RU2749156C9 - Method for obtaining adhesive culture of neural stem/progenitor cells of olfactory nose of mammals for treatment of spinal cord injuries - Google Patents

Abstract

FIELD: biotechnology and medicine.SUBSTANCE: invention relates to the field of biotechnology and medicine. Disclosed is a method of obtaining an adhesive culture of neural stem / progenitor cells from the olfactory lining of a mammalian nose for the treatment of spinal cord injuries. It includes obtaining a tissue sample from the olfactory lining of a mammal's nose, followed by washing and incubating for 1 hour at 37°C with 3.6 units/ml of dispase II, then the tissue of the olfactory epithelium is mechanically separated from its own plate in DMEM:F12 (1:1) medium with antibiotics and incubated with a solution of 500 U/ml collagenase for 10 min at 37°C, the enzyme is inactivated with medium with serum, resuspended, centrifuged for 2 min at 900 g, cells are placed on plastic and cultured for 5 days in DMEM:F12 medium (1:1) containing 10% FBS, 2 mM L-glutamine and antibiotics, the medium is replaced every two days, formed by the monolayer of olfactory epithelium cells is removed with 0.25% trypsin solution, placed on plastic coated with 50 mcg/ml fibronectin, cultured for 1-2 days in neurobasal medium containing 2 mM L-glut amine, 1% ITS, 2% B27, 100 mcg/ml streptomycin, 100 U/ml penicillin, 60 mcg/ml gentamicin, 50 ng/ml bFGF, 50 ng/ml EGF, the resulting cells are characterized by markers nestin and βIII -tubulin.EFFECT: obtaining an adhesive culture enriched in neural stem / progenitor cells with increased survival in an amount sufficient for further transplantation.4 cl, 14 dwg, 5 ex

Description

FIELD OF TECHNOLOGY

The invention relates to medicine, namely to cellular neurotransplantology, and can be used to treat spinal cord injuries.

LEVEL OF TECHNOLOGY

Today, there is a constant increase in the number of victims of spinal cord injuries, there is a high mortality rate and disability of such patients. As a result of post-traumatic mechanical injuries of the spinal cord, patients partially or completely lose the mobility and sensitivity of the body, not only their physical, but also their psychosocial state deteriorates. Drug treatment and surgery do not always allow achieving the desired results, since traumatic injuries result in the death of a large number of functional neurons. In this regard, cell therapy may become a promising direction for the treatment of spinal cord injuries. Cell transplantation can cause regeneration of axons, facilitate replacement of lost neurons, and reduce the risk of disorders after spinal cord injury through the secretion of neurotrophic factors [1, 2]. Neural stem / progenitor cell (NSPC) transplantation is considered a promising strategy for replenishing lost neurons [3].

The most promising may be the NSPC of the olfactory lining of the nose. Obtaining an olfactory lining is an affordable and safe procedure for patients. These cells are tissue-specific and autologous as they can be obtained from a patient with spinal cord injury and, after growing in culture and directed differentiation, transplanted into the same patient.

The olfactory lining of the nose contains a constantly regenerating neuroepithelium. NSPCs of the olfactory epithelium have multipotency and differentiate into neurons and other cell types, express markers nestin, sox-2 [4]. In experimental studies on the regeneration of the spinal cord after injury, NSPCs are most often used in suspension culture of neurospheres. A series of in vitro experiments have shown that cells forming neurospheres divide rapidly and have a high survival rate [4-6]. Cells of neurospheres after transplantation survive, migrate and integrate into the host's spinal cord, produce NGF and BDNF, promote regeneration and reinnervation of axons, which leads to the restoration of motor functions of the limbs [7, 8].

