CN119950819B - Organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa and preparation method thereof - Google Patents
Organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa and preparation method thereofInfo
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Abstract
The invention relates to the technical field of tissue engineering, in particular to an organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa and a preparation method thereof. The organoid tissue implant of the technical scheme comprises a retina precursor stem cell layer, a retina pigment epithelial cell monolayer cell layer and an electrospinning supporting layer which are sequentially arranged. The retina precursor stem cell layer is formed by solidifying a low-temperature agarose cell suspension containing retina precursor stem cells. The organoid tissue implants of the present protocol can supplement a variety of degenerated, absent cells, and can form interactions between transplanted cells to maintain transplanted cell viability. The three-dimensional retina three-dimensional structure can ensure that the correct nerve loop improves the visual function of a patient. And the scheme provides the electrostatic spinning material to form a support for cells, so that the deformation of later-stage planting pieces is prevented. The technical scheme can solve the technical problem that the organoid tissue implant in the prior art has an unsatisfactory treatment effect on the retinitis pigmentosa, and has ideal popularization and application prospect.
Description
Technical Field
The invention relates to the technical field of tissue engineering, in particular to an organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa and a preparation method thereof.
Background
Retinitis pigmentosa (RETINITIS PIGMENTOSA, RP) is an irreversible blinding eye disease, which is mainly manifested by irreversible death of many neurons of the outer retina and Retinal Pigment Epithelium (RPE) cells, and no effective treatment has yet been achieved. In recent years, in vitro tissue engineering by utilizing a retina organoid technology expands stem cells, and transplantation treatment is carried out, so that the retina organoid technology becomes a potential treatment means. The organoid tissue implant is a membranous tissue similar to retina, and the subretinal space is transplanted with stem cells or stem cell tissue pieces, has better nerve regeneration and integration capability, and is beneficial to repairing retina. However, the above-mentioned methods have various problems in practical applications, such as low survival rate of transplanted cells, unsatisfactory repair of retinal structures, etc., and the effect of the transplantation-related treatment is still further improved. The reasons for the phenomena mainly comprise that the photoreceptor cells and the retinal pigment epithelial cells are seriously damaged in the course of the retinal pigment degeneration, and single retinal pigment epithelial cells or photoreceptor cells are transplanted, so that the key problem that multiple cells (such as photoreceptor cells and retinal pigment epithelial cells) in the late stage of the retinal degeneration are difficult to repair and simultaneously lose is solved. The retina is a multi-layered cellular structure tissue having a precise three-dimensional structure (fig. 1), which can be difficult to form by the graft if a general implantation method is adopted. Previous studies have attempted to implant directly as a single cell suspension, retinal tissue sheets (including fetal retina, organoid tissue sections), but the grafts are randomly packed in the subretinal space, or the folds accumulate to form rosettes, which are randomly structured, severely affecting neuronal function communication and visual signaling (see figures 2 and 3). It can be seen that after the cells or tissue sheets are directly transplanted, the crease and rosette structure formed by the subretinal space is greatly different from the interlayer structure with neat retina, and the regular nerve links with therapeutic significance cannot be formed.
Therefore, how to prepare the organoid tissue implant which meets the application requirements is the key for realizing the effective treatment of the retinal pigment degeneration disease. Most of the retinitis pigmentosa is damaged by multiple cells, the traditional transplanting pieces are of single cell monolayer structures, and the three-dimensional structure of the retinas is difficult to simulate. The prior art has the problems of single transplanted cell type, low survival rate, unsatisfactory retinal structure repair effect and the like, so that the transplanting curative effect is limited, and the problems need to be further solved.
Disclosure of Invention
The invention aims to provide an organoid double-layer three-dimensional tissue implant for treating retinal pigment degeneration, which aims to solve the technical problem that the organoid tissue implant in the prior art has an unsatisfactory treatment effect on retinal pigment degeneration.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
An organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa comprises a retinal precursor stem cell layer, a retinal pigment epithelial cell monolayer cell layer and an electrospinning supporting layer which are sequentially arranged.
Further, the retina precursor stem cell layer is a low Wen Qiongzhi gel layer in which retina precursor stem cells are dispersed, and the solidification temperature of low-temperature agar is 26-30 ℃.
Further, the porosity of the electrospinning supporting layer is 30% -90%, the pore diameter is 0.1-15 μm, the thickness of the electrospinning supporting layer is 5-20 μm, and the electrospinning diameter is 200-1000nm, preferably 500nm.
