Retinal pigment epithelial cell spherical preparation

Abstract

The invention belongs to the field of RPE cell preparations, and relates to a retinal pigment epithelial cell spherical preparation which is obtained by the following steps: step 1, obtaining RPE cells; and 2, inoculating the RPE cells into a cell culture plate by using an inoculation culture medium, and culturing after centrifugal precipitation, so that a plurality of RPE cells are aggregated to form RPE cell balls with uniform sizes. The cell preparation form obtained by the scheme not only can avoid injection leakage of the subretinal space, but also has the advantages of being convenient to inject and uniform in cell expansion, so that preclinical needs are met, and an important basis is provided for preparation of a clinical-grade RPE cell preparation and subsequent further development of clinical research.

Description

Technical Field

[0001] The invention belongs to the field of cell technology, and in particular relates to a retinal pigment epithelial cell spherical preparation. Background Art

[0002] Almost one-third of the signals input to the human brain's sensory system come from the retina, which plays a key role in light perception and transduction. The macula is an elliptical yellowish area located in the middle of the retina, responsible for the perception of most visual information in the human eye. Macular degeneration (MD) is characterized by the degeneration of retinal cells (such as retinal pigment epithelial cells and / or photoreceptor cells), which can lead to severe visual dysfunction. [1,2] MD is mainly divided into Stargardt macular degeneration (SMD) and age-related macular degeneration (AMD), which are more common in children and the elderly, respectively. [3,4] .

[0003] In the eye, the neural retina and retinal pigment epithelium (RPE) cells are important components of the retina. The neural retina contains multiple layers of different types of cells, including the RPE cell layer, and the RPE cell layer is composed of a single layer of RPE cells tightly arranged. In the neural retina, it participates in the formation of the blood-retinal barrier, phagocytizes and transports visual cycle products, and plays a vital role in maintaining photoreceptor cell metabolism and retinal function.

[0004] Clinically, AMD is divided into two types: atrophic (dry) and neovascular (wet). These two types of AMD share common pathological features, including degeneration and death of RPE cells and damage to secondary photoreceptor cells. [5] Therefore, RPE cells play an important role in studying the pathogenesis of retinal diseases and developing alternative treatments; and existing studies have shown that transplanting fetal RPE (fRPE) cells from aborted fetuses or RPE cells differentiated by directed induction of stem cells into the subretinal cavity can effectively alleviate the progression of macular degeneration.

[0005] Due to the complex ethical review procedures and the limited amount of materials, fRPE has great limitations in treating macular degeneration. Therefore, RPE produced by directional induction of pluripotent stem cells has become the first choice for alternative treatment of macular degeneration. It has the characteristics of strong plasticity, convenient in vitro expansion and preparation, safe and controllable quality and quantity, and no need for ethical review procedures.

[0006] There are many forms of RPE cell preparations in existing RPE replacement therapies, mainly divided into RPE cell suspension (RPESuspension), RPE cell patch (RPE Sheet / RPE Patch), and RPE cell strip (RPE Strip). Although cell suspension does not require further preparation and is convenient for administration in the subretinal space, it is easy to leak into the vitreous cavity after injection, and it often forms a multi-layer stack in the fundus. [6] Subsequently, in order to solve the above problems, different scholars have obtained RPE cell patches (biomaterial substrates or autologous patches, etc.) through various means. The RPE patches with polar monolayer and tight arrangement can be transplanted into the subretinal space. However, this method is difficult to operate, and the patches are not easy to fix in the fundus and are easy to float in the fundus, which seriously affects the treatment effect. [7,8] The appearance of RPE cell strips avoids cell leakage after transplantation and is convenient for surgery. However, the shape of the cell strips is difficult to control after injection into the fundus and they tend to curl and stack. [9] .

[0007] Therefore, there is currently a lack of an effective form of RPE cell preparation to ensure that RPE cell preparations can meet clinical treatment needs.

[0008] References:

[0009] 1.Colijn JM, et al.Genetic Risk,Lifestyle,andAge-Related MacularDegeneration in Europe:The EYE-RISK Consortium.Ophthalmology.2021Jul;128(7):1039-1049.doi:10.1016 / j.ophtha.2020.11.024.Epub 2020Nov 28.PMID:33253757.

[0010] 2.Zekavat SM,et al.Photoreceptor Layer Thinning Is an Early Biomarkerfor Age-Related Macular Degeneration:Epidemiologic and Genetic Evidence fromUK Biobank OCT Data.Ophthalmology.2022Jun;129(6):694-707.doi:10.1016 / j.ophtha.2022.02.001.Epub 2022Feb 8.PMID:35149155;PMCID:PMC9134644.

[0011] 3.Runhart EH,et al.Stargardt disease:monitoring incidence anddiagnostic trends in the Netherlands using a nationwide disease registry.ActaOphthalmol.2022Jun;100(4):395-402.doi:10.1111 / aos.14996.Epub 2021Aug 25.PMID:34431609;PMCID:PMC9291619.

[0012] 4.Lee KS,et al.Cellular senescence in the aging retina anddevelopments of senotherapies for age-related macular degeneration.JNeuroinflammation.2021Jan22;18(1):32.doi:10.1186 / s12974-021-02088-0.PMID:33482879;PMCID:PMC7821689.

[0013] 5.Yang S,et al.Functions and Diseases of the Retinal PigmentEpithelium.Front Pharmacol.2021Jul 28;12:727870.doi:10.3389 / fphar.2021.727870.PMID:34393803;PMCID:PMC8355697.

[0014] 6.Schwartz SD,et al.Human embryonic stem cell-derived retinalpigmentepithelium in patients with age-related macular degeneration andStargardt's maculardystrophy:follow-up of two open-label phase 1 / 2studies.Lancet.2015 Feb7;385(9967):509-16.doi:10.1016 / S0140-6736(14)61376-3.Epub 2014 Oct 15.PMID:25458728.

