic desmitis, figure B shows the evolution, 35 days after treatment with a composition according to an embodiment of the present invention . Again, a remarkable improvement was observed in a very short time. Moreover, local calcification of tendons before treatment also disappeared after treatment.

Figure 15 shows statistical results of patients (tendinitis or desmitis) treated with compositions according to embodiments of the present invention . Score of 0 indicates 0% improvement again after 6 weeks of treatment with tenogenic induced MSCs in combination with PRP and score 5 100% improvement ( 1 =20%, 2=40%, 3 =60%, and 4=80%). From this figure it can be seen that having independent veterinarians score 20 of the 25 horses (80%) had a score of 4 or more given 6 weeks after the treatment.

Example 7 : Infected wound after abdominal colic

After a colic operation in an Arabian stallion, the stitches became infected at the level of the linea alba which is at the level of the abdomen . The patient developed an infected skin wound . After 3 months of conservative therapy, the wound still didn't cure and the owners decided to treat with mesenchymal stem cells from the blood of another donor horse. About 4 weeks later, the wound was considered completely cured by the attending veterinarian.

Example 8: Tendon Sheath Problems and Lesions of the ligaments

Three horses with a swollen tendon sheath, already present for 6 months, were treated with an embodiment of the composition described and as a result complete recovery was observed after 2 months.

A patient was suffering from lameness as a result of a lesion to the collateral ligament of the fetlock: less than 2 months after the local injection of a composition according to the invention, the horse was able to walk again normal and no relapse had been noticed.

Example 9: Fractures

A 4-year-old gelding with a difficult healing of the hoof bone fracture after 10 months of conservative treatment locally treated with a composition according to the present invention. Full recovery occurred after 4 months.

Example 10 : Mucous membranes Improvement of a horse with severely inflamed ulcers (polyfolliculaire lymphangitis) in the throat region after 4 weeks of treatment. After 6 weeks, the lesions were completely healed.

Example 11 : Cushinq's syndrome

A 19-year old horse with Cushing's syndrome was treated with 2 consecutive treatments of the composition described above (with 2 month interval). After 2 cycles, the symptoms were improved remarkably. Two more horses were treated in the same way with the same result.

Example 12 : Head Shaking This is a neurological pathology of the central nervous system which leads to continuous shaking of the head. A horse that suffered already for 4 months was treated once. Already in the third week after treatment the symptoms disappeared.

Example 13 : Vascular reconstruction In a horse affected by laminitis (detachment of the lamelles in the foot, with a consequent destruction of the peripheral vasculature in the foot) the pain disappeared after nearly 24 hours after a dose was administered. However, we noticed that this horse relapsed after 3 weeks. Example 14: Hypersensitivity reaction

A horse with diffuse nodules (bumps) on the body caused by a hypersensitivity reaction has been treated by the composition described, resulting in the disappearance of the bumps the following day. This result lasted for 2 months.

Another horse with sweet itch (hypersensitivity caused by culicoides mosquitos) rubbed during the first two months of summer his full mane and tail bald. By a single treatment with the component described this horse was spared of further itchiness during the rest of the summer with full recovery of mane and tail.

Example 15 : Isolation protocol for MSCs from adipose tissue

- adipose tissue was obtained from horses

- tissue was minced, washed and agitated in a phosphate-buffered saline solution to promote phase separation (2 phases)

- the upper phase consisting of minced tissue was digested in a solution comprising 1% BSA and 0.1% collagenase Type I with continuous shaking at 37°C for almost 50 minutes

- the obtained sample was subsequently centrifuged at 260 x g for 5 minutes

- the stromal vascular fraction pellet containing MSCs was cultured in growth medium

- further growth and expansion steps were performed according to the steps described in example 1. Example 16 : Isolation protocol for MSCs from bone marrow

- a bone marrow (BM) aspirate was collected into heparinized syringes using a lOg, 3 inch Silverman bone marrow biopsy needle

- BM aspirates were diluted 1 :3 with medium consisting of DMEM-Ham's F12 medium supplemented with antibiotics

- in a subsequent step, the solution was loaded on a density gradient (e.g. Ficoll- Paque^® PLUS)

- fractioning occurred by centrifugation at 350 x g for 30 minutes at 4°C

- the MSCs enriched fraction was in a subsequent step centrifuged and finally plated on cell plates

- further growth and expansion steps were performed according to the steps described in example 1.

Example 17 : Isolation protocol for MSCs from amniotic fluid

- amniotic fluid samples were obtained using a sterile 18-gauge needle

- each sample was 1/1 diluted in a phosphate buffer, e.g. DPBS containing antibiotics

- the obtained solution was centrifuged for 15 minutes at 470 g, supernatant was removed and obtained pellet resuspended in 5ml DMEM

- cells were loaded on a density gradient (e.g. Percoll), thereby collecting the interphase

- interphase was collected and centrifuged at 470 g for 10 minutes at 25°C

- supernatant was aspirated and the cell pellet was washed and finally resuspended in 1ml growth medium

- further growth and expansion steps were performed according to the steps described in example 1. Example 18 : Eye ulcers:

- Two horses with an eye ulcer that was irresponsive to conservative treatment for at least 1 month were treated subconjunctively as well as intravenously with MSCs. Within 1 week the eye effusion decreased and after 1 week the ulcers started to decrease in size. References

Carrade DD, Affolter VK, Outerbridge CA, Watson JL, Galuppo LD, Buerchler S, Kumar V, Walker NJ, Borjesson DL, 2011a. Intradermal injections of equine allogenic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions. Cytotherapy 13, 1180- 1192.

