Culture medium for retinal pigment epithelial cells and use thereof

Abstract

The application provides a culture medium of retinal pigment epithelial cells and application thereof, and particularly provides a serum-free culture medium for culturing retinal pigment epithelial cells, which is a culture solution containing a basic culture medium and additives, wherein the additives include human platelet lysate (HPL) and heparin, and the culture solution does not contain animal serum. The human retinal pigment epithelial cells cultured by the culture medium provided by the application not only have a high proportion of cells with typical hexagonal cobblestone-like morphology and positive protein marker RPE65 (i.e. RPE65 + retinal pigment epithelial cells) of up to 94.5%.

Description

Technical Field

[0001] This invention relates to the field of cell culture. More specifically, this invention relates to a culture medium for retinal pigment epithelial cells and its applications. Background Technology

[0002] Degenerative retinal diseases are the leading cause of irreversible blindness, including age-related macular degeneration (AMD), retinitis pigmentosa, and Stargardt's disease. Globally, there are approximately 30 to 50 million people with AMD and about 1 million with retinitis pigmentosa. Currently, there are over 20 million people with AMD in my country, and this number is projected to double by 2050.

[0003] Since retinal degenerative diseases primarily manifest as damage to the retinal pigment epithelium (RPE) in the macular region, regenerative medicine, particularly RPE cell replacement therapy, has become a major focus of research. To date, various RPE cell transplantations have been reported, including autologous patient RPE cell transplantation, iris pigment epithelium (IPE) cell transplantation, embryonic and adult RPE cell transplantation, and RPE cell transplantation derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). These cell transplantations have all demonstrated clear therapeutic effects in animal experiments, but each also has its own limitations. Autologous RPE transplantation is a complex procedure with complications such as PVR and AMD recurrence. While IPE has therapeutic effects in certain retinal degeneration models, expression profiling studies of IPE and RPE show that IPE lacks certain key enzymes in the rhodopsin cycle and cannot completely replace the physiological functions of RPE; related research has been decreasing in recent years. The clinical application of embryonic stem cells is ethically controversial, and their safety remains a concern due to potential tumorigenicity. iPSCs are introduced with potential proto-oncogenes during construction, and retroviral integration into the host genome also increases the risk of tumorigenesis; recent studies have also confirmed that iPSCs have a higher tendency to form teratomas than embryonic stem cells. Although some studies have reported reducing or completely removing exogenous transcription factors, using small molecule compounds, or removing them after introduction, the safety of iPSCs before clinical application requires further verification.

[0004] Studies have shown that human RPE cells have low immunogenicity, do not elicit a significant specific immune response from T cells, and can inhibit T cell activation, thus avoiding the use of immunosuppressants. They also possess a high capacity for in vitro proliferation, allowing one human eyeball to be used to treat hundreds of AMD patients, alleviating the limitations on sourcing, economic burden, and ethical controversies associated with the clinical application of human RPE cells. In vivo, human RPE cells can secrete trophic factors and form synaptic connections with the host retina. They also exhibit significantly lower tumorigenicity than stem cell-derived RPE cells and can express a variety of RPE-specific functional markers, making them one of the most promising cell sources for retinal pigment epithelial cell transplantation replacement therapy.

[0005] Previously, the isolation and culture of retinal pigment epithelial cells (RPCs) were conducted in culture systems supplemented with animal serum (primarily FBS). On the one hand, animal serum carries the risk of xenogeneic protein immune rejection, and its composition is unclear, potentially containing animal-derived pathogens. It is susceptible to infection by viruses, mycoplasma, and mad cow disease virus, theoretically leading to the transmission of some pathogens during the production of clinical-grade human cell preparations, thus posing a potential danger in clinical applications. On the other hand, batch-to-batch variations in serum result in significant differences in cell phenotype, proliferation capacity, and long-term cell function between different batches, directly impacting the clinical application of RPCs. Therefore, for large-scale production and widespread clinical application of human RPCs, an in vitro culture method free of animal-derived components is essential. In 2008, David M. Gamm et al. proposed culturing human RPCs in a serum-free medium composed of F12 mixed nutrients and B27, but due to its numerous components, complex preparation, and high cost, it is not suitable for large-scale clinical production.

