Artificial follicles

Abstract

Methods for producing an artificial follicle are disclosed. Also disclosed are artificial follicles produced by such methods, in vitro methods of rejuvenating an oocyte, and methods of treating infertility or improving fertility.

Description

[0001] Artificial Follicles

[0002] This application claims priority from SG10202404018U filed 20 December 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the fields of molecular biology and nucleic acid technology. In particular, the present disclosure relates to the production of artificial follicles. The present disclosure also relates to treatment of infertility.

[0005] Background

[0006] The ovarian follicle is the fundamental functional unit of the ovary and is essential for female fertility. Each follicle comprises an oocyte encased by granulosa cells (GCs), which play a critical role in nurturing the oocyte. The GCs are responsible for providing the necessary energy, nutrients, and a supportive microenvironment required for growth and maturation of the oocyte. This maturation process is crucial for the oocyte to attain competency for fertilization, ultimately resulting in a viable embryo. Moreover, GCs secrete estrogen, a vital hormone that regulates various aspects of reproductive health in women, including the menstrual cycle and overall fertility.

[0007] Aging is a significant factor affecting female reproductive health, leading to a decline in oocyte quality and quantity. It has been recently demonstrated that exposing an aged oocyte to a follicular environment in which the GCs are young, can significantly enhance the quality of the aged oocyte (see Wang et al., (2024) Nat Aging. 4(9):1 194-1210, which is hereby incorporated by reference in its entirety). This discovery may be translated into a cell-based therapeutic approach to rejuvenate aged oocytes, thereby improving fertility outcomes in older women. However, one of the primary challenges in clinical application of this technology is the availability of young follicles.

[0008] Summary

[0009] In a first aspect there is provided a method for producing an artificial follicle, comprising:

[0010] (i) providing a cell suspension comprising follicle somatic cells and an oocyte,

[0011] (ii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex, and

[0012] (iii) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle.

[0013] In some embodiments, the culturing in step (ii) is performed under conditions promoting cell aggregation.

[0014] In some embodiments, step (ii) comprises culturing the cells of the cell suspension in the presence of a solid support that promotes cell aggregation, optionally wherein the solid support is a round-bottomed microwell plate or an inverse pyramidal microwell plate. In some embodiments, the culturing in step (ii) is performed in the presence of Growth Differentiation Factor 9 (GDF9).

[0015] In some embodiments, the first hydrogel bead is a hydrogel comprising 1-4 mg / ml of a basement membrane-derived preparation.

[0016] In some embodiments, step (iii) comprises culturing the cells of the cell suspension for 36 to 60 hours.

[0017] In some embodiments, step (iii) comprises culturing the cells of the cell suspension for about 48 hours.

[0018] In some embodiments, the method further comprises:

[0019] (iv) culturing the artificial follicle from step (iii) in a second hydrogel bead to form an antral follicle.

[0020] In some embodiments, the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation.

[0021] In some embodiments, step (iv) comprises culturing the artificial follicle for 5 or 6 days.

[0022] In some embodiments, the method further comprises:

[0023] (v) culturing the antral follicle from step (iv) to produce a graafian follicle.

[0024] In some embodiments, method further comprises:

[0025] (vi) ovulating a mature oocyte from the graafian follicle from step (v) and isolating the mature oocyte.

[0026] In some embodiments, the oocyte is obtained from, or has been obtained from, a female mammalian subject.

[0027] In some embodiments, the subject is a human.

[0028] In some embodiments, the subject is over 35 years of age.

[0029] In a second aspect there is provided an artificial follicle produced by the methods described herein.

[0030] In a third aspect there is provided an in vitro method of rejuvenating an oocyte comprising:

[0031] (i) providing a cell suspension comprising follicle somatic cells and an aged oocyte,

[0032] (ii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex,

[0033] (iii) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle, and

[0034] (iv) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle. In some embodiments, the culturing in step (ii) is performed under conditions promoting cell aggregation.

[0035] In some embodiments, step (ii) comprises culturing the cells of the cell suspension in the presence of a solid support that promotes cell aggregation, optionally wherein the solid support is a round-bottomed microwell plate or an inverse pyramidal microwell plate.

[0036] In some embodiments, the culturing in step (ii) is performed in the presence of Growth Differentiation Factor 9 (GDF9).

[0037] In some embodiments, the first hydrogel bead is a hydrogel comprising 1-4 mg / ml of a basement membrane-derived preparation.

[0038] In some embodiments, step (iii) comprises culturing the cells of the cell suspension for 36 to 60 hours.

[0039] In some embodiments, step (iii) comprises culturing the cells of the cell suspension for about 48 hours.

[0040] In some embodiments, the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation.

[0041] In some embodiments, step (iv) comprises culturing the artificial follicle for 5 or 6 days.

[0042] In some embodiments, the method further comprises:

[0043] (v) culturing the antral follicle from step (iv) to produce a graafian follicle.

[0044] In some embodiments, the method further comprises:

[0045] (vi) ovulating a mature oocyte from the graafian follicle from step (v) and isolating the mature oocyte.

[0046] In some embodiments, the aged oocyte is obtained from, or has been obtained from, a female mammalian subject.

[0047] In some embodiments, the subject is a human.

[0048] In some embodiments, the subject is over 35 years of age.

[0049] In a further aspect there is provided an artificial follicle comprising an oocyte and somatic cells, wherein the somatic cells are obtained or obtainable from a follicle somatic cell suspension.

[0050] In yet a further aspect there is provided a method of treating infertility or improving fertility, comprising:

[0051] (i) obtaining an oocyte from a subject,

[0052] (ii) producing a cell suspension comprising the oocyte of step (i) and follicle somatic cells, (iii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex,

[0053] (iv) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle,

[0054] (v) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle; and

[0055] (vi) a) implanting the follicle obtained in stage (v) to treat infertility or improve fertility, or b) culturing the antral follicle to produce a graafian follicle, ovulating a mature oocyte from the graafian follicle and isolating the mature oocyte.