However, during therapy using neurospheres, it is difficult to analyze their cellular composition, namely, to assess the percentage of NSPCs and neurons formed from them, morphological analysis of transplanted cells, and determination of the total number of cells. There are no methods for obtaining pure populations of NSPCs in vitro, and their development is impossible, since NSPCs in vitro are capable of differentiating into mature neurons, as well as glial cells and oligodendrocytes [9, 10]. Since in order to increase the efficiency of cell transplantation, it is necessary to assess the purity of the culture and the number of cells of the injected drug, it is necessary to develop protocols for obtaining two-dimensional adhesive NSPC cultures, which will make it possible to characterize the cell preparation according to these parameters. As is known, βΙΙΙ-tubulin is expressed in immature and mature olfactory neurons in vivo; nestin is a marker of NSPC [11, 12]. Thus, by revealing nestin and βΙΙΙ-tubulin - positive cells in the obtained adhesive cultures, it is possible to determine the percentage of NSPCs and neurons differentiating from them, i.e. all cells of neuronal differentiation. To date, there is no single protocol for obtaining an adhesive NSPC culture of the olfactory lining. Also, insufficient data have been accumulated on the influence of cultivation conditions on the proliferation and differentiation of these cells.

There are various methods for obtaining a culture of NSPC from the olfactory lining of the nose of mammals.

There is a known method of obtaining NSPC in a suspension culture of neurospheres from the olfactory lining of the human nose [13], which consists in the fact that the tissue of the olfactory lining is dissociated mechanically and then treated for 60 min at 37 ° C with a solution of enzymes: collagenase, dispase, DNase I and hyaluronidase in Dulbecco's modified Eagle's medium and Ham's substrate F-12 (DMEM / F12). The resulting cells were grown on plates coated with poly (2-hydroxyethyl methacrylate) to prevent their attachment to plastic, in DMEM / F 12 medium containing 10% fetal bovine serum (FBS), B27 supplement, 20 ng / ml of basic fibroblast growth factor , 20 ng / ml epidermal growth factor and antibiotic - antimycotic solution. Neuronal differentiation was detected using immunofluorescent staining for specific neuronal markers Tuj 1, MAP2.

The main disadvantage of this method is that when obtaining a primary culture of cells, the lamina propria was not separated from the olfactory epithelium, which does not allow obtaining a purer culture of NSPK, since the lamina propria contains many other types of cells. The disadvantage of this method is the treatment with a whole cocktail of enzymes, namely collagenase, dispase, DNase I and hyaluronidase, which can affect the survival of cells when receiving a primary culture. Determination of neuronal differentiation cells only by markers of mature neurons Tujl, MAP2 does not allow identifying a whole pool of their precursors, namely NSPCs and immature neurons, which undoubtedly can be significant when using this culture for cell therapy in spinal cord injuries. As a result of using this method, NSPCs are obtained in the form of a suspension culture, which significantly complicates their use for cell therapy.

There is also known a method of obtaining NSPK from the tissue of the human olfactory lining of the nose [14], which consists in the fact that the tissue of the olfactory lining obtained from patients with spinal cord injuries was incubated for 45 min at 37 ° C in a dispase II solution of 2.4 units / ml. Then the olfactory epithelium was separated from the underlying epithelial lamina propria. Then the mechanically dissociated olfactory epithelium was incubated in 0.25 mg / ml collagenase for 10 min at 37 ° C. The resulting cells in HAM / F12 medium containing 10% FBS and penicillin / streptomycin were plated on plastic pretreated with poly-L-lysine. Primary neurospheres were obtained after 7-10 days. The cells obtained from the dissociation of these neurospheres were cultivated in the form of adhesive cultures under various conditions: on uncoated or coated plastic with collagen IV (5 μg / cm ² ), fibronectin (10 μg / cm ² ), laminin (3.5 μg / cm ² ), poly-L-lysine (2 μg / cm ² ), or polyornithine (10 μg / cm ² ). After 5 days in culture, cell proliferation was different depending on the culture conditions. On uncoated plastic, cell density was lower in serum-free DMEM compared to medium with 10% FBS, fibroblast growth factor-2 (FGF2), or transforming growth factor alpha (TGFα). Cell density was lowest when cells were grown in serum medium on polylysine and polyornithine. Laminin, fibronectin and collagen did not affect cell density compared to uncoated plastic. Cells obtained by dissociation of neurospheres were cultured on uncoated plastic for 5 days in serum-free and serum-free DMEM medium supplemented with insulin, transferrin, sodium selenite (ITS), 25ng / ml ciliary neurotrophic factor (CNTF), 50 ng / ml nerve growth factor (NGF) and 0.1 and 1 μg / ml retinoic acid. Various combinations of the medium shifted the differentiation of NSPCs in the astroglial, oligodendroglial, or neuronal directions. The authors have shown that the largest proportion of neurons (25.33%) detected by βΙΠ-tubulin expression was obtained by adding NGF to serum-free DMEM medium.

