Purification of culture medium from retinal pigment epithelial (RPE) cells

Abstract

To create a cell specific medium or a cell-free therapeutic agent to improve cell proliferation, cell proliferation rate, viability, and differentiation.SOLUTION: Provided is a method for creating a cell line specific medium or a cell-free therapeutic agent, including: a step for culturing immature RPE cells on a growth support structure, in which the RPE cells show optimal cryo-preservation viability characteristics selected from (i) RPE cells with no pigmentation, (ii) non-polarized or partially polarized RPE cells, (iii) RPE cells with no mature cobblestone-like form, (iv) RPE cells with a gene expression level lower than a mature cell level or with a specific gene expression lacking, and (v) RPE cells with a semi-crowded single layer structure; a step for recovering a supernatant culture solution containing a secretion factor from the culturing step; and a step for refining the culture solution and refining the medium or the cell-free therapeutic agent.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 728,646, filed on September 7, 2018, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Description of Rights to Inventions Made Under Federally Sponsored Research and Development Not applicable

[0003] 1. Field of the Invention This application generally relates to devices and methods for implanting an ultrathin substrate into a target tissue, where the ultrathin substrate is suitable for seeding stem cells for stem cell therapy, microbubbles, and injected gels for drug delivery, among other therapeutic treatments. The ultrathin substrate can further be used for the renewable culturing of cells and cryopreservation after seeding the cells.

Background Art

[0004] 2. Description of Related Art The range of human diseases involving cell loss or damage is very broad and includes, but is not limited to, eye diseases, neurodegenerative diseases, endocrine diseases, cardiovascular diseases, and cancer. Cell therapy involves the use of cells to treat diseased or damaged tissues. It has been rapidly emerging at the forefront of technologies that are prepared to treat many diseases, especially those that affect individuals who do not respond to conventional pharmacological treatments. Many of these diseases would benefit from long - term, focused treatment of the target area, which would reduce systemic side effects. However, certain drugs, such as protein therapeutics, are expensive, costing thousands of dollars per vial and requiring ongoing, repeated treatments.

[0005] In fact, many of the diseases that are candidates for cell therapy are not life - threatening but are associated with a loss of normal physiological function. For example, eye diseases often involve functional degeneration of various eye tissues that affect vision, thereby affecting the quality of life of many individuals.

[0006] The mammalian eye is a specialized sensory organ capable of converting incident photons, focused by the anterior optical system (cornea and lens), into neurochemical signals. This process of light transmission enables vision by sending action potentials via the optic nerve to higher-order cortical centers. The retina of the eye contains photoreceptors that sense various levels of light and interneurons that relay signals from these photoreceptors to retinal ganglion cells. These photoreceptors are the most metabolically active cells in the eye (if not in the body) and are metabolically and functionally supported by retinal pigment epithelial (RPE) cells. These RPE cells are located in a single layer of the eye and are crucial for visual acuity.

[0007] Many medical conditions, including trauma to the eye, infection, degeneration, vascular abnormalities, and inflammatory problems, can impair or completely eliminate an individual's ability to perceive visual images. The central part of the retina, known as the macula, is responsible for central vision, fine visualization, and color discrimination. Macular function can be adversely affected by other medical conditions, including age-related macular degeneration (wet or dry), diabetic macular edema, idiopathic choroidal neovascularization, high myopic macular degeneration, or advanced retinitis pigmentosa.

[0008] Age-related macular degeneration typically causes loss of vision in the central part of the visual field (macula) due to retinal damage. It is the leading cause of vision impairment in older adults (>50 years). Macular degeneration can occur in both "wet" and "dry" forms.

[0009] In the dehydrated state, cell fragments (drusen) accumulate between the RPE cell layer and the choroid, adversely affecting the RPE cells, leading to their dysfunction, degeneration, and ultimately death. Retinal photoreceptor cells, which depend on viable RPE cells to perform vital support functions, become dysfunctional and die after the pathological condition of the RPE cells.

[0010] In more severe moist morphologies, newly formed blood vessels from the choroid infiltrate the space behind the macula, and these newly formed vessels are fragile and often leak blood—thereby causing the death of photoreceptors and their supporting cells.

[0011] While diseases that cause damage to specific cells or tissues are clear candidates for cell therapy, there is still a need in the art for improved methods of cell therapy, including methods, substrates, and devices that improve the effectiveness of cell therapy, as well as methods and compositions that enable the long-term preservation of functional and viable cells used in such treatments. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent No. 8,808,687 [Non-patent literature]

[0013] [Non-Patent Document 1] Kashani AH, Lebkowski JS, Rahhal FM et al. A bioengineered retinal pigment epithelial monolayer for advanced, dry AMD. Sci. Transl. Med. 2018;10:435, eaao4097. [Overview of the project] [Problems that the invention aims to solve]

[0014] In various embodiments, the present invention generally relates to methods and compositions for cryopreservation (also known as "freezing") of cells grown on a substrate. In particular, it relates to methods and compositions for cryopreservation of cells seeded and / or grown on a polymer substrate. In specific applications, cells are retinal pigment epithelial (RPE) cells, photoreceptor cells, stem cells, predifferentiated cells, differentiated versions of such cells, or combinations thereof, regardless of cell differentiation, origin, or culture history. [Means for solving the problem]

[0015] To address the need for improved long-term storage of cell-containing compositions used in cell therapy, several embodiments provide a method for cryopreserving cells on a substrate, comprising: exposing a substrate on which a cell composition is seeded to a temperature ramp-down phase having a desired temperature decrease rate; moving the substrate on which the cells are seeded to a desired intermediate temperature range for a first period; and maintaining the substrate on which the cells are seeded to a desired storage temperature range for a second period, thereby obtaining cryopreserved cells on the substrate that are suitable for long-term storage and use in cell therapy after thawing.

[0016] In various embodiments, the present invention provides modified steps of specific cryopreservation methods and procedures to accommodate various cell characteristics. Furthermore, specific cell characteristics and substrate characteristics are selected at various stages of the cryopreservation process to improve the viability of cells in a substrate seeded with cells for effective therapeutic purposes and to improve transplantation success.

[0017] The present invention introduces a novel method and apparatus that offers numerous advantages, including beneficial results for cryogenic storage of substrates, including higher cell viability, minimal damage to the substrate, and an overall simplified process for producing substrates seeded with cells suitable for cell proliferation and direct transplantation.

[0018] In another embodiment, the present invention introduces a method for intentionally suppressing melanin formation. Such a method may be made possible by certain items included in a kit.

[0019] The present invention also introduces a novel method for forming cell-specific media to improve cell proliferation, cell growth rate, viability, and differentiation. In certain embodiments, such cell-specific media can be further adapted for direct injection, such as intravitreous (IVT) delivery, to areas of a substrate seeded with specific cells or already transplanted cells.

[0020] A system of one or more computers can be configured to perform a particular operation or action by installing in the system, during operation, software, firmware, hardware, or a combination thereof that causes the system to perform the action. One or more computer programs can be configured to perform a particular operation or action by including instructions that, when executed by a data processing apparatus, cause the apparatus to perform the action. A general aspect includes a method of cryopreserving cells on a substrate, the method including the step of providing a biocompatible polymer substrate seeded with a monolayer of immature retinal pigment epithelial (RPE) cells, the polymer substrate providing a cell-seeding surface. The method of cryopreserving the cells also includes the step of identifying i) when a monolayer of immature RPE cells reaches 90% to 99% confluent (also referred to as "confluent") on the substrate and ii) when most of the immature RPE cells are not fully pigmented. The method of cryopreserving the cells also includes, once identified, exposing the substrate seeded with the cells to a controlled rate of temperature decrease of from about -1 °C / minute to about -30 °C / minute until a first temperature below -20 °C is reached. Other embodiments of this aspect include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the actions of the method.

