RU2576842C2 - Method of producing myoblasts, use of gingival biopsy material, myoblasts preparation for treating pathologies of muscular tissue and method for production thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to biotechnology, and can be used for producing myoblasts of oral mucosa of an individual. That is ensured by biopsy of oral mucosa and treatment of biopsy material for washing, milling, enzymatic splitting gingival tissue proteolytic enzymes. Further, method includes culturing fibroblast-like cells recovered from disaggregated tissue on a suitable medium for producing culture of myoblasts. Method includes performing immunophenotypic testing of myoblast culture for immunophenotype and exclusion of viral and bacterial contamination.

EFFECT: use of present method enables to obtain a culture of myoblasts from a readily available source - human oral mucosa.

2 cl, 1 ex, 2 dwg

Description

The invention relates to the field of biopharmacology, biotechnology and regenerative medicine and describes a new source and method for isolating human myoblasts.

Mammalian myoblasts are a unique type of cell capable of in vivo division, directed migration, followed by fusion with the formation of muscle tubes (syncytium) and the rapid loss of antigens of the main histocompatibility complex of class I. This combination of properties makes myoblasts an almost ideal object for research in order to use these cells in the field of cell therapy and genetic engineering in the treatment of muscle diseases. Studies have shown that myoblast transplantation can increase the maximum contractility of muscle tissue in patients with Duchenne myopathy (Duchenne muscular dystrophy). Methods are being developed for the treatment of severe heart failure, which consists in treating this serious disease using “sheets” of autologous myoblasts isolated from the skeletal muscles of the patient [1].

As a result, there is an increase in the life expectancy of patients, an improvement in its quality, a delay in the moment of confinement to a wheelchair in the most serious forms of diseases.

It is known that the main source of satellite cells and myoblasts are the skeletal muscles of humans and animals [2]. It has also been described that multipotent mesenchymal stromal cells (MMSCs) of bone marrow and adipose tissue with the addition of certain factors can differentiate in the myogenic direction [3, 4], which has found application in cell replacement therapy (for cardiomyoplasty, treatment of incontinence, myopathy, and other muscle diseases).

One example of the use of myoblasts for the treatment of muscle diseases is Duchenne disease, a hereditary disease characterized by progressive muscle dystrophy and the second most common fatal hereditary disease.

Thus, patent EP 1407788 describes a method for treating Duchenne disease using a specialized cell processor for producing myoblasts, which involves the use of cells to transfer a normal set of genes to defective cells in order to repair the patient’s genome. The invention includes the steps of collecting a muscle tissue biopsy, isolating myoblasts, culturing them in vitro to obtain the necessary therapeutic dose, and injecting into the damaged muscle tissue.

WO 1996018303 describes a method for treating hereditary, degenerative diseases of varying severity in mammals using native, genetically or phenotypically transformed myogenic cells, including myoblasts, myotubes, young muscle fibers and modified cell lines, including the stages of culturing myogenic cells; sequential administration of a therapeutically effective dose of immunosuppressants and said myogenic cells to a recipient, thereby achieving a therapeutic effect.

At the same time, in patent EP 1048724 it is noted that the successful use of myoblasts requires the use of cell lines capable of actively proliferating, differentiating, in vitro culturing, while maintaining the characteristics of the tissue cells from which they were isolated. The disadvantage of this method is that human myoblasts isolated from muscle tissue by disaggregating it under the action of enzymes, in contrast to rodent myoblasts (mice and rats), are not capable of multiple division under cultivation conditions, and their proliferative ability decreases with age and especially severely in patients with various types of muscular dysfunctions (for example, Duchenne disease), which greatly complicates their use for therapeutic purposes. To solve this problem, the patent describes a method for producing stable lines of human muscle cells, which includes the following stages:

a) processing the primary culture of muscle cells using glucocorticoids;

b) transfer to a cell culture treated according to a) at least one nucleotide vector of non-retroviral nature, capable of “immortalizing” these cells;

c) conducting further cultivation of the obtained cell culture.

All of the above inventions suggest the stage of taking a muscle tissue biopsy, obtaining a primary cell culture and subsequent growth (in vitro cultivation) of myogenic cells, while the biopsy material obtained surgically as a fragment of skeletal muscle tissue is always indicated as a source of such cells. At the same time, it should be recognized that this source of muscle tissue and the method of its collection has a number of disadvantages and limitations. So, the sampling of biopsy material of muscle tissue requires quite a serious surgical intervention, characterized by a high degree of trauma, the conduct of local or general painkillers, which leads to temporary disability and a relatively long rehabilitation period.

