Laminin cell culture matrices and regimens

Abstract

The present invention provides an ear nerve progenitor cell (ONP) culture medium comprising a protein matrix consisting of laminin (211), laminin (521) and laminin (111) wherein the amounts of laminin (211), laminin (521) and laminin (111) in the ear nerve progenitor cell (ONP) culture medium are defined by the ratio X: Y: Z of (laminin 211): (laminin 521): (laminin 111), wherein X is from about 1 to about 10, Y is from about 1 to about 5, and Z is from about 1 to about 5, and methods of culturing cells using the same.

Description

Technical Field

[0001] The present invention relates to methods of expanding a population of otic neural progenitor cells (ONPs), and compositions useful in the methods. Background Art

[0002] Deafness is a disease with a high incidence worldwide, mainly caused by the loss of sensory hair cells and their associated spiral ganglion neurons (SGNs). Among all forms of deafness, auditory neuropathy is particularly worrying. This disease, which is mainly characterized by damage to SGNs while hair cells are relatively well preserved, is the cause of hearing loss in a considerable number of patients. Although the loss of hair cells can be partially avoided by cochlear implants, there is no conventional treatment for the loss of sensory neurons because poor innervation limits the potential performance of the implant. Using stem cells to restore damaged sensory circuits is a potential therapeutic strategy.

[0003] Protocols have been developed to induce differentiation of human embryonic stem cells (hESCs) using signals such as FGF3 and FGF10, which are involved in the initial formation of the ear glial plate (Chen et al. (2012) Nature, 490 (7419): 278-82. doi: 10.1038 / nature11415). Ear progenitor cells have also been generated from induced pluripotent stem cells (iPSCs) (Boddy et al. (2020) (2020) Stem Cells Int. Article ID 3692937, p1-10). The induced ear progenitor cells are able to differentiate into hair cell-like cells and auditory neurons that exhibit the expected electrophysiological properties in vitro. In addition, when transplanted into an auditory neuropathy model, ear neural progenitor cells engraft, differentiate, and significantly improve auditory evoked response thresholds. However, the FGF3 / 10 induction method is inefficient and can only produce about 20% of the desired cell types.

[0004] Laminin is a heterotrimeric extracellular matrix molecule that forms a superstructured meshwork in the basement membrane and elsewhere. Laminin interacts with integrin receptors and plays a key role in regulating cell differentiation programs and maintaining tissue homeostasis in animals. Early studies (e.g., Cosgrove et al. (1997) Hearing Research 105: 159-170) demonstrated that there is a wide range of laminin meshwork in the developing cochlea and the adult cochlea, which is mainly related to the basement membrane, but the composition of the laminin chains of these meshworks is not explained. Rodgers et al. (Rodgers et al. (2001) Hearing Research 158:39-50) studied the laminin isoform composition in the cochlear basilar membrane and showed that the laminin composition varies depending on its location in the cochlear basilar membrane and the developmental stage, and showed that laminin 211, laminin 221, laminin 311 and laminin 321 (laminin-2, -4, -6 and -7) can be present in the spiral ganglion from postnatal day 7 (P7) to adulthood in mice, and laminin 211 is observed from the earliest time point P2.

[0005] Laminin (also referred to herein as "LN") was first isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and is a commonly used matrix for culturing cells. TM Specific laminins such as mouse laminin 111 have been used as a matrix for cell culture. Paccola Mesquita et al. (Paccola Mesquita et al. (2019) Stem Cells International Article ID 9704945) demonstrated the use of laminin 521 as a matrix for the expansion of iPSCs in a closed cell culture system.

[0006] The number of high-quality cells required to make an adult-sized bioartificial organ exceeds one billion. Implanting ONPs to treat sensory neuron loss may require fewer cells, perhaps 10 cells per dose. 5 However, the production of suitable batches suitable for treating multiple patients requires the generation of tens of millions of relevant cells. Methods and matrices that allow ONPs to expand while maintaining their neural potential are needed. For otic progenitor cells used in clinical settings, methods and compositions that can expand ONPs in a cell culture system with a clear chemical composition and no animal-derived components are needed. Summary of the invention

[0007] According to a first aspect of the present invention, there is provided an ear neural progenitor cell (ONP) culture substrate (culture substrate) (also referred to herein as a matrix), which comprises a protein matrix composed of laminin 211, laminin 111 and laminin 521, wherein the amount of laminin 211, laminin 111 and laminin 521 in the ear neural progenitor cell (ONP) culture substrate is defined by a ratio X:Y:Z of (laminin 211): (laminin 111): (laminin 521). X can be from about 1 to about 10. Y can be from about 1 to about 5. Z can be from about 1 to about 5.