The technical scheme also provides a preparation method of the organoid double-layer three-dimensional tissue implant for treating the retinitis pigmentosa, which comprises the following steps in sequence:
s1, preparing a low-temperature agarose cell suspension containing retina precursor stem cells;
S2, preparing a single-layer tissue slice of retinal pigment epithelial cells;
And S3, inoculating the low-temperature agarose cell suspension on a single-layer tissue slice of the pre-cooled retinal pigment epithelial cells to obtain the organoid tissue implant.
Further, in S1, in the low temperature agarose cell suspension, the density of the retinal precursor stem cells is 1X 10 5 -2×106 cells/ml, preferably 1X 10 6 cells/ml;
The retinal precursor stem cells were obtained by the following method:
Culturing embryonic stem cells into retina organoids by induction, taking the neural retina structures of the retina organoids, and obtaining retina precursor stem cells after enzymolysis and flow cytometry separation, wherein the retina precursor stem cells comprise at least one of a Crx positive photosensitive precursor or other retina precursor cells;
Preferably, the retinal precursor stem cells are Crx positive photoreceptor precursor cells;
Preferably, the retina organoids are organoids obtained by induction culture of embryonic stem cells for 60 days;
More preferably, the embryonic stem cells are subjected to induction culture by a retinal organoid differentiation medium containing Y-27632 factor to form embryoid bodies, the embryoid bodies are subjected to induction culture by a retinal organoid differentiation medium containing BMP4, then the medium is gradually replaced by a retinal organoid differentiation medium, and the retinal organoids are formed by culture;
The retina organoid differentiation medium is prepared by adding KOSR with a final concentration of 10%, G1utamax with a final concentration of 1%, lipid concentrate with a final concentration of 1%, thioglycerol with a final concentration of 450 μm, penicillin-streptomycin diabody with a final concentration of 1% into a basal medium, wherein the basal medium is formed by mixing an IMDM medium and a Ham's F-12 medium in equal proportion;
The long-term medium was DMEM/F12-Glutamax medium containing 1% N2, 10% fetal bovine serum, 0.5. Mu.M retinoic acid, 0.2mM taurine, 1% penicillin-streptomycin diabodies.
Further, in S1, the concentration of cryoagarose in the cryoagarose cell suspension is 1-2%, preferably 1.5%;
The preparation of the low-temperature agarose cell suspension is carried out by using an RPC cell culture medium, wherein the RPC cell culture medium is formed by adding 2% of B27, 1% of N2, 10ng/ml of bFGF, 2mM of glutamine and 1% of penicillin-streptomycin double antibody into a DMEM/F12 basal medium.
Further, in S2, retinal pigment epithelial cells are inoculated on the electrospinning support layer and cultured using a retinal pigment epithelial cell differentiation medium to obtain a single-layer tissue sheet of retinal pigment epithelial cells;
Preferably, the inoculation amount of the retinal pigment epithelial cells is 1×10 5cells/cm2, and the composition of the retinal pigment epithelial cell differentiation medium is koDMEM medium 77%, KOSR 20%, glutamax-1 1%, MEM NEAA 1%, and 2-mercaptoethanol 1%, and the culture time period of the culture using the retinal pigment epithelial cell differentiation medium is 20 days.
Further, in S3, a single layer tissue sheet of retinal pigment epithelial cells having a surface area of 0.33cm 2 is inoculated with 30-50. Mu.l of a low-temperature agarose cell suspension, and the thickness of the organoid tissue graft is 50-100. Mu.m.
The technical scheme also provides application of the organoid tissue implant in preparing materials for treating the retinal pigment degeneration diseases.
The technical scheme also provides application of the low-temperature agarose in preparation of the organoid tissue implant for treating the retinitis pigmentosa, wherein the low-temperature agarose is a raw material of a retina precursor stem cell layer of the organoid tissue implant, and the retina precursor stem cell layer is formed by solidifying low-temperature agarose cell suspension containing retina precursor stem cells.
The technical principle and beneficial effects of the technical scheme are as follows:
In the technical scheme, the three-dimensional bionic retina tissue slice consisting of multiple layers of cells is successfully constructed. The tissue sheet not only supplements a variety of cell types that are deficient 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, the three-dimensional tissue slice helps to reconstruct the correct neural loop, significantly improving the visual function of the patient.
The present invention first attempted to integrate a plurality of different types of transplanted cells into a unitary graft, i.e., organoid tissue graft. The prior art is not provided with relevant solutions in the face of challenges of ensuring stable and easy-to-handle grafts between various cells, ensuring effective fusion between cells after transplantation to form ordered structures, avoiding curling deformation of transplanted cells and preventing tissue lesions after transplantation. Through a series of intensive experimental researches, we find that a method for wrapping retina precursor stem cells by using low-temperature agarose gel can construct a double-layer cell organoid tissue implant with stable structure, and is convenient for subsequent transplanting operation.
