Laminin Cell Culture Substrate and Procedure

Abstract

The present invention provides a culture substrate for otic progenitor (ONP) cells, 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 culture substrate for otic progenitor (ONP) cells are defined by the ratio X:Y:Z of (laminin-211):(laminin-521):(laminin-111), where X is about 1 to about 10, Y is about 1 to about 5, and Z is about 1 to about 5, and a method for culturing cells using the cell culture substrate.

Description

[Technical Field]

[0001] The present invention relates to methods for expanding a population of otic progenitor (ONP) cells, and compositions for use in such methods. [Background technology]

[0002] Hearing loss, a global disease with a high prevalence, is primarily caused by the loss of sensory hair cells and their associated spiral ganglion neurons (SGNs). Among all forms of hearing loss, auditory neuropathy is particularly noteworthy. This condition, primarily defined by the relative preservation of hair cells but the loss of SGNs, accounts for a significant proportion of hearing-impaired individuals. While hair cell loss can be partially addressed with cochlear implants, there is no standard treatment for sensory neuron loss, as insufficient nerve conduction limits the expected performance of the implant. Restoring damaged sensory circuits using stem cells is a promising therapeutic strategy.

[0003] Protocols have been developed to induce differentiation from human embryonic stem cells (hESCs) using signals such as FGF3 and FGF10, which are involved in the early specification of the otic placode (Chen et al. (2012) Nature, 490(7419):278-82. doi: 10.1038 / nature11415). Otic progenitor cells have also been produced from induced pluripotent stem cells (iPSCs) (Boddy et al. (2020) Stem Cells Int. Article ID 3692937, pp. 1-10). In vitro, these otic progenitor cells can differentiate into hair cell-like cells and auditory neurons that exhibit promising electrophysiological properties. Furthermore, when transplanted into an auditory neuropathy model, cochlear neural progenitor cells engraft, differentiate, and significantly improve auditory evoked response thresholds. However, the FGF3 / 10 induction method is inefficient, yielding only approximately 20% of the desired cell types.

[0004] Laminin is a heterotrimeric extracellular matrix molecule that forms a superstructure network in the basement membrane and other sites. Laminin interacts with integrin receptors and plays an important 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 extensive laminin networks, primarily associated with the basement membrane, in both the developing and adult cochlea, but did not address the laminin chain composition of these networks. The composition of laminin isoforms in the cochlear basilar membrane was studied by Rodgers et al. (2001) Hearing Research 158: 39-50, who showed that the composition of laminins varies depending on their location in the cochlear basilar membrane and the developmental stage. Laminin-211, laminin-221, laminin-311, and laminin-321 (laminins-2, -4, -6, and -7) were suggested to be present in the spiral ganglion of mice from postnatal day 7 (P7) through adulthood, with laminin-211 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 component of Matrigel™, a commonly used substrate for cell culture. Certain laminins, such as mouse laminin 111, have been used as substrates for cell culture. Paccola Mesquita et al. (2019) Stem Cells International Article ID 9704945) demonstrated the proliferation of iPSCs in a closed cell culture system using laminin 521 as a substrate. Summary of the Invention [Problem to be solved by the invention]

[0006] The number of high-quality cells needed to engineer an artificial organ the size of an adult human is more than one billion. ONP implants to treat sensory neuron loss may require fewer cells, perhaps as few as 10 per implant. 5 The number of cells can range from 100 to 1000. However, producing a suitable batch for treating multiple patients would require generating tens of millions of such cells. Methods and substrates that allow for the growth of ONPs while maintaining neuronal function are needed. Methods and compositions that allow for the growth of ONPs in chemically defined, animal-origin-free cell culture systems are needed for the clinical use of otic precursors. [Means for solving the problem]