[0006] Therefore, there is an urgent need in this field to develop a culture medium free of animal serum (such as fetal bovine serum). Summary of the Invention

[0007] This invention provides a culture medium free of animal serum (such as fetal bovine serum).

[0008] The first aspect of the present invention provides a serum-free culture medium for culturing retinal pigment epithelial cells, wherein the culture medium is a culture medium containing a basal medium and additives, wherein the additives include human platelet lysis buffer (HPL) and heparin, and the culture medium does not contain animal serum.

[0009] In another preferred embodiment, the animal serum includes: human serum and non-human mammal serum.

[0010] In another preferred embodiment, the animal serum includes fetal bovine serum.

[0011] In another preferred embodiment, the culture medium is not supplemented with any fetal bovine serum or fetal serum from other livestock.

[0012] In another preferred embodiment, the serum-free culture medium for culturing retinal pigment epithelial cells has one or more characteristics selected from the group consisting of:

[0013] (a) In the culture medium, the concentration (v / v) of the platelet lysis buffer (HPL) is 5%-20%, preferably 8-20%, more preferably 10-18%, and even more preferably 10-15%.

[0014] (b) In the culture medium, the concentration of heparin is 1-8 U / ml, preferably 1.5-6 U / ml, more preferably 2-5 U / ml;

[0015] The ratio of platelet lysis buffer (HPL) to heparin is 1 ml: 5-50 U, preferably 1 ml: 10-40 U, ​​and more preferably 1 ml: 15-30 U.

[0016] In another preferred embodiment, the basal culture medium comprises the following components:

[0017] MEM medium (α-modification), N1 supplement, L-glutamate, antibiotics (such as penicillin-streptomycin), non-essential amino acids, taurine, hydrocortisone, and triiodothyronine.

[0018] In another preferred embodiment, the concentrations of the components in the basal culture medium are as follows:

[0019]

[0020] The concentrations are calculated based on the total volume of serum-free retinal pigment epithelial cell culture medium.

[0021] A second aspect of the present invention provides a culture medium kit for serum-free culturing of retinal pigment epithelial cells, comprising the following components:

[0022] (i) Basic culture medium;

[0023] (ii) Human platelet lysis buffer (HPL); and

[0024] (iii) Heparin.

[0025] In another preferred embodiment, the kit comprises:

[0026] (i) a first mixture comprising human platelet lysis buffer (HPL) and basal culture medium; and

[0027] (ii) Heparin.

[0028] In another preferred embodiment, the ratio of platelet lysis buffer (HPL) to heparin is 1 ml: 5-50 U, more preferably, 1 ml: 10-40 U, ​​and even more preferably, 1 ml: 15-30 U.

[0029] A third aspect of the present invention provides a method for preparing a serum-free culture medium for culturing retinal pigment epithelial cells as described in the first aspect of the present invention, comprising the steps of:

[0030] (a) The basal culture medium and additives are mixed to form a culture medium, which is the culture medium containing basal culture medium and additives as described in the first aspect of the present invention, wherein the additives include human platelet lysis buffer (HPL) and heparin.

[0031] Furthermore, the method does not include the step of adding serum.

[0032] A fourth aspect of the present invention provides a method for in vitro expansion of retinal pigment epithelial cells, comprising the steps of:

[0033] (a) Under suitable growth and serum-free conditions, retinal pigment epithelial cells are cultured in vitro in a serum-free culture medium for culturing retinal pigment epithelial cells as described in the first aspect of the invention to obtain expanded retinal pigment epithelial cells.

[0034] In another preferred embodiment, the serum-free condition means that no fetal bovine serum or fetal serum from other livestock is added, nor is human serum added.

[0035] In another preferred embodiment, in step (a), the concentration of the expanded retinal pigment epithelial cells is 5 × 10⁻⁶. 5 -20×10 5 8 × 10⁻⁶ cells / ml, preferably 8 × 10⁻⁶. 5 -15×10 5 per ml.

[0036] In another preferred embodiment, the method further includes (b): identifying the amplified retinal pigment epithelial cells.

[0037] In another preferred embodiment, the identification includes: morphological identification, RPE65. + Biomarker identification, cell concentration measurement, or a combination thereof.

[0038] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0039] In another preferred embodiment, the method further includes: mixing the expanded retinal pigment epithelial cells with a pharmaceutically acceptable carrier to prepare a biological agent.

