WO2015081410A1 - Procédé de culture de cellules souches mésenchymateuses adultes, trousse, composition et utilisations - Google Patents

Procédé de culture de cellules souches mésenchymateuses adultes, trousse, composition et utilisations Download PDF

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WO2015081410A1
WO2015081410A1 PCT/BR2014/050033 BR2014050033W WO2015081410A1 WO 2015081410 A1 WO2015081410 A1 WO 2015081410A1 BR 2014050033 W BR2014050033 W BR 2014050033W WO 2015081410 A1 WO2015081410 A1 WO 2015081410A1
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mesenchymal stem
stem cells
tissue
adult mesenchymal
degenerate
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Portuguese (pt)
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João Antonio Pêgas HENRIQUES
Asdrubal FALAVIGNA
Manuela FIGUEIRÓ
Mariana Roesch ELY
Israel Silveira DE AGUIAR
Denise Cantarelli MACHADO
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Fundação Universidade De Caxias Do Sul
Uniao Brasileira De Educacao E Assistencia
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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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)

Definitions

  • the present invention describes a process of culturing adult mesenchymal stem cells, said process comprising the steps of: a) incubating degenerate ex vivo tissue in culture medium; b) isolation of adult mesenchymal stem cells from the medium obtained in step a); and c) culturing adult mesenchymal stem cells isolated in step b).
  • the present invention also provides a kit for culturing adult mesenchymal stem cells comprising: a) container of nonstick material; b) suitable culture medium; and c) suitable container for cell culture.
  • the present invention also relates to a composition comprising the adult mesenchymal stem cells obtained by said process and the use of the adult mesenchymal stem cells obtained by said process for the manufacture of a composition for the treatment of degenerate intervertebral disc discopathy.
  • the present invention is in the fields of embryology, cell biology and regenerative medicine.
  • DDD Degenerative disc disease
  • intervertebral disc (IVD) degeneration is considered an irreversible phenomenon.
  • Treatment options basically involve conservative treatment (physiotherapy, nerve root block) and surgical strategies (disc excision and arthrodesis), which are effective only in symptomatic relief and can actually accelerate the degenerative process at adjacent levels (Acosta et al. , 2005).
  • the human spine is made up of 23 DIVs that separate the vertebrae and provide flexibility. They account for 20-30% of the length of the spine and increase in size from progressing from the cervix to the lumbar spine. In addition, the flexibility of the IVD serves to provide stability and load support during exercise.
  • the structure of the IVD basically consists of a central part called the pulposus nucleus (NP), derived from the notochord, which is surrounded by derivatives of a fibrous ring (AF) of mesenchymal tissue (Roughiey, 2004).
  • NP pulposus nucleus
  • AF fibrous ring
  • NP is rich in proteoglycans and water
  • AF is rich in collagen, especially of types I, II, VI and IX (Feng et al., 2006; Risbud et al., 2004).
  • composition of IVD varies with the level of the spine: the collagen content in the nucleus being higher in the cervical discs and lower in the lumbar discs, while the proteoglycan content shows a contrary tendency (Scott et al., 1994).
  • proteoglycan aggregates are important for water retention, as restricting water flow influences tissue response to column loads (Feng et al., 2006).
  • tissue response to column loads Freng et al., 2006.
  • proteolytic degradation Jahnke et al., 1988; Johnstone et al., 1995.
  • the ability to withstand the compressive load decreases.
  • collagen fibers are oriented in layers around the NP.
  • the main function of this structure is to retain NP by retaining and distributing the load exerted on this tissue during various types of exercise (Feng et al., 2006).
  • Nishimura et al. (1998) performed autologous NP tissue transplantation on rat anucleated discs, where it was shown to slow the progression of degeneration (Nishimura et al., 1998).
  • this cellular source has practical limitations in the clinic due to the need to damage the adjacent disc, probably inducing degeneration at this level.
  • adult mesenchymal stem cells (MSCs) are acquired by bone marrow aspiration, and are better able to adapt to the disk environment and thus achieve a differentiated state suitable for matrix synthesis by longer period (Acosta et al., 2005).