The main disadvantage of this method is that the authors obtained adhesive cultures from primary neurospheres, the stage of obtaining which (7-10 days) significantly lengthens the time of obtaining adhesive cultures. Determination of neuronal differentiation cells only by the marker of immature and mature neurons βIII-tubulin does not allow identifying their precursors - NSPCs, which are also important for cell therapy in spinal cord injuries. In addition, in the resulting adhesive culture, the proportion of neurons (25.33%) is not large, this may be due to the fact that growth factors were used on cultures grown on plastic without the use of matrices.

Of the known methods, the closest in technical essence to the proposed method is a method for obtaining NSPK from the rat's own plate [11], which consists in the fact that the tissue of the olfactory lining was treated with 2.4 units / ml dispase II for 30 minutes at 37 ° C, then the olfactory epithelium was mechanically separated from its own plate, after which the olfactory epithelium was incubated with 0.05% trypsin-EDTA in Ringer's solution with a low calcium content for 5-10 minutes at 37 ° C and then treated for 15 minutes at 37 ° C a solution of enzymes collagenase, hyaluronidase and trypsin inhibitor (1, 1.5, 0.1 mg / ml, respectively) in Ringer's solution, then resuspended 10-20 times to separate the cells. The cell suspension was centrifuged at 200 × g for 10 minutes and the cell pellet was resuspended and seeded in DMEM / F12 culture medium (1: 1) containing 2% FBS, N2 supplement and 25 ng / ml epidermal growth factor (EGF) on plastic coated with poly-D-lysine. Further, globose basal cells were isolated from the obtained cultures on a sorter using specific antibodies, which were further cultured as an adhesive culture.

The disadvantage of this method is the treatment of the tissue of the olfactory epithelium with a whole cocktail of enzymes, namely trypsin, collagenase, hyaluronidase, which may affect the survival of cells when receiving a primary culture. The most significant disadvantage of the known method is the production of only one type of cells, namely globose basal cells, which are precursors of olfactory receptor neurons, which can significantly reduce the therapeutic efficacy of such a cell preparation.

The technical problem to be solved by the proposed invention is to create a method for obtaining an adhesive culture of neural stem / progenitor cells from the mammalian olfactory lining, eliminating the disadvantages of the previous analogs.

SUMMARY OF THE INVENTION

The technical result of the claimed invention consists in the possibility of obtaining an adhesive culture enriched in neural stem / progenitor cells, in an amount sufficient for further transplantation and having a high survival rate.

The specified technical result is achieved by the proposed method of obtaining NSPK from the olfactory lining of the nose of mammals for the treatment of spinal cord injuries, including the stages at which: receive tissue samples from the olfactory lining of the nose of a mammal; wash them; incubate tissue with 3.6 units / ml dispase II for 1 hour at 37 °; transfer tissue into DMEM: F12 medium (1: 1) with antibiotics and mechanically separate the olfactory epithelium from its own lamina; the tissue of the olfactory epithelium is incubated with a solution of 500 U / ml collagenase for 10 minutes at 37 ° C, the enzyme is inactivated with a medium with serum, resuspended and centrifuged for 2 minutes at 900 g; cells are plated on plastic and cultured for 5 days in DMEM: F 12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin and 60 μg / ml gentamicin, the medium is changed every two days; After the formation of a monolayer, the cells of the olfactory epithelium are removed with 0.25% trypsin solution, placed on plastic coated with 50 μg / ml fibronectin and cultured for 1-2 days in neurobasal medium containing 2 mM L-glutamine, 1% ITS, 2% B27 , 100 μg / ml streptomycin, 100 U / ml penicillin, 60 μg / ml gentamicin, 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor (EGF), the resulting cells are characterized by markers nestin- and βIII-tubulin. If necessary, after obtaining tissue samples and until the start of the experiment to obtain a primary culture, tissue samples of the mammalian olfactory lining of the nose are transported in a nutrient medium DMEM: F12 (1: 1) and stored at + 4 ° C for no more than 2 hours. Washing of tissue samples of the olfactory lining is carried out in DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 U / ml penicillin at least 3 times. Mechanical separation of the olfactory epithelium from the lamina propria is performed by means of a microspatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 U / ml penicillin.

