Compatibility method of retinal pigment epithelial cell cryopreservation preparation

Abstract

The invention relates to a compatibility method of a retinal pigment epithelial cell cryopreservation preparation, which comprises the steps of dilution, balancing, washing and compatibility: 1) a dilution step: preparing a diluted solution which comprises a balanced salt solution (for perfusion), preferably, the diluted solution further comprises a compound amino acid injection; 2) a balancing step: resuscitating cells in the RPE cell cryopreservation preparation, then mixing the cells with the prepared diluted solution, and standing for balancing; (3) washing: centrifuging the solution, absorbing and discarding the supernate, adding a balanced salt solution (for perfusion), resuspending the cells, centrifuging, and absorbing and discarding the supernate; and 4) a compatibility step: adding a balanced salt solution (for perfusion) into the cell precipitate, and adjusting the cell to a required density according to a living cell density result.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and specifically to a method for preparing cryopreservation formulations for retinal pigment epithelial (RPE) cells. Background Technology

[0002] Vision is a crucial source of information about the external environment for humans and most animals. Retinal degeneration is a leading cause of blindness, severely impacting patients' quality of life. Retinal pigment epithelial cell transplantation has become a major area of ​​research and treatment.

[0003] Currently, there are generally two solutions for the clinical use of RPE cell preparations: one is to use fresh preparations, but this is generally limited by the stability issues of cell preparations and cannot be used immediately. It often requires in vitro culture and expansion followed by clinical injection within hours, making long-term storage impossible and limiting the shelf life of the cell preparation. The other is to use cryopreserved preparations for direct infusion after thawing, but this inevitably results in a large single infusion of dimethyl sulfoxide (DMSO), which can easily lead to safety risks. Furthermore, another problem is that the cell viability is insufficient after cryopreservation, limiting biological function.

[0004] Previous literature has not addressed the immediate application issues of the aforementioned RPE cell preparations. Other types of cell preparations involve reconstituted cells at high cell density and in small quantities before clinical administration, followed by thawing of the frozen preparations and reconstitution at a fixed density before use. However, these methods generally do not consider cell damage caused by a sudden drop in osmotic pressure after reconstitution. Existing research in the industry has not adequately considered clinical reconstitution solutions for RPE cell preparations suitable for ophthalmic injection, thus affecting the immediate use requirements of cell drugs.

[0005] To address the aforementioned issues, there is an urgent need for a washing and formulation method for RPE cell cryopreservation preparations to fully remove DMSO residues, ensure cell viability and biological function, and be suitable for retinal injection.

[0006] CN115869251A discloses a simple method for preparing retinal pigment epithelial cells. This patent provides a method for preparing fresh RPE cells into an injection solution, but the injection solution provided by this method is not ready for use and does not allow sufficient time for safety release.

[0007] CN111741735A discloses a composition of retinal pigment epithelial cells. This patent proposes a method for preparing human RPE cells that can be directly administered to subjects after thawing. The preparation requires the addition of 0.5%-15% DMSO. However, the RPE cells prepared by this method contain DMSO, which may pose a safety risk to the subjects.

[0008] Therefore, there is still a need in the field for washing and compatibility methods for RPE cell cryopreservation formulations that can effectively remove DMSO while ensuring cell viability and biological function. Summary of the Invention

[0009] This invention addresses the safety risks associated with direct infusion of ready-to-use cryopreserved RPE cell formulations leading to DMSO, as well as the issues of insufficient cell viability and limited biological function after cryopreservation. It provides a method for preparing cryopreserved RPE cell formulations for ophthalmic injection. After washing and preparing the cryopreserved RPE cell formulation using the method described in this application, the cell viability is greater than 90% and the biological activity is stable, facilitating further clinical application.

[0010] In one aspect, this application relates to a washing and compatibility method for RPE cell cryopreservation formulations, the method comprising dilution, equilibration, washing, and compatibility steps:

[0011] 1) Dilution step: Prepare the dilution solution;

[0012] 2) Equilibration step: Resuscitate the cells in the RPE cell cryopreservation formulation, then mix the cells with the prepared dilution solution and allow them to stand to equilibrate;

[0013] 3) Washing steps: After centrifuging the above solution and discarding the supernatant, add washing solution, resuspend the cells, centrifuge again, and discard the supernatant; and

[0014] 4) Preparation steps: Add the preparation solution to the cell pellet and adjust the cell density to the required level based on the live cell density results.

