Organoid bilayer three-dimensional tissue grafts for treating retinitis pigmentosa and methods of making the same

Abstract

The present application relates to the technical field of tissue engineering, and particularly relates to an organoid double-layer three-dimensional tissue graft for treating retinal pigment degeneration and a preparation method thereof.The organoid tissue graft of the technical scheme comprises a retinal precursor stem cell layer, a retinal pigment epithelial cell monolayer cell layer and an electrospun silk support layer arranged in sequence.The retinal precursor stem cell layer is formed by solidification of a low-temperature agarose cell suspension containing retinal precursor stem cells.The organoid tissue graft of the scheme can supplement a plurality of degenerative missing cells, can form interaction between transplanted cells and can maintain the activity of the transplanted cells.The three-dimensional retinal stereoscopic structure formed can guarantee correct nerve circuit and improve the visual function of the patient.The electrospun silk material provided by the scheme forms support for cells, and prevents deformation of the graft in the later period.The technical scheme can solve the technical problem that the treatment effect of the organoid tissue graft of the prior art on retinal pigment degeneration is not ideal, and has an ideal popularization and application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to an organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa and a preparation method thereof. Background Art

[0002] Retinitis Pigmentosa (RP) is an irreversible blinding eye disease, which is mainly manifested by the irreversible death of multiple neurons and retinal pigment epithelial (RPE) cells in the outer retina. There is no effective treatment yet. In recent years, the use of retinal organoid technology for in vitro tissue engineering to expand stem cells and perform transplantation therapy has become a potential treatment method. Organoid tissue grafts are a thin film-like tissue similar to the retina. Stem cells or stem cell tissue sheets transplanted into the subretinal space have good neural regeneration and integration capabilities, which are conducive to repairing the retina. However, there are many problems in the actual application of the above methods, such as: low survival rate of transplanted cells, unsatisfactory repair of retinal structure, etc., and the transplantation-related efficacy needs to be further improved. The main reasons for the above phenomenon include: photoreceptor cells and retinal pigment epithelial cells are severely damaged in the course of retinitis pigmentosa, and it is difficult to repair the key problem of the simultaneous loss of multiple cells (such as photoreceptor cells and retinal pigment epithelial cells) in the late stage of retinal degeneration by transplanting a single retinal pigment epithelial cell or photoreceptor cell. The retina is a multi-layered cell structure tissue with a precise three-dimensional structure ( Figure 1 ), if the general transplantation method is used, it will be difficult for the graft to form the above retinal structure. Previous studies have attempted to directly transplant single cell suspensions and retinal tissue slices (including fetal retina and organoid tissue slices), but the grafts are randomly accumulated in the subretinal space, or wrinkles gather to form rosettes, with a chaotic and irregular structure, which seriously affects the functional connection of neurons and the conduction of visual signals (see Figure 2 and Figure 3 ). It can be seen that after direct transplantation of cells or tissue sheets, the wrinkled and rosette structures formed in the subretinal space are very different from the neat interlayer structure of the retina, and cannot form regular neural connections with therapeutic significance.

[0003] Therefore, the key to effectively treating retinitis pigmentosa is to prepare organoid tissue grafts that meet application requirements. Retinitis pigmentosa is often caused by the combined damage of multiple cells. Traditional grafts are single-cell, single-layer structures that are difficult to simulate the three-dimensional structure of the retina. Existing technologies suffer from problems such as a single transplanted cell type, low survival rate, and unsatisfactory retinal structural repair, resulting in limited transplant efficacy. These problems urgently need to be further addressed. Summary of the Invention

[0004] The purpose of the present invention is to provide an organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa, so as to solve the technical problem that the prior art organoid tissue implant has unsatisfactory therapeutic effect on retinitis pigmentosa.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] An organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa comprises a retinal progenitor stem cell layer, a retinal pigment epithelial cell monolayer and an electrospinning support layer arranged in sequence.

[0007] Furthermore, the retinal progenitor stem cell layer is a low-temperature agar gel layer in which retinal progenitor stem cells are dispersed; and the solidification temperature of the low-temperature agar gel is 26-30°C.

[0008] Furthermore, the porosity of the electrospinning support layer is 30%-90%; the pore size is 0.1-15 μm; the thickness of the electrospinning support layer is 5-20 μm; the electrospinning diameter is 200-1000 nm, preferably, the electrospinning diameter is 500 nm.

[0009] This technical solution also provides a method for preparing a double-layer three-dimensional organoid tissue implant for treating retinitis pigmentosa, comprising the following steps in sequence:

[0010] S1: Prepare a cryogenic agarose cell suspension containing retinal progenitor stem cells;

[0011] S2: Preparation of a monolayer of retinal pigment epithelial cells;

[0012] S3: Inoculate the low-temperature agarose cell suspension onto a pre-cooled monolayer of retinal pigment epithelial cells to obtain organoid tissue explants.