In addition, cells obtained from muscle tissue, in turn, also have a number of disadvantages that significantly limit their use for cell therapy, namely, they have a limited lifespan, are not capable of long-term division in vitro; Moreover, the described properties tend to be aggravated with the patient’s age and are especially pronounced in patients with muscular dystrophies, which makes the use of autologous cell therapy for such patients extremely problematic.

The problem solved by the present invention is a significant simplification of cell therapy in patients with muscle diseases through the use of myoblasts obtained from an easily accessible source by conducting a less traumatic procedure.

The technical solution of the present invention is to obtain myogenic cells (myoblasts, myoblast precursors) from a new, previously unknown source, unexpectedly discovered by the authors during the cultivation of fibroblast-like cells isolated from the mucous membrane of the oral cavity (gums) of a person.

The method consists of the following main stages:

I. Gum biopsy.

II. Biopsy processing to produce myoblast progenitor cells. Processing the biopsy specimen involves traditional procedures for washing, grinding and enzymatically cleaving the gum tissue with proteolytic enzymes that can be easily selected by a specialist. Usually, collagenase preparations are used for this.

III. The progenitor cells isolated from the disaggregated tissue are cultured on a suitable medium to produce a myoblast culture.

IV. Myoblast culture further:

1) tested for immunophenotype and the exclusion of viral and bacterial contamination;

2) cryopreserved and stored in a cryobank;

3) defrost (if necessary.

In another aspect, the invention relates to a method for producing a cell preparation of myoblasts for the treatment of pathologies of muscle tissue.

The resulting myoblasts can be transferred to any physiologically / pharmacologically acceptable carrier / excipient, for example, 0.9% physiological sodium chloride solution for injection, Ringer's solution, with or without autologous serum (0.5-10%). The number of cells can be selected depending on the nosology and is usually 1 × 10 ⁶ -5 × 10 ⁸ cells per 1 ml.

The method and treatment regimen depends on the condition of the patient, the severity of the pathology of muscle tissue and is chosen to achieve the maximum therapeutic effect. Possible routes of administration: intramuscular, intracoronary, intramyocardial.

Brief description of the drawings:

Fig. 1. The formation of numerous multinucleated myotubes (intravital imaging, optical microscopy).

Fig. 2. Immunocytochemical detection of myo D1 (A), sk-myosin (B) and sk-actin (C) in a culture of myoblasts isolated from the mucous membrane of the human oral cavity (gums): a - staining with specific monoclonal antibodies in combination with antispecies antibodies, labeled with dye Alexa488; b - staining of cell nuclei with DAPI reagent; c - combination of images of the result of fluorescent labeling and cell nuclei. The light segment in the photographs corresponds to 20 microns.

A detailed description of the technical solution.

Biopsy specimen (bipsia)

A 2-3 mm ³ gum biopsy was obtained from healthy donors under local anesthesia with 2% lidocaine solution. The age of donors is 25-55 years. An oral mucosal biopsy was immediately placed in a labeled sterile container with transport medium (DMEM / F12).

The biomaterial was delivered to the laboratory in a sterile polymer container containing a transport medium at a temperature of + 4-8 ° C in transport hermetically sealed isothermal containers for no more than 24 hours.

Gum Biopsy Treatment

After the biomaterial was delivered to the laboratory, it was sterilely transferred to a Petri culture dish, washed with a Hanks solution with an antibiotic (gentamicin), and then washed three times with Versen's solution. The biopsy sample was crushed using a sterile scalpel, a disaggregating 0.1% (366 units / mg) type I or II collagenase solution (Sigma) was added, and incubated for 1-1.5 hours at 37 ° C.

Isolation of myoblast precursors and cultivation

After incubation, the tissue suspension was intensively pipetted and centrifuged for 10 minutes at 300 g, the supernatant was removed, and the precipitate was diluted with culture medium (DMEM / F12 1: 1 with the addition of 10% human cord blood serum (SEC) and 10% autologous serum (AS) either DMEM / F12 1: 1 with the addition of 10% SEC and 10% serum of cow embryos (CEC) from NPE PanEco (Russia) or FBS Defined (HyClone, USA), or DMEM / F12 1: 1 with the addition of 20% SEC of the company NPP "PanEco" (Russia) or FBS Defined (HyClone, USA) and 40 μg / ml gentamicin were resuspended and transferred to a culture vial with a tight Stu 3-5 × 104 cells / ^cm2. The cells were cultured at + 37 ° C under 5% CO2. The replacement of the culture medium was performed every 3-4 days, cultures were passaged when they reach 50% confluency. Visual inspection of the morphology and growth culture was using phase contrast microscopy, cell cultures of 1–10 passages were used for studies.