[0007] According to a first aspect of the present invention, there is provided a culture substrate (also referred to herein as a substrate) for otic progenitor (ONP) cells, comprising a protein matrix consisting of laminin-211, laminin-111, and laminin-521, wherein the amounts of laminin-211, laminin-111, and laminin-521 in the culture substrate for otic progenitor (ONP) cells are defined by the 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 a population of otic neural progenitor (ONP) cells, comprising (a) culturing the cells for one passage on a first cell culture substrate consisting of laminin-211, laminin-111, and laminin-521, and then (b) culturing the cells for a further passage 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. In 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] Laminin ratio refers to the ratio of LN211:LN521:LN111 unless otherwise specified. When the ratio is expressed in terms of X, Y, and Z, X is LN211, Y is LN521, and Z is LN111 unless otherwise specified. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows colonies exhibiting the characteristic morphology of ONP (A) and OEP (B). Figure 1(A) shows ONP. Figure 1(B) shows OEP. The scale bar represents 200 μm. [Figure 2] Figure 2 shows the cumulative cell doublings of three separate batches of ONPs over 35 days. ONPs remain proliferative through multiple passagings, resulting in approximately 15 cumulative cell doublings over 30 days. Cumulative cell doublings of three different batches of ONPs grown on the matrices shown in Table 2. Each point on the plot represents a passage. ONP batch 1 is indicated by a circle. ONP batch 2 is indicated by a square. ONP batch 3 is indicated by a triangle. [Figure 3]Figure 3 shows that seeding the same ONP population onto a laminin mixture with a higher percentage of LN211 immediately after sorting affects adhesion and morphology. For Figures 3a-d, the left panel (LHS) shows ONP cells seeded onto a substrate with a 1:1:1 laminin ratio immediately after sorting, while the right panel (RHS) shows ONP cells seeded onto a substrate with a 6:1:1 laminin ratio (i.e., the percentage of LN211 is increased from one-third (1 / 3) to three-quarters (3 / 4)) immediately after sorting. Figure 3a shows ONP cells 2 days after seeding. Figure 3b shows ONP cells 4 days after seeding. Figure 3c shows ONP cells 6 days after seeding. Figure 3d shows ONP cells 7 days after seeding. Scale bars represent 400 µm. [Figure 4] Figure 4 shows that seeding ONPs onto a laminin mixture containing a high percentage of LN211 immediately after sorting affects adhesion and morphology. Figures 4A and 4B show ONP cells seeded onto a substrate containing a 6:1:1 mixture of laminins, LN211:LN521:LN111, immediately after sorting. Figure 4A shows cells 5 days after seeding. Figure 4B shows cells 8 days after seeding. Scale bars represent 400 μm. [Figure 5] Figure 5 shows that seeding ONPs growing on a 6:1:1 matrix affects cell morphology and attachment. Figure 5 shows ONPs growing on a 6:1:1 matrix after transfer to either the same matrix (left-hand side (LHS) panel) or a 100% LN211 matrix (right-hand side (RHS) panel). Figures 5a-d show the differences in ONPs over time. Figure 5a) shows cells 6 hours after seeding; Figure 5b) shows cells 1 day after seeding; Figure 5c) shows cells 3 days after seeding; and Figure 5d) shows cells 4 days after seeding. Scale bars represent 400 µm. [Figure 6]Figure 6 shows that the impaired cell morphology and adhesion caused by seeding growing ONPs on 100% LN211 can be reversed by seeding the cells on a 6:1:1 laminin substrate. Figure 6A shows ONPs one day after seeding on 100% LN211. Figure 6B shows these cells after one week of passage on 100% LN211 (B). Figure 6C shows these cells after one week of passage on a 6:1:1 laminin substrate (C). Returning the cells to the 6:1:1 ratio mixture restores adhesion and cell morphology. Scale bar represents 1000 μm. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides substrates and methods that can be used in processes that comply with Good Manufacturing Practice (GMP), the minimum standard that pharmaceutical manufacturers must meet for their manufacturing processes in order to be certified by the European Medicines Agency and comparable regulatory authorities. The inventors surprisingly found that ONP growth can be improved by changing the combination of laminin isoforms used as a substrate during growth. LN211 is a laminin present during cochlear development. Growth is achieved by gradually increasing the percentage of LN211 present in the substrate. Complete replacement of the laminin mixture with 100% LN211 can also impair growth. Using a combination of LN211, LN511, and LN111 in the substrate results in more efficient growth. Growth is most efficient when the percentage of LN211 is gradually increased over a small number of passages (e.g., 2-4 passages, e.g., 3 passages). ONPs cultured on substrates comprising LN211, LN511, and LN111 and cultured using the growth methods described herein maintain proliferative activity. ONPs cultured on substrates comprising LN211, LN511, and LN111 and cultured using the growth methods described herein maintain good morphology and neuronal function.

