RU2676142C2 - Method for obtaining lining cells from the olfactory lining of mammals for the treatment of spinal cord injuries - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, specifically to cellular neurotransplantology, and can be used in medicine to treat spinal cord injuries. Method for obtaining lining cells from the olfactory lining of mammals for the treatment of spinal cord injuries includes the steps of obtaining tissue samples from the nasal olfactory lining of mammal; incubation of tissue samples with dispase II at a concentration of 2 mg/ml for 1 hour at 37 °C; mechanical separation of the olfactory epithelium from the proper plate; grinding and cultivation of the proper plate in the form of an explant culture for 14 days in DMEM:F12 (1:1); incubation of the formed monolayer with a 0.05 % trypsin solution for 2 minutes at 37 °C. Cultivation of the resulting cells in DMEM:F12 (1:1) medium containing 500 ng/ml hydrocortisone for 3 days. Characterize the resulting cells by p75NTR and GFAP markers.EFFECT: invention provides a pure culture of the lining cells in a sufficient number for further transplantation with high survival rate.4 cl, 8 dwg, 3 ex

Description

FIELD OF TECHNOLOGY

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

BACKGROUND

Traumatic injuries of the spinal cord are an urgent problem of modern neurobiology and neurosurgery due to the constant increase in the number of victims of such injuries of the central nervous system, high mortality and disability of victims. In most cases, spinal cord injuries are the result of direct mechanical injuries that lead to both partial and complete loss of mobility and sensitivity of the body. Traumatic injuries of the spinal cord lead to consequences that have a serious impact on the quality of life of patients: not only their physical, but also psychosocial state is deteriorating. Drug treatment and surgical intervention do not always achieve the desired results, since traumatic injuries result in the death of a large number of functional neurons. In this regard, cell therapy can be a promising area for the treatment of spinal cord injuries. Cell transplantation can cause axon regeneration, contribute to the replacement of lost neurons, and reduce the risk of disorders after spinal cord injury through the secretion of neurotrophic factors (1).

A large number of experimental works and clinical studies have been carried out using various types of cells: embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, Schwann cells. The use of these types of cells in traumatic injuries of the spinal cord in many cases gave a positive therapeutic effect with the restoration of sensory and motor functions of the spinal cord. However, the use of certain cellular preparations is associated with certain methodological and ethical problems. The use of others is ineffective or leads to side effects, such as the formation of teratomas or the occurrence of neuropathic pain. All this makes it difficult to choose the optimal cell preparation for the treatment of spinal cord injuries. The most promising can be cells of the olfactory lining of the nose. Obtaining the olfactory lining is an accessible and safe procedure for patients. These cells are tissue-specific and autologous, since they can be obtained from a patient with a spinal cord injury and after transplantation in culture and directed differentiation are transplanted to the same patient.

Own plate of the olfactory nasal lining can be a source of parietal cells, the regenerative potential of which is actively studied in spinal cord injuries (2, 3). Having simultaneously the properties of Schwann cells and astrocytes, which proves their co-expression of the astrocytic marker - acid glial fibrillar protein (GFAP) and oligodendrocyte - low affinity nerve growth factor receptor (p75NTR), they are able to perform various functions: create a microenvironment for neurons, promote axon growth and remyelination of nerve fibers (4). It was also shown that transplanted parietal cells are able to migrate through the glial scar and thus facilitate axon growth during regeneration. In experimental work on transplantation of parietal cells with spinal cord injuries, positive results were obtained. It was shown that there is a restoration of the motor functions of the limbs of rats (5), a decrease in the area of damage, axon regeneration and remyelination of nerve fibers (6). Several clinical trials have been performed that have demonstrated that transplantation of autologous parietal cells is a safe procedure for treating patients (7). An improvement in both motor and sensory function was demonstrated in patients with spinal cord injuries after transplantation of embryonic lining cells of the human olfactory lining (8). The ability to maintain neuronal regeneration allows considering parietal cells as a source for cell therapy of spinal cord injuries.

There are various methods for obtaining parietal cells from the human olfactory lining.

There is a method of obtaining parietal cells from autopsy material of the human olfactory lining (WO 2007/069927 A2, 06/21/2007), namely, that the tissue of the olfactory lining was treated with dispase II, then the olfactory epithelium was mechanically separated from its own plate, after which the samples of their own plate were subjected additional enzymatic treatment with collagenase N. Cells obtained from the plate itself were cultured in suspension cultures for 7 or 14 days, then transplanted onto culture plates rytye poly-L- lysine. After 4-11 days of cultivation in a Dulbecco-modified Eagle medium and Ham substrate F-12 (DMEM / F-12), with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg / ml streptomycin, 100 units / ml penicillin, the number of parietal cells in the culture was evaluated by immunofluorescence by expression of the p75NTR marker.

