RU2486603C1 - Method for simulating tissue structure of human retina - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: eyeball is enucleated in autopsy, washed with an alcohol solution and Hank's solution with antibiotics. An anterior segment of the eyeball is removed along a dentate line. A vitreous body and neural retina are separated from a retinal pigment epithelium (RPE). That is followed by the devitalising treatment of the separated retina for creating a cell-free retinal frame. This frame is washed in a nutrient medium with antibiotics. Then, the frame is transferred into a slide chamber, and colonised with the RPE cells. For producing an RPE cell culture, an eyecup is filled with Hank's solution with EDTL. Then, the native RPE cells are collected. A part of the RPE cells is transferred into a sterile medium for recovery, and centrifuged. Then, the recovered cells are resuspended in a growth medium, pipetted, bottled and cultured with the medium changed to form a primary culture. Then, the primary culture cells are passaged. These cells are cultured in culture flasks. The passage 1-5 cultured cells are removed as monolayer cultures from the culture surface, and the cultured RPE cells are directed as a suspension for colonisation pf the cell-free retinal frame. In this case, retinal inspection and devitalising treatment control are conducted by microscopy. Monitoring the cell cultures is ensured by immune histochemical test, PCR and microscopy.EFFECT: method provides creating a three-dimensional PRE cell culture model with the specified parameters of number, density and tissue differentiation due to phased controlling the modelling process.14 cl, 2 ex, 3 tbl, 28 dwg

Description

The invention relates to biotechnology, can be used in tissue engineering and cell transplantology in order to obtain the retinal tissue structure for the study of cellular and molecular mechanisms of retinal regeneration and in vitro drug testing, as well as for retinal restoration in various neurodegenerative diseases.

Today, age-related macular degeneration (AMD) is one of the most common and poorly studied eye diseases, threatening a significant decrease in vision, blindness and disability. AMD is characterized by a gradual decrease in central vision in people older than 50-60 years, leading to a significant loss of vision in old age. There are two main forms of AMD - “dry” and “wet”. In 9 out of 10 cases of AMD there is a "dry" form. It is usually characterized by a slow progressive decrease in vision. A “wet” form of AMD develops in about 1 out of 10 cases and can cause serious loss of vision in a fairly short period of time - sometimes over several months. Treatment of AMD (macular degeneration) remains an urgent problem in modern ophthalmology.

A known method of treating a “wet” form of age-related macular degeneration, including intravitreal administration of the drug Avastin [E.V. Boyko, O.V. Filokhina, S.V. Sosnovsky and others. The first experience of using Avastin in the treatment of the wet form of age-related macular degeneration, Modern Technologies for the Treatment of Vitreoretinal Pathology - 2007. Collection of scientific articles based on materials of a scientific and practical conference (Moscow, March 23, 2007) edited by H.P. Takhchidi. Moscow, 2007, S. 54-58]. Avastin is a recombinant monoclonal antibody that selectively binds to and neutralizes biologically active vascular endothelial growth factor (VEGF), inhibiting the interaction of VEGF with its receptors on the surface of endothelial cells, which leads to a decrease in the growth of newly formed vessels. The disadvantage of this method is the short duration of action, which requires repeated injections of the drug.

There is a method of treating age-related macular degeneration of the retina, according to which a partial vitrectomy of the premacular part of the vitreous body is performed, detachment of the posterior hyaloid membrane is induced without its removal, and Avastin is injected into the formed retrogyaloid space [patent RU No. 2368359, IPC A61F 9/00].

There is a method of treating age-related macular degeneration of the retina [RU patent No. 2408335, IPC A61F 9/00]. The method includes laser exposure and the administration of retinalamine, which is a lyophilisate obtained by acetic acid extraction from the retina of cattle or pigs, containing a complex of low molecular weight peptides.

However, all these treatment methods are aimed only at maintaining vision and preventing its deterioration.

The achievements of cell biology at the end of the 20th century became the basis for the development of biomedical technologies. They allowed the use of cultured human cells in medicine to restore the structural and functional activity of damaged tissues in situations where traditional methods of treatment are ineffective or powerless.

There is a method of treating a “dry” form of age-related macular degeneration of the retina, in which the macular region of the retina is irradiated with low-intensity laser radiation and after 3 hours, autologous cultured mesenchymal stem cells of the bone marrow of the patient are intravenously administered [patent RU 2333737, IPC A61F 9/00].

A known method of treating a “dry” form of age-related macular degeneration of the retina [patent RU 2375016, IPC A61F 9/00]. The method consists in intravitreal administration of a suspension of autologous cultured mesenchymal stem cells of a patient’s own bone marrow.

It is believed that precursor cells isolated outside the retina and transplanted by systemic injection or injection subretinally or into the vitreous cavity of the adult eye can integrate into the retina and thereby improve its function.

However, the disadvantage of the known methods is that the results obtained are mostly ambiguous, marginal or negative and indicate that simple transplantation of adult non-retinal precursors into a pathologically modified retina is not capable of leading to significant long-term clinical improvements.

In the last decade, the idea of transplanting healthy cells of retinal pigment epithelium (RPE) has been intensively studied as therapeutic therapy [Sommer F. Hyalocytes in Tissue Engineering First Steps Towards a Cell-based Vitreous Substitute, Dissertation, 2006, p.14-15]. Attempts to use RPE cell layers without any supporting matrix are associated with several drawbacks. First, it is more difficult to control cells during transplantation [Lu L., Nyalakonda K., Kam L., Bizios R., Goepferich A., Mikos A.G. Retinal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from synthetic biodegradable polymers // Biomaterials. - 2001. - Vol.22. - P.291-297]. In addition, non-specific enzymatic treatment (for example, the use of trypsin / EDTA) of cultured RPE layers leads to a significant decrease in retinoid metabolism [Recum N.A., Okano T., Kim S.W., Bernstein P.S. Maintenance of retinoid metabolism in human retinal pigment epithelium cell culture // Exp. Eye Res. - 1999 .-- Vol.69. - P.97-107].

One of the areas of biotechnology, which is engaged in the creation of biological substitutes for tissues and organs, is tissue engineering. Tissue engineering - the creation of new tissues and organs for the therapeutic reconstruction of a damaged organ by delivering support structures, cells, molecular and mechanical signals for regeneration to the desired area. At the first stage of creating a tissue engineering construct, we select our own or donor cell material (biopsy or autopsy), select tissue-specific cells and cultivate them.

One of the central tasks in the treatment of pathologically altered retinas is the identification of the optimal cell type in order to actively control the reparative function of the retina. The ideal cell type should be cells that are completely determined by the fate of retinal cells and still retain the ability to expand in vivo or in vitro. In addition, the ability to differentiate into functional specialized tissue is important for the type of cell of interest. Since epithelial cells usually do not survive in suspension, RPE cells must attach to the surface in order to avoid apoptosis [Tezel TN, Del Priore L.V. Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium // Graefes. Arch. Clin. Exp. Ophthalmol. - 1997 .-- Vol. 235. - R.41-47].

Therefore, in addition to cell culture, a tissue-engineering construct or transplant includes a special carrier (matrix). Matrices can be made of various biocompatible materials. The cells of the obtained culture are applied to the matrix, after which such a three-dimensional structure is transferred to a bioreactor with a nutrient medium, where it is incubated for a certain time. When forming complex spatial structures, a special matrix is required that repeats the shape of the tissue being restored. The biomaterials used to obtain the matrices must be biologically inert and, after being transferred to the body, must ensure the localization of the cellular material applied to them in a specific place. To create tissues and organs, synthetic materials, materials based on natural polymers (chitosan, alginate, collagen), as well as biocomposite materials are used. Biodegradable synthetic biomaterials based on polymers of organic acids, for example, lactic (PLA, polylactate) and glycolic (PGA, polyglycolide), were one of the first in tissue engineering. Matrices based on organic acids formed the basis for the creation of such organs and tissues as skin, bone, cartilage, tendon, muscles (striated, smooth and cardiac), small intestine, etc.

A group of researchers led by A.G. Mikos proposed the use of biodegradable polymer films as temporary substrates for cultured RPE cells and the subsequent transplantation of these polymer-cell complexes into the subretinal space. However, cultured RPE cells on these films made from a combination of lactic and glycolic acid (PLGA) lost their characteristic cubic morphology during 7 days of cultivation [Thomson RC, Giordano GG, Collier JH, Ishaug SL, Mikos AG, Lahiri-Munir D. , Garcia CA Manufacture and characterization of poly (alpha-hydroxy ester) thin films as temporary substrates for retinal pigment epithelium cells // Biomaterials. - 1996 .-- Vol.17. - P.321-327].

In order to maintain the normal morphology and function of RPE cells in vitro, the researchers developed a new degradable microsubstrate from PLGA and a copolymer blocker made of polyethylene glycol (PEG) and PLA using microcontact printing. The surfaces of this film, consisting of adhesive (PLGA) and non-adhesive (PEG / PLGA) domains, allowed the cells to adhere and spread, and were allowed to maintain a differentiated cell phenotype for an 8-hour study period. The polymer substrate was supposed to facilitate cell handling during transplantation and ensure the correct orientation of the transplant in the subretinal space [Lu L., Kam L., Hasenbein M., Nyalakonda K., Bizios R., Goepferich A., Young J.F., Mikos A.G. Retinal pigment epithelial cell function on substrates with chemically micropatterned surfaces // Biomaterials. - 1999 .-- Vol.20. - R.2351-2361]. Although PLA and PLGA are biodegradable, these materials have disadvantages: a change in the pH of the surrounding tissues during cleavage in the body and insufficient mechanical strength, which makes it impossible to use them as a universal material for matrices and substrates.

It is known to use amniotic membranes that modulate the proliferation and differentiation of RPE cells in culture [Ohno-Matsui K., Ichinose S., Nakahama K., Yoshida T., Kojima A., Mochizuki M., Morita I. The effects of amniotic membrane on retinal pigment epithelial cell differentiation // Mol. Vis - 2005 .-- Vol.11. - P.1-10]. During transplantation, amniotic membranes act as an appropriate substrate for epithelization and have been widely used in ophthalmology for the treatment of persistent epithelial defects [Dua H.S. Amniotic membrane transplantation // Br. J. Ophthalmol. - 1999. - Vol. 83. - R.748-752]. [Stanzel B.V., Espana E.M., Grueterich M., Kawakita T., Parel J.M., Tseng S.C., Binder S. Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture // Exp. Eye Res. - 2005. - Vol.80. - P.103-112.] It was shown that amniotic membranes deprived of the epithelium increase the formation of a monolayer from RPE with tight contacts. They were recommended to be used as a matrix containing a basement membrane in order to facilitate the transplantation of RPE in the treatment of AMD.

Isolation of RPE cells using enzymes (such as type 3 collagenase / hyaluronidase) and subsequent passage on a thermally sensitive surface is an appropriate method to preserve the metabolic activity of cultured RPE cells for transplantation [von Recum N.A., Okano T., Kim SW, Bernstein PS Maintenance of retinoid metabolism in human retinal pigment epithelium cell culture // Exp. Eye Res. - 1999 .-- Vol.69. - P.97-107].

Extracellular matrix proteins, including collagen structures, are successfully used as a substrate for cultivation. However, it is not possible to create an artificial volumetric design from collagen that repeats the structure of any organ, including the retina.

