EP1616002A2

EP1616002A2 - Method for obtaining mastocyte lines from porcine tissue and method for production of heparin type molecules

Info

Publication number: EP1616002A2
Application number: EP04742485A
Authority: EP
Inventors: Jean-Marc Guillaume; Werner Dittrich; Sandrine Perez; Christine Andreoni; Romain Paillot
Original assignee: Aventis Pharma SA
Current assignee: Aventis Pharma SA
Priority date: 2003-04-14
Filing date: 2004-04-13
Publication date: 2006-01-18
Also published as: JP2006522597A; US20080064095A1; WO2004092356A3; FR2853663B1; FR2853663A1; US20050181484A1; WO2004092356A2; AR043862A1; TW200506059A

Abstract

The invention relates to a method for obtaining cultures or lines of mastocytes. The invention further relates to a method for production of molecules of the heparin type, comprising the culturing of cultures or lines of mastocytes.

Description

Method for obtaining mast cell lines from pig tissue and method for producing heparin-like molecules

The present application relates to a process for obtaining cultures or lines of mast cells. It also relates to a process for the production of heparin-like molecules comprising the cultivation of cultures or lines of mast cells.

Mast cells are cells of the immune system, derived from hematopoietic precursors, which are involved in the inflammatory response, especially in the phenomena of allergy and hypersensitivity. They are localized in the connective tissue, especially in the skin, intestinal and respiratory mucosa.

Mast cells are in the form of rounded cells with a diameter of between about 5 to 25 μm and have a rounded, single, central or eccentric nucleus. They are also characterized by the presence of numerous meta-chromatic cytoplasmic granulations.

These granules contain various molecular species with proinflammatory activity such as histamine, serotonin, proteoglycans such as heparin or chondroitin sulfate, enzymes, cytokines and chemoattractants of eosinophils and neutrophils. These species are released during mast cell activation.

After activation, a secondary response is set up during which the synthesis of mediators such as leukotrienes, prostaglandins, PAF (platelet activating factor), interleukins (IL4, IL5, IL6, IL10, IL12 and IL 13) takes place. , cytokines (TGF beta, IFN gamma, GM-CSF) and chemokines (MCP-1, IL8, RANTES). All of these species participate in the triggering of an inflammatory process and in the establishment of a specific immune response dependent on T lymphocytes. Mast cell cultures have already been obtained in humans and mice, but the state of the art does not provide a description of such established cultures or lines in pigs.

Razin et al (J. Biol. Chem., 257, 7229-7236, 1982) describe obtaining mouse mast cells using culture media containing IL3.

Wang et al (Cire. Res., 84, 74-83, 1999) describe the isolation of serous mast cells obtained from pleural and peritoneal cavities of rats. Various molecular species are produced, but only when mast cells are co-cultivated with rat aortic smooth muscle cells. Application W099 / 26983 describes similar work, and is very imprecise as to the application to other species.

Cell lines have been established in mice (Montgomery et al, Proc.

Natl. Acad. Sci. USA, 89, 11327-11331, 1992 and application WO90 / 14418) but from mastocytomas. These tumors are extremely rare in pigs.

In humans, obtaining mast cell cultures has proven difficult. It was first permitted by the use of a co-culture system with fibroblasts (Ishizaka et al, Current Opinion in Immunology, 5, 937-943, 1993).

Other authors then succeeded in obtaining mast cells from intestinal cells and in maintaining these cells in culture for approximately 6 months in the presence of SCF (Bischoff et al, J. Immunol., 159, 5560-5567, 1997).

When it was measured, the authors of these various articles reported only a low production of heparin-type compounds.

In pigs Emery et al (Experimental Hematology, 24, 927-935, 1996) maintained, for 7 weeks, cell cultures obtained from bone marrow. However, it appears that the cultures obtained are mixtures of various cell types and not homogeneous cultures or mast cell lines. In addition, these cultures contain cells which are not differentiated into homogeneous mast cell cultures.

Ashraf et al (Veterinary Parasitology; 29, 134-158, 1988) isolated pig mast cells from the intestinal mucosa without maintaining cultures amplifiable. In addition, the characterization of the isolated mast cells reveals the absence of heparin.

Heparin belongs to the family of glycosaminoglycans (GAG), which includes linear polysaccharides containing a repetition of a disaccharide sequence consisting of an amino sugar (D-glucosamine or galactosamine) and an uronic acid (D-glucuronic or L-iduronic). In the case of heparin, which belongs, with heparan sulfate, to the subfamily of glucosaminoglycans, the amino sugar is D-glucosamine. Uronic acid is either glucuronic acid (GIc) or iduronic acid (Ido). Glucosamine can be N-acetylated, N-sulfated and O-sulfated.

Conventionally, the term "heparin" denotes highly sulphated polysaccharides in which more than 80% of the glucosamine residues are N-sulphates and the number of O-sulphates is greater than that of the N-sulphates. The sulfate / carboxylate ratio is generally greater than 2 for heparin.

However, the structure of heparin is actually very heterogeneous, and there are chains that can contain very different relationships.

Like all GAGs, heparin is synthesized in the form of a proteoglycan mainly by mast cells.

The first step in the synthesis of heparin is the formation of the serglycine protein nucleus, consisting of a repetition of serine and glycine residues.

The heparin chain is extended by adding a tetrasaccharide, then by successive additions of glucosamines and uronic acids, regularly alternated.

The proteoglycan thus formed undergoes numerous sequential transformations:

N-deacetylation, N-sulfation, epimerization of D-glucuronic acid, and

O-sulphation.

However, this complete maturation takes place only on part of the proteoglycan, which generates a great structural variability of the heparin, responsible for its heterogeneity.

The polysaccharide chains are then cleaved from serglycine by an endoglucuronidase. These chains then have a molecular weight between 5000 and 30,000 Da. They form complexes with basic proteases and are thus stored in the granules of mast cells. Heparin is excreted only during degranulation of mast cells.

Heparin plays an important biological role, especially in hemostasis, and is very widely used in therapy, in particular as an anticoagulant and antithrombotic agent.

Most of the heparin used is isolated from the intestinal mucosa of the pig, from where it is extracted by proteolysis, followed by purification on anion exchange resin (for review on the different processes for preparing heparin , cf. DUCLOS, “Heparin: fabrication, structure, properties, analysis”; Ed. Masson, Paris, 1984).

Analysis of the disaccharide composition of pig heparin after depolymerization and chromatography makes it possible to differentiate heparin from other glycosaminoglycans. There are in particular eight main disaccharides (Figure 6). The sulfated disaccharides, ls, Ils, Ils, IVs, are proportionally the most abundant with predominantly ls, the amount of which is greater than 40%, and preferably greater than 50%. Next in order of abundance come the disaccharides Ils, llls and IVs. The ratio between the disaccharides ls and Ils is between 3 and 8.

We can observe a heterogeneity in the composition of heparin between batches from batches of animals of different origins. This heterogeneity is likely to generate variability in biological activity.

In addition, the use of animals as a source of heparin constitutes a risk due to the possible presence of viruses capable of being transmitted to humans. In addition, the supply of raw material can be irregular.

-The present invention proposes to overcome these drawbacks and to overcome the supply problems in terms of quantity and quality, by using a source of raw material more easily controllable. The applicant has shown that it is possible to produce in large quantities from mast cell cultures, heparin with properties comparable to those of heparin extracted from porcine intestinal mucus and reproducible.

The Applicant has also demonstrated that the genes encoding three proteins of importance for the production of heparin-like molecules or the independence of mast cells with respect to growth factors, present in pigs sequences different from those of other species.

The subject of the present invention is a process for obtaining cultures or lines of mast cells comprising the cultivation of a population of stem cells from bone marrow of young pigs or fetuses, in a medium comprising at least approximately 0.2 ng / ml of interleukin-3 (IL-3) preferably porcine (preferably at least 0.5 ng / ml, even more preferably at least 2 ng / ml), at least about 8 ng / ml of Stem Cell Factor (SCF ) preferably porcine (preferably at least 20 ng / ml, even more preferably at least 80 ng / ml) and at least approximately 0.1 ng / ml of interleukin-4 (IL-4) preferentially porcine (preferably at least 0, 5 ng / ml, even more preferably at least 1 ng / ml), 10 ng / ml of interleukin-6

(IL-6) preferably porcine (preferably at least 50 ng / ml, even more preferably at least 100 ng / ml) and / or 1 ng / ml of G-CSF preferentially porcine (preferably at least 5 ng / ml, even more preferably at least 10 ng / ml).

Thus, the obtaining medium contains a combination of IL4, ILδ and G-CSF separately, two by two or all three together in a medium containing IL3 and SCF.

Although these various factors are preferably of porcine origin, that is to say that their sequence is deduced from that of the corresponding factor in pigs, it is possible to replace at least one of them by a factor of another origin.

Thus interleukin 4 (IL-4), although preferably of porcine origin, can also be of murine or human origin. According to one mode of implementation of this process, the pigs from which the stem cells are produced have between approximately 2 days and approximately 6 weeks. However, the process can be applied to cells from embryos or older pigs. Advantageously, the cells are kept in the medium for at least about 30 days.

The present invention further relates to cultures and lines of pig mast cells capable of being obtained by said method. The term “mast cells” is understood to mean cells which, among other characteristics, have metachromatic cytoplasmic granules containing heparin-type molecules and proteases such as tryptase, and express on their surfaces receptors such as the SCF receptor called c-kit or still the IgE receptor. The term “culture” here generally designates a cell or a set of cells cultured in vitro. A culture developed directly from a cell or tissue sample taken from an animal is called "primary culture". The term “lineage” is used from the moment at least one passage, and generally several consecutive passages in subculture have been successfully carried out, and designates any culture which results therefrom (SCHAEFFER, In Vitro Cellularand Developmental Biology, 26 , 91-101, 1990).