[0008] The present invention also provides a method for expanding an otic neural progenitor cell (ONP) population, the method comprising (a) culturing cells on a first cell culture substrate consisting of laminin 211, laminin 111, and laminin 521 for one passage; then (b) culturing cells on a second cell culture substrate consisting of laminin 211, laminin 111, and laminin 521 for further passage, wherein the amount of laminin 211 in the second cell culture substrate is increased relative to the amount of laminin 111 and laminin 521 in the first cell culture substrate. In an optional step (c), the cells are cultured on a third cell culture substrate consisting of laminin 211, laminin 111, and laminin 521, wherein the amount of laminin 211 in the third cell culture substrate is increased relative to the amount of laminin 111 and laminin 521 in the second cell culture substrate.

[0009] Unless otherwise stated, laminin ratios refer to the ratio of LN211:LN521:LN111. Unless otherwise stated, when ratios are expressed as X, Y, and Z, X is LN211, Y is LN521, and Z is LN111. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Characteristic morphology of ONP (A) and OEP (B) colonies is shown. Figure 1 (A) shows ONP. Figure 1 (B) shows OEP. Scale bar represents 200 μm.

[0011] Figure 2The cumulative cell doublings of three different batches of ONP in 35 days are shown. ONP keeps proliferating during continuous passage, with a cumulative cell doubling of about 15 times in 30 days. The cumulative population cell doublings of three different batches of ONP are amplified according to the matrix shown in Table 2. Each point in the figure represents a passage. ONP batch 1 is represented by a circle. ONP batch 2 is represented by a square. ONP batch 3 is represented by a triangle.

[0012] Figure 3 It was shown that plating the same ONP population directly onto a laminin mixture containing a higher proportion of LN211 after sorting affects attachment and morphology. Figure 3 a to Figure 3 d, The left (LHS) image shows ONP cells plated directly onto a matrix with a laminin ratio of 1:1:1 after sorting, and the right (RHS) image shows ONP cells plated directly onto a matrix with a laminin ratio of 6:1:1 after sorting (i.e., the ratio of LN211 increased from one-third (1 / 3) to three-quarters (3 / 4)). Figure 3 a shows ONP cells 2 days after plating. Figure 3 b shows ONP cells 4 days after plating. Figure 3 c shows ONP cells 6 days after plating. Figure 3 d shows ONP cells 7 days after plating. Scale bar represents 400 μm.

[0013] Figure 4 It was shown that plating ONPs directly onto laminin mixtures containing a higher proportion of LN211 after sorting affects attachment and morphology. Figure 4 A and Figure 4 B shows ONP cells plated directly after sorting onto a matrix containing a mixture of LN211:LN521:LN111 at a laminin ratio of 6:1:1. Figure 4 A shows cells 5 days after seeding. Figure 4 B shows cells 8 days after seeding. Scale bar represents 400 μm.

[0014] Figure 5 It was shown that plating expanded ONPs on 6:1:1 matrix onto 100% LN211 matrix affects cell morphology and attachment. Figure 5 Shown are ONPs amplified on a matrix with a 6:1:1 laminin ratio, followed by transfer to the same matrix (left (LHS) panel) or 100% LN211 matrix (right (RHS) panel). Figure 5 a to 5d show the difference in ONP with increasing time period. Figure 5 a) shows cells 6 hours after plating; Figure 5 b) shows cells 1 day after plating; Figure 5 c) shows cells 3 days after plating; Figure 5 d) Shows cells 4 days after plating. Scale bar represents 400 μm.

[0015] Figure 6 It was shown that defects in cell morphology and attachment caused by plating expanded ONPs on 100% LN211 can be reversed by plating cells on a matrix with a laminin ratio of 6:1:1. Figure 6 A shows ONPs 1 day after plating on 100% LN211. Figure 6 B shows these cells after one week of passage on 100% LN211 (B). Figure 6 C shows these cells one week after passage on a matrix with a laminin ratio of 6:1:1 (C). Returning the cells to the mixture with a ratio of 6:1:1 restored attachment and cell morphology. Scale bar represents 1000 μm. DETAILED DESCRIPTION

[0016] The present invention provides a matrix and method that can be used in a process that complies with good manufacturing practice (GMP), which is the minimum standard that drug manufacturers must meet in their production process to obtain certification from the European Medicines Agency and equivalent regulatory agencies. The inventors unexpectedly found that the expansion of ONPs can be improved by changing the combination of laminin isomers used as a matrix during the expansion process. LN211 is a laminin present during cochlear development. Amplification can be achieved by gradually increasing the proportion of LN211 present in the matrix. When the laminin mixture is completely replaced by 100% LN211, amplification is also affected. When a combination of LN211, LN511 and LN111 is used in the matrix, amplification is more efficient. When the proportion of LN211 is gradually increased in a few passages (e.g., 2 to 4 passages, such as 3 passages), amplification is most efficient. ONPs cultured on a matrix comprising LN211, LN511 and LN111 described herein and cultured using the amplification method described herein maintain the ability to proliferate. ONPs cultured on the matrix comprising LN211, LN511 and LN111 described herein and cultured using the expansion method described herein maintained good morphology and neural potential.