[0012] Laminins contain three disulfide-linked polypeptides designated α, β, and γ. Each polypeptide is found in multiple isoforms, designated by the corresponding Greek letter followed by a number (e.g., α2, β1, and γ1). Standard laminin nomenclature now refers to laminins by their isoform composition and lists only the numbers. Thus, laminin containing α2, β1, and γ1 is laminin 211 (Aumailley (2013) Cell Adhesion & Migration 7, 48-55), also referred to herein as LN211. Table 1 lists laminins along with their composition and corresponding alternative names that have been used in the literature.

[0013] Table 1. Types of laminin [Table 1]

[0014] The laminin used in the substrates and methods provided herein can be recombinant. That is, the laminin can be a recombinant protein, e.g., overexpressed and purified using techniques well known in the art. The laminin is preferably of human origin. Laminin can be commercially available, e.g., from BioLamina (human recombinant laminin), or can be prepared using standard methods in the art. The amount of laminin in a cell culture substrate is typically measured per unit area (e.g., cm 2 The total concentration of laminin is measured in micrograms per cm. 2 and preferably about 1 μg / cm 2 The total laminin concentration is the sum of the concentrations of all laminin isoforms combined.

[0015] Otic progenitor cells (also referred to herein as otic precursors) are cells that can further differentiate into either hair cells (otic epithelial precursor cells, OEPs) or auditory neuron cells (otic neural precursors, ONPs). OEPs and ONPs each have characteristic morphologies that can be used to distinguish them from each other. OEPs are larger than ONPs, typically exceeding approximately 12 μm in size. OEPs exhibit distinct cell-cell junctions. OEPs form epithelioid islands. ONP cells are typically smaller than OEPs, typically ranging in size from approximately 8 μm to approximately 15 μm. ONPs exhibit cytoplasmic projections. ONPs form loosely connected colonies. ONP cells have denser chromatin than OEP cells. ONPs can be identified by their morphology (Figure 1). The otic placode is the primordium of the hearing organ. The otic precursor state can be identified by the expression of markers associated with the early otic placode during 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. Otic progenitors may express two or more of SOX2, FOXG1, PAX2, and PAX8, such as SOX2 and PAX8, FOXG1 and PAX8, or PAX2 and PAX8. Human otic progenitor cells can be identified as cells bearing 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, for example, using fluorescent methods or by qPCR. Suitable methods for preparing such cells are described in WO 2016 / 156831 and WO 2018 / 051092. The ONP cells may be human ONP cells. ONP batches typically express one or more markers selected from the group including SSEA1 and ITGA4. ONP cells with neural function may express one or more markers selected from the group including SSEA1 and GD2. During ONP proliferation, 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 from pluripotent cells by differentiation. ONP expansion refers to the expansion of ONP cells that have the potential to become auditory neuron cells without differentiation into auditory neuron cells.

[0016] Prior to expansion, e.g., during ONP production, cells (ONP cells and / or differentiating cells) are typically cultured on a substrate containing laminin-521 (LN521) and laminin-111 (LN111), e.g., in a Y:Z ratio of LN521:LN111, e.g., Y=about 1 and Z=about 1. During expansion, the laminin composition of the substrate is gradually changed from that used during ONP production to a mixture containing LN211, such that laminins LN211, LN521, and LN111 are present in the ratio X:Y:Z of LN211:LN521:LN111. X can be about 1 to about 10. X can be 2-6, 3-6, 4-6, or 5-6. X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Y can be from about 1 to about 5. Y can be from 1 to 3, or 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.

