Retinal pigment epithelial cell proliferation

Abstract

Methods and compositions for expanding RPE cells using a suspension cell support matrix are provided herein. Pharmaceutical compositions containing the RPE cells and methods of treating ocular disorders or diseases using the RPE cells are also provided. TIFF2024527986000049.tif98170

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 226,741, filed July 28, 2021, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background The retinal pigment epithelium (RPE) is a monolayer of neuroepithelial-derived pigment cells located on Bruch's membrane between photoreceptor outer segments (POS) and the choroidal vasculature. The RPE monolayer is crucial for photoreceptor function and health. Dysfunction, damage and loss of RPE cells are hallmarks of certain ocular diseases and disorders, such as age-related macular degeneration (AMD), inherited macular degeneration including Best's disease (early-onset vitelliform macular dystrophy) and subtypes of retinitis pigmentosa (RP). Transplantation of RPE (and photoreceptor cells) into the retina of individuals affected by such diseases can be used as a cell replacement therapy in retinal diseases in which the RPE is degenerated.

[0003] Human fetal and adult RPE have been used as donor sources for allogeneic transplantation. However, practical problems in obtaining sufficient tissue supplies and ethical concerns regarding the use of tissue from aborted fetuses limit the widespread use of these donor sources. Given the limitations in the supply of adult and fetal RPE grafts, the possibility of alternative donor sources has been investigated.

[0004] Human pluripotent stem cells offer significant advantages as a source of RPE cells for transplantation. The multipotent developmental potential of human pluripotent stem cells allows them to differentiate into true functional RPE cells, and considering the infinite self-renewal potential of human pluripotent stem cells, human pluripotent stem cells may serve as an infinite donor source of RPE cells. Indeed, it has been demonstrated that human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) can differentiate into RPE cells in vitro, attenuate retinal degeneration, and maintain visual function after subretinal transplantation. Thus, hESCs may be an unlimited source for producing RPE cells for cell therapy.

[0005] However, the number of patients requiring treatment with RPE cells is predicted to exceed 200 million worldwide. These sheer numbers pose manufacturing challenges, as the required industrial allogeneic lot sizes must be increased to billions of RPE cells. Manufacturing platforms for anchorage-dependent cell types such as RPE cells traditionally use two-dimensional culture methods. These platforms are labor intensive, have an extremely large footprint, and consume excessive resources, making them suboptimal for industrial manufacturing. Being uncontrolled open systems, there is also a high risk of contamination and lot-to-lot variation.

[0006] The present disclosure addresses these and other shortcomings in the fields of regenerative medicine and RPE cell therapy. Summary of the Invention

[0007] Described herein are methods for expanding retinal pigment epithelial (RPE) cells, pharmaceutical compositions of RPE cells, and methods for treating ocular disorders using pharmaceutical compositions produced using the methods. In some embodiments, the disease is age-related macular degeneration (AMD). In some embodiments, the disease is hereditary macular degeneration, including Best's disease (early onset vitelliform macular dystrophy), or a subtype of retinitis pigmentosa (RP).

[0008] In one aspect, provided herein is a method for expanding retinal pigment epithelial (RPE) cells, the method comprising: providing a population of RPE cells differentiated from pluripotent stem cells; inoculating the population of RPE cells into a medium comprising a first suspension cell support matrix, such as a microcarrier; and expanding the population of RPE cells on the first suspension cell support matrix in a dynamic suspension state to provide a population of expanded RPE cells.

[0009] In one aspect, provided herein is a pharmaceutical composition containing retinal pigment epithelial (RPE) cells produced by a method for expanding RPE cells, the method comprising: providing a population of RPE cells differentiated from pluripotent stem cells; inoculating the population of RPE cells into a medium comprising a first suspension cell support matrix; and expanding the population of RPE cells on the first suspension cell support matrix in a dynamic suspension state to provide a population of expanded RPE cells.

[0010] In one aspect, provided herein is a method for treating an ocular disorder or disease, the method comprising transplanting a pharmaceutical composition containing retinal pigment epithelium (RPE) cells produced by a method for expanding RPE into retinal tissue of a patient in need of treating an ocular disorder or disease, the method comprising: providing a population of RPE cells differentiated from pluripotent stem cells; inoculating the population of RPE cells into a medium comprising a first suspension cell support matrix; and expanding the population of RPE cells on the first suspension cell support matrix in a dynamic suspension state to provide a population of expanded RPE cells. [Brief description of the drawings]

[0011] The technology described herein may be more fully understood by reference to the following drawings, which are for illustrative purposes only.

[0012] [Figure 1]FIG. 1 is a series of images of RPE cells during passage 1 (P1) of RPE proliferation. Magnification is 10x. Images show (from left to right): RPE cells 4 days after seeding, 7 days after seeding, 8 days after seeding, and 9 days after seeding (day of harvest). RPE cells begin to show a polygonal monolayer on day 4 after seeding and reach a dense polygonal morphology on day 9 (day of harvest).

[0013] [Diagram 2] Figure 2 shows the morphological assessment of the indicated MCS study showing an outer layer of RPE cells on the surface of the microcarriers (MCs). Representative phase images (4x objective) of RPE cells attached to MCs near the end of passage (days indicated).

[0014] [Diagram 3] Figure 3 is a graph showing a comparison of dissolved oxygen percent (DO%) trends during multiple RPE cell growth runs in a single-use bioreactor (SUB) over a period of approximately 10-12 days. The third fed batch of MCS14 was started on day 10, four days prior to harvest. (MCS14 data up to day 6 was lost due to technical reasons.)

[0015] [Figure 4] FIG. 4 is a series of images showing the evolution of MC-cell population density throughout study MCS11B. Although an inoculated cell density of 120×103 cells / cm2 is targeted, the cells are not uniformly distributed on the MCs. This uneven distribution leads to some of the MCs eventually congregating more densely, resulting in more mature RPE cells congregating those MCs over time. Arrows point to the heterogeneously congregated MCs in samples taken on different days. Variation in MC-cell density is evident as early as 4 days post-inoculation. From left to right: day 4, day 4 (zoomed), day 14, and day 14.

[0016] [Diagram 5] FIG. 5 is a flow chart illustrating one embodiment of a large scale RPE cell production process.

[0017] [Figure 6] FIG. 6 is a microscopic image of microcarrier particles coated with RPE cells and stained with PMEL17 and DAPI.

[0018] [Figure 7] Figure 7 shows the growth of RPE cell monolayers on microcarriers (top row) and T175 flasks (bottom row). Microscopic images were taken 3, 7, and 14 days after seeding of each culture.

[0019] [Figure 8] 8 is a schematic diagram of an exemplary method for generating an intermediate cell bank (ICB) from differentiated RPE cells. As shown, at the end of differentiation (P0), cells are harvested and frozen in aliquots. Cells from the ICB can be used to inoculate microcarriers in large-scale bioreactors for expansion of RPE cells.

[0020] [Figure 9] Figure 9 shows two example schematics for the production of RPE cells. The top schematic shows the RPE cell growth phase without the use of an intermediate cell bank (ICB). The bottom schematic shows the RPE cell growth phase with an intermediate cell bank (ICB).

[0021] [Figure 10] FIG. 10 is a flow chart showing an example of a method of differentiation and expansion for a large-scale OPREGEN® cell production process, including the use of microcarriers (MC) and intermediate cell banks (ICB).

[0022] [Figure 11] FIG. 11 is a flow chart illustrating an exemplary method for differentiating and expanding RPE cells from hESCs.

[0023] [Figure 12] FIG. 12 is a flow chart illustrating an exemplary RPE cell expansion method using an intermediate cell bank (ICB).

[0024] [Figure 13A] Figures 13A and 13B show the results of an exemplary RPE cell potency assay. Figure 13A shows microscopic images of high-potency vs. low-potency mature RPE samples. High-potency mature RPE cells show a confluent, uniform polygonal monolayer morphology. Low-potency mature RPE cells show a subconfluent, heterogeneous morphology with holes. [Figure 13B] The left panel of Figure 13B shows a bar graph plot of transepithelial resistance (TEER, Ω cm2) for (1) high-potency and (2) low-potency RPE cells at day 14. The bar graph in the right panel shows the PEDF:VEGF polarized secretion ratio for (1) high-potency and (2) low-potency mature RPE cells.

[0025] [Figure 14] FIG. 14 shows scatter plots of flow cytometry (FCM) analysis of different stages of RPE cell proliferation and maturation, where cells are double labeled with CRALBP_FITC_488 and PMEL_AlexaFlour_647. The top left scatter plot shows the cell population during hESC proliferation. The top right scatter plot shows the cell population at the end of differentiation. The bottom left scatter plot shows cells during RPE proliferation. The bottom right scatter plot shows cells at the end of the RPE proliferation and maturation process. Cells at the end of the proliferation / maturation process are >95% RPE cells.

[0026] [Figure 15] FIG. 15 shows, from left to right, a bar graph of RPE purity / identity biomarker expression (CRALBP / PMEL17) during OPRGEN® production, a bar graph of RPE maturation biomarker (PEDF) secretion during OPRGEN® production, and mature RPE cell morphology at the end of the differentiation, proliferation, and maturation processes.

[0027] [Figure 16A]Figures 16A and 16B show EXP27A preliminary screening study of the six indicated microcarriers for assessing RPE adhesion for 1 day. Figure 16A is a representative phase image of RPE cells attached to all MC types in the presence of 20% HS. [Figure 16B] Figure 16B is a graph showing % adhesion to each MC type at all tested HS concentrations at 24 hours, calculated by % of cells harvested out of total seeded cells per well. (Bars from left to right in each set: 20% HS; 5% HS; 0.5% HS; 0% HS)

[0028] [Figure 17] Figure 17 is an image showing an EXP27A preliminary screening study of the six indicated microcarriers for evaluating RPE attachment and proliferation over a seven day period. The images show that cells reached confluence and polygonal states in all MC types.

[0029] [Figure 18] Figure 18 is a bar graph showing the highest yield achieved by seeding RPE cells on Star-Plus MC. (Bars from left to right in each set: 20% HS; 5% HS; 0.5% HS; 0% HS)

[0030] [Figure 19A] FIG. 19A is a bar graph showing total cells calculated for spinner flasks seeded at the indicated cell densities.

[0031] [Figure 19B] FIG. 19B is a bar graph showing calculated cell yields for spinner flasks seeded at the indicated cell densities.

[0032] [Figure 20A]Figures 20A and 20B are data comparing two feeding regimes along RPE cell growth in spinner flasks. P1 spinner flasks (fed with 1 / 2 medium exchange) and T175 flasks were harvested and seeded into either control T flasks or spinner flasks for P2. Two spinner flasks derived from the P1 spinner flask were used to test two feeding regimes, fed-batch and 1 / 2 medium exchange. The spinner flask receiving fed-batch had a higher yield (Figure 20A). The control P1 spinner flask (EXP 27E MC) and two P2 spinner flasks (EXP27F MC 1A / 2A) showed similar post-thaw purity (%CRALBP / PMEL17) values ​​in flow cytometry studies (Figure 20B). [Figure 20B] See legend to Figure 20A.

[0033] [Figure 21A] Figures 21A and 21B are graphs outlining the results of a study in which the feeding regimen was 1 / 2 medium exchange compared to a study using fed-batch. Analysis shows the yield (Figure 21A) and total cells harvested per cm2 at the end of passaging (Figure 21B). [Figure 21B] See legend to Figure 21A.

[0034] [Figure 22] FIG. 22 is an image showing the growth of RPE cells on various microcarriers at 3, 10, and 12 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description After reading this description, it will be apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, not all of the various embodiments of the invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only and are not limiting. Thus, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the invention described below.

[0036] Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0037] The detailed description of the present invention has been divided into various sections for the convenience of the reader only, and disclosure appearing in any section may be combined with disclosures from another section. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the invention.

[0038] To meet the needs of a rapidly expanding patient population, what is needed is large-scale production of retinal pigment epithelial (RPE) cells. It has been demonstrated that human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) can be consistently differentiated into functional RPE cells in vitro. The use of such RPE cells in patients with age-related macular degeneration (AMD) in clinical trials shows promising functional recovery. Disclosed herein is a platform for RPE growth that uses a suspending cell support matrix or microcarrier (MC). The properties of MCs, such as being suspended in solution while providing a surface on which adherent cells can grow, make them ideal for growing adherent cell cultures in a closed and controlled environment. Another advantage of using MCs for large-scale production is the greatly increased surface area to volume ratio over traditional static culture processes. Thus, cell density can be increased while reducing the required footprint.

[0039] Described herein is a method that uses the potential of RPE cells growing on a suspension cell support matrix (such as, but not limited to, Star-Plus Microcarriers by SOLOHILL®) to grow hESC-derived RPE in a large-scale closed and controlled environment. For example, in the closed system described herein, differentiated RPE cells can be inoculated into a single bioreactor containing a suspension cell support matrix that is screened for optimal RPE yield and quality. Cellular oxygen consumption can be automatically monitored and controlled, as can pH, metabolites and temperature. Nutritional feeding regimens can be performed in a fed-batch mode, with fresh medium and glucose added as needed. All operations, including suspension cell support matrix and medium addition, cell sampling, harvesting and filtration, can be performed in a controlled and closed environment using tubing welding of disposable bags until the final product of cell suspension in freezing medium is automatically dispensed into cryovials. Controlled large-scale freezing of thousands of vials, up to about 2300 vials per hour freezing session, can be achieved.

[0040] definition The term "treating" or "treatment" refers to any indication of success in treating or ameliorating an injury, disease, pathology, or condition, including any objective or subjective parameter, such as alleviation, mitigation, attenuation of symptoms, or making the injury, pathology, or condition more tolerable to the patient, slowing the rate of decline or debilitation, reducing the severity of debilitation at the end point of decline, improving the patient's physical or mental health, etc. Treating or ameliorating symptoms can be based on objective or subjective parameters, including the results of a physical exam, a neuropsychiatric exam, and / or a psychiatric evaluation. The term "treating" and conjugations thereof can include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing. As used herein (and as well understood in the art), "treating" or "treatment" also broadly includes any approach to obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), prevention of the spread or transmission of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the condition, reduction in recurrence of the disease, and remission, whether partial or complete, detectable or undetectable. In other words, "treatment" as used herein includes cure, amelioration, or prevention of the disease. Treatment may prevent the onset of the disease, prevent the spread of the disease, alleviate the symptoms of the disease, completely or partially eliminate the underlying cause of the disease, shorten the duration of the disease, or a combination thereof.