[0015] 7.da Cruz L,et al.Phase 1 clinical study of an embryonic stem cell-derivedretinal pigment epithelium patch in age-related maculardegeneration.Nat Biotechnol.2018 Apr;36(4):328-337.doi:10.1038 / nbt.4114.Epub2018 Mar 19.PMID:29553577.

[0016] 8.Mandai M,et al.Autologous Induced Stem-Cell-Derived Retinal CellsforMacular Degeneration.N Engl J Med.2017 Mar 16;376(11):1038-1046.doi:10.1056 / NEJMoa1608368.PMID:28296613.

[0017] 9.Nishida M,et al.Human iPS cell derived RPE strips for securedelivery ofgraft cells at a target place with minimal surgical invasion.SciRep.2021 Nov2;11(1):21421.doi:10.1038 / s41598-021-00703-x.PMID:34728664;PMCID:PMC8563929.

[0018] 10.Iwasaki Y,et al.Differentiation / Purification Protocol for RetinalPigmentEpithelium from Mouse Induced Pluripotent Stem Cells as a ResearchTool.PLoSOne.2016 Jul 6;11(7):e0158282.doi:10.1371 / journal.pone.0158282.PMID:27385038;PMCID:PMC4934919.

[0019] 11.Mandai M,et al.Autologous Induced Stem-Cell-Derived Retinal CellsforMacular Degeneration.N Engl J Med.2017 Mar 16;376(11):1038-1046.doi:10.1056 / NEJMoa1608368.PMID:28296613.

[0020] 12.Sugita S,et al.Successful Transplantation of Retinal PigmentEpithelialCells from MHC Homozygote iPSCs in MHC-Matched Models.Stem CellReports.2016 Oct 11;7(4):635-648.doi:10.1016 / j.stemcr.2016.08.010.Epub 2016Sep 15.PMID:27641649;PMCID:PMC5063629.

[0021] 13. Wu W, et al. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures-a new donor for cell therapy. Oncotarget.2016Apr 19;7(16):22819-33.doi:10.18632 / oncotarget.8185.PMID:27009841; PMCID: PMC5008403. Summary of the invention

[0022] In order to solve the above technical problems, the present application provides a spherical preparation of retinal pigment epithelial cells, which can avoid leakage during injection into the subretinal cavity and has the advantages of convenient injection and uniform cell expansion.

[0023] The present invention develops a retinal pigment epithelial cell spherical preparation, and the retinal pigment epithelial cell spherical preparation is obtained by the following method:

[0024] Step 1: Obtain RPE cells;

[0025] Step 2: Inoculate the RPE cells in a cell culture plate using an inoculation medium, and culture them after centrifugation to allow a number of RPE cells to aggregate to form RPE cell spheres of uniform size.

[0026] As an improved technical solution of the present application, the RPE cells are fRPE, or RPE cells obtained by differentiation of pluripotent stem cells.

[0027] As an improved technical solution of the present application, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells; the pluripotent stem cells are human pluripotent stem cells, mouse pluripotent stem cells, or monkey pluripotent stem cells.

[0028] As an improved technical solution of the present application, step one also includes: after obtaining the RPE cells, culturing the RPE cells with RPE culture medium for 0-10 days.

[0029] As an improved technical solution of the present application, the inoculation culture medium contains 1-100 μM Y27632.

[0030] As an improved technical solution of the present application, the inoculation culture medium includes an additive; the additive contains 0.25-5% of any one of FBS, KSR, HSA, BSA, recombinant BSA, or recombinant HSA.

[0031] As an improved technical solution of the present application, the additive further includes:

[0032] Either N2(1×) or B27(1×);

[0033] 1-100 mM of any one of Nicotinamide, Niacin, Niacin hydrochloride or L-Tryptophan;

[0034] 10-200 nM EDN3; 1-100 ng / mL FGF2 or EGF;

[0035] 1-100 μM of any one of SB431542, A 83-01, Lefty-A or LY2157299, and 1-100 μM of T3.

[0036] As an improved technical solution of the present application, the additive includes a dosage of 1-100 μM CHIR-99021.

[0037] As an improved technical solution of the present application, the inoculation culture medium includes a basal culture medium; the basal culture medium is any one of MEM, DMEM, Ham's F12, DMEM / F12, RPMI-1640, MEMα, IMDM, and E6.

[0038] As an improved technical solution of the present application, in step 2, the RPE cells are cultured by suspension inoculation.

[0039] As an improved technical solution of the present application, in step 2, when the RPE cells are cultured based on the inoculation medium, the initial inoculation amount of the RPE cells per well is 300-5000 cells, and the diameter of the RPE cell sphere that can be formed is 50-400 μm.

[0040] As an improved technical solution of the present application, the RPE cell preparation has the following properties: several RPE cell spheres are plated in a single well of the same cell culture plate for 1-8 days, no adhesion occurs between the RPE cell spheres, and each RPE cell sphere can achieve adherence and expansion.

[0041] The beneficial effects of the present invention are:

[0042] 1. This application proposes the concept of RPE cell spheres and develops for the first time a preparation process for obtaining RPE cell spheres, which can form RPE cell spheres in vitro and have the potential to automatically extend into a single layer of RPE cell layers in vivo / in vitro.

[0043] 2. The RPE cell sphere of the present application has the characteristics of avoiding leakage of subretinal cavity injection and convenient injection; at the same time, it can also ensure that RPE cells can expand evenly. Among them, the reason for avoiding leakage is: when selecting RPE cell suspension, because the diameter of RPE cells themselves is between 15-17μm, and the inner diameter of the surgical injection needle is between 100-200μm, RPE cells are prone to leak from the injection point after the injection, and the diameter of the RPE cell sphere of the present application is not less than 50μm, compared with a single RPE cell, it is not easy to leak after injection. Convenient injection: Compared with RPE cell patches / strips, its surgical injection method is more convenient.