  • MSCs Mesenchymal stem cells
  • MSC can be inserted into chondrogenic or perhaps discogenic pathways, and are capable of expressing agrecane and type II collagen in large quantities (Anderson et al., 2005). Another advantage is that MSC can be obtained from many sources, including bone marrow and fat, without significant morbidity or immunogenic response and can be easily expanded in cultures (Bartholomew et al., 2002).
  • MSCs are known to be able to differentiate into chondrocyte cells (Wakitani et al., 2002). Thus, because NP and chondrocytes have similar characteristics, it is reasonable to assume that MSCs may also be able to differentiate into NP-type cells (Horner et al., 1976; Gruber et al., 1997).
  • MSCs are capable of differentiating into NP-like cells that could be used in cell-based tissue engineering therapies for IVD regeneration (Yamamoto et al., 2004; Richardson et al. ., 2006; Vadala et al., 2008; Le Visage et al., 2006; Gruber et al., 2010; Sobajima et al., 2008; Svanvik et al., 2010; Yang et al., 2008).
  • Blanco et al. (2010) performed the isolation and characterization of MSCs from a degenerated human disk. They found that degenerate NP contains MSCs and that these cells are extremely similar to those found in the bone marrow. These findings suggest that DDD could be treated by cell therapy by injecting differentiated MSCs grown in NP-like cells and stimulating MSCs already present in NP (Blanco et al., 2010).
  • Bertolo et al. studied the immunosuppressive effect of MSCs on IVD fragments of patients with DDD. They found that 70% of patients had a reduction in IgG production and that peripheral blood lymphocyte proliferation was also decreased when MSCs were present (Bertolo et al., 201 1). This effect on reducing inflammation shows that the potential role of MSCs for DDD goes beyond the ability to repopulate IVD.
  • Bendtsen et al. (201 1) induced disc degeneration in minipigs and injected CTM-free hydrogels and autologous CTM-loaded hydrogels. They then found that MSC and hydrogel therapy are able to partially regenerate IVD and maintain perfusion and permeability of the vertebral end plate and subchondral bone (Bendtsen et al., 201 1).
  • Orozco et al. (201 1) injected autologous MCT in 10 patients with confirmed DDD, and they observed that after MCT injection the patient had a rapid improvement in pain and disability within 3 months, followed by a modest improvement within 6 to 10 months. 12 months after injection. There seemed to be no improvement in the height of the disc. However, the water content of the disc was significantly elevated after 12 months. This author also pointed out that the results are important, as the intervention is simpler, more conservative, preserves normal biomechanics and does not require surgery or hospitalization of the patient (Orozco et al., 201 1).
  • DID degenerated intervertebral disc
  • DNA repair In the laboratory the DID goes through a mechanical dissociation step, where the material is sectioned into two stages: a mechanical one, where it is cut into small portions using a number 10 scalpel, and another enzymatic, where dissociation occurs through the enzymes acting.
  • the material is dissociated by incubating for 2 hours with
  • the literature reports dissociation using one or more enzymes and in varying concentrations. Also, the type of collagenase may be different in the literature.
  • the enzymes are inactivated with DMEM / F12 culture medium supplemented with 10% SFB and 1% P / S.
  • the material is fed through sterile Pasteur falcon pipettes and filtered through filters fitted with 40 ⁇ nylon membrane falcon tubes (for removal of tissue debris). The filtered material is centrifuged at 1200rpm for 10 minutes. The precipitate is resuspended with 1 ml DMEM / F12 supplemented with 10% SFB and 1% P / S.
  • the obtained cells are plated in 1 well of a 24-well plate with 0.02ng / pL EGF (Epidermal Growth Factor) and 0.02 ng / L FGF (Fibroblast Growth Factor) and the culture is incubated in humidified greenhouse at 37 ° C with 5% CO 2 .
  • EGF Epimal Growth Factor
  • FGF Fibroblast Growth Factor
  • the attempt to establish culture occurs by maintaining the culture every 3 days with the addition of a further 500 ⁇ _ DMEM / F12 medium supplemented with 10% SFB and 1% P / S and 0.02ng / Ml_ EGF (Epidermal Growth Factor) and 0.02 ng / ⁇ FGF (Fibroblast Growth Factor).