[0056] Description

[0057] Folliculoqenesis and oocyte maturation

[0058] The present disclosure relates to the production of artificial follicles. The term ‘follicles’ as used herein refers to ovarian follicles. Ovarian follicles are cellular aggregations found in the ovaries of females. Ovarian follicles facilitate the maturation of the oocyte and subsequent ovulation ( / .e. the release of ovum from the ovary). The ovarian follicle comprises an oocyte (germ cell) surrounded by layers of somatic follicular cells (granulosa and theca cells) and a basement membrane. The somatic follicular cells undergo cell division during development of the follicle and the oocyte.

[0059] Granulosa cells (GCs) play an important role in oocyte development and quality. The role of granulosa cells in folliculogenesis is reviewed in Cavalcanti et al., Rev Assoc Med Bras (2023) 69(6):e20230175. It has been recently demonstrated that exposing an aged oocyte to a follicular environment comprising young GCs can enhance the quality of the aged oocyte (e.g. due to higher fidelity of meiotic chromosome segregation). See Wang et al., (2024) Nat Aging. 4(9):1194-1210, which is hereby incorporated by reference in its entirety.

[0060] The maturation or development of the ovarian follicle is known as follicular development or ‘folliculogenesis’. Folliculogenesis refers to the development and maturation of the ovarian follicle from the primordial stage to the primary stage, secondary stage, pre-antral stage, antral stage and graafian stage. Folliculogenesis is reviewed e.g. in Palma et al., (2012) Scientific World Journal. 2012:938138, which is hereby incorporated by reference in its entirety.

[0061] The first stage of follicular development is the primordial follicle. A human primordial consists of an oocyte encapsulated by approximately 10 squamous (thin and flat) pre-granulosa cells. A finite supply of primordial follicles are formed during gestation of the female, and cannot be replenished once lost. Primordial follicle oocytes are diploid and meiotically arrested at the diplotene stage of prophase of meiosis I. They are characterized by the presence of an intact germinal vesicle (GV); the nucleus of the oocyte. The primordial follicle develops into a ‘primary follicle’. During the transition to primary follicles, the cells surrounding the oocyte differentiate from squamous pre-granulosa to cuboidal granulosa cells.

[0062] The next stage is called a ‘secondary follicle’. Secondary follicles comprise 2 or more layers of granulosa cells and the presence of a theca cell layer. Theca cells (also sometimes called thecal cells) are endocrine cells in the ovary which comprise large amounts of smooth endoplasmic reticulum. Thecal cells produce androgens in response to luteinising hormone (LH), which may then be converted into estrogen in the neighbouring granulosa cells. The function of theca cells is described e.g. in Young et al., Reproduction. (2010) 140(4):489-504, which is hereby incorporated by reference in its entirety.

[0063] Secondary follicles also comprise an oocyte with a completely formed zona pellucida. The zona pellucida is an extracellular matrix that surrounds the plasma membrane of the oocyte. Thin cytoplasmic filaments (follicular cell processes) termed transzonal projections (TZPs) become detectable when the zona pellucida is laid down, and the TZPs subsequently increase in number as oocyte growth progresses. TZPs mediate contact between the granulosa cells and the oocyte. Granulosa cells provide metabolic support to the oocyte via TZPs, and they also play a role in maintaining meiotic arrest of the oocyte. The function of TZPs is described in Clarke. Mol Reprod Dev. (2022) 89(11):509-525, which is hereby incorporated by reference in its entirety. The number and quality of TZPs can be used to assess the quality or age of a follicle. For example, an aged ovarian follicle may be indicated by the presence of poorly formed TZPs or a decreased number of TZPs. As used herein, the term ‘aged’ with respect to an ovarian follicle refers to an ovarian follicle that has deteriorated in quality. As such, an aged ovarian follicle is an ovarian follicle that is incapable or less capable of developing into a mature follicle to induce ovulation of an oocyte.

[0064] Secondary follicles then develop into ‘pre-antral follicles’ (also called ‘early antral’). Sometimes this stage of folliculogenesis is encompassed by the term ‘secondary follicles’. In pre-antral follicles, the granulosa cells continue to increase and acquire receptors for FSH (follicle-stimulating hormone). This may also be termed the start of the gonadotropin-responsive phase. Gonadotropins are glycoprotein hormones which include FSH and LH. FSH stimulates the granulosa cells to proliferate and produce estrogen.

[0065] Antral stage (early and late) follicles (also called ‘tertiary follicles’) are characterized by the presence of a follicular fluid-filled cavity adjacent to the oocyte, known as the antrum. During antrum formation, undifferentiated GCs differentiate into functional groups including cumulus cells (CC), which surround the oocyte, and mural granulosa cells (MGCs), which surround the antrum.

[0066] ‘Graafian follicles’ are the final stage of folliculogenesis prior to ovulation (also sometimes called ‘late antral follicles’, ‘pre-ovulatory follicles’ or ‘mature ovarian follicles’). The graafian follicle is a specific form of antral follicle. LH triggers the final stages of oocyte maturation. The oocyte achieves meiotic resumption, undergoing GV breakdown. At the completion of the first meiotic division, two cells are formed, a large secondary oocyte and a small polar body (a haploid cell formed at the same time as the mature oocyte). The polar body is extruded and the ovulated oocyte becomes arrested once more at metaphase II until after fertilization. Accordingly, the terms ‘mature oocyte’, ‘ovulated oocyte’ and ‘ovum’ as used herein refer to a metaphase II arrested oocyte or egg that has undergone meiotic maturation and has the ability to undergo fertilization.

[0067] ‘Ovulation’ as used herein refers to the release of a mature oocyte from an ovarian follicle. During ovulation, the follicle ruptures to release the mature oocyte and the GCs and theca cells differentiate into the corpus luteum.

[0068] Artificial Follicles

[0069] The present disclosure relates to a novel method for generating artificial follicles. The term ‘artificial follicles’ as used herein refers to ovarian follicles which are non-naturally occurring. The article follicles according to the present disclosure are synthetic, and may be described as being ‘man-made’. Artificial follicles according to the present disclosure are preferably not produced in vivo in a subject, and are instead produced in vitro / ex vivo.

[0070] Artificial follicles may also be described as being ‘follicle-like’. The methods described herein relate to the production of artificial follicles using cell suspensions comprising follicle somatic cells and oocytes.

