WO2020234910A1 - Procédé d'expansion de cellules souches adultes à partir de sang total - Google Patents

Procédé d'expansion de cellules souches adultes à partir de sang total Download PDF

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Publication number
WO2020234910A1
WO2020234910A1 PCT/IT2020/050126 IT2020050126W WO2020234910A1 WO 2020234910 A1 WO2020234910 A1 WO 2020234910A1 IT 2020050126 W IT2020050126 W IT 2020050126W WO 2020234910 A1 WO2020234910 A1 WO 2020234910A1
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Prior art keywords
blood
sample
stem cells
mcsf
prp
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PCT/IT2020/050126
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English (en)
Inventor
Marco Polettini
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Thankstem S.R.L.
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Priority to CN202080037624.4A priority Critical patent/CN113924361A/zh
Priority to AU2020277774A priority patent/AU2020277774A1/en
Priority to MX2021014118A priority patent/MX2021014118A/es
Priority to BR112021023101A priority patent/BR112021023101A2/pt
Priority to EP20743864.9A priority patent/EP3973051A1/fr
Publication of WO2020234910A1 publication Critical patent/WO2020234910A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)

Definitions

  • Embodiments described here concern a method for expanding adult stem cells from whole blood, in particular but not only peripheral blood, of adult mammals, and the corresponding application in the medical field, particularly in the human or veterinary field, for the therapeutic treatment of lesions, external lesions, internal lesions, lesions of tendons, lesions of ligaments, lesions of cartilage, bone fractures, as well as therapeutic and/or preventive treatment of chronic and/or acute inflammatory pathologies, neurological and neurodegenerative pathologies, cardiac pathologies, tumorous pathologies, autoimmune pathologies, ophthalmic pathologies and pathologies of genetic origin.
  • expansion means the process to increase the number of cells, either by cell division or, as in the specific case described and claimed here, by “de differentiation” or“de-programming”, that is to say, the process by which some cells in the blood are re -transformed into stem cells following suitable in vitro treatment, as will be seen hereafter.
  • stem cells have proven to be long, laborious, expensive, with limited results and sometimes side effects.
  • the former derive from blastocysts of 8 days, the adult ones on the other hand can be obtained mainly from bone marrow, adipose tissue, muscular tissue, from peripheral blood and from the umbilical cord, etc.
  • stem cell is in constant evolution. For all these cells, embryonic (ES) and adult, both hematopoietic (HSC) and also mesenchymal (MSC) (Kuwana M., et al., 2003), different genetic markers have been identified, some of which are common to many cell types (Condomines M, et ah, 2006; Kang WJ, et al., 2006; Zhao Y., et al., 2003; Rabinovitch, M. et al., 1976).
  • ES embryonic
  • HSC hematopoietic
  • MSC mesenchymal
  • PSCs pluripotent stem cells
  • stem cells of embryonic derivation were pluripotent and also qualifiable and quantifiable, therefore suitable for an experimental trial, but ethical questions and above all the contraindications due to the production of tumors caused them to be set aside. Therefore, today adult stem cells are preferred.
  • the adult stem cells of another individual very frequently give serious rejection phenomena because they are not recognized as“self’. This mainly affects umbilical cord stem cells that are used almost exclusively as allogeneic stem cells.
  • Induced pluripotent stem cells produced by a process that transfers, both through viruses and also not through viruses, the pluripotency factors from embryonic stem cells to adult stem cells are cells that are not suitable for treatment, due to the contraindications similar to those found with embryonic stem cells and the high costs.
  • stem cells obtained from peripheral blood through a process called“apheresis” or“leukapheresis” is accepted.
  • the stem cells are extracted from the blood, collected, and then inoculated into patients suffering from some leukemic pathologies, immediately after a chemo or radiotherapy. These stem cells are hematopoietic, therefore they inter-react exclusively with pathologies of the blood.
  • the stem cells that are being introduced onto the market today to treat different pathologies are adult stem cells and mainly mesenchymal stem cells obtained from bone marrow and fat.
  • they have a number of limitations:
  • This method which initially requires a step of isolating the monocyte and then an expansion step in a culture medium, is very long, about 15-20 days, to obtain a significant number of stem cells, and does not allow to obtain pluripotent stem cells.