[0041] As used herein, "treating" and "treatment" include prophylactic treatment. The treatment method includes administering a therapeutically effective amount of an active agent to a subject. The administration step may consist of a single administration or may include a series of administrations. The length of the treatment period will depend on various factors, such as the severity of the symptoms, the age of the patient, the concentration of the active agent, the activity of the composition used in the treatment, or a combination thereof. It will also be understood that the effective amount of an agent used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regimen. Changes in dosage may occur and may be evident by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, a composition is administered to a subject in an amount and for a period sufficient to treat the patient. In some embodiments, the treating or treatment is not a prophylactic treatment.

[0042] The term "prevent" refers to a reduction in the occurrence of disease symptoms in a patient. As noted above, prevention may be complete (no detectable symptoms) or may be partial, such that fewer symptoms are observed than would occur without treatment.

[0043] "Patient" or "subject in need" refers to an organism suffering from or susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer and other non-mammals. In some embodiments, the patient is a human.

[0044] An "effective amount" is an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect for which the composition is administered, treat a disease, reduce enzymatic activity, increase enzymatic activity, reduce a signal transduction pathway, or reduce one or more symptoms of a disease or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Reduction" of one or more symptoms (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms. A "prophylactically effective amount" of a drug (e.g., a cell described herein) is an amount of drug that, when administered to a subject, has an intended prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or a symptom thereof. A complete preventive effect may not necessarily occur by administration of one dose, but only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more doses. As used herein, "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of the antagonist. As used herein, "function-disrupting amount" refers to the amount of antagonist required to disrupt the function of an enzyme or protein compared to the absence of the antagonist. The exact amount will vary depending on the purpose of the treatment and can be ascertained by the skilled artisan using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0045] For any composition described herein, a therapeutically effective amount can be initially determined from cell culture assays. The target concentration is the concentration (e.g., cell concentration or cell number) of one or more active compositions that can achieve the methods described herein, measured using methods described herein or known in the art.

[0046] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models.For example, human dosages can be formulated to achieve concentrations that have been found to be effective in animals.Dosages in humans can be adjusted by monitoring the effectiveness of the composition and adjusting dosages upwards or downwards, as described above.Adjusting dosages to achieve maximum efficacy in humans based on the above and other methods is well within the capabilities of those skilled in the art.

[0047] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent sufficient to ameliorate a disorder, as described above. For example, for a given parameter, a therapeutically effective amount may exhibit at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least a 100% increase or decrease. The therapeutic effect may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold or greater effect over a control.

[0048] Dosage may vary depending on the requirements of the patient and the composition employed. In the context of the present disclosure, the dosage administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient over an extended period of time. The size of the dosage will also be determined by the existence, nature and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the skill of the physician. Generally, treatment is initiated with smaller dosages that are less than the optimal dosage of the composition. This dosage is then increased by small increments until the optimal effect is reached according to the situation. Dosage and intervals can be individually adjusted to provide a level of the administered composition that is effective for the particular clinical indication being treated. This provides a treatment plan that is commensurate with the severity of the individual's condition.

[0049] "Co-administer" means that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapies. The compositions provided herein can be administered alone or co-administered to a patient. Co-administration is meant to include simultaneous or sequential administration of the compositions individually or in combination (multiple compositions). Thus, the preparations can also be combined with other active substances, if desired (e.g., to reduce metabolic degradation).

[0050] "Control" or "control experiment" is used according to its plain ordinary meaning and refers to an experiment in which the experimental subjects or reagents are treated as in a similar experiment, except for the omission of an experimental procedure, reagent, or variable. In some instances, a control is used as a standard of comparison in evaluating the experimental effect. In some embodiments, a control is a measurement of the activity of a protein in the absence of a composition described herein (including aspects and examples).

[0051] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of active agents to and by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin; carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine and dyes. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compositions of the present disclosure. Those of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0052] As used herein, a "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells of plant and animal origin, such as mammalian, insect (e.g., spodoptera) and human cells. Cells can be useful when they are naturally non-adherent or have been treated to prevent them from adhering to surfaces, for example, by trypsinization.

[0053] As used herein, "stem cells" refer to cells that can remain in an undifferentiated state (e.g., pluripotent or multipotent stem cells) in culture for extended periods of time until induced to differentiate into other cell types (e.g., fully differentiated cells) with specific specialized functions. In some embodiments, "stem cells" include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells, and hematopoietic stem cells. In some embodiments, RPE cells are generated from pluripotent stem cells (e.g., ESCs or iPSCs).

[0054] As used herein, "induced pluripotent stem cells" or "iPSCs" are cells that can be generated from somatic cells by genetic manipulation of somatic cells, for example, by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, and gastric epithelial cells with transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007, 1(1): 39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb14. (Epub ahead of print); IH Park, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451: 141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell 2007;131:861-872]. Other embryonic-like stem cells can be generated by nuclear transfer into oocytes, fusion with embryonic stem cells, or nuclear transfer into zygotes when the recipient cell is arrested in mitosis. Additionally, iPSCs can be generated using non-integrative methods, for example, by using small molecules or RNA.

[0055] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm) or can remain undifferentiated. The phrase "embryonic stem cells" includes cells obtained from embryonic tissues (e.g., blastocysts) formed after conception before implantation of the embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at post-implantation / pre-gastrulation stage (see WO 2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissues at any time during conception, preferably before 10 weeks of conception. In some embodiments, embryonic stem cells are obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts.

[0056] Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos may be expanded to the blastocyst stage. For example, to isolate human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by a procedure in which trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then seeded into tissue culture flasks containing an appropriate medium that allows for its outgrowth. After 9-15 days, the ICM-derived outgrowth is dissociated into clumps by either mechanical dissociation or enzymatic degradation, and the cells are then reseeded in fresh tissue culture medium. Colonies that exhibit undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and reseeded. The resulting ES cells are then routinely split every 4-7 days. For further details on methods for preparing human ES cells, see Reubinoff et al. Nat Biotechnol 2000, May:18(5):559; Thomson et al., [U.S. Patent No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4:706, 1989]; and Gardner et al., [Fertil. Steril. 69:84, 1998].

[0057] It is understood that commercially available stem cells may also be used in aspects and embodiments of the present disclosure. Human ES cells may be purchased from the NIH Human Embryonic Stem Cell Registry (www.grants.nih.govstem_cells) or other hESC registries. Non-limiting examples of commercially available embryonic stem cell lines include HAD-C102, ESI, BGO1, BG02, BG03, BG04, CY12, CY30, CY92, CY1O, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES1, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES25, HUES26, HUES27, HUES28, CyT49, RUES3, WAO1, UCSF4, NYUES1, NYUE S2, NYUES3, NYUES4, NYUESS, NYUES6, NYUES7, UCLA1, UCLA2, UCLA3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES62, HUES63, HUES64, CT I, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBRS, WIBR6, HUES45, Shef3, Shef6, BINhem19, BJNhem20, SAGO1, and SAOO1.

[0058] In some embodiments, the embryonic stem cell line is HAD-C102 or ESI.

[0059] Furthermore, ES cells have been used in mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127:224-7], rat [lannaccone et al., 1994, Dev Biol. 163:288-92], rabbit [Giles et al. 1993, Mol Reprod Dev. 36:130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 30 36:424-33], and several livestock species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43:255-60; Wheeler 1994, Reprod Fertil Dev. 6:563-8; Mitalipova et al. al., 2001, Cloning. 3:59-67] and other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci US A. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55:254-9].

[0060] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days after fertilization, at the pre-gastrulation stage. Before culturing the blastocysts, the zona pellucida is digested to expose the inner cell mass [e.g., by Tyrode's acid solution (Sigma Aldrich, St Louis, MO, USA)]. The blastocysts are then cultured as whole embryos in vitro for at least 9 days (preferably up to 14 days) after fertilization (i.e., before the gastrulation event) using standard embryonic stem cell culture methods.

[0061] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves removing a single cell from an embryo during the in vitro fertilization process. The embryo is not destroyed in this process.

[0062] EG (embryonic germ) cells can be prepared from primordial germ cells obtained from a fetus at about 8-11 weeks of gestation (for a human fetus) using laboratory techniques known to those skilled in the art. The genital ridges are dissociated, cut into small pieces, and then broken down into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks containing the appropriate medium. The cells are cultured with daily changes of medium until cell morphology consistent with EG cells is observed (typically after 7-30 days or 1-4 passages). For further details regarding methods of preparing human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95:13726, 1998] and U.S. Patent No. 6,090,622, which is incorporated herein by reference in its entirety.

[0063] Yet another method for preparing ES cells is by parthenogenesis, a process that also does not destroy the embryo.

[0064] ESCs or other pluripotent stem cells may be grown without feeders prior to the differentiation step. For example, feeder cell-free systems can be used for the culture of ES cells. Such systems utilize matrices supplemented with serum replacement, cytokines, and growth factors (including IL6 and soluble IL6 receptor chimeras) as an alternative to the feeder cell layer. Stem cells can be grown on a solid surface such as an extracellular matrix (e.g., MATRIGEL™, laminin, or vitronectin) in the presence of a culture medium, e.g., Lonza L7 system, mTeSR, StemPro, XFKSR, NUTRISTEM®). Unlike feeder-based cultures, which require the simultaneous growth of feeder cells and stem cells and can result in a mixed cell population, stem cells grown in a feeder-free system are easily separated from the surface. The culture medium used for the growth of stem cells contains factors that effectively inhibit differentiation and promote the growth of stem cells, such as MEF-conditioned medium, bFGF, etc. The feeder-free culture medium TeSR™-E8™ is not used in the methods and protocols described and exemplified herein.

[0065] In some embodiments, after expansion, the pluripotent stem cells, e.g., ESCs, are subjected to directed differentiation on an adherent surface (without intermediate generation of spheroids or embryoid bodies). See, e.g., WO 2017 / 072763, which is incorporated by reference in its entirety for all methods, cells, reagents, compositions, and all other information disclosed therein.

[0066] Thus, according to one aspect of the present disclosure, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells subjected to directed differentiation on the adherent surface are undifferentiated pluripotent stem cells (PSCs), e.g., ESCs, and express markers of pluripotency. For example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells are Oct4+TRA-1-60+. Undifferentiated PSCs may express other pluripotency markers such as NANOG, Rex-1, alkaline phosphatase, Sox2, TDGFβ, SSEA-3, SSEA-4, SSEA-5, OCT4, TRA-1-60, and / or TRA-1-81.

[0067] In one exemplary differentiation protocol, non-differentiated pluripotent stem cells are differentiated toward the RPE cell lineage on an adhesive surface using a suspended cell support matrix, such as microcarriers (MC), in dynamic suspension. For example, SoloHill® Star-Plus Microcarriers can be used. Differentiation inducers such as members of the transforming growth factor beta (TGFβ) superfamily (e.g., TGF1, TGF2 and TGF3 subtypes, as well as activins (e.g., activin A, activin B and activin AB), nodal, anti-Mullerian hormone (AMH), several bone morphogenetic proteins (BMPs), such as BMP2, BMP3, BMP4, BMP5, BMP6 and BMP7, and homologous ligands including growth differentiation factors (GDFs)) can be used.

[0068] According to some embodiments, differentiation inducers such as nicotinamide (NIC) may be used at concentrations of about 1-100 mM, 5-50 mM, 5-20 mM, and for example, 10 mM. The concentrations may be any value or subrange within the recited ranges, including the endpoints.

[0069] NIC, also known as "niacinamide" or NA, is an amide derivative form of vitamin B3 (niacin) that is believed to preserve and improve beta cell function. NIC is essential for growth and the conversion of food to energy and is used in the treatment of arthritis and the treatment and prevention of diabetes. NA has the chemical formula C6H6N 20 and has the following structure:

[0070] TIFF2024527986000002.tif47128

[0071] According to some embodiments, the nicotinamide is a nicotinamide derivative or a nicotinamide mimetic. The term "derivative of nicotinamide (NA)" as used herein refers to a compound that is a chemically modified derivative of natural NA. In one embodiment, the chemical modification can be the substitution of the pyridine ring of the basic NA structure (through the carbon or nitrogen member of the ring) through the nitrogen or oxygen atom of the amide moiety. When substituted, one or more hydrogen atoms may be replaced with a substituent and / or the substituent may be bonded to the N atom to form a quadrivalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes substituted or unsubstituted nicotinamide. In another embodiment, the chemical modification can be the deletion or substitution of a single group, for example to form a thiobenzamide analog of NA, all as would be understood by one skilled in organic chemistry. Derivatives in the context of the present invention also include nucleoside derivatives of NA (e.g., nicotinamide adenine). Various derivatives of NA have been described, some of which are also described in relation to the inhibitory activity of PDE4 enzyme (WO 03 / 068233; WO 02 / 060875; GB2327675A) or as VEGF receptor tyrosine kinase inhibitor (WO 01 / 55114). For example, the method of preparing 4-aryl-nicotinamide derivatives (WO 05 / 014549). Other exemplary nicotinamide derivatives are disclosed in WO 01 / 55114 and EP2128244. Each of these references is incorporated herein by reference in its entirety.

[0072] Nicotinamide mimetics include modified forms of nicotinamide and chemical analogues of nicotinamide that reproduce the effect of nicotinamide in differentiation and maturation of RPE cells from pluripotent cells.Exemplary nicotinamide mimetics include benzoic acid, 3-aminobenzoic acid, and 6-aminonicotinamide.Another class of compounds that can act as nicotinamide mimetics are inhibitors of poly(ADP-ribose) polymerase (PARR).Exemplary PARP inhibitors include 3-aminobenzamide, iniparib (BSI201), olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP9722, MK4827, and BMN-673.

[0073] Further contemplated differentiation inducers include, for example, Noggin, Wnt antagonists (Dkk1 or IWR1e), Nodal antagonists (Lefty-A), retinoic acid, taurine, GSK3b inhibitors (CHIR99021) and Notch inhibitors (DAFT).

[0074] The term "retinal pigment epithelium" or "RPE", also known as the "pigmented layer of the retina", refers to the pigmented layer of cells on the outer retina. The RPE layer is located between Bruch's membrane (the inner choroid) and the photoreceptor cells. The RPE is an intermediate for providing nutrients to the retina and aids in many functions including retinal development, light absorption, secretion of growth factors, and mediating the immune response of the eye. Dysfunction of the RPE can result in vision loss or blindness in conditions including retinitis pigmentosa, diabetic retinopathy, West Nile virus, and macular degeneration.

[0075] As used herein, the phrase "marker of mature RPE cells" refers to an antigen (e.g., a protein) that is elevated (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in mature RPE cells compared to non-RPE cells or immature RPE cells.