  • this conventional DID stem cell isolation process allows cultivation for about 8 months, resulting in a confluence of only 40% of 1 well from a 24-well plate ( Figure 08 - A and B) .
  • Document PI0514387 discloses a method for isolating stem cells / progenitors from the umbilical cord amniotic membrane, comprising separating the amniotic membrane from other components of the umbilical cord in vitro, culturing tissue from amniotic membrane under conditions that allow cell proliferation and isolate stem / progenitor cells from tissue cultures.
  • Isolated stem cells may have properties similar to embryonic stem cells and may be used for various therapeutic purposes.
  • Document PI0706373 "USE OF MESENCHIMAL STEM CELLS FOR TREATMENT OF GENETIC DISEASES AND DISORDERS” discloses a method of treating a disease or genetic disorder comprising administering mesenchymal stem cells in an amount effective to treat the disease or the genetic disorder in the animal.
  • PI0706070 discloses methods of expanding ex vivo mesenchymal stem cells, wherein the method comprises: (a) sowing cells containing the mesenchymal stem cells on a substrate such that a low density of mesenchymal stem cells stick to the substrate; (b) culturing the mesenchymal stem cells in said substrate; (c) removing expanded mesenchymal stem cells from the substrate; (d) pealing the removed mesenchymal stem cells onto the same or a different substrate; and (e) repeat steps (b) - (d) until the desired number of expanded mesenchymal stem cells is reached.
  • Document PI0802241 discloses a method for the treatment of autoimmune diseases, allergic responses, cancer, inflammatory diseases or fibrosis, and said method promotes wound healing, repair of epithelial damage and angiogenesis in a organ or tissue of an animal by administering mesenchymal stem cells in an effective amount.
  • US 2013/108593 discloses a system and method for mesenchymal stem cell transplantation, in particular a system and method for autologous percutaneous transplantation of bone marrow mesenchymal and progenitor helper cells for degenerate intervertebral discs or joints.
  • US 2005/1 18228 discloses materials and methods for augmenting and / or repairing intervertebral discs by means of stem cell material.
  • the present invention has numerous advantages over the literature cited above, as it has been found that the replacement of the mechanical and enzymatic dissociation steps by the ex vivo tissue incubation step Degenerate in culture medium in non-stick material provides desirable technical effects for adult mesenchymal stem cell cultivation processes.
  • Advantages include: obtaining a much larger number of adult mesenchymal stem cells in a shorter period of time; reduction in process costs arising from the need to apply enzymes to the process; simplified and effective methodology; reduction in the interference of other cell types in adult mesenchymal stem cell culture as the process described here allows the prevalent obtainment of adult mesenchymal stem cells with high cell differentiation and renewal power; enabling the use of adult mesenchymal stem cells in regenerative medicine to treat conditions involving, for example, intervertebral disc degeneration.
  • MSCs are a key point in cell therapy and regenerative medicine
  • their large-scale application in clinical practice is made impossible by stem cell isolation and cultivation processes.
  • Adult mesenchymal cells require time and high investment to achieve a satisfactory amount of adult mesenchymal stem cells that have desirable effects on cell therapy.
  • the invention described herein presents, in one aspect, a process of culturing adult mesenchymal stem cells from degenerate tissue comprising the steps of: a) incubation of degenerate ex vivo tissue in suitable culture medium in non-stick container;
  • the degenerate ex vivo tissue is derived from at least one tissue selected from the group consisting of: intervertebral disc, adipose tissue, bone marrow, periosteum, muscle tissue or parenchymal organs.
  • the degenerate ex vivo tissue is preferably derived from intervertebral disc, adipose tissue or bone marrow.
  • the degenerate ex vivo tissue is preferably derived from the degenerate intervertebral disc (DID).
  • DID degenerate intervertebral disc
  • step a) comprises a minimum incubation period in suitable culture medium to 80% confluence.
  • step c) comprises culturing the cells in suitable culture medium for a period of at least 3 days.