[0013] Furthermore, in S1, the density of retinal progenitor stem cells was 1×10 5 -2×10 6 cells / ml, preferably 1×10 6 cells / ml;

[0014] Retinal progenitor stem cells are obtained by the following method:

[0015] Embryonic stem cells are induced and cultured into retinal organoids, and the neural retinal structures of the retinal organoids are obtained, followed by enzymatic hydrolysis and flow cytometry sorting to obtain retinal progenitor stem cells; the retinal progenitor stem cells include at least one of Crx-positive photoreceptor progenitors or other retinal progenitor cells;

[0016] Preferably, the retinal progenitor stem cells are Crx-positive photoreceptor progenitor cells;

[0017] Preferably, the retinal organoids are organoids obtained by culturing embryonic stem cells for 60 days;

[0018] More preferably, the embryonic stem cells are induced and cultured in a retinal organoid differentiation medium containing Y-27632 factor to form embryoid bodies; the embryoid bodies are induced and cultured in a retinal organoid differentiation medium containing BMP4, and then the medium is gradually replaced with a retinal organoid differentiation medium to form retinal organoids; next, the retinal organoids are cultured in a long-term culture medium until neural retinal structures appear; and the culture is continued for 60 days to obtain 60-day retinal organoids;

[0019] Retinal organoid differentiation medium was prepared by adding KOSR to a final concentration of 10%, Giutamax to a final concentration of 1%, lipid concentrate to a final concentration of 1%, monothioglycerol to a final concentration of 450 μM, and penicillin-streptomycin to a final concentration of 1% to the basal medium; the basal medium was prepared by mixing IMDM medium and Ham's F-12 medium in equal proportions.

[0020] The long-term culture medium was DMEM / F12-Glutamax medium containing 1% N2, 10% fetal bovine serum, 0.5 μM retinoic acid, 0.2 mM taurine, and 1% penicillin-streptomycin.

[0021] Further, in S1, in the cryogenic agarose cell suspension, the concentration of cryogenic agarose is 1-2%, preferably 1.5%;

[0022] The low-temperature agarose cell suspension was prepared using RPC cell culture medium; the RPC cell culture medium was a cell culture medium formed by adding 2% B27, 1% N2, 10 ng / ml bFGF, 2 mM glutamine, and 1% penicillin-streptomycin double antibody to DMEM / F12 basal culture medium.

[0023] Further, in S2, retinal pigment epithelial cells are seeded on the electrospun support layer and cultured using a retinal pigment epithelial cell differentiation medium to obtain a monolayer tissue sheet of retinal pigment epithelial cells;

[0024] Preferably, the seeding amount of retinal pigment epithelial cells is 1×10 5 cells / cm 2 The composition of the retinal pigment epithelial cell differentiation medium is: 77% koDMEM medium, 20% KOSR, 1% Glutamax-1, 1% MEM NEAA, and 1% 2-mercaptoethanol. The culture duration using the retinal pigment epithelial cell differentiation medium is 20 days.

[0025] Furthermore, in S3, 0.33 cm 2 30-50 μl of low-temperature agarose cell suspension is inoculated on a monolayer tissue piece of retinal pigment epithelial cells with a surface area of ​​​​the organoid tissue explant; the thickness of the organoid tissue explant is 50-100 μm.

[0026] This technical solution also provides an application of an organoid tissue implant in the preparation of materials for treating retinitis pigmentosa.

[0027] This technical solution also provides an application of low-temperature agarose in the preparation of organoid tissue implants for treating retinitis pigmentosa, wherein the low-temperature agarose is the raw material for the retinal progenitor stem cell layer of the organoid tissue implant; the retinal progenitor stem cell layer is formed by solidifying a low-temperature agarose cell suspension containing retinal progenitor stem cells.

[0028] The technical principle and beneficial effects of this technical solution are:

[0029] In this technical solution, we successfully constructed a three-dimensional biomimetic retinal tissue sheet composed of multiple layers of cells. This tissue sheet not only replenishes multiple cell types lost due to degeneration but also promotes interactions between transplanted cells, thereby maintaining their activity and enhancing functional integration. By mimicking the three-dimensional structure of the natural retina, this three-dimensional tissue sheet helps reconstruct correct neural circuits and significantly improve patients' visual function.

[0030] This invention represents the first attempt to integrate multiple different cell types into a single, integrated graft—an organoid tissue implant. Existing technologies lack solutions to the challenges of ensuring a stable and easily manipulated graft, ensuring effective fusion of cells after transplantation to form an ordered structure, preventing curling and deformation of the transplanted cells, and preventing post-transplant tissue pathology. Through a series of in-depth experiments, we discovered that using cryogenic agarose gel to encapsulate retinal progenitor stem cells allows for the creation of a stable, bilayered organoid tissue implant, facilitating subsequent transplantation.