[0040] In another preferred embodiment, the biological agent (i.e., cell preparation) is injectable.

[0041] In another preferred embodiment, the biological agent is an intraocular injection.

[0042] The fifth aspect of the present invention provides the use of the culture medium described in the first aspect of the present invention or the kit described in the second aspect of the present invention for culturing retinal pigment epithelial cells.

[0043] In another preferred embodiment, the use is non-therapeutic and non-diagnostic.

[0044] The sixth aspect of the present invention provides the use of a composition or mixture comprising platelet lysis buffer and heparin for preparing a serum-free culture medium for culturing retinal pigment epithelial cells as described in the first aspect of the present invention.

[0045] In another preferred embodiment, the ratio of platelet lysis buffer (HPL) to heparin is 1 ml: 5-50 U, more preferably, 1 ml: 10-40 U, ​​and even more preferably, 1 ml: 15-30 U.

[0046] The seventh aspect of the present invention provides a retinal pigment epithelial cell, which is an expanded retinal pigment epithelial cell obtained by the method described in the fourth aspect of the present invention.

[0047] In another preferred embodiment, among the cell population composed of expanded retinal pigment epithelial cells, cells positive for the protein marker RPE65 (i.e., RPE65 cells) + The proportion of retinal pigment epithelial cells is ≥93%, preferably ≥94%, such as 94-96%, based on the total number of cells in the said cell population.

[0048] The eighth aspect of the present invention provides a pharmaceutical composition comprising the retinal pigment epithelial cells described in the seventh aspect of the present invention and a pharmaceutically acceptable carrier, and the pharmaceutical composition is in the form of an injection.

[0049] In another preferred embodiment, the dosage form is an intraocular injection.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0051] Figure 1 The human retinal pigment epithelial cells obtained by centrifugation after trypsin digestion in this invention are shown.

[0052] Figure 2 The invention demonstrates P0 generation human retinal pigment epithelial cells cultured in HPL and heparin according to the present invention.

[0053] Figure 3 Images of P2 and P3 generation human retinal pigment epithelial cells cultured under four different culture methods are shown.

[0054] Figure 4 The results of ELISA detection of VEGF content in P2 generation human retinal pigment epithelial cells under four culture methods are shown.

[0055] Figure 5 The invention demonstrates P2 generation human retinal pigment epithelial cells cultured for 4 weeks in HPL and heparin cultures and in fetal bovine serum cultures.

[0056] Figure 6 The growth curves of P3 generation human retinal pigment epithelial cells under four culture methods are shown.

[0057] Figure 7 The results show a comparison of the expression marker RPE65 in P3 generation human retinal pigment epithelial cells cultured under HPL and heparin and P3 generation cells cultured under fetal bovine serum by flow cytometry.

[0058] Figure 8 The study showed that P3 generation human retinal pigment epithelial cells rounded and detached when cultured in a medium containing only platelet lysis buffer and no heparin. Detailed Implementation

[0059] Through extensive and in-depth research, and after numerous screenings and tests, the inventors unexpectedly discovered for the first time that, under serum-free conditions, adding platelet lysis buffer and heparin to the basal culture medium produces a medium that can very effectively culture and expand human retinal pigment epithelial cells in vitro. Not only is the number of expanded retinal pigment epithelial cells significantly higher than those using fetal bovine serum (FBS), but the expanded retinal pigment epithelial cells also exhibit better morphology and more stable quality (superior to cells expanded using FBS). Experimental results show that human retinal pigment epithelial cells cultured and expanded using the FBS-free medium of this invention not only possess a typical hexagonal cobblestone morphology but also express a large amount of the retinal pigment epithelial cell-specific protein marker RPE65. Based on this, the present invention was completed.

[0060] retinal pigment epithelial cells

[0061] Retinal pigment epithelial (RPE) cells play a crucial and multifaceted role in the ten layers of the retina, including nourishing and supporting photoreceptor cells and participating in the establishment of the blood-retinal barrier. RPE cell degeneration is a major contributing factor and pathological change to many irreversible blinding eye diseases, including age-related macular degeneration (AMD), retinal pigment epithelial cell degeneration (RP), and Startgardt's disease.

