WO2015081410A1

WO2015081410A1 - Adult mesenchymal stem cell culture method, kit, composition and uses

Info

Publication number: WO2015081410A1
Application number: PCT/BR2014/050033
Authority: WO
Inventors: João Antonio Pêgas HENRIQUES; Asdrubal FALAVIGNA; Manuela FIGUEIRÓ; Mariana Roesch ELY; Israel Silveira DE AGUIAR; Denise Cantarelli MACHADO
Original assignee: Fundação Universidade De Caxias Do Sul; Uniao Brasileira De Educacao E Assistencia
Priority date: 2013-12-05
Filing date: 2014-12-05
Publication date: 2015-06-11
Also published as: BR102013031330A8; BR102013031330B1; BR102013031330A2

Abstract

The present invention describes a new adult mesenchymal stem cell culture method comprising the steps of: (a) incubating degenerated ex-vivo tissues in a culture medium; (b) isolating adult mesenchymal stem cells from the medium obtained in step (a); and (c) cultivating the adult mesenchymal stem cells isolated in step (b). The method further dispenses with the mechanical and enzymatic dissociation steps that are usually applied in the prior art. The present invention also provides an adult mesenchymal stem cell culture kit comprising: (a) a container made of an anti-adhesive material; (b) a suitable culture medium; and (c) a suitable cell culture container. The present invention additionally describes a composition comprising the adult mesenchymal stem cells obtained by the above-mentioned method, and a pharmaceutically acceptable carrier. The present invention further describes the use of the adult mesenchymal stem cells obtained by the above-mentioned method for producing a composition for the treatment of diskopathies involving degenerated intervertebral disks.

Description

Patent Invention Descriptive Report

MESENQUIMAL stem cell cultivation process

ADULTS, KIT, COMPOSITION AND USES

Field of the Invention

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.

Background of the Invention

Degenerative disc disease (DDD) is a common and natural process of the human spine. This degeneration occurs progressively and may affect the biomechanics, stability and neurological function of the spine (Roh et al., 2005).

In the United States (USA), about 300,000 surgeries are performed per year due to DDD, and costs related to surgical procedures rise $ 50 billion each year (Koebbe et al., 2002).

Statistical data reveals the severity of the disease, with a total of 1, 5 million disc surgeries performed worldwide each year (Peul et al., 2007). Every year about 5,000 people have sciatica (also known as sciatica), a leg pain due to irritation or sciatic nerve compression due to disc disorders (Bakhsh, 2010), which are the leading cause of chronic low back pain in the modern world (Katz et al., 1999).

Currently, 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).

Recent studies have demonstrated the potential of stem cell treatment to regenerate or repopulate degenerate DIV (Haufe et al., 2006; Orozco et al., 201 1; Yoshikawa et al., 2010).

Pathophysiology of Disc Degeneration

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 is rich in proteoglycans and water, while AF is rich in collagen, especially of types I, II, VI and IX (Feng et al., 2006; Risbud et al., 2004).

The 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).

In mature NPs, proteoglycan aggregates are important for water retention, as restricting water flow influences tissue response to column loads (Feng et al., 2006). With aging, especially in the nucleus, there is a decrease in the aggregates of proteoglycans and an increase in unaggregated agrecane due to proteolytic degradation (Jahnke et al., 1988; Johnstone et al., 1995). Thus, with age, the ability to withstand the compressive load decreases.

In the fibrous ring, 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).

During life, many changes occur in the composition of the extracellular matrix, including: notochordal cell loss, mesenchymal cell senescence, loss of vascularization and calcification of vertebral plates, which alters or decreases the ability of disc cells to synthesize (Roughley, 2004) . Nuclear cell density decreases with age during life, while annular cellularity reaches a plateau after age 50 (Vernon-Roberts et al., 2008). Normal turnover capacity is impaired in the disc matrix and degeneration products accumulate (Roughley, 2004).