The difference between the claimed method and the previously known from the prior art lies in the fact that the tissue of the olfactory lining is treated with the enzyme dispase II in order to mechanically separate the olfactory epithelium from its own lamina in order to obtain a purer culture of NSPC. In order not to subject the tissue of the olfactory epithelium to additional enzymatic treatment in the claimed invention, only one enzyme is used - collagenase, and not a cocktail of enzymes, the use of which can negatively affect the survival of cells when obtaining a primary culture. To obtain a larger number of cells, the primary culture of the olfactory epithelium is grown for 5 days. This invention compares favorably with the presented ones, since it does not contain the stage of primary neurospheres, the stage of obtaining which (7-10 days) significantly lengthens the time of obtaining adhesive cultures, and significantly (more than three times) exceeds the content of cells of neuronal differentiation. In this invention, the percentage of cells of neuronal differentiation when cultured on fibronectin in neurobasal medium is 79.11%, while in Murrell W. et al. - 25.33%. In addition, the resulting NSPC culture was more accurately characterized by the simultaneous expression of two markers nestin- and βIII-tubulin, which makes it possible to identify all cells of neural differentiation, namely, immature and mature neurons and their precursors, NSPCs, which is extremely important for further use in cell therapy. The differences between the claimed method and those previously known from the prior art are that the timing of cultivation of an adhesive culture (within 1-2 days) with the highest percentage of NSPC is determined, i.e. when the culture is the cleanest for further transplantation.

An additional advantage of the invention is the possibility of autologous use of NSPCs when taking the olfactory lining material from patients with spinal cord injury, obtaining NSPCs from this material and further cell transplantation to the same patients.

BRIEF DESCRIPTION OF DRAWINGS

The proposed method is illustrated by drawings, where

in fig. 1 - Primary culture of cells of the olfactory epithelium of the olfactory lining of rats (phase contrast microscopy);

in fig. 2 - NSPCs obtained from the olfactory epithelium of rats (II - a, b, c, d) were identified by the simultaneous expression of markers Sox2 (IIc) and Nestin (IIa);

in fig. 3 - Adhesive culture of rat olfactory epithelium under different cultivation conditions: a - on laminin in DMEM: F12, b - on laminin in neurobasal medium, c - on fibronectin in DMEM: F12 medium, d - on fibronectin in neurobasal medium, scal bar- 50 μm, (× 100).

in fig. 4 - a - Cells in adhesive cultures stained with antibodies to βΙΙΙ-tubulin, b - cells in adhesive cultures stained with antibodies to nestin. The kernels are colored with DAPI. (× 630), seal bar - 10 μm;

in fig. 5 - The number of cells in different cultivation conditions, **** P <0.0001;

in fig. 6 - Percentage of nestin- and βΙΙΙ-tubulin-positive cells under different cultivation conditions, * P <0.05, ** P <0.01;

in fig. 7 - Percentage of nestin - and sox2 - positive cells in the adhesive culture of NSPK;

in fig. 8 - Primary culture of cells of the olfactory epithelium of the human olfactory lining (phase contrast microscopy);

in fig. 9 - NSPCs obtained from the human olfactory epithelium (I - a, b, c, d) were identified by the simultaneous expression of markers Sox2 (Ic) and Nestin (Ia);