  • the method is less invasive than the other methods used so far for collecting stem cells, painless (as opposed to apheresis) and economically convenient.
  • the present Applicant has found that the various processing and manipulation steps to which the stem cells are subjected during the execution of the method described in WO-A-2008/034370, such as the elimination of red blood cells, the purification of the stem cells with respect to other blood components, the obtaining of a larger quantity of pluripotent stem cells with respect to hematopoietic and mesenchymal stem cells, the culturing thereof, the differentiation into other cell types, can stress the adult stem cells thus obtained, leaving them alive but having less effectiveness and reduced potential energy and information capacity.
  • stem cells In the veterinary field there are different techniques and apparatuses to produce stem cells, in particular to concentrate stem cells from fat, from bone marrow and to obtain growth factors.
  • a first obstacle for stem cells is the difficulty of harvesting; in fact, as stated, to obtain them from bone marrow it is necessary to drill a bone or penetrate the sternum, and to obtain them from fat a real surgical operation with stitches can be provided, or, even if this intervention were not required, it would always constitute an invasive technique.
  • this system can also be improved to arrive at the use of stem cells in human clinical practice, to overcome obstacles linked to the shipment of the sample to the laboratory, the subsequent de-programming with MCSF and the re sending to the facilities where the therapeutic treatment takes place.
  • Another limitation can be the complete purification of the stem cells obtained from blood with the method described in WO-A-2008/034370 which allows greater safety for a possible allogeneic inoculation, the safety of which however still needs to be proven.
  • purifications and processes carried out to eliminate red blood cells and the passage of cells in a sorter (for qualification) create considerable stress on the stem cells obtained, causing them to lose part of their healing capacity. Therefore, there is strong need to reduce to a minimum the manipulation of the blood to obtain effective stem cells and thus achieve better results.
  • the international application WO-A-2015/170291 in the name of the present Applicant is also known, in which a method for expanding adult stem cells from blood is described, which comprises growth and de-programming of the adult blood stem cells of a drawn blood sample, by in vitro treatment of the blood sample with MCSF and ozonization of the blood sample.
  • Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.
  • a method for expanding adult stem cells from blood which overcomes the limitations of the state of the art and eliminates the defects present there, comprises:
  • PRP Platelet Rich Plasma
  • the present invention allows, therefore, to obtain pluripotent adult stem cells from the blood sample, without manipulating the blood sample, without adding, or only in a limited manner, components that have an origin that is external or extraneous to the organism and without risk of possible side effects, because for the growth and deprogramming, that is, the expansion, of the adult blood stem cells present in the blood sample, the PRP-based blood derivative product is used, which contains autologous MCSF, advantageously derived from the same blood sample.
  • Applicant has found that the quantity of autologous MCSF present in the growth factors which are released by the lysis of the platelets contained in the PRP obtained according to the method described here, allows growth and de programming of the adult stem cells present in the sample of whole blood subjected to the treatment in vitro. In this way, it is possible to largely, if not completely, replace MCSF of a different derivation from the autologous one that is used in the state of the art.
  • the subsequent use of the blood sample thus treated, in which the expanded stem cells are present is comparable in practice with an autologous blood transfusion, thereby overcoming risks and contraindications due to possible side effects which can be attributed to a growth factor for example of bacterial origin, and therefore extraneous to the organism, as well as overcoming many bureaucratic and regulatory limitations that could hinder the application of treatment to patients.
  • the sample of whole blood is a sample of autologous blood of a patient.
  • the lysis of the platelets releases the growth factors, among which MCSF is advantageously included, contained in the platelets of the PRP.
  • the lysis of the platelets is caused by centrifuging the first portion of the sample of whole blood.
  • the method uses the plasma obtained by the centrifugation, in which, as a result of the lysis of the platelets, the growth factors contained in the platelets, among which MCSF is advantageously included, have been released.
  • the blood sample for the purposes of growth and de-programming of the adult blood stem cells present in the blood sample, not only to the autologous PRP-based blood derivative product which includes autologous MCSF, but also to MCSF of a different derivation, typically obtained from bacterial cells.
  • the use of autologous MCSF contained in the PRP-based blood derivative product obtained as described above according to the present method decreases the quantity of MCSF of extraneous origin, in particular bacterial, and therefore extraneous to the organism, necessary for growth and de-programming of adult stem cells present in the blood sample being treated.
