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    • 专利摘要:
    Procedure to prolong the half-life of neonatal neural progenitor cells with suramine. The present invention relates to an in vitro procedure for the culture and expansion of neural progenitor cells, preferably isolated from a postnatal animal, more preferably from the subventricular area of the brain. In particular, the method of the present invention is based on the pharmacological treatment of neural progenitor cells in culture with suramine. The method of the invention allows to increase the survival of neural progenitor cells for their subsequent clinical use in cell therapy. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2734733A1
    申请号:ES201830557
    申请日:2018-06-07
    公开日:2019-12-11
    发明作者:Delgado Alejandro Herrera；Loro Angel Manuel Pastor；Matarredona Esperanza Rodríguez
    申请人:Universidad de Sevilla；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] The present invention falls within the field of neurology and regenerative cell therapy of the nervous system, specifically within the procedures for the improvement of in vitro culture or expansion of neural progenitor cells from neonatal animals, preferably isolated from the subventricular area of the brain. These procedures allow the ex vivo population of said neural progenitor cells to be amplified, which can be differentiated into the different types of neural lineage cells (neurons, astrocytes and oligodendrocytes), so they are of interest for clinical use in regenerative cell therapy. of the nervous system
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007]
    [0008] Neural progenitor cells are characterized by their capacity for self-renewal and differentiation to different types of cells of neural origin: neurons, astrocytes and oligodendrocytes. They are the cells from which all neurons, astrocytes and oligodendrocytes of the central nervous system are formed during embryonic development. After the birth of the individual, this population of progenitor cells is restricted to only two regions of the brain, namely the subgranular area of the dentate gyrus of the hippocampus and the subventricular area adjacent to the lateral ventricles (Álvarez-Buylla, A. and García-Verdugo , JM, 2002, J. Neurosci. 22, 629-634; Gage, FH, 2000, Science 287, 1433-1438). Progenitor cells in the brain's subventricular zone of neonatal rats can be isolated and cultured in the form of spherical aggregates called neurospheres (Reynolds, BA and Weiss, S., 1992, Science 255, 1707-1710). These cultures of neurospheres allow to have a population of progenitor cells that can be used in cell therapy. Thus, there have been reports of beneficial therapeutic applications of neonatal neural progenitor cell implants from neurosphere cultures in animal models of stroke, head trauma, spinal cord injuries or axotomy (Hicks, AU, et al., 2007, Neuroscience 146 , 31-40; Koutsoudaki, PN, et al., 2016, Glia 64: 763-779; Morado-Díaz, CJ, et al., 2014, J. Neurosa. 34, 7007-17).
    [0009]
    [0010] The neural progenitor cells obtained after birth provide important advantages for use in cell therapy with respect to embryos, since on the one hand the ethical considerations related to experimental work with embryos are avoided and, on the other, the risk of teratomas develop from implanted neural cells. This risk in the generation of teratomas also limits the use of immortalized cell lines in cell therapy. Neonatal or adult neural progenitor cells, however, have some limitations to their use in regenerative therapy. One of them is the need to have a relatively high number of animals to obtain an adequate density of cells to be implanted. Another is the fact that these cells lose proliferative capacity and viability during their ex vivo expansion , which limits the time window of their use for clinical applications to the first subcultures after their isolation. This is why the improvement of these cultures to successfully amplify this cell population, as well as delay their senescence, would expand the possibility of using these cells and reduce the number of animals to be sacrificed to obtain them.
    [0011]
    [0012] Suramin is a blocking agent for P2 type purinergic receptors that is used in both basic and clinical research due to the multiple effects derived from its mechanism of action. Neural progenitor cells express P2 purinergic receptors both in their neurogenic niche of the subventricular zone, and when grown as neurospheres (Messemer, N., et al., 2013, Neuropharmacology 73, 122-137). It has been reported that treatment with suramin reduces both migration and proliferation of neural progenitor cells of the subventricular zone during embryonic development (Liu, X., et al., 2008, Proc. Natl. Acad. Sci. USA 105, 11802-7). However, there are no studies on the effect of suramin in this same population of postnatal cells.