This circumstance initiated studies aimed at developing a method that allows one to obtain decellular organ matrices while maintaining their spatial organization. The technology, which provides for the devitalization of allograft tissue by prolonged soft treatment with detergent solutions, leads to the destruction of all cellular elements of the donor, leaving the connective tissue skeleton of the organ unchanged. The acellularity of the resulting framework ensures its non-immunogenicity, and the absence of cross-linking does not prevent its colonization by the recipient's cells. In addition, the spatial features of the substrate and the chemical composition of the matrix serve as additional stimuli for their maintenance of proliferation, self-organization, and differentiation functions. Three-dimensional models (3D matrix), which are tissue-engineering structures, are by far the best models for assessing the behavior and differentiation of predetermined cells.

A known method of obtaining a connective tissue skeleton of a blood vessel. The method allows you to effectively wash the cells of the vessel without damaging collagen and elastic fibers [Egorova MV, Rogovskaya Yu.V., Ivanov AV, Andreev S.L., Akhmedov Sh.D., Afanasyev S.A., Economical technology for producing a cell-free matrix of the arterial vessel of animals and humans, Cellular technologies in biology and medicine, No. 2, 2011, p.110-113].

A known method for the extraction of cellular components from body tissues and structures that should be used as material for implantation. Decellularization improves biocompatibility and reduces the immunological response against tissue transplant [application GB No. 2375771, IPC A61L 27/26].

A known method of combining cultured osteogenic cells and a three-dimensional carrier material by immobilizing cells on the surface of the carrier material (demineralized bone matrix). The invention provides a tissue-engineering equivalent of bone, including the full combination of osteogenic cells differentiated in vitro and a three-dimensional carrier material made from demineralized bone matrix [RU patent No. 2414916, IPC A61K 35/32].

A known gel model of the extracellular matrix for studying the development and differentiation of embryonic SC (ESCs) in vitro and a method for studying the mechanisms of development of ESCs [CN application No. 101711890, IPC A61L 27/38]. The application describes a three-dimensional model of differentiation and development of ESCs, the introduction of ESCs into this model in different cell densities, and the detection of the phenomenon of formation of embryoid bodies.

Given the key role of RPE in the preservation and viability of the entire retina, transplantation of RPE can solve the problem of AMD.

To find out the biological complexity of the retinal organogenesis and its increasing intersection with the regenerative function of the retina, as well as to decide the feasibility of introducing into the clinical practice of cell transplants based on RPE cells, new model systems are needed that can be used to analyze individual variations of precursor cell types, including the number of cultured RPE cells, extracellular matrix signals, two- and three-dimensional structures, and tissue function with respect to their effects on the global the physiological function of the intact organ. In particular, it is necessary to develop a method for modeling the reparative function of the retina to assess the behavior and differentiation of cultured RPE cells during preclinical studies.

The problem to which the invention is directed, is to develop a method for modeling the tissue structure of the retina of the human eye.

The technical result of the invention is the creation of an ex vivo three-dimensional model of RPE cell culture with predetermined parameters of quantity, density and tissue differentiation on a biologically inert matrix that does not impair cell culture growth and differentiation.

To solve this problem, a method for modeling the tissue structure of the human retina by colonizing the acellular retinal skeleton with retinal pigment epithelium cells is that the cell-free retinal skeleton is obtained by devitalizing the retina from an eyeball enucleated by autopsy by sequentially washing it in an alcohol solution at a concentration of 70 % and in a cold solution of Hanks with antibiotics, removal along the dentate line of the anterior segment of the eye block, separating the vitreous body and the neural retina from the retinal pigment epithelium and isolating the retina, cutting it in the optic nerve, the isolated retina for devitalizing treatment is placed in 10-15 ml of 10 mm EDTA in DMEM for 20 hours at 4 ° C, then treated with a 3% solution of Triton X-100 for 2 hours at 4 ° C and washed in distilled water, then the retina is sequentially placed in a 0.025% solution of DNase I in 1 M NaCl solution for 1 hour at 4 ° C and 4% sodium deoxycholate for 1-2 hours at tamper ature 22 ° C and then washed in distilled water to remove detergent and cell debris, the eye cup is filled with cold Hanks solution with EDTA and after 15-30 minutes the cells of the native retinal pigment epithelium are collected, part of the collected cells are transferred to TRI®-Reagent lysis solution from calculation of 1 ml of solution per 50-100 mg of tissue, pipette to a homogeneous solution and the lysate of cells of the native retinal pigment epithelium is incubated for 5-7 minutes at a temperature of 22 ° C, another part of the collected cells is transferred to a sterile medium for population consisting of DMEM / F12 nutrient medium (1: 1) supplemented with 15% fetal calf serum, 2 mM L-glutamine and antibiotics and centrifuged for 5-7 min at 600-800 rpm, then the cells are resuspended in full culture medium consisting of DMEM / F12 nutrient medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% non-essential amino acid solution, 1% N2 neural supplement solution, 20 ng / ml the main growth factor for fibroblasts and antibiotics, and after pipetting, they are distributed in culture bottles without h cover and cultivate at a temperature of 37 ° C and 5% CO ₂ , after 5-7 days carry out the first change of medium and continue to cultivate with a change of medium after 2-3 days until the cells reach a confluent monolayer and form a primary culture, then passaging of primary cells culture using a solution of trypsin-versene (1: 3), then the cells are placed in culture bottles and in slide chambers at a density of 1 - 2 × 10 ⁴ cells / cm ² and cultured in cells of culture bottles 1-5 passage in adhesive monolayer cultures, commits Neural directions are removed from the culture surface with trypsin-versene solution (1: 3) and, in the form of a suspension of cultured retinal pigment epithelial cells, sent to colonize the cell-free retinal scaffold, and the cell-free retinal scaffold is washed in DMEM / F12 culture medium before colonization (1 : 1) with antibiotics, transferred to a slide chamber and filled with a complete culture medium, and the colonization of the acellular skeleton of the retina is carried out by diffusion deposition eniya suspension of cultured retinal pigment epithelial cells in a density of 4 -. 5,5 × April ¹⁰ cells / cm ² in complete culture medium of the same composition, and culturing at a temperature of 37 ° C and 5% CO ₂ with medium change after 2-3 days before the cultured cells colonize the acellular skeleton of the retina and form a monolayer of cells on its surface to obtain the tissue structure of the retina of the eye, which is sent to study the localization of cultured cells and to an immunohistochemical (IHC) assessment of the stump differentiation potential iruemyh cells in cell-free frame of the retina.

In a particular embodiment, to study the localization of cultured cells, the nuclei of the cells are stained with Hoechst 33342 vital dye for luminescence microscopy.

In another particular embodiment, to study the localization of cultured cells, the cell-free cell-free retinal framework is fixed with 4% paraformaldehyde after 12-25 days, washed with phosphate-buffered saline (PBS, pH 7.4), and carried out with alcohols with an ascending concentration of 70, 96 and 100% -th alcohol, poured into paraffin, made histological sections and stained with hematoxylin and eosin for light microscopy.

In another particular variant of IHC, the differentiation potential of cultured cells in the acellular skeleton of the retina is assessed by immunoperoxidase staining using murine monoclonal antibodies to CD31, CD34, tyrosinase, CK7, CK8, CK18, CK19, vimentin, GFAP, S100 and NSE.

In another particular embodiment, penicillin-streptomycin antibiotics are used in standard concentrations - penicillin 100 U / ml and streptomycin 100 μg / ml.

In another particular embodiment, the cell-free retinal framework after removal of detergent and cell debris is placed in antibiotic culture medium DMEM / F12 at a temperature of 4-8 ° C for 2-20 days until fixation with 4% paraformaldehyde or colonization by cultured cells.

In another particular embodiment, the fixed acellular skeleton of the retina is washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96 and 100% alcohol, embedded in paraffin, and histological sections are made that are mounted on glass slides and stained with hematoxylin and eosin to control the adequacy of the devitalizing treatment using light and / or luminescent microscopy.

In another particular embodiment, after incubation, the lysate of the cells of the native retinal pigment epithelium is stored at a temperature of minus 20 ° C until the polymerase chain reaction (PCR) - PCR evaluation.

In another particular embodiment, primary culture cells and cultured cells of passage 1-5 are sent for PCR evaluation.

In another particular embodiment, for the PCR evaluation, the level of expression of genes encoding transcription factors Oct4 (POU5F1), Nanog, Pax6 and differentiation markers Musashi 1, β tubulin III, RPE65 by total RNA isolation and cDNA synthesis is determined, and the level of gene expression is assessed by intensity luminescence of bands obtained by electrophoretic separation of PCR products in a 1% agarose gel on a gel analyzer.

In another particular embodiment, cell cultivation in slide chambers is carried out in 1-, 2-, 4- or 8-well slide chambers, after 2-3 days, when the cells reach a subconfluent layer, they are fixed with chilled acetone and sent to the IHC for potential assessment differentiation of cells.

In another particular embodiment, the IHC assesses the differentiation potential of cultured cells by immunoperoxidase and immunofluorescence staining using primary mouse monoclonal and rabbit polyclonal antibodies, Pax6, Sox2, vimentin, Nestin, β tubulin III, nNOS, tyrosine hydroxylase, NF GFAP 200 and 200 kAP 200 , CNPase, Iba, N-cadherin, Connexin-43, RPE65, CRALBP, Recoverin, PanCK, Macrophage antigen, Fibronectin, Factor Von Willebrand, Ki67, as well as EnVision ™ polymer conjugated with horseradish peroxidase, (Dako, Denmark) or secondary antibodies: SFX kit with goat antibodies pro ive mouse labeled with Alexa Fluor® 488, antibody against rabbit immunoglobulins conjugated to the fluorochrome Texas Red, diluted in 0.1% solution of bovine serum albumin.

In another particular embodiment, the cell-free retinal skeleton transferred to the slide chamber is filled with a complete culture medium consisting of DMEM / F12 culture medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% a solution of interchangeable amino acids, 1% solution of a neural additive N2, 20 ng / ml of the main fibroblast growth factor and antibiotics.

In another particular embodiment, the acellular retinal skeleton transferred to the slide chamber is filled with a complete culture medium consisting of DMEM / F12 culture medium (1: 1), supplemented with 10% fetal calf serum, 2 mM L-glutamine and antibiotics, and placed in a CO ₂ incubator having a temperature of 37 ° C, 5% CO ₂ in air to balance the pH of the medium.

The invention is illustrated in the following figures.

Figure 1 presents a simulation circuit of the tissue structure of the retina of the human eye.

Figure 2 presents the histological preparation of the neural part of the retina of an adult eye in close connection with the vitreous body, fixation with 4% paraformaldehyde, paraffin section (control). Stained with hematoxylin and eosin, a large-scale increase in X200.

A is a cross section

B is a longitudinal section.

Figure 3 presents the cell-free retina of an adult eye immediately after devitalizing treatment, phase contrast.

A - large-scale increase in X40,

B - large-scale increase in X200.

Figure 4 presents the acellular retina of an adult eye at the border with the acellular skeleton of the vitreous body, paraffin section. Stained with hematoxylin and eosin, a large-scale increase in X400.

A - 2 days after devitalizing treatment,

G-D - 20 days after devitalizing treatment.