in fig. 10 - Adhesive culture of NSPK from the human olfactory lining, cultured on fibronectin in neurobasal medium for 2 days (phase contrast microscopy);

in fig. 11 - MRI image of the formation of a post-traumatic cyst of the spinal cord 4 weeks after spinal cord injury (the area of the cyst is indicated);

in fig. 12 - The dynamics of the restoration of the motor activity of the hind limbs of rats after transplantation of human and rat NSPCs into cysts of 4 weeks of formation, is presented by changes in points (delta BBB). The graph shows mean values with standard deviations;

in fig. 13 - MRI imaging of post-traumatic spinal cord cysts before (left) and 4 weeks after transplantation of 200 thousand NSPC rats (right);

in fig. 14 - Survival of human NSPC in cysts 4 weeks after transplantation. A - overlapping B, C, D. Cell migration to the nearby tissues of the spinal cord is observed (marked with an arrow). B - staining of nuclei with DAPI, C - staining with antibodies to b-III-tubulin, D - PKH26-labeled NSPK. The area of the cyst is marked with an asterisk.

CARRYING OUT THE INVENTION

The method is illustrated by the following examples.

Example 1.

The NSPCs of the olfactory lining of the nose of rats are prepared in a known manner. Wistar rats are anesthetized by intraperitoneal administration of a 1: 1 Relanium / ketamine mixture (1 μl / g). Then the animals are decapitated. Next, the skull is sectioned in the sagittal plane at the level of the nasal septum and the material of the animal's olfactory lining is taken. Then the tissue is washed in DMEM: F12 (1: 1) at least 3 times. Then the tissue is incubated with 3.6 units / ml dispase II for 1 hour at + 37 ° C, after which the olfactory epithelium is mechanically separated from its own plate. Mechanical separation of the olfactory epithelium from the lamina propria is performed by means of a microspatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 U / ml penicillin. The resulting tissue of the olfactory epithelium is transferred into 15 ml. a test tube with a solution of 500 U / ml collagenase, incubated for 10 minutes at 37 ° C, inactivate the enzyme with medium with serum, resuspended and centrifuged for 2 minutes at 900 g. The resulting cells are plated on plastic and cultured for 5 days in DMEM: F12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin and 60 μg / ml gentamicin in an incubator at 5% CO ₂ , + 37 ° C and maintaining a humidity of 95% (figure 1). In this case, the change of Wednesday is carried out every two days. The resulting primary culture of the rat olfactory epithelium is characterized by conventional markers sox-2 and nestin for the presence of neural / stem progenitor cells. Sox-2 and nestin-positively stained cells are detected, their percentage is 5-7% of the total number of cells (Fig. 2). Cells are counted in 10 fields of view in 3 independent experiments. The total number of cells is determined by counting the nuclei, and the nuclei are visualized using DAPI.

After the formation of a monolayer, the cells of the olfactory epithelium are washed with a Versene solution 3 times, the cells are incubated with a 0.05% trypsin solution for 2 minutes at 37 ° C, the suspension with cells is transferred into a test tube, to inactivate trypsin, DMEM: F12 (1: 1) medium with 10 % FBS, resuspended and then centrifuged for 2 minutes at 900g. The supernatant is removed, the pellet is resuspended, and then the resulting cells are cultured under various conditions to select the optimal method for obtaining an adhesive culture enriched with NSPC in an amount sufficient for further transplantation: on fibronectin in DMEM: F12 medium, on fibronectin in neurobasal medium (Neurobasal), on laminin in DMEM: F12, on laminin in neurobasal environment. Cells of the same density are plated on plastic coated with laminin (0.5 μg / ml) or fibronectin (50 μg / ml). Cell cultivation is carried out in serum-free DMEM / F12 and neurobasal media, with the same additives - 1% ITS, 2% B27, 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor (EGF), 2 mM L β-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin and 60 μg / ml gentamicin (Fig. 3). On the 5th day of cultivation, the cells are removed with 0.25% trypsin solution and counted in the Goryaev chamber. In other adhesive cultures obtained in a similar way, detection of nestin- and βIII-tubulin positive cells is carried out (Fig. 4).