[0021] The implementation may include one or more of the following features: In the method, the substrate on which the cells are seeded reaches a temperature lower than the temperature representing the latent heat release of the seeded cells. In the method, the surface is substantially parallel to a monolayer of immature RPE cells seeded on the substrate to a degree sufficient to induce nucleation and efficient temperature compensation in response to the latent heat release of the seeded cells. In the method, further comprising the step of maintaining the substrate on which the cells are seeded at a first temperature, wherein the first temperature is -20°C to about -100°C after a controlled rate of temperature decrease for a first period to obtain temperature uniformity. In the method, further comprising the step of maintaining the cells at a storage temperature lower than the first temperature, within 50°C of the first temperature, for a second period to obtain cryogenically preserved cells. In the method, further comprising the step of maintaining the substrate on which the cells are seeded at a first temperature, wherein the first temperature is -20°C to about -100°C after a controlled rate of temperature decrease for a first period. The method may also include a step of performing a second controlled rate of temperature reduction to ultimately maintain the cells at a storage temperature below -196°C for a second period, thereby obtaining cryopreserved cells. In the method, the second period is 24 hours to 60 months. In the method, the second controlled rate of temperature reduction is approximately -1°C / min to approximately -30°C / min. In the method, the cell monolayer has a cell seeding density of 200,000 to 700,000 cells per milliliter of cell suspension, or 100,000 to 350,000 cells per square centimeter of substrate surface. In the method, more than 50% of the RPE cells have a cobblestone morphology. In the method, the RPE cells are unpigmented or partially pigmented. In the method, the cells are RPE cells exhibiting cryopreservation viability characteristics, selected from (i) RPE cells that are unpigmented or partially pigmented, (ii) RPE cells that are attached but unpolarized, partially polarized, or fully polarized, (iii) RPE cells that have or do not have a mature cobblestone morphology, (iv) RPE cells whose gene expression levels are lower than those of mature cells or that lack specific gene expression, and (v) RPE cells that are dense or have a semi-dense monolayer structure of more than 90%.In the method, the substrate has one or more properties selected from (i) the coefficient of thermal expansion of the substrate, (ii) the elastic parameter of the substrate, (iii) the thickness of the substrate, (iv) surface modification, and (v) shear force resistance, wherein the properties help to improve the viability of seeded cells and the functionality of the substrate during low-temperature storage and thawing. In the method, the biocompatible polymer comprises parylene. In the method, the identification step comprises determining whether at least one or more apical secretions, basal secretions (also called "basal"), or nonpolar specific secretions of immature RPE cells are at a lower level than that of mature RPE cells. In the method, the apical secretions comprise αβ crystallin, hyaluronan, matrix metallopeptidase (MMP)-9, pigment epithelial-derived factor (PEDF), transforming growth factor (TGF)-β, a tissue inhibitor of metalloproteinase (TIMP)-I, or mechano-growth factor (MGF)-E8. In the method, the basal secretion includes cystatin c, endothelin i, fibroblast growth factor (FGF) 5, or vascular endothelial growth factor (VEGF). In the method, the nonpolar specific secretion includes brain-derived neurotrophic factor (BDNF), complement factor H (CFH), ciliary neurotrophic factor (CNTF), fibrin 3 / 5, fibroblast growth factor (FGF) 2, heparin-binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-I, leukemia suppressor (LIF), matrix metalloproteinase (MMP)-9, nerve growth factor (NGF), and tropoelastin. In the method, the identification step includes determining that the gene expression of at least one or more RPE65, REX1, EIF2B2, SERF2, and UBE2R2 in immature RPE cells is lower than that in mature RPE cells. In the method, the substrate includes a thin region configured to allow cells to diffuse nutrients through it. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0022] A common embodiment includes a method for producing a substrate on which transplantable cells are seeded, the method comprising the step of providing a biocompatible polymer substrate on which a monolayer of immature retinal pigment epithelial (RPE) cells are seeded, wherein the polymer substrate provides a cell seeding surface. The method also comprises the step of cryogenically preserving the cells on the substrate by exposing the cell-seed substrate to a controlled temperature reduction rate of about -1°C / min to about -30°C / min. The method also comprises the step of moving the cell-seed substrate to a temperature below 4°C, thereby obtaining cryogenically preserved or dormant cells. The method also comprises the step of thawing the cells on the cryogenically preserved substrate by warming the cell-seed substrate to a target temperature using a temperature ramp-up heating rate, thereby obtaining thawed cells seeded on the substrate, wherein the thawed cells retain viability and / or functionality after thawing. The method also comprises the step of culturing the seeded cells for an additional period to reach a mature state before transplantation. Other embodiments of this model include a corresponding computer system, apparatus, and computer programs stored in one or more computer storage devices, each configured to perform the actions of this method.

[0023] The implementation form may include one or more of the following features. The method further includes culturing the cells seeded on the thawed substrate in a first medium containing a basal medium supplemented with a combination including at least one or more bovine serum albumin (BSA), activin A, hepatocyte growth factor (FGF), insulin-like growth factor (IGF) 1, dickkopf-related protein 1 (DKK1), and noggin. The method further includes culturing the cells seeded on the thawed substrate in a first medium containing a basal medium supplemented with the supernatant derived from previously cultured RPE cells, and subsequently culturing the cells in a second medium containing a basal medium supplemented with insulin-like growth factor (IFG) 1, dickkopf-related protein 1 (DKK1), and noggin. In the method, the supernatant of the first medium is derived from a culture of immature RPE cells including RPE cells that (i) are not pigmented or partially pigmented, (ii) are non-polarized or partially polarized RPE cells, (iii) have or do not have a mature cobblestone-like morphology of at least 50%, (iv) have gene expression levels lower than those of mature cells or lack specific gene expression, and (v) exhibit optimal cryopreservation viability characteristics selected from quasi-confluent or confluent monolayer RPE cells. In the method, the supernatant of the first medium is derived from a culture of RPE cells exhibiting mature characteristics selected from (i) pigmented RPE cells, (ii) polarized RPE cells, (iii) RPE cells having a mature cobblestone-like morphology, (iv) RPE cells having gene expression levels corresponding to those of mature RPE cells, and (v) RPE cells having a confluent monolayer structure. The implementation form of the described technology may include hardware, a method or process, or computer software on a computer-accessible medium.

[0024] A general embodiment includes a method for preparing a cell line-specific medium or cell-free therapy, the method comprising the steps of culturing immature RPE cells on a growth support structure, wherein the RPE cells exhibit optimal cryopreservation viability characteristics selected from (i) non-pigmented RPE cells, (ii) non-polarized or partially polarized RPE cells, (iii) RPE cells that do not have a mature cobblestone morphology, (iv) RPE cells whose gene expression levels are lower than those of mature cells or which lack specific gene expression, and (v) RPE cells with a semi-dense monolayer structure. The method also includes the steps of recovering a culture medium containing secreted factors. The method also includes the steps of purifying the culture medium to prepare a medium or cell-free therapy. Other embodiments of this embodiment include a corresponding computer system, apparatus, and computer program recorded on one or more computer storage devices, each configured to perform the actions of the method.