    [0013]
    [0014] Neural progenitor cells are, therefore, a population of interest for use in cell therapy, not only for their ability to generate the various cells of the neural lineage (neurons, astrocytes and oligodendrocytes) but also for their ability to produce neuroprotective factors. or for exercising important functions immune. Normally these cells are obtained from the brain of embryos, as it is when they have their maximum proliferative capacity and are able to integrate better into the host tissue after implantation. This population of cells persists in only two regions of the brain after birth, the subgranular zone of the dentate gyrus of the hippocampus and the subventricular zone adjacent to the lateral ventricles. The number of progenitor cells in this case, however, is much smaller than in the embryonic state and also their proliferation capacity is restricted. That is why the development of a mechanism that allows amplifying, as well as improving the survival and expandability of these neural progenitor cells after birth would be of interest to limit the use of embryonic tissue as a source of cells and facilitate possibilities of its use in cell therapy.
    [0015]
    [0016] In summary, although it is possible to isolate and maintain in neural progenitor cells obtained from the brain of neonatal and adult animals, these cells have a limited half-life and expandability and after several reseeding they lose self-renewal capacity. Therefore, it is an essential objective in the technical field the development of optimized culture procedures that allow to amplify in vitro the population of neonatal and adult neural progenitor cells until obtaining a high number of healthy and viable cells that can be used in cell therapy . These culture procedures should also increase the half-life or survival of the cells without losing, via subcultures, their viability, proliferation capacity, self-renewal and differentiation to the main neural cell types. The development of these improved culture procedures would thus limit the use of embryonic tissue as a source of the cells, minimize the number of animals to be sacrificed to obtain them, and facilitate, improve and expand the scope of therapeutic application of these cells.
    [0017]
    [0018] DESCRIPTION OF THE INVENTION
    [0019]
    [0020] The present invention relates to a method in which an action of the suramin compound is described in neonatal neural progenitor cell cultures, from the subventricular area of the brain.
    [0021] The procedure described here is an improvement with respect to other procedures described to date in the state of the art, since, with this treatment it is possible to increase the survival of the cultured neural progenitor cells and amplify the population thereof until obtaining a adequate number of healthy and viable cells for later use in cell therapy, with the consequent interest for its application for therapeutic purposes. In addition, this procedure avoids the use of embryos and reduces the number of neonatal animals from which to obtain the cells.
    [0022]
    [0023] In particular, the process described in the present invention is based on the pharmacological treatment of the neural progenitor cells in culture with the suramin compound at a concentration of between 100 and 300 pM, and preferably 200 pM. As the results of the examples described below show, this treatment significantly increases the half-life or survival of these cells, maintaining their proliferative and self-renewal capacity throughout the subcultures and thus successfully amplifying this cell population. Likewise, treatment with suramin induces a decrease of, preferably, 75% in cell death due to apoptosis of the cells in culture. Adult neural progenitor cells treated with suramin have a capacity of expansion throughout the subcultures significantly higher than those not treated with this compound, and also maintain multipotency because they are capable of generating the different cell types corresponding to the neural lineage when subsequently seeded on adherent substrate. Therefore, a new effect of this drug is described that is of interest to amplify and increase the half-life of neural progenitor cells obtained from neonatal animals and maintained in culture as neurospheres.
    [0024]
    [0025] The procedure described here allows, therefore, to increase the number of effective subcultures of said cells maintaining the conditions of cell viability and preserving their proliferation capacity and multipotentiality. In addition, the degree of cell death by apoptosis is reduced, preferably by approximately 75%, with the method of the invention.
    [0026]
    [0027] Therefore, in a first aspect, the present invention relates to the use of suramin as a reagent or supplement for the improvement in obtaining, survival and in vitro expansion of neural progenitor cells.
    [0028]
    [0029] "Suramin" or "sodium suramin" is the chemical compound of CAS number 145-63-1 and molecular formula C51H40N6O23S6. It is a P2 receptor antagonist and rianodin receptor agonist that is commercially available.
    [0030]
    [0031] In a preferred embodiment, the suramin is at a concentration of between 100 and 300 p, M, and more preferably at a concentration of 200 p, M, when used in the context of the present invention.
    [0032]
    [0033] "Neural progenitor cells", "neural precursor cells" or "neural stem cells" are those cells of the nervous system that lack late differentiation markers and have the ability to proliferate, self-renew and differentiate into neural lineages, such as neurons, astrocytes or oligodendrocytes. They are the cells from which all the cells of the nervous system are generated during embryonic development. In the adult mammalian brain, however, its location is restricted to two zones, the subventricular zone adjacent to the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus.