The present invention further relates to cultures or lines of porcine mast cells characterized in that they produce heparin-type molecules having a ratio between the disaccharides Ils and III close to that of porcine heparin. The term heparin-type molecules is understood to mean highly sulphated polysaccharides in which more than 80% of the glucosamine residues are N-sulphates and the number of O-sulphates is greater than that of N-sulphates. Advantageously, such cultures or lines produce heparin-like molecules having a relationship between the disaccharides Ils and llls included. between 0.5 and 5 (preferably between 2.5 and 2.5, even more preferably between 1, 3 and 1, 9) and / or heparin-like molecules having a ratio between the disaccharides ls and Ils of between approximately 3 and 8 ( preferably between 4 and 7 even more preferably between 5 and 7).

Established cultures or lines of porcine mast cells according to the present invention can also be characterized in that they produce, at least 0.1 μg of heparin-type molecules / 10 ⁶ cells (preferably at least 1 μg, even more preferably at least 10 μg).

Advantageously, such cultures or lines produce heparin-type molecules in which the amounts of disaccharides ls are greater than the amounts of disaccharides They, the amounts of disaccharides They are greater than the amounts of disaccharides llls, and the amounts of disaccharides llls are greater to the quantities of disaccharides IVs,

According to another advantageous embodiment, such cultures or lines produce heparin-type molecules having relationships between the disaccharides ls, Ils, llls and IVs close to those of heparin.

Advantageously, such cultures or lines produce heparin-like molecules comprising at least 30% of disaccharides ls (preferably at least 40%, even more preferably at least 50%). Advantageously, such cultures or lines produce heparin-like molecules having an anti-Xa activity greater than at least 10 IU / mg (preferably at least 20 IU / mg) and / or having an anti-Ia activity greater than at least minus 10 IU / mg (preferably at least 20 IU / mg).

According to a preferred embodiment of the invention, such lines are the lines of porcine mast cells deposited with the Collection of Cultures of Microorganisms of the Institut Pasteur (CNCM) 28 rue du Docteur Roux, 75724 Paris cedex 15, France , on April 09, 2003 respectively under n ° 1-3010, I-3011, 1-3012, 1-3013, 1-3014. These lines, deposited with the CNC, have the advantage of having been obtained from pigs meeting health requirements in line with the use of products derived from their cells in human therapy. These pigs come from protected farms, free from specific pathogenic organisms (IOPS) in pigs.

Nucleic acids, comprising genes coding for factors capable of improving the characteristics of the cultures and lines according to the present invention can be introduced into these cells. The term nucleic acid is used to refer to DNA or RNA. Advantageously, if is a complementary or genomic DNA.

Such factors can either promote cell growth or modulate the composition of the biological molecules they produce, and in particular the composition of heparin-like molecules.

They may be genes coding for immortalizing proteins such as the antigen T of the simian virus 40 (SV40), the proteins E6 and E7 of the human papilloma virus HPV, the proteins E1A of the adenovirus, the proteins EBNA2 of the virus. Epstein-Barr or Tax proteins from the HTLV-1 virus. The nucleic acid encoding the telomerase catalytic subunit, TERT, can also be used as an immortalizing gene.

The AgT of the SV-40 virus will preferably be used, the DNA sequence complementary to this antigen is available in GenBank under the reference NC_001669 ..

They may also be genes coding for proteins allowing cells to proliferate, for example G-CSF, SCF and the interleukins (IL-3, IL-4 and IL-6).

Recently, the study of murine and human mastocytomas has made it possible to identify mutations or deletions of the c-kit gene responsible for the constitutive activation of the c-kit receiver. Expression of the mutated c-kit gene at the level of V814 in immature mast cells IC2 induces the transformation of these cells, namely the acquisition of growth independent of SCF and of tumorigenic potential (Pia et al, Blood, 87 (8), 3117-3123, 1996). A nucleic acid comprising such a mutated gene can be introduced into these cells.

They can be genes coding for proteins such as ser / gly or enzymes acting on the sulfation of heparin-like molecules. Such an enzyme can be an O-sulfatase, such as a 3-O-sulfatase. or a 6-0 sulfatase.

Advantageously, such an enzyme is 3 O-sulfatase-1 (3-OST-1), preferably 3 O-sulfatase-1 from pigs.

The nucleic acids comprising these genes can be introduced into these cells by any method known to a person skilled in the art and in particular by transfection, by nucleoporation or by electroporation. Retroviral vectors carrying these genes can also be used to transfect these cells.

In the context of the present invention, the applicant has demonstrated that the introduction of a nucleic acid encoding a 3-OST, and in particular 3-OST-1, makes it possible to modulate the composition of heparin-like molecules of mast cells , regardless of the type of mast cell of porcine origin. The Applicant therefore does not intend to limit this object of its invention to the mast cells obtained by the process described above. The present application therefore relates to any mast cell of porcine origin into which a nucleic acid encoding a 3-OST has been introduced.

The Applicant has also determined the sequences of three proteins of porcine origin that can be used for the implementation of the present invention and the nucleotide sequences encoding these proteins.

Thus the subject of the present application is a protein of porcine origin of the c-kit type, characterized in that it has a C-terminal end having the sequence SEQ ID N ° 3. Such a protein can comprise a sequence having an identity of at least 99% with the sequence SEQ ID No. 2. Preferably, such a protein has a glutamine (Q) at position 40 and / or a lysine (K) at position 173. The present invention further relates to a polynucleotide or a nucleic acid, comprising a sequence coding for a protein porcine origin type c-kit. Such a nucleic acid can comprise a sequence having an identity of at least 99% with the sequence SEQ ID No. 1. Obtaining the complete sequence of the porcine c-kit was not evident from the state of the art.

The present application further relates to a protein of porcine origin having a 3-O-sulfatase activity. Such a protein can comprise a sequence having an identity of at least 95%, preferably at least 97%, and even more preferably at least 99%, of amino acid identity with a protein of sequence SEQ ID N 5.

The present invention further relates to a polynucleotide or a nucleic acid, comprising a sequence coding for a protein of porcine origin exhibiting 3-OST activity. Such a nucleic acid may comprise a sequence having an identity of at least 95%, preferably at least 97%, and even more preferably at least 99%, of identity in nucleotides with a nucleic acid of sequence SEQ ID # 4.

Obtaining the porcine 3-OST sequence was not obvious from the state of the art. Isolated porcine 3-OST is capable of exhibiting unexpected properties, and in particular of exhibiting better activity in porcine mast cells compared to 3-OST of other species known to those skilled in the art.

The present application also relates to a protein of porcine origin having a 6-O-sulfatase activity. Such a protein can comprise a sequence having an identity of at least 90%, preferably at least 95%, and even more preferably at least 99%, of amino acid identity with a protein of sequence SEQ ID N 7. The present invention has in in addition to the subject a polynucleotide or a nucleic acid, comprising a sequence coding a protein of porcine origin having a 6-OST activity. Such a nucleic acid may comprise a sequence having an identity of at least 95% preferably of at least 97%, and even more preferably of at least 99%, of identity in nucleotides with a nucleic acid of sequence SEQ ID # 6.

Obtaining the porcine 6-OST sequence was not obvious from the state of the art. Isolated porcine 6-OST is capable of exhibiting unexpected properties, and in particular of exhibiting better activity in porcine mast cells compared to 3-OST of other species known to those skilled in the art.

In addition, the present application relates to nucleic acids which hybridize under high stringency conditions with a nucleic acid of sequence SEQ ID No. 1, SEQ ID No. 4 or of sequence SEQ ID No. 6.

The “percentage of identity” between two nucleotide or amino acid sequences, within the meaning of the present invention, can be determined by comparing two optimally aligned sequences, through a comparison window. The part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.

The percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of the sequences for the comparison can be achieved by computer using known algorithms contained in the package of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN. By way of illustration, the percentage of sequence identity may be carried out using the BLAST software (BLAST versions 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using only the default parameters (S. F Altschul et al, J. Mol. Biol. 1990 215: 403-410, S. F Altschul et al, Nucleic Acids Res. 1997 25: 3389-3402). Blast searches for sequences similar / homologous to a reference “query” sequence, using the algorithm of AltschuI et al. The request sequence and the databases used can be peptide or nucleic, any combination being possible.

By “high stringency hybridization conditions” within the meaning of the present invention, the following conditions will be understood:

1- Pre-hybridization of membranes and:

-Mix: 40 μl salmon sperm DNA (10 mg / ml) + 40 μl human placental DNA (10 mg / ml) - Denature 5 min at 96 ° C, then immerse the mixture in ice.

- Remove the SSC 2X and pour 4 ml of formamide mix into the hybridization tube containing the membranes.

- Add the mixture of the two denatured DNAs.

- Incubation at 42 ° C for 5 to 6 hours, with rotation. 2- Competition of the labeled probe:

- Add 10 to 50 μl of Cot I DNA to the labeled and purified probe, depending on the amount of repetitions.

- Denature 7 to 10 min at 95 ° C.

- Incubate at 65 ° C for 2 to 5 hours. 3- Hybridization:

- Remove the pre-hybridization mix.

- Mix 40 μl salmon sperm DNA + 40 μl human placental DNA; denature 5 min at 96 ° C, then immerse in ice. - Add to the hybridization tube 4 ml of formamide mix, the mixture of the two DNAs and the labeled probe / denatured Cot I DNA.

- Incubate 15 to 20 hours at 42 ° C, with rotation. 4- Washes: - Wash at room temperature in SSC 2X, to rinse.

- 2 times 5 minutes at room temperature SSC 2X and SDS 0.1% at 65 ° C.

- 2 times 15 minutes at 65 ° C SSC 1X and 0.1% SDS at 65 ° C. Wrap the membranes in Saran and expose.

The hybridization conditions described above are suitable for hybridization, under high stringency conditions, of a nucleic acid molecule of variable length from 20 nucleotides to several hundred nucleotides.

It goes without saying that the hybridization conditions described above can be adapted as a function of the length of the nucleic acid for which hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art. .

Suitable hybridization conditions can, for example, be adapted according to the teaching contained in the work of HAMES and HIGGINS (1985, "Nucleic acid hybridization: a practical approach", Hames and Higgins Ed.,

IRL Press, Oxford) or in the work by F. AUSUBEL et al (1989, Current

Protocols in Molecular Biology, Green Publishing Associates and Wiley

Interscience, N.Y).