The disadvantage of this method is the additional enzyme treatment with collagenase H, which may affect cell survival upon receipt of the primary culture. Another disadvantage of this invention is that parietal cells obtained from autopsy samples are applicable only for allogeneic transplantation. The number and survival of cells from autopsy material may be lower than from other sources. The cultivation of cells in suspension cultures is a more time-consuming method than the cultivation of adhesive cultures.

There is also a method of obtaining parietal cells from tissue of olfactory epithelium of adult patients (RU 2394593 C2, 07.20.2010), which consists in the fact that the tissue of the olfactory lining obtained from patients with spinal cord injuries was crushed and incubated in trypsin solution (0.05%) and ethylenediaminetetraacetic acid (EDTA) (0.02%) for 40 minutes at 36.5 ° C. Cells were cultured in 12-well plates on a polylysine-laminin substrate for 10-15 days in an environment of the following composition: DMEM / F12, 10% FBS, 2 mM L-glutamine, 0.8% glucose, a mixture of insulin, transferrin and sodium selenite (1: 100), HEPES buffer (10 mM), human growth factors neuregulin1-beta1 / heregulin1-beta1, epidermal growth factor (2 ng / ml). After the formation of a dense monolayer, the cells were subcultured into vials with polylysine, a laminin substrate. After 3-4 passages, the cells were removed and the resulting suspension was either used for transplantation or frozen using a cryoprotectant (10% serum, 90% EDTA) and stored in liquid nitrogen at -70 ° C. For transplantation, cryopreserved cells were thawed and their viability was determined using trypan blue staining.

The main disadvantage of this method is that upon receipt of the primary cell culture, the own plate was not separated from the olfactory epithelium, which does not allow to obtain a pure culture of parietal cells.

Of the known methods, the closest in technical essence to the proposed method is a method for producing parietal cells from a person’s own plate (WO 2001/030982 A1, 05/03/2001), namely, that the tissue of the olfactory lining was treated with dispase II, then the olfactory epithelium was mechanically separated from of the own plate, after which the samples of the own plate were subjected to additional enzymatic treatment with collagenase 1, after which the cells of the own plate were cultured on fibronectin or poly - L - lysis not in DMEM medium containing 50 mg / ml gentamicin, 10% FBS. After 10 days, parietal cells were visualized by p75 marker.

The most significant disadvantage of this method is the lack of a stage of cell mass growth in primary culture, before cell cultivation on poly - L - lysine, which significantly reduces the number of cells obtained. As well as additional treatment with collagenase 1, the enzyme may affect the survival of the resulting cells.

The technical problem to which the proposed invention is directed is to create a method for producing a preparation of parietal cells from the olfactory lining of a mammal, eliminating the disadvantages of previous analogues.

SUMMARY OF THE INVENTION

The technical result of the claimed invention lies in the possibility of obtaining a pure culture of parietal cells in a quantity sufficient for further transplantation and having high survival rate.

The specified technical result is achieved by the proposed method for obtaining parietal cells from the olfactory lining of mammals for the treatment of injuries of the spinal cord, including the steps of: receiving tissue samples from the olfactory lining of the nose of a mammal; washed them; incubated tissue with dispase II at a concentration of 2 mg / ml for 1 hour at 37 ° C; tissue is transferred to DMEM: F12 (1: 1) with antibiotics and the olfactory epithelium is mechanically separated from its own plate; own plate is crushed and cultured as an explant culture for 14 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 of gentamicin, a change of medium is performed every three days; after the formation of a monolayer, the cells are incubated with a 0.05% trypsin solution for 2 minutes at 37 ° C, the enzyme is inactivated with serum medium, resuspended and centrifuged for 2 minutes at 900 g; the cells are placed on a plastic coated with 0.01% poly-L-lysine and cultured in DMEM: F12 (1: 1) containing 10% FBS, 2 mM L-glutamine, 1% insulin, transferrin, sodium selenite ( ITS), 100 μg / ml streptomycin, 100 u / ml penicillin and 500 ng / ml hydrocortisone, for 3 days; the resulting cells are characterized by p75NTR and GFAP markers. If necessary, after obtaining tissue samples and until the start of the experiment on obtaining the primary culture, tissue samples of the olfactory nose of the mammal are transported in a nutrient medium DMEM: F12 (1: 1) and stored at + 4 ° C for no more than 2 hours. The washing of the tissue samples of the olfactory lining is carried out in DMEM: F12 (1: 1) with antibiotics 100 μg / ml streptomycin and 100 units / ml penicillin at least 3 times. The olfactory epithelium is mechanically separated from its own plate by means of a micro spatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 units / ml penicillin.