[0017] Laminin contains three disulfide-linked polypeptides, called α, β, and γ. Each polypeptide exists in many isomers, represented by numbers after the corresponding Greek letters, such as α2, β1, and γ1. Standard laminin nomenclature now refers to laminins according to their isomeric composition, referring only to the numbers. Therefore, the laminin containing α2, β1, and γ1 is laminin 211 (Aumailley (2013) Cell Adhesion & Migration 7, 48-55), also referred to as LN211 in this article. Table 1 provides a list of laminins and their compositions and corresponding alternative names used in the literature.

[0018] Table 1. Laminin types

[0019]

[0020] The laminin used in the matrices and methods provided herein can be recombinant. That is, the laminin can be a recombinant protein, for example, a protein that has been overexpressed and purified using techniques known in the art. The laminin is preferably human. The laminin can be purchased, for example, from BioLamina (human recombinant laminin), or prepared using standard methods in the art. The amount of laminin in the cell culture matrix is ​​typically expressed per unit area (e.g., cm 2 The total concentration of laminin should be 0.5 μg / cm 2 Up to 2.0 μg / cm 2 , preferably about 1 μg / cm 2 The total laminin concentration is the sum of the concentrations of all laminin isoforms combined.

[0021] Otic progenitor cells (also referred to herein as otic progenitors) are cells that can further differentiate into hair cells (otic epithelial progenitors, OEPs) or auditory neuron cells (otic neuron progenitors, ONPs). OEPs and ONPs each have a characteristic morphology that can be used to distinguish one from the other. OEPs are larger than ONPs, typically with a size greater than about 12 μm. OEPs display well-defined intercellular connections. OEPs form epithelial islands. ONP cells are typically smaller than OEP cells, typically ranging in size from about 8 μm to about 15 μm. ONPs exhibit cytoplasmic protrusions. ONPs form loosely associated colonies. ONP cells have denser chromatin than OEP cells. ONPs can be identified by morphology ( Figure 1). The otic glial plate is the primordium of the auditory organ. The state of the ear progenitor cells can be defined by the expression of markers associated with the ear glial plate in the early stages of development. Suitable markers include SOX2, FOXG1, PAX2, PAX8, SSEA1 (also known as CD15 or Lewis x), GD3, TRA-2-49 (alkaline phosphatase), SSEA4, GD2 and CD141. The ear progenitor cells can express two or more of SOX2, FOXG1, PAX2 and PAX8, such as SOX2 and PAX8, FOXG1 and PAX8, or PAX2 and PAX8. Human ear progenitor cells can be identified as cells having at least two cell surface markers selected from SSEA1 (also known as CD15 or Lewis x), GD3, TRA-2-49 (alkaline phosphatase), SSEA4, GD2 and CD141. Markers can be quantified using methods such as fluorescence or qPCR. Suitable methods for preparing such cells are described in WO 2016 / 156831 and WO 2018 / 051092. ONP cells may be human ONP cells. ONP batches typically express one or more / two or more markers selected from SSEA1, ITGA4. ONP cells with neural potential may express one or more / two or more markers selected from SSEA1 and GD2. During ONP amplification, surface markers may be downregulated (e.g., SSEA1). Other surface markers may be upregulated (e.g., ITGA4). ONP generation refers to the production of ONP cells by differentiation of pluripotent cells. ONP amplification refers to increasing the number of ONP cells having the ability to develop into auditory neuron cells, but not differentiating ONP cells into auditory neuron cells.

[0022] Prior to expansion, e.g., during ONP generation, cells (ONP cells and / or differentiated cells) are typically cultured on a matrix comprising laminin 521 (LN521) and laminin 111 (LN111) in a ratio of Y:Z LN521:LN111, e.g., wherein Y=about 1 and Z=about 1. During expansion, the laminin composition of the matrix is ​​gradually changed from that used during ONP generation to a mixture comprising LN211, such that laminins LN211, LN521, and LN111 are present in a ratio of X:Y:Z (LN211:LN521:LN111). X may be from about 1 to about 10. X may be from 2 to 6, from 3 to 6, from 4 to 6, from 5 to 6. X may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Y may be from about 1 to about 5. Y may be from 1 to 3 or from 1 to 2. Y can be 1, 2, 3, 4 or 5, for example 1. Z can be from about 1 to about 5. Z can be 1, 2, 3, 4 or 5, for example 1.