The proteins which are the subject of the present invention can be obtained by any means known to those skilled in the art. They are nevertheless advantageously obtained by expression of the nucleic acids as described above, coding for these proteins, optionally inserted into expression vectors, in advantageously selected cells, optionally followed by an extraction and a purification which can be total or partial.

The invention also relates to a recombinant vector comprising a nucleic acid according to the invention. Advantageously, such a recombinant vector will comprise a nucleic acid chosen from the following nucleic acids:

a) a nucleic acid coding for a protein having at least 60% amino acid identity with a sequence SEQ ID No 5 or SEQ ID No 7 or a peptide fragment or a variant thereof; b) a nucleic acid comprising a nucleic acid having a sequence SEQ ID No 1, SEQ ID No 4 or SEQ ID No 6, or a fragment or a variant thereof; c) a nucleic acid having at least 60% nucleotide identity with a nucleic acid having a sequence SEQ ID No. 4 or SEQ ID No. 6 or a fragment or a variant thereof; d) a nucleic acid hybridizing, under high stringency hybridization conditions, with a nucleic acid of sequences SEQ ID No. 1, SEQ ID No. 4 or

SEQ ID No. 6, or a fragment or variant thereof.

By “vector” within the meaning of the present invention is meant a circular or linear DNA or RNA molecule which is either in the form of single strand or double strand.

According to one embodiment, the expression vector comprises, in addition to a nucleic acid in accordance with the invention, regulatory sequences making it possible to direct its transcription and / or translation.

According to an advantageous embodiment, a recombinant vector according to the invention will notably comprise the following elements:

(1) elements for regulating the expression of the nucleic acid to be inserted, such as promoters and enhancers;

(2) the coding sequence included in the nucleic acid according to the invention to be inserted into such a vector, said coding sequence being placed in phase with the regulatory signals described in (1); and

(3) appropriate transcription start and stop sequences. In addition, the recombinant vectors according to the invention may include one or more origins of replication in cellular hosts in which their amplification or expression is sought, markers or selection markers.

Cells comprising such nucleic acids and / or expressing such proteins constitute other objects of the present invention. The present application further relates to a process for the production of heparin-like molecules comprising the cultivation of cultures or lines of porcine mast cells as described above.

The mast cells, obtained according to the invention in a medium containing IL-3, SCF and IL-4, have a better disaccharide structure than those obtained in a medium containing only IL-3 and of the CFS. The Applicant has also shown that the addition of IL-4 to the culture medium makes it possible to obtain from mast cells heparin-like molecules having characteristics closer to porcine heparin compared to those obtained using cells. obtained in a medium containing only IL-3 and SCF or containing IL-3 of SCF and IL-6 or IL-3 of SCF and G-CSF.

Also, the present application further relates to a process for the production of heparin-like molecules comprising the cultivation, in a suitable medium, of cultures or lines of porcine mast cells in a culture medium comprising at least about 0.1 ng / ml of IL-4 (preferably at least about 0.5 ng / ml, even more preferably at least about 1 ng / ml).

Mast cells can also be modified to overexpress IL4. Another object of the present application is therefore a process for the production of heparin-like molecules comprising obtaining cultures or lines of porcine mast cells transfected with a nucleic acid coding for IL4, and culturing these cells in a suitable culture medium. Such mast cells constitute in themselves an object of the present application. They can be obtained by any method known to a person skilled in the art and in particular by transfection, by nucleoporation or by electroporation of a nucleic acid comprising a gene coding for IL-4. Retroviral vectors carrying these genes can also be used to transfect these cells. The DNA sequence complementary to IL-4 has been described by Bailey et al (Biochim.Biophys.Acta. 1171 (3), 328-330, 1993).

The cells, lines and cultures according to the present invention can be maintained in culture under the conditions under which they were obtained. They can also be maintained in culture in media comprising reduced amounts of SCF, GM-CSF, IL-3, IL-4 and / or IL-6. They will nevertheless preferably be maintained in a medium containing IL-4.

These mast cells will preferably be cultivated in a defined culture medium (MEM / DMEM, RPMI, IMDM, etc.) supplemented with growth factors, used in combination or individually.

The media can also be supplemented with bovine serum, at a concentration of between 0.5% and 20% (v / v).

The addition of bovine serum to the culture media can be replaced by the use of a culture medium without serum such as AIMV (INVITROGEN) so as to reduce the protein concentration of the medium and the risks associated with the use of compounds of animal origin (KAMBE et al., J. Immunol. Methods, 240, 101-10, 2000). The independence of the cells with respect to the addition of serum and / or the use of growth factors can be obtained by mutation of the cell phenotype by the action of transforming and / or immortalizing agents (TSUJIMURA , Pathology International, 46, 933-8, 1996; PIAO and BERNSTEIN, Blood, 87 (8), 3117-23, 1996).

Mast cells can be cultured using techniques developed for the mass culture of eukaryotic cells, as described for example by GRIFFITHS et al. (Animal Cell Biology, Eds. Spier and Griffiths, Académie Press, London, vol. 3, 179-220, 1986). Bioreactors with a capacity greater than several m ^{3 can be used} as described by PHILIPS et al. (Large Scale Mammalian Cell Culture, Eds. Feder and Tolbert, Académie Press, Orlando, USA, 1985), or by MIZRAHI (Process Biochem, August, 9-12, 1983). The culture can also be carried out in suspension or on micro-support according to the technique described by VAN WEZEL (Nature, 216, 64-65, 1967). One can also use batch culture systems, which are frequently used for eukaryotic cell cultures, due to their greater simplicity of implementation on an industrial scale (VOGEL and TODARO, Fermentation and Biochemical Engineering Handbook, 2 ^nd edition, Noyes Publication, Westwood, New Jersey, USA, 1997). The cell densities obtained with these systems are generally between 10 ⁶ and 5 x 10 ⁶ cells / ml.

The productivity of batch cultures can be advantageously increased by removing part of the cells from the bioreactor (70% to 90%) for the operations of extracting GAGs and isolating heparin and by conserving the remaining cells within the same bioreactor to initiate a new culture. In this so-called repeated batch culture method, it is also possible to distinguish the optimum parameters of the cell growth phase from those allowing a greater accumulation of GAGs and heparin within the cells.

Continuous perfusion-type culture systems, with or without cell retention can also be used (VELEZ et al., J. Immunol. Methods, 102 (2), 275-278, 1987; CHAUBARD et al., Gen. Eng News, 20, 18-48, 2000). In the context of the present invention, it is possible in particular to use perfused culture systems allowing the retention of cells inside the reactor, and resulting in growth and a production greater than that which can be obtained in batch. Retention can be carried out using spin-filter, hollow fiber or solid matrix retention systems (WANG et al., Cytotechnology, 9, 41-49, 1992; VELEZ et al., J Immunol. Methods, 102 (2), 275-278, 1987) The cell densities obtained are generally between 10 ⁷ and 5 x 10 ⁷ cells / ml. The culture in bio-reactors allows, through the use of online measurement sensors, better control of the physico-chemical parameters of cell growth: pH, p02, Red / Ox, growth substrates such as vitamins, amino acids, carbon substrates (for example glucose, fructose, galactose), metabolites such as lactate or ammonia, etc.

It can be envisaged to increase quantitatively and qualitatively the content of heparin-like molecules in mast cells following treatment with sodium butyrate (Nakamura et al (Biochim.Biophys.Acta. 627, 60-70, 1980).

After 3 to 14 days of culture, preferably after 3 to 5 days, the cells are harvested and separated from the culture medium, generally by centrifugation or filtration.

Different centrifugation systems can be used, for example those described by VOGEL and TODARO (Fermentation and Biochemical Engineering Handbook, 2 ^nd Edition, Noyes Publication, Westwood, New Jersey, USA). Alternatively or in combination with centrifugation, separation can be carried out by tangential microfiltration, using membranes whose porosity is less than the average diameter of the cells (5 to 20 μm) while allowing the passage of the other compounds in solution / suspension. . The speed of the tangential flow and the pressure applied to the membrane will be chosen so as to generate little shear force (Reynolds number less than 5000 sec ^"1 ) in order to reduce the clogging of the membranes and preserve the integrity of the cells during l separation operation.

Different membranes can be used, for example, spiral membranes (AMICON, MILLIPORE), flat membranes or hollow fibers (AMICON, MILLIPORE, SARTORIUS, PALL, GF).

One can also choose membranes whose porosity, charge or grafting makes it possible to carry out a separation and a first purification with respect to any contaminants which may be present in the culture medium, such as cellular proteins, DNA, viruses or other macromolecules.

The use of membranes of reduced porosity can also be envisaged in the case where heparin has been released from the intracellular content, by degranulation or lysis of all or part of the mast cells, and is present in the culture medium at the time of l 'separation step. In this case, the separation of the cells is combined with an ultrafiltration step on one or more membranes whose arrangement and porosity makes it possible to concentrate the heparin and to separate it from the other species present in the medium, depending on the size and molecular weight, and possibly electrical charge or biological properties.

In the context of this embodiment, the cut-off threshold for the membranes is preferably between 1000 and 5 Kda. Membrane systems similar to those used for microfiltration can be used, for example, spiral membranes, flat membranes or hollow fibers. One can advantageously use membranes making it possible to carry out a separation and a purification of heparin, because of their loading properties or of the grafting of ligands having an affinity for heparin (for example antibody, ATIII, lectin).

However, it will generally be preferable to use methods of production and harvesting of the cells making it possible to conserve heparin in the intracellular content. The recovery of heparin from mast cells can also be done after degranulation or lysis of the cells.

Degranulation can be caused by the binding of specific ligands to receptors present on the surface of mast cells, for example the binding of allergen-type agents (such as Fc fragment of IgE or analogs of this fragment) to IgE receptors of mast cells .

Other agents can also induce degranulation of mast cells. These agents can be classified into several categories such as agents cytotoxics, enzymes, polysaccharides, lectins, anaphylatoxins, basic compounds (compound 48/80, substance P, etc.), calcium (ionophore A23187, ionomycin, etc.). [D. Lagunoff and T. W. Martin. 1983. Agents that release histamine from mast cells. Ann. Rev. Pharmacol. Toxicol., 23: 331-51]. The use of degranulating agent can be carried out repeatedly on the same cells maintained in culture. In this production mode, productivity is significantly increased by simplifying the harvesting process from the supernatant and by maintaining the cells in culture. In the particular case of the ionophore A23187, the degranulation of the mast cells can be induced for example by treatment of 2.10 ⁶ cells / ml of mast cells with the ionophore A23187 at concentrations between 1 to 100 μg / ml and times of action varying from 1 minute to 4 hours.