When the low temperature agarose gel embedded with the retinal precursor stem cells and the retinal pigment epithelial cells is transplanted to a designated position, the gel gradually melts along with the temperature rise, and the two cells are further in direct contact and interact to finally form an ordered structure. This process ensures an efficient conduction of visual signals in relation to functional links between neurons.
In addition, the electrostatic spinning material is innovatively introduced as a supporting matrix, so that the morphological stability and the structural integrity of the implanted implant are remarkably improved, the deformation is effectively prevented, and the regeneration and repair capability of tissues are further enhanced. Prior to selecting the electrospun materials, the inventors tried various conventional methods including the use of common films, transwell films, etc., but none of these methods achieved the desired effect.
In particular, common films (e.g., polyethylene) and Transwell films do not provide an ideal growth environment for retinal pigment epithelial cells, resulting in the implant being susceptible to crimping and accompanying significant inflammatory responses. In contrast, the retinal pigment epithelial cells exhibit a more desirable growth state after the electrospun material is used as a support layer, and the graft remains flat with a slight inflammatory response, greatly promoting interactions and functional integration between cells.
Notably, the thickness of the electrospun film has a significant impact on the effectiveness of organoid tissue implants. If the electrostatic spinning membrane is too thin, fracture and curling easily occur in the transplanting process, so that the success rate of operation is affected, and when the membrane thickness is too large, more serious peripheral tissue inflammatory reaction can be initiated, blood oxygen supply of choroid is blocked, so that poor cell state of retinal pigment epithelial cells is caused, and even interstitial transformation occurs. Therefore, by precisely controlling the thickness of the electrospun membrane, we not only optimize the mechanical properties of the graft, but also ensure its biocompatibility and functionality, providing a solid guarantee for achieving the optimal therapeutic effect.
Drawings
Figure 1 shows the multi-layered cellular structure of the retina (from literature ：He XY,Zhao CJ,Xu H,Chen K,Bian BS,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.2021Jul 13;16(7):1805-1817.).
Figure 2 shows an optical coherence tomography image (from literature) of the graft resulting in a random accumulation of the graft in the subretinal space using a single cell suspension of the graft ：Occelli LM,Marinho F,Singh RK,Binette F,Nasonkin IO,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.).
FIG. 3 shows the formation of a rugosity and rosette structure in the subretinal space following direct cell or tissue sheet implantation (from 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 3Dretinal sheets into retinal degenerative mice.Stem Cell Reports.2014Apr 24;2(5):662-74.).
FIG. 4 is a microscopic image of typical GFP green fluorescence labeled Crx positive RPCs and hEROs of example 1.
Fig. 5 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.
FIG. 6 is a typical HE staining micrograph of the post-implantation state of the double cell complex tissue sheet of example 1.
FIG. 7 is a three-dimensional reconstructed image of laser confocal measurement of the state of support cells layered after low-temperature agarose solidification in Experimental example 1.
FIG. 8 is an immunofluorescence micrograph of a post-implantation tissue section of a double-layered cell complex tissue sheet formed using different monolayer RPE cell supports of Experimental example 2.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, 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 and the like used are all commercially available. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1 organoid tissue grafts and methods of making
(1) Preparation of low temperature agarose cell suspensions
Agarose obtained from natural sources generally has a gel temperature of 35-40 ℃, at which most organisms or heat sensitive reagents will be deactivated, thus requiring the selection of agarose with a lower gel temperature. In the preparation of the implant, agarose with a low gelation temperature should be used. The low-melting-point agarose (Sigma-Aldrich, A2576, also called low-temperature agarose) using the scheme has the gel strength of 300g/cm 2 at the concentration of 1.5%, the gel temperature of 20 ℃ and the gel point of lower, and is suitable for application requirements.
The retinal precursor stem cells (RPCs) are seeded in RPC cell culture medium to obtain an RPC suspension. The RPC medium comprises 27% of B, 2% of N2 1% of bFGF 10ng/ml, 2% of glutamine, and 1% of penicillin-streptomycin double antibody added to DMEM/F12 basal medium. The DMEM/F12 (D/F12) basal medium is a common medium in the prior art and is formed by mixing DMEM and Ham's F-12 culture mediums in equal proportion. B27 and N2 are conventional supplements in culture medium for supporting the growth of neural cells. bFGF is basic fibroblast growth factor. The low-temperature agarose is dissolved in RPC culture medium to obtain low-temperature agarose solution. Adding the single-cell RPC suspension into a centrifuge tube, centrifuging for 3 minutes at 180g, sucking the supernatant, and re-suspending cells with a low-temperature agarose solution to obtain the low-temperature agarose cell suspension. In the low-temperature agarose cell suspension, RPC cells with the final density of 1 multiplied by 10 5cells/ml、5×105cells/ml、1×106cells/ml、2×106 cells/ml and the final content of low-temperature agarose of 1 percent, 1.5 percent and 2 percent are obtained. All of the above processes were completed at 37 ℃.