[0025] The implementation may include one or more of the following features. The method further includes culturing RPE cells until, before purifying the culture medium, the cells exhibit a maturation characteristic selected from (i) pigmented RPE cells, (ii) polarized RPE cells, (iii) RPE cells having a mature cobblestone morphology, (iv) RPE cells with gene expression levels similar to those of mature cells, and (v) RPE cells with a dense monolayer structure. In the method, purification is achieved by centrifugal filtration, fractionation, size exclusion chromatography, affinity chromatography, size exclusion filtration, or precipitation. The method further includes packing the culture medium supernatant into a syringe. The supernatant is supplemented with a drug medium or diluent. The method further includes culturing photoreceptor cells or photoreceptor progenitor cells. In the method, the growth support structure includes a substrate, a lattice structure, agar, or a hydrogel. In the method, cells grow on, in, or enclosed (also called "encapsulated") a biocompatible substrate or container. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0026] A general embodiment includes a method for treating a disease or disorder, comprising the step of administering to a subject an effective amount of RPE cell-specific medium or cell-free therapy obtained by a method comprising: the method also comprising the step of culturing RPE cells on a growth support structure, wherein the RPE cells exhibit mature characteristics characterized by the detection of RPE65 levels; the method also comprising the step of recovering a culture medium containing secretory factors; and the method also comprising the step of purifying the culture medium to prepare a cell-free therapy. Other embodiments of this embodiment include a corresponding computer system, apparatus, and computer program recorded on one or more computer storage devices, each configured to perform the actions of the method.

[0027] The implementation may include one or more of the following features: The method further includes the step of transplanting RPE cells. The method further includes the step of transplanting RPE cells seeded on a substrate. The method further includes the step of transplanting photoreceptor cells. The method further includes the step of transplanting RPE cells and photoreceptor cells on a substrate. In the method, purification is achieved by centrifugal filtration, fractionation, size exclusion chromatography, affinity chromatography, size exclusion filtration, or precipitation. In the method, the growth support structure may be a substrate, a lattice structure, agar, or a hydrogel. In the method, cells grow on, in, or encapsulated within a biocompatible substrate or container. The implementation of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0028] A general embodiment includes a substrate for cell therapy to generate the external limiting membrane of the biological (or “anatomical”) structure of the retina, comprising a substrate having a curvature and size substantially similar to that of the affected site of the functional external limiting membrane of the patient in a normal or flexible state. The substrate also includes RPE cells and photoreceptor cells seeded on the substrate as independent individual layers that mature after transplantation to generate the external limiting membrane. Other embodiments of this embodiment include a corresponding computer system, apparatus, and computer program recorded on one or more computer storage devices, each configured to perform the actions of the Method.

[0029] The implementation may include one or more of the following features: A substrate coated with culture medium to attract patient progenitor cells after transplantation to generate an outer boundary membrane. The substrate further comprises a culture supernatant produced by the step of culturing RPE cells on a growth support structure. The substrate may also contain RPE cells exhibiting mature characteristics characterized by the detection of RPE65 levels. The substrate may also include a step of recovering the culture medium containing secreted factors. The substrate may also include a step of purifying the culture medium to prepare a culture medium or cell-free therapy. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0030] A general embodiment includes a method for producing a cell-specific medium, comprising the steps of culturing a first cell line in a first medium on a selectively permeable substrate, wherein the first cell line processes the first medium and produces cellular secretions. The method also includes the steps of collecting the cellular secretions. The method also includes the steps of adding the cellular secretions to a second medium to form a cell-specific medium. Other embodiments of this embodiment include a corresponding computer system, apparatus, and computer program stored on one or more computer storage devices, each configured to perform the actions of the method.

[0031] The implementation may include one or more of the following characteristics: In the method, the cell secretion includes a combination of proteins, hormones, enzymes, by-products, and waste products (also known as "waste"). In the method, the cell secretion includes apical secretion from RPE cells containing at least one of the following tissue inhibitors: αβ crystallin, hyaluronan, matrix metalloproteinase (MMP)-9, pigment epithelial-derived factor (PEDF), transforming growth factor (TGF)-β, and metalloproteinase (TIMP)-I. In the method, the cell secretion includes basal secretion from RPE cells containing at least one of cystatin C, endothelin I, fibroblast growth factor (FGF)-5, and vascular endothelial growth factor (VEGF). In the method, the cell secretions include nonpolar specific secretions from RPE cells containing at least one of BDNF, CFH, CNTF, fibrin 3 / 5, FGF 2, HB-EGF, HGF, IGF-I, LIF, MMP-9, NGF, and tropoelastin. The substrate induces polarity in seeded cells, enabling them to secrete apical, basal, and nonpolar specific secretions. The formed cell-specific medium is for the controlled growth of immature RPE cells. The formed cell-specific medium is for the controlled maturation of immature RPE cells into mature RPE cells. The formed cell-specific medium is prepared for intravitreous (IVT) injection. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0032] A general embodiment includes a method for forming a therapeutic composition, comprising the step of culturing a first cell line in a first culture medium on a selectively permeable substrate, wherein the first cell line processes the first culture medium and produces cellular secretions. The method also includes the step of collecting the cellular secretions. The method also includes the step of purifying the cellular secretions by selecting specific cellular secretions. Other embodiments of this embodiment include a corresponding computer system, apparatus, and computer program recorded on one or more computer storage devices, each configured to perform the actions of the method.

[0033] The implementation may include one or more of the following features: The method further comprises the step of centrifugation once or multiple times at different rates and reconstitution to produce one or more pellets of various compositions. The method further comprises fractionation, size exclusion chromatography, affinity chromatography, size exclusion filtration, or precipitation. The method further comprises the step of reconstitution to different concentrations. The formed therapeutic composition does not contain cells. The implementation of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0034] A common embodiment includes a method for reversibly attaching a polymer membrane to another polymer surface, comprising the step of placing a membrane substantially surrounded by a predetermined amount of culture medium on the other polymer surface. The method also includes the step of gradually removing the predetermined amount of culture medium until the flat surface of the membrane contacts the flat surface of the other polymer surface. The method also includes the step of drying to reversibly attach the flat surface of the membrane to the flat surface of the other polymer surface with which it is in contact. Other embodiments of this embodiment include a corresponding computer system, apparatus, and computer program recorded on one or more computer storage devices, each configured to perform the actions of the method.