[0044] 3. The RPE cell spheres of the present application have controllable size and cell number, and the preparation process is highly repeatable, which can meet clinical stability requirements.

[0045] 4. The RPE cell spheres of the present application are suitable for a seeding range of 300-5000 cells during the preparation process, and the diameter of the formed RPE cell spheres is 50-400 μm, corresponding to the ability to obtain RPE cell spheres with a diameter within the clinical application range to meet the needs of different application scenarios.

[0046] 5. The RPE cell spheres of the present application have the function of adhering to the wall and expanding, that is, whether a single or multiple RPE cell spheres are inoculated, they can adhere to the wall and expand in vitro.

[0047] 6. The RPE cell spheres obtained in the present application can maintain the biological characteristics of the RPE cells before sphere formation, such as higher expression of specific markers, purity of RPE cells, and the ability of RPE cells to secrete VEGF. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Attached Figure 1 This is a cell morphology diagram on day D1 in Example 2.

[0049] Attached Figure 2 This is a cell morphology diagram on day D2 in Example 2.

[0050] Attached Figure 3 This is a cell morphology diagram on day D3 in Example 2.

[0051] Attached Figure 4 The cell morphology diagrams are shown in Example 3 when 300 cells and 600 cells were inoculated on days D0, D1 and D2.

[0052] Attached Figure 5 This is the D0 (first day) state diagram for verifying the expansion performance of the RPE cell sphere in Example 4.

[0053] Attached Figure 6This is the D3 (day 3) state diagram for verifying the expansion performance of the RPE cell sphere in Example 4.

[0054] Attached Figure 7 This is the D7 (seventh day) state diagram for verifying the expansion performance of RPE cell spheres in Example 4.

[0055] Attached Figure 8 This is a state diagram of the expanded performance of multiple RPE cell spheres verified in Example 4.

[0056] Attached Fig. 9 This is the effect of RPE cells from different sources on RPE cell sphering in the same process as in Example 5. At this time, the inoculation range of fRPE is 1000 cells-5000 cells.

[0057] Attached Fig.10 This is the first day (D1) of inoculation culture for verifying the effect of the selection of RPE cell culture medium and inoculation culture medium on RPE cell spheroidization in Example 6.

[0058] Attached Fig.11 This is the second day (D2) of inoculation culture for verifying the effect of the selection of RPE cell culture medium and inoculation culture medium on RPE cell spheroidization in Example 6.

[0059] Attached Fig.12 This is the third day (D3) of inoculation culture for verifying the effect of the selection of RPE cell culture medium and inoculation culture medium on RPE cell spheroidization in Example 6.

[0060] Attached Fig.13 This is the day 0 (D0) for verifying the effects of different concentrations of additives in the inoculation medium on RPE cell spheroidization in Example 7.

[0061] Attached Fig.14 This is the first day (D1) of the experiment to verify the effect of different concentrations of additives in the inoculation medium on the spheroidization of RPE cells in Example 7.

[0062] Attached Fig.15 This is the third day (D3) of the experiment to verify the effect of different concentrations of additives in the inoculation medium on the spheroidization of RPE cells in Example 7.

[0063] Attached Fig.16 This is to verify the effect of different selections of inoculation culture media on RPE cell spheroidization in Example 7.

[0064] Attached Fig.17 These are immunofluorescence detection images of the RPE cell sphere adherent growth in Example 8: (a) RPE65; (b) MITF; (c) ZO-1; (d) Tyrosinase and (e) BEST1.

[0065] Attached Fig.18 The RPE65 expression levels of the RPE cells in the four experimental groups and the control group iPSC cells were detected by flow cytometry in Example 8: (a) iPSC (0%), (b) iPSC-RPE-1 (98.52%), (c) iPSC-RPE-2 (99.69%), (d) iPSC-RPE-3 (99.45%), and (e) iPSC-RPE-4 (99.75%).

[0066] Attached Fig.19 The MITF expression levels of the RPE cells in the four experimental groups and the iPSC cells in the control group were detected by flow cytometry in Example 8: (a) iPSC (0%), (b) iPSC-RPE-1 (99.86%), (c) iPSC-RPE-2 (98.45%), (d) iPSC-RPE-3 (97.35%), and (e) iPSC-RPE-4 (98.56%).

[0067] Attached Fig. 20 The PAX6 expression levels of the RPE cells in the four experimental groups and the iPSC cells in the control group were detected by flow cytometry in Example 8: (a) iPSC (0%), (b) iPSC-RPE-1 (98.35%), (c) iPSC-RPE-2 (98.73%), (d) iPSC-RPE-3 (99.12%), and (e) iPSC-RPE-4 (99.22%).

[0068] Attached Fig.21 The expression levels of RPE65, MITF, BEST1 and PAX6 in the four groups of the qPCR detection experiment in Example 8.

[0069] Attached Fig. 22 The expression level of VEGF secretion in Experiment 4 detected by ELISA assay in Example 8. DETAILED DESCRIPTION

[0070] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0071] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0072] definition:

[0073] In order to more clearly understand the present invention, the following terms are defined:

[0074] Retinal pigment epithelial cell spheroid preparation: a preparation with RPE cell spheroids as the active ingredient.

[0075] RPE cells, Retinal Pigment Epithelium cells, are retinal pigment epithelial cells. They are classified as fRPE or RPE cells obtained by differentiation of pluripotent stem cells.