Calculation of the total number of cells shows that there were more cells on fibronectin and laminin in the neurobasal medium (24094 and 23019 cells / ml, respectively) compared to cultures growing on fibronectin and laminin (6871 and 6586 cells / ml, respectively) in DMEM: F12 (Fig. 5). Thus, various matrices used in this work do not affect cell proliferation. At the same time, cultivation in a neurobasal environment promotes an increase in proliferation. Cell proliferation was maximal when cultured on both matrices in neurobasal medium.

When an adhesive culture was obtained using a known method, it was found that when cultured on fibronectin in neurobasal medium, the percentage of nestin-positive cells was maximum (52.22%) compared to other culture conditions (on fibronectin in DMEM medium: F12 - 16.67% , on laminin in DMEM: F12 - 41%, on laminin in neurobasal medium - 26.88%). The percentage of βΙΙΙ-tubulin-positive cells was maximal and approximately the same when cultured on fibronectin in neurobasal medium and in DMEM: F12 (79.11% and 83.52%, respectively), while when cultured on laminin in neurobasal medium and in DMEM : F12 - much less (60.86% and 63.33%, respectively). At the same time, the DMEM: F12 medium slightly changed differentiation in the neuronal direction, both on fibronectin and laminin (Fig. 6). Cultivation in neurobasal medium on different matrices did not reveal the effect of this particular medium on the percentage of nestin and βΙΙΙ-tubulin - positive cells. However, cultivation in a neurobasal medium on fibronectin showed the best results both in the proliferative activity of cells and in the content of NSPCs and neurons differentiating from them in the culture. Cultivation of adhesive cultures of NSPCs in a neurobasal medium on fibronectin makes it possible to obtain the purest culture, maximally enriched in NSPCs and neurons differentiating from them, in an amount sufficient for further transplantation.

Further, to establish the timing of cultivation with the maximum content of NSPCs, the cells of the olfactory epithelium of rats were cultured on fibronectin in a neurobasal medium. On days 1, 2 and 5 of cultivation, the percentage of nestin and sox2 positive cells was estimated by immunofluorescence. The number of sox2-positive cells was approximately the same, while the largest number of nestin-positive cells was observed in culture on days 1 and 2 of cultivation. Thus, we can recommend for further transplantation adhesive cultures obtained according to our protocol and enriched with NSPK on days 1 and 2 of cultivation (Fig. 7).

Example 2.

The NSPC of the human olfactory lining of the nose is obtained in a known manner.

Tissue samples of the olfactory lining are obtained from the surgical material of the upper and middle nasal passages during elective surgical interventions. The area of the samples required for scientific research is 5-25 mm ² . If necessary, after obtaining tissue samples and until the start of the experiment to obtain a primary culture, tissue samples of the human olfactory lining of the nose are transported in a nutrient medium DMEM: F12 (1: 1) and stored at + 4 ° C for no more than 2 hours. Then the tissue is washed in DMEM: F12 (1: 1) at least 3 times. Then the tissue is incubated with 3.6 units / ml dispase II for 1 hour at + 37 ° C, after which the olfactory epithelium is mechanically separated from its own plate. Mechanical separation of the olfactory epithelium from the lamina propria is performed by means of a microspatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 U / ml penicillin. The resulting tissue of the olfactory epithelium is transferred into 15 ml. a test tube with a solution of 500 U / ml collagenase, incubated for 10 minutes at 37 ° C, inactivate the enzyme with medium with serum, resuspended and centrifuged for 2 minutes at 900 g. The resulting cells are plated on plastic and cultured for 5 days in DMEM: F12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin and 60 μg / ml gentamicin in an incubator at 5% CO ₂ , + 37 ° C and maintaining a humidity of 95% (Fig. 8). In this case, the change of Wednesday is carried out every two days. The resulting primary culture of the rat olfactory epithelium is characterized by conventional markers sox-2 and nestin for the presence of neural / stem progenitor cells. Sox-2 and nestin-positively stained cells are detected, their percentage is 5-7% of the total number of cells (Fig. 9). Cells are counted in 10 fields of view in 3 independent experiments. The total number of cells is determined by counting the nuclei, and the nuclei are visualized using DAPI.