DIV degeneration and its structural changes result from this continuous catabolism occurring in the extracellular matrix and being associated with the inability to replace damaged collagen fibers and agrecane with new molecules during life. IVD seems to be incapable of intrinsic repair in adults, but the ability to induce such biological repair is a goal of many physicians (Guyer et al., 2003).

Regenerative Medicine in the Treatment of Intervertebral Disc Degeneration

Currently, surgical and non-surgical therapies to control IVD degeneration focus only on symptom relief. In recent years, as the understanding of cellular and molecular events involved in disc degeneration has improved, the idea of manipulating the cellular contents of the disc to achieve a beneficial outcome has grown (Freemont et al., 2002). One treatment option is the use of substances to stimulate existing disc cells to increase proteoglycan production. However, due to the relative acceleration of the degenerate disc, it may not be sufficient to achieve a good result. One option for this problem is to introduce into cells that are capable of producing the appropriate matrix in degenerate discs, representing an attempt to recover the biomechanical properties of the disc (Acosta et al., 2005).

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). However, this cellular source has practical limitations in the clinic due to the need to damage the adjacent disc, probably inducing degeneration at this level. In contrast, 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).

Characteristics of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are undifferentiated multipotent cells that have the ability to differentiate into a number of cell types, including bone, cartilaginous, adipose, muscle and tendon cells, depending on signals supplied to the biological environment.

It has been established that 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).

In Vitro Stem Cell Studies and Disc Degeneration 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).

Risbud et al. (2004) demonstrated that rat MSCs, when exposed to hypoxia and beta-growth transforming factor, are able to differentiate into a NP-compatible phenotype by signaling mitogen-activated protein kinase (Risbud et al., 2004). .

Using different methods, other authors have also shown that 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).

One possibility to explain why some patients with DDD have more pain than others is the development of intradiscal inflammation. Bertolo et al. (201 1) 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.

Experimental stem cell studies and disc degeneration After demonstrating that MSCs are able to differentiate into NP-like disc cells, in vivo studies were needed to evaluate the efficacy and safety of this therapeutic option in degenerate discs.

Crevensten et al. (2004) studied the viability of injected MSCs in rat coccid discs, and 14 days after injection a decrease in the number of fluorescence-labeled MSCs was observed. However, 28 days after injection, the number of MSCs rose to the initial number of 100% viable cells (Crevensten et al., 2004).

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).

Another author performed the transplantation of human CTMa into DDD minipigs and found that the cells survived in the porcine disc for at least 6 months and expressed typical markers of chondrocyte differentiation, suggesting differentiation to disc-like cells, which demonstrates the possibility of xenograft (Henriksson et al., 2009).

Some authors have also reported increased disc height with the use of MCT compared to control groups (Yang et al., 2010; Feng et al., 201 1; Sakai et al., 2006).

In vivo studies in rats and minipigs have been able to demonstrate that MSCs are able to survive and differentiate when injected into degenerate IVD and have the potential to restore their normal function and structure. However, these results are limited by their degeneration models that trigger rapid degeneration of IVD, which are not equivalent to the slow degeneration that occurs in human disc degeneration (Rousseau et al., 2007). Furthermore, the tail disc of rats and pigs is not subjected to the same static and dynamic load as human discs (Na et al., 2006; Lotz et al., 2006).

Stem Cell Clinical Studies and Disc Degeneration There are some clinical studies with DDD stem cells in the literature. Haufe and Mork (2006) found no improvement in the lower back after one year in 10 patients with DDD who were injected with hematopoietic stem cells obtained from the patient's bone marrow precursors. However, these authors did not perform any stem cell culture or expansion prior to injection. In addition, they also subjected patients to a 2-week course of hyperbaric oxygen therapy following cell injection (Haufe et al., 2006).

Yoshikawa et al. (2010) studied the effect of MSC in two patients with DDD, finding that 2 years after the initial procedure, symptoms improved in both patients and visual analogue scale scores for lower back pain decreased by 38% in one case, and 18% in the second patient. In both patients, after 2 years, radiography and computed tomography confirmed that the intervertebral vacuum phenomenon improved (Yoshikawa et al., 2010).