[0076] Among them, fRPE, fetal RPE, the source is: the source is obtained by separation and culture of the eyeball of aborted fetus, the specific method is the existing technology, and the method based on includes but is not limited to Chinese patent CN201310552005.0 "Isolation and Culture Method of Human Fetal Retinal Pigment Epithelial Cells". Due to the complex ethical review procedures and the limitation of the amount of materials, fetalRPE has great limitations in the treatment of macular degeneration. For this reason, the present application also studies the method of preparing RPE cell preparations by RPE cells obtained by differentiation of pluripotent stem cells. The RPE formed by directed induction of pluripotent stem cells has the characteristics of strong plasticity, convenient in vitro expansion preparation, safe and controllable quality and quantity, and no need for ethical review procedures, becoming the first choice for alternative treatment of macular degeneration.

[0077] RPE cells differentiated from pluripotent stem cells: RPE cells differentiated from pluripotent stem cells using a specific method. The species of the cells include but are not limited to mouse

[10] , Human

[11] , non-human primates

[12] .

[0078] RPE cell sphere: cell sphere or cell pellet that appears in the text, the definition of sphere: cells in a single well are evenly aggregated and clump together in a spherical shape, and there are no scattered cells or small cell clusters around.

[0079] Expansion: It is the process in which RPE cells disperse and proliferate to the surrounding monolayer after the RPE cell spheres adhere to the wall.

[0080] N2 (1×) means: a medium containing N2 at a volume concentration of 1% obtained by diluting commercially available N2 (100×) 100 times; the medium used for dilution is the basal medium in the inoculation medium.

[0081] B27 (1×) means: a B27 medium containing 1% by volume concentration obtained by diluting commercially available B27 (50×) 100 times; the medium used for dilution is the basal medium in the inoculation medium.

[0082] Centrifugation, normal cell centrifugation process parameters are all OK, such as 300g-500g, 5min-10min. In the embodiment of this application, for the purpose of specific description, the selected parameters are 300g, 5min.

[0083] KSR, Knockout serum replacement: is a chemically defined FBS-free medium used to replace FBS in cell culture.

[0084] In the text, paraformaldehyde, BSA, recombinant BSA, HSA, and recombinant HSA are all mass concentrations (%); FBS, KSR, Triton-X100, and non-essential amino acid solution are all volume concentrations (%).

[0085] Example 1. Preparation method of RPE cell spheres.

[0086] Method 1: The obtained RPE cells are cultured in RPE cell culture medium to obtain RPE cell suspension:

[0087] (1) Obtaining RPE cells, wherein the RPE cells are fRPE cells or RPE cells differentiated from pluripotent stem cells.

[0088] (2) RPE cells are cultured in RPE cell culture medium for 1-10 days to obtain RPE cells in good condition. The purpose is to ensure that the RPE cells for inoculation are in good condition.

[0089] (3) RPE cells were digested using commercial digestion solution, and after neutralization with culture medium, the supernatant was discarded by centrifugation to obtain RPE cell pellet.

[0090] (4) Each well is inoculated with 300-5000 RPE cells as the initial inoculum: After resuspending the RPE cell pellet with inoculation medium, the RPE cell suspension is inoculated into a cell culture plate, centrifuged and placed in an incubator for culture. This time is D0.

[0091] (5) Inoculation and culture method: RPE cells are cultured in a cell culture plate for 3 days, which is D3.

[0092] Method 2: Directly obtain RPE cell suspension:

[0093] (1) Obtaining an RPE cell suspension, wherein the RPE cell suspension is an fRPE cell suspension or an RPE cell suspension obtained by differentiation of pluripotent stem cells.

[0094] (2) Each well is inoculated with 300-5000 RPE cells as the initial inoculum: the RPE cell suspension is inoculated into the cell culture plate, and after centrifugation, it is placed in an incubator for culture. This time is D0.

[0095] (3) Inoculation and culture method: RPE cells are cultured in a cell culture plate for 3 days, which is D3.

[0096] The inoculation culture medium includes: basic culture medium and additives.

[0097] The basic culture medium includes but is not limited to any one of MEM, DMEM, Ham's F12, DMEM / F12, RPMI-1640, MEMα, IMDM, E6 or E8.

[0098] An optional embodiment of the additive is that the additive comprises the following components:

[0099] Component I: any one of 0.25-5% FBS, 0.25-5% KSR, 0.25-5% HSA, 0.25-5% BSA, 0.25-5% recombinant BSA, or 0.25-5% recombinant HSA;

[0100] Component II: either N2 (1×) or B27 (1×);

[0101] Component III: 1-100 mM of any one of Nicotinamide, Niacin, Niacin hydrochloride or L-Tryptophan;

[0102] Component IV: 10-200 nM EDN3;

[0103] Component V: 1-100 ng / mL of either FGF2 or EGF;

[0104] Component VI: 1-100 μM of any one of SB431542, A 83-01, Lefty-A or LY2157299;

[0105] Component VII: and 1-100 μM T3.

[0106] Another optional embodiment of the additive is that the additive includes the following components:

[0107] Component I: any one of 0.25-5% FBS, 0.25-5% HSA, 0.25-5% BSA, 0.25-5% recombinant BSA, or 0.25-5% recombinant HSA;

[0108] Component II: 1-100 μM CHIR-99021.

[0109] As an improvement to the above two embodiments, the additive further comprises Y27632, and the amount of Y27632 is 1-100 μM, so as to make the RPE cell sphere more compact.

[0110] Example 2 Verification of process parameters in the preparation method of RPE cell spheres.

[0111] In this embodiment, the inoculation culture medium is selected from one of the above combinations: 5% recombinant BSA, B27 (1×), 25 mM Niacin, 60 nM EDN3, 50 ng / mL EGF, 5 μM A83-01 and 30 μL / mL T3, and the basal culture medium is MEMα.

[0112] Condition 1: Choose whether to join Y27632; Combine Figure 1-Figure 3 By comparison, it can be concluded that when other conditions are the same, adding only 10 μM Y27632 to the inoculation medium or not adding Y27632 does not affect whether the RPE cells can form spheres, but only affects the compactness of the RPE cell spheres.