[0076] As used herein, the phrase "marker of RPE progenitor cells" refers to an antigen (e.g., a protein) that is elevated (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in RPE progenitor cells compared to non-RPE cells.

[0077] According to some embodiments, the RPE cells have a morphology similar to that of native RPE cells that form the pigmented epithelial cell layer of the retina, e.g., the cells may be pigmented and have a characteristic polygonal shape.

[0078] According to yet another embodiment, the RPE cells are capable of treating diseases such as macular degeneration.

[0079] According to a further aspect, the RPE cells meet at least one, two, three, four or all of the requirements listed herein above.

[0080] The term "disease" or "condition" refers to a state or health condition of a patient or subject that can be treated with the compositions or methods provided herein. Age-related macular degeneration, or AMD, is a progressive, chronic disease of the central retina and is the leading cause of vision loss worldwide. Most vision loss occurs in the later stages of the disease due to one of two processes: neovascularization ("wet" AMD) and geographic atrophy (GA, "dry"). In GA, progressive atrophy of the retinal pigment epithelium, choriocapillaris, and photoreceptor cells occurs. While the dry form of AMD is more common (85-90% of all cases), it can progress to the "wet" form, which, if left untreated, results in rapid and severe vision loss. The estimated prevalence of AMD is 1 in 2,000 in the United States and other developed countries. This prevalence is expected to increase with the proportion of older people in the general population. Risk factors for the disease and Contributing factors include both environmental and genetic factors. The pathogenesis of the disease involves abnormalities in four functionally interrelated tissues: the retinal pigment epithelium (RPE), Bruch's membrane, choriocapillaris, and photoreceptor cells. However, impaired RPE cell function is an early and critical event in the molecular pathway that leads to clinically relevant AMD changes. Currently, there is no approved treatment for dry AMD. Preventive measures include vitamin / mineral supplements. These reduce the risk of developing wet AMD but do not affect the onset of geographic atrophy (GA) progression.

[0081] A non-limiting list of diseases for which the effect of treatment may be measured according to the methods provided herein includes retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), geographic atrophy (GA), Best's disease, retinal detachment, gyrate atrophy, total choroidal atrophy, pattern dystrophies and other dystrophies of the RPE, Stargardt's disease, RPE and retinal damage due to damage caused by any one of actinic, laser, inflammatory, infectious, radiation, neovascular or traumatic insults, retinal dysplasia, retinal atrophy, retinopathies, macular dystrophies, cone dystrophies, cone-rod dystrophies, honeycomb retinal dystrophies (Malattia nephrectomy), and the like. Leventinese), Doyne honeycomb dystrophy, Sorsby dystrophy, pattern / butterfly dystrophy, Best disease, North Carolina dystrophy, central ring choroidal dystrophy, angioid streaks, toxic maculopathy, pathological myopia, retinitis pigmentosa and macular degeneration. In some embodiments, the disease is dry AMD. In some embodiments, the disease is GA.

[0082] "Geographic atrophy" or "GA" or "atrophic retina", also known as dry age-related macular degeneration (AMD) or advanced dry AMD, is an advanced form of age-related macular degeneration that can result in progressive and irreversible loss of the retina (photoreceptors, retinal pigment epithelium, choriocapillaris) that can result in loss of visual function over time.

[0083] In some embodiments, the RPE deficiency may be due to one or more of: advanced age, smoking, unhealthy body weight, low antioxidant intake, or cardiovascular disorder. In other embodiments, the RPE deficiency may be due to a congenital abnormality. "Retinal pigment epithelial cells," "RPE cells," and "RPE," which may be used interchangeably as the context permits, refer to cells of a cell type that resembles, for example, functionally, epigenetically, or by expression profile, the native RPE cells that form the pigment epithelial cell layer of the retina (e.g., when transplanted, administered, or delivered into the eye, they exhibit functional activity similar to that of the native RPE cells).

[0084] As used herein, the term "OpRegen" refers to a lineage-restricted human RPE cell line. RPE cells are induced under differentiation medium supplemented with activin A, transforming growth factor beta (TGF-b) family and nicotinamide to enrich the RPE population. OPREGEN® is a single cell suspension formulated in ophthalmic balanced salt solution (BSS Plus) or as a ready to administer (RTA) thaw and inject (TAI) formulation in a CryoStor® 5.

[0085] As used herein, the term "intermediate cell bank" or "ICB" refers to a stock of cells that are frozen in aliquots at an intermediate stage of production. In some embodiments, the intermediate cell bank referred to herein comprises RPE cells that have been frozen after differentiation of PSCs into RPE cells, but prior to expansion of the RPE cells. The ICB can be thawed and used to inoculate cultures for expansion of RPE cells, e.g., expansion on a suspension cell support matrix. Thawing and inoculation can be days, weeks, months, or years after freezing of the cells.

[0086] As used herein, the term "suspended cell support matrix" refers to a suspended support matrix that allows adherent cells to grow in dynamic or static cell culture and remain in suspension with gentle mixing. An example of a suspended cell support matrix is ​​a microcarrier.

[0087] As used herein, the term "microcarrier" or "MC" refers to a suspension support matrix that allows adherent cells to grow in dynamic or static cell culture and remain in suspension with gentle mixing. Microcarriers can be composed of, but are not limited to, polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or combinations thereof. Microcarriers can be coated with biological support matrices, including but not limited to laminin, matrigel, collagen, polylysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenascin, dextran, peptides, or combinations thereof. Many different types of microcarriers are commercially available, including but not limited to HyQSphere (HyClone), Hillex (SoloHill Engineering), and Low Concentration Synthemax® II (Corning) brands. Microcarriers can be made from cross-linked dextran, such as Cytodex brand (GE Healthcare). Microcarriers can be spherical and smooth, have a microporous surface such as CYTOPORE brand (GE Healthcare), and / or be rod-shaped carriers such as DE-53 (Whatman). Microcarriers can be impregnated with magnetic particles that can aid in cell separation from beads (e.g., GEM particles from Global Cell Solutions). Chip-based microcarriers such as μHex products (Nunc) provide a flat surface for cell growth while maintaining the high surface-to-volume ratio of traditional microcarriers. Microcarrier properties can significantly affect growth rate and cell multipotency or pluripotency.

[0088] In some embodiments, the microcarrier (MC) concentration is about 10 cm 2 In some embodiments, the MC concentration is 10 cm 2In some embodiments, the MC concentration is about 1 cm 2 / mL ~ approx. 30cm 2 / mL, or approximately 1 cm 2 / mL ~ approx. 20cm 2 / mL, or approximately 1 cm 2 / mL ~ approx. 10cm 2 / mL, or approximately 1 cm 2 / mL ~ approx. 5cm 2 In some embodiments, the MC concentration is about 1 cm 2 / mL, or approximately 2 cm 2 / mL, or approximately 3 cm 2 / mL, or approximately 4 cm 2 / mL, or approximately 5 cm 2 / mL, or approximately 6 cm 2 / mL, or approximately 7 cm 2 / mL, or approximately 8 cm 2 / mL, or approximately 9 cm 2 / mL, or approximately 10 cm 2 / mL, or approximately 11 cm 2 / mL, or approximately 12 cm 2 / mL, or approximately 13 cm 2 / mL, or approximately 14 cm 2 / mL, or approximately 15 cm 2 / mL, or approximately 16 cm 2 / mL, or approximately 17 cm 2 / mL, or approximately 18 cm 2 / mL, or approximately 19 cm 2 / mL, or approximately 20 cm 2 In some embodiments, the MC concentration is between 5 and 10 cm 2 / mL.

[0089] In some embodiments, the suspension cell support matrix is ​​uncoated. In some embodiments, the suspension cell support matrix is ​​untreated. In some embodiments, the suspension cell support matrix is ​​treated or coated to promote cell adhesion. Surface chemical modifications can improve cell adhesion and include, but are not limited to, applying positive or negative charges or coating with extracellular matrix proteins such as laminin or vitronectin.

[0090] As used herein, the term "bioreactor" refers to any system capable of supporting a biologically active environment. Bioreactors can be open or closed, anaerobic or aerobic. Bioreactors can be continuous flow or stationary flow. Bioreactors can be fed continuously or by batch. Bioreactors can be monitored for, for example, levels of dissolved oxygen (DO), pH, and gas flows including N2, O2, CO2, and air. Bioreactors can include means for mixing or agitating the cell suspension, for example, by any type of impeller or agitator within the bioreactor, or by a rocking platform to provide dynamic culture conditions. Bioreactors can be disposable.

[0091] As used herein, the term "population doubling level" refers to the total number of times that cells in a given population have doubled during in vitro culture. The mathematical expression of population doubling is log2 (viable cells harvested / viable cells seeded). For example, if 1 million viable cells are seeded and 8 million viable cells are harvested, then log2(8 / 1)=3. That is, the average cell population doubling is 3.

[0092] Methods for RPE cell proliferation Embodiments herein relate generally to methods for the expansion of retinal pigmented epithelial (RPE) cells that involve the use of a suspension cell support matrix, such as a microcarrier.

[0093] In some embodiments, the RPE cells are differentiated from human embryonic stem cells (hESCs). In some embodiments, the RPE cells are differentiated from human pluripotent stem cells (iPSCs).

[0094] According to a particular embodiment, differentiation is performed by: (a) propagation of feeder-free hESCs (FF hESCs) in a highly controlled culture system; (b) FF monolayer-directed differentiation of cells obtained from step a) in a medium containing a member of the TGFβ superfamily (e.g., activin A) and a differentiation inducer (e.g., nicotinamide); (c) RPE propagation on gelatin-coated vessels; (d) second passage RPE cultured on a suspension cell support matrix. Step (a) may be performed in the absence of a member of the TGFβ superfamily (e.g., activin A). Non-limiting examples of the production of RPE cells from pluripotent stem cells can be found in WO 2021 / 242788, WO 2017 / 021973, WO 2017 / 021972, WO 2017 / 017686, WO 2016 / 108239, WO 2016 / 108239, WO 2016 / 108239, WO 2008 / 129554, WO 2006 / 070370, WO 2019 / 028088, WO 2021 / 242788, WO 2020 / 223226 and WO 2013 / 114360. Each of these references is incorporated herein by reference in its entirety for all compositions, reagents and cells, as well as all methods, methods of making and methods of using the compositions, reagents and cells, and all other information disclosed therein.

[0095] In some embodiments, the medium in step (a) is completely devoid of TGFβ superfamily members. In other embodiments, the level of TGFβ superfamily members in the medium is less than 20 ng / mL, less than 10 ng / mL, less than 1 ng / mL, or even less than 0.1 ng / mL. The concentration can be any value or subrange within the recited range, including the endpoints.

[0096] The above protocol may be followed by a validation procedure, which may include the following criteria: (a) highly pure RPE cells with a purity of >95% as measured by CRALBP / PMEL17 flow cytometry; (b) net transepithelial electrical resistance (TEER) >100 Ω / cm 2 and generation of a polarized monolayer after thawing with polarized secretion of PEDF and VEGF; and / or (c) no residual stem cells, e.g., hESCs, lacking TRA-1-60 / Oct-4 as measured by flow cytometry, confirmed by the high accuracy Fuzzy C-Mean (FCM) method.

[0097] The cell suspension obtained using the above protocol can then be transplanted, for example, into a patient in need of this. The cells can repopulate large areas, locate the required damaged space and grow by constrained RPE cell proliferation until contact inhibition is reached. The cells can generate a mature polarized RPE layer with barrier function for batch release (BR) capacity measured by TEER and polarized PEDF and VEGF secretion. The resulting cell suspension is ready to be thawed and injected into the patient, no cell preparation is required before surgery.

[0098] The above protocol is suitable for 5 x 10 cells per 3 L bioreactor. 9 Approximately 2500 vials with RPE cells can be obtained. The number of bioreactors and the volume of the bioreactors can be scaled up relatively easily.

[0099] According to some embodiments, at least 50%, 60%, 70%, 80%, 85%, 87%, 89%, 90%, or 95% of the cells express cellular retinaldehyde binding protein (CRALBP) as measured by immunostaining. For example, 95-100% of the cells express CRALBP. The percentages can be any value or subrange within the recited ranges, including the endpoints.

[0100] In another embodiment, at least 50%, 60%, 70%, 80%, 85%, 87%, 89%, 90%, or 95% of the cells express the cellular melanocyte lineage-specific antigen GP100 (PMEL17) as measured by immunostaining. For example, 95-100% of the cells express PMEL17. The percentages can be any value or subrange within the recited ranges, including the endpoints.

[0101] In one aspect, provided herein is a method for expanding retinal pigment epithelial (RPE) cells, comprising: providing a population of RPE cells; inoculating the population of RPE cells into a medium containing a first suspension cell support matrix; and expanding the population of RPE cells on the first suspension cell support matrix in a dynamic suspension state to provide a population of expanded RPE cells.In some embodiments, the population of RPE cells is differentiated from pluripotent stem cells before providing.

[0102] In one embodiment, the population of RPE cells is grown on a solid surface under static conditions. In some embodiments, the solid surface is a culture plate. In some embodiments, the solid surface is a culture flask. In some embodiments, the solid surface is a multi-well culture dish.

[0103] In one embodiment, the population of RPE cells is grown on a solid surface under dynamic conditions prior to the first seeding step, hi some embodiments, the solid surface contains a second suspended cell support matrix.

[0104] In some embodiments, the solid surface is coated. In some embodiments, the solid surface is coated with laminin, matrigel, collagen, polylysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenascin, dextran, peptides, or combinations thereof. In some embodiments, the solid surface is coated with laminin. In some embodiments, the solid surface is coated with matrigel. In some embodiments, the solid surface is coated with collagen. In some embodiments, the solid surface is coated with polylysine. In some embodiments, the solid surface is coated with poly-L-lysine. In some embodiments, the solid surface is coated with poly-D-lysine. In some embodiments, the solid surface is coated with vitronectin. In some embodiments, the solid surface is coated with fibronectin. In some embodiments, the solid surface is coated with tenascin. In some embodiments, the solid surface is coated with dextran. In some embodiments, the solid surface is coated with a peptide.

[0105] In some embodiments, the population of RPE cells is provided from an intermediate cell bank. In some embodiments, the population of RPE cells is grown on a solid surface under static conditions for one passage prior to providing the RPE cells.

[0106] In embodiments, the first suspension cell support matrix is ​​uncoated. In embodiments, the suspension cell support matrix is ​​coated.

[0107] In some embodiments, differentiation of the population of RPE cells from pluripotent stem cells comprises: i) growing the pluripotent stem cells on a solid surface under conditions that maintain the pluripotency of the pluripotent stem cells to provide expanded pluripotent stem cells; and ii) differentiating the expanded pluripotent stem cells in a medium comprising a differentiation inducer and optionally a growth factor for a period of time to provide the population of RPE cells.