After the formation of a subconfluent monolayer, the cells were washed with Versen solution, then removed from the surface of the culture dish with Versen solution with 0.25% trypsin, resuspended in the culture medium and explanted into a larger culture dish for subsequent cultivation.

Immunophenotypic analysis of fibroblast-like gum cells. The resulting cell cultures were plated on coverslips and after 2 days immunofluorescence analysis was performed. The expression of proteins characteristic of fibroblast-like cells — type I, III collagen, elastin and vimentin — was determined using primary monoclonal and secondary antibodies labeled with Alexa488 (Life Technologies, USA). Cells were visualized using an Axioplan 200 microscope with an Axiocam HRm camera using AxioVision software (Carl Zeiss, Germany).

For cytofluorimetric analysis, the cell suspension was fixed with 1% paraformaldehyde solution in phosphate-buffered saline (pH 7.4) and fluorescent antibodies to CD34PE, CD45FITC, CD73PE, CD90APC, CD324FITC, cytokeratins 14, 15, 16, 19 (BDPharmingen, USA) were used CD105 Alexa488 (Life Technologies, USA), according to the recommendations of the manufacturers.

Induction of myogenic differentiation. To induce myogenic differentiation, the cells were scattered with a high density of 5 × 105 / cm2, cultured in DMEM / F12, 20% FBS (HyClone, USA) to 90-100% of the confluent monolayer, then the medium was replaced by low glucose DMEM (HyClone, USA) supplemented with 2% horse serum (BioInd, Israel) and incubated at 37 ° C and 5% CO ₂ until myotube-like structures appeared.

In cell culture, myogenic cells go through the following stages: cell adhesion to the substrate, activation and proliferation of myoblast progenitor cells; the formation of myoblasts with their subsequent merger into multinucleated myotubes; the formation of myofibrils, characterized by contractile activity (Fig. 1)

Testing, cryopreservation and storage of myoblasts.

Before freezing, cells were seeded based on the preparation of a subconfluent monolayer.

A sterile cryoprotective solution (freezing medium), consisting of 10% DMSO, DMEM / F12 and 40% FBS, was prepared in an ice bath in order to maintain a temperature of + 4 ° C.

The cell culture was washed three times with Versen's solution and trypsinized at 37 ° C, 5% CO ₂ for 10 minutes. The cell suspension was centrifuged for 200 g, 10 min. The supernatant was removed, the cells were resuspended in Hanks solution, after which the cells were counted to the Goryaev chamber.

Part of the cells were selected for:

1) immunophenotypic analysis (in order to confirm the myogenic nature of the cells), for this, the cell suspension was plated on coverslips and cultured in DMEM / F12 medium with 10-20% FBS (without passaging) for 21 days, then fixed in 4% p PFA (SIGMA Product No. 158127) and immunofluorescence analysis was performed using primary mouse monoclonal antibodies: DAKO Actin (Sarcomeric) Product No. M087401-2, DAKO MyoD1 Product No. M351201-2, Invitrogen Myosin (skeletal muscle) Product No. 18-0105; and secondary rabbit antibodies labeled with fluorochrome Alexa488 - Molecular Probes Alexa Fluor 488 Rabbit Anti-Mouse IgG Product No. A-11059. Cell nuclei were additionally stained with DAPI reagent - SIGMA Product No. 32670-F (Fig. 2).

Evaluation of the effectiveness of myogenic differentiation. To assess the effectiveness of the induction of myogenic differentiation, cells sown on slides were fixed with 4% paraformaldehyde. The cultures were then incubated with primary mouse antibodies specific for human skeletal myosin (AbD Serotec, UK), actin (Thermo Scientific, USA) and MyoD1 nuclear phosphoprotein inducing myogenesis (DAKO, USA), followed by staining with rabbit secondary antibodies labeled with fluorescein (FDT) ( ) (Life Technologies, USA). Images were taken using an Axioplan 2 microscope, an Axiocam HRc camera, and AxioVision software (Carl Zeiss, Germany). Then, using the ImageJ program (National Institutes of Health, USA), the percentage of specifically colored area in the photographs was isolated and measured in relation to the entire field. For each case, 25 independent visual fields were analyzed and averaged values were given for all patients (Fig. 2).