[0113] Condition 2: On D0 (the day of RPE cell inoculation), choose whether to centrifuge the inoculated RPE cells to precipitate the RPE cells; the centrifugation parameters can be: speed 300g, 5min. Figure 1-Figure 3 It can be compared that: when other conditions are the same, centrifugation is easier to form pellets than non-centrifugation. It is also verified that the applicant has also verified that the normal cell centrifugation process parameters, such as 300g-500g, 5min-10min, can produce the same effect as in this embodiment.

[0114] Condition 3: Initial cell seeding amount: This example compares the effects of 1000 cells, 2000 cells and 5000 cells seeding amount on RPE cell spheres. Figure 1-Figure 3 The conclusion is that different inoculation amounts do not affect spheroid formation, but affect the diameter of RPE cell spheres.

[0115] Table 1 Effect verification results of Example 2 (RPE cells obtained by differentiation of pluripotent stem cells)

[0116]

[0117]

[0118] In Table 1, + represents addition / centrifugation, and - represents no addition / no centrifugation; tightness and looseness are relative conclusions drawn only when a single parameter is changed under the same conditions.

[0119] The comprehensive conclusion is: the relevant drawings are as follows Figure 1-Figure 3(RPE cells differentiated from pluripotent stem cells). At D0, centrifugation had a greater impact on the spherical morphology of RPE cells. RPE cells could aggregate after centrifugation, while RPE cells were in a dispersed state before centrifugation.

[0120] However, under the same conditions, centrifugation made the cells aggregate into spheres on days D1-D3; and under centrifugation, the cell spheres with Y27632 added were more rounded and more regular than those without Y27632. This shows that the combination of centrifugation and Y27632 is the best choice, and spheroids can be formed when 1000-5000 cells are inoculated.

[0121] When the same number of cells were inoculated, the diameter of the spheres under centrifugation and Y27632 conditions was smaller, the cell boundaries were clearer, and the cell surface was smoother and tighter, indicating that the spheres were more compact.

[0122] Example 3: Verification of the spheroidization ability of RPE cells at low cell inoculation amounts in the method for preparing RPE cell spheres.

[0123] In this example, RPE cells were cultured in RPE cell seeding medium (with Y27632), centrifuged, and cultured for 3 days, and the relevant dosages were the same as in Example 2. The only difference from Example 2 was that the RPE cell seeding amount was 300 cells and 600 cells.

[0124] The inoculation medium of the present invention is selected from one of the above combinations: 5% recombinant BSA, B27 (1×), 25 mM Niacin, 60 nM EDN3, 50 ng / mL EGF, 5 μM A83-01 and 30 μL / mL T3, and the basal medium is MEMα.

[0125] Operation method: Choose whether to add Y27632 to the obtained RPE cells, and then choose whether to centrifuge, speed 300g, 5min. Use cell culture plates, culture conditions are Non-Treated, that is, non-adherent, suspension culture of RPE cells.

[0126] 10 μM Y27632 is added to this solution, which acts to tighten the cells.

[0127] Conclusion Figure 4 , RPE cells can still form spheres at inoculation levels of 300 cells and 600 cells.

[0128] Example 4 verifies the expansion performance of RPE cell spheres.

[0129] 1. Inoculation verification of single RPE cell spheres.

[0130] In Example 2, the inoculation culture medium: 5% recombinant BSA, B27 (1×), 25mM Niacin, 60nM EDN3, 50ng / mL EGF, 5μM A83-01 and 30μL / mL T3, whose basal culture medium is MEMα, and the RPE cell spheres obtained after 3 days of culture are used to verify the expansion performance of the RPE cell spheres.

[0131] RPE cell spheres obtained with an initial inoculation amount of 1000-5000 cells were selected for plating, adherence culture, and the cell adhesion was observed about one week after inoculation.

[0132] Combination Figure 5-Figure 7 The day of RPE cell sphere inoculation was recorded as D0.

[0133] Combination Figure 5 It can be seen that the RPE cell spheres obtained by centrifugation combined with the use of Y27632 have a tendency to expand outward at D0, but the whole is still in a spherical state; and gradually expand with the increase of days; combined with Figure 5-Figure 7 The initial inoculation amount of RPE cell spheres during the inoculation stage is 1000-2000 cells. RPE cell spheres have good expansion performance.

[0134] In summary, this embodiment has verified the effect of the above parameters on the cell state of RPE cell spheres after they are spheroidized and plated; at the same time, the applicant has also verified that the RPE cell spheres obtained with an initial inoculation amount of 300-600 cells and 600-1000 cells also have good expansion performance when plated.

[0135] 2. Verification of simultaneous inoculation of several RPE spherical preparations.

[0136] In order to simulate the in vivo RPE cell sphere attachment state in vitro, the present application adopted several small spheres for verification in the same well.

[0137] In Example 2, the inoculation culture medium: 5% recombinant BSA, B27 (1×), 25mM Niacin, 60nM EDN3, 50ng / mL EGF, 5μM A83-01 and 30μL / mL T3, whose basal culture medium is MEMα, and the RPE cell spheres obtained after 3 days of culture are used to verify the expansion performance of the RPE cell spheres.

[0138] The RPE cell spheres obtained with an initial inoculation amount of 1500 cells were selected for plating, adherence culture, and the cell adhesion was observed about one week after inoculation.

[0139] The day of plating was recorded as D0, and the cell morphology was observed about one week after inoculation. This example also verified the cell morphology after inoculation after multiple cell spheres were placed at the same time, as shown in the attached Figure 8 .

[0140] According to the attached Figure 8 It can be seen that: D0 is the state of just inoculating into the well plate, and the cells have not begun to expand. On D1, the cells on the outer edge of the sphere begin to detach from the sphere and expand by adhering to the wall. As time goes by, the cells will expand to the surrounding. On D5 and D6, the surface of the cell sphere continues to flatten, and the cells that detach from the sphere will gradually form a single layer or pseudo-multilayer arrangement.