[0023] In the first passage of expansion, LN211 is usually included in the matrix. The laminin ratio is usually changed from Y:Z (wherein Y=about 1 and Z=about 1) to X:Y:Z (wherein X=about 1, Y=about 1 and Z=about 1), such as 1:1:1 LN211:LN521:LN111. Therefore, the ear neural progenitor cell (ONP) culture matrix for inoculating cells in the first culture step (0 passage (p0)) of expansion usually includes a protein matrix containing human laminin 211, human laminin 521 and human laminin 111, wherein the amount of laminin 211, laminin 521 and laminin 111 in the ear neural progenitor cell (ONP) culture matrix is ​​defined by the ratio X:Y:Z of (laminin 211): (laminin 521): (laminin 111), wherein X=about 1, Y=about 1 and Z=about 1. In subsequent culture steps (e.g., the second step, the first passage (p1)), the ratio X:Y:Z of the otic neural progenitor cell (ONP) culture substrate is generally X=about 2, Y=about 1, and Z=about 1. In subsequent culture steps (e.g., the third step (p2), the fourth step (p3), etc.), the ratio X:Y:Z of the otic neural progenitor cell (ONP) culture substrate is generally X=about 6, Y=about 1, and Z=about 1 (e.g., 6:1:1). The otic neural progenitor cell (ONP) culture substrate may have a ratio X:Y:Z, wherein X is 1, 2, 3, 4, 5, or 6; Y is about 1; and Z is about 1. The otic neural progenitor cell (ONP) culture substrate may have a ratio X:Y:Z, wherein X is about 2 to about 6; Y is about 1; and Z is about 1.

[0024] In a method of expanding a population of otic neural progenitor cells (ONPs), the method may include the step of culturing cells on a first cell culture substrate, the first cell culture substrate consisting of laminin 211, laminin 111, and laminin 521 in a ratio of (laminin 211): (laminin 521): (laminin 111) X1: Y: Z. The method may also include the step of culturing cells on a second cell culture substrate for further passage, the second cell culture substrate consisting of laminin 211, laminin 111, and laminin 521 in a ratio of (laminin 211): (laminin 521): (laminin 111) X2: Y: Z, wherein X2 is greater than X1. Thus, the amount of laminin 211 in the second cell culture substrate is increased relative to the amount of laminin 111 and laminin 521 in the first cell culture substrate. The method may further include the step of culturing the cells on a third cell culture substrate for further passage, the third cell culture substrate being composed of laminin 211, laminin 111 and laminin 521 in a ratio X3:Y:Z of (laminin 211):(laminin 521):(laminin 111), wherein X3 is greater than X2. Thus, the amount of laminin 211 in the third cell culture substrate is increased relative to the amount of laminin 111 and laminin 521 in the second cell culture substrate.

[0025] Any of the cell culture substrates described herein may be used in methods of expanding a population of otic neural progenitor cells.

[0026] The expansion method may include culturing ONP cells on a cell culture substrate composed of laminin 111 and laminin 521 for one passage, and then culturing in a matrix containing LN211, optionally wherein laminin 111 and laminin 521 are human laminin 111 and human laminin 521. Therefore, the method may also include an initial step before step (a): culturing ONP cells on a cell culture substrate composed of laminin 111 and laminin 521 for one passage, and then transferring the cells to the cell culture substrate of step (a). In this step, the cells typically reach a confluence of 80% to 90% (e.g., 90% confluence), for example, after 3 to 10 days of culture.

[0027] In the culture methods described herein, ONP cells are typically seeded initially at about 10% confluence, e.g., about 20,000 cells / cm 2 Up to 40,000 cells / cm 2 .

[0028] The terms "passaging" and "culturing step" are used interchangeably herein. The time between each passaging / culturing step is typically about 1 week, such as 3 to 10 days, for example 5 to 9 days. The cells can be cultured for about 1 week or until confluence is reached before the next culturing step. The cells are typically cultured at 37°C. The cells are kept at 5% CO 2 The cells are cultured in an environment. The cells are usually passaged when the cell confluence is 80% to 100%, preferably when the cell confluence is 80% to 90%, for example when the cell confluence is about 90%. Passaging and culturing are terms known in the art. When the cells are passaged, they are usually harvested from the culture container. After removing the liquid culture medium, the cells are separated from each other and from the container using a cell dissociation buffer (e.g., containing trypsin). The cells are washed and centrifuged (e.g., 100g to 200g for 5 to 10 minutes), then suspended in fresh culture medium for counting, and inoculated into a container with a laminin matrix as described herein for culture (re-culture).

[0029] The product of the method for amplifying an otic neural progenitor cell (ONP) colony is an amplified ONP cell colony, wherein the ONP cell has good morphology and / or neural potential. The colony can be located in the same container, or can be separated in two or more containers. The population doubling time and cumulative population doubling are calculated using the initial number of cells seeded, the number of cells grown, and the length of time between passage / cultivation steps. For example, the population size can undergo 5 cumulative population doublings (in 10 days (a week)). Figure 2 ). The cumulative population doubling can be 2 to 4 times, or 2 to 3 times, such as about 3 times. Preferably, the cumulative population doubling is greater than 2 times, such as 2 to 4 times. The population doubling time can be less than 50 hours, such as 35 to 45 hours. The population doubling time can be about 38 hours.