  • the culture medium employed in at least one of steps a) and / or c) is Dulbecco's modified Eagle type (DMEM) supplemented with fetal bovine serum (SFB) at a concentration ranging from 5%. v / va 15% v / v; and at least one antibiotic selected from the group consisting of: streptomycin and penicillin, in a concentration ranging from 0.1% w / v to 2% w / v.
  • DMEM Dulbecco's modified Eagle type
  • SFB fetal bovine serum
  • the culture medium employed in at least one of steps a) and / or b) is additionally supplemented with at least one growth factor chosen from the group consisting of: EGF and FGF, in a concentration ranging from 0 .01 ng / ⁇ . at 0.1 ng / ⁇ ..
  • said process further comprises step d) of differentiating the adult mesenchymal stem cells obtained in step c) into bone and / or adipose tissue cells.
  • the present invention provides a kit for culturing adult mesenchymal stem cells from degenerate tissue, comprising:
  • the kit further comprises at least one growth factor-containing reagent.
  • the present invention provides a composition comprising adult mesenchymal stem cells obtained by process as defined above and pharmaceutically acceptable carrier.
  • the present invention provides for the use of process-obtained adult mesenchymal stem cells in the manufacture of a composition for the manufacture of a composition for regenerating biological tissues.
  • the biological tissue is preferably degenerate intervertebral disc, adipose tissue or bone marrow.
  • Figure 1 Image of degenerated intervertebral disc cultivation in Petri dishes with DMEM medium supplemented with 10% SFB and 1% P / S plus growth factors after 4 days of cultivation (10X magnification).
  • FIG 2 images of degenerated intervertebral disc cultivation in Petri dishes with DMEM medium supplemented with 10% SFB and 1% P / S plus growth factors as mentioned in the text above.
  • A) and (B) show two-week cultivation with many attached mesenchymal stem cells (4x magnification).
  • C) shows cultivation after 1 week; and
  • D shows the two-week culture with many mesenchymal stem cells adhered with tissue imaging. Under the biological material, the number of cells adhered is much larger (10X increase).
  • Blue arrow stem cells not yet attached; Red arrow: adhered stem cells (with fibroblastic morphology); Black arrow: biological material (degenerated intervertebral disc).
  • Figure 3 images of mesenchymal stem cell culture isolated from degenerate intervertebral disc in bottles (evidence of plastic adhesion and fibroblastic characteristic typical of mesenchymal stem cells) (10X magnification).
  • Figure 4 images of degenerated intervertebral disc culture after three days of bottle plating (so that the culture had high cell confluence for characterization - about 90%) (10X magnification).
  • Figure 6 Differentiation of mesenchymal stem cells obtained from degenerated intervertebral disc to adipocytes stained with Oil Red O (Sigma-Aldrich).
  • a and B demonstrate differentiated mesenchymal stem cells into adipocytes.
  • C) and D express negative control of mesenchymal stem cells for staining (10X increase).
  • Figure 7 Differentiation of mesenchymal stem cells obtained from degenerated intervertebral disc for osteocytes stained with Alizarin Red S (Sigma-Aldrich).
  • a and B are differentiated mesenchymal stem cells into osteocytes.
  • C) and D show negative control of mesenchymal stem cells for staining (10X increase).
  • Figure 8 cells isolated by the conventional method plated in 1 well of a 24 well plate with DMEM / F12 medium supplemented with 10% SFB and 1% P / S. Then, the obtained cells are plated in 1 well of a 24-well plate with 0.02ng / ⁇ l Epidermal Growth Factor (EGF) and 0.02 ng / L Fibroblast Growth Factor (FGF).
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • the present invention provides a process of culturing adult mesenchymal stem cells comprising the steps of:
  • step b) culture of adult mesenchymal stem cells isolated in step b).
  • Such process does not require the application of the mechanical and enzymatic dissociation steps, usually employed in the state of the art, so that obtaining large amount of adult mesenchymal stem cells for cultivation from degenerate ex vivo tissue is possible through the steps to be ) and b).
  • Incubation of said degenerate ex vivo tissue in culture medium promotes the release of said adult mesenchymal stem cells from degenerate tissue into the culture medium in which said tissue is contained.