[0015] In some implementations, the RPE cell cryopreservation formulation is used for cryopreservation in... RPE cells in CS5 cell cryopreservation medium, with an RPE cell concentration of 1-5 × 10⁻⁶. 6 Cells / ml

[0016] In some embodiments, during the dilution step, the diluting solution comprises one or more of the following: a balanced salt solution (for perfusion), sodium lactate Ringer's solution, a compound electrolyte injection, sodium chloride injection, or glucose injection. Preferably, the diluting solution comprises a balanced salt solution (for perfusion).

[0017] In some embodiments, the dilution step includes preparing a diluent solution, which is one or more of the following: balanced salt solution (for perfusion), sodium lactate Ringer's solution, compound electrolyte injection, sodium chloride injection, or glucose injection. After dilution, the cell viability is greater than 85% after standing at room temperature for 1-4 hours. Therefore, the above solutions can be used as candidate solutions for compatibility. In some embodiments, sodium lactate Ringer's solution and balanced salt solution (for perfusion) show better cell viability and stability after dilution of RPE cells and standing at room temperature for 0-4 hours. Balanced salt solution (for perfusion) is a safe intraocular perfusion solution for intraocular medication; therefore, it is preferable to use balanced salt solution (for perfusion) as a compatibility solution.

[0018] In some embodiments, the dilution step includes preparing a diluent solution comprising one or more of a balanced salt solution (for perfusion), sodium lactate Ringer's solution, compound electrolyte injection, sodium chloride injection, or glucose injection. Preferably, the diluent solution further comprises a compound amino acid injection. In some embodiments, the compound amino acid injection is added at a rate (v / v%) of 2%-10%. In specific embodiments, the compound amino acid injection is added at a rate (v / v%) of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In a preferred embodiment, the compound amino acid injection is added at a rate (v / v%) of 5%.

[0019] Therefore, in some embodiments, the diluent is one or more of the following: a balanced salt solution (for perfusion), sodium chloride injection, sodium lactate Ringer's injection, or compound electrolyte injection; or a balanced salt solution (for perfusion), sodium chloride injection, sodium lactate Ringer's injection, or compound electrolyte injection containing 2-10% compound amino acid injection.

[0020] In some implementations, the diluted solution is prepared fresh for each use during the dilution step.

[0021] In some embodiments, during the equilibration step, cells in the RPE cryopreservation formulation are revived using a cell resuscitator, and the revived RPE cryopreservation formulation is then transferred to a biosafety cabinet. When diluting the RPE cryopreservation formulation, the DMSO is diluted exothermically and the osmotic pressure suddenly decreases, which can lead to cell damage. Therefore, allowing the formulation to stand for a certain period after dilution may help improve cell viability. Thus, in some embodiments, during the equilibration step, the diluent solution is allowed to stand at a suitable temperature for 0-4 hours after diluting the RPE cryopreservation formulation.

[0022] In some embodiments, during the equilibration step, after thawing the cells in the RPE cell cryopreservation formulation, the cells are mixed with a dilution solution and incubated at a suitable temperature to equilibrate and improve cell viability. In specific embodiments, the suitable temperature is 10-30°C, for example, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C, or any range between any two of the above values, for example, 10-15°C, 10-20°C, 15-20°C, 15-25°C, 20-25°C, or 25-30°C. In a preferred embodiment, the suitable temperature is room temperature (15-25°C).

[0023] In some embodiments, after thawing cells from the RPE cell cryopreservation formulation, the cells are mixed with a dilution solution and allowed to equilibrate at a suitable temperature for 0.5-5 hours to improve cell viability. In specific embodiments, after thawing cells from the RPE cell cryopreservation formulation, the cells are mixed with a dilution solution and allowed to equilibrate at a suitable temperature for, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours, preferably 1 hour.

[0024] In some embodiments, after diluting the RPE cryopreservation formulation with different dilution solutions and allowing it to stand at room temperature for 1-4 hours, the cell viability was significantly improved and all exceeded 90%. In a preferred embodiment, to reduce the time spent on washing and preparation, the standing time was 1 hour.