Carrade DD, Owens SD, Galuppo LD, Vidal MA, Ferraro GL, Librach F, Buerchler S, Friedman MS, Walker NJ, Borjesson DL, 2011b. Clinicopathologic findings following intra-articular injection of autologous and allogenic placentally derived equine mesenchymal stem cells in horses. Cytotherapy 13, 419-430.

Fang BJ, Song YP, Lin QD, Zhang YL, Cao Y, Zhao RCH, Ma YF, 2007. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft- vs. -host disease in two children. Pediatric Transplantation 11, 814-817.

Giovannini S, Brehm W, Mainil-Varlet P, Nesic D, 2008. Multilineage differentiation potential of equine blood-derived fibroblast-like cells. Differentiation; research in biological diversity 76, 118-129.

Guest DJ, Ousey JC, Smith MRW, 2008. Defining the expression of marker genes in equine mesenchymal stromal cells. Stem Cells and Cloning : Advances and Applications 1, 1-9.

Hoynowski SM, Fry MM, Gardner BM, Leming MT, Tucker JR, Black L, Sand T, Mitchell KE, 2007. Characterization and differentiation of equine umbilical cord- derived matrix cells. Biochemical and Biophysical Research Communications 362, 347-353.

Koch TG, Thomsen PD, Betts DH, 2009. Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells. Cytotherapy 11, 443-447. Koerner J, Nesic D, Romero JD, Brehm W, Mainil-Varlet P, Grogan SP, 2006. Equine peripheral blood-derived progenitors in comparison to bone marrow- derived mesenchymal stem cells. Stem cells 24, 1613-1619. Martinello T, Bronzini I, Maccatrozzo L, Iacopetti I, Sampaolesi M, Mascarello F, Patruno M, 2010. Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood. Tissue engineering. Part C, Methods 16, 771-781.

Radcliffe CH, Flaminio MJ, Fortier LA, 2010. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Stem cells and development 19, 269-282.

Richardson LE, Dudhia J, Clegg PD, Smith R, 2007. Stem cells in veterinary medicine— attempts at regenerating equine tendon after injury. Trends in biotechnology 25, 409-416. Ringden O, Uzunel M, Rasmusson I, Remberger M, Sundberg B, Lonnies H, Marschall HU, DIugosz A, Szakos A, Hassan Z, Omazic B, Aschan J, Barkholt L, Le Blanc K, 2006. Mesenchymal stem cells for treatment of therapy-resistant graft- versus-host disease. Transplantation 81, 1390-1397. Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN, 2000. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Research 2, 477-488.

Claims

1. A method for obtaining mesenchymal stem cells (MSCs) from mammalian tissue or body fluid, comprising isolating said MSCs from tissue or body fluid and expanding said isolated MSCs in multiple cultivation passages, characterized in that the cell doubling log (CDL) per 24 hours of the MSCs is between 0.25 and 3.2 during expansion.

2. Method according to claim 1, characterized in that the cell doubling index (CDI) per 24 hours of the MSCs is between 1 and 3 during expansion.

3. Method according to any of the preceding claims, characterized in that after expansion, MSCs of a suspension diameter between 10 to 100 μηι are selected.

4. Method according to claim 3, characterized in that said selection involves a filtration step.

5. Method according to any of the claims 3 or 4, characterized in that said cells have a concentration between O.OlxlO⁶ and lOxlO⁶ cells/ml prior to said selection.

6. Method according to any of the preceding claims, characterized in that expansion of said MSCs occurs by at least 5 cultivation passages, preferably at least 10 passages.

7. Method according to any of the preceding claims, characterized in that cultivated MSCs form colonies, whereby the percentage of colony forming units per number of seeded cells is between 50% and 250%, more preferably between 70% and 140%.

8. Method according to any of the preceding claims, characterized in that said MSCs are expanded in expansion medium comprising a low glucose medium, up to 20% serum and antibiotics.

9. Method according to any of the preceding claims, characterized in that said MSCs are cultured in growth medium prior to the first passage, said growth medium is a low glucose growth medium supplemented with antibiotics, serum and dexamethasone for suppressing the differentiation of said MSCs.

10. Method according to claim 9, characterized in that MSCs are seeded in said growth medium at a density of at least 10 x 10⁴ cells/cm².

11. Method according to any of the preceding claims, characterized in that said MSCs originate from blood, bone marrow, fat tissue or amniotic fluid.

12. Method according to any of the preceding claims, characterized in that said MSCs originate from blood, by preference peripheral blood.