  • a degenerate tissue has a low amount of viable cells, so modification in the process steps results in desirable and superior technical effects compared to the state of the art, which is to obtain a much larger number of cells from a small sample. , with much greater purity than the techniques usually employed.
  • the present invention also provides a kit for culturing adult mesenchymal stem cells from degenerate tissue comprising: container of nonstick material; suitable culture medium; and container suitable for cell cultivation.
  • the kit further comprises at least one growth factor-containing reagent.
  • the present invention provides a composition comprising adult mesenchymal stem cells obtained by process as defined above and pharmaceutically acceptable carrier.
  • the present invention provides for the use of process-obtained adult mesenchymal stem cells in the manufacture of a composition for the manufacture of a composition for regenerating biological tissues.
  • the biological tissue is preferably degenerate intervertebral disc, adipose tissue, bone marrow or knee cartilage.
  • MSCs Middle Mesenchymal Stem Cells
  • adult mesenchymal stem cells is to be understood as undifferentiated multipotent cells that have the ability to differentiate into a number of cell types, including bone, cartilage, fat, muscle. and tendons, depending on the signals provided to the biological or culture environment.
  • MSCs used in the present invention have no limitation on genetic inheritance and / or its origin. MSCs are present, for example, in small amounts in perivascular regions of all adult tissues, including the intervertebral disc, bone marrow (MO), adipose tissue, periosteum, muscle tissue, parenchymal organs, umbilical cord, synovial tissue, among others. Characteristic markers of MSCs include, but are not limited to, SH2, SH3, SH4, CD10, CD13, CD29, CD44, CD54, CD73, CD90, CD105 and CD166; the MSCs being negative for CD1 1 b, CD14, CD19, CD31, CD34, CD38, CD40 or CD45 markers.
  • degenerate ex vivo tissue is to be understood as the section or sample of a biological tissue obtained by surgical method such as biopsy. Degeneration corresponds to changes in tissue morphology and composition that result in loss of function, cellularity and / or integrity of the affected tissue. Importantly, degenerate ex vivo tissue does not undergo any mechanical or enzymatic dissociation process prior to undergoing incubation step a).
  • Non-limiting examples of biological tissues, in any degree of degeneration, compatible with the realization of the present invention consist of: intervertebral disc, adipose tissue, bone marrow, periosteum, muscle tissue and parenchymal organs.
  • the degenerate ex vivo tissue is derived from degenerate intervertebral disc.
  • the term "Incubation of degenerate ex vivo tissue” should be understood to be the packaging of degenerate ex vivo tissue in a container containing culture medium under usual conditions known in the art.
  • “usual incubation conditions” is meant the conditions commonly employed in incubation / culture of mesenchymal stem cells, such as humidified greenhouse with 5% CO 2 at 37 ° C.
  • incubation of degenerate ex vivo tissue occurs for a period of time comprising a minimum incubation period in suitable culture medium to at least 80% confluence.
  • non-stick container should be understood to be a suitable container for incubation of degenerate ex vivo tissue that is unfavorable to the adherence of adult mesenchymal stem cells, being essentially inert and biologically inactive. .
  • the non-stick container should allow said cells to remain in suspension contained in the culture medium employed in the incubation step.
  • a non-limiting example of non-stick material to adult mesenchymal stem cells from which the non-stick container can be made is glass.
  • suitable culture medium should be understood as a medium that provides the essential substances for cell growth, as well as controlling the in vitro growth of adult mesenchymal stem cell cultures. Ways of Culture and reagents employed in cell culture are well known in the art.
  • Non-limiting examples of culture media suitable for incubation or transport of degenerate ex vivo tissue samples include Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F12 medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Non-limiting examples of culture media suitable for culturing adult mesenchymal stem cells are: Dulbecco's Modified Eagle Medium (DMEM), DMEM / F12 Medium, RPMI Medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • F12 Medium DMEM / F12 Medium
  • RPMI Medium fetal serum
  • the media may be supplemented with fetal serum as well as antibiotics, growth factors, amino acids, inhibitors and the like, common in the prior art.