[0025] In the washing step, after centrifuging the solution and discarding the supernatant, the washing solution is added, the cells are resuspended, centrifuged again, and the supernatant is discarded.

[0026] In some embodiments, the solution is centrifuged at 500-1000×g for 2-10 minutes during the washing step. In a preferred embodiment, the solution is centrifuged at 800×g for 5 minutes during the washing step.

[0027] In some implementations, the cells are washed once, twice, or three times during the washing step, preferably once.

[0028] In some embodiments, during the washing step, the washing solution comprises one or more of the following: a balanced salt solution (for perfusion), sodium lactate Ringer's solution, compound electrolyte solution, sodium chloride solution, or glucose solution. In some embodiments, during the washing step, the washing solution further comprises 2-10% compound amino acid solution. Therefore, in some embodiments, the washing solution comprises a balanced salt solution (for perfusion) containing 2-10% compound amino acid solution, sodium chloride solution, sodium lactate Ringer's solution, or compound electrolyte solution. In a preferred embodiment, the washing solution is a balanced salt solution (for perfusion) or a balanced salt solution (for perfusion) containing 5% compound amino acid solution.

[0029] In the preparation step, the preparation solution is added to the cell pellet, and the cells are gently resuspended by aspiration. Cell viability and viable cell density are determined using the AO / PI method. Then, based on the viable cell density results, the cell suspension is adjusted to the desired density, for example, 1.5 × 10⁻⁶. 6 per ml.

[0030] In some embodiments, during the reconstitution step, the reconstitution solution comprises one or more of the following: balanced salt solution (for perfusion), sodium lactate Ringer's solution, compound electrolyte injection, sodium chloride injection, or glucose injection. In some embodiments, during the reconstitution step, the reconstitution solution further comprises 2-10% compound amino acid injection. Therefore, in some embodiments, the reconstitution solution comprises a balanced salt solution (for perfusion) containing 2-10% compound amino acid injection, sodium chloride injection, sodium lactate Ringer's solution, or compound electrolyte injection. In a preferred embodiment, the reconstitution solution is a balanced salt solution (for perfusion) or a balanced salt solution (for perfusion) containing 5% compound amino acid injection.

[0031] In another aspect, stability studies are conducted on the formulated RPE cell suspension. Specifically, the stability studies include placing the formulated RPE cell suspension at room temperature or low temperature and sampling at different time points to detect cell viability and biological function. The room temperature is, for example, 10-30°C, preferably 15-25°C. The low temperature is, for example, 0-10°C, preferably 2-8°C. In some embodiments, the RPE cryopreservation formulation is placed at room temperature or low temperature for a period of time after formulation. In some embodiments, the RPE cryopreservation formulation is placed at 15-25°C or 2-8°C for 0-8 hours after formulation.

[0032] In some embodiments, after washing the RPE cell cryopreservation formulation according to the method of the present invention and placing it at room temperature (15-25°C) or 2-8°C for 0-8 hours, the cell viability is not less than 90%, and the melanin content and PEDF secretion are stable. Therefore, the stability time for use after formulation is not less than 8 hours.

[0033] In some implementations, different solutions are used at different stages of the washing process.

[0034] In some embodiments, a balanced salt solution (for perfusion) + 5% compound amino acid injection is used in the dilution step, a balanced salt solution (for perfusion) + 5% compound amino acid injection is used in the washing step, and a balanced salt solution (for perfusion) + 5% compound amino acid injection is used in the compatibility step.

[0035] In some implementations, a balanced salt solution (for perfusion) is used in the dilution step, in the washing step, and in the compatibility step.

[0036] In some implementations, a balanced salt solution (for perfusion) + 5% compound amino acid injection is used in the dilution step, a balanced salt solution (for perfusion) is used in the washing step, and a balanced salt solution (for perfusion) is used in the compatibility step.

[0037] In a preferred embodiment, a balanced salt solution (for perfusion) + 5% compound amino acid injection is used in the dilution step. In another preferred embodiment, a balanced salt solution (for perfusion) + 5% compound amino acid injection is used in the dilution step, a balanced salt solution (for perfusion) is used in the washing step, and a balanced salt solution (for perfusion) is used in the compatibility step.