    [0034]
    [0035] Neural progenitor cells can be isolated and are capable of proliferating in vitro forming multipotent neurospheres with the ability to self-renew.
    [0036]
    [0037] In another preferred embodiment, the neural progenitor cells referred to in the present invention are derived from neonatal rats, and preferably from rats 6 or 7 days old.
    [0038]
    [0039] In the context of the present invention, "neonatal" means an animal that is not in its embryonic or fetal stage, but has already been born, and is in the first days of life (6 or 7 days old).
    [0040]
    [0041] The neural progenitor cells that are cultured in the present invention can come, but are not limited to, from rodents or other mammals. Preferably, the neural progenitor cells referred to in the present invention are from the Rattus norvegicus (rat) species, more preferably from rats 6 or 7 days old.
    [0042] In another preferred embodiment, the neural progenitor cells that are cultured in the present invention come from the subventricular area of the brain.
    [0043]
    [0044] The "subventricular zone", ZSV, is the tissue located adjacent to the wall of the lateral ventricles of the central nervous system in mammals and is made up of different types of cells, including neural progenitor cells. It is one of the areas where Neurogenesis takes place in the brain of adult mammals.The subventricular zone is therefore a niche of neural stem cells for the adult neurogenesis process.It houses the largest population of proliferative cells in the adult brain of rodents, monkeys and humans.
    [0045]
    [0046] By way of example and without limitation, neural progenitor cells can be isolated from the subventricular area of the brain of a neonatal or adult animal by isolation of all or part of said area and subsequent enzymatic and / or mechanical ex vivo treatment by the procedures described in Talaverón, R., et al., 2013, PLoS One 8: e54519; Talaverón, R., et al., 2014, Glia 62, 623-638; o Talaverón, R., et al., 2015, Front. Cell Neurosci. 9, 411. After said treatment the resulting cell suspension is cultured in a culture medium suitable for the formation of neurospheres as will be explained later in the present description.
    [0047]
    [0048] Another aspect of the invention relates to a process, hereafter "process of the invention", for the culture, expansion, proliferation, amplification, obtaining or production in vitro of neural progenitor cells, preferably adults, comprising the following steps :
    [0049]
    [0050] to. prepare a culture of neurospheres from an isolated population of neural progenitor cells, and
    [0051] b. add suramin to the culture of step (a).
    [0052]
    [0053] In a preferred embodiment of the process of the invention, the neural progenitor cells are derived from neonatal rats, and preferably from rats 6 or 7 days old.
    [0054] In another preferred embodiment, the neural progenitor cells come from the subventricular zone of the brain.
    [0055]
    [0056] "Neurospheres" means spherical-looking aggregates of cells that are formed in culture, preferably in suspension culture, from the sowing of neural progenitor cells and in the presence of certain mitogenic factors, such as the epidermal growth factor ( EGF) and the basic fibroblast growth factor (FGF-2).
    [0057]
    [0058] Preferably, the base culture medium employed in step (a) in the present invention consists of Dulbecco's Modified Eagle's Medium; Nutrient Mixture F-12 (DMEM-F12) supplemented with EGF and FGF-2. More preferably, said culture medium is also supplemented with B-27 and Glutamax®, both commercially available products. Even more preferably, said culture medium is also supplemented with antibiotics and antifungals. The antibiotics that could be used in this culture medium are those commonly used for in vitro culture of mammalian cells, such as but not limited to, penicillin and streptomycin, and as an antifungal, amphotericin.
    [0059]
    [0060] Thus, in another preferred embodiment of the process of the invention, the culture of step (a) takes place in the presence of a culture medium supplemented with EGF and FGF-2.
    [0061]
    [0062] In another preferred embodiment of the process of the invention, the cultivation of step (a) takes place at a temperature of 37 ° C, 5% CO2.