Mast cell lysis can be induced, for example, by osmotic shock using hypotonic or hypertonic solutions, by thermal shock (freezing / thawing), by mechanical shock (for example sonication or pressure variation), by action of agents chemical (NaOH, THESIT ™, NP40 ™, TWEEN 20 ™, BRIJ-58 ™, TRITON X ™ -100, ...) or by enzymatic lysis (papain, trypsin, ...) or by a combination of two or more of these methods.

To extract and purify heparin from the cell lysate, separate the polysaccharide chains from the serglycine nucleus, and separate the heparin chains from the other GAGs present in the extraction medium, methods similar to those used in the part of the extraction and purification of heparin from animal tissues, which are known in themselves, and described in general works, such as the manual of DUCLOS, cited above).

By way of nonlimiting examples, to separate heparin from nucleic acids and cellular proteins, and to dissolve it, that is to say to break the bonds with the serglycine nucleus:

- the cell lysate can be subjected to one or more enzymatic digestions (pronase, trypsin, papain, etc.); - the heparin-protein bonds can be hydrolyzed in an alkaline medium, in the presence of sulfates or chlorides;

- it is also possible to carry out a treatment in an acid medium (for example with cold trichloroacetic acid) to destroy the nucleic acids and proteins originating from the cells, supplemented by the use of an ionic solution which makes it possible to dissociate the interactions GAGs- proteins.

It is also possible to carry out an extraction with guanidine, after enzymatic hydrolysis; to purify the dissolved heparin, it can for example be precipitated by potassium acetate, by quaternary ammonium, by acetone, etc.

These purification steps can advantageously be supplemented or replaced by one or more chromatography steps, in particular anion exchange chromatography.

The present invention also relates to the heparin preparations capable of being obtained from mast cell cultures by implementing a method according to the invention.

The heparin preparations according to the invention, which have biological properties comparable to those of the heparin preparations obtained in the prior art from animal tissues, can be used in all the usual applications of heparin.

FIGS. 1 A to 1 H illustrate the anti-tryptase labeling of the mast cells obtained after 3 weeks of culture, respectively under conditions C1 to C8. The shaded and clear peaks correspond respectively to the controls (without antibody) and to the cells obtained under conditions C1 to C8.

FIGS. 2A to 2H illustrate the labeling of the IgE receptors of the mast cells obtained after 5 weeks of culture, respectively under conditions C1 to C8. The hatched, shaded and clear peaks correspond respectively to unlabeled cells (non-mast cell swine cells), to control cells and to cells obtained under conditions C1 to C8. FIGS. 3A to 3H illustrate the anti-tryptase labeling of the mast cells obtained after 7 weeks of culture, respectively under conditions C1 to C8. The shaded and clear peaks correspond respectively to the controls and to the cells obtained under conditions C1 to C8.

Figures 4 A to 4H illustrate the FGF labeling of mast cells obtained after 8 weeks of culture, respectively under conditions C1 to C8. The shaded and clear peaks correspond respectively to the controls and to the cells obtained under conditions C1 to C8.

FIG. 5 illustrates the growth of the cultures under the different conditions C1 to C8 in the first 7 weeks of culture.

FIG. 6 represents the chemical structures of the disaccharides ls, Il llls and IVs corresponding to the disaccharides N sulfated by heparin, as well as the homologous acetylated disaccharides la lia, llla et.lva.

Figure 7 illustrates the reactor growth of mast cells obtained under conditions C1, C7 and C8.

The present invention is illustrated by the following implementation examples. These examples are purely illustrative and should not be considered as limiting.

EXAMPLES

EXAMPLE 1 Isolation of Mast Cell Populations from Bone Marrow of Young Pigs and Obtaining Lines

The animals used for the samples come from protected breeding, free from specific pathogenic organisms (IOPS) of the pig (MERIAL SA Lyon France) The sternum of piglets aged four and six weeks, respectively PI and PHI are aseptically removed and then transported in one sterile container in the laboratory to be decontaminated and rinsed successively with a solution of pure bleach diluted 1/100 in PBS buffer (Phosphate Buffer Saline pH 7.4) then in PBS. The sternum is then cut, then the bone marrow is aspirated using a syringe to be then diluted in PBS. The medullary suspension is sieved on a sterile pad; diluted in 40 ml of PBS and then centrifuged for 10 minutes at 400 g. The cell pellet is taken up in 5 ml of PBS and then purified on 5 ml of Ficoll (Dutscher) (1100 g × 10 '). The ring containing the medullary cells is recovered then rinsed twice in PBS (14ml, 400gx10min) then taken up in 2 ml of PBS to be counted, approximately 1x 108 total cells per sternum.

After counting, the cells are centrifuged then taken up at a concentration of 1-3 x10 ⁶ cells / ml in 6-well culture plates and 4 ml per well in the medium containing the following components: MEMD (Invitrogen) Fetal calf serum 15% (PAA Laboratories) Penicillin (Sigma) 100 IU / ml, 100 μg / ml Streptomycin (Sigma) 2ng / ml porcine r-IL3 (Biotransplant) 80ng / ml r-porcine r-SCF (Biotransplant). As soon as the cells are cultured, the cells are cultured in the culture medium described above supplemented with cytokines (recombinant porcine IL4 1 ng / ml R1 D Systems; recombinant porcine IL6 100ng / ml, R&D Systems; recombinant human G-CSF 10ng / ml) as shown in Table 1 below.

Table 1: cell culture conditions

(+ medium with; - medium without cytokine)

The culture plates are incubated at 38 ° C +/- 0.5 ° and under an atmosphere containing 5% CO 2. Twice a week and for eight weeks, the medium of each well is renewed with fresh medium. The characterization of the mast cell phenotype of the isolated cells is carried out from week 2 and then at regular intervals (week 3, week 5 and week 7)

Pig mast cell lines, obtained under some of the conditions indicated above, were deposited with the Collection of Cultures of Microorganisms of the Institut Pasteur (CNCM) on April 09, 2003.

These lines deposited under the numbers 1-3010, 1-3011, 1-3012, 1-3013, 1-3014 were respectively obtained under conditions C1, C2, C3, C4 and C5 described in Table 1.

Confirmation of the mast cell phenotype of the cells in each culture condition is demonstrated by the detection in fluorocytometry of the presence of specific markers such as IgE receptor, and tryptase. The detection of labeling with FGF is also carried out for the revelation of the site of attachment of FGF on heparin of mast cells.

Tryptase labeling

1 ml of cell suspension of each condition is taken. Each sample is rinsed once by centrifugation in PBS buffer, then the cells are resuspended in 400 μl of PBS buffer containing 0.5% BSA (Bovine Serum Albumin) and 0.01% sodium azide.

The cells are then permeabilized at 4 ° C. in cytofix / cytoperm solution (Pharmingen), 200 μl per sample and incubated for 25 minutes. After two rinses in permawash buffer (Pharmingen) the samples are incubated for 30 minutes at 4 ° C with 1 μg of murine monoclonal antibody specific for tryptase (Mouse anti-human Mast cell tryptase; Chemicon) After three rinses in permawash buffer, the labeling is revealed by incubation for 25 minutes with an anti-mouse immunoglobulin conjugate labeled with FITC (FITC-conjugates Affinity pure Goat Anti Mouse IgG; Jackson Immunoresearch) Duplicates of samples are produced according to the same procedure except for the incubation with the anti tryptase antibody in order to be able to subtract during the analysis the fluorescence due to the non-specific binding of the FITC-labeled conjugate.

At the end of the last incubation, the cells are rinsed twice in permawash buffer, then resuspended in cold PBS buffer supplemented with 1% formaldehyde (Sigma) The analysis in cytofluorimetry is carried out on FACS (Faxcalibur Becton Dickinson)

IgE receptor labeling

About 2x10 ⁵ cells of each condition are removed, rinsed twice in PBS and then incubated with 2 μg for 10 ⁶ cells of canine IgE (Monoclonal Canine IgE; Bethyl) The samples are incubated for 3 h 30 min at 37 ° C, then rinsed twice in PBS . After resuspending in PBS, the samples are then incubated for 30 minutes at 4 ° C with 5 μg for 10 ⁶ cells of goat anti-canine IgE antibody (Goat anti Dog IgE affinity purified; Bethyl)

After incubation, the samples are rinsed again twice in PBS, then incubated for 30 minutes with an anti-goat conjugate labeled with FITC (Donkey anti Goat / Sheep FITC; Serotec) After two rinses in PBS buffer, the samples are returned in suspension and fixed in a buffer supplemented with 1% formaldehyde. As before, duplicate samples are also produced by omitting the incubation with IgE in order to subtract during the analysis the fluorescence due to the non-specific binding of the FITC-labeled conjugate. A sample of non mastocytic pig cells (PIR) is also analyzed under the same conditions in order to confirm the specificity of the labeling.

Marking of the FGF fixation site. The cell culture samples to be analyzed are distributed in a 96-well plate, conical bottom, at the rate of 0.2.10 ⁶ per well and then centrifuged at 1400 rpm, 4 min. The cell pellet is rinsed in 100 μl of PBS buffer containing 5 g / l of bovine albumin and then centrifuged at 1400 rpm, 4 min. Two successive rinses are carried out under the same conditions.

The cell pellets are diluted in a Cytofix Cytoperm fixing / permeabilization buffer (Pharmingen), rinsed in 100 μl of Perm / Wash buffer (Pharmingen) and then centrifuged at 1400 rpm, 4 min, 4 ° C. Three successive rinses are carried out under the same conditions.

The cell pellets are diluted in 100 μl of Perm / Wash buffer containing 172 ng / ml of FGF basic (R&D Systems) and incubated for 30 minutes in ice. The cells are rinsed in 100 μl of Perm / Wash ™ buffer and then centrifuged at 1400 rpm, 4 min, 4 ° C. Three successive rinses are carried out under the same conditions.