[0071] The present disclosure also provides artificial follicles produced by the methods described herein. The artificial follicles produced through this method have a three-dimensional structure and mimic the natural follicular environment, facilitating the growth and maturation of oocytes.

[0072] The various aspects of the present disclosure find utility in reproductive biology, with a particular focus on assisted reproductive technologies (ART). ‘ART’ as used herein refers to procedures that aim to achieve pregnancy. Typically, ART refers to procedures in which eggs or embryos (and optionally sperm) are handled, e.g. in vitro fertilization (IVF). It has been previously demonstrated that exposing an aged oocyte to a young follicular environment can significantly enhance the quality of the aged oocyte (see Wang et al., Supra, which is hereby incorporated by reference in its entirety). However, one of the primary challenges in clinical application of this discovery is the availability of young follicles.

[0073] The present disclosure addresses the challenge of forming functional follicles using a cell suspension of follicle somatic cells (e.g. granulosa cells). This approach avoids the use of native follicles, which may be difficult to isolate and limited in availability. The technologies described herein make the process of in vitro development and rejuvenation of oocytes using a follicle-like somatic environment more accessible and feasible for clinical application and research.

[0074] The ability to produce high-quality, mature oocytes from artificial follicles underscores the potential of this method to improve fertility outcomes. It ensures that the oocytes are competent for fertilization, increasing the chances of successful embryo development and pregnancy in ART procedures. The methods described herein therefore find application in enhancing IVF success rates, especially for women over 35; preserving fertility; supporting reproductive research and drug development; enabling personalized fertility treatments; and providing educational tools fortraining professionals in reproductive technologies. Methods of making a follicle

[0075] The methods described herein relate to production of artificial follicles using cell suspensions comprising follicle somatic cells (e.g. granulosa cells) and oocytes. A schematic of an exemplary method according to the present disclosure is shown in Figure 1. In some embodiments, artificial follicles may be produced essentially as described in Example 1.1 herein.

[0076] Disclosed herein are methods of making an artificial follicle comprising:

[0077] (i) providing a cell suspension comprising follicle somatic cells and an oocyte,

[0078] (ii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex, and

[0079] (iii) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle, and optionally

[0080] (iv) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral follicle.

[0081] Follicle Somatic Cells

[0082] Follicle somatic cells (also called somatic follicular cells) are non-reproductive (non-germ) cells which surround the oocyte during folliculogenesis as described hereinabove. In some embodiments, the follicle somatic cells are granulosa cells or theca cells. In some embodiments, the follicle somatic cells are derived from Induced Pluripotent Stem (iPS) cells.

[0083] Methods described herein comprise providing a cell suspension comprising an oocyte and follicle somatic cells. In some embodiments, in step (i) of the process of the present disclosure, a cell suspension comprising an oocyte and follicle somatic cells is provided.

[0084] Granulosa Cells

[0085] In some embodiments, the follicle somatic cells are granulosa cells (GCs). Accordingly, in some embodiments, methods described herein comprise providing a cell suspension comprising an oocyte and granulosa cells. In some embodiments, in step (i) of the process of the present disclosure, a cell suspension comprising an oocyte and granulosa cells is provided.

[0086] GCs are somatic cells (non-reproductive cells) which surround the oocyte during folliculogenesis as described hereinabove. GCs play an important role in oocyte development and quality. Granulosa cells have been shown to originate from Gonadal Ridge Epithelial-Like cells (see e.g. Hummitzsch et al., PLoS One. 2013;8(2):e55578, which is hereby incorporated by reference in its entirety). GCs may be identified via expression of marker genes including FSHR, CYP19A1 and AMH (see e.g. Hatzirodos et al., PLoS One. (2015) 16;10(3):e0119800, which is hereby incorporated by reference in its entirety).

[0087] Pre-granulosa cells are squamous and encapsulate the oocyte in a primordial follicle. In some embodiments, the GCs provided for use in methods described herein are pre-granulosa cells.

[0088] There are two types of granulosa cells: cumulus cells (CCs), which surround the oocyte; and mural granulosa cells (MGCs), which line the walls of the follicles and surround the antrum. CCs and MGCs serve different functions during folliculogenesis (see e.g. Dompe et al., Cells. (2021) 5;10(6):1396, which is hereby incorporated by reference in its entirety). In some embodiments, the granulosa cells provided for use in methods described herein are undifferentiated GCs. In some embodiments, the granulosa cells provided for use in methods described herein are CCs. In some embodiments, the granulosa cells provided for use in methods described herein are MGCs. In some embodiments, the granulosa cells provided for use in methods described herein are a combination of any of undifferentiated GCs, CCs and MGCs (e.g. a combination of CCs and MGCs).

[0089] The follicle somatic cells (e.g. GCs) used in the methods of the present disclosure may be provided as a single cell suspension.

[0090] Herein, a “cell suspension” refers to composition comprising cells in a liquid medium. As used herein, the term “single cell suspension” refers to a cell suspension in which individual cells are provided in a liquid medium in which none of the cells, or substantially none of the cells, are provided as aggregates.

[0091] Aggregates comprise two or more cells (e.g. 3, 4, 5, 10, or more cells) that are physically associated with one another, and may also sometimes be referred to as ‘clumps’ of cells.

[0092] In a single cell suspension according to the present disclosure, fewer than 15%, e.g. one of <10%, <5%, <4%, <3%, <2% or <1% of the cells of the single cell suspension may be provided in the form of an aggregate. In a single cell suspension according to the present disclosure, more than 85%, e.g. one of >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% of the cells of the single cell suspension may be not provided in the form of an aggregate.

[0093] Accordingly, in some embodiments, the methods described herein comprise providing a single cell suspension of follicle somatic cells and one or more oocytes, which are combined to provide a cell suspension comprising an oocyte and follicle somatic cells. In some embodiments, the methods described herein comprise providing a single cell suspension of GCs and one or more oocytes, which are combined to provide a cell suspension comprising an oocyte and GCs.