[0030] The cell culture medium added to the cell on the cell culture matrix can include at least one growth factor. The cell culture medium can include at least one growth factor selected from FGF, EGF and IGF (e.g., IGF-I and IGF-2, e.g., IGF-1). The cell culture medium can include FGF (e.g., bFGF), EGF and IGF-1. Growth factors are preferably recombinant growth factors. If present, the concentration of each growth factor in the culture medium is generally 10ng / ml to 30ng / ml, e.g., 20ng / ml. Growth factors can be human growth factors. The cell culture medium can include Dulbecco's modified Eagle's medium (DMEM). The cell culture medium can include L-glutamine, glucose, sodium pyruvate and sodium bicarbonate (e.g., DMEM including 4mM L-glutamine, 4500mg / L glucose, 1mM sodium pyruvate and 1500mg / L sodium bicarbonate). The cell culture medium can include B27 neuron culture supplement (e.g., B27 supplement, Thermofisher, catalog number 17504044).

[0031] Preferred features of the second and subsequent aspects of the invention are the same as for the first aspect mutatis mutandis.

[0032] Implementation

[0033] The following numbered clauses describe embodiments of the cell matrix compositions and methods described herein.

[0034] Item 1. An otic neural progenitor cell (ONP) culture matrix comprising a protein matrix composed of laminin 211, laminin 521 and laminin 111, wherein the amount of laminin 211, laminin 521 and laminin 111 in the otic neural progenitor cell (ONP) culture matrix is ​​defined by a ratio X:Y:Z of (laminin 211):(laminin 521):(laminin 111), wherein X is about 1 to about 10; Y is about 1 to about 5; and Z is about 1 to about 5.

[0035] Item 2. The otic neural progenitor cell (ONP) culture matrix according to Item 1, wherein X is about 2 to about 6; Y is about 1; and Z is about 1.

[0036] Item 3. The otic neural progenitor cell (ONP) culture matrix according to Item 1 or Item 2, wherein laminin 211, laminin 111 and laminin 521 are human laminin 221, human laminin 111 and human laminin 521.

[0037] Item 4. The otic neural progenitor cell (ONP) culture matrix according to any one of items 1 to 3, wherein laminin 211, laminin 111 and laminin 521 are recombinant.

[0038] Item 5. A method for expanding a population of otic neural progenitor cells (ONPs), the method comprising: (a) culturing cells on a first cell culture substrate consisting of laminin 211, laminin 111, and laminin 521 for one passage; and subsequently (b) culturing the cells again on a second cell culture substrate consisting of laminin 211, laminin 111, and laminin 521 for further passage, wherein the laminin 111 and laminin 521 in the first cell culture substrate used in step (a) are greater than the laminin 111 and laminin 521 in the second cell culture substrate used in step (b). (c) repeating step (b), wherein cells are cultured on a third cell culture substrate consisting of laminin 211, laminin 111 and laminin 521, and wherein the amount of laminin 211 in the third cell culture substrate used in step (c) is increased relative to the amount of laminin 111 and laminin 521 in the second cell culture substrate used in step (b).

[0039] Item 6. A method according to Item 5, wherein the amount of laminin 211, laminin 111 and laminin 521 in the cell culture matrix of step (a) is defined by a ratio X:Y:Z of (laminin 211):(laminin 111):(laminin 521), wherein X is about 1 to about 10; Y is about 1 to about 5; and Z is about 1 to about 5.

[0040] Item 7. The method according to Item 5 or Item 6, wherein laminin 211, laminin 111 and laminin 521 are (i) human laminin 221, human laminin 111 and human laminin 521 and / or (ii) recombinant.

[0041] Clause 8. The method according to any one of Clauses 5 to 7, wherein the ONP cells are human cells.

[0042] Clause 9. A method according to any one of clauses 5 to 8, wherein the method further comprises an initial step before step (a): culturing ONP cells on a cell culture matrix consisting of laminin 111 and laminin 521 for one passage, and then transferring the cells to the cell culture matrix of step (a), optionally wherein laminin 111 and laminin 521 are human laminin 111 and human laminin 521.

[0043] Clause 10. The method according to any one of clauses 5 to 8, wherein a cell culture medium comprising at least one growth factor selected from the group consisting of FGF, EGF and IGF is added to the cells on the cell culture substrate.