[0108] In some embodiments, the solid surface is a culture plate. In some embodiments, the solid surface comprises a second suspension cell support matrix. In some embodiments, the pluripotent stem cells are grown in dynamic culture.

[0109] In some embodiments, the method step ii) comprises differentiating the expanded pluripotent stem cells. In some embodiments, the pluripotent stem cells are expanded on a third suspended cell support matrix in dynamic culture. In some embodiments, the expanded pluripotent stem cells from step i) remain attached to the second suspended cell support matrix in step ii). In some embodiments, the method step ii) comprises differentiating the expanded pluripotent stem cells on the culture plate in static culture.

[0110] In embodiments, the pluripotent stem cells are grown into a monolayer attached to the second suspension cell support matrix and / or the third suspension cell support matrix. In some embodiments, the pluripotent stem cells are grown into a monolayer attached to the second suspension cell support matrix. In some embodiments, the pluripotent stem cells are grown into a monolayer attached to the third suspension cell support matrix. In some embodiments, the pluripotent stem cells are grown into a monolayer attached to the second and third suspension cell support matrices.

[0111] In some embodiments, the conditions for maintaining the pluripotency of the pluripotent stem cells do not include feeder cells. In some embodiments, the conditions for maintaining the pluripotency include a feeder cell population.

[0112] In embodiments, the differentiation-inducing agent is nicotinamide.

[0113] In some embodiments, the growth factor is a member of the TGFβ family. In some embodiments, the member of the transforming growth factor B (TGFβ) superfamily is TGF1, TGF2 and TGF3 subtypes, as well as activin (e.g., activin A, activin B and activin AB), nodal, anti-Mullerian hormone (AMH), some bone morphogenetic proteins (BMPs), e.g., BMP2, BMP3, BMP4, BMP5, BMP6 and BMP7, and homologous ligands including growth differentiation factors (GDFs). In a specific embodiment, the member of the transforming growth factor-B (TGFβ) superfamily is activin A.

[0114] In embodiments, the first suspended cell support matrix, the second suspended cell support matrix, and / or the third suspended cell support matrix are comprised of polystyrene, surface modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or combinations thereof. In some embodiments, the suspended cell support matrix is ​​comprised of polystyrene. In some embodiments, the suspended cell support matrix is ​​comprised of surface modified polystyrene. In some embodiments, the suspended cell support matrix is ​​comprised of chemically modified polystyrene. In some embodiments, the suspended cell support matrix is ​​comprised of cross-linked dextran. In some embodiments, the suspended cell support matrix is ​​comprised of cellulose. In some embodiments, the suspended cell support matrix is ​​comprised of acrylamide. In some embodiments, the suspended cell support matrix is ​​comprised of collagen. In some embodiments, the suspended cell support matrix is ​​comprised of alginate. In some embodiments, the suspended cell support matrix is ​​comprised of gelatin. In some embodiments, the suspended cell support matrix is ​​comprised of glass. In some embodiments, the suspension cell support matrix is ​​composed of DEAE-dextran.

[0115] In some embodiments, the first suspended cell support matrix, the second suspended cell support matrix, and / or the third suspended cell support matrix are uncoated. In some embodiments, the first suspended cell support matrix, the second suspended cell support matrix, and / or the third suspended cell support matrix are coated. In some embodiments, the suspended cell support matrix is ​​coated with laminin, matrigel, collagen, polylysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenascin, dextran, peptides, or combinations thereof. In some embodiments, the suspended cell support matrix is ​​coated with laminin. In some embodiments, the suspended cell support matrix is ​​coated with matrigel. In some embodiments, the suspended cell support matrix is ​​coated with collagen. In some embodiments, the suspended cell support matrix is ​​coated with polylysine. In some embodiments, the suspended cell support matrix is ​​coated with poly-L-lysine. In some embodiments, the suspended cell support matrix is ​​coated with poly-D-lysine. In some embodiments, the suspension cell support matrix is ​​coated with vitronectin. In some embodiments, the suspension cell support matrix is ​​coated with fibronectin. In some embodiments, the suspension cell support matrix is ​​coated with tenascin. In some embodiments, the suspension cell support matrix is ​​coated with dextran. In some embodiments, the suspension cell support matrix is ​​coated with a peptide.

[0116] In embodiments, the first suspended cell support matrix, the second suspended cell support matrix, and / or the third suspended cell support matrix are spherical, elliptical, rod-shaped, disc-shaped, porous, non-porous, smooth, flat, or a combination thereof. In some embodiments, the suspended cell support matrix is ​​spherical. In some embodiments, the suspended cell support matrix is ​​elliptical. In some embodiments, the suspended cell support matrix is ​​rod-shaped. In some embodiments, the suspended cell support matrix is ​​disc-shaped. In some embodiments, the suspended cell support matrix is ​​porous. In some embodiments, the suspended cell support matrix is ​​non-porous. In some embodiments, the suspended cell support matrix is ​​smooth. In some embodiments, the suspended cell support matrix is ​​flat.

[0117] In embodiments, the first suspended cell support matrix, the second suspended cell support matrix, and the third suspended cell support matrix are the same. In some embodiments, at least two of the first suspended cell support matrix, the second suspended cell support matrix, and the third suspended cell support matrix are the same. In some embodiments, the first suspended cell support matrix, the second suspended cell support matrix, and the third suspended cell support matrix are different.

[0118] In some embodiments, the suspending cell support matrix is ​​a microcarrier. In some embodiments, the first suspending cell support matrix is ​​a first microcarrier. In some embodiments, the second suspending cell support matrix is ​​a second microcarrier. In some embodiments, the third suspending cell support matrix is ​​a third microcarrier.

[0119] In some embodiments, the population of RPE cells has a population doubling level of 2-4 during P0 (initial proliferation phase after differentiation or seeding from an ICB), 2-3 during P1 (first passage after P0), and 1-2 during P2 (second passage).

[0120] In some embodiments, the proliferation of RPE cells described herein can result in an increase in the number of cells proliferated over a given time.For example, without ICB, in a year, two different batches can be propagated at the same place, and four batches per set can be propagated. In contrast, with ICB, in a year, ten batches per set of the same size can be propagated, for example, three times the amount without two batches being propagated at the same time. In some embodiments, the number of RPE cells at the end of proliferation is about two times higher with ICB than without it. In some embodiments, the number of RPE cells at the end of proliferation is about three times higher with ICB. In some embodiments, the number of RPE cells at the end of proliferation is about four times higher with ICB. In some embodiments, the cost of RPE proliferation is lower with ICB than without ICB.

[0121] In embodiments, the conditions for growth of RPE cells include maintaining the % dissolved oxygen above 30%.

[0122] In some embodiments, the conditions for the proliferation of RPE cells include an initial growth medium volume starting at about 50% of the total system growth chamber volume. In some embodiments, a growth medium volume of about 10% to about 25%, e.g., about 16.6%, of the total system growth chamber volume is added periodically. In some embodiments, a growth medium volume is added every 2 to 4 days.

[0123] In some embodiments, the RPE cells are characteristic of mature RPE cells. In some embodiments, mature RPE cells are greater than 95% double positive for cellular retinaldehyde binding protein (CRALBP) and pre-melanosome protein (PMEL17) as measured by flow cytometry. In some embodiments, mature RPE cells have a net transepithelial electrical resistance (TEER) >100 Ω / cm after thawing. 2 and produces a polarized monolayer with polarized secretion of PEDF and VEGF.

[0124] In some embodiments, the mature RPE cells are cryopreserved and ready to be administered to a subject upon thawing. In some embodiments, the RPE cells are cryopreserved in a cryopreservation medium. In some embodiments, the cryopreservation medium comprises a cryoprotectant, such as glycerol, sucrose, dimethylsulfoxide (DMSO) or other suitable cryoprotectant. In some embodiments, the cryoprotectant comprises glycerol. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant comprises DMSO. In some embodiments, the cryoprotectant comprises dextran.

[0125] In some embodiments, the cryopreservation medium comprises about 0.1% to about 40% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 0.1% to about 30% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 0.1% to about 20% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 0.1% to about 10% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 0.1% to about 5% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 1% to about 40% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 1% to about 30% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 1% to about 20% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 1% to about 10% cryoprotectant. In some embodiments, the cryopreservation medium comprises about 1% to about 5% cryoprotectant. The percentages may be measured as weight of cryoprotectant per volume of medium. The percentages may be measured as volume of cryoprotectant per volume of medium. The percentages may be any value or subrange within the recited range, including the endpoints.

[0126] In some embodiments, mature RPE cells contain less than 0.01% pluripotent stem cells as determined by high resolution flow cytometry (FCM) and are negative for TRA-1-60 / Oct-4 as measured by flow cytometry.

[0127] In some embodiments, RPE cells generate polygonal monolayers at every passage during expansion until post-differentiation formulation and after injection as a cell suspension.

[0128] In some embodiments, one or more steps of the method are performed in a single-use bioreactor. In some embodiments, the expansion of the RPE cells is performed in a single-use bioreactor. In some embodiments, the differentiation of the RPE cells is performed in a single-use bioreactor.

[0129] Treatment Method Aspects of the present specification relate generally to methods, compositions and devices for treating ocular diseases and illnesses, including retinal conditions such as macular degeneration.

[0130] In one aspect, a method for treating an ocular disorder or disease, comprising implanting a pharmaceutical composition containing retinal pigment epithelial (RPE) cells generated from the method of expanding RPE cells described herein into the retinal tissue of a patient in need of treating the ocular disorder or disease.

[0131] In some embodiments, the ocular disorder or disease is age-related macular degeneration (AMD), hereditary macular degeneration including Best's disease (early onset vitelliform macular dystrophy), or a subtype of retinitis pigmentosa (RP). In some embodiments, the ocular disorder or disease is age-related macular degeneration (AMD). In some embodiments, the ocular disorder or disease is hereditary macular degeneration. In some embodiments, the ocular disorder or disease is Best's disease (early onset vitelliform macular dystrophy). In some embodiments, the ocular disorder or disease is a subtype of retinitis pigmentosa (RP).

[0132] In some embodiments, the population of RPE cells is ready for immediate use in patients based on a product release decision that includes determining that the mature RPE cells are greater than 95% double positive for cellular retinaldehyde binding protein (CRALBP) and pre-melanosome protein (PMEL17) as measured by flow cytometry; determining that the mature RPE cells generate a polarized monolayer after thawing having a net transepithelial electrical resistance (TEER) of >100 Ω / cm2 and polarized secretion of PEDF and VEGF; and / or that the mature RPE cells contain less than 0.01% pluripotent stem cells as confirmed by high resolution flow cytometry (FCM) and are negative for TRA-1-60 / Oct-4 as measured by flow cytometry.

[0133] In some embodiments, RPE cells generate polygonal monolayers at every passage during expansion until post-differentiation formulation and after injection as a cell suspension.

[0134] In some embodiments, the concentration of mature RPE cells is in the range of 10,000 to 500,000 cells per 50 to 200 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 15,000 to 300,000 cells per 50 to 200 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 25,000 to 250,000 cells per 50 to 200 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 50,000 to 250,000 cells per 50 to 200 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 10,000 to 500,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 15,000 to 300,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 25,000 to 250,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 50,000 to 250,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 10,000 to 500,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 15,000 to 300,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 25,000 to 250,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 50,000 to 250,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 10,000 to 500,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 15,000 to 300,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 25,000 to 250,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is in the range of 50,000 to 250,000 cells per 150 microliters.In some embodiments, the concentration of mature RPE cells ranges from 10,000 to 500,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells ranges from 15,000 to 300,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells ranges from 25,000 to 250,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells ranges from 50,000 to 250,000 cells per 200 microliters. The concentration may be any value or subrange within the recited range, including the endpoints.

[0135] In some embodiments, the concentration of mature RPE cells is about 25,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 50,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 75,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 100,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 125,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 150,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 175,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 200,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 225,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 250,000 cells per 50 microliters. In some embodiments, the concentration of mature RPE cells is about 25,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 50,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 75,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 100,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 125,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 150,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 175,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 200,000 cells per 100 microliters.In some embodiments, the concentration of mature RPE cells is about 225,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 250,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 25,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 50,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 75,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 100,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 125,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 150,000 cells per 100 microliters. In some embodiments, the concentration of mature RPE cells is about 175,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 200,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 225,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 250,000 cells per 150 microliters. In some embodiments, the concentration of mature RPE cells is about 25,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells is about 50,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells is about 75,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells is about 100,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells is about 125,000 cells per 200 microliters. In some embodiments, the concentration of mature RPE cells is about 150,000 cells per 200 microliters.In some embodiments, the concentration of mature RPE cells is about 175,000 cells per 200 microliters.In some embodiments, the concentration of mature RPE cells is about 200,000 cells per 200 microliters.In some embodiments, the concentration of mature RPE cells is about 225,000 cells per 200 microliters.In some embodiments, the concentration of mature RPE cells is about 250,000 cells per 200 microliters.

[0136] In some embodiments, the pharmaceutical composition is formulated to be thawed and injected into a subject without cell preparation prior to injection.

[0137] In one embodiment, transplantation is performed via delivery of cells into the subretinal space through a small retinal opening or by direct injection following pars plane vitrectomy surgery.

[0138] In certain embodiments, administration may include delivery of the RTA therapeutic cell composition to the subretinal space in the macular region via a cannula through a small retinotomy following vitrectomy. Depending on the cell dose, a total volume of 50-100 µL of cell suspension may be implanted in areas at potential risk for GA enlargement.

[0139] In some embodiments, a single surgical procedure is performed in which the RTA therapeutic cell composition is delivered through a small retinotomy after vitrectomy into the subretinal space created in the macular region along the area of ​​GA and the border between the better preserved extrafoveal retina and the RPE layer, if GA is present. After placement of the eyelid retractor, a standard three-port vitrectomy can be performed. This may include placement of a 23G or 25G injection cannula and two 23G or 25 / 23G ports (trocars). A core vitrectomy can then be performed with a 23G or 25G instrument, followed by peeling of the posterior vitreous surface. The RTA therapeutic cell composition can be injected into the subretinal space at a predetermined site in the posterior pole, preferably penetrating the retina in an area that is still relatively preserved near the border of GA, if GA is present.

[0140] Pharmaceutical Compositions In some aspects, the present disclosure is a cell therapy comprising retinal pigment epithelial (RPE) cells derived from pluripotent cells. Such cell therapy includes, but is not intended to be limited to, OPREGEN®.