[0035] The implementation may include one or more of the following features: In the method, the polymer surface is the bottom surface of a cell culture plate or well. In the method, the culture medium is Dulbecco phosphate-buffered saline (dpbs). In the method, the drying step is carried out by baking in a desiccator, temperature / humidity controlled chamber, oven, or standing in a controlled room such as a cleanroom. In the method, the polymer membrane is parylene. The method further includes the step of introducing a predetermined amount of culture medium to separate a flat surface of the membrane from the flat side in contact with another polymer surface. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium. [Brief explanation of the drawing]

[0036] The above will be more readily understood from the following detailed description of the present invention, especially when considered in conjunction with the drawings. [Figure 1] This flowchart illustrates the low-temperature storage process from substrate preparation to transplantation, based on one embodiment. [Figure 2] This graph, based on one embodiment, shows the release of latent heat by cells seeded on a substrate during a controlled temperature ramp-down process for low-temperature storage. [Figure 3] Based on one embodiment, here is an example of a substrate for cell seeding that emphasizes a patterned thin surface for nutrient diffusion, and a support structure that enables cell proliferation. [Figure 4] Based on one embodiment, this flowchart illustrates the process of pigment deposition in RPE cells, specifically (A) immature RPE cells without pigment deposition, (B) premelanosomes (with visible lines), (C) melanosomes (with pigment deposited on the lines, where both lines and pigment are visible), and (D) mature RPE cells (filled with lines covered by pigment). [Figure 5] This is a photograph of an exemplary cube-like, rounded stone-like form. [Figure 6] This is a photograph of a disordered morphology containing a mixture of cobblestone-like and fibroblast-like forms. [Figure 7] Based on one embodiment, this is a photograph of a substrate exhibiting uniform cell proliferation, close to dense (90-99% density) or 100% dense. It is indistinguishable to the naked eye, and imaging technology is required to determine the actual density ratio. [Figure 8] Based on one embodiment, this is a photograph of a substrate that exhibits heterogeneous cell proliferation and is only about 70% dense. [Figure 9] This graph illustrates the difference in PEDF secretion between immature RPE cells and mature RPE cells, based on one embodiment. [Figure 10]This graph illustrates various gene expression values ​​(Values) measured by RT-qPCR based on one embodiment. In particular, the ranges for mature RPE cells are RPE65 ΔCt≧-3.0 and REX1 ΔCt≦-8.0. The geometric mean of the reference genes (EIF2B2, SERF2, UBE2R2) is Ct≦32.0. Several other genes, including MAP2, S100A4, TYRP1, and LIN28A, are tested for informational purposes only. [Figure 11] This flowchart illustrates a manufacturing process for a target supernatant containing specific cell-secreted factors, which can be used for injection as IVT or as a supplemental culture medium for subsequent culture, based on one embodiment. [Figure 12] This is a simplified diagram of one embodiment highlighting the interaction between photoreceptors and RPE cells on one or more substrates. [Figure 13] This flowchart illustrates a process for reversibly attaching a membrane to a cell culture well, based on one embodiment. Such a method is useful for immobilizing substrates for automated substrate coating, or for cell seeding processes that depend on the immobilized X and Y axis orientation. Attachment is reversible by introducing a predetermined amount of culture medium onto the membrane. [Figure 14] These are various comparative images, based on one embodiment, comparing the overall cell viability after cryopreservation and transplantation, along with highlighted specific gene expression. [Figure 15] These are multiple images, based on one embodiment, showing the differences in cell morphology one day after thawing due to the use of different cryoprotective agents, compared to unfrozen control cells. [Figure 16] These are multiple images, based on one embodiment, showing differences in cell morphology one week after thawing due to different cryoprotective agents used, compared to unfrozen control cells. [Figure 17] These are six graphs of gene expression obtained from a single sample, comparing non-cold storage and cold storage, based on one embodiment. [Figure 18] A graph of metabolism obtained by a colorimetric metabolic assay is shown based on one embodiment. [Modes for carrying out the invention]

[0037] This invention generally relates to a substrate on which cells are seeded, a cell evaluation process, and a method for enhancing the viability of cell seeding membranes by seeding on the substrate and cryogenic storage processes. Unlike conventional single cells or cell clusters, cell seeding membranes require special consideration to enhance viability after cryogenic storage and thawing. In particular, by making the following adaptations, the results show not only increased cell viability but also increased health of surviving cells (e.g., metabolic activity, lifespan), including after transplantation.

[0038] Low-temperature storage optimization: Substrate / Membrane In some embodiments, the substrate comprises a biocompatible polymer that functions as a seeding surface. In some embodiments, the substrate comprises parylene in combination with other materials, the other materials being either biodegradable or non-biodegradable. In some embodiments, the substrate is treated to have one or more properties that improve the viability of seeded cells. For example, in some embodiments, the substrate further comprises a coating to improve cell adhesion to the substrate. In some embodiments, the coating comprises one or more layers of Matrigel, Vitronectin, Fibronectin, and Retronectin, or derivatized parylene. Other cell culture media and various combinations thereof known to those skilled in the art are substituted. In other embodiments, other coatings or surface modifications are used to achieve improved cell adhesion to the substrate and / or improve the durability and / or viability of cells and the substrate during and after the cryogenic storage process. For example, in some embodiments, the coating improves cell viability during, after, or both cryogenic storage. In another embodiment, the cell proliferation surface of the substrate is treated with oxygen to create a hydrophilic cell proliferation surface. Other surface treatments include oxygen plasma treatment, chemical etching, and additional polymer deposition. The additional polymer deposition offers advantages in terms of its elasticity (e.g., elongation at break (%)) compared to parylene-C, and its high oxygen gas permeability (cc.mm / m²). 2Regarding parylene (N), it is particularly useful when using different parylene types depending on their functional requirements, such as parylene-N (Specialty Coating Systems, KISCO Corporation). Furthermore, functionalized parylene can be deposited on the surface and conferred with active chemical functional groups, including, but not limited to, (1) R-hirudin, a protein that has anticoagulant properties and thereby enhances the blood compatibility of transplants; (2) amine functional groups that covalently bond fibronectin to the surface to improve osteoblast adhesion; and (3) pNIPAM chains (Tan et al. 2010) that form a hydrophobic surface that promotes tissue adhesion.

[0039] In some embodiments, the properties of the substrate include one or more of the thermal expansion coefficient of the substrate, the elastic parameters of the substrate, or the thickness of the substrate. In some embodiments, the substrate contains parylene and is selectively permeable, and its properties include the thickness of the substrate, which is selected to allow nutrients to pass through the substrate. The substrate may also be non-permeable and depend solely on the thickness of the substrate in terms of its permeability properties. Thus, when transplanted to a target site after thawing, the substrate allows appropriate nutrients to pass to the cells and / or cell waste to pass away from the substrate. In some embodiments, the thickness is selected so that the thermal expansion coefficient of the substrate is such that adverse effects on seeded cells are reduced. The thickness is particularly important in embodiments where cells to be cryopreserved are seeded between two or more materials (e.g., sandwiched, fixed, or other embodiments), where the expansion of the cells or cryoprotectant used may increase during freezing, resulting in shear, torsion, or other stresses that may damage the substrate and / or cells. In some embodiments, the material and thickness are selected so that they have thermal energy release properties that do not interfere with the latent heat release of seeded cells. In one embodiment, the material composition is selected to increase shear force resistance, such as a hexagonal honeycomb pattern of the support structure that may be encountered during low-temperature storage and thawing procedures.