Figure 5 presents the cell culture of the retinal pigment epithelium of the eye of an adult 2nd passage. Immunoperoxidase staining, nuclei stained with hematoxylin. Large-scale increase in X200.

A - Nestin marker - brown staining,

B - marker β tubulin III - brown staining,

B - marker NF 68 and 200 kDa - brown staining.

Figure 6 presents the cell culture of the retinal pigment epithelium of the eye of an adult of the 2nd passage. Immunofluorescence study, nuclei stained with DAPI (blue glow). Scale bar 200 µm.

A - marker nNOS - red glow,

B - marker GFAP - red glow,

In - marker CNPase - green glow.

Figure 7 presents the results of PCR analysis, illustrating the expression pattern of genes RPE65, Oct4, Nanog, Musashi 1, Pax6, β Tubulin III in primary (PC) and passivated (P1-P4) adhesive cell cultures of retinal pigment epithelium of an adult human eye, as well as in controls, in the cell culture of postnatal fibroblasts (K) and native retinal pigment epithelium (H). As a gene for normalization, RPL19 was used.

On Fig-10 presents the colonization of the acellular retina and the acellular skeleton of the vitreous body of an adult eye with autologous cells of the retinal pigment epithelium of the 2nd passage.

On Fig on the 7th day, phase contrast.

A - clusters of rounded cells on the surface of the acellular skeleton of the retina, large-scale increase in X100,

B - rounded formations interconnected by elongated cells, a large-scale increase in X200.

In Fig.9 - on the 7th day, phase contrast.

A - rounded formations interconnected by elongated cells, a large-scale increase in X100,

B - two subpopulations, a large-scale increase in X100.

Figure 10 - on the 12th day, a monolayer of retinal pigment epithelial cells on the surface of the acellular skeleton of the retina, phase contrast, large-scale increase in X100.

11 - on the 12th day, the nuclei of the cells of the retinal pigment epithelium, forming a monolayer on the surface of the acellular skeleton of the retina, are stained with vital nuclear dye Hoechst 33342, luminescent microscopy, large-scale increase in X100.

On Fig-16 presents histological sections of a cell-free skeleton of the retina of an adult, intimately associated with a cell-free skeleton of the vitreous body; cell-free scaffolds are colonized by autologous cells of the retinal pigment epithelium of the 2nd passage, stained with hematoxylin and eosin.

On Fig presents

A - conglomerates of cells of the retinal pigment epithelium of a rounded shape on the vitreous skeleton, a fragment with one of the cell conglomerates, a large-scale increase in X40

B - fragment increase, large-scale increase in X400.

In Fig.13 - the cells of the retinal pigment epithelium populated the entire inner border membrane of the retina bordering the posterior hyaloid membrane of the vitreous, a large-scale increase in X100.

On Fig - a monolayer of a cuboid shape of the cells of the retinal pigment epithelium, tightly adjacent to each other; visible migration of cells into the vitreous skeleton, a large-scale increase in X400.

On Fig - cells of the retinal pigment epithelium of a flattened shape, populated the inner border membrane of the retina adjacent to the posterior hyaloid membrane of the vitreous body, a large-scale increase in X400.

In Fig.16 - conglomerate of cells of the retinal pigment epithelium on the surface of the vitreous skeleton, a large-scale increase in X200.

On Fig-28 presents the acellular cell structure of the retina of an adult, intimately associated with the cell-free framework of the vitreous body; cell-free scaffolds are colonized by autologous cells of the retinal pigment epithelium of the 2nd passage. Immunoperoxidase staining on serial paraffin sections.

On Fig - without primary antibodies (control), a large-scale increase in X200.

On Fig - negative staining for marker endothelial cells CD31.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X200,

B - in cell conglomerates located on the acellular skeleton of the vitreous body, a large-scale increase in X200.

On Fig - negative staining for endothelial cell marker CD34.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X400,

B - on the surface of the acellular retina and in cell conglomerates, a large-scale increase in X100.

On Fig - negative staining for marker cells synthesizing melanin, tyrosinase.

A - on the surface of the acellular retina, a large-scale increase in X200,

B - in cell conglomerates located on the acellular skeleton of the vitreous body, a large-scale increase in X200.

On Fig - brown (positive) staining of dedifferentiated cells of the retinal pigment epithelium on the marker of the SK7 epithelium.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X200,

B - on the surface of the acellular retina and in cell conglomerates, a large-scale increase in X200.

On Fig - brown (positive) staining of migrating cells of the retinal pigment epithelium on the epithelial marker SK19.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X100,

B - on the surface of the acellular retina in cell conglomerates, a large-scale increase in X200.

In Fig.23 - brown (positive) staining of differentiated cells of the retinal pigment epithelium on the marker of the SK8 epithelium.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X200

B - on the surface of the acellular retina in cell conglomerates, a large-scale increase in X400.

On Fig - brown (positive) staining of differentiated cells of the retinal pigment epithelium on the epithelial marker SK18.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X100

B, C, D - on the surface of the acellular retina and in cell conglomerates, a large-scale increase in X200.

On Fig - brown (positive) staining for glime cell marker vimentin.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X200,

B - on the surface of the acellular retina, a large-scale increase in X200.

On Fig - brown (positive) staining for marker differentiated glial cells S100.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X200,

B - on the surface of the acellular retina and in cell conglomerates, a large-scale increase in X40.

On Fig - brown (positive) staining for the neural marker NSE.

A - on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, a large-scale increase in X200,

B - on the surface of the acellular retina and in cell conglomerates, a large-scale increase in X40.

On Fig - staining for a marker of glial cells GFAP.

A, B - negative staining in the cells of the retinal pigment epithelium, located on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, large-scale increase in X200,

B, D - negative staining in the cells of the retinal pigment epithelium located on the surface of the inner border membrane of the acellular retina bordering the acellular skeleton of the vitreous body, and brown (positive) staining in the acellular skeleton of the retina (in the form of a network), large-scale increase in X200,

D - brown (positive) staining in individual cell conglomerates, large-scale increase in X100,

E - brown (positive) staining in individual cell conglomerates, large-scale increase in X400.

The figures show:

1 - hyaloid stroma of the vitreous body;

2 - neural retina;

3 - connection of the inner border membrane of the retina with the posterior hyaloid membrane of the vitreous body;

4 - vascular framework;

5 - skeleton of the neural part of the retina, consisting of the basement membranes of Mueller cells;

6 - vitreous frame;

7 - the inner border membrane of the retina;

8 - a subpopulation of cells of the retinal pigment epithelium attached to the surface of the acellular skeleton of the retina;

9 - a subpopulation of cells of the retinal pigment epithelium, spread on the surface of the culture plastic and formed a monolayer;

10 - a monolayer of cuboid shape of cells closely adjacent to each other;

11 - a layer of cells of a flattened shape.

Modeling of the tissue structure of the retina of the human eye (three-dimensional model) was carried out according to the scheme shown in figure 1.

Obtaining a cell-free retinal framework from cadaveric material was carried out by purifying it from functioning cells (devitalizing treatment).

Adult eyeballs were enucleated by autopsy, freed from surrounding tissues and washed once in 70% alcohol and 3 times in cold Hanks solution with antibiotics in standard concentration - penicillin 100 Units / ml and streptomycin 100 μg / ml. The anterior segment of the eyeball was removed in a circular incision behind the dentate line, which was well defined under normal lighting, as the boundary between the dark and light areas. The vitreous body and the neural retina were separated from the retinal pigment epithelium (RPE) and isolated by cutting the neural retina in the optic nerve.

The isolated retina was processed according to the Carlson method [Carlson E.S. 1989. Fenestrated subendothelial basement membranes in human retinal capillaries. Invest. Ophthalmol. Vis Sci. 30 (9): 1923-1932]. The entire retina was placed in 10-15 ml of 10 mM EDTA in DMEM for 20 hours at 4 ° C. Then the retina was treated with 3% Triton X-100 solution for 2 hours at 4 ° C, washed several (3-4) times in distilled water. Then it was successively placed in a 0.025% solution of DNase I in a 1 M NaCl solution for 1 hour at 4 ° C, then in 4% sodium deoxycholate for 1-2 hours at a temperature of 22 ° C and then placed several (3-5) times in distilled water in order to remove detergent and cell debris.

All solutions were pre-sterilized through a 0.22 μm filter.

The retina was monitored using an Olympus SKX31 inverted microscope (Japan), and photographing with an Olympus DP70 camera (Japan).

After sequential treatment with detergents and DNase, the retina was reduced in size and, when examined using phase contrast microscopy, it was a cell-free framework consisting of extracellular matrix fibers, components of the inner boundary membrane of the retina (collagen fibrils and basement membranes of Mueller cells) and basement membranes of capillaries.

The cell-free retina framework, after washing in distilled water to remove detergent and cell debris, was placed in antibiotic culture medium DMEM / F12 at a temperature of 4-8 ° C for 2-20 days until fixation with 4% paraformaldehyde or colonization with cultured cells.

The fixed acellular skeleton of the retina was washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96 and 100% alcohol, embedded in paraffin, made histological sections, which were mounted on glass slides and stained with hematoxylin and eosin to control the adequacy of devitalizing treatment using light and / or luminescent microscopy.

Getting native RPE.

To obtain a native RPE, the eyeball of the eyeball after filling the neural retina was filled with cold Hanks solution with EDTA and after 15-30 minutes the process of collecting cells of the native RPE was started. Throughout the procedure, the eye cups were on ice in a Hanks solution. Controlling the process under a binocular magnifier, 200 μl of the tip was carried out along the layer and at the same time the cells separated from the Bruch membrane were aspirated.

A part of RPE cells was transferred into sterile tubes, the supernatant was removed and filled with TRI®-Reagent lysis solution (Sigma, USA) at the rate of 1 ml of TRI®-Reagent solution per 50-100 mg of tissue, pipetted to a homogeneous solution, and the lysate was incubated for 5 min at a temperature of 22 ° C, and then transferred to sterile tubes and stored at a temperature of minus 20 ° C until molecular analysis of total RNA isolation and cDNA synthesis was performed (PCR assessment).

Another part of the RPE cells was sent to obtain cell cultures.

Obtaining cell cultures of RPE.

For cultivation, RPE cells were transferred to a sterile medium to isolate the following composition: DMEM / F12 medium (1: 1) supplemented with 15% fetal calf serum, 2 mM L-glutamine and antibiotics penicillin-streptomycin at standard concentration, and centrifuged for 5 -7 min at 600-800 rpm Next, RPE cells were resuspended in a complete culture medium, of the following composition: DMEM / F12 nutrient medium (1: 1), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% essential amino acid solution, 1% neural solution N2 additives (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and antibiotics penicillin-streptomycin in standard concentration, then, after soft pipetting, they were evenly distributed in 25 cm ² uncoated culture bottles (Greiner, Germany) and cultivated at 37 ° C and 5% CO ₂ . After 5-7 days, the first change of medium was carried out and continued to cultivate with a change of medium after 2-3 days until the cells reached a confluent monolayer and the formation of the primary culture.