[0038] In another aspect, the present invention also relates to the treatment of various ocular diseases with RPE cells obtained by the method described in the present invention, including but not limited to retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD).

[0039] This application relates to a washing and compatibility method for RPE cell cryopreservation formulations, the method comprising dilution, equilibration, washing, and compatibility steps:

[0040] 1) Dilution step: Prepare a diluent solution, wherein the diluent solution contains a balanced salt solution (for perfusion), preferably, the diluent solution further contains a compound amino acid injection, more preferably, the diluent solution is a balanced salt solution (for perfusion) + 5% compound amino acid injection;

[0041] 2) Equilibration step: Resuscitate the cells in the RPE cell cryopreservation formulation, then mix the cells with the prepared dilution solution and allow them to stand to equilibrate;

[0042] 3) Washing steps: After centrifuging the above solution and discarding the supernatant, add balanced salt solution (for perfusion), resuspend the cells, centrifuge again, and discard the supernatant; and

[0043] 4) Preparation steps: Add balanced salt solution (for perfusion) to the cell pellet, and adjust the cell density to the required level based on the live cell density results.

[0044] The method described in this application is applicable to the preparation of primary RPE cryopreserved cells. Furthermore, using commercially available sodium chloride injection, balanced salt solution (for perfusion), or buffered salt solution from other manufacturers as the preparation solution can achieve the same effect. Using commercially available compound amino acid injection from other manufacturers as a diluent additive or preparation solution additive can also achieve the same effect.

[0045] The following are examples of commercially available balanced salt solutions (for perfusion), sodium lactate Ringer's solution, compound electrolyte injection, sodium chloride injection, glucose injection, and compound amino acid injection:

[0046] Balanced salt solutions (for perfusion) are available in the following drug categories: National Drug Approval Number HJ20171363, National Drug Approval Number H20233038, National Drug Approval Number H20183074, National Drug Approval Number H20183404, National Drug Approval Number H20233247, and National Drug Approval Number H20233285.

[0047] Sodium lactate Ringer's injection is available in the following drug approval numbers: H19983144, H20044961, H20044239, H20033736, H20064929, and H20023666.

[0048] The following compound electrolyte injections are already on the market: National Drug Approval Number H2000047, National Drug Approval Number H20133245, National Drug Approval Number H20173380, National Drug Approval Number H20113035, National Drug Approval Number H20123411, National Drug Approval Number H20113476, etc.

[0049] Sodium chloride injections that have been marketed include: National Drug Approval Numbers H12020025, H65020253, H63020182, H62021250, H61020100, and H61020021.

[0050] The following are the approved drug names for glucose injection: H12020022, H61020032, H32024826, H20013100, H22023070, and H20056816.

[0051] The following compound amino acid injections are already on the market: National Drug Approval Number H19993803, National Drug Approval Number H19993299, National Drug Approval Number H20054765, National Drug Approval Number H19999094, National Drug Approval Number H20033620, National Drug Approval Number H20103092, etc.

[0052] Unless otherwise indicated, all reagents used in this invention are commercially available. All instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus.

[0053] Technical effects achieved by the present invention

[0054] The RPE cell preparation prepared by washing according to the method of the present invention can be placed at room temperature (15-25℃) or 2-8℃ for 0-8h with a cell viability of not less than 90%, stable melanin content and PEDF secretion, and no DMSO residue was detected in the supernatant.

[0055] This invention can be implemented through the following exemplary embodiments; however, those skilled in the art will understand that this invention is not limited to these exemplary embodiments. Those skilled in the art can make corresponding modifications to the exemplary embodiments of this invention as needed, and the modified embodiments are also included within the scope of this invention.

[0056] 1. A washing and compatibility method for a cryopreservation formulation of retinal pigment epithelial (RPE) cells, the method comprising dilution, equilibration, washing, and compatibility steps.

[0057] 1) Dilution step: Prepare the dilution solution;

[0058] 2) Equilibration step: Resuscitate the cells in the RPE cell cryopreservation formulation, then mix the cells with the prepared dilution solution and allow them to stand to equilibrate;

[0059] 3) Washing steps: After centrifuging the above solution and discarding the supernatant, add washing solution, resuspend the cells, centrifuge again, and discard the supernatant; and

[0060] 4) Preparation steps: Add the preparation solution to the cell pellet and adjust the cell density to the required level based on the live cell density results.