[0017] At the first propagation passage, LN211 is typically included in the matrix, and the laminin ratio is typically changed from Y:Z (where Y=about 1 and Z=about 1) to X:Y:Z (where X=about 1, Y=about 1, and Z=about 1 (e.g., 1:1:1 LN211:LN521:LN111)). Therefore, in the first culture step (passage 0 (p0)) of expansion, the ONP cell culture substrate used to seed the cells typically contains a protein matrix consisting of human laminin-211, human laminin-521, and human laminin-111, where the amounts of laminin-211, laminin-521, and laminin-111 in the ONP cell culture substrate are defined by the X:Y:Z ratio of (laminin-211):(laminin-521):(laminin-111), where X = about 1, Y = about 1, and Z = about 1. In the next culture step (e.g., the second step, passage 1 (p1)), the X:Y:Z ratio of the ONP cell culture substrate is typically X = about 2, Y = about 1, and Z = about 1. The X:Y:Z ratio of the culture substrate for otic progenitor (ONP) cells in subsequent culture steps (e.g., the third step (p2), the fourth step (p3), etc.) is typically X = about 6, Y = about 1, and Z = about 1 (e.g., 6:1:1). The culture substrate for otic progenitor (ONP) cells may have an X:Y:Z ratio in which X is 1, 2, 3, 4, 5, or 6; Y is about 1; and Z is about 1. The culture substrate for otic progenitor (ONP) cells may have an X:Y:Z ratio in which X is about 2 to about 6; Y is about 1; and Z is about 1.

[0018] In a method for expanding a population of otic neural progenitor (ONP) cells, the method may include culturing the cells on a first cell culture substrate consisting of laminin-211, laminin-111, and laminin-521 in an X1:Y:Z ratio of (laminin-211):(laminin-521):(laminin-111). The method may further include culturing the cells for further passages on a second cell culture substrate consisting of laminin-211, laminin-111, and laminin-521 in an X2:Y:Z ratio of (laminin-211):(laminin-521):(laminin-111), where X2 is greater than X1. Thus, the amount of laminin-211 in the second cell culture substrate is increased compared to the amount of laminin-111 and laminin-521 in the first cell culture substrate. The method may further include culturing the cells for further passages on a third cell culture substrate consisting of laminin-211, laminin-111, and laminin-521 in an X3:Y:Z ratio of (laminin-211):(laminin-521):(laminin-111), where X3 is greater than X2. Thus, the amount of laminin-211 in the third cell culture substrate is increased compared to the amount of laminin-111 and laminin-521 in the second cell culture substrate.

[0019] Any of the cell culture substrates described herein can be used in methods for expanding a population of cochlear neural progenitor cells.

[0020] The expansion method may include culturing ONP cells for one passage on a cell culture substrate consisting of laminin-111 and laminin-521, where optionally the laminin-111 and laminin-521 are human laminin-111 and human laminin-521, prior to culturing on the substrate comprising LN211. Thus, the method may further include, prior to step (a), an initial step of culturing ONP cells for one passage on a cell culture substrate consisting of laminin-111 and laminin-521, and then transferring the cells to the cell culture substrate of step (a). In this step, the cells typically reach 80-90% confluence (e.g., 90% confluence), for example, after 3-10 days of culture.

[0021] In the culture methods described herein, ONP cells are typically cultured at about 10% confluence, e.g., about 20,000 to 40,000 cells / cm. 2 It is seeded to start with.

[0022] As used herein, the terms "passage" and "culturing" are used interchangeably. The time between each passage / culturing step is usually about one week, for example, 3 to 10 days, e.g., 5 to 9 days. Cells can be cultured for about one week or until confluent before the next culturing step. Cells are usually cultured at 37°C. Cells are cultured in a 5% CO2 environment. Cells are usually passaged when they become 80 to 100% confluent, preferably when they become 80 to 90% confluent, for example, when they are about 90% confluent. Passage and culturing are terms well known in the art. When passaged, cells are usually harvested from the culture vessel. After removing the liquid medium, the cells are detached from each other and from the vessel using a cell detachment buffer (e.g., containing trypsin). The cells are washed and centrifuged (e.g., at 100-200 g for 5-10 minutes), then suspended in fresh medium, counted, and seeded into a container containing the laminin substrate described herein for culture (reculture).

[0023] The product of the method for expanding a population of otic neural progenitor (ONP) cells is an expanded population of ONP cells, which have good morphology and / or neural function. The population may be in the same container or may be split into two or more containers. Cell doubling time and cumulative cell doublings were calculated using the number of cells initially seeded, the number of cells suspended, and the length of time between passaging / culturing steps. For example, a population may undergo five cumulative cell doublings over a 10-day (1-week) period (Figure 2). The cumulative cell doublings may be 2-4, or 2-3, e.g., about 3. More than two cumulative cell doublings are preferred, e.g., 2-4. The cell doubling time may be less than 50 hours, e.g., 35-45 hours. The cell doubling time may be about 38 hours.