[0040] In one embodiment, where two or more layers of different cells are arranged on a membrane, a second substrate layer may be placed between such cell layers to promote isolated growth within separate layers. For example, if the substrate on which the cells are seeded consists of a specific order of the first substrate layers, the first layer of cells consisting of RPE cells, including the basal side, is in contact with the substrate, the second layer of cells consisting of photoreceptor cells is in contact with the apical surface of the first layer of cells, and the second substrate layer may be placed between the first and second layers of cells. In one embodiment, two cell layers are grown simultaneously on the first substrate layer, with the first layer of RPE cells being seeded first on the substrate, and the second layer of photoreceptor cells being seeded at a subsequent time on the substrate above the RPE cells. Such a time may be 1 to 10 days later, thereby allowing the RPE cells to primarily adhere to the substrate first. In other embodiments, retinal progenitor cells, epithelial cells, other ophthalmic cells, stem cells, or reprogrammed cells that will differentiate into other cell types may consist of one or more layers. Other embodiments may include genetically modified cells that specifically generate certain factors (e.g., proteins, growth factors, antibodies) to promote specific cellular signaling and reprogram adjacent layers. In various embodiments, RPE stem cells may be modified to secrete neurotrophic factors (e.g., PEDF, CNTF, BDNF) that further support the survival of photoreceptor cells. In other embodiments, overexpression of surface integrins improves RPE adhesion to Brooke membranes, increases melanin and reduces the risk of AMD, and overexpression of receptors required for phagocytosis increases the clearance of liopfiscin and other metabolic waste that can interfere with cell layer health.

[0041] In other embodiments, each cell layer grows independently on separate substrates. In this embodiment, it may be beneficial to make the second substrate biodegradable or to consist of a gelatinous growth medium that will be dissolved or degraded and / or absorbed by one or more adjacent cell layers after stacking and transplantation. The growth medium may consist of specific growth factors (e.g., bovine serum albumin (BSA), activin A, fibroblast growth factor (FGF), insulin-like growth factor (IGF1), human Dickkopf WNT signaling pathway inhibitor (DKK1), and noggin) or antibodies (complementary to the functional groups embedded in the parylene substrate, as described below) to improve the adhesion of one or more layers.

[0042] Temperature compensation (Appendix X) provided during controlled rate storage for nucleation control, initiation of liquid-to-crystalline state change, and latent heat release is known to improve the viability of cells after cryopreservation and thawing. In many embodiments, the substrate is oriented parallel to the seeded cells by the composition of the substrate and the seeding area. Thus, the substrate is in close contact and / or proximity to all seeded cells, thereby enabling uniform nucleation of all cells simultaneously and efficient temperature compensation in response to the latent heat release of the seeded cells. This is a particularly beneficial factor for embodiments in which the seeded cells grow to be nearly dense (e.g., >98%) or a dense monolayer. Thereafter, during the cryopreservation process, the substrate efficiently induces nucleation without requiring other methods known in the art, including seeding of ice crystals or other nucleating agents, mechanical vibration, electrofreezing, etc., which would adversely affect the uniform cell layer formed on the substrate. The specific methods of nucleation described above can be used in conjunction with a substrate that merely assists the nucleation process, since the cells are in a dense or nearly dense orientation. The substrate composition and its spatial relationship to the cells thus contribute more beneficially to the viability of cryopreserved cells, in addition to temperature compensation provided by controlled rate freezing during the process of latent heat release by the seeded cells. Latent heat release is partially dependent on the cell line, but primarily on the composition of the cryopreservation culture medium used. In other words, a substrate surface substantially parallel to a monolayer of seeded cells effectively functions as a heat sink, efficiently transferring heat during cryopreservation and thawing, including during latent heat release from the seeded cells.

[0043] An ideal substrate may also possess beneficial properties, such as those found in the substrate described in U.S. Patent No. 8,808,687. These substrate properties include a cell proliferation surface to promote the attachment and formation of a cell monolayer, a periphery that inhibits cell proliferation (e.g., the periphery of the substrate does not consist of a thinned membrane portion for sufficient nutrients for cell proliferation affinity and waste transport, and / or the periphery consists of a raised edge), and enable mechanical manipulation and implantation adjacent to the target tissue.

[0044] In certain embodiments, the substrate is designed to have an optimal non-planar normal state that matches the desired transplantation site. While the substrate can be manipulated during culture and cryogenic storage to maintain a planar shape for easier handling, ease of cell seeding by automated machinery, and improved cell viability, a non-planar shape may be beneficial after transplantation. In embodiments where the seeded substrate contains RPE cells and is transplanted to adequately cover geographic atrophic areas within the retina, the substrate is optimally curved to match the radius of curvature of the retina within the eye. This curvature induces parallel growth of the outer limiting membrane (ELM), which indicates the ultrastructure of photoreceptors and the restoration of adjacent visual function. The RPE monolayer and photoreceptors formed parallel to the substrate act as an adhesive for progenitor cells, forming the ELM after maturation. In comparison, injection of RPE cells in isolation, cell gels, and suspensions with indistinct shapes has shown inadequate clinical outcomes.

[0045] A method for reversibly attaching a substrate / membrane to a tissue culture vessel. Small substrate / film (100mm 2In embodiments where a thin substrate / membrane (less than 50 microns) is required for subsequent transplantation, the minimum mass makes it prone to floating in a given volume of culture medium, making it difficult to maintain the substrate in a standard culture well, thereby causing the substrate to move around and potentially dislodge the seeded cells. One way to prevent such movement of the substrate is to reversibly adhere the membrane to the tissue culture plate by evaporation drying. This may seem counterintuitive, as many cell culture plates have additional features that minimize evaporation (e.g., the evaporation moat filled with sterile water or culture medium in the ThermoFisher Scientific Nunc Edge 2.0 96-well cell culture plate). In one embodiment, the membrane is first placed in the wells of the tissue culture plate with the evaporating solution (e.g., Dulbecco phosphate-buffered saline (DPBS)). The DPBS is slowly removed from the well until the membrane is flat at the bottom of the well and a small amount of liquid residue remains between the bottom of the membrane and the surface of the bottom of the well. After repeating this for all other applicable wells, the tissue culture plate is dried. Various drying methods include baking in a desiccator, temperature / humidity controlled chamber, or oven, or leaving the solution in a controlled room such as a cleanroom. During this drying phase, residual DPBS evaporates, thereby causing the membrane to adhere to the bottom of the wells, which in turn facilitates the application of cell growth coatings (e.g., vitronectin) and cell seeding. By restricting movement within the wells, automated cell seeders can be used for uniform seeding, which requires known X, Y, and Z axes and is typically limited to two-dimensional functionality. After cell seeding, the membrane is reversibly attached by introducing DPBS into the wells, allowing the membrane to be removed from the bottom of the wells and moved to another well for culture. In other embodiments, various other solutions that do not negatively alter pH, salinity, or other cell growth factors can be used instead of DPBS.

[0046] Optimization of cryopreservation: Biological evaluation of cells In embodiments where cells seeded on a substrate are stored at low temperatures, the optimal low-temperature storage viability characteristics differ at each manufacturing stage.

[0047] First, at cell seeding, defined as day 0, cells are optimally selected to be immature cells that readily adapt to the seeding process and maintain viability after inoculation. In embodiments of RPE cells, these characteristics include (i) non-polarization or partial polarization, (ii) minimal to mild pigmentation or lack of melanin, and (iii) seeding at a seeding density of <100% (e.g., 50-90%, more ideally 70-80%).