[0061] 2. The method described in Implementation Scheme 1, wherein the diluent comprises one or more of the following: balanced salt solution (for perfusion), sodium lactate Ringer's solution, compound electrolyte injection, sodium chloride injection, or glucose injection.

[0062] Preferably, the diluting solution comprises a balanced salt solution (for perfusion), and more preferably, the diluting solution further comprises a compound amino acid injection.

[0063] 3. The method described in Implementation Scheme 2, wherein the compound amino acid injection is added at a ratio (v / v%) of 2%-10%, preferably, the compound amino acid injection is added at a ratio (v / v%) of 5%.

[0064] 4. The method according to any one of embodiments 1-3, wherein in the equilibration step, after the cells in the RPE cell cryopreservation preparation are thawed, the cells are mixed with a dilution solution and allowed to stand at a suitable temperature to equilibrate in order to improve cell viability.

[0065] 5. The method described in Implementation Scheme 4, wherein the suitable temperature is 10-30℃, preferably 15-25℃.

[0066] 6. The method described in implementation scheme 4 or 5, wherein the cells are allowed to stand for equilibration for 0.5-5 hours to improve cell viability, preferably for 1 hour.

[0067] 7. The method of embodiment 1, wherein the washing solution comprises one or more of the following: balanced salt solution (for perfusion), sodium lactate Ringer's injection, compound electrolyte injection, sodium chloride injection, or glucose injection; preferably, the washing solution comprises balanced salt solution (for perfusion).

[0068] 8. The method of embodiment 7, wherein the washing solution further comprises a compound amino acid injection, wherein the compound amino acid injection is added at a ratio (v / v%) of 2%-10%, preferably, the compound amino acid injection is added at a ratio (v / v%) of 5%.

[0069] 9. The method described in Implementation Scheme 1, wherein in the washing step, the cells are washed once, twice, or three times, preferably once.

[0070] 10. The method described in Implementation Scheme 1, wherein in the compatibility step, the compatibility solution comprises one or more of the following: balanced salt solution (for perfusion), sodium lactate Ringer's injection, compound electrolyte injection, sodium chloride injection, or glucose injection; preferably, the compatibility solution comprises balanced salt solution (for perfusion).

[0071] 11. The method of embodiment 10, wherein the compatibility solution further comprises a compound amino acid injection, wherein the compound amino acid injection is added at a ratio (v / v%) of 2%-10%, preferably, the compound amino acid injection is added at a ratio (v / v%) of 5%.

[0072] 12. The method of any one of the foregoing embodiments, wherein the dilution step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection, the washing step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection, and the compatibility step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection; or

[0073] A balanced salt solution (for perfusion) is used in the dilution step, in the washing step, and in the preparation step; or

[0074] The dilution step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection; the washing step uses a balanced salt solution (for perfusion); and the compatibility step uses a balanced salt solution (for perfusion).

[0075] Preferably, the dilution step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection, the washing step uses a balanced salt solution (for perfusion), and the compatibility step uses a balanced salt solution (for perfusion).

[0076] 13. Use of RPE cells obtained by the method of any one of the foregoing embodiments in the preparation of a medicament for treating eye diseases, wherein the eye diseases include, but are not limited to, retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD). Example

[0077] Example 1 - Washing and compatibility method of RPE cell cryopreservation formulation

[0078] 1.1 Experimental Materials

[0079] RPE cells were differentiated from human embryonic stem cells (from the National Stem Cell Resource Bank) in our laboratory.

[0080] RPE cell cryopreservation solution The CS5 was purchased from BioLife Solutions, Inc.

[0081] AO / PI (RE010212) was purchased from Shanghai Ruiyu Biotechnology Co., Ltd.

[0082] The balanced salt solution (for perfusion) was purchased from Alcon Research LLC, import drug registration certificate number HJ20171363.

[0083] The compound amino acid injection (National Drug Approval Number H19993803) was purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd.