  • the method provides that an anticoagulant is present in the sample of whole blood that is made available.
  • the method provides the ozonization of the blood sample before the division into portions, or of the blood sample while it is subjected, or after it has been subjected, to the in vitro treatment, or a combination of these possibilities.
  • the ozonization has a sterilizing effect of possible contaminations exogenous and/or endogenous, of the blood sample being treated.
  • a pathogen is present in a blood sample that is subjected to treatment with the PRP-based blood derivative product that includes autologous MCSF
  • the introduction/inoculation, in a biological tissue to be treated, of the blood sample treated according to the method described here could create problems due to the presence of the pathogen.
  • the ozonization has an advantageous catalytic effect on the growth and de programming, that is, the expansion, of the adult stem cells present in the blood sample.
  • the method provides that the ozonization of the blood sample is carried out before the sample of whole blood is treated in vitro with the PRP-based blood derivative product which includes autologous MCSF. In this way, the growth and de-programming of adult blood stem cells is carried out in the already ozonized blood sample.
  • the method provides that the ozonization of the blood sample is carried out during the in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF.
  • treatment with the PRP-based blood derivative product which includes autologous MCSF can be carried out on the blood sample during ozonization.
  • the method provides that the ozonization of the blood sample is carried out after the in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF.
  • the treatment with the PRP-based blood derivative product which includes autologous MCSF can be carried out on the blood sample before it is ozonized.
  • the method provides that the ozonization provides the blood sample with a mixture of 0 2 -0 3 .
  • the method provides a blood/0 2 -0 3 mixture volumetric ratio equal to 1 : 1.
  • the method provides a quantity of 0 2 -0 3 mixture in the blood sample greater than, or equal to, about 1 mcg/1.
  • the method provides that the quantity of 0 2 -0 3 mixture in the blood sample can be selected in a range from about 1 mcg/ml to about 42 mcg/ml.
  • the method provides to add an anticoagulant to the blood sample.
  • the method provides to use a kit for collecting blood which includes at least a container able to contain at least the blood drawn, containing at least the PRP-based blood derivative autologous which includes autologous MCSF.
  • the method provides that the quantity of blood sample made available and treated is comprised between 10 ml and 1000 ml, in particular from 10 ml to 500 ml, more particularly from 15 ml to 250 ml, even more particularly from 20 ml to 150 ml.
  • a possible example of the sample is 100 ml.
  • the method provides a growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product, containing autologous MCSF, comprised between 4 hours and 120 hours, in particular between 24 hours and 96 hours, more particularly between 24 hours and 72 hours, even more particularly between 36 hours and 72 hours.
  • Embodiments described here also concern a blood sample containing adult stem cells which is obtainable with a method according to the present description.
  • the blood sample is provided for use in therapeutic and/or preventive treatment of pathologies, for example by autologous introduction/inoculation, locally or intravenously.
  • the blood sample is provided for use in therapeutic treatment, including therapy for lesions, external lesions, internal lesions, tendon lesions, ligament lesions, cartilage lesions, bone fractures, the therapy and/or the prevention of chronic and/or acute inflammatory pathologies, neurological and neurodegenerative pathologies, cardiac pathologies, tumorous pathologies, autoimmune pathologies, ophthalmic pathologies and genetic pathologies.
  • a blood sample is provided for use in a treatment which provides intravenous or intra-arterial or local administration (for example subcutaneous, intramuscular or intra-tissue) of the blood sample treated with the PRP-based blood derivative product which includes autologous and ozonized MCSF.
  • a blood sample is provided for use in a treatment which provides intravenous or intra-arterial or local administration (for example subcutaneous, intramuscular or intra-tissue) of the blood sample treated with the PRP-based blood derivative product which includes autologous MCSF and the systemic ozonization of the patient.
  • intravenous or intra-arterial or local administration for example subcutaneous, intramuscular or intra-tissue
  • PRP-based blood derivative product which includes autologous MCSF and the systemic ozonization of the patient.
  • Embodiments described here also concern a kit including at least a container containing a blood sample containing adult stem cells obtainable with a method according to the present description.
  • the present description also includes the intervals that derive from uniting or overlapping two or more intervals described, unless otherwise indicated.