[0048] In embodiments where RPE cells seeded on a substrate are cryogenically preserved, RPE cells are selected and cryogenically preserved if they exhibit optimal cryogenic viability characteristics after seeding. Such characteristics are observed 2 to 10 days after seeding, and in most embodiments, 6 to 8 days after seeding. Optimal cryopreservation viability characteristics observed immediately before cryopreservation include: (i) RPE cells that are unpigmented or minimally pigmented (including depigmented RPE cells, RPE cells with altered pigmentation due to spontaneous or induced gene mutations in the pigmentation pathway, or chemical induction of altered pigmentation or a combination thereof); (ii) unpolarized or partially polarized RPE cells; (iii) RPE cells in which several fibroblast morphologies may be acceptable, but more than 50% have a mature cobblestone morphology; (iv) RPE cells with gene expression levels lower than those of mature cells or lacking specific gene expression; and (v) RPE cells with a subconfluent (less than 100%) or subconfluent (more than 90%) monolayer structure, illustrating the characteristics of the epithelial layer, such as positive adhesion and growth on the substrate and the formation of tight junctions.

[0049] One characteristic of RPE cells with optimal cryopreservation viability is non-pigmentation or minimal / partial pigmentation. RPE cells contain many melanosomes, pigment granules extending from the apical region to the central part of the cell. Certain RPE cells, such as those adjacent to the macular region, are more heavily pigmented. Non-pigmented or partially pigmented RPE cells may be cells that are not fully differentiated, not isolated, and / or not purified. Various methods can be employed to depigment RPE cells, including chemical removal (e.g., melanogenesis inhibitors), modification of growth medium, and interaction with physiologically adjacent cell types. In one embodiment, a melanogenesis inhibitor, propylthiouracil (PTU), is used to prevent multi-step conversion from tyrosine to dopaquinone and eumelanin. Such methods that inhibit melanogenesis and melanin deficiency during cryopreservation promote RPE cell survival after thawing. PTU may be replaced with various substances known in the art to block or restrict melanogenesis-related pathways.

[0050] Another characteristic of optimally cryopreserved RPE cells is that they are unpolarized, partially polarized, or fully polarized. Polarized RPE cells distinguish between the apical (corresponding to the retinal side of the RPE cell) and basal (corresponding to the choroidal side of the RPE cell) orientations, mimicking physiological characteristics including apical microvilli, distinct tight junctions, membrane transport capacity, and melanocyte pigmentation. Polarization can be distinguished visually or by measured ratios of apical, basal, and nonpolarity-dependent cell secretions.

[0051] Another characteristic of optimally cryopreserved RPE cells is the observation of a cobblestone morphology. Differentiated, isolated, and / or purified RPE cells take on the appearance of a cuboidal, cobblestone morphology.

[0052] An additional characteristic of optimally cryopreserved RPE cells is gene expression levels that are lower than those of mature cells or lack specific gene expression. This characteristic can be tested by RNA or other nucleic acid expression, protein expression, lipid expression, glycosylation patterns, immunohistochemical staining, or electrophysiological characteristics. Secondary measurement techniques, such as secretome level measurement, can also be performed as an alternative or additional measure.

[0053] Another characteristic of optimal cryopreserved viability RPE cells is a semi-dense monolayer configuration. Denseness is achieved when cells grow completely to the available portion and reach contact inhibition. After reaching density, many cell types, including mammalian cell types, exhibit different characteristics compared to semi-dense. The ideal cell seeding density for monolayer RPE cells on a substrate is 2.0 × 10⁵ to 7.0 × 10⁵ cells per milliliter of cell suspension, or 1.0 × 10⁃ to 4.0 × 10⁃ cells per square centimeter of substrate surface, or 1.0 × 10⁵ to 3.5 × 10⁵ cells per well of a standard 48-well cell culture plate. Once density is reached, the cell seeding density approaches 1.0 × 10⁶ as the cells proliferate, as can be understood from the standard characteristic proliferation pattern of cultured cells following the logarithmic growth phase. Cell density can be measured by image analysis, spectrometry, electrical / impedance analysis, and other more invasive / destructive processes. Monolayer formation is promoted compared to multicellular layers by the flat cell seeding surface of the substrate and the matching of the substrate's nutrient / waste transport ratio to support the monolayer.

[0054] Optimal cryogenic viability of RPE cells is achieved 3–10 days after seeding RPE cells onto the substrate, at which point the cells have not yet reached 100% density and are not fully differentiated on the substrate. While optimal cryogenic viability of RPE cells can be obtained within this time window, it may vary depending on the cell line used, growth medium, substrate characteristics, culture conditions, and their combinations. Therefore, one or more of the above characteristics of optimal cryogenic viability of RPE cells should be qualitatively and quantitatively re-examined before cryogenically storing the substrate on which the cells have been seeded.

[0055] The characteristics of the substrate and cell line described above should be considered in order to create an optimal cryogenic storage and thawing protocol to maximize cell viability and functionality, as well as the integrity of the substrate.

[0056] The viability of cells thawed after cryopreservation can be increased by using various methods, including various combinations of cryoprotective substances and their removal.

[0057] Optimal post-thaw RPE cells should maintain nearly 100% density, less than 30% dead cells (ideally less than 10%), and retain a cobblestone morphology. Ideally, PEDF secretion levels should be 5–12 ng / mm³ on days 5–10 after thawing. 2 / 24 hours, optimally around 9.6 ng / mm 2 The time interval is 24 hours. In some embodiments, further gene expression testing is completed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to confirm that gene expression is within the range. In embodiments of RPE cells, key gene expressions are RPE65 ΔCt ≥ -3.0, REX1 ΔCt ≤ -8.0, and geometric mean Ct ≤ 32.0 for reference genes (EIF2B2, SERF2, UBE2R2), or comparable to or similar to mature RPE cells as understood in the literature.

[0058] Optional cell culture improvement: supernatant In various embodiments, cell culture has been shown to produce many cytochemical products, including those used in paracrine, autocrine, endocrine, and intercellular electrical signaling. These chemical products can be found in cell cultures and, after extraction and classification, have potential value as cell culture additives. Cellular secretions may include proteins, hormones, enzymes, by-products, waste products, or combinations thereof. The raw combinations of factors obtained, selected combinations thereof, or purified forms are hereafter collectively referred to as “target supernatants.”

[0059] Further development of cell therapies aimed at replenishing RPE lost in geographic atrophy (GA) is of great interest. One such therapy currently in Phase I / IIa clinical trials involves CPCB-RPE1, a subretinal graft containing polarized human embryonic stem cell (hESC)-derived RPE (PRPE) grown on an ultrathin parylene membrane (Kashani AH, Lebkowski JS, Rahhal FM et al. A bioengineered retinal pigment epithelial monolayer for advanced, dry AMD. Sci. Transl. Med. 2018;10:435p., eaao4097). CPCB-RPE1 can restore the PRPE monolayer and support photoreceptor (PR) preservation even across the graft boundary in animal studies, suggesting that PRPE-derived soluble factors play an important paracrine role. Therefore, in addition to the graft itself, the unique set of essential trophic and signaling factors secreted by the polarized pigmented monolayer of PRPE can also maintain PR survival and function.

[0060] Therefore, the use of soluble trophoblasts derived from PRPE cells to promote PR rescue, survival, and repair holds great promise. Several researchers have demonstrated the ability of RPE-derived conditioning media or specific RPE trophoblasts to reduce retinal cell loss in in vitro and in vivo models. Combinations of growth factors and conditioning media have been reported to promote PR proliferation and differentiation in retinal explants. Specifically, the use of neurotrophic growth factors such as BDNF and GDNF has been used as supplemental nutrients in retinal sheet transplantation. Similarly, LaVail et al. showed that multiple growth factors, cytokines, and neutrophin slowed damaged PR in a mild Royal College of Surgery (RCS) rat model. Nevertheless, single or limited combinations of growth factors have shown limited success on their own, as they target only a single pathogenesis.