    [0063]
    [0064] In a particular embodiment, the neurosphere culture is prepared, in step (a) of the process of the invention, according to the protocol described in Talaverón, R., et al., 2013, PLoS One 8: e54519; Talaverón, R., et al., 2014, Glia 62, 623-638; o Talaverón, R., et al., 2015, Front. Cell Neurosci. 9, 411, which is detailed below. The subventricular zone is isolated from 4 rats of six to seven days and is subjected to an enzymatic and mechanical treatment described in the aforementioned articles, after which the resulting cell suspension is sown in a T25 bottle in culture medium with a defined composition (DMEM-F12 added from supplement B-27, 2 mM Glutamax®, 100 units / ml penicillin, 100 pg / ml streptomycin, 0.25 pg / ml amphotericin and supplemented with EGF at 20 ng / ml and FGF-2 at 10 ng / ml) and kept in an incubator at 37 ° C and 5% CO2. At 48-72h, neurospheres will begin to form from the neural progenitor cells of the culture and thereafter a subculture of the cells will have to be performed every 72-96h. For this, the contents of the bottle are centrifuged at 150g for 5 minutes, the pellet is resuspended in 200 pl of the culture medium and the neurospheres are mechanically broken down by successive aspirations through a p200 pipette tip. 5 ml of medium are added and centrifuged again. After removing the supernatant, the pellet is resuspended in 1 ml of culture medium, the resulting cells are counted in a Neubauer chamber and then seeded again at a rate of 15,000 cells / cm2 with 5 ml of fresh medium.
    [0065]
    [0066] In another preferred embodiment, the process of the invention further comprises a step (c) after step (b), which comprises the realization of additional subcultures, preferably of at least two additional subcultures, consecutive from the culture obtained after the stage (to).
    [0067]
    [0068] In another preferred embodiment, the neurospheres generated in the culture of stage (a) and in each of the subcultures of stage (c) are broken down, preferably mechanically, prior to the realization of the next subculture.
    [0069]
    [0070] The aforementioned disintegration can be carried out, for example but not limited to, by one or several aspirations of the neurosphere culture, for example with the help of a glass Pasteur pipette with the tip polished to the fire or with the plastic tip of an automatic pipette p200 .
    [0071]
    [0072] In a more preferred embodiment, each subculture of the intermediate stage (c) takes place for a time between 48 and 96 hours, and preferably 72 and 96 hours.
    [0073]
    [0074] The culture medium used in the subcultures of intermediate stage (c) is preferably the same as previously described and used in stage (a).
    [0075]
    [0076] The cells seeded at the beginning of each of these subcultures of stage (c) are taken from the previous culture or subculture once it has finished.
    [0077] In another preferred embodiment, the cells are seeded, for the realization of each subculture, in an initial amount of 10,000-15,000 cells / cm 2 of culture support surface, and preferably 15,000 cells / cm 2.
    [0078]
    [0079] In another preferred embodiment, the suramin is added at the beginning of each subculture of step (c), that is, every 72 to 96 hours.
    [0080]
    [0081] In another preferred embodiment, the suramin is added in the process of the invention in a final concentration of between 100 and 300 p, M, and preferably 200p, M, from a stock 100 times more concentrated.
    [0082]
    [0083] After the culture of stage (a) and / or subcultures of intermediate stage (c) of the process of the invention, optionally, a quality control, sterility and cellular safety can be carried out. Likewise, the cells obtained in different densities can be distributed to be administered, stored and / or transported.
    [0084]
    [0085] In another preferred embodiment, the process of the invention further comprises an additional step (d) comprising the storage, more preferably the cryopreservation, of all or part of the neural progenitor cells obtained. Even more preferably this storage takes place in sterile containers, preferably in vials, of equal or different densities with each other. The "cryopreservation" can be carried out between, preferably, -80 ° C and -196 ° C.
    [0086]
    [0087] In another preferred embodiment, the process of the invention takes place in a clean room, that is, under controlled conditions of sterility and safety that allow the subsequent safe clinical use of the resulting cells.
    [0088]
    [0089] Thus, the process of the present invention is based on the pharmacological treatment of neural progenitor cells in culture with suramin. The method of the invention allows to obtain healthy neural progenitor cells in a number suitable for subsequent clinical use in neural cell therapy. In addition, these cells maintain proliferative capacity and multipotentiality for more subcultures than those that do not receive the treatment and have greater survival and viability. This procedure also allows avoiding the use of embryonic tissue as a source of neural progenitor cells and reducing the number of animals Neonatals needed to obtain the cultures.
    [0090]
    [0091] Another aspect of the invention relates to a kit, hereafter "kit of the invention", which comprises all the reagents, supports and means necessary to carry out the process of the invention as described above. Preferably, said kit comprises at least: (i) a culture medium as defined above, and (ii) suramin.Preferably, said kit further comprises, but without any limitation, antibiotics, antifungals or any other agent to prevent contamination of the cultured cells, one or more sterile containers or supports for containing and / or culturing the cells and / or one or more vials for the storage and / or cryopreservation of the cells, said kit may further comprise the instructions for carrying out the procedure. of the invention.