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).

Available Mesenchymal Stem Cell Cultivation Protocols

currently

A noteworthy point is the amount of viable mesenchymal stem cells present in a sample of degenerate tissue, which is much smaller compared to healthy tissue. Currently available MSC cultivation processes are costly and time-consuming as they involve mechanical and enzymatic dissociation steps, which often They tend to destroy tissue cells by the aggressive action of enzymes, considerably decreasing the yield of the process when employed in the cultivation of mesenchymal stem cells obtained from degenerate tissue.

In addition to the fact that the cell yield obtained is very small, the percentage of properly characterized mesenchymal stem cells further reduces this number. These factors hinder the application of MSCs in

Regenerative medicine for the treatment, for example, of diseases

Degenerative Intervertebral Disc.

The study by Vadalà et al., 2008, describes the methodology currently employed for culturing MSC from degenerated intervertebral disc tissue, as described below.

After removal of the degenerated intervertebral disc (DID), it is washed 3x in sterile physiological solution and placed in DMEM / F12 culture medium supplemented with 10% SFB and 1% P / S (the choice by this medium may vary according to literature, always correlating with the highest amount of nutrients).

The material is transported to the Laboratory of Genomics, Proteomics and

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.

Thus, the material is dissociated by incubating for 2 hours with

0.2 mg / ml Pronase (Sigma-Aldrich) under stirring in a humidified oven at 37 ° C with 5% CO ₂ . After this period the material is incubated ON with

1 mg / ml Collagenase Type IA (Sigma-Aldrich) also under stirring in a humidified oven at 37 ° C with 5% CO ₂ .

It is important to report that the literature reports dissociation using one or more enzymes and in varying concentrations. Also, the type of collagenase may be different in the literature. After this period the enzymes are inactivated with DMEM / F12 culture medium supplemented with 10% SFB and 1% P / S. At this time the material is fed through sterile Pasteur falcon pipettes and filtered through filters fitted with 40 µιη 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. Then 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 .

The attempt to establish culture occurs by maintaining the culture every 3 days with the addition of a further 500μΙ_ DMEM / F12 medium supplemented with 10% SFB and 1% P / S and 0.02ng / Ml_ EGF (Epidermal Growth Factor) and 0.02 ng / μί FGF (Fibroblast Growth Factor).

In short, 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) .

Patented search in the scope of stem cell cultivation

The search in patent literature has pointed out some prior art documents which will be described below.

Document PI0514387 "ISOLATION, CULTIVATION AND USES OF STEM CELLS / PROGENITORS" 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 "MESENQUIMAL STEM CELL CULTIVATION METHOD" 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 "MESENQUIMAL STEM CELLS AND THEIR USES" 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.

From what is clear from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity in the state of the art.

Although many advances have been made and many experimental models have shown that 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.

There remains, therefore, the need for a process of culturing adult mesenchymal stem cells that results in a larger number of adult mesenchymal stem cells, while not requiring large amounts of biological tissue sample.

Summary of the Invention

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;

b) isolation of adult mesenchymal stem cells from the medium obtained in step a); and

c) culture of adult mesenchymal stem cells isolated in step b).

In a preferred embodiment, 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.

In a more preferred embodiment, the degenerate ex vivo tissue is preferably derived from intervertebral disc, adipose tissue or bone marrow.

In a more preferred embodiment, the degenerate ex vivo tissue is preferably derived from the degenerate intervertebral disc (DID).

In a preferred embodiment, step a) comprises a minimum incubation period in suitable culture medium to 80% confluence.

In a preferred embodiment, step c) comprises culturing the cells in suitable culture medium for a period of at least 3 days.

In a preferred embodiment, 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.

In a preferred embodiment, 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 / μΐ. at 0.1 ng / μΐ ..

In a preferred embodiment, said process further comprises step d) of differentiating the adult mesenchymal stem cells obtained in step c) into bone and / or adipose tissue cells. In another aspect, the present invention provides a kit for culturing adult mesenchymal stem cells from degenerate tissue, comprising:

a) container of nonstick material;

b) suitable culture medium; and

c) container suitable for cell culture.