[0031] When this cryogenic agarose gel, containing retinal progenitor stem cells and retinal pigment epithelial cells, is transplanted into the designated location, the gel gradually melts as the temperature rises, allowing the two cells to come into direct contact and interact, ultimately forming an ordered structure. This process ensures functional connections between neurons and the effective transmission of visual signals.

[0032] Furthermore, this solution innovatively incorporates electrospinning materials as a support matrix, significantly improving the morphological stability and structural integrity of the graft after implantation, effectively preventing deformation and further enhancing the tissue's regeneration and repair capabilities. Prior to selecting electrospinning materials, the inventors had attempted various traditional methods, including using conventional films and Transwell membranes, but none of these methods achieved the desired results.

[0033] Specifically, ordinary films (such as polyethylene) and Transwell membranes cannot provide an ideal growth environment for retinal pigment epithelial cells, causing the grafts to easily curl and deform, accompanied by a significant inflammatory response. In contrast, using electrospun materials as a support layer allows retinal pigment epithelial cells to grow in a more ideal state, with the grafts remaining flat and the inflammatory response being minimal, greatly promoting cell-to-cell interaction and functional integration.

[0034] It is worth noting that the thickness of the electrospun membrane has a significant impact on the effectiveness of organoid tissue implants. If the electrospun membrane is too thin, it is prone to breakage and curling during the transplantation process, affecting the success rate of the surgical operation; if the membrane thickness is too large, it may trigger a more severe inflammatory response in the surrounding tissue, hindering the blood oxygen supply to the choroid, and further leading to poor cell status of the retinal pigment epithelium and even cell-mesenchymal transition. Therefore, by precisely controlling the thickness of the electrospun membrane, we not only optimize the mechanical properties of the transplant, but also ensure its biocompatibility and functionality, providing a solid guarantee for achieving the best treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The multi-layered cell structure of the retina is demonstrated (from the literature: He XY, Zhao CJ, Xu H, Chen K, BianBS, Gong Y, Weng CH, Zeng YX, Fu Y, Liu Y, Yin ZQ. Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells. Stem Cell Reports. 2021 Jul 13; 16(7): 1805-1817.).

[0036] Figure 2The optical coherence tomography image shows that the transplantation of single cell suspension resulted in the disordered accumulation of the graft in the subretinal space (from the literature: Occelli LM, Marinho F, Singh RK, Binette F, NasonkinIO, Petersen-Jones SM. Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model. J Vis Exp. 2021Aug 5; (174): 10.3791 / 61683.).

[0037] Figure 3 The results show that wrinkles and rosette structures are formed in the subretinal space after direct transplantation of cells or tissue sheets (from the literature: Assawachananont J, Mandai M, Okamoto S, Yamada C, Eiraku M, Yonemura S, Sasai Y, Takahashi M. Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice. Stem Cell Reports. 2014 Apr 24; 2(5): 662-74.).

[0038] Figure 4 These are typical microscopic images of Crx-positive RPCs and hEROs labeled with GFP green fluorescence in Example 1.

[0039] Figure 5 This is an optical coherence tomography image of the double-layer cell complex tissue sheet of Example 1 after implantation into the subretinal space of an animal.

[0040] Figure 6 This is a typical HE-stained microscopic picture of the double-layer cell complex tissue sheet after transplantation in Example 1.

[0041] Figure 7 This is a laser confocal three-dimensional reconstruction image of the layered state of supporting cells after the low-temperature agarose solidified in Experimental Example 1.

[0042] Figure 8 These are immunofluorescence microscopic images of tissue sections after transplantation of a double-layer cell complex tissue sheet formed using different single-layer RPE cell supports in Experimental Example 2. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

[0044] Example 1: Organoid tissue implants and preparation methods

[0045] (1) Preparation of low-temperature agarose cell suspension

[0046] The gelation temperature of agarose obtained from natural raw materials is generally 35-40°C. At this temperature, most organisms or heat-sensitive reagents will be inactivated, so it is necessary to select agarose with a lower gelation temperature. In the process of preparing the explant, agarose with a lower gelation temperature should be used. The low-melting point agarose (Sigma-Aldrich, A2576, also known as low-temperature agarose) used in this protocol has a gel strength of 300g / cm at a concentration of 1.5%. 2 , gelling temperature is 20℃, the gelling point is low, which is suitable for application requirements.