    [0092]
    [0093] Another aspect of the invention relates to the use of this kit of the invention to carry out the process of the invention.
    [0094]
    [0095] The neural progenitor cells obtained after the process of the invention have functional and structural characteristics that differentiate them from other isolated neural progenitor cells, cultured, obtained and / or purified by methods other than the process of the invention. It is commonly known in the art that the gene expression profiles of cells and, as a consequence their cellular functions, differ depending on the protocol used for their culture and expansion in vitro. Thus, the process of the invention employs specific culture conditions and reagents, in particular the use of suramin, which affect the gene expression profile and the properties of the neural progenitor cells finally obtained. In this sense, and as explained earlier in the present description, these cells obtained at the end of this procedure have: i) increased survival; ii) a longer half-life, and (iii) a preserved multipotentiality throughout subcultures, with respect to other neural progenitor cells obtained by other ex vivo expansion procedures where suramin is not used. Therefore, said cells are necessarily different from other neural progenitor cells obtained by other prior art procedures.
    [0096]
    [0097] Therefore, another aspect of the invention relates to a population of cells Neural progenitors obtained or obtainable by the method of the invention, hereafter referred to as the "cell population of the invention".
    [0098]
    [0099] The cell population of the invention can be administered directly to an individual for regeneration or nerve cell repair in pathologies, diseases or clinical conditions associated with the nervous system, preferably central, or they can be added to pharmaceutical compositions for use in cell therapy.
    [0100]
    [0101] Thus, another aspect of the invention relates to a pharmaceutical composition, "pharmaceutical composition of the invention", which comprises the cell population of the invention, preferably in a therapeutically effective cell density. More preferably, this pharmaceutical composition further comprises at least one Pharmaceutically acceptable carrier as long as it is not incompatible with the survival and functionality of the cells of the invention comprised in the pharmaceutical composition.
    [0102]
    [0103] The term "therapeutically effective density" refers to the density of cells of the invention that produces the desired effect. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex, pathological condition or tolerance of the individual to whom the composition of the invention is to be administered.
    [0104]
    [0105] The pharmaceutical composition of the invention may be adapted for application by a "device suitable for the injection of cells into a tissue." This would be any device or instrument that may be useful for the injection of cells into blood circulation or into a tissue. Examples of such devices or instruments are, but are not limited to, syringes, vials, catheters, needles, cannulas, or in general any instrument that can be used in cell therapy, including those known in the state of the art.
    [0106]
    [0107] Another aspect of the invention relates to the cell population of the invention for use in cell therapy. Preferably, this aspect of the invention relates to the cells or cell population of the invention for use in the treatment and / or prevention of diseases, pathologies or clinical conditions related to the nervous system, preferably with the central nervous system. In one embodiment more preferred, the diseases, pathologies or clinical conditions related to the nervous system are selected from the list consisting of: neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, etc., stroke or ischemia, craniocerebral trauma, axotomies, pigmentary retinosis , nerve injuries such as spinal cord injuries, or the like
    [0108]
    [0109] By "cell therapy" or "cytotherapy" is meant the therapy in which cellular material or cells are administered to an individual, in the context of the present invention the living cells or cell population of the invention.
    [0110]
    [0111] The pharmaceutical composition to which the invention relates could be administered, but without limitation, by systemic transplantation or by local injection into the affected tissue.
    [0112]
    [0113] In general, the pharmaceutical composition to which the invention relates is for the treatment and / or prevention of any clinical situation in which it is necessary to restore or regenerate all or part of the functionality of the nervous system, preferably central, in an individual with some type of lesion or pathology of the nervous system.
    [0114]
    [0115] In another preferred embodiment, the individual presenting the disease, pathology or pathological condition associated with the nervous system is a mammal, more preferably a primate, even more preferably a human.