[0062] Clinically, apart from wet AMD, which can be controlled with anti-VEGF drugs such as Avastin and Lucentis, there are no effective treatments for dry AMD, RP, and Startgardt's disease. Therefore, regenerative medicine RPE cell transplantation has become a major direction in therapeutic research. However, large-scale production of functionally stable human RPE cells that meet GMP standards requires serum-free and animal-derived culture media.

[0063] Platelet lysis fluid

[0064] Human platelet lysate (HPL) is a cell culture additive containing high concentrations of growth factors and cytokines.

[0065] In this invention, the raw material is platelets supplied by a blood center in the United States that have undergone comprehensive testing and are traceable. Produced under GMP conditions, it boasts extremely high safety and batch-to-batch stability. According to the cell culture safety standards published by the US FDA, it meets Level 2 and is suitable for human clinical use.

[0066] Platelet lysis buffer is typically prepared using conventional methods. It is currently commercially available and can be purchased from Helios.

[0067] In this invention, platelet lysis buffer and heparin can also be added together to the culture medium to culture human retinal pigment epithelial cells.

[0068] Culture medium (or culture solution)

[0069] Typically, culture media for retinal pigment epithelial cells contain animal serum (such as fetal bovine serum). In this invention, platelet lysis buffer and heparin are added to a basal culture medium that does not contain fetal bovine serum to culture human retinal pigment epithelial cells.

[0070] The composition of the basal culture medium is not particularly limited. In a preferred embodiment, the basal culture medium includes the following components:

[0071] MEM medium (α-modification), N1 supplement, L-glutamic acid, penicillin-streptomycin, non-essential amino acids, taurine, hydrocortisone, triiodothyronine.

[0072] Furthermore, the concentration of each component in the basal culture medium is not particularly limited. In a preferred embodiment, the concentrations of each component in the basal culture medium are as follows:

[0073]

[0074]

[0075] In this invention, the concentration of platelet lysis buffer is not particularly limited, but a preferred concentration (v / v) of platelet lysis buffer is 5-20%, more preferably 8-20%, more preferably 10-18%, and even more preferably 10-15%.

[0076] In this invention, the concentration of heparin is not particularly limited, but a preferred concentration of heparin is 1-8 U / ml, more preferably 1.5-6 U / ml, and even more preferably 2-5 U / ml.

[0077] In this invention, the ratio of platelet lysis buffer to heparin is not particularly limited. A preferred ratio is 1 ml: 5-50 U, more preferably, 1 ml: 10-40 U, ​​and even more preferably, 1 ml: 15-30 U.

[0078] Retinal pigment epithelial cells cultured using the culture medium of this invention not only exhibit a typical hexagonal cobblestone morphology and can be passaged to P3 or higher, but also possess strong secretory function, can express large amounts of protein markers specific to retinal pigment epithelial cells, and can secrete pigment. This culture medium is superior to fetal bovine serum and can be used to culture retinal pigment epithelial cells that meet clinical application standards.

[0079] Culture medium kit

[0080] In this invention, a culture medium kit for culturing retinal pigment epithelial cells is also provided, comprising the following components:

[0081] (i) Basic culture medium;

[0082] (ii) Human platelet lysis buffer (HPL); and

[0083] (iii) Heparin.

[0084] In a preferred embodiment, the kit comprises the following components:

[0085] (i) a first mixture comprising human platelet lysis buffer (HPL) and basal culture medium; and

[0086] (ii) Heparin.

[0087] In vitro culture amplification methods

[0088] The present invention also provides a method for expanding retinal pigment epithelial cells in vitro under serum-free conditions, comprising:

[0089] Under suitable growth and serum-free conditions, retinal pigment epithelial cells are cultured in vitro in the serum-free culture medium for culturing retinal pigment epithelial cells described in this invention to obtain expanded retinal pigment epithelial cells.

[0090] In this invention, the serum-free condition refers to the absence of any added fetal bovine serum or fetal serum from other livestock, as well as the absence of added human serum.

[0091] Typically, the culture temperature is 37℃±2℃, preferably 37℃±1℃.

[0092] Typically, the incubation period is 1-30 days, preferably 2-25 days, and even better, 3-20 days, such as 7-20 days, 7-14 days, or 10-15 days.