[0061] Based on the success of these studies and the paracrine effects observed beyond the CPCB-RPE1 boundary, it is hypothesized that different concentrations of multiple trophic factors secreted by this graft could serve as a potential combination therapy for the treatment of human dry progressive non-neovascular AMD (dry AMD). Characterization, in vitro and in vivo results in animal models of retinal dystrophy treated with PRPE-soluble factor (SF) derived from CPCB-RPE1-modified grafts supported the feasibility of this concept.

[0062] A method for generating cell line-specific target supernatants involves culturing target cells using a specific growth medium cocktail on, in, or encapsulated within a biocompatible substrate or container that promotes specific growth, between specific maturation and / or different differentiation stages, thereby amplifying the efficient production of specific factors. The stages can be monitored by periodic evaluation of cells seeded on the substrate by combinations of the above-mentioned visual forms (e.g., cuboid, cobblestone, pigmentation, etc.), specific testing of specific factors (e.g., BDNF, BMP-7, PEDF, TGF, VEGF, etc.) and their concentrations, and whole-cell current-fixed recording to test the cell resting membrane potential (e.g., mV).

[0063] In embodiments of RPE cells, the literature provides various indicators for distinguishing between immature and mature RPE cells. For example, mature RPE cells typically have a resting membrane potential of -40 to -50 mV. However, immature RPE cells have a slightly depolarized resting membrane potential (typically -25 to -35 mV). Furthermore, whole-cell voltage-fixed recordings of mature RPE cells show voltage-activated inward current and voltage-opening sodium channel activity, which are not observed in immature RPE cells (Nymark et al. 2013).

[0064] In one embodiment, RPE cells were seeded on a parylene membrane coated with vitronectin and cultured in XVIVO10 (XVIVO™10, Lonza) at 37°C in a 5% CO2 incubator. The target supernatant (summarized in Figure X) was collected every four days starting on day 28 and continuing until day 40. The period from day 28 to day 40 was particularly interesting because it encompassed specific stages of maturation as desired: nearly dense (over 90%, ideally over 98% membrane coverage), cobblestone morphology, a uniform monolayer distribution across the entire membrane, and minimal to moderate pigmentation (0% to 70% pigmentation). The collection method may vary, but in one embodiment, the culture medium was extracted from the culture wells by pipetting, taking care not to extract cells. In another embodiment, a cell filter may be used to ensure that only the supernatant is extracted.

[0065] Further cell viability and, therefore, the quality of secreted factors can be confirmed by USP sterility and USP mycoplasma tests. Cells can also be tested for gene expression by reverse transcriptase polymerase chain reaction (RT-qPCR) to identify specific genes of interest (e.g., RPE65, REX1, EIF2B2, SERF2, UBE2R2, etc. for RPE cells). The recovered target supernatant can be further tested for the quantity and concentration of specific factors outlined in Figure X. While secreted factors vary depending on the cell line and culture method, multiple samples have shown minimal batch-to-batch variability in supernatant composition when using the same cell line and culture method, thereby demonstrating reproducibility in supernatant generation.

[0066] In one embodiment, the recovered target supernatant may be pooled from multiple days and / or multiple membranes. The target supernatant is then filtered using a 0.2 μm syringe filter system. In various embodiments, the filtered target supernatant can be used in an unconcentrated 1x form, concentrated (e.g., 3x) using an Amicon centrifugal filter device (Milipore Sigma) with a kD cutoff (e.g., 3 kD cutoff), or diluted with a diluent or drug medium. In some embodiments, the target supernatant is first concentrated and then diluted with a specific drug medium (e.g., aqueous, lipophilic solution, suspension) selected for desired medium-dependent factor absorption and improved shelf life of specific factors. Ideally, the target supernatant should be stored at -80°C until further use to extend its shelf life. The target supernatant is then packed into a syringe and optionally supplemented with growth medium or other therapeutic agents. In another embodiment, the target supernatant pellet may be further extracted for further use as a high-concentration or long-term diffusion graft.

[0067] In yet another embodiment, cells can be cultured on a selectively permeable substrate to polarize them and produce specific secretions. For example, polarized RPE cells are known to produce apical secretions, basal secretions, and nonpolar specific secretions. The apical secretion of RPE cells contains αB crystallin, hyaluronan, MMP-9, PEDF, TGF-β, TIMP-I, and MGF-E8. The basal secretion of RPE cells contains cystatin C, endothelin I, FGF-5, and VEGF. The nonpolar specific secretions of RPE cells contain BDNF, CFH, CNTF, fibrin 3 / 5, FGF-2, HB-EGF, HGF, IGF-I, LIF, MMP-9, NGF, and tropoelastin. Other identified trophoblasts include IGFBP-2, IGFBP-3, IGFBP-6, PEDF-AA, and BMP-7.

[0068] The packed supernatant can be periodically injected therapeutically into the target site after transplantation of cell therapy media (e.g., cell solution, cell seeding gel, cell seeded substrate, etc.), whether or not it is an additional supplemental nutrient to support growth, maintain functionality, and enhance the integration of host and / or transplanted cells with the target site tissue. The packed supernatant can also be coated onto cell therapy media before transplantation, added to the media, or coated onto the substrate before cell seeding.

[0069] In other embodiments, after adding the supernatant, the cells are further cultured until they exhibit mature characteristics. In RPE cells, these mature characteristics are selected from (i) pigmented RPE cells, (ii) polarized RPE cells, (iii) RPE cells having a mature cobblestone morphology without neural or fibroblast regions, (iv) RPE cells having the gene expression levels of mature cells, and (v) RPE cells with a dense monolayer structure.

[0070] In embodiments where the cell-seeded support structure is not transplanted, various other cell proliferation surfaces can be used, including substrates, lattice structures, agar, hydrogels, or other cell proliferation surfaces known in the art.

[0071] The supernatant demonstrated therapeutic activity in animal models (i.e., the Immunodeficient Royal College of Surgical Sciences (iRCS) rat model, an approved FDA animal model for the development of drug and cell therapies for geographic atrophy and age-related macular degeneration) that included an anti-inflammatory visual function response (i.e., increased b-wave amplitude as assessed by electroretinography) and preserved retinal structures, including increased photoreceptor survival and function. Furthermore, the supernatant showed greater photoreceptor survival compared to a single-factor PEDF control, which is a known neuroprotective and anti-angiogenic agent, indicating that two or more factors contribute to the target paracrine effect. In addition, despite numerous injections, no evidence of tumorigenicity or endophthalmitis was observed.