After the formation of a monolayer, the cells of the olfactory epithelium are washed with a Versene solution 3 times, the cells are incubated with a 0.05% trypsin solution for 2 minutes at 37 ° C, the suspension with cells is transferred into a test tube, to inactivate trypsin, DMEM: F12 (1: 1) medium with 10 % FBS, resuspended and then centrifuged for 2 minutes at 900g. The supernatant is removed, the pellet is resuspended, and then the resulting cells are placed on plastic coated with 50 μg / ml fibronectin and cultured for 1-2 days in neurobasal medium containing 2 mM L-glutamine, 1% ITS, 2% B27, 100 μg / ml streptomycin, 100 U / ml penicillin, 60 μg / ml gentamicin, 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor (EGF). As a result, an adhesive NSPC culture is obtained, suitable for further transplantation in spinal cord injuries (Fig. 10-phase).

Example 3.

The resulting adhesive cultures of rat and human NSPCs are used to treat experimental spinal cord injuries in rats. The experimental trauma model is a rat post-traumatic spinal cord cyst.

Modeling of post-traumatic cysts of the spinal cord of rats is carried out according to the method developed by us on sexually mature female rats of the Wistar line, weighing from 200 grams. Sterile conditions are maintained during the operation. All manipulations causing pain or other trauma to animals are performed with anesthesia, animals are anesthetized by intraperitoneal administration of a relanium / ketamine mixture (1 μl / g). Before fixing the animal on the operating table, the depth of anesthesia is determined by the severity of the blinking and vibrissa reflexes. Prepare the operating field using an electric razor and rubbing the skin with alcohol. A median incision is made along the spine at the Th9-Th10 level, partial muscle resection on both sides of the spinous process, with insignificant blood secretions, a 3% hydrogen peroxide solution and gauze tampons are used. The Th10 spinous process is removed using surgical nippers, and access to the spinal cord is opened. Impact with a 200 kilodynes impact mechanism (Precision Systems and Instrumentation LLC, Fairfax, VA). The edges of the operating wound are brought together and secured with staples and treated with brilliant green solution. For the general prevention of postoperative infectious complications, 300 μl of cefazolin solution is used intramuscularly. Cyst formation was confirmed by animal tomography (ClinScan, Bruker BioSpin) 4 weeks after surgery (FIG. 11).

In the cavity of the formed cyst, the animals are transplanted with the NSPC of rats. To assess the therapeutic effect, 200 thousand obtained cells are transplanted in 20 μl DMEM: F12 (1: 1) containing 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin. Animals of the control group are injected with 20 μl of DMEM: F12 (1: 1) containing 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin. After transplantation of cells to the site of injury, the restoration of the motor activity of the hind limbs of the rats is assessed. The main tool for assessing the recovery of motor function is the 21-point open field scale developed by D. Basso, Μ. Beattie and J. Bresnahan (BBB), which allows to study the dynamics of recovery of the musculoskeletal system (9). The dynamics of recovery of motor activity in the hind limbs of rats was assessed using the BBB test once a week for 3 weeks. In rats transplanted with the cell preparation, there was a significant improvement in the motor activity of the hind limbs according to the BBB tests (Fig. 12).

Example 4.

The resulting adhesive cultures of rat and human NSPCs are used for transplantation into formed cysts for 4 weeks, and 4 weeks after transplantation, the size of the cysts is assessed using the MRI method. Analysis of MRI images is carried out on sections of the frontal and sagittal projections of the spinal cord of rats with a scanning step of 0.5 mm in the Vidar Dicom Viewer software. An area corresponding to the detected cyst is highlighted in each section. The program automatically calculates the area of the selected area. To determine the volume of the cyst, the average values of the areas of the cysts of the frontal and sagittal projections are considered and multiplied by the scanning step. No statistically significant changes in cyst size were found 4 weeks after NSPC transplantation in rats and humans. Interestingly, some animals with cysts showed a decrease in cyst area volume at 4 weeks after transplantation of 200,000 NSPC rats (FIG. 13). Apparently, we can only talk about the individual ability of rats to regenerate, when in some animals, after cell transplantation, cysts decrease.