Next, confluent primary cell cultures were passaged using trypsin - versene solution (1: 3). Cells were cultured in a density of 1 - 2 × 10 ⁴ cells / cm ² in a complete culture medium, the following composition: DMEM / F12 medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% - replaceable amino acids solution, 1% solution of N2 neural supplement (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and antibiotics penicillin-streptomycin in standard concentration. Cultivation was carried out in vials and in 2 or 4-well slide chambers for cell cultures (BD Falcon ™, BD Biosciences, USA). The slide chamber was a slide for cell growth, to which a plastic separation grid was attached, forming a chamber with a certain number of holes (there are 1-, 2-, 4- and 8-well slide chambers), covered with a lid on top to maintain sterile conditions.

RPE cells cultured on slide chambers were fixed after 2–3 days when they reached the subconfluent layer with chilled acetone. Next, slides with cells were freed from dividing lattices, dried at a temperature of 20 ° C, wrapped in foil and stored in a low-temperature refrigerator at a temperature of minus 70-80 ° C until an IHC for differentiating the resulting cultures was performed.

RPE cells cultured in culture vials grew as adhesive monolayer cultures. RPE cell cultures were monitored using an Olympus SKX31 inverted microscope (Japan).

From one culture vial, cell cultures of RPE 1-5 of the passage committed in the neural direction were removed from the culture surface with trypsin-versene solution (1: 3) and used as a suspension to colonize the cell-free retinal framework in a density of 4 - 5.5 × 10 ⁴ cells / cm ² .

From other culture flasks, cells of the primary culture and RPE cell culture of passage 1-5 were used for PCR assessment of cell differentiation. For this, the cell monolayer was washed once with DPBS (PanEco, Russia), filled with TRI®-Reagent lysis solution (Sigma, USA) at the rate of 1 ml per 25 cm ^{2 of the} culture bottle, the cell lysate was pipetted to a homogeneous transparent state of the solution, the homogenate was incubated for 5 min at a temperature of 22 ° C, and then transferred to sterile tubes and stored at a temperature of minus 20 ° C until total RNA was isolated and cDNA synthesized (PCR for differentiation of cultured RPE cells differentiation).

IHC and PCR assessment of differentiation (committing in the neural direction) of cultured RPE cells was carried out as follows.

IHC assessment of differentiation of cultured RPE cells.

For IHC assessment of differentiation (committing in the neural direction) of cells in primary and passaged cultures, immunoperoxidase and immunofluorescence staining methods were used using primary murine monoclonal and rabbit polyclonal antibodies, Pax6, Sox2, vimentin, Nestin, β tubulin III, ninoxyl hydroxyl, ninoxyl, nFOS, hydroxyl 68 and 200 kDa, GFAP, CNPase, Iba, N-cadherin, Connexin-43, RPE65, CRALBP, Recoverin, PanCK, Macrophage antigen, Fibronectin, Factor Von Willebrand, Ki67, as well as EnVision ™ polymer conjugated with horseradish peroxidase (Dako, Denmark), or secondary antibodies: SFX kit with goat anti-mouse antibodies labeled with Alexa Fluor® 488 (Molecular Probes, USA), rabbit anti-rabbit antibodies conjugated with Texas Red fluorochrome (Jackson ImmunoResearch, USA), diluted in 0.1% bovine serum albumin (BSA, Sigma, USA).

Table 1 shows the characteristics of the antibodies used.

Slides with RPE cells fixed on them were removed from the low-temperature refrigerator, freed from foil and, after condensate evaporation, placed in 0.01 M phosphate-buffered saline (PBS, pH 7.4) (BioloT LLC, Russia) for washing from acetone.

Immunoperoxidase staining of the preparations was carried out according to the following scheme: blocking endogenous peroxidase with Dual Endogenous Enzyme Block from the EnVision ™ kit (Dako, Denmark) for 10 min, blocking non-specific binding to 1% BSA solution (Sigma, USA) for 20 min, incubation with primary antibodies for 17 hours at 4 ° C, incubation with a polymer from the EnVision ™ kit conjugated to horseradish peroxidase for 15 minutes, incubation with a DAB solution for 5-10 minutes. Further, the preparations were stained with hematoxylin, covered with an aqueous non-fluorescent medium (Shandon Immu-Mount ™, Thermo Electron Corporation, USA) and enclosed under a coverslip.

An immunofluorescence study of the preparations included: blocking nonspecific binding with Image-iT ™ FX signal enhancer from the SFX kit (Molecular Probes, USA) for 30 min at 22 ° C, incubation with primary antibodies for 17 hours at 4 ° C, binding of primary antibodies to the corresponding secondary fluorochrome-labeled antibodies for 1 hour at 22 ° C, coating with DAPI nuclear dye medium (Vectashield®, Vector Laboratories, USA) or staining with a Hoechst 33342 nuclear luminescent dye at a concentration uu 5 ug / mL (Sigma, USA) followed by coating with glycerol conclusion preparations under the coverslip.

IHC assessment and photographing was performed using an Olympus ANZ light microscope (Japan).

PCR assessment of differentiation of cultured RPE cells.

For molecular analysis (PCR assessment), total RNA was isolated according to the manufacturer's instructions TRT®-Reagent (Sigma, USA), cDNA synthesis was performed using RevertAid H Minus Kit (Fermentas) using 1 μg of total RNA treated with DNase (Fermentas). For PCR assessment, the expression level of genes encoding transcription factors Oct4 (POU5F1), Nanog, Pax6, and differentiation markers Musashi 1, β tubulin III, RPE65 were determined.

Table 2 presents the characteristics of gene function and the sequence of primers for PCR evaluation.

Amplification conditions: temperature 94 ° C - 45 s, temperature 56 ° C - 45 s, temperature 72 ° C - 1 s, 30-40 cycles. The cDNA libraries were normalized to a large human ribosomal protein with a molecular weight of 19 kDa (RPL19). The level of gene expression was evaluated by the intensity of the luminescence bands obtained by electrophoretic separation of PCR products in a 1% agarose gel on a gel analyzer (BIO-RAD ™ XR, USA).

Colonization of the acellular cell structure of the retina of an adult with cultured RPE cells was carried out as follows.

The cell-free retinal framework was washed several (5-7) times in DMEM / F12 nutrient medium (1: 1) with antibiotics penicillin-streptomycin in standard concentration, transferred to a sterile 1-well slide chamber (BD Falcon ™, BD Biosciences, USA) , filled with 2 ml of one of the following two compositions of the complete culture medium: DMEM / F12 nutrient medium (1: 1), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% solution of essential amino acids, 1% solution neural supplement N2 (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and ntibiotikami penicillin-streptomycin in a standard concentration or culture medium DMEM / F12 (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine and antibiotics penicillin-streptomycin in a standard concentration, and placed in a CO ₂ incubator having a temperature 37 ° C, 5% CO ₂ in air, to balance the pH of the medium.

On a cell-free framework of the human retina, a suspension of cultured RPE cells, both autologous and allogeneic, grown in monolayer cultures, was planted by diffusion deposition. RPE cells were planted in a density of 4 - 5.5 × 10 ⁴ cells / cm ² in 2-3 ml of a complete culture medium of the same composition in which there was a cell-free framework. Cells were cultured in the presence of a cell-free scaffold at a temperature of 37 ° C and 5% CO ₂ , with a change of medium 2-3 days before the cells colonized the cell-free scaffold of the retina and formed a monolayer of cells on its surface to obtain a tissue structure of the retina of the eye, which was sent to study localization cultured cells and on IHC an assessment of the differentiation potential of cultured cells in the acellular retinal framework.

The behavior of RPE cell cultures on the acellular retina was monitored using an Olympus SKX31 inverted microscope (Japan).

When determining the monolayer of RPE cells on the surface of the acellular skeleton of the retina, the nuclei of the latter were stained with vital Hoechst 33342 dye for luminescent microscopy.

The retina frameworks colonized by cultured human RPE cells were fixed after 12-25 days with 4% paraformaldehyde. The fixed preparations were washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96, and 100% alcohol and poured into paraffin.

From paraffin embedded blocks, histological sections 5 μm thick were made on a microtome, which were mounted on slides coated with adhesive. The study of the localization of cultured RPE cells in a cell-free retinal framework was performed on serial paraffin sections stained with hematoxylin and eosin using an Olympus AN3 light microscope (Japan) for light microscopy.

IHC assessment of the differentiation potential of cultured RPE cells in a cell-free retinal framework on serial paraffin sections was performed by immunoperoxidase staining with LSAP kit reagents (Dako, Denmark) using ready-to-use mouse monoclonal antibodies to CD31, CD34, tyrosinase, CK7, CK8, CK18, SK19, vimentinu, GFAP, S100 and NSE (Dako, Denmark), according to the following scheme: heat treatment of dewaxed sections in a microwave oven (power 650-750 W) for 5 min with appropriate solutions for antigen recovery (Dako, Dani ), blocking endogenous peroxidase with a solution from the LSAP kit for 10 min, blocking nonspecific binding with a 1% BSA solution for 20 min, incubation with primary antibodies for 17 hours at 4 ° C, incubation with secondary biotinylated antibodies from the LSAP kit for 20 minutes, incubation with streptavidin conjugated with horseradish peroxidase from the LSAP kit for 15 minutes, incubation with DAB solution for 5-10 minutes. Further, the preparations were stained with hematoxylin, covered with an aqueous medium (Faramount, Dako, Denmark) and placed under a coverslip.

The invention is illustrated by the following examples.

Example 1

As a control, in assessing the adequacy of devitalizing treatments of other retinas, a retina without devitalizing treatment was used, which was obtained as follows. The eyeball was freed from the surrounding tissues and washed once in 70% alcohol and 3 times in a cold Hanks solution with antibiotics in standard concentration - penicillin 100 U / ml and streptomycin 100 μg / ml. The anterior segment of the eyeball was removed in a circular incision behind the dentate line, which is well defined under normal lighting, as the boundary between the dark and light areas. The vitreous and the neural retina were separated from the RPE and isolated by trimming the neural retina in the optic nerve. After extraction, the whole retina was placed in 4% paraformaldehyde for fixation. The fixed retina was washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96, and 100% alcohol and embedded in paraffin. Histological sections 5 μm thick were made on a microtome from a paraffin-embedded block, which were mounted on glass slides and stained with hematoxylin and eosin (Fig. 2A, B).

The cell-free retinal framework was obtained by devitalizing the enucleated eyeball during autopsy as follows.

Adult eyeballs (female, 50 years old) were freed from surrounding tissues and washed once in 70% alcohol and 3 times in cold Hanks solution with antibiotics penicillin-streptomycin in standard concentration. The anterior segment of the eye was removed in a circular incision behind the dentate line. The vitreous and the neural retina were separated from the RPE and isolated by trimming the neural retina in the optic nerve.

The retina from one eyeball after extraction was completely placed in 10 mM EDTA (in DMEM medium) with a volume of 15 ml for 20 hours at a temperature of 4 ° C. Then the retina was treated with 3% Triton X-100 solution for 2 hours at 4 ° C, washed 3 times with distilled water. Then it was sequentially placed in a 0.025% solution of DNase I in a 1 M NaCl solution for 1 hour at 4 ° C, then transferred to 4% sodium deoxycholate for 1 hour at 22 ° C and washed 3 times with distilled water to remove detergent and cell debris. All solutions were pre-sterilized through a 0.22 μm filter. After such sequential processing, the retina was examined in phase contrast using an Olympus SKX31 inverted microscope (Japan) (Fig. 3A, B). Then it was stored for 20 days at 4 ° C in DMEM / F12 medium with antibiotics penicillin-streptomycin in standard concentration until fixation with 4% paraformaldehyde. The fixed retina was washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96, and 100% alcohol and embedded in paraffin.