[0141] By contrast, no matter it is to inoculate a RPE cell ball or multiple RPE cell balls simultaneously, no sticking occurs between the cell balls, and the RPE cells are uniformly expanded all around. The present embodiment only selects one of the initial inoculum amounts to carry out the performance verification of several RPE spherical preparations, and in actual operation, the applicant has also verified the RPE cells of multiple initial inoculum amounts such as 300, 600, 1000, 1500, 2000, 3000, 5000. The RPE cells of multiple initial inoculum amounts, when adopting multiple RPE cell balls to carry out bed boarding, all adopt multiple RPE cell balls to carry out bed boarding with the present embodiment and have the same technical effect.

[0142] Example 5

[0143] Effect of RPE cells from different sources in the same process on RPE cell spheroidization: The process parameters in this example are: 5% recombinant BSA, B27 (1×), 25mM Niacin, 60nM EDN3, 50ng / mL EGF, 5μM A83-01 and 30μL / mL T3, and the basal culture medium is MEMα.

[0144] The inoculation amounts for stage 2 were 1000 cells, 1500 cells, 2000 cells, 3000 cells, 4000 cells, and 5000 cells.

[0145] The source of RPE cells is fRPE cells.

[0146] like Fig. 9 It is shown that by culturing fRPE cells using the process of the present application, compact RPE cell spheres can be obtained at different inoculation doses.

[0147] Example 6

[0148] Verify the influence of the selection of RPE cell culture medium and inoculation medium on RPE cell spheroidization: In this example, only the RPE cell culture medium and inoculation medium were changed between the various schemes to verify the influence on cell spheroid formation. Other process parameters remained the same.

[0149] RPE cell culture medium selection: high glucose DMEM + 20% Knockout serum replacement (KSR) + 1% non-essential amino acid solution + 1mM L-Glutamine + 0.1mM β-mercaptoethanol

[13] .

[0150] The inoculation culture medium was selected as follows: 5% recombinant BSA, B27 (1×), 25 mM Niacin, 60 nM EDN3, 50 ng / mL EGF, 5 μM A 83-01 and 30 μL / mL T3, and the basic culture medium was MEMα.

[0151] In the following scheme, the cell inoculation amount in the stage 2 inoculation process is 1000 cells, and the inoculation volume is 100 μL. RPE cells are all derived from RPE cells differentiated from pluripotent stem cells. First, the RPE cells are cultured with the above two culture media, and then the cells are digested to obtain RPE cell suspension, and then the above two culture media are used to inoculate and produce cell spheres.

[0152] Scheme 1: Phase 1, culture with RPE cell culture medium for 2 days; Phase 2, use RPE cell culture medium for spheroid inoculation culture; and perform three parallel experiments;

[0153] Scheme 2: In stage 1, the cells were cultured in RPE cell culture medium for 2 days; in stage 2, the cells were cultured in inoculation medium for spheroids; and three parallel experiments were performed.

[0154] Scheme 3: In stage 1, the cells were cultured in inoculation medium (the inoculation medium was used for RPE cell culture at this time) for 2 days; in stage 2, the cells were cultured in RPE cell culture medium for spheroid inoculation; and three parallel experiments were performed;

[0155] Scheme 4: In stage 1, the cells were cultured in an inoculation medium (the inoculation medium was used for RPE cell culture at this time) for 2 days; in stage 2, the cells were cultured in an inoculation medium for spheroids; and three parallel experiments were performed.

[0156] After inoculation, the morphology of RPE cells in scheme 1-4 groups were photographed on the 1st, 2nd and 3rd days respectively. The experiment found that no cell spheroids were formed in scheme 1 and scheme 3 groups, while cell spheroids were formed in scheme 2 and scheme 4 groups.

[0157] The results showed that the preparation of RPE cell spheroid preparations was only related to the inoculation medium used in stage 2, and had nothing to do with the medium used during the RPE cell culture in stage 1. Moreover, the inoculation medium formula of the present invention could form cell spheres of uniform size. Fig.10 (Day 1 after inoculation) - Fig.12 (3rd day of culture after inoculation).

[0158] Example 7

[0159] Verify the effect of the selection of additives in the seeding medium on the sphere formation of RPE cells.

[0160] Scheme 1, the cell inoculation amount is 1000 cells, and the inoculation medium selected in Example 2 is: 10μM Y27632, recombinant BSA, B27 (1×), 25mM Niacin, 60nM EDN3, 50ng / mL EGF, 5μM A83-01 and 30μL / mL T3, and the basic medium is MEMα. Among them, the amount of recombinant BSA is selected as: 0.01%, 0.25%, 0.5%, 1%, 2.5%, 5%. Figures 13 to 15 The results showed that the culture medium containing 0.25%-5% BSA could form RPE cell spheres.

[0161] Scheme 2, the cell inoculation amount is 1000 cells, and the inoculation medium selection is: 10μM Y27632, 5% recombinant BSA, 10μM CHIR-99021 dosage. Fig.16 Under the same other conditions, the same amount of recombinant BSA, and different types of additives, RPE cell spheres can also be formed.

[0162] In this embodiment, only one of the initial inoculum amounts (1000 cells) is selected to verify the performance of the additive. In actual operation, the applicant has also verified RPE cells with multiple initial inoculum amounts of 300, 600, 1500, 2000, 3000, 5000, etc., and the results are similar to the conclusions drawn when the inoculum amount of 1000 cells is selected.

[0163] Example 8

[0164] Immunofluorescence detection of RPE cell sphere adhesion growth.

[0165] After the cultured cells reached fusion, the supernatant was discarded and the cells were washed once with DPBS. 4% paraformaldehyde was added and incubated at room temperature for 15 minutes. Then, the cells were washed 3 times with DPBS, each time for 2 minutes. 0.2% Triton-X100 was added for permeabilization for 10 minutes. The cells were blocked with DPBS containing 1% bovine serum albumin (BSA) at room temperature for 1 hour.