[0024] The cell culture medium added to cells on a cell culture substrate may contain at least one growth factor. The cell culture medium may contain at least one growth factor selected from the group consisting of FGF, EGF, and IGF (e.g., IGF-I and IGF-2, e.g., IGF-1). The cell culture medium may contain FGF (e.g., bFGF), EGF, and IGF-1. The growth factor is preferably a recombinant growth factor. When present in the medium, the concentration of each growth factor in the medium is typically 10 to 30 ng / ml, e.g., 20 ng / ml. The growth factor may be a human growth factor. The cell culture medium may contain Dulbecco's Modified Eagle's Medium (DMEM). The cell culture medium may contain L-glutamine, glucose, sodium pyruvate, and sodium bicarbonate (e.g., DMEM containing 4 mM L-glutamine, 4500 mg / L glucose, 1 mM sodium pyruvate, and 1500 mg / L sodium bicarbonate). The cell culture medium may include B27 neuronal cell culture supplement (e.g., B27 supplement, Thermofisher, catalog number 17504044).

[0025] Preferred features for the second and subsequent aspects of the invention are as for the first aspect, mutatis mutandis.

[0026] Embodiment The following numbered sections describe embodiments of the cell substrate compositions and methods described herein.

[0027] Item 1 1. A culture substrate for otic progenitor (ONP) cells, 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 ONP cell culture substrate are defined by a ratio of (laminin-211):(laminin-521):(laminin-111) X:Y:Z, where X is about 1 to about 10; Y is about 1 to about 5; and Z is about 1 to about 5.

[0028] Section 2 Item 2. A culture substrate for inner ear progenitor (ONP) cells according to Item 1, wherein X is about 2 to about 6; Y is about 1; and Z is about 1.

[0029] Section 3 Item 3. A culture substrate for otic neuroprogenitor (ONP) cells according to Item 1 or 2, wherein laminin 211, laminin 111, and laminin 521 are human laminin 221, human laminin 111, and human laminin 521.

[0030] Section 4 Item 4. The culture substrate for otic neuroprogenitor (ONP) cells according to any one of Items 1 to 3, wherein laminin-211, laminin-111, and laminin-521 are recombinant.

[0031] Section 5 (a) culturing the cells for one passage on a first cell culture substrate comprising laminin-211, laminin-111, and laminin-521; and thereafter (b) culturing the cells for one passage on a second cell culture substrate comprising laminin-211, laminin-111, and laminin-521, wherein the amount of laminin-211 in the second cell culture substrate used in step (b) is increased compared to the amount of laminin-111 and laminin-521 in the first cell culture substrate used in step (a). replanking the cells for passaging; and optionally, (c) repeating step (b), wherein the 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 compared to the amount of laminin-111 and laminin-521 in the second cell culture substrate used in step (b).

[0032] Section 6 Item 6. The method of item 5, wherein the amounts of laminin-211, laminin-111, and laminin-521 in the cell culture substrate in step (a) are defined by the ratio X:Y:Z of (laminin-211):(laminin-111):(laminin-521), where 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.

[0033] Section 7 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.

[0034] Section 8 Item 8. The method according to any one of Items 5 to 7, wherein the ONP cells are human cells.

[0035] Section 9 9. The method of any one of items 5 to 8, further comprising, prior to step (a), an initial step of culturing ONP cells for one passage on a cell culture substrate consisting of laminin-111 and laminin-521, and thereafter transferring the cells to the cell culture substrate of step (a), wherein optionally the laminin-111 and laminin-521 are human laminin-111 and human laminin-521.