  • the present description also includes the intervals that can derive from the combination of two or more values taken at different points, unless otherwise indicated.
  • the blood can be whole blood, more particularly peripheral whole blood, that is, blood present in the blood vessels (arteries, veins, capillaries).
  • the release and spread of growth factors also continues, including autologous MCSF, present in the platelets subjected to lysis, which therefore further enrich the blood sample thus treated.
  • a centrifugation operation of the first portion of the sample of whole blood can be used to obtain the lysis of the platelets, which causes the separation of the plasma and the lysis of the platelets.
  • the centrifugation operation is also advantageously the operation with which the PRP is obtained.
  • the lysed platelets allow to release the growth factors contained therein, advantageously including MCSF, into the blood plasma.
  • the plasma rich in platelets and growth factors, including autologous MCSF constitutes, in these embodiments, the PRP-based blood derivative autologous product.
  • the present invention provides to use mainly, instead of MCSF, or other growth factors, of extraneous origin, in particular derived from bacterial cells, MCSF contained in the growth factors released thanks to the lysis of the platelets of the PRP, that is, natively contained in the blood sample made available.
  • the present invention provides to use the PRP, whose platelets, subjected to lysis, release various growth factors, including MCSF and others, into the plasma obtained for example by centrifugation, as a PRP-based blood derivative autologous product.
  • the growth factors, including MCSF, present and released in the PRP whose platelets are subjected to lysis are advantageously obtained from autologous blood, that is, from the same patient, and are put back into contact with the sample of whole blood for a certain period of time to allow the de-programming and growth of the adult stem cells contained therein.
  • the use mainly of MCSF present in autologous growth factors released in the PRP thanks to the lysis of platelets, and not of bacterial origin excludes, or minimizes, any minimum operation of manipulating the blood.
  • the present invention which provides to use autologous growth factors contained in the PRP-based blood derivative product, including autologous MCSF, instead of using exclusively MCSF, or other growth factors, of extraneous origin to the organism, in particular obtained from bacterial cells, both avoids or considerably reduces interventions on and manipulations of the blood sample, such as the addition of an extraneous component obtained from bacterial cells, and also avoids a potential risk of possible side effects due to the use of an extraneous substance.
  • the lysis of the platelets present in the PRP obtained from the first portion of the blood sample can be obtained with any one of the known procedures.
  • obtaining the PRP- based blood derivative product where as a consequence of the lysis the growth factors, including MCSF, present in the platelets, have been released, can occur preferably by centrifugation of the portion of the sample of whole blood.
  • the PRP-based blood derivative product can be obtained by centrifugation of the portion of blood.
  • the method described herein may include for instance a single centrifugation.
  • the centrifugation can be carried out for a time from 3 to 9 minutes, in particular from 5 to 7 minutes, with a speed from 800 to 1200 rpm (revolutions per minute), in particular from 900 to 1100 rpm.
  • the method may include a single centrifugation of 6 minutes at a speed of 1000 rpm.
  • the method provides that an anticoagulant is present in the sample of whole blood made available.
  • the anticoagulant made available is preferably sodium citrate.
  • the portion used to obtain the PRP can be about half of the total blood sample made available.
  • the portion of the blood sample can in any case be chosen according to the volume of the blood sample made available.
  • MCSF of an extraneous origin in particular of bacterial origin, but advantageously in a smaller quantity than that provided in the state of the art, since autologous MCSF is already present, present natively in the autologous growth factors released in the PRP as a consequence of the lysis of the platelets.
  • it is in any case possible to reduce the quantity of MCSF extraneous to the organism, reducing possible side effects.
  • the method can allow to supplement the PRP-based blood derivative product with non-autologous growth factors, for example MCSF of bacterial origin.
  • non-autologous growth factors for example MCSF of bacterial origin.
  • a preferred supplementation growth factor can be MCSF of bacterial origin at a concentration, for example, between 0.5 nM and 4 nM, in particular from 1 nM to 4nM.