[0166] After aspirating BSA, add primary antibodies without washing: mouse monoclonal MITF antibody (the antibody is diluted 1:100 with diluent), rabbit monoclonal BEST1 antibody (the antibody is diluted 1:250 with diluent), rabbit monoclonal Tyrosinase antibody (the antibody is diluted 1:100 with diluent), rabbit monoclonal RPE65 antibody (the antibody is diluted 1:100 with diluent) and rabbit monoclonal ZO-1 antibody (the antibody is diluted 1:100 with diluent); incubate at 4°C overnight.

[0167] Wash with PBS 3 times, 5 minutes each time, then add secondary antibodies: 546-labeled goat anti-rabbit IgG antibody (the antibody is diluted with diluent at 1:1000), 546-labeled goat anti-mouse IgG antibody (the antibody is diluted with diluent at 1:1000), FITC-labeled goat anti-rabbit IgG antibody (the antibody is diluted with diluent at 1:1000) and FITC-labeled goat anti-mouse IgG antibody (the antibody is diluted with diluent at 1:1000) and incubate at room temperature for 2 hours. Then wash with DPBS 3 times, 5 minutes each time, then add DAPI staining solution and incubate at room temperature in the dark for 15 minutes. Finally, wash with DPBS 3 times, 5 minutes each time, and observe and take pictures directly under an inverted fluorescence microscope.

[0168] The adherent cells were plated and cultured with cell pellets formed by inoculation of 1500 cells. Immunofluorescence detection was performed on the 8th day. RPE cells specifically expressed RPE65, MITF, ZO-1, Tyrosinase and BEST1.

[0169] RPE cell spheres adherent growth immunofluorescence see attached Fig.17 , respectively detected are (a) RPE65; (b) MITF; (c) ZO-1; (d) Tyrosinase and (e) BEST1 immunofluorescence. The above cells can detect positive fluorescence expression of RPE65; MITF; ZO-1; Tyrosinase and BEST1, indicating that the adherent cells are the RPE cells required by this application.

[0170] Example 9

[0171] RPE cell sphere detection: RPE cells with seeding amounts of 2000 cells, 1500 cells, 1000 cells and 600 cells were inoculated and cultured for 3 days, and the expression of specific markers of RPE cells and the secretion of functional protein VEGF were detected by flow cytometry, qPCR and ELISA. iPSC was set as a negative control group, that is, a total of 5 groups, including 4 experimental groups:

[0172] iPSC-RPE-1: RPE spheres formed by seeding 2000 cells;

[0173] iPSC-RPE-2: RPE spheres formed by seeding 1500 cells;

[0174] iPSC-RPE-3: RPE spheres formed by seeding 1000 cells;

[0175] iPSC-RPE-4: 600 cells were seeded to form RPE spheres.

[0176] And negative control group 1:

[0177] iPSC: negative control group.

[0178] In the following experiments, the number of experimental cells used in each group was consistent to ensure the stability of the experiments.

[0179] Flow Cytometry:

[0180] After collecting the RPE cell spheres, the cells were digested and the RPE cell suspension digested into single cells was washed once with DPBS and incubated with 4% paraformaldehyde at room temperature for 10 minutes; washed once with DPBS, centrifuged and discarded the supernatant and then added with 0.2% Triton-X100 for permeabilization for 10 minutes; washed once with DPBS, centrifuged and discarded the supernatant and added with 1% bovine serum albumin (BSA) in DPBS for blocking at room temperature for 30 minutes.

[0181] After centrifugation and discarding BSA, PAX6 was incubated with FITC-labeled PAX6 antibody at room temperature for 0.5 hours, then washed once with PBS, and after discarding the supernatant, DPBS was added and analyzed under the flow cytometer system.

[0182] For MITF and RPE65, after absorbing BSA, primary antibodies were added directly without washing: mouse monoclonal MITF antibody (the antibody was diluted 1:100 with diluent) and rabbit monoclonal RPE65 antibody (the antibody was diluted 1:100 with diluent), and incubated at 4°C overnight.

[0183] Wash once with PBS, then add secondary antibodies: FITC-labeled goat anti-mouse IgG antibody (the antibody is diluted with diluent at 1:1000), PE-labeled goat anti-rabbit IgG antibody (the antibody is diluted with diluent at 1:1000) and incubate at room temperature for 1 hour. Then wash once with PBS, discard the supernatant and add DPBS for sample analysis under the flow cytometer system.

[0184] The expression levels of RPE65, MITF and PAX6 in the RPE of the experimental 4 groups and the iPSC cells of the control group were detected by flow cytometry. It was found that the expression of RPE-65 in the RPE of the experimental 4 groups was about 99%, as shown in the attached figure. Fig.18 ,(a) iPSC(0%),(b) iPSC-RPE-1(98.52%),(c) iPSC-RPE-2(99.69%),(d) iPSC-RPE-3(99.45%),(e) iPSC-RPE-4(99.75%);The expression of MITF in RPE of the four experimental groups was also similar and the expression was high, with the highest being 99.86%. For details, see the attached Fig.19 , (a) iPSC (0%), (b) iPSC-RPE-1 (99.86%), (c) iPSC-RPE-2 (98.45%), (d) iPSC-RPE-3 (97.35%), (e) iPSC-RPE-4 (98.56%); the PAX6 expression of RPE in the four experimental groups was also similar, and the expression was high, with the highest being 99.22%, as shown in the attached figure. Fig. 20 , (a) iPSC (0%), (b) iPSC-RPE-1 (98.35%), (c) iPSC-RPE-2 (98.73%), (d) iPSC-RPE-3 (99.12%), (e) iPSC-RPE-4 (99.22%). None of the iPSC control groups expressed the same protein.