[0044] Example

[0045] Example 1: Preparation of ONP

[0046] ONPs can be differentiated from hESCs and iPSCs that generate ear progenitor cells by plating iPSCs in Dulbecco's modified Eagle's medium: Ham's F12 (DMEM / F12) (abbreviated as DFNB) (both from Life Technologies, UK), FGF3 and FGF10 (both 50ng / ml) (both from R&D Systems, UK) supplemented with 1x N2 and B27 on laminin-coated tissue culture plastic containers (LN521:LN111 with a laminin ratio of 1:1). Wnt inhibitor IWR-1 (10μM) is also present. Cells were cultured for 8 days. On day 9, the culture medium was replaced with DFNB supplemented with FGF3 and FGF10 (both 25ng / ml) and Wnt activator BIO (2mM). Throughout the differentiation process, cells were plated on laminin-coated tissue culture plastic containers (LN521:LN111 with a laminin ratio of 1:1). Human recombinant laminin (LN521-02; LLN111-02; LN211-02) was purchased from BioLaminin. Cells can be further differentiated along the hair cell or auditory neuron lineages, and cultures can be manually purified by removing cells lacking the relevant characteristic progenitor morphology to specifically enrich for the otic epithelial progenitor (OEP) phenotype or the otic neuronal progenitor (ONP) phenotype.

[0047] Example 2: Identification of ONP and OEP

[0048] ONPs and OEPs can be differentiated from hESCs and iPSCs from undifferentiated colonies plated as a monolayer on laminin-coated flasks, for example, according to Example 1 and as described by Chen (2012) and Boddy (2020). The differentiation process induces two morphologically distinct ear populations ( Figure 1 ). Otic epithelial progenitor cells (OEP) present a flat phenotype with large cytoplasm and formed epithelial-like islands. Otic epithelial progenitor cells (OEP) show well-defined intercellular junctions. Otic neural progenitor cells (ONP) are smaller than OEPs, have more dense chromatin, and present cytoplasmic processes.

[0049] According to the co-expression of ear markers PAX8, PAX2, FOXG1 and SOX2, ear neural progenitor cells can be identified by quantitative microscopy. The cells were first fixed with phosphate buffered saline (PBS) containing 4% paraformaldehyde at room temperature for 15 minutes, and then blocked with a PBS solution containing 0.1% Triton-X, 5% donkey serum and 1% bovine serum albumin. The following primary antibodies can be used: SOX2 (1:100, Millipore), FOXG1, PAX2 (all 1:100, Abcam, USA), PAX8 (1:100, Santa Cruz), POU4F1 (BRN3A, 1:100, Chemicon) and B-tubulin III (1:100, Sigma). Suitable secondary antibodies include anti-mouse, anti-goat or anti-rabbit Alexa Fluor 488 and 568 (Molecular Probes, Life Technologies, UK). Cell nuclei can be counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma). Cells can be imaged on an EVOS FL cell imaging system or using the IN Cell Analyzer 2000 system platform (GE Healthcare). Quantitative immunofluorescence analysis can be performed on the IN Cell Analyzer using the Developer Toolbox. Cells can be exposed to, for example, a combination of PAX8 antibodies and antibodies raised against one of the other markers (PAX2, SOX2, or FOXG1) to identify co-expression in individual cells. Highly expressing cells can be defined as cells with fluorescence intensities above the 75th percentile for each antibody. Cell populations that are highly positive (co-expressing) for both markers can be identified as otic progenitor cells.

[0050] Example 3: Purification of ONP

[0051] ONPs can be selected manually. ONPs can be purified using FACS using the markers mentioned herein and methods known to those skilled in the art, for example by fluorescent automated cell sorting using SSEA1 and CD141 antibodies, using the methods described in WO2018 / 051092, which is incorporated herein in its entirety.

[0052] After sorting, ONP cells are typically cultured on a matrix comprising laminin 521 (LN521) and laminin 111 (LN111), with the ratio of LN521:LN111 being, for example, 1: 1. Between eight and ten days after sorting, the confluence of the culture dish / flask in which ONPs were initially seeded reaches about 80% to 90%.

[0053] Example 4: Transition to a different laminin matrix

[0054] ONPs purified by cell sorting (e.g. as taught in WO2018 / 051092 using SSEA1 as a cell surface marker) were cultured from p0 (i.e. immediately after sorting) to p6, at which time the cells were frozen. Cells were passaged essentially when the confluence reached about 90%. Cells were separated using 0.5x TrypLE Select. After the first few cycles of expansion, the cells were switched to growth on a matrix containing a higher ratio of LN211. At p1, the ratio of LN211:521:111 increased from 1:1:1 to 2:1:1; at p2, the ratio increased to 6:1:1, and this ratio was used from then on (Tables 2 and Figure 2 ).

[0055] Table 2. Composition of laminin matrices used for expansion of ONPs.

[0056]

[0057]

[0058] Figure 2 Cumulative population cell doublings of three different batches of ONPs are shown, expanded according to the matrix shown in Table 2. Each point in the graph represents one passage. These growth curves demonstrate that ONPs can maintain proliferation capacity using the matrix composition described herein.

[0059] It is critical to convert ONPs to the appropriate laminin composition to support efficient expansion. LN211 needs to be added stepwise. LN211 is used in combination with other laminins. When ONPs are plated into a 6:1:1 mixture immediately after sorting, cells initially attach. However, cells do not spread, and colonies grow upward into three-dimensional spheres ( Figure 3 and Figure 4 ). The population doubling time and cumulative population doubling were calculated using the number of cells initially seeded, the number of cells increased, and the length of time between cycles. Compared to the 1:1:1 mixture, population doublings and cell yields were greatly reduced (Table 3).