[0093] Expanded retinal pigment epithelial cells and their preparations

[0094] The retinal pigment epithelial cells cultured using the method of this invention not only have a typical hexagonal cobblestone morphology and can be passaged to P3 or higher, but also have strong secretory function, can express large amounts of protein markers specific to retinal pigment epithelial cells, and can secrete pigment, making them superior to retinal pigment epithelial cells prepared using fetal bovine serum.

[0095] The retinal pigment epithelial cells prepared or expanded using this invention can be mixed with conventional pharmaceutically acceptable carriers to prepare biological agents or cell preparations. Typically, the concentration of expanded retinal pigment epithelial cells in the preparation is 1 × 10⁻⁶. 4 -1×10 6 5 x 10⁻⁶ cells / ml, preferably 5 x 10⁻⁶. 4 -5×10 5 per ml.

[0096] Preferably, the biological agent or cell preparation is an injectable preparation, especially an injectable preparation suitable for intraocular injection.

[0097] The main advantages of this invention include:

[0098] 1. The culture medium and method of the present invention can be efficiently expanded, and the number of retinal pigment epithelial cells obtained under the same conditions is about 4 times that of the culture medium using fetal bovine serum.

[0099] 2. The retinal pigment epithelial cells amplified using the culture medium and method of this invention exhibit good morphology and stable quality. Among them, cells positive for the protein marker RPE65 (i.e., RPE65 cells) + The proportion of retinal pigment epithelial cells (RPE65) was as high as 94.5%, significantly higher than that of RPE65 cells expanded using fetal bovine serum-based culture medium. + The proportion of retinal pigment epithelial cells.

[0100] 3. All materials used in the culture medium of the present invention are non-animal derived and serum-free, which greatly improves the safety of cells in clinical applications.

[0101] 4. Since no serum is used, the quality stability of the retinal pigment epithelial cells prepared by this invention is improved (including batch-to-batch stability), which helps to prepare retinal pigment epithelial cells that meet clinical application standards on a large scale.

[0102] 5. The human retinal pigment epithelial cells cultured in the culture medium of this invention have a much greater in vitro proliferation capacity than those cultured with fetal bovine serum, KnockOut Serum Replacement, and human AB serum. A small amount of raw material cells (derived from cells from a single eyeball) can be used to treat hundreds of patients, effectively solving the problem of donor shortage in transplantation surgery.

[0103] 6. Human retinal pigment epithelial cells cultured using the culture medium of this invention exhibit a typical hexagonal cobblestone pattern, can be passaged to P4 or higher, possess strong secretory function, can express large amounts of protein markers specific to retinal pigment epithelial cells, and can secrete pigment. This culture medium is superior to fetal bovine serum, serum substitutes (KnockOut Serum Replacement), and human AB serum culture and can be used to culture retinal pigment epithelial cells that meet clinical application standards.

[0104] 7. This invention is low in cost. The human platelet lysate and other components in the culture medium are all inexpensive culture reagents. It does not use expensive cytokines, thus avoiding the high treatment costs that patients would have to pay for transplantation surgery, which is conducive to large-scale clinical application.

[0105] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0107] Material

[0108] MEM culture medium (α-modification) Purchased from Sigma, part number M-4526 N1 supplements Purchased from Sigma, part number N6530 L-glutamic acid Purchased from Invitrogen, part number 12360-038 Penicillin-Streptomycin Purchased from Gibico, product number 15140-122 Non-essential amino acids Purchased from Sigma, part number M-7145

[0109] Example 1 Culture Medium NO.1

[0110] Add 5 ml of platelet lysis buffer and 2000 U / L (2 U / ml) of heparin sequentially to 45 ml of basal culture medium to obtain the culture medium for human retinal pigment epithelial cells of the present invention. It can be stored at 4°C for 1 month. The concentration of heparin is 2 U / ml, the concentration (v / v) of platelet lysis buffer is 10%, and the concentration ratio of platelet lysis buffer to heparin is 1 ml: 20 U.

[0111] The basal culture medium is prepared according to the following formula:

[0112]

[0113] Example 2: Culture Medium No. 2

[0114] Add 5 ml of fetal bovine serum to 45 ml of basal culture medium to obtain the culture medium for human retinal pigment epithelial cells of the present invention, which can be stored at 4°C for 1 month.