In a preferred embodiment, the kit further comprises at least one growth factor-containing reagent.

In another aspect, the present invention provides a composition comprising adult mesenchymal stem cells obtained by process as defined above and pharmaceutically acceptable carrier.

In another aspect, 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.

In a preferred embodiment, the biological tissue is preferably degenerate intervertebral disc, adipose tissue or bone marrow.

Brief Description of the Figures

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).

Figure 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 5 - Flow cytometry results obtained.

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 / µl Epidermal Growth Factor (EGF) and 0.02 ng / L Fibroblast Growth Factor (FGF). (A) consists of cells isolated by the conventional method highlighted, and (B) are cells isolated by the conventional method with greater confluence obtained after 8 months of maintaining the culture in the same plate (20X increase).

Detailed Description of the Invention

As previously described, the present invention provides a process of culturing adult mesenchymal stem cells comprising the steps of:

a) incubation of degenerate ex vivo tissue in culture medium; b) isolation of adult mesenchymal stem cells from the medium obtained in step a); and

c) 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.

In another aspect, 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. In a preferred embodiment, the biological tissue is preferably degenerate intervertebral disc, adipose tissue, bone marrow or knee cartilage.

The following are some of the terms that are presented throughout the patent application.

Adult Mesenchymal Stem Cells (MSCs)

In the context of this patent application, the term "adult mesenchymal stem cells (MSCs)" 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.

The 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

In the context of the present application, the term "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.

In a preferred embodiment, the degenerate ex vivo tissue is derived from degenerate intervertebral disc.

Incubation of degenerate ex vivo tissue

In the context of the present application, 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. By "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.

In a preferred embodiment, 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.

Nonstick Material Container

In the context of the present patent application, the term "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

In the context of the present patent application, the term "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.

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. 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

In the context of the present patent application, the term "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

In the context of the present patent application, the term "growth factor" should be understood as a substance capable of stimulating cell growth, proliferation and / or differentiation. Examples of 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).

In a preferred embodiment, growth factors are employed at a concentration ranging from 0.01 ng / μΐ. at 0.1 ng / μΐ .. Isolation of adult mesenchymal stem cells

In the context of the present patent application, the term "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.

Importantly, in the context of the present invention, 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.

Adult Mesenchymal Stem Cell Culture

The term "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. By "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. In a preferred embodiment, 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.

Container suitable for cell culture

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.

Pharmaceutically acceptable vehicle

In the context of the present application, 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.

Example 1. Preferred Realization

The examples shown herein are intended solely to exemplify one of the numerous ways of carrying out the invention, however, without limiting the scope thereof.

The method of isolating adult mesenchymal stem cells of the present invention involves the degenerate intervertebral disc (DID) of patients suffering from DDD. Such discs are obtained during spinal surgery of these patients with DDD and with painful and deficient symptoms refractory to clinical treatment.

Once the DID is removed, it 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). This container was kept at 37 ° C in a humidified greenhouse with 5% CO ₂ until collection began in the operating room. All DID is maintained in DMEM medium (supplemented with 5-20% SFB, preferably 10% SFB, and 1% P / S) and then the collected material was stored in a sterile petri dish. To this material were added 15ml of DMEM medium supplemented with 5-20% SFB, preferably 10% SFB, 1% P / S and growth factors at concentrations of 0.02ng ^ L (EGF and FGF-Sigma-Aldrich).

Subsequently, 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.

In small bottles, plating occurred with the addition of 1 ml of DMEM medium, supplemented with 5-20% SFB, preferably 10% SFB, and 1% P / S, plus 3 ml of medium containing the cells that were on the plate. Petri (small bottles), and growth factors at concentrations of 0.02ng ^ L (EGF and FGF-Sigma-Aldrich). These bottles were then incubated for 3 days in a humidified greenhouse at 37 ° C with 5% CO ₂ .