[0047] Retinal progenitor stem cells (RPCs) were inoculated into RPC cell culture medium to obtain RPC suspension. The composition of RPC culture medium is as follows: B27 2%, N2 1%, bFGF 10ng / ml, glutamine 2mM, and penicillin-streptomycin 1% were added to DMEM / F12 basal culture medium. DMEM / F12 (D / F12) basal culture medium is a commonly used culture medium in the prior art, which is a mixture of DMEM and Ham's F-12 culture media in equal proportions. B27 and N2 are conventional supplements in the culture medium, used to support the growth of nerve cells. bFGF is basic fibroblast growth factor. Low-temperature agarose was dissolved in RPC culture medium to obtain low-temperature agarose solution. The single-cell RPC suspension was added to a centrifuge tube and centrifuged at 180g for 3 minutes. After the supernatant was removed, the cells were resuspended in low-temperature agarose solution to obtain a low-temperature agarose cell suspension. In the low-temperature agarose cell suspension, the final density of RPC cells was 1×10 5 cells / ml、5×10 5 cells / ml, 1×10 6 cells / ml, 2×10 6 cells / ml; the final content of low-temperature agarose was 1%, 1.5%, and 2%. The above process was completed at 37°C.

[0048] Among them, the source of retinal progenitor stem cells (RPC) is cells derived from 60-day retinal organoids. Embryonic stem cells are induced into retinal organoids through multi-step culture, which is a conventional method in the prior art. Embryonic stem cells are first cultured under the induction of Y-27632 factor to form embryoid bodies, and then the embryoid bodies are induced to form retinal organoids under the induction of BMP4. After the formed retinal organoids are enzymatically hydrolyzed by the conventional method of the prior art, RPC cells can be isolated and obtained. More specifically, the culture of 60-day retinal organoids (human embryonic stem cell derived retinal organoids, hEROs) and the method for obtaining RPC are as follows:

[0049] Human embryonic stem cells (H9-hESC, hESC) in good culture status were washed with PBS and digested into single cells with Tryple (containing 20 μM Y27632 and 0.05 mg / ml DNase I) in an incubator. Digestion was terminated with PBS and then centrifuged at 180 g for 2 minutes to collect the cell pellet. The cell pellet was resuspended in 1 ml of retinal organoid (RO) differentiation medium. RO differentiation medium, i.e. gfCDM(+) medium, is specifically: the basal medium is formed by mixing IMDM medium and Ham's F-12 medium in equal proportions, and then a serum-free substitute KOSR with a final concentration of 10%, a final concentration of 1% of Gibco 11905-031, a final concentration of 450 μM of monothioglycerol, and a final concentration of 1% of PS (penicillin-streptomycin double antibody) are added to the basal medium.

[0050] Then, the final cell viability of the cell suspension was counted (more than 90% active cells), the cell concentration was adjusted to 120,000 cells / ml, and a final concentration of 20 μM Y-27632 factor (Cas: 146986-50-7) was mixed. The mixed cell suspension was plated into a low-cell-adhesion V-shaped 96-well plate at 100 μL / well. The cells will aggregate into embryoid bodies (EBs). On the sixth day, the EBs were completely replaced with RO differentiation medium containing 1.5 nM BMP4 (bone morphogenetic protein 4). Thereafter, half of the RO induction medium without BMP4 (i.e., half of the medium was replaced with gfCDM(+)) every three days. On day 18, the formed hEROs were aspirated using a Pasteur pipette and transferred to a low-viscosity 10-cm diameter culture dish. The long-term culture medium (abbreviated as LTCM, specific components: 1% N2, 10% fetal bovine serum (FBS), 0.5 μM retinoic acid (Sigma), 0.2 mM taurine (Sigma), 1% PS, and the basic culture medium was DMEM / F12-Glutamax medium (Gibco)) was completely replaced every 3 days until the neural retina (NR) structure appeared (25-30 days).

[0051] hEROs were induced to differentiate for 60 days using long-term induction medium (LTCM) and infected with Crx-GFP-AAV virus (a conventional tool virus in the prior art) at a viral volume of 1 μL / RO. One week later, Crx-positive RPCs were clearly marked with GFP green fluorescence. Figure 4 Typical microscopic images of Crx-positive RPCs and hEROs labeled with GFP green fluorescence are shown. Under a stereomicroscope, NR structures were aseptically isolated using a 1ml syringe needle. The NR was transferred to a 15ml centrifuge tube, and the supernatant was discarded. The NR was digested with 1ml of Trypsin at 37°C for 25 minutes, then the digestion reaction was terminated (pipetting 2-3 times every 5-8 minutes using a Pasteur pipette). After centrifugation at 180g for 5 minutes, the supernatant was discarded, and the cells were resuspended and Crx-positive RPCs were isolated by fluorescence flow cytometry.