[0115] The basal culture medium is prepared according to the following formula:

[0116]

[0117] Example 3: Culture Medium No. 3

[0118] Add 5 ml of human AB serum to 45 ml of basal culture medium to obtain the culture medium for human retinal pigment epithelial cells of the present invention, which can be stored at 4°C for 1 month.

[0119] The basal culture medium is prepared according to the following formula:

[0120]

[0121] Example 4: Culture Medium No. 4

[0122] Add 7.5 ml of KnockOut Serum Replacement sequentially to 45 ml of basal culture medium to obtain the culture medium for human retinal pigment epithelial cells of the present invention, which can be stored at 4°C for 1 month.

[0123] The basal culture medium is prepared according to the following formula:

[0124]

[0125] Example 5: Culture Medium No. 5

[0126] Add 5 ml of platelet lysis buffer to 45 ml of basal culture medium to obtain the culture medium for human retinal pigment epithelial cells of the present invention, which can be stored at 4°C for 1 month. The concentration (v / v) of the platelet lysis buffer is 10%.

[0127] The basal culture medium is prepared according to the following formula:

[0128]

[0129]

[0130] Example 6

[0131] Patients who voluntarily donate their eyes must be free of infectious diseases, retinitis pigmentosa, and have no family history of ocular tumors. Using autoclaved ophthalmic surgical scissors, the muscle tissue surrounding the eyeball wall is removed in PBS. The eyeball is rinsed twice with PBS, 5 minutes each time. The cornea and iris are removed 1-2 mm along the limbus, and the lens and vitreous humor are also removed. The eye cup is squeezed to gently expel the retina. The eye cup is cut into petal shapes using ophthalmic surgical scissors and flattened. Starting from the edge of the black retinal pigment epithelium, the retinal pigment epithelium is separated from the choroid layer little by little using ophthalmic surgical forceps. The torn retinal pigment epithelium is pipetted into a 15 ml centrifuge tube, and 2 ml of 0.25% trypsin is added. The tube is incubated at 37°C for 10 minutes. The retinal pigment epithelium tissue is repeatedly pipetted until digested and separated. 4 ml of culture medium is added, and the tube is centrifuged at 1000 rpm for 5 minutes to obtain black retinal pigment epithelial cells. Figure 1 After centrifugation, discard the supernatant and resuspend the cells in 4 ml of the above culture medium to obtain a cell suspension for inoculation. Used in Examples 7-17.

[0132] Example 7 uses the culture medium No. 1 from Example 1 to culture human retinal pigment epithelial (RPE) cells.

[0133] The cell suspension prepared in Example 6 was seeded into six-well plates pre-coated with matrix gel, with each well containing 2 ml of culture medium No. 1 (Example 1), and the seeding volume was 3 × 10⁻⁶. 5 1.5 × 10⁻⁶ holes / hole 5 (pcs / ml).

[0134] Change the medium the day after inoculation, and then every two days. Once the cells have reached confluence, passage them: add 1 ml of 0.25% trypsin to each well of a six-well plate and incubate at 37°C for 3 minutes. Once the cells become rounded and suspended, immediately add 2 ml of culture medium to stop the digestion. Repeatedly pipette the culture plate to help suspend the cells, and then aspirate the cells into a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant, resuspend the cells in culture medium, and count them. (The text abruptly ends here, so the translation stops as well.) 5 1.5 × 10⁻⁶ holes / hole 5 Cells were seeded at a concentration of (cells / ml) on six-well culture plates prepared with laminin or matrix gel.

[0135] The subsequent cultivation and propagation methods are the same as above.

[0136] P2 generation retinal pigment epithelial cells were cultured in human platelet lysates (HPL) and heparin for 2 weeks before observation and identification.

[0137] The results are as follows Figure 2-7 As shown, the results indicated fewer fibrotic cells in platelet lysate and heparin cultures, and the retinal pigment epithelial cells were well-defined hexagonal paving stones. Figure 2 The secretion of VEGF was detected by ELISA, and the secretion level of cells cultured in platelet lysate and heparin was found to be approximately 800 pg / ml. Figure 4 During passaging, pigments can be clearly observed in cells cultured with platelet lysate. Figure 5 ).