Wherein the retinal precursor stem cells (RPCs) are 60 day retinal organoid-derived cells. The induction of embryonic stem cells into retinal organoids by multi-step culture is a conventional manner of the prior art. The embryonic stem cells are firstly cultured under the induction of Y-27632 factor to form embryoid bodies, and then the embryoid bodies are induced by BMP4 to form retina organoids. The RPC cells can be isolated after enzymolysis of the formed retinal organoids in a manner conventional in the art. More specifically, the 60 day retinal organoids (human embryonic STEM CELL DERIVED RETINAL organoids, hEROs) were cultured and RPC obtained as follows:
Human embryonic stem cells (H9-hESC, hESC) in good culture were washed with PBS, and the human embryonic stem cells (hESC) were digested with Tryple (containing 20. Mu. M Y27632 and 0.05mg/m1 Dnase I) into single cells, placed in an incubator at 37℃and digested with PBS, followed by 180g centrifugation for 2min to collect cell pellet, and cell pellet was resuspended with 1ml of retinal organoid (Retinal Organoid, RO) differentiation medium. RO differentiation medium, namely gfCDM (+) medium, is specifically formed by mixing IMDM medium and Ham's F-12 medium in equal proportion, then adding serum-free substitute KOSR with the final concentration of 10%, G1utamax with the final concentration of 1%, lipid concentrate (Lipid Concentrate, gibco 11905-031) with the final concentration of 1%, thioglycerol (monothioglycerol) with the final concentration of 450 mu M and PS (penicillin-streptomycin diabody) with the final concentration of 1% into the basal medium.
Then, the final cell viability (more than 90% active cells) of the cell suspension was counted, the cell concentration was adjusted to 120000cells/ml, and the final concentration was mixed to 20. Mu. M Y-27632 factor (Cas: 146986-50-7), and the mixed cell suspension was inoculated into a V-type 96-well plate with low cell adhesion according to 100. Mu.L/well cell suspension, and the cells were collected into Embryoid Bodies (EBs). On day six EBs were completely exchanged for RO differentiation medium containing 1.5nM BMP4 (bone morphogenic protein 4), followed by half-exchanging RO induction medium without BMP4 every 3 days (i.e. half-exchanging with gfCDM (+). On day 18, the resulting hEROs was pipetted into a low viscosity 10cm diameter petri dish and the long term medium (LTCM; specific composition: 1% N2,10% Fetal Bovine Serum (FBS), 0.5. Mu.M retinoic acid (sigma), 0.2mM taurine (sigma), 1% PS, basal medium DMEM/F12-Glutamax medium (gibco)) was completely changed every 3 days until the Neuroretina (NR) architecture appeared (25-30 days).
HEROs continued to use long-term induction medium (LTCM)) induced differentiation to 60 days, infection with Crx-GFP-AAV virus (a prior art conventional tool virus) at a viral load of 1 μl/RO, and it was clearly observed that Crx positive RPC would be GFP green fluorescent-labeled after one week (figure 4 shows a microscopic image of typical Crx positive RPCs and hEROs labeled GFP green fluorescent-labeled). NR structures were aseptically isolated under a stereomicroscope using a 1ml syringe tip section, NR was transferred to a 15ml centrifuge tube, the supernatant was discarded, and the digestion reaction was stopped after digestion with 1ml trypsin Trype at 37℃for 25 minutes (during which time the papanicolaou tube was used for pipetting 2-3 times every 5-8 minutes). 180g centrifugation for 5min, discarding supernatant, re-suspending cells, and performing fluorescence flow type sorting to obtain RPC of Crx positive cells.
(2) Preparation of Retinal Pigment Epithelial (RPE) monolayer tissue sheet (retinal pigment epithelial cell monolayer cell layer + electrospun support layer)
The degradable polylactic acid electrostatic spinning fiber membrane with better biocompatibility is customized (R-JD-0229 of Siamirili biotechnology Co., ltd.). The porosity of the electrospinning supporting layer is 30% -90%, the aperture is 0.1-15 mu m, the electrospinning diameter is 200-1000nm, preferably 500nm, the thickness of the whole electrospinning membrane is about 5-20 mu m (preferably about 10 mu m), and after the electrospinning membrane is cultured to form an RPE single cell layer, the total thickness of the cell-added implant is about 50-100 mu m. The electrostatic spinning used in the prior art is usually fibroin/polycaprolactone/gelatin composite spinning, and the problems of irregular membrane fiber microstructure, uneven membrane strength, poor transparency and the like are easy to occur, so that conventional electrostatic spinning is not selected in the technical scheme. The polylactic acid used in the technical scheme is a novel biodegradable material and has good biocompatibility and degradability. The high polymer polylactic acid material has regular microstructure, mechanical property and physical property superior to those of the mixed material, good glossiness and transparency, and is beneficial to in-vitro and in-vitro experimental observation, and the influence on visual function after transplantation can be reduced.