[0052] (2) Preparation of retinal pigment epithelial (RPE) monolayer tissue sheets (retinal pigment epithelial cell monolayer + electrospun support layer)

[0053] Customized biodegradable polylactic acid electrospun fiber membrane with better biocompatibility (R-JD-0229 from Xi'an Ruixi Biotechnology Co., Ltd.). The porosity of the electrospinning support layer is 30%-90%; the pore size is 0.1-15μm; the electrospinning diameter is 200-1000nm, preferably 500nm, and the thickness of the overall electrospinning membrane is about 5-20μm (preferably about 10μm). After the electrospinning membrane is cultured to form an RPE single cell layer, the total thickness of the cell-added implant is about 50-100μm. The electrospinning used in the prior art is usually silk protein / polycaprolactone / gelatin composite spinning, which is prone to problems such as irregular membrane fiber microstructure, uneven membrane strength, and poor transparency. Therefore, conventional electrospinning is not selected in this technical solution. The polylactic acid used in this technical solution is a new type of biodegradable material with good biocompatibility and degradability. The polymer polylactic acid material has a regular microstructure, and its mechanical and physical properties are superior to those of mixed materials. It also has good gloss and transparency, which is conducive to in vivo and in vitro experimental observations and can reduce the impact on visual function after transplantation.

[0054] After human embryonic stem cells (hESCs) are cultured and reach super fusion (100%), the original embryonic stem cell growth medium is removed, the hESCs are washed once with koDMEM medium (Knockout DMEM), and then differentiation medium with a concentration of 20% serum replacement is added for differentiation induction until pigment foci appear (25-35 days), i.e., human RPE cells.

[0055] Among them, the composition of the RPE cell differentiation medium is: koDMEM (A1286101) 77%, serum-free substitute KOSR (A3020902 / 12618013) 20%, Glutamax-1 (A128660-01) 1%, MEM NEAA (Gibco, 11140-050) 1%, 2-mercaptoethanol (Gibco) 1%. koDMEM is a conventional serum-free DMEM culture medium in the prior art. It is specially designed to support serum-free culture of stem cells and can provide the nutrients required by stem cells. KOSR is a serum substitute used to supplement the missing serum components in the culture medium to support cell proliferation and differentiation. It helps maintain the undifferentiated state of stem cells and is suitable for long-term culture. Glutamax is a stable glutamine derivative that can reduce pH fluctuations and provide a more stable cell culture environment. MEM non-essential amino acid solution (MEM NEAA) contains all non-essential amino acids.

[0056] Tryple digested single-cell RPE cells, according to 1×10 5 cells / cm 2RPE cells were seeded onto a custom electrospun membrane sheet and induced to differentiate at 37°C. After about a week, the RPE cell density reached 100%. After 20 days of continuous culture, a polarized monolayer of RPE cells was obtained, resulting in a monolayer of retinal pigment epithelial (RPE) tissue.

[0057] (3) Preparation of double-layer cell complex tissue slices

[0058] The retinal progenitor stem cell cryogenic agarose solution (cold agarose cell suspension) was inoculated into a mold containing a single-layer tissue sheet of retinal pigment epithelial cells (RPE) (pre-cooled in an ice bath) to allow it to contact the retinal pigment cell layer. The cryogenic agarose RPC suspension solidified upon cooling to form an RPC+RPE double-layer cell complex tissue sheet.

[0059] The specific operation method is as follows: the RPE single layer tissue piece (preferably with an area of ​​0.33 cm) obtained in step (2) is placed on the surface of the RPE single layer tissue piece. 2 ) is placed on an ice bath device in advance, and then the low-temperature agarose cell suspension is inoculated onto the RPE monolayer tissue slice. The amount of low-temperature agarose cell suspension is preferably 30μl-50μl (the preferred amount for experimental animal rabbits). The RPC cells are brought into contact with the RPE cell layer, and the low-temperature agarose RPC suspension is solidified when cooled. After standing for 2-5min (time range), an RPC+RPE double-layer complex tissue slice is formed. The tissue slices obtained in this scheme are, from top to bottom: an RPC cell layer wrapped in low-temperature agarose, an RPE monolayer cell layer, and an electrospinning support layer. 30μl-50μl of low-temperature agarose cell suspension is inoculated, and the thickness of the double-layer cell complex tissue slice formed is 50-100μm. Typical microscopic images of double-layer cell complex tissue slices can be seen in Figure 5 (Use 50 μl of low-temperature agarose cell suspension, and the RPC cell density in the suspension is 1×10 6 cells / ml, the content of low-temperature agarose is 1.5%; the electrospun membrane used is preferably around 10 μm. This thickness condition matches the anatomical characteristics of subretinal space transplantation in rabbit eyes. If transplantation is required in other animals or humans, the thickness of the double-layer cell complex tissue sheet can be determined based on the actual anatomical characteristics of subretinal space transplantation. The thickness can be adjusted by adjusting the amount of low-temperature agarose cell suspension (and the ratio to the RPE single-layer tissue sheet).