[0072] Optimized cryopreservation protocol and considerations Cryo-hibernation protocol In one embodiment, an alternative cryogenic resting protocol to a standard cryogenic preservation protocol has shown increased viability of thawed cells seeded on a substrate. The cryogenic preservation protocol is as follows: Following an initial controlled temperature ramp-down phase, once a first temperature is reached that is lower than the latent heat release of the seeded cells (0°C to 20°C), the cells are kept at the first temperature for a first period. The first temperature is any temperature lower than the latent heat release temperature and may be -20, -30, -40, -50, -60, -70, -80, -90, or -100°C. The first period is 12 hours, 1 day, 7 days, or 28 days and helps to acclimate the cells to the cryogenic preservation state without abrupt temperature changes. This acclimatization further prevents the formation of micro-tears in the substrate that may be caused by temperature drops (-1°C / min or more). After the first period, the cryogenically preserved cells are transferred and maintained at the preservation temperature for a second period. In most cases, the storage temperature is, for convenience, -196°C (e.g., the temperature of liquid nitrogen), but it may be the same temperature as the resting temperature. In some embodiments, the cell-seeded substrate is also moved to a second temperature by a controlled temperature ramp-down phase with a rate of decrease of approximately -1°C to approximately -30°C per minute, or any rate within this range, until the second temperature is obtained. This particular temperature ramp-down helps to ensure a uniform temperature decrease of the various elements (i.e., the substrate, at least one cell type, the cryoprotection solution, and the cryogenic storage container).

[0073] In certain embodiments, the cells are stored in a set-temperature freezer and carrying case at a first temperature until thawed. This requires a portable set-temperature freezer, but the long resting temperature increases the viability of the cells after thawing because the temperature difference during the thawing process is smaller than the temperature difference from -196°C (e.g., the temperature of liquid nitrogen), thereby significantly reducing the temperature change zones that cause thawing of the substrate on which the batch cells are seeded, as well as small, variable ice crystal formation patterns and different cryoprotection material removal rates within each individual substrate.

[0074] Optimized for low-temperature storage: RPE cell lines In specific embodiments of hESC-RPE cells, various combinations of cryopreservation protocols were tested, and specific cell lines were optimized. Although specific cell lines were tested, similar results are expected for other RPE cell lines, given that the key factors of the cell lines and substrate properties described above were considered.

[0075] RPE cells from CPCB-RPE1 grafts exhibited optimal cryopreservation viability characteristics, including (i) unpigmented RPE cells (including depigmented RPE cells), (ii) unpolarized or partially polarized RPE cells, (iii) RPE cells with a predominantly mature, cobblestone morphology, (iv) RPE cells with gene expression levels lower than those of mature cells or lacking specific gene expression, (v) RPE cells with a semi-dense monolayer structure, or combinations thereof. This optimal combination of cryopreservation viability characteristics was obtained 3 to 12 days after seeding on a transplantable substrate (7 days being the median between batches).

[0076] The CPCB-RPE1 cell line demonstrated optimal low-temperature viability characteristics of the substrate, including the thermal expansion coefficient of the substrate, the elastic parameters of the substrate, the thickness of the substrate, and the substrate implantation size, all of which were taken into consideration in the substrate design.

[0077] Various cell lines were tested with different combinations of cryoprotectant and freezing rate. The most viable combination was the use of CS-10 (manufacturer: BioLife Solutions, Bothell, Washington, USA) with a DMSO concentration of 10% and freezing rates ranging from -5°C / min to 30°C / min. In comparison, DMSO concentrations of 2% and 5% and freezing rates of -1°C to -3°C resulted in lower cell viability.

[0078] Next, the combinations were evaluated using various methods, including phase-contrast imaging of cells one day and one week after thawing, cell gene expression, cell viability staining, and Alamal Blue metabolism.

[0079] [Table 1]

[0080] The terms “substantially” or “about” mean ±10% (e.g., by mass or volume), and in some embodiments, ±5%. Throughout this specification, “one embodiment,” “an example,” “one embodiment,” or “an embodiment” means that a particular feature, structure or property described in relation to that embodiment is included in at least one embodiment of the present art. Thus, the appearance of the expressions “in one embodiment,” “in an example,” “in one embodiment,” or “in an embodiment” in various places throughout this specification does not necessarily mean that all of them refer to the same embodiment. Furthermore, a particular feature, structure, routine, step, or property can be combined in any suitable way in one or more embodiments of the art. The headings described herein are for convenience only and are not intended to limit or imply any limitation or interpretation of the scope or meaning of the claimed art.

Claims

1. A method for preparing a therapeutic composition, A step of culturing RPE cells on a biocompatible polymer substrate that allows nutrients to pass through to the cells, wherein the RPE cells form a subconfluent monolayer on the substrate; A process of freezing a subconfluent monolayer of cells on a substrate; A process of thawing a subconfluent monolayer of cells on a substrate; A process of inducing cells thawed in a liquid culture medium to secrete apical pigment epithelial-derived factor (PEDF); A step of recovering the supernatant containing PEDF from the liquid culture medium; The process of purifying cytoplasm containing PEDF from the supernatant to prepare a therapeutic composition. Methods that include...

2. The method according to claim 1, further comprising the step of culturing RPE cells until, before purifying the cell secretions, the cells exhibit maturation characteristics selected from (i) pigmented RPE cells, (ii) polarized RPE cells, (iii) RPE cells having a mature cobblestone morphology, (iv) RPE cells having gene expression levels similar to those of mature cells, and (v) RPE cells with a dense monolayer structure.

3. The method according to claim 1, wherein purification is achieved by centrifugal filtration, fractionation, size exclusion chromatography, affinity chromatography, size exclusion filtration, or precipitation.

4. The method according to claim 1, further comprising the step of filling a syringe with the therapeutic composition.

5. The method according to claim 4, wherein the therapeutic composition is supplemented with a drug medium or diluent.

6. The method according to claim 1, further comprising the step of culturing photoreceptor cells or photoreceptor progenitor cells.

7. The method according to claim 1, wherein the biocompatible polymer substrate comprises parylene.

8. The method according to claim 1, wherein the cells are enclosed in a biocompatible container.

9. The method according to claim 1, wherein RPE cells are cryopreserved when they reach 90% to 99% confluence on the substrate.

10. The method according to claim 1, further comprising the step of removing the cryoprotectant from the cells after thawing.

11. The method according to claim 1, further comprising the step of adding the cell secretion as a cell-specific medium to a second culture medium for RPE cells.

12. The method according to claim 11, wherein the cell secretion comprises a combination of proteins, hormones, enzymes, by-products and waste products.

13. The method according to claim 11, wherein the cell secretion comprises apical secretion from RPE cells containing at least one of αB crystallin, hyaluronan, matrix metalloproteinase (MMP)-9, pigment epithelial-derived factor (PEDF), transforming growth factor (TGF)-β, and metalloproteinase (TIMP)-I tissue inhibitors.

14. The method according to claim 11, wherein the cell secretion comprises basal secretion from RPE cells containing at least one of cystatin C, endothelin I, fibroblast growth factor (FGF) 5, and vascular endothelial growth factor (VEGF).

15. The method according to claim 11, wherein the cell secretion comprises a nonpolarity-specific secretion from RPE cells containing at least one of BDNF, CFH, CNTF, fibrin 3 / 5, FGF 2, HB-EGF, HGF, IGF-I, LIF, MMP-9, NGF, and tropoelastin.

16. The method according to claim 11, wherein the substrate induces cell polarity so that apical, basal, and nonpolar specific secretions can be secreted.

17. The method according to claim 11, wherein the cell-specific medium is for the controlled growth of immature RPE cells.

18. The method according to claim 11, wherein the cell-specific medium is for controlled maturation of immature RPE cells to mature RPE cells.

19. The method according to claim 11, wherein the cell-specific culture medium is formulated for intravitreous (IVT) injection.

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