After culturing human embryonic stem cells (hESCs) and removing the growth medium of the original embryonic stem cells when the fusion degree reaches super fusion (100%), washing the hESCs once by koDMEM medium (Knockout DMEM), adding a differentiation medium with 20% serum replacement concentration for differentiation induction until pigment focus appears (25-35 days), namely human RPE cells.
Wherein the RPE cell differentiation medium comprises koDMEM (A1286101) 77%, serum-free substitute KOSR (A3020902/12618013) 20%, glutamax-1 (A128660-01) 1%, MEM NEAA (Gibco, 11140-050) 1%, and 2-mercaptoethanol (Gibco) 1%. koDMEM is a conventional serum-free DMEM medium of the prior art, which is designed to support serum-free culture of stem cells and to provide nutrients required by the stem cells. KOSR is a serum replacement for supplementing the missing serum components in the medium, supporting cell proliferation and differentiation. It helps to maintain the undifferentiated state of stem cells and is suitable for long-term culture. Glutamax is a stable glutamine derivative that reduces pH fluctuations and provides a more stable cell culture environment. The MEM nonessential amino acid solution (MEM NEAA) contains all nonessential amino acids.
Single cell RPE cells digested with Tryple RPE cells were seeded onto custom-made electrospun membranes at a density of 1x 10 5cells/cm2 and induced differentiation was continued at 37 ℃. The density of RPE cells can reach 100% for about one week, and polarized single-layer RPE cells can be obtained by continuous culture for 20 days, thus obtaining the single-layer tissue sheet of retinal pigment epithelial cells (RPE).
(3) Preparation of double-layer cell complex tissue slice
The retinal precursor stem cells are inoculated into a single-layer tissue sheet mould (advanced low-temperature ice bath) containing retinal pigment epithelial cells (RPE) by a low-temperature agarose solution (low-temperature agarose cell suspension), and are contacted with a retinal pigment cell layer, and the low-temperature agarose RPC suspension is solidified when being cooled to form an RPC+RPE double-layer cell complex tissue sheet.
The specific operation method is that the RPE monolayer tissue sheet (preferably with the area of 0.33cm 2) obtained in the step (2) is placed on ice bath equipment in advance, and then the low-temperature agarose cell suspension is inoculated on the RPE monolayer tissue sheet, wherein the dosage of the low-temperature agarose cell suspension is preferably 30-50 mu l (which is the preferred dosage for experimental animal rabbits). And (3) enabling the RPC cells to be in contact with the RPE cell layer, solidifying the low-temperature agarose RPC suspension when the suspension is cooled, and standing for 2-5min (time range) to form the RPC+RPE double-layer complex tissue sheet. The tissue slice obtained by the scheme comprises an RPC cell layer wrapped by low-temperature agarose, an RPE monolayer cell layer and an electrospinning supporting layer from top to bottom. The double-layer cell complex tissue piece formed by inoculating 30-50 mu l of low-temperature agarose cell suspension has the thickness of 50-100 mu m. A typical micrograph of a double-layered cell complex tissue sheet can be seen in FIG. 5 (50. Mu.l of low temperature agarose cell suspension is used, with RPC cell density of 1X 10 6 cells/ml in suspension, low temperature agarose content of 1.5%, preferably about 10 μm for the electrospun membrane used). The thickness condition can be matched with the implantation anatomical characteristics of the subretinal space of the rabbit eye, if implantation is needed in other animals or human bodies, the thickness of the double-layer cell complex tissue piece can be determined according to the implantation anatomical characteristics of the actual subretinal space, and the thickness adjustment can be realized by adjusting the dosage of the low-temperature agarose cell suspension (and the proportion of the low-temperature agarose cell suspension to the RPE single-layer tissue piece).
(4) Transplantation of double-layer cell complex tissue pieces
The double-layer cell complex tissue slice is cut to have proper mechanical strength and compatibility. The area of the double-layer cell complex tissue piece is trimmed (preferably: 1×2mm 2) to make it suitable for implantation in subretinal space of experimental animals. The disease model gray rabbit is subjected to a general anesthesia post-vitrectomy operation, sterilizing air is injected into a subretinal cavity to separate nerve epithelium from pigment epithelium, a double-layer cell complex tissue sheet is implanted into a gap through special equipment, and silicone oil is injected into a vitreous cavity to reset retina. Agarose is gradually melted and absorbed under the condition of body temperature, and the transplanted cells are integrated with the retina tissue of a receptor, so that the curative effect is exerted. It can be seen that the complex tissue pieces are stable in the animal's subretinal space and integrate with the recipient's inner retinal tissue (fig. 6).