Example 5.

The resulting adhesive cultures of human NSPCs are used for transplantation into formed cysts for 4 weeks, and 4 weeks after transplantation of 200 thousand cells, their survival and migration to nearby tissues are studied. To study the survival of NSPCs in the spinal cord after transplantation into cysts, an intravital membrane dye PKH26 (Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling was obtained from Merck / Sigma-Aldrich, USA) is used. PKH26 - labeled human NSPCs are transplanted into spinal cord cysts at 4 weeks of formation. The cells are injected in 10 μl of DMEM / F-12 medium (1: 1). Control animals are injected with the same amount of medium without cells. The survival rate of transplanted NSPCs was studied for 4 weeks. 4 weeks after transplantation, the animals are removed from the experiment, the spinal cord is fixed in 4% formaldehyde. Using a vibrotome (Thermo Fisher Scientific), serial sections of the spinal cord in the area of injury are obtained. The slice thickness is 40 µm. To visualize the spinal cord tissue, primary antibodies to the marker of mature neurons b-III-tubulin (1: 300) and secondary antibodies labeled with Alexa Fluor 488 (1: 500) are used; visualization of the nuclei is performed using DAPI. 4 weeks after transplantation, NSPCs are detected in the area around post-traumatic cysts of the spinal cord (Fig. 14). Within 4 weeks, human NSPCs survive in spinal cord cysts and migrate to nearby tissues. The high survival rate and the possibility of obtaining NSPCs from the olfactory lining of patients to create an autologous preparation make it possible to consider them as a promising material for the treatment of patients with post-traumatic spinal cysts.

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Claims (12)

1. A method of obtaining an adhesive culture of neural stem / progenitor cells from the olfactory lining of the nose of mammals for the treatment of spinal cord injuries, including the stages at which obtaining tissue samples from the olfactory lining of the mammal's nose; wash them; the tissue is incubated with 3.6 units / ml dispase II for 1 hour at 37 ° C; the tissue is transferred to DMEM: F 12 (1: 1) medium with antibiotics and the olfactory epithelium is mechanically separated from its own plate; the tissue of the olfactory epithelium is incubated with a solution of 500 U / ml collagenase for 10 min at 37 ° C, the enzyme is inactivated with a medium with serum, resuspended and centrifuged for 2 min at 900 g; cells are plated on plastic and cultured for 5 days in DMEM: F 12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin and 60 μg / ml gentamicin, the medium is changed every two days; after the formation of a monolayer, cells of the olfactory epithelium are removed with 0.25% trypsin solution, placed on plastic coated with 50 μg / ml fibronectin, and cultured for 1-2 days in neurobasal medium containing 2 mM L-glutamine, 1% ITS, 2% B27, 100 μg / ml streptomycin, 100 U / ml penicillin, 60 μg / ml gentamicin, 50 ng / ml bFGF, 50 ng / ml EGF; the resulting cells are characterized by the markers nestin and βIII-tubulin. 2. The method according to claim 1, characterized in that, if necessary, after obtaining tissue samples and before the start of the experiment to obtain a primary culture, tissue samples of the olfactory lining of the mammalian nose are transported in a nutrient medium DMEM: F 12 (1: 1) and stored at + 4 ° С no more than 2 hours 3. The method according to claim 1, characterized in that the tissue samples of the olfactory lining are washed in DMEM: F 12 (1: 1) medium with antibiotics 100 μg / ml streptomycin and 100 U / ml penicillin at least 3 times. 4. The method according to claim 1, characterized in that the mechanical separation of the olfactory epithelium from the lamina propria is performed by means of a microspatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 U / ml penicillin.

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