The cell pellets are diluted in 100 μl of Perm / Wash buffer containing 1 μg of anti-FGF basic mouse monoclonal antibody (R&D Systems) coupled with biotin and incubated for 30 minutes in ice. The cells are rinsed in 10 μl of Perm / Wash buffer and then centrifuged at 1400 rpm, 4 min, 4 ° C. Three successive rinses are carried out under the same conditions.

The cell pellets are diluted in 100 μl of Perm / Wash buffer containing a solution of streptavidin peridinin chlorophyll-a protein and incubated for 20 minutes, in ice, in the dark. The cells are rinsed in 100 μl of Perm / Wash ™ buffer and then centrifuged at 1400 rpm, 4 min, 4 ° C. Three successive rinses are carried out under the same conditions. The pellet is diluted in 150 μl of cold PBS buffer containing 5 g / l of bovine albumin, 0.01% sodium azide, 1% formaldehyde. The presence of intra cytoplasmic labeling is detected by cytofluorimetry. Duplicate samples are produced according to the same procedure with the exception of incubation with the anti-FGF antibody in order to be able to subtract during the analysis the fluorescence due to the non-specific binding of the FITC-labeled conjugate. A sample of non mastocytic porcine cells (IRP) is also analyzed under the same conditions in order to confirm the specificity of the labeling.

Figures 1, 2, 3 and 4 present the results of the phenotypic characterization of the mast cells obtained respectively after 3, 7 and 8 weeks of culture.

A positive and specific labeling of the cells with respect to markers of mast cells, IgE receptor, and tryptase is observed, as well as the detection of the intracellular binding of FGF. The detection carried out on the cells from the third week of culture is positive for the presence of tryptase. The cultures in conditions 1 to 5 are homogeneous, and contain 100% of mast cells, as revealed by labeling of the IgE receptor from week 5. On week 7, the cultures in conditions C1 to C5 and C7 are 100% homogeneous , the homogeneity of the cultures under conditions C6 and C8 is greater than 50%.

Analysis in electron microscopy

The characterization of the isolated cells was also completed by observation by electron microscopy. The cells have a characteristic morphology of mast cells with many granulations, with a large eccentric nucleus, an irregular contour.

Cell proliferation during isolation At regular intervals, culture samples for each condition (C1 to C8) are taken and counted under the microscope after dilution in PBS buffer supplemented with 0.4% trypan blue.

A decrease in cell concentration is observed during the first four weeks of culture (S1 to S4) corresponding to the death and lysis of the medullary cells not stimulated by SCF and to the passage from a heterogeneous culture to an essentially mast cell culture. From the fifth week (S5) we observe a proliferation of cultures correlated with a more intense marking specific markers of mast cells. The proliferation is significantly greater for the culture conditions comprising NL4 (FIG. 5).

Characterization of the heparin content of cultures in HPLC

After 15 weeks of culture and amplification, samples were taken to analyze the proteoglycan composition of the mast cells according to the protocol described by Linhardt et al (Biomethodes, 9, 183-197, 1997) The samples are treated as follows:

Proteolysis: The cell samples, 2 × 10 ⁶ cells, are centrifuged and rinsed twice in PBS buffer. Each pellet is taken up in 100 μl of distilled water added with 10 μl of alkalase (Novozymes) then heated for 5 hours at 60 ° C. with stirring. Then the samples are diluted with 200 μl of Tris 10mM buffer pH 7.0 (Prolabo) 0.5M NaCl (Prolabo) before being centrifuged for 10 minutes at 10,000 Trs / min. The proteolysis stage makes it possible to release the intracellular content and to dissociate the protein-polysaccharide bonds.

Extraction: The supernatant of each sample is purified by ion exchange on SAX quaternary ammonium resin in 96-well plate format, 100 mg / 2 ml (Thermohypersil). After fixing and washing in Tris pH7 buffer, 0.5M NaCl, Glyco-aminoglycans (GAGs) are eluted with 500 μl of Tris pH7.0 buffer, 3M NaCl.

Desalting / concentration: The samples are then desalinated on a permeation gel column (NAP-5, Pharmacia)

After elution in a volume of 1 ml, the samples are concentrated by lyophilization, then taken up in 130 μl of distilled water.

Depolymerization. For the HPLC analysis, the GAGs are depolymerized by a mixture of heparinases I, II and III (Grampian enzymes) Each heparinase solution is adjusted to 0.5 IU / ml in phosphate buffer. The heparinase solution I, II, III is prepared by mixing 1/3 volume by volume of each heparinase solution. For 100 μl of sample to be analyzed, 15 μl of the heparinase mixture is added and 10 μl of acetate buffer containing 0.73 ml of 100% acetic acid (Prolabo) 12.5 mg of bovine albumin (Sigma) and 39.5 mg of calcium acetate (Prolabo) for 30 ml of distilled water.

HPLC analysis.

The samples are then analyzed by HPLC on a Waters spherisorb SAX 5 μm, 250 × 3 mm, Thermohypersil column. 50 μl of sample are injected by analysis, the mobile phase buffer is composed of 2.5mM of Sodium Di Hydrogeno Phosphate (Na2HP04, Prolabo) whose pH is adjusted to 2.9 with ortho-phosphoric acid (H3P04, Prolabo ). The elution of the disaccharides constituting the GAGs extracted from the cell samples is carried out in a gradient from 0 to 100% in 50 minutes of 2.5 mM Na2HP04 buffer and 1 M of perchlorate (NaCI04, Prolabo) The disaccharides are detected by their retention time and compared to a standard heparin sample (Aventis) in UV at 234 nM.

Analysis of cell cultures after 15 weeks of culture reveals the presence in significant quantities of heparin-like compounds in the cells, confirming the specifically mast cell nature of the isolated cultures. We find indeed the major disaccharides constituting heparin as described by Linhardt et al (Biomethodes, 9, 183-197, 1997). These disaccharides are mainly represented by ls, They llls and IVs (chemical structures represented in FIG. 6) corresponding to the disaccharides N sulfated from heparin. We also find the acetylated homologous disaccharides IIIa, IIIa and IVa (FIG. 6).

Table 2 shows the compositions obtained for each culture. A reproducible modulation of the disaccharide structure is observed as a function of the presence or absence of IL4, this modulation is mainly observed on the percentage of the disaccharides They and IIIs. EXAMPLE 2 Cultivation of the lines and analysis of the production of heparin-like molecules

The disaccharide profile of the isolated mast cells was analyzed for three conditions of culture media (C1, C7 and C8). The cells were amplified in suspension and cultured in a 100 ml spinner.

Cellular culture.

The initial cell density is 2 × 10 ⁵ cells per ml, the cells are incubated under an atmosphere of 5% CO 2 at 37 ° C. and counting at regular intervals is carried out under the microscope.

Samples of the cultures thus produced are taken during the counts for analysis in HPLC of the content of heparin-type polysaccharides and measurement of the biological activity anti lia and anti Xa. Under these conditions, the maximum cell density is between 4 and 6.10 ⁵ cells / ml with a doubling time in the exponential phase between

24 and 48 hours.

Figure 7 illustrates the growth of mast cells on the 14th passage.

Analysis of polysaccharides.

HPLC analysis of the samples for three days of harvest (D4, D7 and D10) shows for all cultures a heparin-like profile of the polysaccharides with an effect of IL4 on the relative percentage of the disaccharides Ils and IIIs. The productivity of the cultures is significant, ranging between 2 and 12 μg for 10 ⁶ cells.

By comparison, the cultures of murine mast cell lines such as the MST cells described by Montgomery et al (Proo.Natl. Acad. Sci., 89, 11327-11331, 1992) or the human mast cell line HMC1 ( Butterfield et al Leuk Res, 12 (4), 345-355,1988) exhibit a productivity of heparin-like compounds 20 to 200 times lower than the porcine lines which are the subject of the present invention. (Tables 3 and 4) Biological activity of polysaccharides:

The inactivation of factors Xa and lia is characteristic of heparin and makes it possible to differentiate it from heparan sulfate and dermatan. The method used is that described in the monograph of the European Pharmacopoeia 3rd edition (1997).

The reaction takes place in three stages:

1: ATI II + heparin [ATI II - Heparin]

2: [ATIII - Heparin] + Excess factor [ATIII-Heparin-Factor] + Residual factor

3: Residual factor + Chromophore substrate Para-nitroaniline colored The amount of Paranitroaniline released is inversely proportional to the amount of heparin.

The anti-Xa or Anti-lla quantity is measured relative to a calibration line established with the SPIM standard (International Heparin Standard). The sensitivity of the method is 0.006IU / ml.

The biological activity is expressed in IU / mg taking into account the quantification of the disaccharides obtained by HPLC.

The analysis performed on the 10 day of harvest after the end of the growth phase reveals an anti-Xa and anti-Ila biological activity of between 10 to 25 IU / mg.

It is noted that for this stage of the culture, that the ratio between the two activities is close to 1, which is the characteristic ratio of the heparin obtained from the extraction from the intestinal mucosa of pigs.

The results obtained by measuring the inactivation of factors Xa and lia are summarized in Table 5.

Table 5: measurement of the inactivation of factors Xa and lia

EXAMPLE 3 Genetic modification of isolated cells

Mast cells can be genetically modified by the introduction of an exogenous nucleic acid using for example transfection, electroporation, nucleoporation or infection techniques which will result in transient or stable expression of the nucleic acid introduced. In the case of stable expression, DNA can be integrated into the cell genome or maintained as an episome.

1 Transfection by nucleoporation and electroporation.

Stably transfected cells can be obtained using the nucleoporation method described below, by applying, 24 to 72 hours after nucleoporation, a selection pressure (hygromycin, geneticin, blasticidin, puromycin or zeocin. Resistance to the agent selection is conferred by the integration of the plasmid carrying the gene of interest and the resistance gene.

nucleoporation

This method is used preferentially because it makes it possible to target DNA directly in the nucleus.