[0036] Section 10 Item 9. The method according to any one of Items 5 to 8, wherein a cell culture medium containing 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. [Example]

[0037] Example 1. Preparation of ONPs Oncogene-derived progenitor neurons (ONPs) were differentiated from hESCs and iPSCs by seeding them on laminin-coated tissue culture plastic (laminin ratio 1:1, LN521:LN111) in Dulbecco's Modified Eagle Medium: Ham's F12 (DMEM / F12) supplemented with 1xN2 and 1xB27 (abbreviated as DFNB) (both Life Technologies, UK), FGF3, and FGF10 (both 50 ng / ml) (both R&D Systems, UK). The Wnt inhibitor IWR-1 (10 μM) was also present. Cells were cultured for 8 days. On day 9, the medium was changed to DFNB supplemented with FGF3 and FGF10 (both 25 ng / ml) and the Wnt activator BIO (2 mM). Throughout the differentiation period, cells were plated on laminin-coated tissue culture plastic (laminin ratio 1:1, LN521:LN111). Human recombinant laminin was purchased from BioLaminin (LN521-02; LLN111-02; LN211-02). Cells could be further differentiated along either the hair cell or auditory neuron lineage, and cultures were manually purified to remove cells lacking the relevant characteristic precursor morphology and specifically enriched for either otic epithelial precursor (OEP) or otic neuron precursor (ONP) phenotypes.

[0038] Example 2: Identification of ONPs and OEPs ONPs and OEPs can be differentiated from hESCs and iPSCs, for example, according to Example 1, or from undifferentiated colonies seeded as monolayers on laminin-coated flasks, as described in Chen (2012) and Boddy (2020). The differentiation process leads to two morphologically distinct types of otic colonies (Figure 1). OEP cells exhibit a flattened phenotype, large cytoplasm, and epithelioid islands. OEP cells exhibit distinct cell-cell junctions. OEP cells are smaller than OEPs, have denser chromatin, and possess cytoplasmic processes.

[0039] Cochlear nerve precursors can be identified by quantitative microscopy based on the co-expression of the otic markers PAX8, PAX2, FOXG1, and SOX2. Cells were fixed in phosphate-buffered saline (PBS) with 4% paraformaldehyde for 15 minutes at room temperature and then blocked with PBS 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, UK), 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 Alexa Fluor 568 (Molecular Probes, Life Technologies, UK). Nuclei can be counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma). Cells can be imaged using either the EVOS FL Cell Imaging System or the IN Cell Analyzer 2000 system platforms (GE Healthcare). Quantitative immunofluorescence can be performed on the IN Cell Analyzer using the Developer Toolbox. Cells can be contacted with, for example, a PAX8 antibody in combination with an antibody against one of the other markers (PAX2, SOX2, or FOXG1), and co-expression in individual cells can be confirmed. Highly expressing cells can be defined as cells with a fluorescence intensity above the 75th percentile for each antibody, independently. A cell population highly positive (co-expressing) for two markers can be identified as an otic precursor.

[0040] Example 3. Purification of ONP ONPs can be manually selected, or purified by FACS using the markers referenced herein and methods well known to those of skill in the art, for example, by Fluorescent Automated Cell Sorting using SSEA1 and CD141 antibodies and the methods described in WO 2018 / 051092, which is incorporated herein in its entirety.

[0041] After sorting, ONP cells are typically cultured on a substrate containing laminin 521 (LN521) and laminin 111 (LN111), e.g., a 1:1 ratio of LN521:LN111. Eight to ten days after sorting, the plates / flasks initially seeded with ONPs reach ~80-90% confluence.

[0042] Example 4: Migration to different laminin matrices ONPs purified by cell sorting as instructed (e.g., as in WO 2018 / 051092 using SSEA1 as a cell surface marker) were cultured from p0 (i.e., immediately after sorting) to p6, at which point they were frozen. Passages were primarily performed when cells reached approximately 90% confluence. Cells were detached with 0.5x TrypLE Select. Over the first few growth cycles, cells were transitioned to growth on a matrix containing a high proportion of LN211. At p1, the ratio was increased from 1:1:1 to 2:1:1, LN211:52:1:11, and at p2, this ratio was increased to 6:1:1, and this was used from then on (Table 2 and Figure 2).

[0043] Table 2. Laminin matrix combinations used for growth of ONPs. [Table 2]

[0044] Figure 2 shows the cumulative cell doublings of three different batches of ONPs grown on the matrices shown in Table 2. Each point on the plot represents a passage. These growth curves demonstrate that ONPs can maintain proliferation using the matrix compositions described herein.