[0060] (4) Transplantation of double-layer cell complex tissue sheets

[0061] The double-layer cell complex tissue slice is cut to have appropriate mechanical strength and compatibility. The double-layer cell complex tissue slice is trimmed to the size of 1×2 mm. 2), making it suitable for the operation of implantation into the subretinal space of the corresponding experimental animals. The disease model gray rabbit underwent vitrectomy surgery after general anesthesia, and sterilized air was injected into the subretinal space to separate the neural epithelium from the pigment epithelium. After the double-layer cell complex tissue sheet was implanted into the gap through special equipment, silicone oil was injected into the vitreous cavity to reposition the retina. Agarose gradually melted and absorbed under body temperature, and the transplanted cells integrated with the recipient's retinal tissue to exert therapeutic effects. It can be seen that the complex tissue sheet is stable in the subretinal space of the animal and integrated with the inner retinal tissue of the recipient ( Figure 6 ).

[0062] In late stage retinitis pigmentosa, RPE cells and photoreceptors are damaged, and single RPE cells (or membrane sheets) have limited effects, and retinal progenitor cells (RPCs) need to be supplemented at the same time. In addition, effectively maintaining the RPE monolayer structure and its epithelial polarity will also promote the survival of transplanted cells, improve the retinal transplant microenvironment, and establish effective phagocytic function to support the repair or reconstruction of inner retinal photoreceptor function. Therefore, this technical solution has adopted the RPC and RPE cell layer inoculation integration scheme.

[0063] Experimental Example 1: Screening of low-temperature agarose concentration

[0064] This experiment tested the gelation time of different concentrations of low-temperature agarose (1%, 1.5%, and 2%) in an ice bath. It can be seen that 1.5% concentration agarose can remain in a liquid state at conventional culture temperature and can quickly gel and set in an ice bath in about 2 minutes, which is sufficient to support us to complete the cell seeding operation. After gelation, it can maintain a good gel shape to support the cell stratification state.

[0065] The monolayer RPE cells on the membrane were labeled with Dil dye, and RPCs were infected with adeno-associated virus using the CAG promoter to express green fluorescent protein. A low-temperature agarose cell suspension was prepared according to the method of Example 1, and the RPC cell density was controlled to be 1×10 6 cells / ml, with a low-temperature agarose content of 1.5%, and inoculated onto a single RPE tissue sheet, allowing the RPC cells to contact the RPE cell layer. The sheet was then placed in an ice bath for gelation to form an RPC+RPE double-layer complex tissue sheet. Observation was performed using a confocal microscope. Figure 7 After solidification, low-temperature agarose can maintain a good gel-shaped state to support the cell stratification state (RPE red, RPC green).

[0066] Experimental Example 2: Screening of the Support Layer for Bilayer Cell Complex Tissue Sheets

[0067] This technical solution ultimately uses electrospinning as the support layer for the double-layer cell complex tissue sheet. Prior to this, the materials of the support layer were screened, including ordinary films (polyethylene), Transwell membranes, etc.

[0068] For the case of using ordinary film (polyethylene):

[0069] The optimal method for preparing a double-layer cell complex tissue sheet in Example 1 was used to obtain a tissue sheet, except that the electrospun membrane in Example 1 was replaced with a common film. In vitro and in vivo experiments on the prepared film revealed that choroidal blood oxygen and nutrients could not pass through and RPE cells could not survive in vivo. In addition, the common film did not provide sufficient support for the cells, resulting in curling, wrinkling and deformation in the later stage. Figure 8 A, Red RPE cell explant curled after transplantation).

[0070] For use with Transwell membranes:

[0071] The Transwell membrane is the porous membrane in the conventional Transwell culture device in the prior art. The main components of the Transwell device include an upper chamber (Insert) and a lower chamber (Well). The bottom of the upper chamber is a membrane with micropores, which can be made of polycarbonate, polyester or other materials. The lower chamber refers to the culture well below the Transwell device. The best method for preparing a double-layer cell complex tissue sheet in Example 1 was used to obtain the tissue sheet, except that the electrospinning membrane in Example 1 was replaced with a Transwell membrane. The Transwell membrane has a certain physical rigidity, is not easy to curl and deform, and has small pores through which nutrients and the like can pass to ensure cell survival. At the beginning of the study, the inventor wanted to use the above advantages of the Transwell membrane to prepare tissue sheets. However, in actual animal in vivo experiments, it was found that the effect of the Transwell membrane was not ideal, specifically: the Transwell membrane caused stimulation to the tissue, aggravated the inflammatory response, hindered the blood oxygen supply to the choroid, and the RPE cells were in poor condition, resulting in interstitial cell transition. See for details. Figure 8 C, Severe inflammatory response, with a large number of green Müller cells activated and glialized, and red RPE cells with disordered morphology and mesenchymal transition and loss of function.

[0072] Unlike ordinary films (polyethylene) and Transwell membranes, the electrospinning method of this solution is used as the support layer. The RPE cells grow in an ideal state, and the implants do not curl or deform, and the inflammatory response is not obvious. The experimental results are shown in Figure 8 B (the electrospinning membrane is preferably around 10 μm).