Histological sections 5 μm thick were made on a microtome from a paraffin-embedded block, which were mounted on glass slides and stained with hematoxylin and eosin (Fig. 4B, C, D).

This retina was used to control the adequacy of devitalizing processing and the possibility of long-term storage. Microscopic examination of histological sections showed that after sequential treatment with detergents and DNase, the retina was a cell-free framework consisting of extracellular matrix fibers of the inner retinal border membrane and vitreous fibrils woven into it, which indicates the adequacy of devitalizing processing. The absence of microbial contamination of the culture medium during long-term storage of the cell-free retinal framework, indicating the preservation of the sterility of the biomaterial, allows it to be used after a long period of waiting necessary to increase cell mass.

The retina from the second eyeball after extraction was completely placed in 10 mM EDTA (in DMEM medium) with a volume of 10 ml for 20 hours at a temperature of 4 ° C. Then the retina was treated with 3% Triton X-100 solution for 2 hours at 4 ° C, washed 3 times with distilled water. Then it was sequentially placed in a 0.025% solution of DNase I in 1 M NaCl solution for 1 hour at 4 ° C, then transferred to 4% sodium deoxycholate for 1 hour at 22 ° C and washed 3 times with distilled water to remove detergent and cell debris. After such sequential treatment with detergents and DNase, the retina was stored for 20 days at 4 ° C in DMEM / F12 medium with antibiotics penicillin-streptomycin in standard concentration until it was colonized with cultured RPE cells.

After extraction of the neural retinas, two eye cups were filled with cold Hanks solution with EDTA, and after 15 min, the process of collecting cells of the native RPE was started. Throughout the procedure, the eye cups were on ice in a Hanks solution. Controlling the process under a binocular magnifier, 200 μl of the tip was carried out along the layer and at the same time the cells separated from the Bruch membrane were aspirated.

Native RPE cells collected from one eyeball were transferred into sterile tubes, the supernatant was removed and filled with TRI ^® -Reagent lysis solution (Sigma, USA) at the rate of 1 ml of TRI ^® -Reagent solution per 100 mg of tissue, pipetted to a homogeneous solution the cell lysate was incubated for 5 min at a temperature of 22 ° C, transferred to sterile tubes and stored at a temperature of minus 20 ° C until total RNA was isolated and cDNA was synthesized (PCR assessment).

In the cells of the native RPE during PCR assessment revealed expression at the major level of the RPE65 gene, which is a marker of RPE cells (Fig.7). The protein product of the gene is involved in the phototransduction cycle in the interaction of RPEs with photoreceptors. In addition, in the native RPE, expression of the Oct4 pluripoten gene (POU5F1) and neural stem cell markers Musashi 1 and Pax6 at a minor level were observed, which suggests the presence of a small population of cells with the properties of neural stem-progenitor cells in the native tissue.

Native RPE cells collected from the second eyeball were transferred to a sterile medium to isolate the following composition: DMEM / F12 medium (1: 1) supplemented with 15% fetal calf serum, 2 mM L-glutamine and standard concentrations of penicillin-streptomycin antibiotics and centrifuged for 5 minutes at 800 rpm. RPE cells were resuspended in a complete culture medium of the following composition: DMEM / F12 culture medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% essential amino acid solution, 1% neural supplement solution N2 (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and antibiotics of penicillin-streptomycin in standard concentration, then, after soft pipetting, they were evenly distributed in sterile plastic bottles with an area of 25 cm ² without coating (Greiner, Germany) and cultivated temperature of 37 ° C and 5% CO _2. After 5-7 days, the first change of medium was carried out and continued to cultivate with a change of medium after 2-3 days until the cells reached a confluent monolayer and the formation of the primary culture.

The confluent primary culture was passaged using trypsin-versene solution (1: 3). Cells were cultured in a density of 1 - 2 × 10 ⁴ cells / cm ² in a complete culture medium, the following composition: DMEM / F12 medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% - with a replaceable amino acid solution, a 1% solution of N2 neural supplement (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and penicillin-streptomycin antibiotics in standard concentration. Cultivation was carried out in vials and in 2- or 4-well slide chambers for cell cultures (BD Falcon ™, BD Biosciences, USA). RPE cells on slide chambers after 2-3 days when they reached the subconfluent layer were fixed with chilled acetone. Next, the glass slides with cells were freed from dividing lattices, the glass slides were dried at 20 ° C, wrapped in foil and stored in a low-temperature refrigerator at a temperature of minus 70-80 ° C until an IHC of the obtained cultures was evaluated.

Cell cultures during IHC assessment were positive for proneural (Pax6), neural (Nestin (Fig.5A), β tubulin III (Fig.5B), nNOS (Fig.6A), TH, NF 68 and 200kDa (Fig.5B), N-cadherin, Recoverin) and glial (vimentin, GFAP (Fig.6B), CNPase (Fig.6B)) markers, as well as a marker of PanCK epithelial cells. IHC assessment and photographing was performed using an Olympus AN3 light microscope (Japan).

RPE cells in vials grew in the form of adhesive monolayer cultures. RPE cell cultures were monitored using an Olympus SKX31 inverted microscope (Japan).

From one culture bottle, cells of the primary culture and RPE cell culture of passage 1-5 were used for PCR assessment. For this, the cell monolayer was washed once with DPBS (PanEco, Russia), filled with TRI ^® -Reagent lysis solution (Sigma, USA) at the rate of 1 ml per 25 cm ^{2 of the} culture bottle, the cell lysate was pipetted to a homogeneous transparent state of the solution, the homogenate was incubated for 5 min at a temperature of 22 ° C, and then transferred to sterile tubes and stored at a temperature of minus 20 ° C until total RNA was isolated and cDNA was synthesized.

PCR assessment of cultured adult RPE cells compared to native RPE showed a sharp drop in the expression level of the RPE65 gene (Fig. 7), a marker of RPE cells involved in the phototransduction cycle when they interact with photoreceptors, which indicates the beginning of dedifferentiation. In the cells of primary and passivated cultures, expression of stem marker-progenitor cell marker genes was simultaneously detected: Oct4 (POU5F1), Nanog, Musashi 1 and Pax6.

The presence of expression of the neural markers β tubulin III, Pax6, and Musashi 1 indicated the neural potential of RPE cell cultures.

Thus, the results of the IHC assessment and molecular genetic analysis showed that under the conditions of RPE cell cultivation, the eyes of an adult undergo strong changes, they are capable of dedifferentiation and development along the neural pathway.

From another culture vial, cell cultures of RPE of the 2nd passage (20 days of cultivation), switched in the neutral direction, were removed from the culture surface with trypsin-versene solution (1: 3) and in the form of a suspension in a density of 5.5 × 10 ⁴ cells. / cm ^{2 was} used to colonize an autologous cell-free retinal framework stored for 20 days at 4 ° C in DMEM / F12 medium.

Before applying the cultured RPE cells, the cell-free retina was examined under an inverted microscope for microbial contamination. The absence of microbial contamination of the culture medium during long-term storage of the cell-free retinal skeleton, which indicates the preservation of the sterility of the biomaterial, made it possible to use it after a long waiting period necessary to build up autologous cell mass. The cell-free retina was washed 5 times in DMEM / F12 nutrient medium (1: 1) with antibiotics penicillin-streptomycin in standard concentration and transferred to a sterile 1-well slide chamber (BD Falcon ™, BD Biosciences, USA), filled with 2 ml full culture medium consisting of DMEM / F12 medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% essential amino acid solution, 1% N2 neural supplement solution (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (OFF) and penicillin-streptomycin antibiotics in standard concentrations, and placed in a CO ₂ incubator having a temperature of 37 ° C, 5% CO ₂ in air for balancing pH environment.

A suspension of autologous RPE cells of the 2nd passage in the amount of 470 thousand (density 5.5 × 10 ⁴ cells / cm ² ) in 3 ml of a complete culture medium of the same composition as the cell-free framework was planted on the acellular skeleton of the retina. Cells were cultured at a temperature of 37 ° C and 5% CO ₂ , with a change of medium after 2-3 days.

On the 2nd day, when studying with a phase contrast microscope, the cells were divided into 2 subpopulations: some cells were spread on the surface of the slide chamber, while others were attached to the surface of the cell-free matrix, but remained rounded.

On the 7th day, on the surface of the acellular skeleton of the retina, clusters of cells of a rounded shape were determined (Fig. 8A; Fig. 9B), as well as rounded formations interconnected by elongated cells (Fig. 8B; Fig. 9A).

On the 12th day, a monolayer was determined on the surface of the acellular skeleton of the retina (Fig. 10), the nuclei of which were stained with the vital dye Hoechst 33342 (Fig. 11).

Fixation of a cell-free retinal framework colonized by cultured cells with 4% paraformaldehyde was carried out after 12 days. The fixed preparations were washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96, and 100% alcohol and poured into paraffin.

From paraffin embedded blocks, histological sections 5 μm thick were made on a microtome, which were mounted on slides coated with adhesive.

The study of the localization of cultured RPE cells in the cell-free retina was performed on serial paraffin sections stained with hematoxylin and eosin using an Olympus AN3 light microscope (Japan). RPE cells have populated the entire inner border membrane of the retina bordering the posterior hyaloid membrane of the vitreous. At the same time, they formed both a monolayer of a cuboid shape of cells closely adjacent to each other (Fig. 13, Fig. 14) and a layer of cells of a flattened form (Fig. 13; Fig. 15).

In addition, RPE cells formed round-shaped RPE conglomerates both on the surface of the acellular matrix of the retina and on the surface of the vitreous (in the absence of the retina) (Fig. 12A, B, Fig. 16).

The IHC assessment of the differentiation of cultured RPE cells that populated the acellular retina was carried out by immunoperoxidase using ready-to-use murine monoclonal antibodies to CD31, CD34, tyrosinase, CK7, CK8, CK18, CK19, vimentin, GFAP, S100 and NSE (Dako, Denmark).

RPE cells that populated the retina were negative for endothelial cell markers (CD31 (Fig. 18A, B), CD34 (Fig. 19A, B)) and a melanocyte marker (tyrosinase (Fig. 20A, B)). Positive staining was noted on markers differentiated (CK8 (Fig. 23A, B) and CK18 (Fig. 24A, B, C, D)) and cultured (CK (Fig. 21A, B) and CK19 (Fig. 22A, B)) RPE cells, as well as glial cells (GFAP (Fig. 28A, B, C, D, D, E), S100 (Fig. 26A, B), vimentin (Fig. 25A, B)) and neuronal (NSE) (Fig. .27A, B) markers. GFAP-positive staining was determined as a network in rounded formations (FIG. 28B, E).

Negative staining for endothelial cell markers (CD31 and CD34) indicates both the absence of newly formed vessels and the adequacy of devitalizing treatment (death of retinal vascular endothelial cells after sequential treatment with detergents and DNase).

Negative staining for tyrosinase indicates the absence of cells that synthesize melanin, including melanocytes. These data once again confirm the data obtained by IHC analysis of cultured RPE cells about the absence of contamination of RPE cell cultures by cells from the underlying choroid (fibroblasts, melanocytes, endothelial cells and pericytes).