The differences of the claimed method from previously known from the prior art are that the tissue of the olfactory lining was treated with only one enzyme, Dispase II, in order to mechanically separate the olfactory epithelium from its own plate in order to obtain a cleaner culture of parietal cells. In order not to subject the tissue of its own plate to additional enzyme treatment, the claimed invention employs an explant culture method, when the own plate is subjected to mechanical grinding and the obtained tissue pieces are placed in culture dishes in a culture medium.

To obtain more cells, the primary culture was expanded for 14 days. Then, to obtain parietal cells, the obtained primary culture was transplanted onto plastic or cups coated with poly-L-lysine in medium (DMEM: F12 (1: 1), with 10% FBS, 2 mM L-glutamine, 100 pg / mL streptomycin, 100 μ / mL penicillin, 1% ITS and 500 ng / ml hydrocortisone). Hydrocortisone increases the sensitivity of cells to growth factors contained in serum, which allows to obtain a larger number of cells. In addition, the resulting culture of human parietal cells was more accurately characterized by the simultaneous expression of two p75NTR and GFAP markers.

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

BRIEF DESCRIPTION OF THE DRAWINGS

The proposed method is illustrated by drawings,

where in FIG. 1 - The primary culture of the own plate of the olfactory lining of the person obtained by the method of explant culture (phase contrast microscopy);

in FIG. 2 - Culture of human parietal cells obtained by culturing on poly-L-lysine (phase contrast microscopy);

in FIG. 3 - Culture of human parietal cells, characterized by immunofluorescence method for the simultaneous expression of GFAP and p75NTR markers;

in FIG. 4 - Primary culture of the own plate of the olfactory lining of rats obtained by the method of explant culture (phase contrast microscopy);

in FIG. 5. - Culture of rat parietal cells obtained by culturing on poly-L-lysine (phase contrast microscopy);

in FIG. 6. - Culture of rat parietal cells, characterized by immunofluorescence method for the simultaneous expression of GFAP and p75NTR markers;

in FIG. 7. - MRI image of the formation of a post-traumatic cyst of the spinal cord 4 weeks after spinal cord injury, the cyst is indicated by an arrow;

in FIG. 8. - Restoration of the motor activity of the hind limbs of rats after transplantation of rat parietal cells. In the experimental and control group presents average data for three animals.

DETAILED DESCRIPTION OF THE INVENTION

The method is illustrated by the following examples.

Example 1

The parietal cells of the human olfactory lining are obtained in a known manner.

Samples of tissue of the olfactory lining are obtained from the surgical material of the upper and middle nasal passages during planned surgical interventions. The area of samples required for scientific research is 5-25 mm ² . If necessary, after obtaining tissue samples and before the start of the experiment on obtaining the primary culture, tissue samples of the olfactory lining of the human nose are transported in a nutrient medium DMEM: F12 (1: 1) and stored at + 4 ° C for no more than 2 hours. Then the fabric is washed in DMEM: F12 (1: 1) at least 3 times. Then the tissue is incubated with dispase II at a concentration of 2 mg / ml for 1 hour at + 37 ° C, after which the olfactory epithelium is mechanically separated from its own plate. The olfactory epithelium is mechanically separated from its own plate by means of a micro spatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 units / ml penicillin. The resulting tissue of the plate itself was transferred to a Petri dish with DMEM: F12 (1: 1) culture medium containing 10% FBS, 2 mM L-glutamine and antibiotics. Own plate is crushed and cultured as an explant culture for 14 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 of gentamicin, in an incubator at 5% CO ₂ , + 37 ° C and maintaining a humidity of 95% (Fig. 1). At the same time, the medium is changed every three days.