1 to 2.10 ⁶ mast cells, in the exponential phase, preferably after 3 or 4 days of culture, are centrifuged at 1000 rpm 5 minutes and taken up in 100 μl of nucleofection solution (Amaxa, Kit 8351). 2 to 4 μg of pcDNA3.1-eGFP, plasmid coding for GFP, are then added to the cell suspension. The cells are then transferred to the electroporation cuvette and subjected to an electric shock by the use of a specific program (such as U14, T20 and T22 AMAXA) The cells are then transferred into 2 ml of complete medium preheated to 37 ° C. and then incubated at 37 ° C., 5% CO 2.

24 to 48 hours after transfection, the cells are harvested to be fixed with 1% paraformaldehyde (Prolabo). For this, the entire culture is centrifuged for 5 min at 1000 rpm. After removal of the supernatant, the cells are washed in 4 ml of 1x PBS and then centrifuged again. The cell pellet is then taken up in 1 ml of 1% paraformaldehyde. The cells thus fixed are then analyzed with a cytometer (Cytomics FC 500, Beckman Coulter) The nucleoporation conditions described above make it possible to transfect pig mast cells with a transfection efficiency of between 30 to 50% while obtaining good cell viability , greater than 50% •}

Electroporation 1 to 5.10 ⁶ cells, in the exponential phase, are brought into contact with 1 to 30 μg of DNA. The cells, transferred to a 4 mm electroporation cuvette, are incubated for 5 min in ice before being electroporated at a voltage between 150 V and 400 V with a capacitance of 500 or 960 μF (Gene Puiser II, Biorad) After electroporation, the cells are again incubated for 5 min in ice to finally be transferred to 5 ml of complete culture medium and incubated at 37 ° C., 5% CO 2.

The process of selecting cells which have integrated the transgene stably uses the same technique as described above using the resistance conferred by the integration of the plasmid on a selection agent.

2 Transfection with viral vectors, use of pantropic retro-viral vectors.

As an alternative to the transfection methods by electroporation and nucleoporation, recombinant and deleted retro-viral vectors can be used for replication. One can for example use pseudo-typed vectors with the envelope glycoprotein of the vesicular stomatitis virus (VSV-G) which allows the production of pantropic retro-viral vectors capable of infecting porcine cells.

In this mode of transfection, the retro-viral vector carrying the gene of interest to be expressed or integrated into the porcine mast cell is first produced using the packaging cell such as GP-293 (Clontech protocol ref PT 3132-1), which possesses the genetic elements for the production of the vector (gag and poï) with the exception of the gene for the production of the pseudotyped envelope protein (env-VSV-G).

During the production of the retro-viral vector, the packaging cells are co-transfected with the plasmid coding for the envelope gene VSV-G and a retro-viral plasmid coding for the gene of interest under the control of a promoter with or without a selection gene.

In practice, GP-293 cells are cultured 48 hours to 72 hours before transfection in order to be in the exponential growth phase. On the day of the transfection, the culture medium is changed with fresh medium (15-20 ml for 10 ⁶ cells), then 1 to 2 ml of solution containing the mixture of the plasmid VSV-G (5 to 20 μg for 10 ⁶ cells ) and the plasmid carrying the gene of interest (10 to 30 μg for 1 Q ⁶ cells), in pH7 calcium phosphate buffer are added dropwise to the culture medium (1 to 2 ml (Promega)

The cells are then incubated again for 16 to 24 hours at 37 ° C or preferably at a temperature between 32 and 35 ° C. The culture medium is changed again with fresh medium. The cells are incubated for an additional 48 hours at 32-35 ° C. At the end of the incubation period, the culture supernatant containing the neoformed retro-viral vectors is harvested. Part of the packaging cell infection supernatant is aliquoted and frozen at -80 ° C, the other part is mixed with the mast cell culture medium in exponential growth phase. In practice, the mast cells in culture are centrifuged and resuspended in a medium containing 50% of fresh medium and 50% of infection supernatant. Mast cells are incubated for 24 hours at 32-35 ° C, then the medium is changed again with fresh medium.

48 to 72 hours after infection of the mast cells, samples are taken to analyze by cytofluorimetry in the case of the infection control with the fluorescent reporter gene GFP (Green Fluorescent Protein) or by PCR for infections with the gene of interest . By cytofluorimetry the measurement of the transfection efficiency is greater than 20% of the total cells.

The populations of transfected cells are then selected by adding to the culture medium the cytotoxic agent (Hygromycin, puromycin, G418,

Zeocyne) for which only mast cells transfected with the retroviral vector carrying the gene of interest and the resistance gene continue to grow. By this method, the gene of interest is stably integrated and is expressed in a stable manner.

After selection and cloning of the populations, the selection agent can be removed from the culture medium while retaining the expression of the gene of interest. The retroviral vector produced in this way does not replicate in the host mast cell and therefore there is no production of replicative vectors.

Alternatively, it is also possible to use vectors for which the expression is subjected to an induction of the promoter regulating the expression of the gene of interest by a compound added at the desired time in the culture medium.

EXAMPLE 4 Isolation of the porcine c-kit gene

The porcine c-kit gene was isolated by 3'-RACE using as RNA source, total RNA isolated from a culture of pig liver mast cells according to the procedure previously published (Piu et al, CR Acad. Sci. Paris, 316, 772-779, 1993).

The reverse transcription of 2 μg of total RNA into cDNA was carried out according to the protocol of the kit 573 'RACE (Roche), using as an primer an oligodT called OligodT anchor primer of sequence SEQ ID N ° 8 ^5' gac cac gcg tat cga tgt cga ctt ttt ttt ttt ttt ttv ^{3 '} . The cDNA is then amplified by PCR using the protocol of the Expand High fidelity System kit (Roche).

The PCR was carried out on 1 μl of cDNA, with the primer sense C15203 hybridizing specifically in the 5 ′ non-coding region of the porcine c-kit gene (nucleotides 24 to 42 relative to the published porcine c-kit sequence , GenBank ref AJ223228) of sequence SEQ ID N ° 9 ⁵ gga att ce cga gag cag gaa cgt gga aag gag ³ and the antisense primer named PCR anchor primer of sequence SEQ ID N ° 10 ⁵ gac cac gcg tat cga tgt cga c ^{3 '} hybridizing specifically in 3' at the level of the oligo dT primer. 10 cycles then 25 PCR cycles were applied (cycle 1: 15 sec denaturation at 94 ° C, 45 sec hybridization at 55 ° C and 4 min elongation at 68 ° C, cycle 2: 15 sec denaturation at 94 ° C, 45 sec hybridization at 60 ° C and 4 min elongation at 68 ° C).

The PCR product obtained is purified on 1% TBE1x agarose gel, using the Quiaquick gel extraction kit (Quiagen).

A second PCR, identical to the first, is carried out on 1/30 ^th of the purified PCR product, by applying 25 thermal cycles (15 sec of denaturation at 94 ° C, 45 sec of hybridization at 60 ° C and 4 min of elongation at 68 ° C). At the end of the second PCR, the PCR product is again purified to clone it into a pGEMTeasy vector according to the pGEM-T Easy vector System protocol (Promega).

The pig gene sequence is then partially determined by sequencing. The nucleotide sequence obtained is the sequence SEQ IDN ° 1. The deduced protein sequence is the sequence SEQ IDN ° 2. This sequence SEQ ID No. 1 shows differences compared to the sequence published under the reference AJ 223228. Indeed, the C-terminal end has 9 additional amino acids and the following differences in the nucleotide sequence have been observed, leading to the modification of two amino acids: Modifications (compared to the published sequence AJ 223228): nt 2371 → g,: H → Q nt 351 a → t nt 523 a → c nt 606 c → t nt 609 g → a nt 635 g → a ,: R → K nt 639 c → t nt 663 c → g nt 669 c → a nt 2016 a → g nt 2865 c → t

EXAMPLE 5 Isolation and Sequencing of the 3 'Coding Sequence of the Pig 3- OST Gene

The partial sequence of the porcine gene coding for 3-OST is available in an EST bank (GenBank accession number BF075483). Alignment of this sequence with the human sequence shows that about 650 bp of the 3 'coding region is missing.

The missing portion of the porcine 3-OST gene was identified by combining RT-PCR and 3'-RACE using as RNA source, porcine RNA isolated according to the protocol of the Trizol kit (Invitrogen)

The reverse transcription of 2 μg of total RNA into cDNA was carried out according to the protocol of the First Strand Synthesis Sytem kit (Invitrogen), using as primer a mixture of the oligonucleotides BS02 and BS03 of respective sequences SEQ ID N ° 11 5 ' -GCA GCA GCC ACG TCG GG-3 'and SEQ ID N ° 12 5'-TCA GTG YCA GTC RAA TGT TC-3'.

2 μl of these cDNAs were then amplified by PCR in the presence of a sense primer BS05 of sequence SEQ ID No. 13 5'-CGG NGA CCG CCT NAT CAG-3 'and of an antisense primer BS06 of sequence SEQ ID N ° 14 5'-TCA GTG YCA GTC RAA TGT TC-3 'with KOD hot start Polymerase (Novagen). After the 30 thermal cycles (15 sec of denaturation at 98 ° C, 30 sec of hybridization at 60 ° C and 30 sec extension at 68 ° C.), the amplified fragment of 277 bp was cloned into the vector pCR-Blunt II TOPO (Invitrogen, Zero Blunt TOPO PCR Cloning kit) and then sequenced. The sequence of this fragment was used to generate 2 primers BS21 and BS22 to isolate, by 3'-RACE, the entire 3 'region.

In the context of 3'-RACE, 1 μl of porcine liver RNA was reverse transcribed into cDNA according to the protocol of the First Strand Synthesis Sytem kit from Invitrogen, using as oligodT CDSIII of sequence SEQ ID N ° 15 (5'-ATT CTA GAG GCC GAG GCG GCC GAG ATG T ₃₀ VN-3 ').

The 3 ′ region of the gene coding for 3-OST was then amplified by 2 successive PCRs. The first PCR was carried out on 2 μl of cDNA, obtained previously, with the sense primer BS21 of sequence SEQ ID No. 16 5'-GCA CGC CCA GAT CGA CGC C-3 'and an antisense primer CDSIII. 30 thermal cycles were applied (10 sec of denaturation at 94 ° C, 30 sec of hybridization at 60 ° C and 120 sec of elongation at 68 ° C). The second PCR was then carried out under the same conditions as the first PCR with - 1 μl of product resulting from the first PCR using the primer sense BS22 of sequence SEQ ID No. 17 5'-CAA ACT CCT CAA TAA ACT GCA CG-3 'and the anti-sense primer CDSIII.