[0073] In this technical solution, the final design uses an electrospun membrane: the porosity of the electrospun support layer is 30%-90%; the pore size is 0.1-15μm; the electrospinning diameter is 200-1000nm, preferably 500nm, the thickness of the entire electrospun membrane is about 5-20μm, and the total thickness of the cell implant is 50-100μm, preferably 50μm. If the thickness of the electrospun membrane is <5μm, it is easy to break and curl during the transplantation process. If the thickness of the electrospun membrane is >20μm, the surrounding tissue will experience an aggravated inflammatory response, which will hinder the blood oxygen supply to the choroid, the RPE cells will be in poor condition, and the cells will undergo mesenchymal transition.

[0074] Comparative Example 1:

[0075] Prior art CN113766937A (complex comprising neural retina retinal pigment epithelial cells and hydrogel and its manufacturing method), a complex of neural retina and RPE cells is prepared using hydrogel (non-agarose). The complex is a complex comprising neural retina, RPE cell sheet and hydrogel (melting point is 20 ° C -40 ° C), and a neural retinal layer at least comprising a photoreceptor layer is formed in the neural retina, and the photoreceptor layer at least comprises one or more cells selected from the group consisting of photoreceptors, photoreceptor precursor cells and retinal progenitor cells. The entirety of the neural retina and RPE cell sheet is embedded in the hydrogel, and the tangential directions of the respective surfaces of the neural retina and RPE cell sheet are roughly parallel, and the apical surface of the neural retina is opposite to the apical surface of the RPE cell sheet, and the two are isolated and do not contact by the hydrogel.

[0076] However, the complex obtained by the technical solution in this comparative example is not suitable for transplantation. Even when the hydrogel is not melted, its mechanical strength is not enough to support the three-dimensional structure of the implant, resulting in difficulty in delivery during transplantation or curling and deformation in the body after transplantation, thereby losing the therapeutic effect. In contrast, the present technical solution supports RPE cells by an electrospinning method and drips an agarose suspension containing RPC cells thereon, ensuring that the implant is easy to deliver and does not curl, thereby maintaining its therapeutic value. The optimized electrospinning implant has sufficient mechanical strength, so that the photoreceptor precursor cell suspension can be solidified by cold on the monolayer retinal pigment epithelium, which is beneficial to delivery during transplantation.

[0077] The retinal pigment epithelium (RPE) in a healthy human body is a regular single-layer cell structure, responsible for engulfing the membrane disk of the outer segment of the photoreceptor cell and maintaining the visual cycle. However, in this comparative example, the RPE cells in the hydrogel membrane failed to form a single-layer structure, but were embedded in the hydrogel by roughly cut pigment cell clumps, which led to disordered cell polarity and failed to exert the desired functional effect. In contrast, this technical solution uses electrospinning technology to form a single-layer structure of RPE cells, which can more effectively exert the therapeutic effect.

[0078] In addition, in this comparative example, retina and RPE are respectively wrapped in gel and then stacked together, due to the isolation effect of hydrogel, lack direct contact between the two, lack the interaction between cells, and the synergistic treatment effect is therefore greatly reduced, and the significance of cell combination transplantation is lost. And in this technical scheme, the agarose suspension containing RPC cells is added dropwise on the monolayer structure of RPE cells, and after being solidified by cold, it is ensured that RPC cells and RPE cells fully contact and interact. This design not only maintains healthy three-dimensional structure, but also promotes the synergistic effect between two layers of cells, strengthens therapeutic effect together.

[0079] In summary, the organoid tissue implant of this patented technical solution is different from the existing technology. A single layer structure of RPE cells is formed on the polylactic acid electrospun fiber membrane. On this basis, an agarose suspension of RPC cells is added dropwise to form a composite implant. The above structure and preparation method not only ensure the mechanical properties of the implant, but also ensure that the RPE cell single layer and RPC cells can interact with each other, and finally form an orderly structure (see Figure 6 ). This process ensures functional connections between neurons and the effective transmission of visual signals.

[0080] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A two-layered three-dimensional organoid tissue implant for treating retinitis pigmentosa, characterized by: The method comprises a retinal progenitor stem cell layer, a retinal pigment epithelial cell monolayer and an electrospinning support layer which are arranged in sequence; The retinal progenitor stem cell layer is a low-temperature agar gel layer in which retinal progenitor stem cells are dispersed; the low-temperature agar gel has a solidification temperature of 26-30°C; the retinal progenitor stem cell layer melts at body temperature, and the retinal progenitor stem cells and retinal pigment epithelial cells directly contact and interact with each other, ultimately forming an orderly structure to ensure functional connections between neurons and effective transmission of visual signals; The porosity of the electrospinning support layer is 30%-90%; the pore size is 0.1-15 μm; the thickness of the electrospinning support layer is 5-20 μm; the electrospinning diameter is 200-1000 nm; and the electrospinning support layer is a degradable polylactic acid electrospinning fiber membrane.