[0060] Table 3. Effect of matrix composition on cell growth parameters when transferred directly into a 6:1:1 mixture after sorting

[0061]

[0062] The effect of switching the expanded ONP (p4) to 100% LN211 was explored using cells in a 6:1:1 mixture. However, cells had difficulty attaching and colonies displayed a "spiky" morphology ( Figure 5). The number of population doublings of cells on 100% LN211 substrates was reduced (Tables 4 and 5) and showed a longer population doubling time (Table 5). Expansion efficiency was higher on substrates with a laminin ratio of 6:1:1.

[0063] Table 4. Effect of matrix composition on cell growth parameters when ONPs amplified in a 6:1:1 mixture were transferred to pure matrix containing 211 only.

[0064] cell LN Ratio Population multiplication Harvested cells ONP p4 6:1:1 2.64 3,770,000 ONP p4 Contains LN211 only 1.34 1,520,000

[0065] Table 5. Experiments similar to those shown in Table 4, showing population doublings and population doubling times.

[0066]

[0067]

[0068] Subsequently, cells harvested from the 100% LN211 matrix were replated on either 100% LN211 or the 6:1:1 mixture. Cells replated on 100% LN211 retained their unusual spiky morphology, whereas cells plated on the 6:1:1 mixture regained their characteristic appearance, indicating that the alterations caused by pure LN211 are reversible ( Figure 6 ).

[0069] Example 5: Amplification of ONPs in culture

[0070] All measurements refer to a single T12.5cm 2 Flask (e.g., Corning Falcon vented flask, VWR International catalog number 353107) required volume. ONP cells are typically cultured on a matrix comprising laminin 521 (LN521) and laminin 111 (LN111), for example, at a ratio of 1:1 for LN521:LN111. During expansion, the laminin composition of the matrix used in the ONP generation process is gradually changed to a mixture containing gradually introduced LN211, for example, from a ratio of 1:1 for LN521:LN111 to a ratio of 1:1:1 for LN211:521:111. LN211 is a laminin in the development of the cochlea. This transition progressed from a 1:1:1 mixture of LN211:521:111 used to plate cells immediately after sorting (passage 0 (p0)), to a 2:1:1 mixture of LN211:521:111 used from subsequent splits (p0 to >p1), to a 6:1:1 mixture of LN211:521:111 used from the next passage onwards (p1 to >p2 and beyond).

[0071] Thaw aliquots of laminin 211, laminin 521, and laminin 511 at 4°C for at least 30 minutes. The concentration of the laminin stock solution is 100 μg / ml. Measure 1 ml of Ca 2+ and Mg 2+ Dulbecco's Phosphate Buffered Saline (DPBS) (Model D8662, stored at 4°C) was added to a cold 50 ml tube. Laminin was added to the DPBS. The buffer and laminin were quickly and thoroughly mixed using a cold P1000 pipette tip and then transferred to a T12.5 flask, making sure the flask remained horizontal at all times. Gently tilt the flask to ensure even coverage. The flask was placed horizontally at 4°C and incubated overnight.

[0072] In the first amplification step (p0->p1), 62.5 μl LN211, 31.25 μl LN521 and 31.25 μl LN111 were added in a ratio of 2:1:1. In subsequent amplification steps (p1->p2 and onwards), 93.75 μl LN211, 15.625 μl LN521 and 15.625 μl LN111 were added in a ratio of 6:1:1.

[0073] Cell seeding and passaging protocols

[0074] Warm up a stock solution of 4 ml DPBS- / -, 2 ml OSCFM, and 1 ml 0.5x TrypLE Select overnight. Dilute the 1x stock solution 1:1 with DPBS- / -. Rinse the laminin-coated flask with cold DPBS+ / +, then rinse twice with 2 ml RTDPBS- / -. Add 1 ml warmed OSCFM medium and place the flask in a 37°C incubator.

[0075] To harvest the cells, remove the old medium from the culture vessel and wash the cells twice with 2 ml DPBS (- / -). Detach the cells using cell dissociation buffer (TrypLE Select, ThermoFisher) per T12.5 (12-well plate, 0.5 ml per well; 24-well plate, 0.25 ml per well). Incubate the flask at 37°C for 3 minutes and then check for detachment under an inverted microscope. If the cells do not detach easily, the culture dish can be incubated for a total of up to 5 minutes and checked regularly for detachment.