In advanced retinal pigment degeneration disease, both RPE cells and photoreceptor cells are damaged, and single RPE cells (or patches) have limited effect and need to be supplemented with Retinal Precursor Cells (RPCs) at the same time. In addition, the effective maintenance of the RPE monolayer structure and the polarity of the epithelium thereof will also promote the survival of transplanted cells, improve the retinal transplantation microenvironment, establish effective phagocytic function support for the repair or reconstruction of the inner retinal photoreceptor cell function. Therefore, the technical scheme adopts an integration scheme of RPC and RPE cell layer inoculation.
Experimental example 1 screening of Low temperature agarose concentration
The experimental example detects the gel time of low-temperature agarose (1%, 1.5% and 2%) with different concentrations in an ice bath, and can show that the agarose with the concentration of 1.5% can keep a liquid state at a conventional culture temperature, and can be rapidly gelled and shaped for about 2min under the ice bath condition, so that the experimental example is sufficient for supporting the completion of cell inoculation operation, and can maintain a better gel shaping state after the gel so as to support a cell layering state.
Marking single-layer RPE cells on a diaphragm by using a Dil dye, infecting RPC by using CAG promoter adeno-associated virus to enable the RPC to express green fluorescent protein, preparing low-temperature agarose cell suspension according to the method of the example 1, controlling the density of the RPC cells to be 1 multiplied by 10 6 cells/ml, inoculating the low-temperature agarose to the RPE single-layer tissue slice, enabling the RPC cells to be contacted with the RPE cell layer, and placing the RPC cells in an ice bath environment for gelation to form the RPC+RPE double-layer complex tissue slice. The observation was performed by confocal microscopy. As a result, referring to fig. 7, the low temperature agarose after solidification can maintain a better gel-shaped state to support the cell layered state (RPE red, RPC green).
Experimental example 2 screening of support layer of double-layered cell Complex tissue sheet
According to the technical scheme, electrostatic spinning is finally adopted as a supporting layer of the double-layer cell complex tissue sheet, and materials of the supporting layer are screened before the supporting layer, wherein the supporting layer comprises a common film (polyethylene), a Transwell film and the like.
For the case of using a common film (polyethylene):
The tissue sheet was obtained by the optimal method for preparing a double-layered cell complex tissue sheet of example 1, except that the electrospun film of example 1 was replaced with a common film. In vitro and in vivo experimental researches are carried out on the prepared film, and the result shows that choroidal blood oxygen and nutrition cannot pass through RPE cells in vivo and cannot survive, and the common film is insufficient in support of cells, and the film is deformed in a later curling and folding way (fig. 8A, curling of the red RPE cell implant after transplantation occurs).
For the case of using a Transwell membrane:
The Transwell membrane is a membrane with holes in a conventional Transwell culture device in the prior art. The main components of a Transwell apparatus include an upper chamber (Insert) and a lower chamber (Well). The bottom of the upper chamber is a membrane with micropores, which may be made of polycarbonate, polyester or other materials. The lower chamber refers to the culture well below the Transwell apparatus. The tissue pieces were obtained by the optimal method for preparing double-layered cell complex tissue pieces of example 1, except that the electrospun membrane of example 1 was replaced with a Transwell membrane. The Transwell membrane has certain physical rigidity, is not easy to deform by curling, and has small holes, nutrients and the like which can pass through to ensure the survival of cells. The inventors have at the beginning of their study devised to use the above-mentioned advantages of Transwell membranes for the preparation of tissue sheets. However, in practical animal experiments, the effect of the Transwell membrane is found to be not ideal, and the Transwell membrane is particularly used for stimulating and aggravating inflammatory response to tissues, blocking choroidal blood oxygen supply, leading to poor RPE cell state and generating interstitial transformation. As shown in FIG. 8C, the inflammatory response is severe and a large number of green Muller cells activate gliosis and red RPE cells lose function from morphologically disturbed interstitial transformation.
Unlike common films (polyethylene) and Transwell films, the electrospinning of this approach was used as a support layer, the RPE cell growth state was ideal, no curling of the implant occurred, the inflammatory response was not apparent, and the experimental results were shown in fig. 8B (electrospun films preferably around 10 μm).