[0138] After P3 passage, cells cultured in platelet lysate and heparin showed good proliferation. Figure 3 P3 generation cells were treated with 1×10⁻⁶ cells. 4 Cells were seeded per well in a 24-well plate, with 0.5 ml of culture medium per well. The medium was changed and cells were counted every two days, and a growth curve was plotted. Figure 6 The results showed that after two weeks of culture, the number of cells obtained from platelet lysis buffer and heparin culture was as high as 4.5 × 10⁻⁶. 50.5 ml per well. Flow cytometry analysis of P3 generation cells revealed that RPE65, a cell-specific protein marker, was present in 94.5% of the cell population expanded using platelet lysate culture. The results showed that cells expressing RPE65 (i.e., high-quality retinal pigment epithelial cells) constituted up to 94.5% of the cell population. Figure 7 ).

[0139] In summary, the results show that the serum-free culture medium of the present invention produces human retinal pigment epithelial cells with a typical hexagonal morphology, strong in vitro proliferation capacity, secretory function, and the ability to express protein markers specific to retinal pigment epithelial cells under in vitro culture. RPE cells cultured using this method can be applied clinically.

[0140] Example 8 uses the culture medium from Example 2 to culture human retinal pigment epithelial cells.

[0141] The experimental method was the same as in Example 7, except that the culture medium No.2 prepared in Example 2 was used.

[0142] The results showed that there were fewer fibrotic cells cultured in fetal bovine serum (FBS), and the retinal pigment epithelial cells were well-defined hexagonal basalt cells resembling cobblestones. ELISA analysis of VEGF secretion revealed that the secretion level in cells cultured in platelet lysate and heparin was approximately 800 pg / ml. Figure 4 During passage, pigments can be clearly observed in cells cultured with fetal bovine serum. Figure 5 ).

[0143] After P3 generation, cells cultured in fetal bovine serum proliferated well. Figure 3 P3 generation cells were treated with 1×10⁻⁶ cells. 4 Cells were seeded per well in a 24-well plate, with 0.5 ml of culture medium per well. The medium was changed and cells were counted every two days, and a growth curve was plotted. Figure 6 The results showed that after two weeks of culture, the number of cells obtained from fetal bovine serum culture was 1.1 × 10⁻⁶. 5 0.5 ml / well. Flow cytometry analysis of P3 generation cells revealed that 90.6% of the cells in the expanded cell population cultured in fetal bovine serum expressed RPE65. Figure 7 ).

[0144] However, fetal bovine serum is animal serum, and the composition of serum products is uncertain and fluctuates greatly. Fetal bovine serum has a potential risk of prion infection and increases the risk of immune rejection, so it cannot be used clinically.

[0145] Example 9: Human retinal pigment epithelial cells were cultured using the culture medium from Example 3.

[0146] The experimental method was the same as in Example 7, except that the culture medium No.3 prepared in Example 3 was used.

[0147] The results showed that retinal pigment epithelial cells cultured with human AB serum had indistinct edges and numerous fibrotic cells. ELISA analysis of VEGF secretion revealed that cells cultured with human AB serum had the lowest VEGF secretion level, at 673.2 pg / ml. Figure 4 During passage, cytochrome secretion was reduced in human AB serum culture.

[0148] After passage 3, cells cultured with human AB serum gradually detached. Figure 3 ).

[0149] The results showed that RPE cells cultured using this method had unclear edges, many fibrotic cells, very low VEGF secretion, low cytochrome secretion, cell detachment after 3 passages, poor stability, and could not be used.

[0150] Example 10 uses the culture medium from Example 4 to culture human retinal pigment epithelial cells.

[0151] The experimental method was the same as in Example 7, except that the culture medium No.4 prepared in Example 4 was used.

[0152] The results showed that retinal pigment epithelial cells cultured with the knockout serum replacement exhibited a large number of fibrotic cells. ELISA analysis of VEGF secretion revealed that the secretion level of cells cultured with human AB serum was 892.9 pg / ml. Figure 4 During passage, cytochromes were clearly visible in cells cultured with KnockOut Serum Replacement.

[0153] After P3 generation, cells cultured with the knockout serum replacement gradually detached. Figure 3 ).

[0154] The results showed that RPE cultured using this method produced a large number of fibrotic cells, which gradually detached after three passages, indicating poor stability and unsuitability for application.