2) biosafety testing (to exclude viral and bacterial contamination):

- PCR (HIV-1 and -2, HBV, HCV, CMV, HSV 1, 2 and 6, EBV, Toxoplasma gondii, Mycoplasma hominis);

- bacteriological examination for sterility (exclusion of bacterial and fungal microflora).

The remaining cells were re-centrifuged, the cell pellet was resuspended in freezing medium at a concentration of 2 × 106 cells in 1 ml, after which the cell suspension was transferred to cryovials. Cryovials were marked: sample number, date, type of biomaterial, number of cells, number of passages.

Cellular material was subjected to program freezing to -80 ° C at a rate of 1 ° C / min and then transferred to storage in a cryostorage with liquid nitrogen. After that, cryovials were placed in quarantine cryogenic storage until the results of biosafety testing were obtained.

Permanent storage of samples with a single identification number was carried out in a cryogenic repository (with negative biosafety testing results). The shelf life of the cells is not limited.

Defrosting myoblasts

The cryovial with myoblasts was thawed, the cell suspension was transferred to a centrifugation tube, cold DMEM / F12 culture medium (+ 4 ° C) was added (dropwise), and then centrifuged for 5 min at 300 g, 4 ° C. The supernatant was removed, the cells were resuspended in DMEM / F12 culture medium. The washing procedure from DMSO was repeated twice.

Example

The volunteer (after signing the informed consent) was taken a biopsy sample of the oral mucosa from the retro-molar space with a diameter of 3 mm ³ . Before receiving the primary cell culture, the biopsy was washed with solutions of antibiotics and antimycotics (penicillin 100 u / ml, streptomycin 100 μg / ml, gentamicin 200 μg / ml, amphotericin B 25 μg / ml) in DMEM / F12 medium. Then the biopsy was transferred to DMEM / F12 medium containing 2% fetal calf serum (ETS), 40 μg / ml gentamicin and type II collagenase (Sigma). The biopsy sample was incubated at 37 ° C, incubated for 1-1.5 hours at a temperature of 37 ° C. The obtained cell pellet was resuspended in a culture medium - DMEM / F12, 10-20% ETS (PanEco), 40 μg / ml gentamicin and plated in T 25 culture bottles (Nunc). The vials were placed in a CO ₂ incubator (37 ° C, 5% CO ₂ ) and cultured for 2 weeks until a primary culture was obtained. As the subconfluent layer grew and reached, the cells were trypsinized and transferred to a new larger area (T-75, T-175 "Nunc") culture vial. Upon reaching a subconfluent monolayer, a medium with 10-20% ETS was replaced with a serum-free medium. Before removing the cells and obtaining the finished suspension, the cells were kept for at least 8-14 hours at 37 ° C. At the end of the incubation, the cells were removed from the culture plastic by means of a 0.25% trypsin-EDTA solution, washed by three centrifugation and resuspension in 0.9% physiological sodium chloride solution, and then suspended in an amount of 20 × 106 cells / ml in 0.9 % solution of sodium chloride for injection. The resulting myoblast preparation should be stored at a temperature of (+ 4 ° C) - (+ 8 ° C) and should be used within 24 hours from the date of preparation.

Bibliography:

1. Miyagawa S, et al. Impaired myocardium regeneration with skeletal cell sheets-a preclinical trial for tissue-engineered regeneration therapy. Transplantion. 2010; 90: 364-72.

2. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961 Feb; 9: 493-5.

3. Pittenger MF, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284 (5411): 143-7.

4. Zuk P.A. et. al. Multilinege cells from human adipose tissue: implication for cell-based therapies. Tissue Eng. 2001; 7: 211-28.

Claims (2)

1. The method of obtaining myoblasts by culturing fibroblast-like cells isolated from the mucous membrane of the human oral cavity, including:
a) a biopsy from the mucous membrane of the human oral cavity;
b) processing of the biopsy specimen, which includes washing, grinding, enzymatic cleavage of the gum tissue by proteolytic enzymes;
C) the cultivation of fibroblast-like cells isolated from disaggregated tissue on a suitable medium to obtain a culture of myoblasts;
d) immunophenotypic testing of myoblast culture for immunophenotype and the exclusion of viral and bacterial contamination. 2. The method of producing myoblasts according to claim 1, where after stage g) cryopreservation of myoblasts is performed.

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