In the technical scheme, the electrostatic spinning membrane is finally designed and used, wherein the porosity of an electrostatic spinning supporting layer is 30% -90%, the aperture is 0.1-15 mu m, the electrostatic spinning diameter is 200-1000nm, preferably 500nm, the thickness of the integral electrostatic spinning membrane is about 5-20 mu m, and the total thickness of the cell-added implant is 50-100 mu m, preferably 50 mu m. The electrospun membrane with a thickness of less than 5 μm is easy to break and curl during the implantation process. The thickness of the electrostatic spinning membrane is more than 20 mu m, so that the peripheral tissues have the phenomenon of aggravating inflammatory reaction, the choroidal blood oxygen supply is blocked, the RPE cell state is poor, and the interstitial transformation occurs.
Comparative example 1:
Prior art CN113766937a (complex comprising neural retina pigment epithelial cells and hydrogel and method of manufacturing the same) a complex of neural retina and RPE cells was prepared using hydrogel (not agarose). The complex is a complex comprising a neural retina, an RPE cell sheet, and a hydrogel (melting point 20 ℃ to 40 ℃) and the neural retina has formed therein a neural retina layer comprising at least an optic cell layer comprising at least one or more cells selected from the group consisting of an optic cell, an optic cell precursor cell, and a retinal precursor cell. The whole of the neural retina and the RPE cell sheet is embedded in the hydrogel, the tangential directions of the surfaces of the neural retina and the RPE cell sheet are approximately parallel, the top end surface of the neural retina is opposite to the top end surface of the RPE cell sheet, and the neural retina and the RPE cell sheet are isolated from contact by the hydrogel.
However, the complex obtained by the technical scheme in this comparative example is not suitable for transplantation. Even in the case of unmelted hydrogels, their mechanical strength is insufficient to support the three-dimensional structure of the implant, resulting in difficult delivery during implantation or curling deformation in the body after implantation, thereby losing therapeutic effect. In contrast, the present solution supports RPE cells by electrospinning and drops 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 electrostatic spinning implant has enough mechanical strength, so that the photosensitive precursor cell suspension can be solidified on the single-layer retinal pigment epithelium in a cooling way, and the delivery in the transplanting process is facilitated.
Retinal Pigment Epithelium (RPE) in healthy humans is a regular single-layer cellular structure responsible for phagocytizing the membrane discs of the outer segments of photoreceptor cells, maintaining the visual circulation. However, in this comparative example, RPE cells in the hydrogel film do not form a monolayer structure, but rather pigment cell clusters cut by roughness are embedded in the hydrogel, which causes a disturbance in cell polarity and fails to exert a functional effect as intended. In contrast, the technical scheme utilizes the electrostatic spinning technology to form a monolayer structure of RPE cells, and can more effectively play a role in treatment.
In addition, in this comparative example, the retina and RPE were individually encapsulated in a gel and then superimposed together, and due to the isolation of the hydrogel, there was no direct contact between the two, and there was no interaction between the cells, thus the synergistic therapeutic effect was greatly compromised, and the significance of cell combination transplantation was lost. In the technical scheme, agarose suspension containing RPC cells is dripped on a monolayer structure of the RPE cells, and after solidification under cold conditions, the sufficient contact and interaction between the RPC cells and the RPE cells are ensured. The design not only maintains a healthy three-dimensional structure, but also promotes the synergistic effect between two layers of cells, and jointly enhances the therapeutic effect.
In summary, unlike the prior art, the organoid tissue implant according to the present patent application forms a monolayer structure of RPE cells on the polylactic acid electrospun fiber membrane, and on this basis, the composite implant is formed by dropping agarose suspension of RPC cells. The structure and the preparation method ensure the mechanical property of the implant and the interaction between the single cell layer of the RPE cell and the RPC cell, and finally form an ordered structure (see figure 6). This process ensures an efficient conduction of visual signals in relation to functional links between neurons.
The foregoing is merely exemplary embodiments of the present application, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present application, and these should also be considered as the scope of the present application, which does not affect the effect of the implementation of the present application and the utility of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (9)
1. The organoid double-layer three-dimensional tissue implant for treating the retinitis pigmentosa is characterized by comprising a retinal precursor stem cell layer, a retinal pigment epithelial cell monolayer cell layer and an electrospinning supporting layer which are sequentially arranged;
the retina precursor stem cell layer is a low Wen Qiongzhi gel layer in which retina precursor stem cells are dispersed, the solidification temperature of low-temperature agar gel is 26-30 ℃, the retina precursor stem cell layer is melted under the condition of body temperature, and the retina precursor stem cells and the retina pigment epithelial cells are in direct contact and interact to finally form an ordered structure so as to ensure functional connection among neurons and effective conduction of visual signals;
the porosity of the electrospinning supporting layer is 30% -90%, the aperture is 0.1-15 mu m, the thickness of the electrospinning supporting layer is 5-20 mu m, the electrospinning diameter is 200-1000nm, and the electrospinning supporting layer is a degradable polylactic acid electrospinning fibrous membrane.