  • the Platelet Rich Plasma (PRP)-based blood derivative autologous product contains the following autologous growth factors: b-NGF (Nerve Growth Factor beta), EGF (Epidermal Growth Factor), FGF-2 (basic Fibroblast Growth Factor), FGF-4 (Fibroblast Growth Factor 4), FGF-6 (Fibroblast Growth Factor 6), FGF-7 (Fibroblast Growth Factor 7), GCSF (Granulocyte Colony- Stimulating Factor), GDNF (Glial cell-derived neurotrophic factor), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), HB-EGF (Heparin-binding EGF-like Growth Factor), HGF (Hepatocyte Growth Factor), IGF-I (Insulin-like Growth Factor), IGF-II (Insulin-like Growth Factor 2), MCSF (Macrophage Colony Stimulating Factor).
  • b-NGF Neve Growth Factor beta
  • the Platelet Rich Plasma (PRP)-based blood derivative autologous product also contains the following autologous growth factors: NT-3 (Neurotrophin-3), NT-4 (Neurotrophin-4), PDGF-AA ( Platelet Derived Growth Factor- AA), PDGF-AB (Platelet Derived Growth Factor- AB), PDGF-BB (Platelet Derived Growth Factor-BB), SCF (Stem Cell Factor), TGF-a (Transforming Growth Factor-a), TGF-b (Transforming Growth Factor-b), ⁇ R-b2 (Transforming Growth Factor- b2), T01 -b3 (Transforming Growth Factor ⁇ 3), VEGF (Endothelial Growth Factor); VEGF-D (Endothelial Growth Factor-D).
  • NT-3 Neurotrophin-3
  • NT-4 Neurotrophin-4
  • PDGF-AA Platelet Derived Growth Factor- AA
  • PDGF-AB Platelet Derived Growth Factor
  • the PRP-derived blood derivative product which includes autologous MCSF, possibly supplemented with growth factors extraneous to the organism, for example bacterial, is combined and amalgamated to treat, in vitro, the corresponding portion of remaining whole blood.
  • growth factors contained in the lysed platelets continue to be released and to spread in the blood sample.
  • the division of the sample of whole blood can provide that the first portion is about half of the blood sample made available.
  • the remaining part for example about half, can in turn be divided into portions, to each of which the PRP-based blood derivative product obtained can be added, in order to trigger growth and de-programming of the adult blood stem cells present.
  • the treatment can provide to amalgamate the PRP-based blood derivative product with the respective remaining portion of whole blood, which can preferably occur with a circular movement in a glass container.
  • the method provides an in vitro treatment time comprised between 4 hours and 120 hours, in particular between 24 hours and 96 hours, more particularly between 24 hours and 72 hours, even more particularly between 36 hours and 72 hours.
  • the method also provides the ozonization of the blood sample.
  • the present invention therefore allows to obtain pluripotent adult stem cells from the blood sample made available.
  • the stem cells obtained by applying the method described here include the stem cell markers CD90, CD90/34, CD34 and CD117, they also express some intracellular transcription factors strongly linked to pluripotency characteristics (Sox2,Oct3/4 and Nanog) and do not lose the self recognition factors following division or expansion. These stem cells do not give rise to side effects such as phenomena of rejection, infection, development of teratomas once administered in the patient, they are able to differentiate in vivo and therefore behave as pluripotent stem cells.
  • ozonization here means the treatment of the blood sample with ozone, that is, the addition, delivery, administration or mixing of ozone, or of a mixture of oxygen and ozone, to/in the blood sample.
  • Ozone (symbol 0 3 ) is an allotropic form of oxygen, with a triatomic molecule and molecular weight 48. Ozone appears in normal conditions as a blue, acrid smelling gas, with strong oxidizing power. Ozone can act as a disinfectant, deodorant, bactericide, sterilizer, or as an oxidizer in numerous organic syntheses.
  • the ozonization of the blood sample can be carried out before the treatment with the PRP-based blood derivative product.
  • the ozonization of the blood sample can be carried out simultaneously to the treatment with the PRP-based blood derivative product.
  • the ozonization of the blood sample can be carried out after treatment with the PRP-based blood derivative product.
  • an anticoagulant to the blood sample.
  • Heparin, EDTA or sodium citrate are examples of possible anticoagulants.
  • the method according to the present description can provide to use a kit for collecting blood, for the production of pluripotent stem cells according to the method described above, including at least a container, such as a test tube, suitable to contain the blood sample, containing the PRP- based blood derivative product and possibly, if provided, the anticoagulant.