In the native RPE, normal for all mammalian species, including humans, the main intermediate filaments are cytokeratins CK8 and CK18, markers of the simple epithelium, with CK18 being the dominant type. Markers of ductal epithelium CK7 and CK19 are not found in human RPE cells. This specificity makes SK18 an effective marker for identifying RPE cells.

In the culture, RPE cells express SK 7 and SK19, and the expression of SK8 and SK18 either does not change or disappears.

As can be seen from the nature of staining on the cytokeratins of RPE cells of an adult who populated a cell-free retinal framework, SK18 is the dominant type. In the same places where SK18 is localized, SK8, SK7 and SK19 are found in decreasing order. CK19 is expressed in a small number of cells. The presence of cytokeratin-positive staining in all cell formations found in the cell-free matrix indicates the receipt of cell cultures from RPE. The presence of CK18 and CK8 positive cells indicates the differentiation of cultured cells into mature RPE cells. In addition, given the existing evidence that CK19 is expressed in migrating RPE cells, as well as the fact that CK18 can be associated with cell migration, it can be assumed that the final differentiation did not occur and further development can be expected with a longer cultivation. The same idea is confirmed by IHC staining of the acellular retinal framework, colonized by adult RPE cells, on other markers.

In the native retina of an adult, GFAP, a marker of astrocytes and neural stem cells, and vimentin are expressed in Mueller cells and astrocytes. Whereas intracellular Ca ²⁺ -binding regulatory protein S100 is expressed in synapses between neurons (photoreceptor and bipolar cells) of an adult retina.

In vitro human RPE cells acquire pronounced staining for vimentin. Identification of the simultaneous expression of glial markers (vimentin, S100 and GFAP) in cultured RPE cells is associated with the differentiation of cells in the glial direction. Astroglial cells are the most numerous cells in the nervous tissue. They form a three-dimensional network, which is the supporting frame for neurons.

In a study of the development of astrocytes in the subventricular zone of the postnatal brain of an adult, it was shown that S100 is a marker of mature glial cells (astrocytes), which is long expressed after the end of GFAP expression. S100 is colocalized with intermediate GFAP filaments and vimentin and is involved in their phosphorylation.

NSE or γ (neuron-specific enolase, gamma enolase) is a glycolytic enzyme involved in glucose metabolism. The NSE enzyme is present in cells of neuroectodermal origin, including differentiated RPE cells, brain neurons, and peripheral nervous tissue. In cultured cells, RPE NSE disappears.

As can be seen from the example, the IHC staining of adult RPE cells that populated the cell-free retinal skeleton showed a reaction to cytokeratins (CK7, 8, 18, 19), neural (NSE) and glial (vimentina, S100 and GFAP) markers. In cells located on the surface of the inner boundary membrane, staining for cytokeratins is detected, which indicates their similarity to native RPE cells, however, the simultaneous detection of expression of NSE, vimentin and S100 with cytokeratins indicates incomplete cell differentiation. GFAP is not detected in these cells. Expression of this protein is observed in fibrous structures, as well as in cell formations collected in round conglomerates. This indicates that dedifferentiated human RPE cells under conditions of 3D cultivation on a devitalized retinal matrix are determined in the neural direction.

Table 3 presents data comparing the localization of IHC markers in the neural retina and in RPE cells in vivo and in vitro.

RPE cells populated the inner border membrane of the retina bordering the posterior hyaloid membrane of the vitreous body, where, firstly, they formed a monolayer of cuboid shape of cells closely adjacent to each other, according to morphology and IHC markers corresponding to differentiated RPE cells of the native retina. Secondly, RPE cells formed a layer of cells of a flattened shape, located more discharged, in shape resembling glial cells of the neural retina. Thirdly, RPE cells formed conglomerates that were found both on the surface of the acellular skeleton of the retina and on the surface of the vitreous (in places where there is no retina). In microscopic and IHC studies, these conglomerates were a matrix of the neural part of the retina populated by RPE cells.

As can be seen from the example, the cell-free retinal framework 20 days after devitalizing treatment was successfully colonized by autologous cultured RPE cells. The study also showed that cultured RPE cells of the 2nd passage have pronounced potencies, they can differentiate (return to the differentiated state of RPE) and transdifferentiate into cells of the neural retina.

Thus, the colonization of the acellular matrix of the retina by viable autologous cultured RPE cells, which showed specific migration and differentiation, indicates that the tissue structure of the human retina was modeled.

Example 2

Adult eyeballs (m, 63 years old) were freed from surrounding tissues and washed once in 70% alcohol and 3 times in a cold Hanks solution with antibiotics in standard concentration - penicillin 100 Units / ml and streptomycin 100 μg / ml. The anterior segment of the eyeballs was removed in a circular incision behind the dentate line, which is well defined under normal lighting, as the boundary between the dark and light areas. The vitreous and the neural retina were separated from the RPE and isolated by trimming the neural retina in the optic nerve.

After extraction, the retina of the eyeballs was completely placed in 10 mM EDTA (in DMEM) with a volume of 10 ml for 20 hours at a temperature of 4 ° C. Then the retina was treated with a 3% solution of Triton X-100 for 2 hours at a temperature of 4 ° C, washed 4 times with distilled water. Then they were successively placed in a 0.025% solution of DNase I in 1 M NaCl solution for 1 hour at 4 ° C, then transferred to 4% sodium deoxycholate for 1 hour at 22 ° C and washed 3 times with distilled water to remove detergent and cell debris. All solutions were pre-sterilized through a 0.22 μm filter. Then they were stored for 2 days at 4 ° C in DMEM / F12 medium with antibiotics penicillin-streptomycin in standard concentration until 4% paraformaldehyde was fixed in one retina and the second retina was colonized.

The fixed retina was washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96, and 100% alcohol and embedded in paraffin. Histological sections 5 μm thick were made on a microtome from a paraffin-embedded block, which were mounted on glass slides and stained with hematoxylin and eosin. This retina was used to control the adequacy of devitalizing processing (figa).

After processing, the retinas were examined in phase contrast using an Olympus SKX31 inverted microscope (Japan) and photographed.

As a study using phase contrast microscopy showed, the retinas after sequential processing were a cell-free framework consisting of capillary basement membranes, extracellular matrix fibers, components of the inner retinal border membrane (collagen fibrils and Muller cell basal membranes) and posterior hyaloid fibrils woven into it vitreous membranes, which indicates the adequacy of devitalizing treatment.

After extraction of the neural retinas, two eye cups were filled with cold Hanks solution with EDTA and after 30 min the process of collecting cells of native RPE was started. Throughout the procedure, the eye cups were on ice in a Hanks solution.

Controlling the process under a binocular magnifier, 200 μl of the tip was carried out along the layer and at the same time the cells separated from the Bruch membrane were aspirated.

Native RPE cells collected from one eyeball were transferred into sterile tubes, the supernatant was removed and filled with TRI ^® -Reagent lysis solution (Sigma, USA) at the rate of 1 ml of TRI ^® -Reagent solution per 50 mg of tissue, pipetted to a homogeneous solution the cell lysate was incubated for 7 min at a temperature of 22 ° C, transferred to sterile tubes and stored at a temperature of minus 20 ° C until a PCR assessment of cell differentiation was performed.

In the cells of the native RPE during PCR assessment, expression at the major level of the RPE65 gene, which is a marker of RPE cells, was detected. In addition, in the native RPE, expression of the Oct4 pluripoten gene (POU5F1) and markers of neural stem cells Musashi 1 and PaxG at a minor level were observed, which suggests the presence of a small population of cells with the properties of neural stem-progenitor cells in the native tissue.

Native RPE cells collected from the second eyeball were transferred to a sterile medium to isolate the following composition: DMEM / F12 medium (1: 1) supplemented with 15% fetal calf serum, 2 mM L-glutamine and standard concentrations of penicillin-streptomycin antibiotics and centrifuged for 7 minutes at 600 rpm. RPE cells were resuspended in a complete culture medium, of the following composition: DMEM / F12 medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% essential amino acid solution, 1% neural supplement solution N2 (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and antibiotics of penicillin-streptomycin in standard concentration, then, after soft pipetting, they were evenly distributed in sterile plastic bottles with an area of 25 cm ² without coating (Greiner, Germany) and cultivated temperature of 37 ° C and 5% CO _2. After 5-7 days, the first change of medium was carried out and continued to cultivate with a change of medium after 2-3 days until the cells reached a confluent monolayer and the formation of the primary culture.

Then, the confluent primary culture was passaged using trypsin - versene solution (1: 3). Cells were cultured in a density of 1 - 2 × 10 ⁴ cells / cm ² in a complete culture medium, the following composition: DMEM / F12 medium (1: 1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% - with a replaceable amino acid solution, a 1% solution of N2 neural supplement (Gibco, Invitrogen, USA), 20 ng / ml of the main fibroblast growth factor (RPF) and penicillin-streptomycin antibiotics in standard concentration. Cultivation was carried out in vials and in 2- or 4-well slide chambers for cell cultures (BD Falcon ™, BD Biosciences, USA).

RPE cells on slide chambers after 2-3 days when they reached the subconfluent layer were fixed with chilled acetone. Next, the glass slides with cells were freed from the separation gratings, the glass slides were dried at a temperature of 20 ° C, wrapped in foil and stored in a low-temperature refrigerator at a temperature of minus 70-80 ° C until an IHC of the obtained cultures was evaluated.

Cell cultures upon IHC assessment were positive for proneural (Pax6), neural (Nestin, β-tubulin III, nNOS, TH, NF 70 and 200kd, N-cadherin, Recoverin) and glial (vimentin, GFAP, CNPase) markers, as well PanCK epithelial cell marker. IHC assessment and photographing was performed using an Olympus AN3 light microscope (Japan). Since cells that can contaminate RPE cell cultures from the underlying choroid, such as fibroblasts, melanocytes, endothelial cells, and pericytes, are cytokeratin-negative, the presence of PanCK staining confirmed the receipt of cell cultures from RPE.

Staining for proneural, neural, and glial markers demonstrates that human RPE cells under culture conditions were committed in the neural direction.

RPE cells in vials grew in the form of adhesive monolayer cultures. RPE cell cultures were monitored using an Olympus SKX31 inverted microscope (Japan).

From culture bottles, cells of the primary culture and RPE cell culture of passage 1-5 were used for PCR assessment. For this, the cell monolayer was washed once with DPBS (PanEco, Russia), filled with TRI ^® -Reagent lysis solution (Sigma, USA) at the rate of 1 ml per 25 cm ^{2 of the} culture bottle, the cell lysate was pipetted to a homogeneous transparent state of the solution, the homogenate was incubated for 5 min at a temperature of 22 ° C, and then transferred to sterile tubes and stored at a temperature of minus 20 ° C until total RNA was isolated and cDNA was synthesized.

PCR assessment of cultured adult RPE cells compared to native RPE showed a sharp drop in the expression level of the RPE65 gene, a marker of RPE cells involved in the phototransduction cycle when they interact with photoreceptors, which indicates the onset of dedifferentiation. In the cells of primary and passivated cultures, expression of stem marker-progenitor cell marker genes was simultaneously detected: Oct4 (POU5F1), Nanog, Musashi 1 and Pax6. The presence of expression of the neural markers β tubulin III, Pax6, and Musashi 1 indicated the neural potential of RPE cell cultures.