2. The organoid double layer three dimensional tissue graft for treating retinitis pigmentosa of claim 1, wherein the electrospun diameter is 500nm.
3. The method for preparing the organoid double-layer three-dimensional tissue implant for treating retinitis pigmentosa according to claim 1 or 2, characterized by comprising the following steps carried out in sequence:
S1, preparing a low-temperature agarose cell suspension containing retina precursor stem cells, wherein the density of the retina precursor stem cells in the low-temperature agarose cell suspension is 1 multiplied by 10 5-2×106 cells/ml, and the concentration of the low-temperature agarose in the low-temperature agarose cell suspension is 1-2%;
s2, inoculating the retinal pigment epithelial cells on the electrospinning supporting layer, and culturing the retinal pigment epithelial cells by using a retinal pigment epithelial cell differentiation medium to obtain a single-layer tissue sheet of the retinal pigment epithelial cells;
s3, inoculating the low-temperature agarose cell suspension on a single-layer tissue sheet of the pre-cooled retinal pigment epithelial cells to obtain an organoid tissue implant;
The organ-like tissue implant melts solidified low-temperature agarose under the condition of body temperature, and the retinal precursor stem cells and the retinal pigment epithelial cells are in direct contact and interact to finally form an ordered structure so as to ensure functional connection among neurons and effective conduction of visual signals.
4. The method for preparing an organoid double-layered three-dimensional tissue graft for treating retinitis pigmentosa according to claim 3, wherein in S1, in a low-temperature agarose cell suspension, the density of retinal precursor stem cells is 1X 10 6 cells/ml;
The retinal precursor stem cells were obtained by the following method:
Culturing embryonic stem cells into retina organoids by induction, taking the neural retina structures of the retina organoids, and obtaining retina precursor stem cells after enzymolysis and flow cytometry separation, wherein the retina precursor stem cells comprise at least one of a Crx positive photosensitive precursor or other retina precursor cells;
The retinal precursor stem cells are Crx positive photoreceptor precursor cells;
the retina organoids are organoids obtained by induction culture of embryonic stem cells for 60 days.
5. The method for preparing an organoid double-layered three-dimensional tissue implant for treating retinitis pigmentosa according to claim 3, wherein in S1, the concentration of cryoagarose in the cryoagarose cell suspension is 1.5%;
The preparation of the low-temperature agarose cell suspension is carried out by using an RPC cell culture medium, wherein the RPC cell culture medium is formed by adding 2% of B27, 1% of N2, 10ng/ml of bFGF, 2mM of glutamine and 1% of penicillin-streptomycin double antibody into a DMEM/F12 basal medium.
6. The method for preparing an organoid double layer three dimensional tissue graft for treating retinitis pigmentosa according to claim 3, wherein in S2,
The inoculation amount of the retinal pigment epithelial cells is 1×10 5cells/cm2, and the composition of the retinal pigment epithelial cell differentiation medium is koDMEM medium 77%, KOSR 20%, glutamax-1 1%, MEM NEAA 1% and 2-mercaptoethanol 1%, and the culture time period of the culture using the retinal pigment epithelial cell differentiation medium is 20 days.
7. The method for preparing a double-layered three-dimensional tissue-like sheet for treating retinitis pigmentosa according to claim 3, wherein 30-50. Mu.l of the low-temperature agarose cell suspension is inoculated onto a single-layered tissue sheet of retinal pigment epithelial cells having a surface area of 0.33cm 2 in S3, and the thickness of the tissue-like sheet is 50-100. Mu.m.
8. Use of an organoid bi-layer three-dimensional tissue graft according to claim 1 or 2 for the treatment of retinitis pigmentosa in the preparation of a material for the treatment of retinitis pigmentosa diseases.
9. The application of the low-temperature agarose in preparing the organoid double-layer three-dimensional tissue implant for treating the retinitis pigmentosa is characterized in that the low-temperature agarose is used as a raw material of a retina precursor stem cell layer of the organoid tissue implant;
In the low-temperature agarose cell suspension, the density of the retinal precursor stem cells is 1 multiplied by 10 5 -2×106 cells/ml, the concentration of the low-temperature agarose is 1-2%, the solidification temperature of the low-temperature agarose is 26-30 ℃, the retinal precursor stem cells are melted under the condition of body temperature, and the retinal precursor stem cells and the retinal pigment epithelial cells are in direct contact and interact to finally form an ordered structure so as to ensure the functional connection between neurons and the effective conduction of visual signals;
The organoid tissue implant is sequentially provided with a retina precursor stem cell layer, a retina pigment epithelial cell monolayer cell layer and an electrospinning supporting layer.
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