The sequencing of the PCR product thus obtained at the end of 3'-RACE made it possible to identify the 3 'sequence of porcine 3-OST as well as around 250 bp of the non-coding region.

Isolation of the complete coding phase of the porcine 3-QST gene.

In order to clone the complete coding phase of porcine 3-OST, a new RT-PCR experiment was carried out using the information obtained in the first step.

The source of RNA is the same as in the previous step. 2 μg of RNA were reverse transcribed into cDNA according to the protocol of the First kit

Strand Synthesis Sytem from Invitrogen, using a dT ₂₄ oligonucleotide as the primer. The gene coding for 3-OST was then amplified by PCR in two stages. The first PCR made it possible to amplify the gene including a portion of the 3 ′ non-coding sequence of the gene, the second PCR then made it possible to amplify the coding sequence using primers compatible with the Gateway system (Invitrogen)

The first PCR was carried out on 2 μl of cDNA with a BS10 sense primer of sequence 5'-AGG CCC GTG ACA CCC ATG AGT-3 'hybridizing specifically in the 5' non-coding region of the porcine 3-OST gene and an antisense BS30 primer of sequence 5'-CAC CTA GTG TAC ACC ACA ATT TAC-3 'hybridizing specifically in 3' at the UTR. 35 thermal cycles were applied (10 sec denaturation at 98 ° C, 30 sec hybridization at 64 ° C and 150 sec elongation at 68 ° C)

A second PCR is then carried out on 1 μl of the PCR product in order to specifically amplify the coding phase. For this, we use the sense primer BS31 of sequence SEQ SD M ° 1S ^{5 '} GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC AGC ATG GCC GCG CTG CTC ^3' and the antisense primer BS32 of sequence SEQ ID N ° 19 ^{5 '} GGG ACC ACT TTG TAC AAG AAA GCT GGG TTT AGT GCC AGT CAA ATG TTC TGC C ^3' . The PCR program used is identical to that used for the first PCR.

The 1 kb PCR product was then cloned according to the procedure of the Gateway cloning technology kit, Invitrogen in the episomal vector pE-IRES-neo2. The sequence of the porcine gene has been verified by sequencing. The nucleotide sequence obtained is the sequence SEQ IDN ° 4. The deduced protein sequence is the sequence SEQ IDN ° 5.

EXAMPLE 6 Identification of the complete coding sequence of the porcine 6-OST gene The partial sequence of the porcine gene (nucleotide 682 to 910 of the human sequence) coding for 6-OST is available in an EST bank (GenBank accession number BE235545)

The complete coding sequence of the 6-OST gene was identified by combining two RT-PCR experiments with 5 'and 3'-RACE experiments using, as the RNA source, porcine RNA isolated from Trizol kit protocol (Invitrogen)

Reverse transcription of 80 ng of total RNA into cDNA was carried out according to the protocol of the First Strand Synthesis Sytem kit (Invitrogen), using as an primer an oligonucleotide dT24.

2 μl of these cDNAs were then amplified by PCR in the presence of a sense primer 386-03 of sequence SEQ ID No. 20 5'-AGA TGA CTG GTC GGG CTG C-3 'and an antisense primer 386 -01 of sequence SEQ ID N ° 21 5'-CAA TGA TRT GGC TCA TGT AGT CC-3 'with KOD hot start Polymerase (Novagen). After the 35 thermal cycles (15 sec of denaturation at 95 ° C, 30 sec of hybridization at 60 ° C and 2 min of elongation at 68 ° C), the amplified fragment of 537 bp was cloned into the vector pCR- Blunt II TOPO (Invitrogen, Zero Blunt TOPO PCR Cloning kit) then sequence.

The sequence of this fragment was used to generate three primers 386-05, 386-19 and 386-20 used for the next PCR and the 3 -RACE.

2 μl of the cDNAs obtained above were amplified by PCR in the presence of a primer sense 386-07 of sequence SEQ ID No. 22 5'-ATG GTT GAG CGC GCC AGC AAG TTC G-3 'and the anti-primer sense 386-05 of sequence SEQ ID No. 23 5'-GGT TAT TGG CCA GGT TGT AGG GGC-3 'with KOD hot start Polymerase (Novagen). After the 30 thermal cycles (15 sec of denaturation at 95 ° C, 30 sec of hybridization at 60 ° C and 1 min of elongation at 68 ° C), the amplified fragment of 718 bp was cloned into the vector pCR- Blunt II TOPO (Invitrogen, Zero Blunt TOPO PCR Cloning kit) then sequence.

The sequence of this fragment was used to generate two primers 386-24, 386-26 used for 5'-RACE. In the context of 3'-RACE, 1 μl of porcine liver RNA was reverse transcribed into cDNA according to the protocol of the First Strand Synthesis Sytem kit from Invitrogen, using as oligodT CDS-C of sequence SEQ ID N ° 24 5'-ATT CTA GAG GCC GAG GCG GCC GAC ATG T ₃₀ VC-3 '.

The 3 ′ region of the gene coding for 6-OST was then amplified by 2 successive PCRs. The first PCR was carried out on 2 μl of cDNA, obtained previously, with the sense primer 386-19 of sequence SEQ ID No. 25 5'-GGA CCT CTT CCA GCA GCG-3 'and the antisense primer CDS-C with Advantage 2 polymerase micx (Clontech). 24 thermal cycles were applied (7 sec of denaturation at 98 ° C, 10 sec of hybridization at 62 ° C and 2 min of elongation at 68 ° C) A second PCR was then performed on 2 μl of product from the first PCR using the sense primer 386-20 of sequence SEQ ID No. 26 5'-GCT ATC AGT ACA AGC GGC AGC -3 'and the antisense primer CDS-C. After the 30 thermal cycles (7 sec of denaturation at 95 ° C, 10 sec of hybridization at 62 ° C and 2 min of elongation at 68 ° C), the amplified fragment of 300 bp was cloned into the vector pCR- Blunt II TOPO (Invitrogen, Zero Blunt TOPO PCR Cloning kit) then sequence. This experiment made it possible to identify the 3 ′ region coding for 6-OST and approximately 32 bp of the non-coding region.

As part of the 5'-RACE, 2 μl of porcine liver RNA was reverse transcribed into cDNA according to the protocol of the First Strand Synthesis Sytem kit from Invitrogen, using as oligonucleotide 386-28 of sequence SEQ ID N ° 27 5'- CCA GGC TCA GCC CCG G-3 '.

The phosphorylated oligonucleotide okib57 of sequence_SEQ ID No. 28 5'-p GTA GGA ATT CGG GTT GTA GGG AGG TCG ACA TTG CC-3 'was grafted into 5' of the cDNA by ligation (RNA ligase, Roche).

The 5 ′ region of the gene coding for 6-OST was then amplified by 2 successive PCRs. The first PCR was carried out on 2 μl of grafted cDNA, with the okib58 sense primer of sequence SEQ ID No. 29 5'-GGC AAT GTC GAC CTC CCT ACA AC-3 'hybridizing to the okib57 primer and 386-24 anti-sense primer sequence SEQ ID No. 30_ 5'-TCA GCC CCG GGC CCG CG-3 'according to the protocol of the Adavantage 2 polymerase mix kit. 24 thermal cycles were applied (10 sec denaturation at 98 ° C, 10 sec hybridization at 64 ° C and 2 min elongation at 72 ° C). A second PCR was then carried out on 0.5 μl of product resulting from the first PCR using the primer meaning okib59 of sequence SEQ ID No. 31_5'- CTC CCT ACA ACC CGA ATT CCT AC-3 'and the primer anti-sense 386-26 of sequence SEQ ID No. 32_5'-GCC CGC GTA CTG GTA GAG G-3 '. After the 40 thermal cycles (10 sec of denaturation at 98 ° C, 10 sec of hybridization at 66 ° C and 2 min of elongation at 72 ° C), the amplified fragment of 170 bp was sequenced. This experiment made it possible to identify the 5 ′ region coding for 6-OST and approximately 14 bp of the non-coding region.

Isolation of the complete coding phase of the porcine 6-QST gene.

In order to clone the complete coding phase of porcine 6-OST, a new RT-PCR experiment was carried out using the information obtained in the first step.

The source of RNA is the same as in the previous step. 2 μg of RNA were reverse transcribed into cDNA according to the protocol of the kit First Strand Synthesis Sytem, using as primer the oligonucleotide dT CDSIII. The codan gene for 6-OST was then amplified by PCR using primers compatible with the Gateway system (Invitrogen). The PCR was carried out on 2 μl of cDNA with a sense primer 386-33 of sequence SEQ ID No. 33_5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AGG ACA ATG GTG ACA CAT GCG GCG GC-3 'and an antisense primer 386-34 of sequence SEQ ID N ^c 34 5'- GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA CCA CTT CTC GAT GAT GTG GCT C-3 '. 35 thermal cycles were applied (5 sec of denaturation at 98 ° C, 20 sec of hybridization at 66 ° C and 1 min 30 sec of elongation at 72 ° C)

The 1 kb PCR product was then cloned according to the procedure of the Gateway cloning technology kit, Invitrogen in the episomal vector pE-IRES-neo2. The sequence of the porcine gene has been verified by sequencing. The nucleotide sequence obtained is the sequence SEQ ID No. 6. The deduced protein sequence is the sequence SEQ ID No. 7.

EXAMPLE 7 Transformation of the lines according to the invention with the porcine c-kit gene

In order to obtain pig mast cells whose growth would be independent of SCF in the long term, mast cells can be transformed with the mutated c-kit gene.