[0045] It is crucial to transfer ONPs to the appropriate laminin composition to support efficient proliferation. LN211 must be introduced in stages. It is used in combination with other laminins. When ONPs are seeded in a 6:1:1 mixture immediately after sorting, cells initially adhere. However, instead of spreading, colonies grow upward and form three-dimensional spheres (Figures 3 and 4). The initial number of cells seeded, the number of cells detached, and the length of time between cycles were used to calculate cell doubling time and cumulative cell doublings. Compared to the 1:1:1 mixture, cell doublings and cell yield were significantly reduced (Table 3).

[0046] Table 3. Effect of matrix composition on cell growth parameters when transferred to a 6:1:1 mixture immediately after sorting. [Table 3]

[0047] The effect of shifting growing ONP(p4) to 100% LN211 was investigated with cells on a 6:1:1 mixture. However, the cells struggled to adhere, and colonies exhibited a "spiky" morphology (Figure 5). Cells on a 100% LN211 matrix exhibited a reduced number of cell doublings (Tables 4 and 5) and a longer doubling time (Table 5). Growth was more efficient on the 6:1:1 laminin substrate.

[0048] Table 4. Effect of matrix composition on cell growth parameters when ONPs growing on a 6:1:1 mixture are transferred onto a pure 211 substrate. [Table 4]

[0049] Table 5. Similar experiments to those shown in Table 4, showing cell doublings and cell doubling times. [Table 5]

[0050] Cells recovered from the 100% LN211 matrix were then replated either on 100% LN211 or on the 6:1:1 mixture. Cells replated on 100% LN211 maintained their abnormal spiny morphology, whereas cells replated on the 6:1:1 mixture reverted to their characteristic appearance, suggesting that the changes induced by pure LN211 were reversible (Figure 6).

[0051] Example 5: Growth of ONPs in culture All measurements are taken from a single T12.5cm 2 This section concerns the volume required for a flask (e.g., Corning Falcon Vented Flask, VWR International Catalog No. 353107). ONP cells are typically cultured on a substrate containing laminin 521 (LN521) and laminin 111 (LN111), e.g., a 1:1 ratio of LN521:LN111. The laminin composition of the substrate used during ONP generation is gradually changed during growth to a mixture containing gradually introduced LN211, e.g., from a 1:1 ratio of LN521:LN111 to a 1:1:1 ratio of LN211:521:111. LN211 is a laminin present during cochlear development. This transition progresses from a 1:1:1 mixture of LN211:521:111 used to seed cells immediately after sorting (passage 0 (p0)), to a 2:1:1 mixture of LN211:521:111 used from subsequent divisions (p0->p1), to a 6:1:1 mixture of LN211:521:111 used from subsequent passages (p1->p2 and beyond).

[0052] Aliquots of laminin-211, laminin-521, and laminin-511 were thawed at 4°C for a minimum of 30 minutes. The laminin stock solution had a concentration of 100 μg / ml.2+ and Mg 2+ One ml of Dulbecco's Phosphate Buffered Saline (DPBS) containing laminin (Cat. No. D8662, stored at 4°C) was measured into a chilled 50 ml tube. Laminin was added to the DPBS. The buffer and laminin were quickly mixed well with a chilled P1000 pipette tip and transferred to a T12.5 flask, ensuring the flask remained level. The flask was gently tilted to ensure even distribution. The flask was incubated overnight at 4°C in a horizontal position.

[0053] In the first expansion step (p0->p1), 62.5 μl of LN211, 31.25 μl of LN521, and 31.25 μl of LN111 were added for a 2:1:1 ratio. In subsequent expansion steps (p1->p2 and beyond), 93.75 μl of LN211, 15.625 μl of LN521, and 15.625 μl of LN111 were added for a 6:1:1 ratio.

[0054] Cell seeding and passaging procedures A stock solution of 4 ml DPBS- / -, 2 ml OSCFM, and 1 ml 0.5x TrypLE Select is warmed overnight. The 1x stock is diluted 1:1 with DPBS- / -. The laminin-coated flask is washed with chilled DPBS+ / +, followed by 2x 2 ml room temperature DPBS- / -. 1 ml of warmed OSCFM medium is added and the flask is placed in a 37°C incubator.