[0141] In one aspect, provided herein are pharmaceutical compositions containing RPE cells generated from the methods of expanding RPE cells described herein.

[0142] In embodiments, the compositions are frozen prior to use in a patient, hi embodiments, the compositions are formulated to be thawed and injected into a subject without cell preparation prior to injection. EXAMPLES

[0143] Example 1 The purpose of this example is to demonstrate membrane formation of cultured RPE cells using the propagation of feeder-free hESCs (FF hESCs) in a highly controlled culture system. Then, feeder-free monolayer-directed differentiation of cells in a medium containing a member of the TGFβ superfamily (e.g., activin A) and a differentiation inducer (e.g., nicotinamide) can be used for RPE propagation on gelatin-coated vessels, and the second passage RPE cultured on microcarriers (MCs) can be used for transplantation. Propagation of FF hESCs can be performed in the absence of a member of the TGFβ superfamily (e.g., activin A).

[0144] RPE cells cultured using the methods described herein can generate polygonal monolayers even when injected as a cell suspension. RPE cells generate such polygonal monolayers at every passage after differentiation and during proliferation until formulation. Figure 1 shows RPE cells during the P1 passage in RPE proliferation, beginning 4 days after seeding, organizing and reaching a dense polygonal morphology at day 9 (harvesting day).

[0145] Example 2 The purpose of this example is to summarize the development of a small-scale process for the expansion of RPE cells in Corning disposable 0.1 L spinner flasks.

[0146] This development in small-scale vessels is a preliminary step before scaling up the growth of RPE cells in bioreactors to produce OPREGEN® in controlled growth conditions. In this method, differentiated RPE were seeded on plastic MCs and grown for 1-2 passages. Several parameters such as type of plastic, concentration of MCs and seeding cell density per area of ​​MCs were tested and optimized.

[0147] RPE cells were grown on MC in spinner flasks. Several conditions were tested in a stepwise fashion compared to control T-flasks. Other process modifications were implemented during each run and documented along the process development. Parameters tested included: Screening for MC types - Pall Sollohil(Star Plus, Plastic Plus, Hillex II)Corning Synthmax(Hi,Low,CellBind); Seeding density - Various cell densities, from 60,000 to 120,000 cells / cm 2 The cells were seeded in a range of 100 μm. Seed Mixing - 40 RPM with 5 minute breaks every 30 minutes, 10 RPM in horizontal tube, constant at 40 RPM; Nutritional regimen - Cells were fed either by medium exchange or a fed-batch regimen. Growth medium was exchanged (half the volume) or added (28-56%) two to three times a week. Decreased serum concentration - follow-up of studies carried out in TC flasks (EXP29 A-C), in which the standard seeding medium of 20% HS / DMEM was replaced by 2% HS / Nut minus / HSA or 0% HS / Nut minus / HAS; Nicotinamide - with or without the addition of nicotinamide to the growth medium; and MC concentration - 5~20cm 2 / mL(360cm 2 Several MC concentrations were tested in spinner flasks ranging from 0.5 to 2 gr / spinner flask. Table 1: Study design TIFF2024527986000003.tif214170TIFF2024527986000004.tif220170

[0148] Testing Procedure: RPE cells were either thawed directly or expanded in T-flasks before being seeded onto different types of MCs (Table 2) in wells of 6-well plates (EXP27A) or in 0.1 L Corning spinner flasks (EXP27 B-C, G-K).

[0149] Non-frozen sources of differentiated RPE cells derived from feeder-free hESCs were grown on Star plus MC (Table 2-1st row) in spinner flasks (EXP27D-F, L).

[0150] Cells were counted using an NC-200 cell counter and yields were calculated by dividing by the number of cells plated.

[0151] Cell morphology and lactate and glucose levels were examined along the passages.

[0152] At the end of passaging, RPE cultures from each condition were harvested and yields were compared to control flasks. Yields and morphology were compared to control T-flasks at one or more serial passages.

[0153] In some experiments, frozen cell samples at the end of the passage were harvested and tested for identity and potency.

[0154] Screening for suitable MC types Table 2 - Microcarriers tested in the EXP27 study. TIFF2024527986000005.tif61169

[0155] Optimal cell density

[0156] The effect of seeding cell density on RPE proliferation was performed in studies EXP27C, EXP27D, EXP27F and EXP27H. Table 3: Summary of results of RPE cell seeding at different densities on Star-Plus MC. TIFF2024527986000006.tif104162 * Yield was calculated by dividing the number of cells harvested on the last day of passage by the number of cells seeded on day 0 of passage. Conclusion: RPE cells grow as efficiently on MCs as on tissue culture flasks at all seeding densities tested.

[0157] Fed-batch vs. 1 / 2 medium exchange The effect of different nutritional regimens on RPE proliferation on MCs was examined in studies EXP27C, EXP27D, EXP27F and EXP27H. Table 4 - Summary of results of testing two feeding regimens along RPE cell growth in spinner flasks to optimize the feeding process TIFF2024527986000007.tif134170 Table 5 - Post-thaw recovery purity and potency (TEER and secreted PEDF and VEGF) tested in study REC#3, RM119. TIFF2024527986000008.tif40170

[0158] Conclusions - The results showed similar values ​​for the two feeding methods. Fed-batch was chosen because it is easier to scale up.

[0159] MC concentration

[0160] The effect of different MC concentrations at seeding on RPE proliferation was examined in the EXP27H and EXP27L studies during P2. Table 6 - MC concentration test P2 RPE cells at approximately 56,000 cells / cm2 (EXP27H) or approximately 88,000 cells / cm2 2 The cells were seeded at a cell density of (EXP27L). TIFF2024527986000009.tif90170 Conclusion: 1. MC concentration is 5-10cm 2 The optimal MC concentration was determined to be in the range of 7.2 cm 2 / mL, i.e., 1 gram of MC (360 cm) in 50 mL inoculation medium in a spinner flask 2 ) was. 2. The most efficient MC concentration is 10 cm, as it allows the highest cell density at the end of passaging. 2 / mL. 20cm 2 Even though twice as many cells were seeded at 10 / mL, the final yield is very close to that achieved with half the MC concentration. Reduced serum in the inoculation medium Table 7: Degraded Tests TIFF2024527986000010.tif144170 Conclusion - As with other studies performed in TC flasks, the 2% HS concentration in Nut / HSA can replace the standard 20% HS / DMEM seeding medium, as it allows for a higher cell density at the end of passaging, resulting in a higher yield and total cells per vessel. Table 8: Presence of Nic during proliferation on MC TIFF2024527986000011.tif161170 Conclusion: The addition of Nic resulted in a 50% increase in the final cell yield at each passage. The results were significant (P<0.05). Conclusions about the developmental stages of RPE growth on MCs in 0.1L spinner flasks: 1. Of the six types of MC tested, Star-Plus MC was found to best support RPE proliferation. 2. RPE cells are 90,000 cells / cm 2 ~120,000 cells / cm 2 This density range is identical to that seen in TC flasks. 3. RPE proliferation was comparable between the two feeding methods. Fed-batch was chosen over 1 / 2 medium exchange because it is easier to scale up when using MC and is more cost-effective. 4. The most efficient MC concentration is 10 cm, as it allows the highest cell density at the end of passaging. 2 / mL. 2 At 100 / mL, even though twice as many cells were seeded, the final yield was very close to that achieved with half the MC concentration. 5. As in previous studies performed in TC flasks, the 2% HS concentration in nut / HSA can replace the standard 20% HS / DMEM seeding medium, since it allows for a higher cell density at the end of passaging. The yield and total cells per vessel were also higher. 6. The addition of Nic resulted in a 50% increase in the final cell yield at each passage. The results were significant (p<0.05).

[0161] Example 3 Further screening of suitable MC types

[0162] In the RPE-Pro-05 study, additional MC types were screened for suitability to support RPE proliferation. The commercially available microcarriers tested in this study are listed in Table 9. Table 9: Further testing of MC suitability for RPE proliferation TIFF2024527986000012.tif95170

[0163] Example 4 The purpose of this example is to summarize the development of a scaled-up process for the growth of RPE cells in an Eppendorf disposable 3 L bioreactor (SUB) BioBlu 3C, monitored by an Eppendorf BioFlo 320 bioprocess system.

[0164] The development of RPE growth on Star Plus microcarriers (MC) in spinner flasks provided a basis for process parameters that could be further applied in preliminary studies for growing RPE cells on microcarriers (MC) under controlled conditions in bioreactors: seeding cell density, seeding agitation speed, MC concentration, and nutrient feeding regime.

[0165] The system utilizes its proprietary software to monitor and control cell culture applications that require continuous control of the varying process parameters required in cell culture, including aeration method (air, O2, CO2, N2), pH, temperature and agitation speed.

[0166] Utilizing a Biowelder to connect the media-containing bags allowed the SUB to be maintained as a "closed system." With the goal of maintaining a closed system capable of supporting significant scale-up, the current filtration procedure for separating RPE cells from MCs and other large particles (matrix and cell aggregates), which was previously performed manually using an open 40 micron mesh, was replaced.

[0167] Materials, devices and cells

[0168] Table 10: Materials TIFF2024527986000013.tif145170

[0169] Table 11 Equipment TIFF2024527986000014.tif39157

[0170] Abbreviations and Definitions CS5-Cryostor 5% DP-formulation FFMD-Feeder-free monolayer differentiation HSA - Human serum albumin ICB - Intermediate Cell Bank MC-Microcarriers Nic-nicotinamide NUTS-Nutristem (cell culture medium) OpRegen® TAI-OpRegen® 'Thaw-and-Inject' RPE-retinal pigment epithelial cells. RPM - Revolutions per minute SF-Spinner Flask SUB-Disposable Bioreactor

[0171] Experimental design

[0172] RPE cells were expanded on T175 flasks from P0 (end of RPE cell differentiation and start of proliferation) to the end of the first passage, P1, days 9-14. Cells were then harvested and maintained at 90,000-120,000 cells / cm. 2 At a cell density of 3.6 to 7.2 cm 2SUB was inoculated with an MC concentration of 1000 / mL. 3-4 days after inoculation, the medium (20% HS / DMEM) was replaced with growth medium Nutristem minus (Nut(-)) with or without HSA. Glucose and lactate levels were measured before medium addition, and glucose was replenished to 2-3 grams / L (plus additional glucose to make up for the weekend according to current consumption).

[0173] As a control for growth on MC, cells were sampled from the SUB approximately 10 minutes after inoculation and transferred to spinner flasks for parallel culture. Other process modifications were tested and implemented during each run and are recorded in Table 12. Table 12: List of study designs in order of execution TIFF2024527986000015.tif23787TIFF2024527986000016.tif239106TIFF2024527986000017.tif23768

[0174] Experimental procedure

[0175] Thawed or ongoing differentiating RPE cell sources were derived from hESCs grown on human umbilical cord or in feeder-free conditions and expanded in T-flasks before seeding on MCs in SUB. Cells were counted using an NC-200 cell counter and yields were calculated by dividing by the number of cells inoculated. Cell morphology as well as lactate and glucose levels were examined along the passage. At the end of the passage, RPE cell cultures for each condition were harvested and yields were compared to control spinner flasks (cultured in 1 / 30 (50 ml) of the SUB inoculation volume and with RPE cells harvested 10 min after inoculation of the SUB). In some experiments, frozen cell samples at the end of the passage were harvested and tested for identity and potency. Table 13 - Cells used in development TIFF2024527986000018.tif70170

[0176] result

[0177] The main findings are summarized below. Morphology is shown in Figure 2. Morphological assessment of the MCS study shown indicates an outer layer of RPE cells on the MC surface. Representative phase image with a 4x objective of RPE cells attached to MCs near the end of passage (days indicated in Figure 2).

[0178] yield Table 14: Cell count and related parameters at inoculation time and final yield estimation TIFF2024527986000019.tif24479

[0179] * For MCS1 and MCS2 the cultures were transferred into spinner flasks and the final indicated yields are obtained from the harvested spinner flasks. ** For MCS7 and MCS8, several samples (3% of total volume each) were taken along the passage. The MC surface areas shown in Table 14 (column 3 - final surface density) are final at the time of harvest; the inoculated MC surface areas were MCS7-10,800 cm 2 , MCS8-8, 208cm 2 It was.

[0180] Glucose and lactate determination Table 15: Glucose consumption rate along passages TIFF2024527986000020.tif117128 Table 16: Lactate consumption rate along passages TIFF2024527986000021.tif111144

[0181] Glucose consumption and lactate production were calculated by subtracting the previous level (nmol / mL) from the current level and dividing the result by the period (days) since the previous measurement. In most cell lines grown in bioreactors, such as CHO cells, lactate production levels increase continuously, but not in RPE cell cultures. A relative decrease in glucose consumption indicated that the cultures had reached confluence, whereas a decrease in lactate production indicates a shift from glycolysis to oxidative phosphorylation, which may accompany lactate consumption by RPE cells. Table 17 TEER measurements (results of modified assay using QC-WIN-11) TIFF2024527986000022.tif51128

[0182] conclusion

[0183] The BioBlu 3C SUB combined with the BioFlo 320 bioprocess system demonstrated reproducibility and robustness in maintaining and supporting the growth of RPE cells on MC in a controlled and monitored environment. The three gas mix algorithm (O2, CO2 and air) is effective in maintaining RPE growth at a 30% DO set point. Agitation was adjusted along the process; run at 22 RPM to achieve sufficient cell attachment inoculum, then agitation was further increased to 29 RPM for complete suspension of MC, and finally agitation was raised to 40 RPM to increase the efficiency of gas dissolution in the culture and promote an increased rate of oxygen consumption by the growing cells. Four independent runs (MCS3, MCS6, MCS7 and MCS8) gave good cell yields at the end of growth in the BioBlu 3C SUB prior to filtration. Filtration using Sartopure 50 μm filters was successful in two runs, while in two other runs, significant loss of cells was observed. Efforts are ongoing to further improve the filtration process. In two of the three runs, thawed vials passed potency assays, indicating that RPE cells grown in SUB on MC retained their properties and biological activity. The optimal harvest date emerged as day 14, so future cell harvests will be performed on days 13-14 to allow for operational flexibility.

[0184] The final process parameters are summarized in Table 18. Table 18: Solution volumes, MC amounts and process parameter set points TIFF2024527986000023.tif192161 Table 19. Data Summary TIFF2024527986000024.tif236166TIFF2024527986000025.tif237157

[0185] Example 5 This example summarizes the development of a scaled-up production process for the growth of RPE cells (for one expansion passage) on MC in a 3 L disposable bioreactor using a Bioflo 320 bioprocess system from Eppendorf, based on previously established process development.