  • a kit of this type it is possible to collect the whole blood, preferably peripheral blood, to rapidly start the growth and production of the stem cells by means of the method described above according to the present description, and therefore make their production much faster.
  • Embodiments described here can provide that the quantity of blood sample that is collected and subjected to the method described here, that is, growth and de-programming with the PRP-based blood derivative product, and possible ozonization of the blood sample, is of a few milliliters, for example comprised between 10 ml and 1000 ml, in particular between 10 ml and 500 ml, more particularly between 15 ml and 250 ml, even more particularly between 20 ml and 150 ml. A possible example is 100 ml.
  • the blood sample once subjected to the in vitro treatment described here, can for example be injected into the circulation of the patient (intravenous or intraarterial administration), which can then possibly be subjected, subsequently, to systemic ozonization treatment.
  • Some embodiments which can be combined with all the embodiments described here, can provide a method as described above which can use any type of collection container in which to introduce the whole blood and the ozone with any type of possible anticoagulant, with any concentration volume of PRP-based blood derivative product which includes autologous MCSF.
  • Some embodiments which can be combined with all the embodiments described here, can provide that ozonization gives the blood sample a mixture of O2-O3.
  • the blood/0 2 -0 3 mixture volumetric ratio can preferably be 1 : 1.
  • the quantity of 0 2 -0 3 mixture in the blood sample may be greater than, or equal to, about 1 mcg/1, in particular selected in a range from about 1 mcg/ml to about 42 mcg/ml, more particularly from about 5 mcg/ml to about 30 mcg/ml, even more particularly from about 10 mcg/ml to about 20 mcg/ml.
  • Embodiments described here provide that, having left the blood in these conditions possibly ozonized, preferably at room temperature, after a certain time, preferably between 4 hours and 96 hours, in particular between 4 hours and 72 hours, more particularly between 4 hours and 48 hours, the whole blood thus obtained, with the component of stem cells obtained from de-programming can be completely inoculated, both systemically (intravenously, intra-arterially), and also locally into or near a diseased tissue.
  • the growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF can be comprised between 12 hours and 120 hours, in particular between 12 hours and 96 hours, more particularly between 12 hours and 72 hours, even more particularly between 12 hours and 36 hours.
  • the growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF can be comprised between 24 hours and 120 hours, in particular between 24 hours and 96 hours, more particularly between 24 hours and 72 hours, even more particularly between 24 hours and 36 hours.
  • the growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF can be comprised between 48 hours and 120 hours, in particular between 48 hours and 96 hours, more particularly between 48 hours and 72 hours, even more particularly between 48 hours and 60 hours.
  • Applicant has hypothesized that the ozonization of the blood sample subjected to growth and de-programming by means of in vitro treatment with a PRP-based blood derivative product which includes autologous MCSF stimulates the process of expansion and de-programming of adult stem cells, so that even after a few hours there is a significant number of useful adult stem cells.
  • Embodiments of the method described here can also provide, in addition to the container where the PRP-based blood derivative product which includes autologous MCSF is present and where the expansion of adult stem cells occurs, the use of a second container to contain the stem cells obtained as described above, for example in the case of intravenous or intra-arterial use, and possibly a third container, of a different size, for local use.
  • the stem cells produced and preserved in the containers can be used immediately, or they can be preserved, for example in liquid nitrogen, to be used subsequently, when there is a need.
  • the ozonization according to variants of the embodiments described here can be carried out on blood samples containing the stem cells, before, during or after these have been expanded and de-programmed with the PRP-based blood derivative product which includes autologous MCSF, contained in any one of the containers mentioned.
  • the blood just taken from the patient can be immediately put into the test tube with the anticoagulant.
  • the anticoagulant can block the beginning of the coagulation.
  • a first portion is removed from this sample, which is processed to obtain the PRP-based blood derivative product which includes autologous MCSF.
  • the latter is put into the remaining blood sample to allow the expansion process to rapidly begin, and to guarantee that the time to start treating the patient is minimized.
  • the sample can possibly be subjected to ozonization according to the present description.
  • an anticoagulant can be added to the blood taken from the patient to block the coagulation of the blood; the blood is subjected to a preservation procedure which does not alter its ability to produce stem cells.