2. The organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa according to claim 1, characterized in that: The electrospun diameter was 500 nm.

3. The method for preparing a double-layer three-dimensional organoid tissue implant for treating retinitis pigmentosa according to claim 1 or 2, characterized in that: The method includes the following steps: S1: Prepare a cryogenic agarose cell suspension containing retinal progenitor stem cells; the density of the retinal progenitor stem cells in the cryogenic agarose cell suspension is 1×10 5 -2×10 6 cells / ml; in the cryogenic agarose cell suspension, the concentration of cryogenic agarose is 1-2%; S2: retinal pigment epithelial cells are seeded on the electrospun support layer and cultured in retinal pigment epithelial cell differentiation medium to obtain a monolayer of retinal pigment epithelial cells; S3: Inoculate the cryogenic agarose cell suspension onto a pre-chilled monolayer of retinal pigment epithelial cells to obtain organoid tissue explants; The organoid tissue implants melt the solidified low-temperature agarose under body temperature conditions; the retinal progenitor stem cells and retinal pigment epithelial cells directly contact and interact with each other, ultimately forming an orderly structure to ensure functional connections between neurons and effective transmission of visual signals.

4. The method for preparing a two-layer three-dimensional organoid tissue graft for treating retinitis pigmentosa according to claim 3, characterized in that: In S1, retinal progenitor stem cells were plated at a density of 1 × 10 6 cells / ml; Retinal progenitor stem cells are obtained by the following method: Embryonic stem cells are induced and cultured into retinal organoids, and the neural retinal structures of the retinal organoids are obtained, followed by enzymatic hydrolysis and flow cytometry sorting to obtain retinal progenitor stem cells; the retinal progenitor stem cells include at least one of Crx-positive photoreceptor progenitors or other retinal progenitor cells; Retinal progenitor stem cells are Crx-positive photoreceptor progenitor cells; Retinal organoids are obtained by culturing embryonic stem cells for 60 days.

5. The method for preparing a double-layered three-dimensional organoid tissue implant for treating retinitis pigmentosa according to claim 3, characterized in that: In S1, the concentration of cryo-agarose in the cryo-agarose cell suspension was 1.5%; RPC cell culture medium was used to prepare low-temperature agarose cell suspension; RPC cell culture medium was a cell culture medium formed by adding 2% B27, 1% N2, 10 ng / ml bFGF, 2 mM glutamine, and 1% penicillin-streptomycin double antibody to DMEM / F12 basal culture medium.

6. The method for preparing a double-layered three-dimensional organoid tissue implant for treating retinitis pigmentosa according to claim 3, characterized in that: In S2, The seeding volume of retinal pigment epithelial cells was 1×10 5 cells / cm 2 The composition of the retinal pigment epithelial cell differentiation medium is: koDMEM medium 77%, KOSR 20%, Glutamax-1 1%, MEM NEAA 1%, 2-mercaptoethanol 1%; the culture duration using the retinal pigment epithelial cell differentiation medium is 20 days.

7. The method for preparing a double-layered three-dimensional organoid tissue implant for treating retinitis pigmentosa according to claim 3, characterized in that: In S3, 0.33 cm 2 30-50 μl of low-temperature agarose cell suspension is inoculated on a monolayer tissue piece of retinal pigment epithelial cells with a surface area of ​​​​the organoid tissue explant; the thickness of the organoid tissue explant is 50-100 μm.

8. Use of the organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa according to claim 1 or 2 in preparing materials for treating retinitis pigmentosa.

9. Use of cryogenic agarose in the preparation of organoid double-layer three-dimensional tissue implants for treating retinitis pigmentosa, characterized in that: The low-temperature agarose is the raw material of the retinal progenitor stem cell layer of the organoid tissue implant; the retinal progenitor stem cell layer is formed by solidifying a low-temperature agarose cell suspension containing retinal progenitor stem cells; In the cold agarose cell suspension, the density of retinal progenitor stem cells was 1×10 5 -2×10 6 cells / ml; in the cryogenic agarose cell suspension, the concentration of cryogenic agarose is 1-2%; the solidification temperature of the cryogenic agarose gel is 26-30°C; the retinal progenitor stem cell layer melts at body temperature, and the retinal progenitor stem cells and retinal pigment epithelial cells directly contact and interact with each other, ultimately forming an orderly structure to ensure functional connections between neurons and effective transmission of visual signals; The organoid tissue implant is sequentially arranged with a retinal progenitor stem cell layer, a retinal pigment epithelial cell monolayer and an electrospun support layer.

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