[0094] In some embodiments, the follicle somatic cells are derived from Induced Pluripotent Stem (iPS) cells. In some embodiments, the follicle somatic cells are granulosa cells derived from iPS cells. Such cells may be described as ‘granulosa-like’ cells. Granulosa-like cells have the phenotypic markers (e.g. expression of marker genes including FSHR, CYP19A1 and AMH) of a granulosa cell. In some embodiments, Granulosa-like cells have the phenotypic markers (e.g. expression of expression of marker genes including FSHR, CYP19A1 and AMH) of a granulosa cell, but have not been derived from a Gonadal Ridge Epithelial-Like cell. The term ‘granulosa cells’ as used herein encompasses granulosa cells or ‘granulosa-like’ cells derived from iPS cells. Accordingly, in some embodiments, the granulosa cells are ‘granulosa-like’ cells.

[0095] In some embodiments, the follicle somatic cells (e.g. GCs) are derived from the same subject as the oocyte (autogeneic). Where follicle somatic cells are referred to herein as being ‘autogeneic’ or ‘autologous’, the subjects from which the follicle somatic cells are derived may be genetically identical to the subject from which the oocyte is derived. That is, follicle somatic cells and an oocyte may be obtained from the same subject prior to production of an artificial follicle according to methods described herein. In some embodiments, the follicle somatic cells and / or the oocyte may have been previously obtained from the same subject. In some cases, the artificial follicle may be subsequently returned to the same subject. In some embodiments, the oocyte may be returned to the subject after fertilization, e.g. via IVF.

[0096] In some embodiments, methods disclosed herein may use follicle somatic cells (e.g. GCs) which are allogeneic with respect to the oocyte. Where follicle somatic cells are referred to herein as being ‘allogeneic’ or ‘non-autologous’ with respect to the oocyte, the subjects from which the follicle somatic cells and the oocyte are derived are not the same. The follicle somatic cells may be obtained from a subject who is genetically non-identical to the subject from which the oocyte is derived. In other words, an artificial follicle is produced using follicle somatic cells obtained from a first subject and an oocyte obtained from a second subject.

[0097] In some embodiments, the follicle somatic cells are ‘young follicle somatic cells’. As used herein, the term ‘young follicle somatic cells’ refers to follicle somatic cells derived from a human female subject under the age of 35 years. In some embodiments, the GCs are ‘young GCs’. As used herein, the term ‘young GCs’ refers to GCs derived from a human female subject under the age of 35 years. In some embodiments, the subject from which the follicle somatic cells are derived is a human female under the age of 35 years. As used herein, the term ‘under the age of 35’ refers to any time before the subject reaches 35 years of age ( / .e. the subject may be age 34 years and 364 days). In some embodiments, the subject from which the oocyte is derived is a young subject. As used herein, a ‘young subject’ refers to a human female subject under the age of 35 years.

[0098] In some embodiments, the artificial follicles produced according to the methods described herein may be referred to as chimeric follicles. The term ‘chimeric follicles’ (or ‘chimeric ovarian follicle’) as used herein refers to follicles having follicle somatic cells (e.g. GCs) and an oocyte obtained from genetically nonidentical subjects. In some embodiments, provided herein are chimeric follicle(s) comprising an oocyte and somatic cells, wherein the somatic cells are obtained or obtainable from a GC suspension.

[0099] It will be appreciated that in some such embodiments, follicle somatic cells (e.g. GCs) may be “off the shelf’. In some embodiments, the follicle somatic cells are cells of a cell line. In some embodiments, the follicle somatic cells are cells which have been immortalised. In some embodiments, the follicle somatic cells are human or non-human mammal follicle somatic cells. In some embodiments, the follicle somatic cells are human follicle somatic cells. In some embodiments, the follicle somatic cells are GCs of a human granulosa cell line. Human granulosa cell lines include COV343 (Cellosaurus Accession: CVCL_2010), KGN (Cellosaurus Accession: CVCL_0375) and HGL5 (Cellosaurus Accession: CVCL_6644).

[0100] Oocytes

[0101] The methods described herein comprise providing a cell suspension comprising an oocyte and follicle somatic cells. Accordingly, the methods described herein comprise providing an oocyte. It will be appreciated that such cell suspensions may comprise more than one oocyte. An oocyte is a female germ cell which may undergo meiotic division to form an ovum.

[0102] The methods described herein may use any of primordial follicle oocytes, primary follicle oocytes, secondary follicle oocytes, pre-antral follicle oocytes or antral follicle oocytes. In some embodiments, the oocyte is a primordial follicle oocyte, primary follicle oocyte, secondary follicle oocyte, pre-antral follicle oocyte or antral follicle oocyte. It will be appreciated that the term ‘primordial follicle oocyte’ refers to an oocyte obtained from a primordial follicle, etc.

[0103] Primordial follicle oocytes are diploid and meiotically arrested in meiosis I. In some embodiments, the oocyte is an oocyte that has not yet completed meiosis I. For example, an oocyte which is meiotically arrested at the diplotene stage of prophase of meiosis I and may be characterized by the presence of an intact germinal vesicle (GV) (also called a primary oocyte). During oocyte maturation, oocytes re-enter meiosis and advance from prophase I to metaphase II of meiosis just prior to ovulation and subsequent fertilization. In some embodiments, the oocyte is a primordial follicle oocyte. Accordingly, in some embodiments, the oocyte is meiotically arrested at the diplotene stage of prophase of meiosis I. Primordial follicle oocytes may also be characterized by the presence of an intact GV. In some embodiments, the oocyte is characterized by the presence of an intact germinal vesicle. Primordial follicle oocytes may also be characterized by having a size of about 10 to 15 pM (see e.g. Lees-Murdock et al., Dev Biol. (2008) 1 ;321 (1):238-50, which is hereby incorporated by reference in its entirety). In some embodiments, the oocyte has a size of about 10 to 15 pM.

[0104] In some embodiments, the oocyte is a primary follicle oocyte. Primary follicle oocytes may also be characterized by having a size of about 16 to 55 pM. In some such embodiments, the oocyte has a size of about 16 to 55 pM.

[0105] In some embodiments, the oocyte is a secondary follicle oocyte. Secondary follicle oocytes may also be characterized by having a size of about 56 to 70 pM. In some such embodiments, the oocyte has a size of about 56 to 70 pM.