The results of the IHC assessment and molecular genetic analysis showed that under the conditions of RPE cell cultivation, the eyes of an adult undergo strong changes, they are capable of dedifferentiation and development along the neural pathway, i.e. commits in the direction of retinal neural cells.

Next, the acellular skeleton of the retina, stored for 2 days at 4 ° C in DMEM / F12 medium, was colonized with a suspension of allogeneic RPE cells of the 5th passage at a density of 4 × 10 ⁴ cells / cm ² . The 5th passage RPE cell cultures obtained from an adult's eyeball (w, 50 years) were used as allogeneic RPE cells for colonizing the cell-free retinal framework. The method of obtaining native RPE cells, the method of obtaining the primary culture and cultures of RPE cells 1-5 of passage, committing RPE cells in the neural direction and removing them from the culture surface with trypsin-versene solution (1: 3) are shown in Example 1.

Before applying the cultured RPE cells, the cell-free retinal framework was washed 7 times in DMEM / F12 nutrient medium (1: 1) with antibiotics penicillin-streptomycin in standard concentration and transferred to a sterile 1-well slide chamber (BD Falcon ™, BD Biosciences, USA ), filled with 2 ml of a complete culture medium consisting of DMEM / F12 nutrient medium (1: 1), supplemented with 10% fetal calf serum, 2 mM L-glutamine and antibiotics, and placed in a CO ₂ incubator having a temperature of 37 ° C , 5% CO ₂ in air, to balance the pH of the medium.

A suspension of allogeneic RPE cells of the 5th passage in the amount of 350 thousand (density 4 × 10 ⁴ cells / cm ² ) in 2 ml of a complete culture medium of the same composition as the cell-free framework was planted on a cell-free framework of the human retina. Cells were cultured at a temperature of 37 ° C and 5% CO ₂ , with a change of medium after 2-3 days.

On the 2nd day, when studying with a phase-contrast microscope, the cells were divided into 2 subpopulations: some cells were spread on the surface of the culture plastic, while others were attached to the surface of the cell-free matrix, but remained round.

On the 14th day, round cells were determined on the surface of the acellular matrix.

Fixation of a cell-free retinal framework colonized by cultured cells with 4% paraformaldehyde was carried out after 25 days. The fixed preparations were washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96, and 100% alcohol and poured into paraffin.

From paraffin embedded blocks, histological sections 5 μm thick were made on a microtome, which were mounted on slides coated with adhesive.

The study of the localization of cultured RPE cells in the acellular retina was performed on serial paraffin sections stained with hematoxylin and eosin using an Olympus AN3 light microscope (Japan). RPE cells formed round conglomerates both on the surface of the acellular matrix of the retina and on the surface of the vitreous (in places where there is no retina).

The differentiation of cultured RPE cells that populated the acellular retina was assessed by immunoperoxidase staining using ready-to-use murine monoclonal antibodies to CD31, CD34, tyrosinase, CK7, CK8, CK18, CK19, vimentina, GFAP, S100 and NSE (Dako, Denmark).

RPE cells that populated the retina were negative for endothelial cell markers (CD31, CD34) and melanocyte marker (tyrosinase). Positive staining was observed on markers of differentiated (CK8 and CK18) and cultured (CK7 and CK19) RPE cells, as well as on glial (vimentin, S100 and GFAP) and neuronal (NSE) markers. GFAP-positive staining was determined as a network in rounded formations.

As can be seen from the example, the cell-free retinal framework was successfully colonized 2 days after devitalizing treatment by allogeneic cultured RPE cells. In the presence of the 3D matrix, allogeneic RPE cells of the 5th passage colonized the acellular retinal framework, forming round conglomerates both on the surface of the acellular matrix of the retina and on the surface of the vitreous (in places where there is no retina).

Cultured RPE cells immobilized on a carrier from a acellular matrix of the retina retain their viability, proliferative and migratory activity, and the given direction of histospecific differentiation for at least 25 days.

Thus, the colonization of the acellular skeleton of the retina by viable allogeneic cultured RPE cells, which showed specific migration and differentiation, indicates that the tissue structure of the human retina was modeled.

The present invention allows to create a method for simulating the tissue structure and / or reparative function of the retina by creating ex vivo a three-dimensional model that has no world analogues and consists of a cell-free human retina matrix that is biologically inert and does not impair the growth and differentiation of the cell culture of retinal pigment epithelium with specified quantity parameters , density and tissue differentiation.

The invention can be used to study the differentiation of cultured cells of the retinal pigment epithelium, to study the effect on the pigment epithelium when various factors are added to the culture medium, to simulate the processes occurring in the pigment epithelium with damage and pathological conditions of the retina, as well as to study the regenerative responses of retinal pigment epithelial cells .

The invention will allow us to determine the direction of further research, which in the future will help create a viable substitute for the human retina and tissue analogues of nerve tissue to replace affected retinal tissues, which can treat and / or prevent eye diseases, such as age-related macular degeneration, both wet and dry, glaucoma , diabetic retinopathy, including diabetic macular edema, choroidal neovascular membrane, uveitis, myopic degeneration, eye tumors, occlusion center retinal vein veins, iris redness, eye neovascularization, central serous retinopathy, eye surface diseases such as dry eye syndrome, central retinal artery occlusion, cystic macular edema and any other degenerative eye disease.

Table 1 Marker Manufacturer Breeding Characteristic one 2 3 four Primary antibodies Pax6 Abcam, ab5790 1: 100 Stem cell marker (early neuronal marker), incl. retina amacrine and progenitor cells Prox1 Abcam, ab11941 1: 200 In embryonic tissues, in the development of the central nervous system, postmitotic young undifferentiated neurons (including horizontal and amacrine retinal cells) Sox2 Millipore, Chemicon, MAB4343 1: 500 In the development of ESCs, a developing central nervous system (including amacrine neurons of the retina) Vimentin Chemicon 1: 4 Neural stem cell marker (progenitor cell marker), including Mueller cells and retinal astrocytes Nestin Abcam, ab6320 1: 100 Neural stem cell marker (including undifferentiated neural cells and Mueller cells) Millipore, MAB5326 1: 200 β tubulin III Abcam, ab7751 1: 500 Neuronal marker (all neurons, axons) nNOS Abcam, ab16650 1: 200 Neuronal marker (including amacrine and ganglion cells of the retina, neural processes of the retinal layers of the retina) Tyrosine hydroxylase (TH) Santa Cruz, sc-73152 1:50 Marker of differentiated neurons (including dopaminergic amacrine retinal cells) Abcam, ab75875 1: 200 NF 68 and 200kDa Abcam, abl5234 1: 100 Differentiated Neuron Marker Gfap Abcam, ab7260 1: 200 Marker of activated or cultured astrocytes (including Mueller cells and retinal astrocytes) Millipore, 04-1031 1: 400 CNPase Abcam, ab6319 1: 200 Oligodendrocyte marker, as well as the outer segments of the retinal photoreceptors Iba Abcam, ab15690 1: 200 Microglia marker N-cadherin Sigma, C3865 1: 200 Marker of cell contacts inherent in neural cells Connexin-43 (Cx43) Sigma, C6219 1: 200 Marker of slotted cell contacts characteristic of the epithelium RPE65 Abcam, ab13 826 1: 250 Retinal pigment epithelium marker CRALBP Abeam 1: 400 Retinal pigment epithelium marker Recoverin Millipore, AB5585 1: 500 Photoreceptor Marker

one 2 3 four Panck Sigma, C2931 1: 400 Epithelial cell marker Macrophage antigen Sigma, M1919 1: 200 Macrophage marker Fibronectin Abcam 1: 500 Extracellular Matrix Protein hSTROl Sigma 1: 100 Mesenchymal cell marker Factor Von Willebrand Sigma 1: 400 Endothelial cell marker Ki67 Santa Cruz, Sc-15402 1:50 Proliferating Cell Marker Secondary antibodies SFX with Anti-mouse Alexa Fluor® 488 Molecular probes 1: 300 Anti-mouse IgG antibodies conjugated with a green fluorescent label Anti-rabbit Texas Red Jackson ImmunoResearch 1: 100 Antibodies against rabbit IgG conjugated with a red fluorescent label

table 2 Gene Function The structure of the primers (sequence) Fragment Length, np Oct4 (POU5F1) Transcription factor, pluripotent status Project 5′atgtgtaagctgcggcccttg3 ′ Sample 5′gtttgaatgcatgggagagcc3 ′ 491 Nanog Transcription factor, pluripotent status Ex.5′gtgtggatccagcttgtccc3 ′ Sample 5′ctgcgtcacaccattgctattc3 ′ 497 Musashi 1 RNA-binding protein that regulates the self-renewal of neural stem cells Project 5′tgaaggagtgtctggtgatgcg3 ′ Sample 5′tgccggttggtggttttgtc3 ′ 310 Pax6 Transcription factor of neural differentiation Project 5′gtcatcaataaacagagttcttc3 ′ Sample 5′cgattagaaaaccatacctgtat3 ′ 424 β tubulin III Neuroblast Microtubule Protein Project 5′cagtgcggcaaccagatcgg3 ′ Sample 5′caggtcagcgttgagctggc3 ′ 720 RPE65 RPE cell protein controlling interaction with retinal photoreceptors Project 5′ctctttgaagttggatctgagcc3 ′ Sample 5′agggcattgtcagtaacctctac3 ′ 254 RPL19 Human ribosomal protein for normalization of cDNA libraries Project 5′agggtacagccaatgcccga3 ′ Sample 5′ccttggataaagtcttgatgatc3 ′ 326

Table 3 Marker Feature, Function Localization in the neural retina and RPE cells in vivo In vitro expression of RPE cells one 2 3 four Oct4 Transcription factor, marker of pluripotent cells There is no data Protein product in RPE cells in vitro not detected Nanog Transcription factor, marker of pluripotent cells There is no data Protein product in RPE cells in vitro not detected Sox2 Marker of embryonic stem and neural progenitor cells (Wang et al, 2006) In proliferating cells and in amacrine neurons of the retina of chickens and mice, absent in RPE In single cells of primary and passivated RPE cultures of an adult Prox1 Transcription factor, controls proliferation of progenitor cells. Marker expressed in the embryonic central nervous system and in postmitotic, but undifferentiated neurons of the retina Horizontal and amacrine retinal cells of the newt, not expressed in cells of native RPE In the cytoplasm of single cells of primary and passivated RPE cultures of an adult Pax6 Transcription factor, controls cell differentiation. Early neuronal marker and marker of neuroepithelium Triton retinal ganglion and amacrine cells, adult amacrine and progenitor cells, are not normally expressed in mature RPE cells In almost all cells of primary and passivated RPE cultures of an adult Nestin Neural SC marker Undifferentiated neural cells and Mueller cells. Not normally expressed in RPE cells In the cells of RPEs of adult rats, in almost all cells of primary and passivated cultures of RPEs of an adult β tubulin III Microtubules in cells perform a supporting function, and also participate in the movement of organelles within the cell, a marker of early neuroblasts Neurons of the normal human retina In RPE cells of adult rats, in RPE cells on day 5 of the primary culture, and in passaged RPE cells growing in a monolayer culture