13. Method according to claim 12, comprising the following steps prior to expansion of said MSCs;

a) the collection of one or more blood samples from donors, in a sample bottle, coated with an anti-coagulant;

b) centrifuging said blood sample(s) thereby obtaining a 3-phase distribution phase consisting of plasma-phase, buffy coat, and erythrocytes-phase;

c) collecting said buffy coat and loading it on a density gradient;

d) collecting of the blood-inter-phase obtained from the density gradient of step c ); and

e) isolating the mesenchymal stem cells from the blood-inter-phase by centrifugation.

14. Method according to claim 13, characterized in that step d) is preceded by a step c'), wherein the buffy coat from step c) is at least 1 :2 is diluted in a physiological buffer.

15. Method according to claim 14 or 15, characterized in that the mesenchymal stem cells are isolated from equine blood.

16. Composition comprising mesenchymal stem cells, characterized in that said composition comprises at least 75% of single cells and whereby said single cells have a suspension diameter between 10 μηη and 100 μιτι.

17. Composition according to claim 16, characterized in said MSCs are positive for at least one marker chosen from the group of CD29, CD44, CD90, CD105, vimentin, fibronectin, collagen type II, Ki67 and CK18.

18. Composition according to the preceding claims, characterized in that said composition has a low or undetectable level of MHC I, MHC II and/or p63 protein expression.

19. Composition according to claims 16 to 18, characterized in that the composition is formulated for intravenous, intra-articular, intramuscular or intra- lesional, intra-arterial, topical, subconjunctival administration or through regional perfusion to mammals.

20. Composition according to any one of the claims 16 to 19, characterized in that the mesenchymal stem cells may be differentiated towards tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, keratinocytes, neurons or fibroblasts.

21. Composition according to any one of the claims 16 to 20, characterized in that the composition is mixed with components selected from the group consisting of platelet- rich plasma (PRP), hyaluronic acid or glycosaminoglycans.

22. Composition according to any one of the claims 16 to 21, characterized in that the MSCs are equine derived.

23. Cell composition obtained by differentiation of the composition according to any of the claims 16 to 22, characterized in that the cells of said cell composition are tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, keratinocytes, neurons or fibroblast.

24. Composition according to any one of the claims 16 to 22 or cell composition according to claim 23, for allogenic administration to a subject.

25. Composition according to any one of the claims 16 to 22 or cell composition according to claim 23 for treatment of trauma which is selected from the group comprising of skin trauma, cartilage trauma, tendon traumas, traumas of ligaments, traumas of bones, traumas of mucus membranes, cysts or fractures.

26. Composition according to any one of the claims 16 to 22 or cell composition according to claim 23, for treatment of neurological and neurodegenerative diseases selected from the group comprising of Cushing's syndrome, difficulty of breathing or paresis of the extremities.

27. Composition according to any one of the claims 16 to 22 or cell composition according to claim 23, for the treatment of acute or chronic inflammatory disease states selected from the group comprising of laminitis, periostitis, gastritis, osteoarthritis, inflammation caused by viral, bacterial, parasitic or mycotic agents in mammals.

28. Composition according to any one of the claim 16 to 22 or cell composition according to claim 23, for the treatment of hypersensitivity reactions.

29. Composition according to any one of the claim 16 to 22 or cell composition according to claim 23, for the treatment of stomach dilatation and torsion- complexes.

30. Composition according to any one of the claims 16 to 22 or cell composition according to claim 23, for the treatment of infertility in mares or precocity in foals.

31. A container for preserving a composition according to any of the claims 16 to 22 or a cell composition according to claim 23, said container comprises an outer compartment and an inner compartment, suitable for receiving said composition or cell composition, whereby said inner compartment is removable from said outer compartment, said outer and inner compartment are each closeable by a cap, characterized in that the ratio between the maximal outer diameter D2 of the inner compartment and the maximal inner diameter Dl of the outer compartment is in the range of between 0.5 and 0.95.

32. Container according to claim 31, characterized in that the ratio between the maximal length I I of the outer compartment and the maximal length L2 of the inner compartment provided with a cap is between 1 and 1.5.

33. Container according to any of the preceding claims, characterized in that in a closed conformation, the cap of the outer compartment is engaged with the cap of the inner compartment.

34. Container according to any of the preceding claims, characterized in that said caps are provided with an internal screw thread.

35. A method for preparing a composition according to any of the claims 16 to 22 or cell composition according to claim 23, being contained in a container according to claims 31 to 34, prior to administering it to a subject, wherein the subject is a mammal and whereby said administration may be intravenous, intraarticular, intramuscular or intra-lesional, intra-arterial, topical, subconjunctival or through regional perfusion, comprising the steps of:

a) thawing said container comprising the composition, frozen at at least -80°C, whereby said thawing is carried out at a temperature between 20°C and 37°C, preferably between 25°C and 37°C, and in a time span of maximal 20 minutes, more preferably maximal 5 minutes; b) aspirating the sample from the container by means of a needle with an inner diameter of at least 0.3mm, preferably at least 0.35mm; and

c) optionally mixing of the composition with components selected from the group consisting of platelet-rich plasma (PRP), hyaluronic acid or glycosaminoglycans.