    [0116]
    [0117] The pharmaceutical composition referred to in the present invention can be administered alone or in combination, simultaneously or sequentially, with other medications or therapeutic agents intended for the treatment and / or prevention of diseases, pathologies or clinical conditions related to the nervous system, preferably with the central nervous system
    [0118]
    [0119] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0120]
    [0121] DESCRIPTION OF THE FIGURES
    [0122]
    [0123] FIG. 1 . It shows the effect of suramin treatment in neurospheres obtained from the subventricular zone of neonatal rats. Appearance of neurospheres grown under control conditions (A) and treated with 200 pM suramin (B) . Bars: 100 pm. (C) Average diameter of control neurospheres and treated with suramin. The percentage with respect to the control value is displayed. The bars show the mean value ± the standard error of the mean (eem). The data collect measures of diameters of neurospheres obtained from 7 different cultures. * p <0.05 (Student's t test). (D) Measurement of the percentage of cell death after successive subcultures or passes (p) (p3, p4, p5, p6, p7) in cultures of control neurospheres and treated with suramin. The data correspond to the mean ± eem, n = 4. * p <0.05 (Student's t test). (E) Percentage of cell death due to apoptosis in cultures of control neurospheres and treated with suramin. The data represent the mean ± eem, n = 7. * p <0.001 (Student's t test).
    [0124]
    [0125] FIG. 2 . It shows the appearance of control and suramin treated neurospheres after six passes of the culture. It is observed that the control neurospheres lose proliferative capacity and adhere to the bottle, something that is not observed in any of the neurospheres of the vials treated with suramin. Bars: 100 pm.
    [0126]
    [0127] FIG. 3: Shows the multipotentiality of neural progenitor cells from cultures of neurospheres treated with 200 pM suramin . Photomicrographs taken on an epifluorescence microscope of cells from the cultures of suramin-treated neurospheres seeded on an adherent substrate and in the absence of mitogens to favor cell differentiation are shown and after seven days in culture. Under these conditions, the neural lineage cells are formed: astrocytes (A, identified by immunocytochemistry for the acidic fibrillar protein of the glia), neurons (B, identified by immunocytochemistry for the doublecortin protein) and oligodendrocyte precursors (PREC. OLIGODEN. ) (C, identified by immunocytochemistry for proteoglycan chondroitin sulfate NG2, the nuclei of the cells present in that field, stained with a specific nucleic acid dye). Bars: 50 pm.
    [0128]
    [0129] EXAMPLES
    [0130]
    [0131] Next, the invention will be illustrated by tests performed by the inventors that demonstrate the effectiveness of the process of the invention.
    [0132]
    [0133] EXAMPLE 1. PROCEDURE FOR GROWING NEONATAL NEUTRAL PROGENITING CELLS AND SURAMINE TREATMENT
    [0134]
    [0135] To apply the proposed invention, cultures of neurospheres from neonatal rats were performed following the protocols already described in detail above (Talaverón, R., et al., 2013, PLoS One 8: e54519; Talaverón, R., et al., 2014 , Glia 62, 623-638; Talaverón, R., et al., 2015, Front. Cell. Neurosa. 9, 411). Briefly, the subventricular zone was isolated from 6 rats of seven days and underwent an enzymatic and mechanical treatment as described in the aforementioned articles, after which the resulting cell suspension was seeded in a T25 bottle in culture medium with a Defined composition (DMEM-F12 added from supplement B-27, 2 mM Glutamax®, 100 units / ml penicillin, 100 pg / ml streptomycin, 0.25 pg / ml amphotericin and supplemented with epidermal growth factor at 20 ng / ml and basic fibroblast growth factor at 10 ng / ml) and kept in an incubator at 37 ° C and 5% CO2. At 48-72h, neurospheres began to form from the neural progenitor cells of the culture and thereafter a subculture of the cells was performed every 72-96h. For this, the contents of the bottle were centrifuged at 150g for 5 minutes, the precipitate was resuspended in 200 pl of the culture medium and the neurospheres were mechanically disintegrated by successive aspirations through a p200 pipette tip. 5 ml of medium was added and centrifuged again. After removing the supernatant, the precipitate was resuspended in 1 ml of culture medium, the resulting cells were counted in a Neubauer chamber and then seeded again at a rate of 15,000 cells / cm2 with 5 ml of fresh medium.
    [0136]
    [0137] For the treatment with suramin a 100X stock (20 mM) of suramin was prepared in milliQ water, filtered and 50 pl aliquots were made that were frozen until utilization. The treatment of the cells with suramin began after performing two subcultures to the initial culture of neurospheres, because that is when the population of progenitor cells is purified and the dead cells are eliminated. To do this, after the pass, T25 bottles with the cell density mentioned above and 5 ml of culture medium were sown to which 50 ^ 1 of a 100X aliquot of the suramin stock was added. This process was repeated every 72-96h. The cells were kept with this treatment alive and in self-renewal for at least 10 subcultures.