one 2 3 four nNOS Ca ²⁺ -calmodulin-sensitive enzyme Amacrine and ganglion cells in the neural processes of the retinal layers of the chicken retina. Native RPE is not contained In D407 line RPE cells, in almost all cells in primary and passivated adult RPE cultures Th Tyrosine hydroxylase, a marker of differentiated dopaminergic neurons Dog dopaminergic amacrine cells In passaged adult RPE cells NF 68 and 200 kDa Cytoskeletal protein, intermediate filaments, marker of differentiated neurons Mammalian Neural Retinal Cells In single cells of primary and passivated RPE cultures of an adult Gfap Cytoskeleton protein, intermediate filaments, marker of astrocytes and neutral stem cells (Eng et al., 1971, 2000; Doetsch, 2003) Mueller cells and human astrocytes In the cells of RPEs of adult rats, in almost all cells of primary and passivated cultures of RPEs of an adult S100 Ca ²⁺ - dependently regulates many functions in cells, is expressed after the end of GFAP expression and characterizes the mature stage of differentiation, a marker of astrocytes and melanocytes In the synapses between neurons (photoreceptor and bipolar cells) of the retina, in the processes of glial cells In cultured rabbit RPE cells, human RPE cells Vimentin Cytoskeletal protein, intermediate filaments, mesenchymal cell marker Mueller cells and astrocytes in humans and dogs. RPE cells of birds, bulls and guinea pigs. In RPEs, amphibians (frogs, xenopuses) and mammals (mice, monkeys, rats, rabbits, fetal and adult humans) are not detected In cultured RPE cells of many species, including adults

one 2 3 four CNPase An enzyme associated with mitochondrial membranes can regulate the distribution of cytoplasmic microtubules (tubulin), a marker of mature oligodendrocytes Outer segments of the photoreceptors of the retina; no RPE data available In a small percentage of cells of primary and passivated RPE cultures of an adult Nse Glycolytic enzyme involved in glucose metabolism RPE cells In cultured cells disappears Recoverin Ca ²⁺ - dependently regulates rhodopsin phosphorylation, a photoreceptor marker Outer and inner segments, as well as axon endings of photoreceptor cells, bipolar and ganglion cells of the retina In primary and passivated cells of adult RPE CRALBP Cellular retinaldehyde-binding protein - metabolism of vitamin A and retinoids, a marker of Mueller cells and RPE cells Mueller cells and RPE cells Single cells in primary and passaged RPE cells of an adult Tyrosinase An oxidoreductase class enzyme involved in melanin synthesis, a marker of melanocytes Human RPE cells in the early stages of its embryonic development, in adult human RPE Express in response to stimulating factors Ck8 Cytoskeletal protein, intermediate filaments, marker of a simple epithelium, marker of differentiated RPE cells RPE cells Expression in cultured adult RPE cells does not change or disappears Ck18 Cytoskeletal protein, intermediate filaments, marker of a simple epithelium, marker of differentiated RPE cells RPE cells Expression in cultured adult RPE cells does not change or disappears

one 2 3 four SC7 Cytoskeleton protein, intermediate filaments, epithelial marker, RPE dedifferentiated cell marker Rabbit RPE cells absent in human RPE cells In cultured adult RPE cells (Alge et al., 2003; Sheridan et al., 2005). SC19 Cytoskeletal protein, intermediate filaments, epithelial marker, RPE migratory cell marker Not in RPE In cultured adult RPE cells N-cadherin Neural cell contacts None in RPE Intercellular contacts in RPE cells CD31 Endothelial cell marker None in the neural retina and RPE None in cultured RPE Cd34 Endothelial cell marker None in the neural retina and RPE None in cultured RPE Ki67 Proliferation marker At the periphery of the retina, RPE cells of adult rats are positive for Ki-67 High mitotic activity in the RPE cell monolayer

Claims (14)

1. The method of modeling the tissue structure of the human retina by colonizing the acellular skeleton of the retina with retinal pigment epithelium cells, the method is that the cell-free skeleton of the retina is obtained by devitalizing the retina from an autopsy eyeball by washing it in succession with an alcohol solution at a concentration of 70% and in a cold solution of Hanks with antibiotics, removal of the anterior segment of the eyeball along the dentate line, separation of the vitreous body and neutral retina from retinal pigment epithelium and retinal excision, cutting it in the optic nerve, the isolated retina for devitalizing treatment is placed in 10-15 ml of 10 mM EDTL in DMEM for 20 h at 4 ° C, then treated with 3% solution of Triton X-100 for 2 hours at 4 ° C and washed in distilled water, then the retina is sequentially placed in a 0.025% solution of DNase 1 in 1 M NaCl solution for 1 hour at 4 ° C and in 4 % sodium deoxycholate for 1-2 hours at a temperature of 22 ° C and then washed in distilled water to remove detergent and cell debris, the eye thicket is filled with cold Hanks solution with EDTA and after 15-30 minutes. cells of the native retinal pigment epithelium are collected, part of the collected cells are transferred to a TRI®-Reagent lysis solution at the rate of 1 ml of solution per 50-100 mg of tissue, pipetted to a homogeneous solution, and the lysate of the cells of the native retinal pigment epithelium is incubated for 5-7 minutes at 22 ° C, another part of the harvested cells is transferred to a sterile isolation medium consisting of DMEM / F12 nutrient medium (1: 1) supplemented with 15% fetal calf serum, 2 mM L-glutamine and antibiotics and centrifuged for 5-7 min at 600-800 rpm, then the cells are resuspended in a complete culture medium consisting of DMEM / F12 nutrient medium (1: 1), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% solution of essential amino acids, 1% solution of neural supplement N2, 20 ng / ml of the main growth factor for fibroblasts and antibiotics, and after pipetting, they are distributed in uncoated culture bottles and cultivated at 37 ° C and 5% CO ₂ , after 5-7 days the first shift is carried out medium and continue to cultivate with a change of medium after 2-3 days to reach the cells of the confluent monolayer and the formation of the primary culture, then the cells of the primary culture are passaged using trypsin-versene solution (1: 3), then the cells are placed in culture bottles and in slide chambers at a density of 1 - 2 · 10 ⁴ cells / cm ² and cultured, cells 1-5 of the passage cultured in the culture bottles in the form of adhesive monolayer cultures, committed in the neural direction, removed from the culture surface with trypsin-versene solution (1: 3) and in the form of a suspension of cultured retinal cells Igmental epithelium is directed to the colonization of the acellular retinal framework, wherein the acellular retinal framework is washed with antibiotics in a culture medium DMEM / F12 (1: 1) before colonization, transferred to a slide chamber and filled with a complete culture medium, and the acellular retinal framework is colonized by diffusion precipitation suspension cultured cells of the retinal pigment epithelium in the density of 4-5.5 x 10 ⁴ cells. / cm ² in complete culture medium of the same composition and cultured Nia at 37 ° C and 5% _CO2, with change of medium after 2-3 days before kolonizirovaniya cultured cells and cell-free frame retinal cells form a monolayer on its surface to obtain retinal tissue structures of the eye, which is sent to study the localization of cultured cells and in IHC assessment of the differentiation potential of cultured cells in the acellular retinal framework. 2. The method according to claim 1, characterized in that in order to study the localization of cultured cells, the nuclei of the cells are stained with Hoechst 33342 vital dye for luminescent microscopy. 3. The method according to claim 1, characterized in that in order to study the localization of cultured cells, the cell-free cell-free retinal framework is fixed with 4% paraformaldehyde after 12-25 days, washed in phosphate-buffered saline (PBS, pH 7.4), carried out according to alcohols with an ascending concentration of 70, 96 and 100% alcohol, poured into paraffin, made histological sections and stained with hematoxylin and eosin for light microscopy. 4. The method according to claim 1, characterized in that the IHC assessment of the differentiation potential of cultured cells in a cell-free retinal framework is carried out by immunoperoxidase staining using murine monoclonal antibodies to CD31, CD34, tyrosinase, CK7, CK8, CK18, CK19, vimentin, GFAP, SIOO and NSH. 5. The method according to claim 1, characterized in that they use antibiotics penicillin-streptomycin in a standard concentration - penicillin 100 Units / ml and streptomycin 100 μg / ml. 6. The method according to claim 1, characterized in that the extracellular skeleton of the retina after removal of detergent and cell debris is placed in a culture medium DMEM / F12 with antibiotics at a temperature of 4-8 ° C for 2-20 days before fixation 4 % paraformaldehyde or colonization with cultured cells. 7. The method according to claim 6, characterized in that the fixed acellular skeleton of the retina is washed in phosphate-buffered saline (PBS, pH 7.4), carried out with alcohols with an ascending concentration of 70, 96 and 100% alcohol, filled in and paraffin made histological sections that are mounted on slides and stained with hematoxylin and eosin to control the adequacy of the devitalizing treatment using light and / or luminescent microscopy. 8. The method according to claim 1, characterized in that after incubation, the lysate of the cells of the native Rinal pigment epithelium is stored at a temperature of minus 20 ° C until PCR assessment. 9. The method according to claim 1, characterized in that the cells of the primary culture and cultured cells 1-5 of the passage are sent for PCR assessment. 10. The method according to claim 9, characterized in that for the PCR assessment determine the level of expression of genes encoding transcription factors Oct4 (POU5F1), Nanog, Pax6 and differentiation markers Musashi 1, β tubulin III, RPE65 isolation of total RNA and cDNA synthesis, the level of gene expression is estimated but the intensity of the luminescence of the bands obtained by electrophoretic separation of PCR products in a 1% agarose gel on a gel analyzer. 11. The method according to claim 1, characterized in that the cultivation of cells in slide chambers is carried out in 1-, 2-, 4- or 8-well slide chambers, after 2-3 days, when the cells reach the subconfluent layer, they are fixed chilled acetone and sent for IHC assessment of the potential for differentiation of cells. 12. The method according to claim 11, characterized in that the IHC assesses the differentiation potential of cultured cells by immunoperoxidase and immunofluorescence staining using primary murine monoclonal and rabbit polyclonal antibodies, Pax6, Sox2, vimnthine, Nestin, β tubulin III, nNOS, tyrosine hydroxyl , NF 68 and 200 kDa, GFAP, CNPasc, Iba, N-cadherin, Connexin-43, RPE65, CRALBP, Rccoverin, PanCK, Macrophage antigen, Fibronectin, Factor Von Willcbrand, Ki67, as well as EnVision ™ polymer conjugated to horseradish peroxidase, (Dako, Denmark) or secondary antibodies: SFX kit with antibody s goat anti-mouse Alexa Fluor® 488-labeled, rabbit anti-immunoglobulin antibody, conjugated with fluorochrome Texas Red, diluted in 0.1% solution of bovine serum albumin. 13. The method according to claim 1, characterized in that the acellular retina frame transferred to the slide chamber is filled with a complete culture medium consisting of DMEM / F12 culture medium (1: 1), supplemented with 10% fetal calf serum, 2 mm L-Glutamine, a 1% solution of non-essential amino acids, 1% solution of neural supplement N2, 20 ng / ml of the main fibroblast growth factor and antibiotics. 14. The method according to claim 1, characterized in that the acellular retina frame transferred to the slide chamber is filled with a complete culture medium consisting of DMEM / F12 culture medium (1: 1) supplemented with 10% fetal calf serum, 2 mm L-glutamine and antibiotics and placed in a CO ₂ incubator having a temperature of 37 ° C, 5% CO ₂ in air to balance the pH of the medium.

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