[0186] OPREGEN® "Thaw-and-Injetct" (OPREGEN® TAI) process development involves the implementation of a large-scale manufacturing process that encompasses inoculation, growth, and harvesting of retinal pigment epithelial (RPE) cells under monitored and controlled conditions. This scaled-up process development is focused on the growth of RPE cells in the terminal (P2) growth phase of RPE cells; the earlier growth phases (P0 and P1) currently remain flask-dependent and unchanged from current practices.

[0187] The basis for this transition from flask-dependent processes to large-scale semi-automated closed systems relies on two fundamental changes. The first is the incorporation of flexibility into benchtop bioprocessing systems, such as Eppendorf's BioFlo 320, to control disposable vessels in cell culture processes. The system utilizes its proprietary software to monitor and control cell culture applications that demand continuous control of the changing process parameters required in cell culture. The second change is the ability to grow RPE cells on microcarriers (MCs), which provide the required surface for RPE cell attachment, replacing the gelatin-coated tissue culture flask surface without affecting the quality attributes and characteristics of OPREGEN®. This developed process also contributes to the overall intent of reducing sterility risks during the OPREGEN® production process.

[0188] Aim and Scope

[0189] We summarize the development of a scaled-up production process for the growth of RPE cells on MC (one passage expansion) in a 3 L disposable bioreactor using Eppendorf's Bioflo 320 bioprocess system. We present and describe the OPREGEN® TAI scaled-up manufacturing process.

[0190] Abbreviations and Definitions BioFlo 320 - Eppendorf's automated monitoring and control bioprocessing system. BLOD - below detection limit Deadband - The span of the input within a specified range within which there is no change in the output. DMEM-Dulbecco's modified Eagle's medium. DO- Dissolved oxygen (%). DS-former. ECM - Extracellular Matrix. Fed-batch - an operating technique in which one or more nutrients are fed (fed) into the bioreactor during cultivation and the product remains in the bioreactor until the end of the run. FF-feeder-free hESCs. FFMD-Feeder-Free Monolayer Differentiation. GRP-group HS-human serum. HSA-human serum albumin. ICB-Intermediate Cell Bank. Inoculation - Transfer of cells into the bioreactor. LM521-Laminin 521. MC-Microcarriers. NUT(-) / HSA-NutriStem (cell culture medium) containing HSA. OpRegen® TAI-OpRegen® "Thaw-and-Inject" (new OpRegen® formulation). PBS - Phosphate Buffered Saline RPE-retinal pigment epithelial cells. RPM- Revolutions per minute. SF-Spinner flask. SLPM-Standard Liters Per Minute. SP - set point. SUB-Disposable bioreactor. Temperature - Temperature. w / v - weight to volume ratio. Table 20. Materials and consumables. TIFF2024527986000026.tif152170 Table 21. Equipment TIFF2024527986000027.tif45141

[0191] Testing Procedure:

[0192] Harvesting of differentiated RPE cells grown on T175 flasks at the end of P1. Harvesting of differentiated RPE cells in the FFMD process grown on T175 flasks coated with recombinant human gelatin (with the exception of MCS9, cells were thawed from the ICB and grown on flasks prior to SUB inoculation). Inoculating the SUB with harvested RPE cells (with the exception of MCS9, which originated from the ICB) from the ongoing RPE FFMD differentiation procedure in flasks for one RPE expansion passage to P2 (with the exception of MCS11A, which was inoculated at P1). The origin of cells for each run is listed in Table 22 below. Table 22: Cell Sources in Development Research TIFF2024527986000028.tif43168 1 Thawed cells from ICB.

[0193] Selection of MC type, number of cells required for inoculation and inoculation parameters (medium volume and agitation speed) were developed using Corning spinner flasks. Inoculation of harvested RPE cells on MCs into SUBs was performed at 100,000–120,000 viable cells / cm, depending on the final yield of RPE cells obtained from harvesting flasks. 2The MCs were optimized for HS. To allow coating of the MCs with HS to improve cell attachment and achieve a homogenous MC suspension in the SUB, the inoculation medium (20% HS / DMEM) and MCs are equilibrated in the SUB for at least 20 min at 37 °C prior to inoculation. Other parameters such as DO, pH, gassing and medium exchange / addition are as established during the MCS1-MCS8 studies.

[0194] Stirring Speed

[0195] Because the spinner flask and the SUB have significantly different impeller and vessel sizes and shapes, the agitation rate was established by applying the "constant impeller tip speed" principle to maintain a relatively constant shear level during the transition from the spinner flask to the SUB while allowing sufficient mixing and oxygen transfer. The "constant tip speed" was calculated according to Equation I below. Tip speed=π×d×N In the formula, d = outer diameter of the impeller (m) N = stirring speed (rpm) Table 23: SUB inoculation conditions TIFF2024527986000029.tif32128

[0196] The inoculation conditions were maintained for 4 days, after which the medium was replaced. Table 24: SUB inoculum size per study TIFF2024527986000030.tif43140

[0197] 1 Approximately 3.3% of the total inoculated viable cell count was harvested from the SUB for various study controls as documented in the protocol.

[0198] Growth of RPE cells on MCs in 3L SUB

[0199] Four days after inoculation, the inoculation medium was replaced with an equal volume (1.5 L) of NUT(-) / HSA, and the attachment of cells to the MC was observed to confirm cell attachment. Growth of RPE cells was performed under a fed-batch nutrient feeding regime and controlled parameters of temperature, DO, pH, agitation, and gassing. The nutrient feeding regime, temperature, and agitation rate have been previously established using T-flasks and SFs, but the environmental parameters - DO, pH, and gassing - could not be scaled up based on previous experience with SFs. These parameter set points were developed and established based on previous MCS1-8 runs and general practice.

[0200] As mentioned above, a scale-up of the agitation speed was established by applying the "constant impeller tip speed" principle in conjunction with our observations of MC behavior, aiming to keep MC suspended in the medium while minimizing or eliminating settling. Immediately after medium exchange with NUT(-) / HSA, the agitation speed is first increased from 22 rpm to 29 rpm on day 4. This increase improves both temperature stability and MC suspension in the SUB, which results in better overall homogeneity of the culture, while keeping shear stress relatively low. Agitation is further increased to 40 rpm on day 7 and kept unchanged for the duration of the process to improve MC suspension in larger volumes. Table 25: Proliferative Phase SP TIFF2024527986000031.tif36128

[0201] The pH set point was set at 7.25 with a dead band of 1.25 and no active regulation. The pH gradually decreased over time during the process, from a relatively high pH of about 7.9 at the inoculation step (mainly contributed to the presence of HS) to about pH 7.3 at the end of growth. The pH was confirmed by using an external analog pH meter to measure samples of the SUB, and the pH standard was amended to the actual measured value if the actual pH value deviated from the BioFlo 320 pH value by more than ±0.05 pH units. The nutrient feeding regime during RPE cell growth was previously established as fed-batch, with glucose addition. On three separate days, at intervals of 2–3 days, the culture is supplemented with 0.5 L of NUT(-) / HSA to a final volume of 3 L. Glucose is supplemented to 2–2.5 gr / L (with a 45% w / v d-glucose solution) based on the actual daily culture glucose levels measured using a metabolite testing device. Table 26 below details the schedule of medium changes, fed-batch regimes and harvests for each run. Table 26: Medium exchange, fed-batch mode and harvest schedule TIFF2024527986000032.tif39161D=Day

[0202] The optimal harvest date was set at day 14, so a minimum growth of 13–14 days was determined to allow for operational flexibility.

[0203] Gassing Regime

[0204] The gassing regimen through both the Overlay and the Sparger is based on a mixture of three gases (air, oxygen and carbon dioxide) actively controlled by the BioFlo system's automated three-gas mix algorithm. The operating range was set based on common industry practice with hESCs. The SUB has two aeration inlets. The primary inlet is a pinhole sintered body through which the three-gas mixture required to maintain oxygen concentration (DO) is supplied. The second inlet is an overlay inlet to maintain headspace aeration. Both inlets have pre-installed 0.2 μm 5 cm disk filters to filter the incoming gas. Small pore sizes, such as in the sintered body, generate small gas bubbles with high surface area, thus improving the gas transfer rate into the medium. However, such high levels of small gas bubbles can result in reduced gas exchange at the liquid headspace boundary and ultimately lead to the formation of bubbles that may even clog the exhaust filter. Gas inflow was very low and no significant foam layer formation was observed during these studies. Table 27: 3 Gas Mix SP TIFF2024527986000033.tif20128

[0205] Vessel pressure affects dissolved gases in the culture, thus affecting pH and DO. A decrease in overall gas pressure can cause a decrease in gas solubility, resulting in an increase in overall gas demand. The SUB is designed to operate under positive pressure, and as per the manufacturer's recommendations, gas pressure within the SUB should not exceed 0.44 barg (6 psig). However, since a positive gas flow is necessary to sustain the culture, but a relatively low gas flux is required during the culture of RPE cells (see lines 20 and 21 of Table 18, and Table 27), no significant pressure increase was expected or observed during these studies. Furthermore, the pre-installed vent filter of the SUB is designed to withstand a maximum pressure of up to 5 bar.

[0206] Inoculation, medium exchange and fed-batch medium addition

[0207] In contrast to growing RPE in flasks, maintaining the SUB as a closed system requires that all medium additions and exchanges throughout the process are performed without exposing the cultures to the outside environment, keeping the cultures sterile. An automated biowelder is used to weld bags containing medium to ports on the SUB in a sterile and reliable manner. Disposable bags are pre-filled with the required medium in a biological safety cabinet before being welded to the SUB.

[0208] collection

[0209] Filtration of RPE cells harvested from T-flasks and SF was established using a mesh of 40 μm grid. The procedure was scaled up and both were 0.15 μm. 2 This procedure was established as the required "closed system" using filter sizes of 50 μm or 60 μm with a surface area of ​​10 μm, which ensures the absence of particles larger than 60 μm in the DS suspension, as these filters have the closest pore size to the mesh of the 40 μm grid.

[0210] To separate the RPE cells from the MC and remove the solution from remaining large cell aggregates and ECM, each SUB was filtered using a 50 μm (SARTOPURE®) or 60 μm (VANGUARD®) high volume filter that maintains a closed system and was quenched immediately after enzymatic incubation with TrypLE Select. Additionally, samples were taken from the SUB at the end of the enzymatic incubation, immediately prior to quenching, and the samples were quenched and filtered using a 40 μm mesh grid to assess the scaled up filtration process and to obtain a yield estimate of the SUB.

[0211] Before filtering the suspension, approximately 0.5 L of quench solution is applied to the filter, and then the incubated suspension is passed through the filter via a peristaltic pump. The cell suspension is filtered intermittently to ensure that the fraction of the cell suspension containing the largest visible aggregates passes through the filter during the final stage of filtration, in order to minimize filter clogging and potential cell loss. This part of the filtration procedure was developed and established following the relatively low cell yields obtained in MCS9 (see Table 28, Estimated Yields vs. Final Yields for MCS9-14).

[0212] result

[0213] The following results represent the scale-up development studies of MCS9, MCS11A, MCS11B, MCS13 and MCS 14. Results were obtained from the following assays: Viability (%), Recovery (%), Purity, HES remaining, Biological activity (potency) and Karyology.

[0214] QC Assay Pass / Fail Criteria

[0215] Viability (%) ≥ 70%. Recovery (%): 100% ± 25% viable cells / ml, calculated relative to a target final batch concentration of 2 x 106 cells / ml. Potency: Net TEER (day 14): > 100 Ω·cm2; Basal VEGF / Apical VEGF ratio (day 14): > 1.00; Apical PEDF / Basal PEDF ratio (day 14): > 1.00 Purity: OpRegen® cells at P2 are ≥ 95.00% double positive for CRALBP and PMEL17. HES Remaining: < 0.01000% of cells are double positive for TRA1-60 and Oct-4. Karyology: < 3 identical deletions or < 2 identical additions to chromosomes.

[0216] DO tendency

[0217] As shown in Figure 3, a sharp drop in DO% is evident immediately after inoculation until the medium is changed. This drop in DO% indicates successful inoculation and survival. MCS9 exhibits higher DO% values ​​during inoculation due to the smaller inoculum size. The second and third fed batches result in an attenuated DO% spike due to the smaller relative proportion of medium added and relatively less air entering the SUB with each fed batch.

[0218] Final DS Yield Table 28: Estimated Yield vs. Actual Final Yield TIFF2024527986000034.tif63170

[0219] At the end of the enzymatic incubation, immediately prior to quenching, samples (60 mL) were taken from each SUB, which were then quenched and filtered using a 40 μm mesh grid to assess the scaled-up filtration process and to obtain yield estimates for the SUB. The average estimated yield for all studies (presented in Table 28) was about 4.09, close to the actual final yield measured in DS after filtration, with the average final yield being about 2.87, which constitutes an average on-filter loss of about 30%. MCA11 A, MCS11 B and MCS13 achieved a final average DS yield of about 3.35 after filtration, which is similar to their respective estimated yields. The final yield of study MCS9, obtained before establishing the requirement to prepare the filters prior to filtration, was about 44% lower than its estimated yield (4.78 vs. 2.68). MCS14, which was subjected to a different fed-batch regime from the other studies, had a final yield 60% lower than expected.

[0220] Assessment of viability (%) and recovery (%)

[0221] Five vials (2 × 10 6 The cells (cells / ml) were thawed and assessed for percent viability and percent recovery. The results are summarized in Table 29. Table 29: Results of percent viability and percent recovery TIFF2024527986000035.tif152170

[0222] Percent viability and percent recovery of all vials from all studies immediately after thawing met the OPREGEN® TAI acceptance criteria (presented in Table 29). Percent viability remained above 90%. Percent recovery was above 78% with an average of 90%.

[0223] Biological activity (potency) evaluation

[0224] The thawed cells were assayed for biological activity and the results are summarized in Table 30. Table 30: Potency assay results TIFF2024527986000036.tif59155

[0225] All studies met the pass / fail criteria of the biological activity assays in both potency methods.

[0226] Purity Assay Results

[0227] The thawed cells were assayed for purity and the results are summarized in Table 31 below. Table 31: Purity (CRALBP / PMEL17) assay results TIFF2024527986000037.tif38128

[0228] RPE cells obtained from studies MCS11A, MCS11B, and MCS13 met the pass / fail criteria of the purity assay, whereas cells from MCS9 and MCS14 did not. Note that the fed-batch format of both MCS9 and MCS14 differed from the other studies. Additionally, both MCS9 and MCS14 gave the lowest final yields, which may have influenced the final cell population.