[0106] In some embodiments, the oocyte is an antral follicle oocyte. Antral follicle oocytes may also be characterized by having a size of about 70 pM. In some such embodiments, the oocyte has a size of about 70 pM.

[0107] As used herein, ‘obtaining’ an oocyte comprises retrieving an oocyte from an ovary. In some embodiments, the oocyte may be extracted in vitro e.g. by dissection of an ovary. In some embodiments, the oocyte may be extracted in vitro from a follicle which was previously obtained from a subject. In some embodiments, the oocyte may be obtained from an ovary in vivo e.g. by needle aspiration. Needle aspiration may comprise puncturing an individual follicle on an ovary and withdrawing a single oocyte through the needle. A vacuum source may be connected to the needle (e.g. via flexible tubing) to draw the oocyte through the needle into a container. In some embodiments, cell suspensions comprising an oocyte and follicle somatic cells for use in the methods described herein may comprise more than one oocyte, wherein the oocytes are obtained from follicles at a non-identical stage of folliculogenesis. In other words, a cell suspension comprising an oocyte and follicle somatic cells may contain any combination of primordial follicle oocyte, primary follicle oocyte, secondary follicle oocyte, pre-antral follicle oocyte or antral follicle oocytes. For example, in some embodiments, a cell suspension for use in the methods described herein comprises primordial follicle oocytes and primary follicle oocytes and follicle somatic cells.

[0108] In some embodiments, the methods described herein additionally comprise the step of obtaining an oocyte from a subject, e.g. a human or non-human mammalian subject. In some embodiments, the methods described herein additionally comprise the step of obtaining an oocyte from a human female subject. Methods of obtaining oocytes from human female subjects (sometimes called ‘egg collection’) are known to the skilled person. In some embodiments, the methods described herein additionally comprise the step of obtaining an oocyte from a human female subject in vivo e.g. by needle aspiration.

[0109] In some embodiments, the methods described herein additionally comprise the step of obtaining an oocyte from a subject prior to step (i) of the methods described herein.

[0110] In some embodiments, the methods described herein additionally comprise the step of obtaining a primordial follicle from a subject in vivo e.g. by needle aspiration. It will be appreciated that in such embodiments, methods may comprise obtaining more than one primordial follicle from the subject. In some embodiments, the method comprises an in vitro step of extracting an oocyte from a primordial follicle which was previously obtained from a subject.

[0111] In some embodiments, the oocyte was previously obtained from a subject e.g. a human or non-human mammalian subject. In some embodiments, the oocyte was previously obtained from a human female subject. In some embodiments, the oocyte was previously obtained from a human female subject in vivo e.g. by needle aspiration.

[0112] In some embodiments, an oocyte is provided which is obtained from a primordial follicle previously obtained from a subject. It will be appreciated that in such embodiments, more than one primordial follicle may have been previously obtained from the subject. In some such embodiments, the method comprises an in vitro step of extracting an oocyte from a primordial follicle which was previously obtained from a subject.

[0113] In some embodiments, the follicle somatic cells (e.g. GCs) and / or the oocyte may have been previously obtained from different subjects. In some cases, the artificial follicle may be subsequently returned to the subject from which the oocyte was derived. In some embodiments, the oocyte may be returned to the subject from which the oocyte was derived after fertilization, e.g. IVF.

[0114] In some embodiments, the subject from which the oocyte is derived is a human female over the age of 20 years. As used herein, the term ‘over the age of 20’ refers to any time after the subject reaches 20 years of age ( / .e. the subject may be age 20 years and 1 day). In some embodiments, the subject from which the oocyte is derived is a human female over the age of 25, 30, 35 or 40 years. In some embodiments, the subject from which the oocyte is derived is a human female over the age of 30 years. In some embodiments, the subject from which the oocyte is derived is a human female over the age of 35 years.

[0115] In some embodiments, the subject from which the oocyte is derived is a geriatric subject. As used herein, a ‘geriatric subject’ refers to a human female subject over the age of 35 years.

[0116] In some embodiments of the methods described herein, the oocyte was previously obtained from a human female subject over the age of 20 years (e.g. from a human female subject over the age of 25, 30, 35 or 40 years). In some embodiments, the methods described herein additionally comprise the step of obtaining an oocyte from a human female subject over the age of 20 years.

[0117] In some embodiments, the oocyte is an aged oocyte. The term ‘aged oocyte’ as used herein refers to an oocyte with reduced competence to develop into a mature oocyte. For example, an aged oocyte may be an oocyte derived from a geriatric subject. In some embodiments, an aged oocyte may be an oocyte derived from an aged follicle.

[0118] In some embodiments, the aged oocyte is an oocyte derived from a subject with a condition characterised by premature oocyte aging. Conditions characterised by premature oocyte aging may be associated with high serum FSH and are reviewed e.g. in Sukur et al., J Turk Ger Gynecol Assoc. (2014) 8; 15(3): 190-6, which is hereby incorporated by reference in its entirety. In some embodiments, the aged oocyte is an oocyte derived from a subject with Premature Ovarian Aging (POA) (also known as Early ovarian ageing (EOA)). EOA may be defined as <5 oocytes harvested in a minimum of two FSH-stimulated cycles ( / .e. standard FSH stimulation during an ART procedure). Normal ovarian ageing (NOA) may be defined as >8 oocytes in at least one FSH-stimulated cycle (see e.g. Christensen et al., Hum Reprod. (2020) 1 ;35(10):2375-2390, which is hereby incorporated by reference in its entirety).

[0119] Phenotypes that can be used to determine the competence of the oocyte to develop into a mature oocyte may comprise but are not limited to morphology of the spindles, alignment of the chromosomes, distribution of mitochondria, chromosome cohesion and level of ATP (see e.g. Hoshino. Reprod Med Biol. (2018) 27;17(4):434-441 , which is hereby incorporated by reference in its entirety).

[0120] The aged oocyte may display spindle abnormalities, chromosomes misalignment, abnormal mitochondria distribution, altered chromosome cohesion, an altered level of ATP, a low rate of successful fertilization and / or a low rate of successful post-fertilization development.