[0055] To harvest the cells, remove the old medium from the culture vessel and wash the cells twice with 2 ml of DPBS (- / -). Detach the cells using cell detachment buffer (TrypLE Select, ThermoFisher) per T12.5 (0.5 ml per well for 12-well plates, 0.25 ml per well for 24-well plates). Incubate the flask at 37°C for 3 minutes before checking for detachment under an inverted microscope. If the cells do not detach easily, incubate the plate for up to 5 minutes total, periodically checking for detachment.

[0056] To collect the cells, for each flask, dilute the lysate with OSCFM and pipette the suspension up and down. Collect the cell suspension and transfer it to a 50 ml centrifuge tube. If any cells remain, wash them off the surface of the vessel with OSCFM again. Centrifuge at 1000 rpm (195 x g) for 5 minutes. Resuspend the pellet in OSCFM. Cell counts can be performed using an automated cell counter (e.g., Biorad TC20). Count the cells present using the TC20 cell counter. Set the gating histogram to 8-22 μm and record the total and viable cell counts. Repeat the procedure for the other slide chamber and average the two counts. If the discrepancy is >10%, repeat the procedure for another cell suspension sample and average the data from the three most consistent counts.

[0057] Record the total number of cells obtained. This value (and the total number of cells initially seeded) will be used to determine cell doubling (PD). Considering that 1 ml of OSCFM is already in the pre-heated flask, approximately 20,000-40,000 cells / cm will be obtained in a total of 2.5 ml of OSCFM per flask. 2 Seed the cells at a density of 250,000-500,000 cells per T12.5 flask. The normal division ratio is 1:3, but always count the cells to accurately determine PD. Return the flask to the incubator and incubate at 37°C and 5.0% CO2. 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 they reach 100% confluence, whichever comes first.

[0058] Table 6. Composition of DFNB medium [Table 6]

[0059] Table 7. Composition of Ootic Stem Cell Complete Medium (OCSFM) [Table 7]

[0060] All documents mentioned in this application are incorporated herein by reference in their entirety.

Claims

1. A culture substrate for otic neuroprogenitor (ONP) cells, comprising a protein matrix consisting of laminin 211, laminin 521, and laminin 111, The amounts of laminin-211, laminin-521, and laminin-111 in the culture substrate for otic neuroprogenitor (ONP) cells are defined by the ratio X:Y:Z of (laminin-211):(laminin-521):(laminin-111): 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; Culture substrate for otic neural progenitor (ONP) cells.

2. X is from about 2 to about 6; Y is about 1; and Z is about 1; A culture substrate for otic neuroprogenitor (ONP) cells according to claim 1.

3. The culture substrate for otic neuroprogenitor (ONP) cells according to claim 1 or 2, wherein laminin 211, laminin 111, and laminin 521 are human laminin 221, human laminin 111, and human laminin 521.

4. The culture substrate for otic neuroprogenitor (ONP) cells according to any one of claims 1 to 3, wherein laminin 211, laminin 111, and laminin 521 are recombinant.

5. (a) culturing cells for one passage on a first cell culture substrate consisting of laminin 211, laminin 111, and laminin 521; and thereafter (b) culturing the cells for further passages 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 compared 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 comprising laminin-211, laminin-111, and laminin-521, wherein the amount of laminin-211 in the third cell culture substrate is increased compared to the amount of laminin-111 and laminin-521 in the second cell culture substrate; 1. A method for expanding a population of otic neural progenitor (ONP) cells, comprising:

6. wherein the amounts of laminin-211, laminin-111, and laminin-521 in the cell culture substrate of step (a) are defined by the ratio X:Y:Z of (laminin-211):(laminin-111):(laminin-521): 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; The method of claim 5.

7. Laminin 211, laminin 111, and laminin 521 (i) human laminin 221, human laminin 111, and human laminin 521; and / or (ii) is recombinant; The method according to claim 5 or claim 6.

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

9. Prior to step (a), the method further comprises culturing ONP cells for one passage on a cell culture substrate consisting of laminin 111 and laminin 521; Optionally, the laminin 111 and the laminin 521 are human laminin 111 and human laminin 521. The method according to any one of claims 5 to 8.

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

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