[0155] Example 11: Human retinal pigment epithelial cells were cultured using the culture medium from Example 5.

[0156] The experimental method was the same as in Example 7, except that the culture medium No. 5 prepared in Example 5 was used.

[0157] The results showed that P3 retinal pigment epithelial cells cultured with platelet lysis buffer alone, without the addition of heparin, exhibited cell rounding and detachment. Figure 8 ).

[0158] The results showed that RPE cultured using this method exhibited cell rounding and detachment, rendering it unsuitable for application.

[0159] Examples 12-17

[0160] Examples 1 and 7 were repeated, except that the amount and ratio of platelet lysis buffer and heparin added to the culture medium were changed to prepare 50 ml culture media No. 12-No. 17.

[0161]

[0162] Culture results showed that, using the aforementioned culture media No. 12 to No. 17, cells cultured with platelet lysate and heparin proliferated well after the P3 generation. The P3 generation cells were cultured at a concentration of 1×10⁻⁶. 4 Cells were seeded per well in 24-well plates, with 0.5 ml of culture medium per well. The medium was changed and cells were counted every two days. After two weeks of culture, the cell count obtained after platelet lysis and heparin culture was 3.0–6.0 × 10⁻⁶. 5 0.5 ml per well. Furthermore, there were fewer fibrotic cells in platelet lysate and heparin cultures, and the retinal pigment epithelial cells were well-defined hexagonal paving stones.

[0163] Flow cytometry analysis of P3 generation cells revealed the presence of the cell-specific protein marker RPE65. + The proportion of retinal pigment epithelial cells is 93-95%.

[0164] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for in vitro non-diagnostic and non-therapeutic expansion of retinal pigment epithelial cells, characterized in that, include: Under suitable growth and serum-free conditions, retinal pigment epithelial cells were cultured in vitro in a serum-free culture medium to obtain expanded retinal pigment epithelial cells. The culture medium is a culture medium containing a basal medium and additives, wherein the additives include human platelet lysis buffer (HPL) and heparin, and the culture medium does not contain animal serum. The serum-free culture medium for culturing retinal pigment epithelial cells has a combination of the following characteristics: (a) In the culture medium, the concentration (v / v) of the platelet lysis buffer (HPL) is 8%-20%; (b) In the culture medium, the concentration of heparin is 1.5-6 U / mL; and (c) The ratio of the platelet lysis buffer (HPL) to the heparin is 1 mL: 10-40 U; Furthermore, the basal culture medium comprises the following components: α-modified MEM medium, N1 supplement, L-glutamic acid, penicillin-streptomycin, non-essential amino acids, taurine, hydrocortisone, and triiodothyronine.

2. The method as described in claim 1, characterized in that, The concentration of the expanded retinal pigment epithelial cells was 5 × 10⁻⁶. 5 -20×10 5 per mL.

3. The method as described in claim 1, characterized in that, The concentration of the expanded retinal pigment epithelial cells was 8 × 10⁻⁶. 5 -15×10 5 per mL.

4. The method as described in claim 1, characterized in that, The method further includes: identifying the amplified retinal pigment epithelial cells.

5. The method as described in claim 1, characterized in that, The method further includes: mixing the expanded retinal pigment epithelial cells with a pharmaceutically acceptable carrier to prepare a biological agent.

6. The use of a non-diagnostic and non-therapeutic serum-free culture medium for culturing retinal pigment epithelial cells, characterized in that, Used for culturing retinal pigment epithelial cells; The culture medium is a culture medium containing a basal medium and additives, wherein the additives include human platelet lysis buffer (HPL) and heparin, and the culture medium does not contain animal serum. The serum-free culture medium for culturing retinal pigment epithelial cells has a combination of the following characteristics: (a) In the culture medium, the concentration (v / v) of the platelet lysis buffer (HPL) is 8%-20%; (b) In the culture medium, the concentration of heparin is 1.5-6 U / mL; and (c) The ratio of the platelet lysis buffer (HPL) to the heparin is 1 mL: 10-40 U; Furthermore, the basal culture medium comprises the following components: α-modified MEM medium, N1 supplement, L-glutamic acid, penicillin-streptomycin, non-essential amino acids, taurine, hydrocortisone, and triiodothyronine.

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