[0121] ‘Spindles’ as used herein refers to meiotic spindles which are microtubules that assemble in a bipolar fashion to segregate chromosomes in two distinct groups. Spindle abnormalities may comprise asymmetrically shaped spindles with more than two spindle poles. As used herein, ‘chromosomes misalignment’ refers to chromosomes not being aligned in the centre of the oocyte during metaphase (also sometimes referred to as ‘chromosomal misalignment on the metaphase plate’).

[0122] As used herein, ‘abnormal mitochondria distribution’ refers to aggregation of mitochondria in the cytoplasmic compartment of the oocyte. Aggregation of mitochondria may also interfere with spindle organization.

[0123] ‘Chromosome cohesion’ refers to the linkage between sister chromatids. As used herein, ‘altered chromosome cohesion’ refers to a decrease or absence of chromosome cohesion relative to an oocyte that is competent to develop into a mature oocyte.

[0124] As used herein, an ‘altered level of ATP’ refers to the level of ATP relative to an oocyte that is competent to develop into a mature oocyte. An aged oocyte may display a reduced level of ATP relative to an oocyte that is competent to develop into a mature oocyte.

[0125] Fertilization occurs when a sperm and an oocyte combine and their nuclei fuse, resulting in a zygote (fertilized oocyte). As used herein, fertilization encompasses both conventional insemination (during which sperm and mature oocytes are brought into contact) and intracytoplasmic sperm injection (ICSI). ICSI comprises direct injection of sperm into the cytoplasm of a mature oocyte. ‘Normal’ fertilization success rates during IVF are 70-80% (see e.g. Kahyaoglu et al., J Assist Reprod Genet. (2014) 31 (9):1155-60, which is hereby incorporated by reference in its entirety). Accordingly, in some embodiments a ‘low rate of successful fertilization’ (also referred to as a low fertilization rate) is successful fertilization of less than 70% of mature oocytes. In some embodiments, a low fertilization rate is a rate of successful fertilization less than 70%, less than 65% less than 60% less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%. In some embodiments, a low rate of successful fertilization is total fertilization failure (TFF), which is the failure of fertilization in all oocytes.

[0126] As used herein, ‘post-fertilization development’, also called embryo development, refers to the development of a single-celled zygote into an embryo, e.g. a cleavage stage embryo or a blastocyst. During early embryonic development, the zygote undergoes several rounds of division (via a 2-cell stage, 4-cell stage, 8-cell stage and 16-cell stage; during which it may be referred to as a cleavage stage embryo) to form a blastocyst. Embryonic development is reviewed e.g. in Mu et al., Fundam Res. (2022) 28;2(6):859-872, which is hereby incorporated by reference in its entirety. A blastocyst is an early embryonic structure which develops about 5- or 6-days post-fertilization. Blastocysts comprise an inner cell mass comprising stem cells which develops into the embryo, a fluid filled cavity and an outer layer of trophoblast cells which subsequently develops into the placenta. Approximately 50% of fertilized embryos successfully progress to the blastocyst stage during IVF. Accordingly, in some embodiments a ‘low rate of successful post-fertilization development’ is development of less than 50% of fertilized embryos to the blastocyst stage. In some embodiments, a low rate of successful post-fertilization development is development of less than 50% less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of fertilized embryos to the blastocyst stage.

[0127] In some embodiments, the oocyte is an aged oocyte which displays or is likely to display one or more of spindle abnormalities, chromosomes misalignment, abnormal mitochondria distribution, altered chromosome cohesion, an altered level of ATP, a low rate of successful fertilization and / or a low rate of successful post-fertilization development. An oocyte may be considered ‘likely to display’ one or more of these characteristics if other oocytes obtained from the same subject have been determined to display one or more of spindle abnormalities, chromosomes misalignment, abnormal mitochondria distribution, altered chromosome cohesion, an altered level of ATP, a low rate of successful fertilization and / or a low rate of successful post-fertilization development.

[0128] GDF9

[0129] Growth differentiation factor 9 (GDF9) is an oocyte-specific member of the TGF-p family. GDF9 is thought to be required for ovarian folliculogenesis and ovulation. The role of GDF9 is ovarian function is reviewed e.g. in Belli et al., Vitam Horm. (2018) 107:317-348, which is hereby incorporated by reference in its entirety.

[0130] In some embodiments, the methods described herein comprise culturing cells in the presence of GDF9. In the methods described herein, ‘culturing cells in the presence of GDF9’ may comprise culturing cells in cell culture medium comprising GDF9. In other words, GDF9 may be provided in cell culture medium comprising GDF9. Accordingly, in some embodiments, culturing cells in the presence of GDF9 comprises culturing cells in cell culture medium (e.g. cell culture medium as described hereinbelow) comprising GDF9 (e.g. recombinant human GDF9).

[0131] The GDF9 is preferably mammalian. In some embodiments, GDF9 is recombinant mammalian GDF9 (e.g. recombinant mouse, rat or sheep GDF9). In some embodiments, the GDF9 is human GDF9. In some embodiments, GDF9 is recombinant human GDF9 (also called ThGDF9’ or ‘rhGDF-9’). In other words, in some embodiments, GDF9 is a recombinant human GDF9 peptide. Recombinant human GDF9 is readily available to the skilled person (e.g. R&D Systems, Catalog number: 8266-G9).

[0132] In some embodiments, the methods described herein comprise culturing the cells of the cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte in the presence of GDF9. In the methods described herein, GDF9 may be provided in cell culture medium comprising GDF9. Accordingly, in some embodiments, culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 comprises culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in cell culture medium (e.g. cell culture medium as described hereinbelow) comprising GDF9 (e.g. recombinant human GDF9).

[0133] Formation of oocyte-follicle somatic cell complexes

[0134] Methods described herein comprise culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte (optionally in the presence of GDF9) to form an oocyte-follicle somatic cell complex. In other words, the methods comprise culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte under conditions suitable for formation of an oocyte-follicle somatic cell complex.