For large bottles, plating occurred with the addition of

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). The plated bottles were then incubated for 3 days in a humidified greenhouse at 37 ° C with 5% CO ₂ .

After this incubation period, a confluence of 80 to 90% of stem cells was observed in the culture bottles (Figures 3 and 4). Thus, after 7 days of withdrawing the sample from the IDD patient, adherence of numerous fibroblastic cells can already be achieved.

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.

It is interesting and important to note that in the present preferred embodiment 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).

Table 1. Flow cytometry results relative to specific surface antigens cited by Dominici et al., 2006

Results Dominici et al.,

CD Antibody

obtained 2006

CD11 b 0.51 (Negative) Negative

CD14 1, 36 (Negative) Negative

CD19 1,28 (Negative) Negative

CD34 0.66 (Negative) Negative

CD45 7.97 (Negative) Negative

CD73 98.13 (Positive) Positive

CD90 98.45 (Positive) Positive

CD105 70.9 (Positive) Positive

HLA DR 0.21 (Negative) Negative

Thus, 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.

Cell differentiation in two differentiated tissues was another proof in the study proposed by Dominici et al., 2006. Thus, mesenchymal stem cells were differentiated for bone and adipose tissue. Cells were grown under three different culture conditions: Control medium: DMEM and 10% SFB and 1% P / S.

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).

Adipocyte staining occurred with cell fixation by 4% paraformaldehyde for 40 minutes, washed once with PBS and stained with

5 minutes with Oil Red O (Sigma-Aldrich).

Osteocyte staining, in turn, occurred by washing (once) the culture with PBS and adding Alizarin Red S (Sigma-Aldrich) for 5 minutes, according to established protocols.

Cell differentiation images are valid only if negative stained mesenchymal stem cells are identified, thus proving a true power of cell differentiation.

(Dominici et al., 2006). Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments within the scope of the appended claims.

Claims

CULTURAL PROCESS OF MESENQUIMAL stem cells, ADULT, KIT, COMPOSITION AND USES

1 . Cultivation process of adult mesenchymal stem cells from degenerate tissue characterized by comprising the steps of:

(a) incubating degenerate ex vivo tissue in a suitable culture medium in a non-stick container;

c) culture of adult mesenchymal stem cells isolated in step b).

Method according to Claim 1, characterized in that the degenerate ex vivo tissue is derived from at least one tissue selected from the group consisting of: adipose tissue, bone marrow, periosteum, muscle tissue or parenchymal organs.

Process according to Claim 1 or 2, characterized in that the degenerate ex vivo tissue is preferably derived from intervertebral disc, adipose tissue or bone marrow.

Process according to any one of claims 1 to 3, characterized in that step a) comprises a minimum incubation period in a suitable culture medium until a confluence of 80%.

Process according to any one of claims 1 to 4, characterized in that step c) comprises culturing the cells in suitable culture medium for a period of at least 3 days.

Process according to any one of claims 1 to 5, characterized in that 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 / v to 15% v / v; and at least one antibiotic selected from the group consisting of: streptomycin or penicillin, in a concentration ranging from 0.1% w / v to 5% w / v.

Process according to any one of claims 1 to 6, characterized in that 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 / μί to 0,1 ng / μΐ ..

A method according to any one of claims 1 to 7, further comprising step d) of differentiating adult mesenchymal stem cells obtained in step c) into bone and / or adipose tissue cells.

An adult mesenchymal stem cell culture kit from degenerate tissue by a process as defined in any one of claims 1 to 8, comprising:

a) container of nonstick material;

b) suitable culture medium; and

c) container suitable for cell culture.

Kit according to claim 9, characterized in that it further comprises at least one growth factor-containing reagent.

1 1. Composition comprising adult mesenchymal stem cells obtained by process as defined in any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

Use of adult mesenchymal stem cells obtained by process as defined in any one of claims 1 to 8, characterized in that it is for the manufacture of a composition for regeneration of biological tissues.

Use according to claim 12, characterized in that the biological tissue is preferably degenerate intervertebral disc, adipose tissue, bone marrow or knee cartilage.