[0084] 1.2 Instruments and Equipment

[0085] Shanghai Ruiyu Biotechnology Co., Ltd.'s fully automated cell fluorescence analyzer: Countstar Rigel S2;

[0086] Suzhou Antai Air Technology Co., Ltd. Biosafety Cabinet: BSC-1304IIA2;

[0087] Hangzhou Comers Technology Co., Ltd. Cell Resuscitation Instrument: ThawHome-AB;

[0088] Thermo Fisher Scientific centrifuge: ST40.

[0089] 1.3 Compatibility Methods

[0090] (1) Preparation of dilution solution: In the biosafety cabinet, take 19ml of balanced salt solution (for perfusion) and add 1ml of compound amino acid injection to prepare a dilution solution. Prepare the solution immediately before use.

[0091] (2) Cell resuscitation: Take out one vial of RPE cryopreservation preparation, revive it with a cell resuscitation device and transfer it to a biosafety cabinet.

[0092] (3) Dilution equilibration: Transfer all RPE cells from the cryopreservation tube to a 15ml centrifuge tube, slowly add 9ml of dilution solution along the tube wall, and mix gently. Incubate at room temperature (generally 15-25℃) for 1 hour to equilibrate.

[0093] (4) Washing

[0094] a) Centrifuge at 800×g for 5 minutes, slowly aspirate the supernatant using a suction aid, and remove any remaining supernatant with a 200μl pipette.

[0095] b) Add 5 ml of balanced salt solution (for perfusion), gently aspirate and resuspend the cells.

[0096] c) Centrifuge at 800×g for 5 minutes, slowly aspirate the supernatant using a suction aid, and remove any remaining supernatant with a 200μl pipette.

[0097] (5) Compatibility

[0098] a) Add 300 μl of balanced salt solution (for perfusion) to the cell pellet, gently aspirate and resuspend the cells, and determine cell viability and viable cell density using the AO / PI method.

[0099] b) Based on the viable cell density results, adjust the cell suspension to 1.5 × 10⁻⁶. 6 per ml.

[0100] 1.4 Stability Study

[0101] Three batches of prepared RPE cell suspensions were incubated at room temperature and 2-8℃ for 8 hours, respectively, and samples were taken at different time points to detect cell viability and biological function. The results are shown in Tables 1 and 2.

[0102] Table 1 Results of Clinical Compatibility Stability Study – Room Temperature Placement

[0103]

[0104]

[0105] Table 2 Results of Clinical Compatibility Stability Study – Placement at 2-8℃

[0106]

[0107] Clinical compatibility stability studies have shown that after recombining the RPE cryopreserved formulation, the cell viability remains at no less than 90% when stored at room temperature (15-25℃) or 2-8℃ for 0-8 hours, and the melanin content and PEDF secretion remain stable. Therefore, the stability period after recombining is no less than 8 hours.

[0108] Example 2 - Study on Washing Compatibility Methods

[0109] 2.1 Experimental Materials

[0110] All RPE cells were differentiated from human embryonic stem cells (from the National Stem Cell Resource Bank) in our laboratory.

[0111] AO / PI (RE010212) was purchased from Shanghai Ruiyu Biotechnology Co., Ltd.

[0112] The balanced salt solution (for perfusion) was purchased from Alcon Research LLC, import drug registration certificate number H20171363.

[0113] The sodium lactate Ringer's injection (National Drug Approval Number H19983144) was purchased from Shanghai Baxter Medical Supplies Co., Ltd.

[0114] The compound electrolyte injection (National Drug Approval Number H20000475) was purchased from Shanghai Baxter Medical Supplies Co., Ltd.

[0115] Sodium chloride injection (National Drug Approval Number H12020025) was purchased from Otsuka Pharmaceutical Co., Ltd., China.

[0116] The glucose injection (National Drug Approval Number H12020022) was purchased from Otsuka Pharmaceutical Co., Ltd., China.

[0117] The compound amino acid injection (National Drug Approval Number H19993803) was purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd.

[0118] 2.2 Study on Compatibility of Solutions

[0119] 2.2.1 Screening of Compatible Solutions

[0120] RPE cells were resuscitated and diluted with five different incompatible solutions. Cell viability was measured after incubation at room temperature for 0–4 h. Based on the cell viability results after different incubation times, the incompatible solutions that were most effective in maintaining cell viability were selected. The results are shown in Table 3.