For this, it is possible to use the c-kit gene, preferably porcine, carrying a point mutation responsible for the modification of valine 556 into glycine (gene noted c-kit ^G556 ), this mutation is analogous to the c-kit mutants ^G559 in mice and c-kit ^G560 in humans. Alternatively, it is also possible to use the c-kit gene deleted at the level of amino acids TQLPYDH 570 to 576, in mice this deletion is analogous to amino acids 573 to 579, in humans 574-580. Likewise, due to the inter-species conservation of the c-kit gene, murine, human, bovine genes or any other gene having at least 80% homology with the porcine gene can be used. In this case, it is then also possible to use a point mutation responsible for the modification of aspartic acid to valine 814 or 816 respectively in mice and in humans.

The mast cells are transfected by one of the methods described in Example 4, preferably nucleoporation, with an integrative vector in which the coding phase of the mutated c-kit gene is cloned under the control of a strong viral promoter (CMV, RSV) or cellular (EF1α). In addition to the mutated c-kit gene, this vector can also carry a gene coding for resistance to an antibiotic (geneticin, hygromycin, puromycin, etc.)

48 hours after transfection, the cells are counted, centrifuged and seeded at 2.10 ⁵ C / ml in the complete culture medium supplemented with the selection antibiotic. Cells are grown in the presence of selection for 2 to 3 weeks, which makes it possible to eliminate the cells which are not stably transfected. After this selection period, the cells are amplified.

The cells are then genetically analyzed by PCR and RT-PCR in order to verify the integration of the mutated c-kit gene and its expression. The independence of these cells from SCF is demonstrated by comparing the growth of cells transfected with the mutated c-kit gene in a medium without SCF with the growth of cells transfected with the empty vector in a medium with and without SCF.

A variant of this protocol consists in using a vector carrying only the mutated c-kit gene. In this case, the cells are selected 48 hours after transfection without using a selection agent but by seeding the cells at 2.10 ⁵ C / ml in a medium devoid of SCF. Untransfected cells are not able to grow in a medium lacking SCF unlike transfected cells.

EXAMPLE 8 Transfection of the lines according to the invention with the porcine 3QST gene.

In order to increase the biological activity of heparin-like compounds from mast cell cultures, it is possible to stably overexpress the gene coding for 3-OST-1 (3 O-sulfatase-1). use the pig gene. Alternatively, it is possible to use genes from other species encoding the expression of the 3-0 Sulfatase activity and having at least 80% homology with the porcine gene, in particular murine 3-OST-1.

The mast cells are transfected by the nucleoporation method described in Example 4, with an integrative plasmid in which the coding phase of the 3-OST gene has been cloned under the control of a strong viral promoter (CMV, RSV) or cell (EF1α) In addition to the 3-OST gene, this plasmid carries a gene coding for resistance to an antibiotic (geneticin, hygromycin, puromycin, etc.).

48 hours after transfection, the cells are counted, centrifuged and seeded at 2.10 ⁵ C / ml in the complete culture medium supplemented with the selection antibiotic. The cells are cultured in the presence of selection for 2 to 3 weeks, which makes it possible to eliminate the cells which are not stably transfected. After this selection period, the cells are amplified.

These cells are genetically analyzed by PCR and RT-PCR in order to verify the integration of the mutated c-kit gene and its expression.

The functionality of 3-OST is demonstrated by HPLC analyzes of heparin produced by mast cells compared with that produced by untransfected mast cells. Analyzes of the biological activity of the product make it possible to confirm the increase in the biological activity with respect to factor Xa and lia.

Table 2: HPLC analysis of the disaccharides obtained from the cultures isolated after 15 weeks.

Disaccharides% C1 C2 C3 C4 C5 C6 C7 C8 Reference

Standard heparin

IVs 12.9 8.6 9.9 1 Us 1 10.5 9.8 6.6 7.6 4.1 lia 3.9 4.0 4.7 3 9 3.9 4.1 4.2 2.9 4.0

Nia 0.7 2.2 1.2 2.9 0.9 2.4 0.5 1.6 1.1

They 16.3 O.-D 18.2 7.6 16.7 8.0 17.4 8.0 10.6 llls 12.4 23.7 14.0 26.8 12.2 26.2 13.2 24.6 6.4 la 0.3 0.4 0.8 0.5 0.5 0.5 0.4 0.4 1.4 ls 53.6 52.6 51.2 48.2 55.2 49.0 57.7 54.8 55.9

u Ifates / carboxylates 2.36 2.38 2.35 2.31 2.40 2.33 2.46 2.43 2.38

μg / 10 ⁶ cells 3.3 9.4 0.9 14.3 1.6 9.4 2.3 8.6

Table 3: Productivity and disaccharide profile of mast cell lines in culture for different species

Table 4: Disaccharide profile during culture (OJ to D10) culture condition C1

Claims

1. Method for obtaining cultures or lines of mast cells comprising the cultivation of a population of stem cells of bone marrow from young pigs or fetuses, in a medium comprising at least approximately

0.2 ng / ml IL-3, at least about 8 ng / ml SCF and at least about 0.1 ng / ml IL-4, 10 ng / ml IL-6 and / or 1 ng / ml of G-CSF.

2. Method according to claim 1 characterized in that the pigs are between about 2 days and about 6 weeks.

3. Method according to claim 1 characterized in that the cells are maintained in the medium for at least about 30 days.

4. Culture or line of pig mast cells capable of being obtained by a process according to one of claims 1 to 3.

5. Culture or line of porcine mast cells characterized in that it produces heparin-type molecules in which the quantities of disacharides ls are greater than the quantities of disacharides They, the quantities of disacharides They are greater than the quantities of disacharides llls, and quantities of disacharides llls are greater than quantities of disacharides IVs,

6. Culture or line of porcine mast cells according to claim 5 characterized in that it produces heparin-like molecules having relationships between the disacharides ls, Ils, llls and IVs close to those of heparin.

7. Culture or line of porcine mast cells according to claim 5 characterized in that it produces heparin-like molecules comprising at least 30% of disacharides ls.

8. Culture or line of porcine mast cells according to claim 5 characterized in that it produces heparin-like molecules having a ratio between the disacharides Ils and llls close to that of heparin.

9. Culture or line of porcine mast cells according to claim 5 characterized in that it produces heparin-like molecules having a ratio between the disacharides Ils and llls of between 1, 3 and 1, 9.

10. Culture or line of porcine mast cells according to claim 5 characterized in that it produces, at least 0.1 μg of heparin-type molecules / 10 ⁶ cells.

11. Culture or line of porcine mast cells according to claim 5 characterized in that it produces heparin-like molecules having a ratio between the disacharides ls and Ils of between approximately 3 and 8.

12. Culture or line of pig mast cells according to claim 5 characterized in that it produces heparin-like molecules having an anti-Xa activity greater than at least 10 IU / mg.

13. Culture or line of porcine mast cells according to claim 5 characterized in that it produces heparin-like molecules having an anti-lia activity greater than at least 10 IU / mg.

14. Line of porcine mast cells deposited with the Collection of Cultures of Microorganisms of the Pasteur Institute on April 09, 2003 under the n ° 1-3010

15. Line of porcine mast cells deposited with the Collection of Cultures of Microorganisms of the Pasteur Institute on April 09, 2003 under the n ° 1-3011

16. Line of porcine mast cells deposited with the Collection of Cultures of Microorganisms of the Pasteur Institute on April 09, 2003 under the n ° 1-3012

17. Line of porcine mast cells deposited with the Collection of Cultures of Microorganisms of the Pasteur Institute on April 09, 2003 under the number 1-3013.

18. Line of porcine mast cells deposited with the Collection of Cultures of Microorganisms of the Pasteur Institute on April 09, 2003 under the n ° 1-3014

19. Culture or line according to one of claims 5 to 8 characterized in that it is comprises a nucleic acid coding for an immortalizing protein.

20. Culture or line according to one of claims 5 to 8 characterized in that it comprises a nucleic acid coding for the c-kit.

21. Culture or line according to one of claims 5 to 13 characterized in that it comprises a nucleic acid coding for the T antigen.

22. Culture or line according to one of claims 5 to 13 characterized in that it comprises a nucleic acid encoding an enzyme acting on the sulfation of heparin-like molecules.

23. Culture or line according to one of claims 5 to 13 characterized in that it comprises a nucleic acid encoding a 3-OST.

24. A method of producing heparin-like molecules comprising culturing cultures or lines of pig mast cells according to one of claims 4 to 23, or obtained according to one of claims 1 to 3.

25. A method for producing heparin-like molecules comprising culturing, in a suitable medium, cultures or lines of porcine mast cells in a culture medium comprising at least about 0.1 ng / ml of IL-4.

26. A method for producing heparin-like molecules comprising obtaining cultures or lines of pig mast cells overexpressing IL4, and culturing these cells in a suitable culture medium.

27. A method of producing heparin-like molecules comprising obtaining cultures or lines of porcine mast cells transfected with a nucleic acid coding for CIL4, and culturing these cells in a suitable culture medium.

28. Protein of porcine origin of the c-kit type, characterized in that it has a C-terminal end having the sequence SEQ ID No. 3.

29. Protein according to claim 28 characterized in that it comprises a sequence having an identity of at least 99% with the sequence SEQ

ID # 2.

30. Nucleic acid comprising a sequence encoding a protein according to one of claims 28 and 29.

31. Nucleic acid according to claim 30 characterized in that it comprises a sequence having an identity of at least 99% with the sequence SEQ ID No. 1.

32. Protein of porcine origin with 3-O-sulfatase activity.

33. Protein according to claim 32 characterized in that it comprises a sequence having an identity of at least 95% with the sequence SEQ ID No. 4.

34. Nucleic acid comprising a sequence encoding a protein according to one of claims 32 and 33.

35. Nucleic acid according to claim 34 characterized in that it comprises a sequence having an identity of at least 95% with the sequence SEQ ID No. 5.

36. Protein of porcine origin with 6-O-sulfatase activity.

37. Protein according to claim 36 characterized in that it comprises a sequence having an identity of at least 90% with the sequence SEQ

ID # 6.

38. Nucleic acid comprising a sequence encoding a protein according to one of claims 36 and 37.

39. Nucleic acid according to claim 38 characterized in that it comprises a sequence having an identity of at least 95% with the sequence SEQ ID No. 7.

40. Cell comprising a nucleic acid according to one of claims 30,

31, 34, 35, 38 and 39.

41. Cell expressing a protein according to one of claims 28, 29, 32, 33, 36 and 37.