  • fetal serum should be understood as an animal fluid employed to meet the needs of cultured cells by growth factors, hormones, proteins and peptides, nucleosides, lipids and inhibitors.
  • Non-limiting examples of fetal sera that may be employed are: fetal bovine, horse and human serum.
  • growth factor should be understood as a substance capable of stimulating cell growth, proliferation and / or differentiation.
  • growth factors suitable for adult mesenchymal stem cell cultivation include, but are not limited to, EGF and FGF. It should be understood that the growth factors EGF and FGF were used in the preferred embodiment because, based on previous research group experiences, it is adequate to assist mesenchymal stem cell cultivation. Moreover, both are consistent for this type of cells (fibroblastic and epidermal characteristic - high cell replication).
  • growth factors are employed at a concentration ranging from 0.01 ng / ⁇ . at 0.1 ng / ⁇ .. Isolation of adult mesenchymal stem cells
  • Isolation of adult mesenchymal stem cells should be understood as the extraction of the cells suspended in culture medium resulting from step a). Isolation may be performed, for example, with the aid of a sterile Pasteur pipette capable of collecting an aliquot of the culture medium containing the suspended adult mesenchymal stem cells.
  • the step of "isolating adult mesenchymal stem cells from the medium obtained in the incubation step of degenerate ex vivo tissue” does not include any mechanical dissociation procedure (such as cuts in degenerate ex vivo tissue that has been incubated; centrifugation, among others) and / or enzymatic dissociation (use of enzymes such as collagenases).
  • the exclusion of the mechanical and enzymatic dissociation steps promotes significant advantages in the yield of the adult mesenchymal stem cell cultivation process obtained from degenerate tissue, since it is part of a very restricted number of cells to obtain a larger number. properly characterized adult mesenchymal stem cells, enabling the application of cell therapy in the recovery of degenerated tissues.
  • culture is well known in the art, so that in the understanding of the present invention it corresponds to the incubation of adult mesenchymal stem cells isolated in b) in culture medium and the culture medium may be supplemented with fetal serum as well. as antibiotics and growth factors under usual culture conditions.
  • usual culture conditions is meant the conditions commonly employed in incubation / culture of mesenchymal stem cells, such as a humidified greenhouse with 5% CO 2 at 37 ° C.
  • the culture occurs for a period of at least 3 days. At culture times longer than 3 days, the process preferably comprises renewing the culture medium, which consists in removing the culture medium first employed and adding a new culture medium.
  • Suitable containers for cell culture are well known in the art.
  • a non-limiting example of a container suitable for growing adult mesenchymal stem cells is a container made of polymeric material such as plastic.
  • the term "pharmaceutically acceptable carrier” should be understood as a carrier capable of not altering the cellular viability of adult mesenchymal stem cells obtained by said process in a composition.
  • the method of isolating adult mesenchymal stem cells of the present invention involves the degenerate intervertebral disc (DID) of patients suffering from DDD.
  • DID intervertebral disc
  • Such discs are obtained during spinal surgery of these patients with DDD and with painful and deficient symptoms refractory to clinical treatment.
  • DID is washed in a vat with sterile physiological solution 3 times and collected in a Falcon tube containing 15ml of DMEM (Dulbecco's Modified Eagle Medium / Sigma-Aldrich) medium supplemented with 5-20% Bovine Fetal Serum. (SFB), preferably 10% SFB, and 1% Penicillin / Streptomycin (P / S).
  • DMEM Dulbecco's Modified Eagle Medium / Sigma-Aldrich
  • FFB Bovine Fetal Serum.
  • P / S Penicillin / Streptomycin
  • this material was incubated for at least 4 days in a humidified greenhouse at 37 ° C with 5% CO 2 . After said period, the petri dish was analyzed using inverted microscope. At this time, large amount of cells dispersed in the culture medium was observed ( Figures 1 and 2). These cells were plated in small or large bottles to obtain a large amount of mesenchymal stem cells.