After the formation of a monolayer, the cells are washed with Versen's solution 3 times, the cells are incubated with a 0.05% trypsin solution for 2 minutes at 37 ° C, the suspension of the cells is transferred to a test tube, DMEM: F12 medium (1: 1) with 10% FBS is added to inactivate trypsin , resuspended and then centrifuged for 2 minutes at 900g. The supernatant is removed, the precipitate is resuspended in DMEM: F12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 1% insulin supplement, transferrin, sodium selenite (ITS), 100 μg / ml streptomycin, 100 u / ml penicillin and 500 ng / ml hydrocortisone. Next, the obtained cells are placed on a plastic coated with 0.01% poly-L-lysine and cultured in DMEM: F12 (1: 1) containing 10% FBS, 2 mM L-glutamine, 1% insulin, transferrin, selenite sodium (ITS), 100 μg / ml streptomycin, 100 u / ml penicillin and 500 ng / ml hydrocortisone for 3 days (Fig. 2). The resulting cells are characterized by p75NTR and GFAP markers (Fig. 3). The purity of the resulting culture of parietal cells is 95-97%).

Example 2

The parietal cells of the olfactory lining of rats are isolated as described. Wistar rats were anesthetized by intraperitoneal administration of a 1: 1 ratio of Relanium / Ketamine (1 μl / g). Then produce decapitation of animals. Next, a section of the skull is made in the sagittal plane at the level of the nasal septum and the material is collected on the olfactory lining of the animal. Then the fabric is washed in DMEM: F12 (1: 1) at least 3 times. Then the tissue is incubated with dispase II at a concentration of 2 mg / ml for 1 hour at + 37 ° C, after which the olfactory epithelium is mechanically separated from its own plate. The olfactory epithelium is mechanically separated from its own plate by means of a micro spatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 units / ml penicillin. The resulting tissue of the plate itself was transferred to a Petri dish with DMEM: F12 (1: 1) culture medium containing 10% FBS, 2 mM L-glutamine and antibiotics. Own plate is crushed and cultured as an explant culture for 14 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 of gentamicin, in an incubator at 5% CO ₂ , + 37 ° C and maintaining a humidity of 95% (Fig. 4). At the same time, the medium is changed every three days.

After the formation of a monolayer, the cells are washed with Versen's solution 3 times, the cells are incubated with a 0.05% trypsin solution for 2 minutes at 37 ° C, the suspension of the cells is transferred to a test tube, DMEM: F12 medium (1: 1) with 10% FBS is added to inactivate trypsin , resuspended and then centrifuged for 2 minutes at 900g. The supernatant is removed, the precipitate is resuspended in DMEM: F12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 1% insulin supplement, transferrin, sodium selenite (ITS), 100 μg / ml streptomycin, 100 u / ml penicillin and 500 ng / ml hydrocortisone. Next, the obtained cells are placed on a plastic coated with 0.01% poly-L-lysine and cultured in DMEM: F12 (1: 1) containing 10% FBS, 2 mM L-glutamine, 1% insulin, transferrin, selenite sodium (ITS), 100 μg / ml streptomycin, 100 u / ml penicillin and 500 ng / ml hydrocortisone for 3 days (Fig. 5). The resulting cells are characterized by p75NTR and GFAP markers (Fig. 6). The purity of the resulting culture of lining cells is 95-97%).

Example 3

Obtained rat parietal cells are expanded in culture and passage 2-3 cells are used to treat experimental injuries of rat spinal cord. The experimental injury model is post-traumatic rat spinal cysts.

Modeling of post-traumatic cysts of the spinal cord of rats is carried out according to our methodology on sexually mature female Wistar rats weighing 200 grams or more. During the operation, sterile conditions are observed. All manipulations that cause animals pain or other trauma are performed during anesthesia, animals are anesthetized with intraperitoneal administration of a mixture of Relanium / Ketamine (1 μl / g). Before fixing the animal on the operating table, the depth of anesthesia is determined by the severity of blinking and vibris reflexes. Prepare the surgical field using an electric shaver and rubbing the skin with alcohol. A midline incision is made along the spine at the level of Th9-Th10, partial resection of the muscles on both sides of the spinous process, with a slight discharge of blood using a 3% solution of hydrogen peroxide and gauze swabs. The spinous process of Th10 is removed using surgical nippers, giving access to the spinal cord. A 200 kilodynes impact mechanism is produced (Precision Systems and Instrumentation LLC, Fairfax, VA). The edges of the surgical wound are reduced and fixed with fastening brackets and treated with a solution of brilliant green. For the general prevention of postoperative infectious complications, 300 μl of cefazolin solution intramuscularly is used. Cyst formation is confirmed using an animal tomograph (ClinScan, Bruker BioSpin) 4 weeks after surgery (Fig. 7).