[0121] Table 3 Results of the screening study on compatible solutions

[0122]

[0123] Results analysis:

[0124] 1) After dilution and standing for 1-4 hours, the cell viability of sodium lactate Ringer's injection, balanced salt solution (for perfusion), compound electrolyte injection, and sodium chloride injection is greater than 85%, and they can be used as candidate solutions for compatibility.

[0125] 2) After diluting RPE cells with sodium lactate Ringer's injection and balanced salt solution (for perfusion), the cell viability and stability were better after standing at room temperature for 0-4 hours. Moreover, as an intraocular perfusion solution, balanced salt solution (for perfusion) ensures the safety of intraocular medication. Therefore, balanced salt solution (for perfusion) is preferred as the compatibility solution for subsequent studies.

[0126] 2.2.2 Dosage Study of Compound Amino Acid Injection

[0127] Based on the applicant's earlier research, adding amino acids to the resuspension solution can improve the viability of RPE cells after resuspension. A compatibility solution was prepared by adding 2%-10% of a compound amino acid injection to a balanced salt solution (for perfusion), and this solution was used to prepare RPE cryopreservation formulations. After standing at room temperature for 0-4 hours, cell viability was measured to study the effect of different proportions of compound amino acid injection on cell viability. The results are shown in Table 4.

[0128] Table 4. Results of studies involving the addition of compound amino acid injections in different proportions.

[0129]

[0130] The results showed that adding 2%-10% compound amino acid injection to the balanced salt solution (for perfusion) improved cell viability in all RPE cryopreservation formulations. The improvement was most significant with the addition of 5% compound amino acid injection, therefore this was the preferred dosage for the additive.

[0131] 2.2.3 Study on different stages of washing and compatibility solutions

[0132] To simplify the excipient composition and ensure cell viability after preparation, only ophthalmic balanced salt solution (for perfusion) was used during the washing and preparation stages, and cell viability was investigated after incubation at room temperature for 0-4 hours. The results are shown in Table 5.

[0133] Table 5. Results of solutions at different stages of washing compatibility study.

[0134]

[0135] The results showed that, during the dilution phase, using a balanced salt solution (for perfusion) containing 5% compound amino acid injection resulted in significantly higher cell viability compared to the control group without the added compound amino acid injection. After removing the compound amino acid injection during the washing and preparation phases, the cell viability after preparation was similar to that of the control group without removal. Therefore, a balanced salt solution containing 5% compound amino acid injection (for perfusion) is preferred for the dilution phase, and a balanced salt solution (for perfusion) is preferred for both the washing and preparation phases.

[0136] 2.3 Compatibility Study

[0137] 2.3.1 Study on standing time after dilution

[0138] When diluting RPE cryopreservation formulations, DMSO releases heat and its osmotic pressure suddenly decreases, which can lead to cell damage. Therefore, allowing the cryopreservation formulation to stand for a certain period of time after dilution may help improve cell viability.

[0139] The RPE cryopreservation formulation was diluted with different diluents and then allowed to stand at room temperature for 0-4 hours before washing, compatibility testing, and cell viability detection. The effect of the dilution time on cell viability was studied, and the results are shown in Table 6.

[0140] Table 6 Results of the study after dilution and standing at room temperature

[0141]

[0142] The results showed that after diluting the RPE cryopreservation formulation with different dilution solutions and allowing it to stand at room temperature for 1-4 hours, the cell viability was significantly improved, and all exceeded 90%. Therefore, the RPE cryopreservation formulation should be allowed to stand for 1-4 hours after dilution before subsequent washing and preparation. To reduce the time spent on washing and preparation, a standing time of 1 hour is preferred.

[0143] 2.3.2 Study on the number of washes

[0144] The RPE cryopreservation formulation contains 5% DMSO (v / v). The Chinese Pharmacopoeia, Volume III, 0861, "Residual Solvent Determination Method," requires that the residual DMSO content be less than 0.5% (equivalent to 5000 ppm). To determine the number of washes required to meet the pharmacopoeia requirement for DMSO residue after reconstitution, this study performed reconstitution after 1, 2, and 3 washes, and measured cell viability and DMSO residue after room temperature storage. The results are shown in Table 7.

[0145] Table 7. Results of cell viability and DMSO residue detection after different washing cycles.