[0135] The term ‘oocyte-follicle somatic cell complex’ as used herein refers to aggregates of cells comprising an oocyte and follicle somatic cells. In other words, there is cell-to-cell contact between the oocyte and the follicle somatic cells. It will be appreciated that the term ‘oocyte-follicle somatic cell complex’ as used herein refers to oocyte-follicle somatic cell complexes comprising more than one follicle somatic cell.

[0136] In some embodiments, methods described herein comprise culturing the cells of the cell suspension comprising granulosa cells (GCs) and an oocyte (optionally in the presence of GDF9) to form an oocyte- GC complex. In other words, the methods comprise culturing the cells of the cell suspension comprising GCs and an oocyte under conditions suitable for formation of an oocyte-GC complex.

[0137] The term ‘oocyte-GC complex’ as used herein refers to aggregates of cells comprising an oocyte and GCs. In other words, there is cell-to-cell contact between the oocyte and the GCs. It will be appreciated that the term ‘oocyte-GC complex’ as used herein refers to oocyte-GC complexes comprising more than one GC.

[0138] In some embodiments, the methods described herein comprise culturing a cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte under conditions which promote cell aggregation. In other words, the conditions suitable for formation of an oocyte-follicle somatic cell complex are conditions which promote cell aggregation. In the methods described herein, conditions which promote cell aggregation are conditions which promote cell-to-cell contact and thus facilitate formation of cell aggregates. This ensures consistent and reproducible formation of oocyte-follicle somatic cellcomplexes, improving the reliability and efficiency of follicle creation.

[0139] It will be appreciated that ‘culturing the cell suspension’ (e.g. ‘culturing the cell suspension from step (i)’) involves culturing the cells of the cell suspension. It is not required that the cells are provided in suspension during the culturing step. In some embodiments, the method of the present disclosure comprises collecting / harvesting the follicle somatic cells (e.g. GCs) and the oocyte of the cell suspension, e.g. by centrifugation, prior to culturing the cells to form an oocyte-follicle somatic cell complex. Accordingly, in some embodiments, step (ii) comprises collecting the cells of the cell suspension from step (i) (e.g. by centrifugation), and culturing the cells of the cell suspension to form an oocyte-follicle somatic cell complex.

[0140] In some embodiments, conditions which promote cell aggregation may comprise culturing the cells of the cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte in the presence of a solid support which promotes cell aggregation. In other words, culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of a solid support having a 3D structure which facilitates aggregation of the follicle somatic cells and oocyte. As used herein, ‘culturing in the presence of a solid support’ encompasses applying the cells of the cell suspension to the solid support and culturing the cells as described herein. The solid support may be any solid support suitable for in vitrolex vivo cell culture. Suitable solid supports for use in such methods are well known to the skilled person. In some embodiments, a solid support may comprise or consist of polystyrene, polypropylene, polycarbonate, cyclo-olefin, glass or quartz. In some embodiments, a solid support may be a microwell plate (also known as a ‘microplate’, ‘microtiter’ or ‘multiwell’ plate) or microarray plate. In some embodiments, a solid support may be a bead, e.g. a magnetic bead.

[0141] In some embodiments, the solid support is a microwell plate. In other words, the solid support having a 3D structure which facilitates aggregation of the follicle somatic cells (e.g. GCs) and oocyte is a microwell plate. Methods for culturing, including selection of an appropriate microwell plate to facilitate cell aggregation are well known to the skilled person. For example, a round-bottomed (also called ‘U-shaped’ or half-sphere), conical (also called ‘V-shaped’), inverse pyramidal or ‘C-bottom’ microwell plate. In some embodiments, the solid support having a 3D structure which facilitates aggregation of the follicle somatic cells and oocyte is a round-bottomed (also called ‘U-shaped’ or half-sphere), conical (also called ‘V- shaped’), inverse pyramidal or ‘C-bottom’ microwell plate.

[0142] In some embodiments, the solid support having a 3D structure which facilitates aggregation of the follicle somatic cells and oocyte is a round-bottomed (also called ‘U-shaped’ or half-sphere) microwell plate. Round-bottomed microwell plates are known to the skilled person (e.g. ThermoFisher Scientific™ UK, Catalog number 163320 or Scientific Laboratory Supplies (SLS) UK, Catalog number 3788). In other words, in some embodiments, follicle somatic cells and an oocyte are cultured using a plate comprising round-bottomed microwells.

[0143] In some embodiments, the solid support having a 3D structure which facilitates aggregation of the follicle somatic cells and oocyte is an inverse pyramidal microwell plate (also called an ‘inverted’ pyramidal microwell plate). Inverse pyramidal microwell plates are microwell plate having inverse (or inverted) pyramidal shaped microwells and are known to the skilled person (see e.g. Cha et al., Biofabrication. (2017) 14;9(3):035006, which is hereby incorporated by reference in its entirety). In other words, in some embodiments follicle somatic cells and an oocyte are cultured using a plate comprising inverse pyramidal microwells.

[0144] In some embodiments, the inverse pyramidal microwell plate is an AggreWell™ plate. In some embodiments, the AggreWell™ plate comprises microwells that are 400 pm or 800 pm in size (e.g. STEMCELL technologies Catalog Number: 34411). Use of such plates allows for high-throughput production of artificial follicles that can be used for both research and clinical applications.

[0145] The cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte may be cultured under conditions which prevent cell-adhesion. In some embodiments the conditions which prevent cell-adhesion may comprise preparation of a solid support using an anti-adherence rinsing solution. An anti-adherence rinsing solution is a surfactant solution for pre-treating a solid support for cell culture to reduce surface tension and prevent cell adhesion. Selection of a suitable anti-adherence rinsing solutions is within the capabilities of the skilled person (e.g. STEMCELL technologies Catalog Number: 07010).

[0146] In some embodiments, the method of making an artificial follicle comprises the step of preparing the solid support using an anti-adherence rinsing solution. Accordingly, in some embodiments, the cell suspension comprising follicle somatic cells and an oocyte is cultured on a solid support which has been prepared using an anti-adherence rinsing solution prior to the culturing step.

[0147] As used herein, ‘preparing the solid support using an anti-adherence rinsing solution’ comprises applying the anti-adherence rinsing solution to the solid support. It will be appreciated that if the solid support is a microwell plate, ‘preparing the solid support using an anti-adherence rinsing solution’ may comprise applying the anti-adherence rinsing solution to each well of the microwell plate.