[0076] To collect cells, each flask is diluted with a lysis solution containing OSCFM, and the suspension is pipetted up and down. The cell suspension is collected and transferred to a 50ml centrifuge tube. Any residual cells on the surface of the container are further washed repeatedly with OSCFM. Centrifuge at a speed of 1000rpm (195x g) for 5 minutes. Resuspend the precipitated cells with OSCFM. An automatic cell counter (e.g., Biorad TC20) can be used to count the number of cells. TC20 cell counter is used to calculate the cells present. The gated histogram is set to 8μm to 22μm, and the total number and live cell counts are recorded. Repeat this operation for another slide chamber and calculate the average of the two counts. If the difference is greater than 10%, repeat the above operation with another cell suspension sample and take the average of the most consistent 3 readings.

[0077] The total number of cells obtained was recorded. This value (plus the total number of cells initially seeded) was used to determine the population doubling - PD. 2 Up to 40,000 cells / cm 2 Plate at a density of 1:3 (i.e., 250,000 to 500,000 cells in a T12.5 flask) for a total of 2.5 ml OSCFM per flask, with 1 ml of OSCFM already in the pre-warmed flask. A typical split ratio is 1:3, although cells should always be counted to accurately determine the PD. Return the flask to the incubator at 37°C and 5.0% CO. 2 Incubate at 4 °C. Feed the cells every other day by completely replacing the old medium with fresh, pre-warmed OSCFM. Passage the cells once a week or when the cells reach 100% confluence, whichever comes first.

[0078] Table 6. Composition of DFNB medium

[0079] Composition of culture medium Final concentration 40ml capacity (41ml) <![CDATA[DMEM / F-12 containing GlutaMAX TM > 40ml N2 1X 400μl B27 1X 800μl

[0080] Table 7. Composition of complete auris stem cell culture medium (OCSFM)

[0081] Composition of culture medium Final concentration 20ml capacity DFNB 20ml FGF 20ng / ml 4μl (100μg / ml stock solution diluted 1:5,000) EGF 20ng / ml 1μl (400μg / ml stock solution diluted 1:20,000) IGF-I 50ng / ml 10μl (100μg / ml stock solution diluted 1:2,000)

[0082] All documents cited in this application are hereby incorporated by reference in their entirety.

Claims

1. An otic neural progenitor cell (ONP) culture matrix, comprising a protein matrix consisting of laminin 211, laminin 521 and laminin 111, wherein the amounts of the laminin 211, the laminin 521 and the laminin 111 in the otic neural progenitor cell (ONP) culture matrix are defined by a ratio X:Y:Z of (laminin 211):(laminin 521):(laminin 111), wherein: X is from about 1 to about 10; Y is from about 1 to about 5; and Z is from about 1 to about 5.

2. The otic neural progenitor cell (ONP) culture matrix according to claim 1, wherein X is from about 2 to about 6; Y is approximately 1; and Z is approximately 1. 3 . The otic neural progenitor cell (ONP) culture matrix according to claim 1 , wherein the laminin 211 , the laminin 111 , and the laminin 521 are human laminin 221 , human laminin 111 , and human laminin 521 . 4 . The otic neural progenitor cell (ONP) culture matrix according to claim 1 , wherein the laminin 211 , the laminin 111 and the laminin 521 are recombinant.

5. A method for expanding an otic neural progenitor cell (ONP) population, the method comprising: (a) culturing the cells on a first cell culture matrix consisting of laminin 211, laminin 111, and laminin 521 for one passage; and subsequently (b) culturing the cells for further passaging on a second cell culture substrate consisting of laminin 211, laminin 111 and laminin 521, wherein the amount of laminin 211 in the second cell culture substrate is increased relative to the amount of laminin 111 and laminin 521 in the first cell culture substrate; Optionally (c) culturing the cells on a third cell culture substrate consisting of laminin 211, laminin 111 and laminin 521, wherein the amount of laminin 211 in the third cell culture substrate is increased relative to the amounts of laminin 111 and laminin 521 in the second cell culture substrate.

6. The method of claim 5, wherein the amount of laminin 211, laminin 111 and laminin 521 in the cell culture matrix in step (a) is defined by the ratio X:Y:Z of (laminin 211):(laminin 111):(laminin 521), wherein X is from about 1 to about 10; Y is from about 1 to about 5; and Z is from about 1 to about 5.

7. The method according to claim 5 or 6, wherein the laminin 211, the laminin 111 and the laminin 521 are: (i) human laminin 221, human laminin 111 and human laminin 521, and / or (ii) Recombinant.

8. The method according to any one of claims 5 to 7, wherein the ONP cells are human cells.

9. The method according to any one of claims 5 to 8, wherein the method further comprises a step before step (a): culturing the ONP cells on a cell culture matrix consisting of laminin 111 and laminin 521 for one passage, optionally wherein, The laminin 111 and the laminin 521 are human laminin 111 and human laminin 521.

10. The method according to any one of claims 5 to 9, wherein a cell culture medium is added to the cells on the cell culture substrate, the cell culture medium comprising at least one growth factor selected from the group consisting of FGF, EGF and IGF.

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