[0146]

[0147] Note: The limit of quantitation for DMSO detection method is 1000 ppm. Quantities below 1000 ppm will be reported as not detected.

[0148] Based on the cell viability and DMSO residue determination results, after washing 1-3 times, the cell viability after preparation was greater than 90%, and no DMSO residue was detected in the supernatant, which meets the requirements of the Chinese Pharmacopoeia. Therefore, washing 1-3 times is acceptable, with 1 wash being preferred.

[0149] Although exemplary embodiments of the present invention have been shown and described in this application, those skilled in the art will understand that the above embodiments should not be construed as limiting the scope of the present invention, and that changes, substitutions and modifications may be made without departing from the spirit, principles and scope of the present invention.

Claims

1. A washing and compatibility method for a cryopreservation formulation of retinal pigment epithelial (RPE) cells, the method comprising dilution, equilibration, washing, and compatibility steps. 1) Dilution step: Prepare the dilution solution; 2) Equilibration step: Resuscitate the cells in the RPE cell cryopreservation formulation, then mix the cells with the prepared dilution solution and allow them to stand to equilibrate; 3) Washing steps: After centrifuging the above solution and discarding the supernatant, add washing solution, resuspend the cells, centrifuge again, and discard the supernatant; and 4) Preparation steps: Add the preparation solution to the cell pellet and adjust the cell density to the required level based on the live cell density results.

2. The method of claim 1, wherein the diluent comprises one or more of the following: balanced salt solution (for perfusion), sodium lactate Ringer's solution, compound electrolyte injection, sodium chloride injection, or glucose injection. Preferably, the diluting solution comprises a balanced salt solution (for perfusion), and more preferably, the diluting solution further comprises a compound amino acid injection.

3. The method according to claim 2, wherein the compound amino acid injection is added at a ratio (v / v%) of 2%-10%, preferably, the compound amino acid injection is added at a ratio (v / v%) of 5%.

4. The method of any one of claims 1-3, wherein in the equilibration step, after the cells in the RPE cell cryopreservation formulation are thawed, the cells are mixed with a dilution solution and allowed to equilibrate at a suitable temperature to improve cell viability.

5. The method of claim 4, wherein the suitable temperature is 10-30°C, preferably 15-25°C.

6. The method of claim 4 or 5, wherein the cells are allowed to stand for equilibration for 0.5-5 hours to improve cell viability, preferably for 1 hour.

7. The method of claim 1, wherein the washing solution comprises one or more of a balanced salt solution (for perfusion), sodium lactate Ringer's injection, compound electrolyte injection, sodium chloride injection, or glucose injection, preferably, the washing solution comprises a balanced salt solution (for perfusion).

8. The method of claim 7, wherein the washing solution further comprises a compound amino acid injection, wherein the compound amino acid injection is added at a ratio (v / v%) of 2%-10%, preferably, the compound amino acid injection is added at a ratio (v / v%) of 5%.

9. The method of claim 1, wherein in the washing step, the cells are washed once, twice or three times, preferably once.

10. The method of claim 1, wherein in the compatibility step, the compatibility solution comprises one or more of a balanced salt solution (for perfusion), sodium lactate Ringer's injection, compound electrolyte injection, sodium chloride injection, or glucose injection, preferably, the compatibility solution comprises a balanced salt solution (for perfusion).

11. The method of claim 10, wherein the compatibility solution further comprises a compound amino acid injection, wherein the compound amino acid injection is added at a ratio (v / v%) of 2%-10%, preferably, the compound amino acid injection is added at a ratio (v / v%) of 5%.

12. The method of any one of the preceding claims, wherein the dilution step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection, the washing step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection, and the compatibility step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection; or A balanced salt solution (for perfusion) is used in the dilution step, in the washing step, and in the preparation step; or The dilution step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection; the washing step uses a balanced salt solution (for perfusion); and the compatibility step uses a balanced salt solution (for perfusion). Preferably, the dilution step uses a balanced salt solution (for perfusion) + 5% compound amino acid injection, the washing step uses a balanced salt solution (for perfusion), and the compatibility step uses a balanced salt solution (for perfusion).

13. Use of RPE cells obtained by the method of any one of the preceding claims in the preparation of a medicament for treating eye diseases, wherein the eye diseases include, but are not limited to, retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD).

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