Rat lining cells are transplanted into the cavity of the formed cyst. To assess the therapeutic effect, 750 thousand obtained cells are transplanted in 20 μl of DMEM: F12 (1: 1) containing 2 mM L-glutamine, 100 μg / ml streptomycin, 100 u / ml penicillin. Control animals were 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, an assessment is made of the restoration of motor activity of the hind limbs of rats. The main tool for assessing recovery of motor function is the 21-point open-field scale developed by D. Basso, M. Beattie, and J. Bresnanan (BBB), which allows studying the dynamics of recovery of the musculoskeletal system (9). The dynamics of restoration of motor activity of the hind limbs of rats is evaluated using the BBB test once a week for 3 weeks. In rats, which transplant a cell preparation, there is a significant improvement in the motor activity of the hind limbs according to the BBB tests (Fig. 8).

Bibliography:

1. Pearse DD, Bunge MB. Designing cell- and gene-based regeneration strategies to repair the injured spinal cord. J Neurotrauma. 2006; 23 (3-4): 438-52.

2. Barnett SC, Riddell JS. Olfactory ensheathing cell transplantation as a strategy for spinal cord repair-what can it achieve? Nat Clin Pract Neurol. 2007; 3: 152-161.

3. Mackay-Sim A St, John JA. Olfactory ensheathing cells from the nose: clinicai application in human spinal cord injuries. Exp Neurol. 2011; 229: 174-180.

4. Borgmann-Winter K, Willard SL, Sinclair D, et al. Translational potential of olfactory mucosa for the study of neuropsychiatric illness. Citation: Transl Psychiatry. 2015; 5, e527.

5. Yamamoto M, Raisman G, Li DQ et al. Transplanted olfactory mucosal cells restore paw reaching function without regeneration of severed corticospinal tract fibers across the lesion. Brain Research. 2009; 1303: 26-31.

6. Richter MW, Fletcher PA, Liu J et al. Lamina propria and olfactory bulb ensheathing cells exhibit differential integration and migration and promote differential axon sprouting in the lesioned spinal cord. J Neurosci. 2005; 25: 10700-10711.

7. Feron F, Perry C, Cochrane J et al. Autologous olfactory ensheathing cell transplantation in human spinal cord injury. Brain. 2005; 128: 2951-2960.

8. Lim PAC, Tow AM. Recovery and regeneration after spinal cord injury: A review ana summary of recent literature. Annals Academy of Medicine Singapore 2007; 36: 49-57.

9. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale lor open field testing in rats. J Neurotrauma. 1995; 12 (1): 1-21.

Claims (12)

1. A method of obtaining parietal cells from the olfactory lining of mammals for the treatment of spinal cord injuries, comprising the steps of: receive tissue samples from the olfactory nose of the mammal; washed them; tissue is incubated with dispase II at a concentration of 2 mg / ml for 1 hour at 37 ° C; the tissue is transferred to DMEM: F12 (1: 1) with antibiotics and the olfactory epithelium is mechanically separated from its own plate; own plate is crushed and cultured as an explant culture for 14 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 of gentamicin, a change of medium is performed every three days; after the formation of a monolayer, cells with a 0.05% trypsin solution are incubated for 2 minutes at 37 ° C, the enzyme is inactivated with serum medium, resuspended and centrifuged for 2 minutes at 900 g; the obtained cells are placed on a plastic coated with 0.01% poly-L-lysine and cultured in DMEM: F12 (1: 1) medium containing 10% FBS, 2 mM L-glutamine, 1% ITS, 100 μg / ml of streptomycin, 100 units / ml of penicillin and 500 ng / ml of hydrocortisone, for 3 days; the resulting cells are characterized by p75NTR and GFAP markers. 2. The method according to p. 1, characterized in that, if necessary, after obtaining tissue samples and before the start of the experiment to obtain the primary culture, tissue samples of the olfactory nose of the mammal are transported in a nutrient medium DMEM: F12 (1: 1) and stored at +4 ° C no more than 2 hours. 3. The method according to p. 1, characterized in that the tissue samples of the olfactory lining are washed in DMEM: F12 (1: 1) with antibiotics 100 μg / ml streptomycin and 100 units / ml penicillin at least 3 times. 4. The method according to p. 1, characterized in that the mechanical separation of the olfactory epithelium from its own plate is performed by means of a micro spatula in a Petri dish with DMEM: F12 medium (1: 1) with antibiotics 100 μg / ml streptomycin and 100 units / ml penicillin.

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