[0185] Real-time fluorescence quantitative polymerase chain reaction (qPCR) detection:

[0186] The total RNA of each group of cells was extracted using an RNA extraction kit (Novozyme, China), and the OD was measured to ensure that the OD 260 / OD280 value of the RNA extracted from each group was between 1.8 and 2.1 to ensure its purity. Then the RNA was reverse transcribed using a reverse transcription kit (Novozyme, China) to obtain cDNA. The reaction system and reaction procedure refer to the product manual. The product cDNA was detected using qPCR technology, and the primer sequences are shown in Table 2. The reaction system refers to the SYBR kit (Bio, USA) manual. After instant centrifugation, the reaction was carried out on the machine, pre-denatured at 95°C for 5 minutes, and reacted for 40 cycles (94°C, 30 seconds; 59°C, 30 seconds; 72°C, 30 seconds). 2μL of the reaction product was aspirated and added with 3μL of SYBRGREEN MIX to mix well and then put on the machine.

[0187] Table 2 Primer sequences

[0188]

[0189] Note: F: Forward primer; R: Reversed primer; the product sequence length corresponding to the primer of GAPDH gene is 197bp; the product sequence length corresponding to the primer of RPE65 gene is 236bp; the product sequence length corresponding to the primer of MITF gene is 276bp; the product sequence length corresponding to the primer of PAX6 gene is 84bp; the product sequence length corresponding to the primer of BEST1 gene is 88bp.

[0190] The expression of RPE65, MITF, PAX6 and BEST1 in RPE cells was detected at the transcriptional level, and GAPDH was used as an internal reference gene to correct and standardize the expression of the target gene.

[0191] The results showed that compared with the iPSC control group, all four groups of the iPSC-RPE experiment expressed RPE65, MITF, BEST1, and PAX6, and the expression levels in the four groups were not much different, indicating the specificity and stability of the formed RPE spheres. Fig.21 .

[0192] ELISA test:

[0193] The human VEGF ELISA kit (Thermo, USA) was used to measure the cell supernatant of the iPSC-RPE experimental group 4 and the iPSC control group cells that were cultured for 2 days. The experimental steps are as follows: the antigen was diluted with the coating solution (the antigen was diluted with the diluent at 1:250) and added to the ELISA plate for overnight coating. The next day, it was taken out and cleaned, and the ELISA working solution was added for blocking treatment at room temperature for 1 hour; after washing, the standard and the corresponding sample were added to each well and incubated at room temperature for 2 hours; after washing, the primary antibody was added and incubated at room temperature for 1 hour (the antibody was diluted with the diluent at 1:250); after washing, the enzyme-labeled secondary antibody was added and incubated at room temperature for 30 minutes (the antibody was diluted with the diluent at 1:250); after washing, the substrate solution (TMB) was added for color development, incubated at room temperature for 10 minutes, and finally, the stop solution (2mol / L H2SO4) was added to each well to stop the reaction, and then the microplate reader was used for detection.

[0194] The experimental results showed that compared with the iPSC control group, the culture supernatants of the four iPSC-RPE experimental groups all expressed a large amount of VEGF (>1500pg / mL), and the VEGF secretion levels of the four groups of RPE cells were not much different, indicating that the formed RPE spheres were specific and stable. Fig. 22 .

Claims

1. A retinal pigment epithelial cell spheroid preparation, characterized in that: The retinal pigment epithelial cell spheroid preparation is obtained by the following method: Step 1: Obtain RPE cells; Step 2: Inoculate the RPE cells in a cell culture plate using an inoculation medium, and culture after centrifugation to allow a number of RPE cells to aggregate to form RPE cell spheres of uniform size.

2. A retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: The RPE cells are fRPE, or RPE cells obtained by differentiation of pluripotent stem cells.

3. A retinal pigment epithelial cell spheroid preparation according to claim 2, characterized in that: The pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells; the pluripotent stem cells are human pluripotent stem cells, mouse pluripotent stem cells, or monkey pluripotent stem cells.

4. A retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: Step one also includes: after obtaining the RPE cells, culturing the RPE cells using RPE culture medium for 0-10 days.

5. A retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: The inoculation medium contains 1-100 μM Y27632.

6. A retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: The inoculation medium includes an additive; the additive comprises any one of 0.25-5% FBS, KSR, HSA, BSA, recombinant BSA, or recombinant HSA.

7. A retinal pigment epithelial cell spheroid preparation according to claim 6, characterized in that: The additives also include: Either N2(1×) or B27(1×); 1-100 mM of any one of Nicotinamide, Niacin, Niacin hydrochloride or L-Tryptophan; 10-200 nM EDN3; 1-100 ng / mL of either FGF2 or EGF; 1-100 μM of any one of SB431542, A 83-01, Lefty-A or LY2157299, and 1-100 μM of T3.

8. A retinal pigment epithelial cell spheroid preparation according to claim 6, characterized in that: The additive includes a CHIR-99021 dosage of 1-100 μM.

9. The retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: The inoculation culture medium includes a basal culture medium; the basal culture medium is any one of MEM, DMEM, Ham's F12, DMEM / F12, RPMI-1640, MEMα, IMDM, and E6.

10. The retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: In step 2, the RPE cells are cultured in suspension.

11. The retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: In step 2, when the RPE cells are cultured based on the inoculation medium, the initial inoculation amount of the RPE cells per well is 300-5000 cells, and the diameter of the RPE cell sphere that can be formed is 50-400 μm.

12. The retinal pigment epithelial cell spheroid preparation according to claim 1, characterized in that: The RPE cell preparation has the following properties: a plurality of RPE cell spheres are plated in a single well of the same cell culture plate for 1-8 days, the RPE cell spheres do not adhere to each other, and each RPE cell sphere can achieve adherence and expansion.

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