[0229] hESC survival assay results

[0230] Thawed cells were assayed for % hESC survival and the results are summarized in Table 32. Table 32: hESC Survival Rate (%) (Oct-4 / TRA1-60) Assay Results TIFF2024527986000038.tif49128

[0231] RPE cells from all five studies met the pass / fail criteria for the % hESC Survival assay (Table 32).

[0232] Karyotype analysis

[0233] Frozen vials of P2 were thawed, subcultured for 2 passages, and fixed for karyotype analysis. The karyotype of cells from each of these studies was normal; no three identical chromosomal deletions or two identical additions were observed. The one exception was MCS11A, where isoq20 was found, a finding that is under investigation.

[0234] Consideration

[0235] To establish a scaled-up process for OPREGEN® production, RPE cells were cultured on MC for one growth passage in 3 L SUB under controlled and monitored conditions. Five consecutive development studies MCS9 / 11A / 11B / 13 / 14 were performed under the proposed scaled-up process parameters that were gradually improved during earlier scale-up studies MCS1-8. These parameters have been shown to result in a robust and reproducible scaled-up process. Examination of DO trends in these studies reveals nearly identical behavior. A sharp drop immediately after inoculation that continues for the first few days indicates successful inoculation and viability (%). DO% then stabilizes at SP30%, followed by intermittent spikes corresponding to medium changes and subsequent fed-batch medium additions. The fed-batch regimen has been shown to result in better final cell yields when fed at 2-3 day intervals (with medium changes, fed-batch mode and harvest schedule as shown in Table 26), whereas a 4 day interval (MCS9 and MCS14) may induce some stress in the cultures that subsequently affects the final population composition in terms of filtration efficiency and RPE maturation.

[0236] During development, priming the filters with quench solution prior to MC-cell separation to minimize cell loss was shown to be an improvement. Furthermore, an improved filtration procedure involving passing the suspension fraction containing large aggregates only at the final stage of filtration, rather than continuously mixing and filtering the entire bulk volume (as performed in MCS9), proved essential to obtain higher cell yields. Regarding the significantly lower final yield obtained in MCS14, it could be attributed to a different nutritional feeding regime, during which the second fed batch was supplemented 4 days after the first fed batch instead of 3 days after. This could have contributed to a greater production of ECM by the RPE cells, ultimately capturing more mature cells during collection and filtration.

[0237] Considering that cells were grown and obtained under the proposed fed-batch regimen (less than 3 days between feedings) and improved filtration procedures (i.e., MCS11A, MCS11B, and MCS13), the developed scaled-up process was shown to be effective and reproducible with a final average cell yield of about 3.35 (see Table 28, the average number of 3.35 is for MCS11A_11B and 13).

[0238] The purity assay results of studies MCS11A, MCS11B and MCS13 (shown in Table 31) met the pass / fail criteria of the assay, whereas MCS9 and MCS14 failed to meet these pass / fail criteria. The reason is due to different parameters in the process of MCS9 and MCS14. Their respective actual FACS diagrams show a broader population of cells exhibiting lower PMEL values, which is typical of less mature (younger) RPE cells. These relatively broader populations of less mature cells than usual (i.e., compared to MCS13) are most likely due to a suboptimal harvesting procedure. As more mature cells produce more ECM, this makes the mature RPE cells less likely to detach from the MC during enzymatic incubation with TrypLe Select compared to the less mature RPE cells. As a result, these mature cells are trapped within ECM aggregates during filtration, and thus, after filtration, the DS is relatively enriched in less mature RPE cells.

[0239] Studies MCS11A, MCS11B and MCS13 met the pass / fail criteria of the purity assay, indicating that optimized fed-batch regimens and filtration procedures are necessary for successful scaled-up RPE expansion. Maturity variations in the final RPE population may result in inherent heterogeneous MC cell densities, likely a result of seeding. MCs, being a 3D stirred cell growth platform, are prone to heterogeneous distribution of cells during seeding, in contrast to 2D static tissue culture flasks (as shown in Figure 4). Diverse maturity of the final population is likely beneficial for RPE cells.

[0240] hESC survival (%) assay results met the OpRegen® TAI pass / fail criteria. All studies met the biological activity (potency) assay pass / fail criteria under both assay methods. Karyotype results in four of the five studies were normal; one study showed an abnormal karyotype (Isoq20 (3 / 50)) which was investigated.

[0241] In summary, the bioprocessing system in general and the scaled-up expansion platform in particular are capable of maintaining and supporting robust RPE cell expansion and reproducible processes by maintaining the required OPREGEN® quality attributes and characteristics.

[0242] conclusion

[0243] The BioBlu 3C SUB combined with the BioFlo 320 bioprocess system demonstrated robustness in maintaining and supporting the growth of RPE cells on MC in the current OPREGEN® process. Under controlled and monitored conditions, 10.8x10 cells were cultured in 3L SUB. 3 cm 2 From culturing RPE cells on MCs with a surface area of ​​about 4x10 9 of OPREGEN® TAI cells (after final filtration) can be obtained. The scaled-up filtration procedure was shown to be effective and reproducible when the fed-batch regimen of the established cell growth process and the improved filtration procedure were implemented, resulting in a final DS cell yield of approximately 3.35. Table 33: Solution volumes, MC amounts and process parameter set points TIFF2024527986000039.tif189161

[0244] Example 6 The purpose of this example is to summarize the OPRGEN® Non-GMP engineering run of the GMP FF seed lot bank.

[0245] The OPREGEN® manufacturing process for commercial production includes the development of a feeder-free and monolayer differentiation (FFMD) process that should be highly controlled with minimal sterility risks, should be robust, and should have less reliance on spontaneous differentiation steps.

[0246] The developed OPREGEN® commercial process relies on four stages of cell differentiation: stage 1 - Feeder-free (FF) human embryonic stem cells grown on Laminin 521 (LN521) for 3 weeks; stage 2 - differentiation of hESCs into RPE cells, performed for 6-7 weeks with a FFMD procedure of hESCs developed based on the current OPREGEN® Thaw and Inject (TAI) process; stage 3 - RPE expansion for 2 passages (P0, P1), currently performed for another 4 weeks on gelatin-coated flasks as was done for OPREGEN® TAI; stage 4 - large-scale culture of RPE cells on microcarriers (MC) in a semi-automated, controlled, closed system (Eppendorf's BioFlo320 console monitoring a BioBlu Single-Use Bioreactor (SUB)).

[0247] Abbreviations and Definitions CoA - Certificate of Analysis CS5-CryoStor5 DMEM - Dulbecco's Modified Eagle Medium DO - Dissolved Oxygen Fed-batch - a cultivation technique in biotechnology processes in which one or more nutrients are fed to the bioreactor during the cultivation and the product remains in the bioreactor until the end of the run. FF - Feeder Free FFB - Feeder Free Bank FFMD-Feeder-free and monolayer differentiation GRP-group hESC - human embryonic stem cells HS-Human serum HSA - Human serum albumin IPC-In-process control LN521-Laminin 521 MC-Microcarriers MCB - Master Cell Bank NIC - Nicotinamide Nut(-)w / HSA - NutriStem minus containing HSA Nut+w / HSA - NutriStem Plus with HSA PDL-Population Doubling Level POC - Proof of Concept QC-Quality Control R&D RPE-Retinal Pigment Epithelium RPM- Revolutions per minute. SD - standard deviation TAI-Thawing and Injection TEER - Transepithelial Electrical Resistance TS-TrypLE Select V-Version w / -Yes With or without

[0248] Procedures and Methods:

[0249] Cells: Frozen and stored in a CCN GMP facility.

[0250] Experimental procedure

[0251] hESC proliferation and thawing

[0252] Thaw one vial of frozen cells and plate at 6,000 viable cells / cm on dishes coated with 5 μg / ml LN521 in Nut+w / HSA medium. 2 hESCs were cultured. When the cell cultures reached >50% confluency, cells were harvested, counted, and plated at 3,500 viable cells / cm for hESC expansion I. 2 The seeds were sown in.

[0253] hESC Expansion I

[0254] hESCs were passaged for one further passage on 5 μg / ml LN521-coated dishes containing Nut+w / HSA medium. When cell cultures reached >50% confluency, cells were harvested, counted, and cultured at 4,000 viable cells / cm for hESC expansion II. 2 Additionally, samples from harvested hESCs were assessed for pluripotency markers.

[0255] hESC Expansion II

[0256] hESCs were seeded on 5 μg / ml LN521-coated dishes containing Nut+w / HSA medium. Cell morphology and confluency were assessed starting from day 6 of culture until the cell cultures reached 80% or more confluency. Additionally, one equivalent flask (cultured under the same conditions) was harvested to test pluripotency markers.

[0257] Differentiation of hESCs into RPE

[0258] NIC I

[0259] At the end of hESC expansion II, the OPREGEN® differentiation process was initiated by changing the medium from Nut+w / HSA to Nut-w / HSA supplemented with 10 mM NIC and culturing the cells at 5% O2, 5% CO2 and 37°C for 2 weeks.

[0260] NIC + Activin A

[0261] Cells were cultured for 2 weeks with Nut-w / HSA supplemented with 10 mM NIC and 140 ng / ml activin A at 5% O2, 5% CO2 and 37° C. At the end of 14 days, media samples were collected for factor secretion studies.

[0262] NIC II

[0263] Cells were cultured for 5 days with Nut-w / HSA supplemented with 10 mM NIC in 5% O2, 5% CO2 and at 37°C until mildly pigmented areas were observed under a binocular microscope. Cells were then cultured for an additional 10 days in normoxia (20% O2, 5% CO2 and 37°C). At the end of 15 days (end of differentiation), the morphology of the differentiating cells was assessed, samples of the medium were collected for factor secretion tests, and cells were harvested using TS at 60,000 viable cells / cm for P0 of the RPE proliferation stage. 2Cells were seeded onto dishes coated with 0.1% rh-gelatin in 20% HS-DMEM at 100° C. Additionally, samples of cells were tested for RPE purity / identity.

[0264] RPE proliferation

[0265] RPE in T175 flasks (P0, P1)

[0266] Cells were cultured in 5% CO2 and 37°C for 4 days in 20% HS-DMEM and for P0 in Nut-w / HSA medium (until 100% confluency and typical morphology of RPE polygons were reached) for an additional 11 days (total of 15 days for P0). At the end of P0, cells were harvested and cultured at 120,000 viable cells / cm. 2 The cells were cultured for an additional 13 days in P1 before being harvested and seeded for P2.

[0267] RPE during SUB (P2)

[0268] The harvested P1 cells were inoculated into the SUB under optimal conditions. The 20% HS-DMEM seeding medium containing MC was equilibrated in the SUB for at least 20 min. Then, the cells were inoculated at 10,800 cm at 37°C. 2 1.26 x 10 per MC 9 viable cells (117,000 viable cells / cm 2Cells were seeded at 1000 x 1000 μl / min; MC; 22 rpm agitation; 30% DO; pH 7.25. After 4 days, 20% HS-DMEM was replaced with Nut(-) w / HSA medium, but the growth conditions of RPE cells remained the same, except for the agitation speed (29 rpm on days 3-7 and 40 rpm on days 7-14). The feeding regime during the P2 growth phase was performed as fed-batch with glucose addition. On three separate days (7, 10 and 12), 0.5 L of Nut(-) w / HSA medium was replenished to the cell culture to a final volume of 3 L. Glucose was replenished to 2.5 g / L (using a 45% w / v d-glucose solution) based on the actual daily culture glucose levels measured using a metabolite tester. At the end of the 13-day culture, the SUB was harvested and RPE cells were filtered using a 60 μm filter (Vanguard) and 2 × 10 6 Cells / vial were stored frozen. Table 34: Batch Release Testing of FFMD Large Scale OPREGEN® Production Process TIFF2024527986000040.tif62137

[0269] result

[0270] In-process management

[0271] Pluripotency. Expression of pluripotency markers (SSEA-5 / TRA-1-60, Oct-4 / Nanog) was examined at the end of each hESC expansion stage and the percentage of double positive cells in each assay was determined by FACS and shown in Table 35 below. Table 35: Expression of pluripotency markers along hESC expansion stages TIFF2024527986000041.tif24170

[0272] Expression of pluripotency markers was high across all hESC expansion stages - the percentage of SSEA-5 / TRA-1-60 double positive cells was over 97.91% and 95.36%, and the % of Oct-4 / Nanog double positive cells was 98.09% and 96.96%. The results showed that the cultured hESCs maintained their pluripotency before the start of the FFMD procedure. The mild decrease in expression of markers observed at the end of hESC expansion step I compared to the end of hESC expansion step II is related to the intended higher seeding density and confluency of cells before the start of FFMD, which is required for the differentiation process.

[0273] Purity and Identity

[0274] The expression of RPE markers (CRALBP / PMEL17) was examined along different stages of FFMD and RPE proliferation. Table 36: RPE differentiation and expression of RPE markers along P0. TIFF2024527986000042.tif17139

[0275] The % of RPE cells is represented by the % of CRALBP / PMEL17 positive cells. At the end of the differentiation stage, the % of RPE cells was 45.96%. After passaging and enrichment of RPE cells at the end of P0, the % of RPE cells was 97.96%, meeting the batch release acceptance criteria (>95.00% RPE cells) at this point.

[0276] residual hESCs

[0277] To determine remaining hESCs, RPE cells were stained for the hESC pluripotency marker TRA-1-60 / Oct-4 at the end of P0. The % of TRA-1-60 / Oct-4 was below the limit of detection (<0.0004%).

[0278] PEDF secretion along the process

[0279] In the previous OPRGEN® TAI V1.1 run, a magnitude increase in PEDF levels secreted into the culture medium was observed at the terminal differentiation stage (end of Activin A, end of differentiation) and early RPE proliferation stage (end of P0), so these IPCs were selected to test for PEDF concentration in the FFMD process and in this non-GMP engineering run-FFMD large-scale OPRGEN® production process. These three points for evaluating PEDF were found to be important, indicative points as IPCs for the FFMD large-scale OPRGEN® production process. PEDF concentration was evaluated in the culture medium at three points: end of NIC+Activin A, end of differentiation (end of NIC II), and end of P0. The results are shown in Table 37. Table 37: Secretion of PEDF into culture medium along the process TIFF2024527986000043.tif20170

[0280] As expected, PEDF levels increased as the cell population became purer and the RPE population became more mature (along the progression of the process). Between the end of the NIC and Activin A steps and the end of the differentiation steps, PEDF levels increased (>10-fold higher). Between the end of differentiation and the end of P0, the increase in PEDF was moderate (>2-fold higher).