  • the blood is taken from where it is stored and is subjected to the stem cell expansion procedure described above, that is, taking a first portion thereof which is processed to obtain the PRP-based blood derivative product which includes autologous MCSF, which is then added to the remaining part of the blood sample, rapidly obtaining the necessary quantity of stem cells.
  • the sample is subjected to ozonization according to the present description.
  • the method according to the present description allows to overcome the disadvantages of the state of the art and entails many advantages.
  • the present invention allows to prepare whole blood and greatly simplify the therapy, avoiding any type of cellular manipulation made in the laboratory, as well as avoiding, according to advantageous variants, the addition of components that are extraneous to the organism, such as growth factors derived from bacterial cells.
  • the present invention eliminates the need or the possibility to carry out treatments for example to eliminate red blood cells, to purify the stem cells with respect to all the other blood components, to obtain a greater quantity of pluripotent stem cells with respect to the other two stem cell components, hematopoietic and mesenchymal, to cultivate them, to differentiate them into other cell types.
  • These additional treatments can normally stress the stem cells obtained, leaving them alive, but with less information and energy potential.
  • the present invention advantageously avoids all further processing and manipulations of the blood sample, so that the adult stem cells present in the whole blood maintain their characteristics better because they are not stressed, and because they can benefit from the presence in the blood of other elements assisting in the regenerative process.
  • Applicant has found that incurable pathologies such as myocardial degeneration already treated with good results with stem cells obtained from blood by de-programming, as described in WO-A-2008/034370, have had a decidedly more positive evolution with stem cells de-programmed in whole blood, avoiding the additional operations and manipulations mentioned above, thus adopting a method that can even consist exclusively of growth and de programming with the PRP-based blood derivative product which includes autologous MCSF, without additional processing steps, including purification, subsequent expansions, or which can include growth and de-programming with the PRP-based blood derivative product which includes autologous MCSF and ozonization.
  • the preparation of stem cells according to the method described here avoids complex laboratory preparations, allowing any hospital, clinic or doctor to prepare stem cells by possessing a simple test tube with the preferably minimum quantity of the PRP-based blood derivative product which includes autologous MCSF obtained as described above.
  • a simple test tube in which to place an adequate quantity of blood sample with the PRP-based blood derivative product which includes autologous MCSF, it is possible to treat and improve even serious pathologies, such as for example the after-effects of a heart attack or Parkinson's disease.
  • a method for expanding adult stem cells from blood can even consist exclusively of growth and de-programming of the adult blood stem cells of a blood sample, by means of in vitro treatment of the blood sample with the PRP-based blood derivative product which includes autologous MCSF.
  • the addition or supply of ozone to the blood sample, deriving from the ozonization treatment of the blood sample can have a catalyzing effect on the de -programming process of the adult stem cells and on the quality of the stem cells obtained and on the their information and energy content, such that it positively influences the cellular regeneration of damaged tissues, as well as giving the product additional safety with regard to sterility.
  • ozone can act as a disinfectant or bactericide.

Abstract

L'invention concerne un procédé d'expansion de cellules souches adultes à partir du sang comprenant la croissance et la déprogrammation des cellules souches sanguines adultes d'un échantillon de sang, au moyen d'un traitement in vitro de l'échantillon de sang avec un produit dérivé du sang à base de PRP qui comprend le MCSF autologue obtenu par lyse de plaquettes dans une partie du même échantillon de sang autologue.
PCT/IT2020/050126 2019-05-22 2020-05-19 Procédé d'expansion de cellules souches adultes à partir de sang total WO2020234910A1 (fr)

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CN202080037624.4A CN113924361A (zh) 2019-05-22 2020-05-19 从全血中扩增成体干细胞的方法
AU2020277774A AU2020277774A1 (en) 2019-05-22 2020-05-19 Method for expanding adult stem cells from whole blood
MX2021014118A MX2021014118A (es) 2019-05-22 2020-05-19 Metodo para expandir celulas madre adultas a partir de sangre total.
BR112021023101A BR112021023101A2 (pt) 2019-05-22 2020-05-19 Método para expansão de células-tronco adultas do sangue total
EP20743864.9A EP3973051A1 (fr) 2019-05-22 2020-05-19 Procédé d'expansion de cellules souches adultes à partir de sang total

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BR112021023101A2 (pt) 2022-01-04
AU2020277774A1 (en) 2021-12-09

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