  • 3ml_ DMEM medium supplemented with 5-20% SFB, preferably 10% SFB, and 1% P / S, with 9ml_ medium with cells in the Petri dish, and growth factors at concentrations of 0, 02ng1 L (EGF and FGF-Sigma-Aldrich).
  • SFB 5-20% SFB
  • P / S 9ml_ medium with cells in the Petri dish
  • growth factors at concentrations of 0, 02ng1 L (EGF and FGF-Sigma-Aldrich).
  • the plated bottles were then incubated for 3 days in a humidified greenhouse at 37 ° C with 5% CO 2 .
  • the aforementioned mesenchymal stem cells have high cellular confluence, especially below the tissue added in the culture plate culture. Petri In the conventional method, this number of cells would be reached only after months of cultivation, in inferior confluences and with a large amount of tissue debris.
  • the estimated confluence of 4 (four) days for the suspended cells is 100% and that, in this period of time, there is no adhered cell.
  • Isolated DID cells have been shown to be mesenchymal stem cells obtained by a less expensive, extremely faster, and much higher yielding technique, as evidenced by the article by Dominici et al., 2006 ( Figures 1-4). Cell confluence obtained was 80% after 3 day incubation of the bottles, and 106 cells were characterized by the expression of specific surface antigens ( Figure 5 and Table 1).
  • cell characterization was performed by FACScalibur (Becton Dickison Immunocytometry Systems, San Jose, USA) using antibodies cited by Dominici et al., 2006. The parameters of the cytometry were not described in detail as they follow the conventional patterns of mesenchymal stem cell characterization.
  • Osteogenic medium DMEM supplemented with 85mg / ml 2-L-ascorbic acid phosphate (Wako Chemicals, Germany) and 5mM b-glycerolphosphate (Sigma-Aldrich, Denmark).
  • Adipogenic Medium DMEM supplemented with 15% SFB and 1% P / S and 100 nM dexamethasoma (Sigma-Aldrich, Denmark).
  • Each culture condition was plated in 8 wells of 24-well plates so that differentiation and control could be evaluated in more than triplicate. This cultivation was performed for 1 month. And medium was added to the cells every 3 days without the addition of growth factors. After this period the adipocytes and osteocytes were properly stained to visualize the differentiation ( Figures 6 and 7).
  • Osteocyte staining occurred by washing (once) the culture with PBS and adding Alizarin Red S (Sigma-Aldrich) for 5 minutes, according to established protocols.

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Abstract

La présente invention concerne un nouveau procédé de culture de cellules souches mésenchymateuses adultes, ledit procédé comprenant les étapes suivantes: a) incubation de tissu ex vivo dégénéré en milieu de culture; b) isolement des cellules souches mésenchymateuses adultes du milieu obtenu dans l'étape a); et c) culture des cellules souches mésenchymateuses adultes isolées dans l'étape b). En outre, ledit procédé permet de supprimer les étapes de dissociation mécanique et enzymatique habituellement utilisées dans la technique. La présente invention concerne également une trousse pour la culture de cellules souches mésenchymateuses adultes, comprenant: a) un récipient de matière anti-adhérente; b) un milieu de culture approprié; et c) un récipient approprié pour la culture cellulaire. Par ailleurs, la présente invention concerne une composition comprenant les cellules souches mésenchymateuses adultes obtenues au moyen du procédé tel que défini antérieurement, et un véhicule pharmaceutiquement acceptable. De plus, la présente invention se rapporte à l'utilisation des cellules souches mésenchymateuses adultes obtenues au moyen du procédé tel que défini antérieurement pour la fabrication d'une composition destinée au traitement de discopathies du disque intervertébral dégénéré.
PCT/BR2014/050033 2013-12-05 2014-12-05 Procédé de culture de cellules souches mésenchymateuses adultes, trousse, composition et utilisations WO2015081410A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111317747A (zh) * 2020-03-24 2020-06-23 北京大学口腔医学院 肠道菌群调节剂与间充质干细胞的组合物及其应用
WO2021189558A1 (fr) * 2020-03-24 2021-09-30 北京大学口腔医学院 Composition de modulateur de la flore intestinale et de cellules souches mésenchymateuses et utilisation associée

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