[0281] Process-driven PDL

[0282] During RPE differentiation, yield values ​​were calculated to determine how many RPE cells were harvested from each hESC seeded for the differentiation process. Table 38: PDL of cells during differentiation and RPE proliferation TIFF2024527986000044.tif20170

[0283] The number of cell doublings along the process was approximately 16, similar to previous runs of the FFMD protocol.

[0284] Batch Release

[0285] Viability (%) and total cell count / vial

[0286] 2×10 6 Frozen vials (MCS11B) at the end of P2 with a concentration of viable cells / mL / vial were thawed and tested for % recovery and % viability. Table 39: Viability (%) and recovery (%) of thawed vials. TIFF2024527986000045.tif17170

[0287] The results met the acceptance criteria of greater than 75% recovery and greater than 70% viability. The relatively low recovery values ​​could be the result of the long DP incubation time (>2 hours) due to the parallel cryopreservation of several groups. Furthermore, cell counting in CS5 just before vialing revealed a low concentration of viable cells (1.75×10 6 The recovery rate was approximately 89% when the post-thaw cell count was normalized to the actual number of cells counted immediately prior to placing in the vial.

[0288] RPE purity / identity (CRALBP / PMEL17) was assessed at batch release. RPE cells at the end of P2 were 96.65% double positive for RPE purity / identity.

[0289] polarization

[0290] A potency assay was performed and the results of polarized cytokine secretion as well as the net TEER of the cells at day 14 are shown in Table 40 below. Table 40: Current formal potency assays. TIFF2024527986000046.tif13170 * According to QC-REP-012 and Figure 1

[0291] In addition, frozen samples were thawed and tested in the modified potency assay. The results are summarized in Table 41. Table 41: Modified direct potency assay. TIFF2024527986000047.tif13170 * According to QC-REP-012 and Figure 1

[0292] Survival of hESCs

[0293] To determine the presence of residual hESCs, cells were thawed and stained for TRA-1-60 / Oct-4. The % of hESCs was below the limit of detection (0.0004%).

[0294] Karyotype analysis

[0295] A frozen vial of P2 was thawed, subcultured for 2 passages, and fixed for karyotype analysis. The cells had a normal karyotype; no triple identical chromosomal deletions or double identical additions were found in any sample.

[0296] Consideration

[0297] Process development for manufacturing OPREGEN® towards a commercial process includes FFMD procedures for FF hESCs in addition to culturing RPE cells in a large-scale system under monitored and controlled conditions. This report summarizes the production process including FFMD and RPE expansion in 3L SUBs of cells manufactured at a CCN GMP facility. The first step of the FFMD process involves expansion of FF hESCs. Cell pluripotency at each of the hESC expansion passages was high, exceeding 95% for all markers tested. As expected, a moderate decrease in pluripotency marker expression was observed between the hESC expansion I and hESC expansion II stages.

[0298] Assessment of RPE purity / identity at the end of differentiation and at the end of P0 demonstrated successful differentiation of cells from the cell bank into 45.96% CRALBP / PMEL17 expressing RPE cells at the end of the differentiation stage. Passaging and culturing of differentiated cells enriched the RPE population, with 97.96% co-expression of CRALBP / PMEL17 markers at the end of P0. These results were within the range of RPE purity at these stages seen in previous FFMD runs. Furthermore, the PEDF concentration in the culture medium increased as differentiation progressed to P0 of the RPE expansion procedure, from 256.19 ng / ml / day at the end of the NIC and Activin A steps to 4,687.02 ng / ml / day at the end of P0, confirming the enrichment and maturation of the RPE population for the marker points selected according to the PEDF selection results.

[0299] Finally, at the end of the process, cells were cultured for P2 passage on MC in a semi-automated, controlled, closed system (Eppendorf BioFlo 320 console) monitoring the BioBLU 3L SUB. At the end of P2, cells were harvested and cryopreserved as OPREGEN® TAI. The RPE cells produced in this developed FFMD and large-scale non-GMP engineering run met all OPREGEN® required pass / fail criteria in all batch release tests performed. Taken together, these results qualify the developed process of the OPREGEN® non-GMP engineering run of the CCN-FFHESC-01 MCB.

[0300] conclusion

[0301] The non-GMP engineering run-FFMD large-scale OPREGEN® production process met OPREGEN® TAI IPC and release criteria; there were no residual hESCs in OPREGEN® TAI and the purity of the RPE cells was not compromised. Furthermore, the cells retained their biological activity and met OPREGEN® acceptance criteria for % viability and % recovery. Table 42 TIFF2024527986000048.tif70170

[0302] Example 5 To grow hESC-derived RPE in a large-scale closed and controlled environment, we developed a method for growing RPE on Star Plus microcarriers (Solohil), as established for fetal-derived RPE. In our closed system, differentiated RPE cells are inoculated into a single bioreactor containing microcarriers screened for optimal RPE yield and quality. Cellular oxygen consumption is automatically monitored and controlled, as are PH metabolites and temperature. The nutritional regimen is performed in fed-batch mode, where fresh medium and glucose are added as needed. All operations, including microcarrier and medium addition, cell sampling and filtration, are performed in a controlled and closed environment by using tube welding of disposable bags until the final product of cell suspension in freezing medium is automatically dispensed into cryovials, after controlled large-scale freezing of up to 2300 thousands of vials per hour freezing session.

[0303] Although the description herein contains many specific details, these should not be construed as limiting the scope of the present disclosure, but merely as providing an illustration of some of the currently preferred embodiments. Thus, it will be understood that the scope of the present disclosure fully encompasses other embodiments that may become apparent to those skilled in the art.

[0304] In the claims, reference to an element in the singular is not intended to mean "one and only one" unless expressly stated, but rather "one or more." All structural, chemical and functional equivalents to the elements of the disclosed embodiments known to those of skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Furthermore, no element, component or method step of the disclosure is intended to be made available to the public, regardless of whether the element, component or method step is expressly recited in the claims. No element in the claims herein should be construed as a "means plus function" element unless the element is expressly recited using the phrase "means for." No element in the claims herein should be construed as a "step plus function" element unless the element is expressly recited using the phrase "step for."

Claims

1. A method for proliferating retinal pigment epithelial (RPE) cells, comprising: a) providing a population of RPE cells differentiated from pluripotent stem cells; and b) inoculating the population of RPE cells into a medium containing a first suspension cell support matrix; and c) proliferating the population of RPE cells on the first suspension cell support matrix in a dynamic suspension state to provide a proliferated population of RPE cells. A method comprising the above steps.

2. The method according to claim 1, wherein the population of RPE cells has been proliferated on a solid surface under static conditions prior to step a).

3. The method according to claim 2, wherein the solid surface includes a culture plate.

4. The method according to claim 1, wherein the population of RPE cells has been proliferated on a solid surface under dynamic conditions prior to step a).

5. The method according to claim 4, wherein the solid surface includes a second suspension cell support matrix.

6. The method according to any one of claims 2 to 5, wherein the solid surface is coated.

7. The method according to any one of claims 2 to 5, wherein the population of RPE cells has been proliferated on the solid surface under static conditions for one passage prior to step a).

8. The method according to any one of claims 2 to 5, wherein the population of RPE cells for proliferation on the solid surface is obtained from an intermediate cell bank.

9. The method according to any one of claims 1 to 5, wherein the suspension cell support matrix includes microcarriers.

10. The method according to claim 1, wherein the population of RPE cells is provided from an intermediate cell bank.

11. The method according to any one of claims 1 to 5, wherein the first suspension cell support matrix is not coated.

12. The differentiation of the population of RPE cells from pluripotent stem cells comprises: i. proliferating pluripotent stem cells on a solid surface under conditions that maintain the pluripotency of the pluripotent stem cells to provide proliferated pluripotent stem cells; and ii. differentiating the proliferated pluripotent stem cells in a medium containing a differentiation inducer and optionally a growth factor for a certain period to provide the population of RPE cells. The method according to any one of claims 1 to 5, comprising the above steps.

13. The method according to claim 12, wherein the solid surface is a culture plate.

14. The method according to claim 12, wherein the solid surface comprises a second suspension cell support matrix and the pluripotent stem cells are grown in dynamic culture.

15. The method according to claim 12, wherein step ii comprises differentiating the expanded pluripotent stem cells on a third suspension cell support matrix in dynamic culture.

16. The method according to claim 15, wherein the expanded pluripotent stem cells from step i remain attached to the second suspension cell support matrix in step ii.

17. The method according to claim 14, wherein the second suspension cell support matrix comprises a second microcarrier.

18. The method according to claim 15, wherein the third suspension cell support matrix comprises a third microcarrier.

19. The method according to any one of claims 1 to 5, wherein at least two of the first suspension cell support matrix, the second suspension cell support matrix, and the third suspension cell support matrix are the same.

20. The method according to any one of claims 1 to 5, wherein the first suspension cell support matrix, the second suspension cell support matrix, and the third suspension cell support matrix are different.

21. The method according to claim 12, wherein step ii comprises differentiating the expanded pluripotent stem cells on a culture plate in static culture.

22. The method according to claim 14, wherein the pluripotent stem cells grow into a monolayer attached to the second suspension cell support matrix and / or the third suspension cell support matrix.

23. The method according to claim 12, wherein the conditions for maintaining pluripotency do not include feeder cells.

24. The method according to claim 12, wherein the conditions for maintaining pluripotency include a feeder cell population.

25. The method according to claim 12, wherein the differentiation-inducing reagent is nicotinamide.

26. The method according to claim 12, wherein the growth factor is a member of the TGFβ family.

27. The method according to any one of claims 1 to 5, wherein the first suspension cell support matrix, the second suspension cell support matrix, and / or the third suspension cell support matrix comprises polystyrene, polystyrene with a modified surface, chemically modified polystyrene, crosslinked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or a combination thereof.

28. The method according to claim 27, wherein the first microcarrier, the second suspension cell support matrix, and / or the third suspension cell support matrix is spherical, elliptical, rod-shaped, disc-shaped, porous, non-porous, smooth, flat, or a combination thereof.

29. The method according to any one of claims 1 to 5, wherein the first suspension cell support matrix, the second suspension cell support matrix, and / or the third suspension cell support matrix is coated with laminin, Matrigel, collagen, polylysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenascin, dextran, peptide, derivatives thereof, or a combination thereof.

30. The method according to any one of claims 1 to 5, wherein the solid surface is uncoated or coated with laminin, Matrigel, collagen, polylysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenascin, dextran, peptide, derivatives thereof, or a combination thereof.

31. The method according to any one of claims 1 to 5, wherein the population of RPE cells has a population doubling level of 2 to 4 between P0, 2 to 3 between P1, and 1 to 2 between P2 in step c).

32. The method according to any one of claims 1 to 5, wherein the population of RPE cells is seeded in the presence of 2% to 20% human serum / DMEM between P0, between P1, and between P2 in step c).

33. The method according to any one of claims 1 to 5, wherein the population of RPE cells is seeded on a solid substrate by dynamic culture between P0, between P1, and between P2 in step c).

34. The population of the RPE cells is i) In engineering c), between P0, between P1, and between P2, 50,000 cells / cm ２ to 120,000 cells / cm ２ of cell density, on a solid substrate by dynamic culture, or ii) the population of the RPE cells is dynamically cultured on a solid substrate having a surface area of 2.5 cm2 / ml to 10 cm2 / ml during P0, during P1, and during P2 in step c) The method according to any one of claims 1 to 5, wherein the method is seeded. **Claim 35**: i) the conditions for growth during step c) include maintaining the dissolved oxygen% above 30%, or ii) the conditions for growth in step c) include an initial growth medium volume starting from 50% of the total growth chamber volume of the system, and a growth medium volume of 16.6% of the total growth chamber volume of the system is added every 2 to 4 days The method according to any one of claims 1 to 5. **Claim 36** The method according to any one of claims 1 to 5, wherein the RPE cells exhibit characteristics of mature RPE cells. **Claim 37** The method according to claim 36, wherein the mature RPE cells are more than 95% double positive for cellular retinaldehyde-binding protein (CRALBP) and premelanosome protein (PMEL17) when measured by flow cytometry. **Claim 38** The mature RPE cells have a net transepithelial electrical resistance (TEER) > 100 Ω*cm ２ The method according to claim 37, wherein a polarized monolayer having a polarized secretion of PEDF and VEGF is generated after thawing. **Claim 39** The method according to claim 38, wherein the mature RPE cells are cryopreserved and can be administered to a subject immediately upon thawing. **Claim 40** The method according to claim 36, wherein the mature RPE cells contain less than 0.01% pluripotent stem cells when confirmed by high-precision flow cytometry (FCM) and are negative for TRA-1-60 / Oct-4 when measured by flow cytometry. **Claim 41** The method according to any one of claims 1 to 5, wherein the dynamic cell growth suspension is performed in a disposable bioreactor. **Claim 42** The method according to any one of claims 1 to 5, wherein the population of the RPE cells grows in the presence of nicotinamide. **Claim 43** A pharmaceutical composition comprising RPE cells generated by the method according to any one of claims 1 to 5 for use in the preparation of a medicament for the treatment of an eye disorder or disease in a patient by transplanting the RPE cells into the patented retinal tissue. **Claim 44** The pharmaceutical composition according to claim 43, wherein the eye disorder or disease is a subtype of age-related macular degeneration (AMD), Best disease (early-onset vitelliform macular dystrophy) including hereditary macular degeneration, or retinitis pigmentosa (RP). **Claim 45** The population of the RPE cells is a) determining that the mature RPE cells are more than 95% double positive for cellular retinaldehyde-binding protein (CRALBP) and premelanosome protein (PMEL17) when measured by flow cytometry; b) the mature RPE cells have a net transepithelial electrical resistance (TEER) > 100 Ω*cm ２ and determining to generate a polarized monolayer having a polarized secretion of PEDF and VEGF after thawing; c) the mature RPE cells contain less than 0.01% pluripotent stem cells when confirmed by high-precision flow cytometry (FCM) and are negative for TRA-1-60 / Oct-4 when measured by flow cytometry The pharmaceutical composition according to claim 43, which can be immediately used in a patient based on a product release determination including the above.

46. The pharmaceutical composition according to claim 43, wherein the concentration of mature RPE cells for transplantation is 100,000 cells per 50 microliters.

47. The pharmaceutical composition according to claim 43, wherein the mature RPE cells for transplantation are injected into the subretinal space of the patient.

48. The pharmaceutical composition according to claim 43, which is formulated so as to be thawed and injected into a subject without cell preparation before injection.

49. A pharmaceutical composition comprising the cells generated by the method according to any one of claims 1 to 5.

50. The pharmaceutical composition according to claim 49, which is formulated so as to be thawed and injected into a subject without cell preparation before injection.

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