    [0138]
    [0139] Once the bottles were seeded with neurosphere cells treated with suramin, the proliferation and degree of cell death of the mimes were monitored and compared against control bottles, which were not treated with suramin, to which the same amount of vehicle (50 ^ l of milliQ water). Thus, 72h after sowing, the neurospheres treated with suramin had a significantly larger diameter than the neurospheres of the control bottles (Figure 1A, B and C). When the successive subcultures were carried out, the number of live and dead cells in each bottle was counted. The bottles treated with suramin always had a significantly higher percentage of living cells and a significantly lower percentage of dead cells than the control bottles (Figure 1D). When evaluating the degree of cell death due to apoptosis using the TUNEL technique, it was shown that suramin-treated cells had a lower apoptosis death rate (p <0.001) than that of untreated bottles (Figure 1E).
    [0140]
    [0141] It was also observed that, after the fifth-sixth subculture, the cells of the control bottles were losing self-renewal capacity and, therefore, ability to continue forming neurospheres and were also dying or beginning to adhere to the surface of the bottle to differentiate (Figure two). However, this did not happen with the cells of the bottles treated with suramin, which did not adhere to the surface of the bottle and maintained their ability to form viable neurospheres at least after ten subcultures (Figure 2).
    [0142]
    [0143] Therefore, it can be concluded that the treatment of the neurospheres obtained from the subventricular zone of neonatal rats with 200 ^ M suramin produces a significant increase in the survival of these cells and in the number of subcultures in which they remain proliferative and viable. .
    权利要求:
    Claims (18)
    [1]
    1. Use of suramin as a reagent or supplement for obtaining and expanding in vitro neural progenitor cells.
    [2]
    2. Use according to claim 1, wherein the suramin is at a concentration of between 100 and 300 p, M.
    [3]
    3. The use according to claim 2, wherein the suramin is at a concentration of 200p, M.
    [4]
    4. Use according to any of claims 1 to 3, wherein the neural progenitor cells are derived from neonatal rats.
    [5]
    5. Use according to any of claims 1 to 4, wherein the neural progenitor cells originate from the subventricular area of the brain.
    [6]
    6. Procedure for in vitro culture of neural progenitor cells comprising the following stages:
    to. prepare a culture of neurospheres from an isolated population of neural progenitor cells, and
    b. add suramin to the culture of step (a).
    [7]
    7. Method according to claim 6, wherein the suramin is added in a concentration of 200p, M.
    [8]
    8. Method according to any of claims 6 or 7, wherein the neural progenitor cells are derived from neonatal rats.
    [9]
    9. Method according to any of claims 6 to 8, wherein the neural progenitor cells come from the subventricular area of the brain.
    [10]
    10. Method according to any of claims 6 to 9, wherein the culture of step (a) takes place in the presence of a culture medium supplemented with epidermal growth factor and basic fibroblast growth factor.
    [11]
    11. Method according to any of claims 6 to 10, wherein the cultivation of step (a) takes place at 37 ° C, 5% CO2 and for a time between 48 and 72 hours.
    [12]
    12. A method according to any of claims 6 to 11, further comprising a stage (c) after stage (b), which comprises the realization of additional consecutive subcultures from the culture obtained after stage (a).
    [13]
    13. Method according to claim 12, wherein each subculture of step (c) takes place for a time between 48 and 96 hours.
    [14]
    14. Method according to any of claims 12 or 13, wherein the neurospheres generated in the culture of stage (a) and in the subcultures of stage (c) are disintegrated prior to the realization of the following subculture.
    [15]
    15. A method according to any of claims 12 to 14, wherein the cells are seeded, for the realization of each subculture, in an amount of 10,000-15,000 cells / cm2 of culture support surface.
    [16]
    16. The method according to any of claims 12 to 15, wherein suramin is added at the beginning of each subculture of step (c).
    [17]
    17. Method according to claim 16, wherein the suramin is added in a final concentration of between 100 and 300. ^ M.
    [18]
    18. Method according to claim 17, wherein the suramin is added in a final concentration of 200. ^ M.
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    同族专利:
    公开号 | 公开日
    ES2734733B2|2020-05-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2008028531A1|2006-09-07|2008-03-13|Neuroprogen Gmbh Leipzig|Method of culturing npcs|
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