[0148] In some embodiments, preparing the solid support using an anti-adherence rinsing solution additionally comprises removing the anti-adherence rinsing solution (e.g. by aspiration) and washing the solid support with a buffer (e.g. by applying and removing the buffer). It will be appreciated that if the solid support is a microwell plate, each well of the microwell plate should be washed with the buffer. Suitable buffers compatible with cell culture methods are known by the skilled person and include e.g. NaCI (saline), TBS (tris-buffered saline) or PBS (phosphate-buffered saline). In some embodiments, the buffer is PBS.

[0149] In some embodiments, the methods disclosed herein comprise preparing a solid support using an antiadherence rinsing solution, and culturing the cells of the cell suspension of step (i) in the presence of the solid support that promotes cell aggregation, to form an oocyte-follicle somatic cell complex.

[0150] In some embodiments, the methods disclosed herein comprise culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 to form an oocyte-follicle somatic cell complex. In other words, the methods comprise culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 and under conditions suitable for formation of an oocyte-follicle somatic cell complex. In some embodiments, the methods disclosed herein comprise culturing the cells of the cell suspension comprising granulosa cells (GCs) and an oocyte in the presence of GDF9 to form an oocyte-GC complex. In other words, the methods comprise culturing the cells of the cell suspension comprising GCs and an oocyte in the presence of GDF9 and under conditions suitable for formation of an oocyte-GC complex.

[0151] In some embodiments, the methods disclosed herein comprise preparing a solid support using an antiadherence rinsing solution, and culturing the cells of the cell suspension of step (i) in the presence of the solid support that promotes cell aggregation and in the presence of GDF9, to form an oocyte-follicle somatic cell complex.

[0152] Cell Culture

[0153] Aspects of the present disclosure relate to methods comprising culturing cells / cell suspensions / cell complexes / follicles in vitro / ex vivo. “Culturing” refers to maintaining the cells / cell suspensions / cell complexes / follicles in an environment ( / .e. under suitable conditions) providing for their continued survival, growth and / or maturation / development.

[0154] Methods for culturing (including generating and / or expanding) populations of cells in vitro / ex vivo - including suitable culture conditions ( / .e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods etc. - are well known to the skilled person. For example, in vitro culture of human follicles and oocytes is reviewed in Yang et al., Front Endocrinol (Lausanne). 2020 Aug 11 ;11 :548, which is hereby incorporated by reference in its entirety.

[0155] It will be appreciated that the cells are cultured under suitable environmental conditions. The cells may be cultured at 28°C to 38°C, e.g. at one of about 28°C, about 28.5°C, about 29°C, about 29.5°C, about 30°C, about 30.5°C, about 31 °C, about 31 ,5°C, about 32°C, about 32.5°C, about 33°C, about 33.5°C, about 34°C, about 34.5°C, about 35°C, about 35.5°C, about 36°C, about 36.5°C, about 37°C, about 37.5°C or about 38°C. The cells may be cultured in 4% to 10% CO2, e.g. 5% to 8% CO2. The cells may be cultured at >90% humidity, e.g. about 95% humidity. The cells may be cultured without agitation, or with agitation. Agitation may be at 75 rpm to 175 rpm, e.g. 90 rpm to 130 rpm, e.g. about 110 rpm. The pH of the cell culture may be between 6.8 to 7.4, e.g. one of about 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3 or 7.4. In some embodiments, the pH of the cell culture is about 7.0. In some embodiments, the pH of the cell culture is about 7.2.

[0156] Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. Accordingly, in some embodiments the cell suspension comprising follicle somatic cells and an oocyte is cultured at, or at about, 37°C.

[0157] The skilled person is able to select suitable culture conditions including appropriate cell culture media. For example, the cell culture medium may be a complete growth medium. As used herein, a ‘complete growth medium’ refers to a culture medium that is enriched to contain all of the growth requirements of the cultured cells or aggregates of cells (e.g. follicle somatic cells and oocyte).

[0158] In some embodiments, the cell culture medium is a cell culture medium suitable for the culture of mammalian cells. Such cell culture media include aMEM (Minimum Essential Medium a), F-12 medium, Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's Modified Eagle Medium (DMEM), and DMEM / F12 medium.

[0159] In some embodiments, the cell culture medium comprises the GlutaMAX™ dipeptide (e.g. Catalog Number:35050061 , ThermoFisher Scientific, UK). In some embodiments, the cell culture medium comprises aMEM Glutamax. In some embodiments, the cell culture medium is a mixture of aMEM Glutamax and F-12 Glutamax.

[0160] In some embodiments, the cell culture medium (e.g. any of the cell culture mediums described herein) comprises follicle-stimulating hormone (FSH). In some embodiments, the cell culture medium comprises 10 mIU / ml to 150 mIU / ml FSH. In some embodiments, the cell culture medium comprises 50 mIU / ml to 120 mIU / ml FSH. In some embodiments, the cell culture medium comprises about 100 mIU / ml FSH. In some embodiments, the cell culture medium comprises 100 mIU / ml FSH.

[0161] In some embodiments, the cell culture medium (e.g. any of the cell culture mediums described herein) comprises fetal bovine serum (FBS). In some embodiments, the cell culture medium comprises 3% to 10% FBS. In some embodiments, the cell culture medium comprises about 5% FBS. In some embodiments, the cell culture medium comprises 5% FBS.

[0162] In some embodiments, the cell culture medium (e.g. any of the cell culture mediums described herein) comprises insulin. In some embodiments, the cell culture medium comprises 3 pg / ml to 7 pg / ml insulin. In some embodiments, the cell culture medium comprises 4 pg / ml to 6 pg / ml insulin. In some embodiments, the cell culture medium comprises about 5 pg / ml insulin. In some embodiments, the cell culture medium comprises 5 pg / ml insulin.

[0163] In some embodiments, the cell culture medium (e.g. any of the cell culture mediums described herein) comprises transferrin. In some embodiments, the cell culture medium comprises 3 pg / ml to 7 pg / ml transferrin. In some embodiments, the cell culture medium comprises 4 pg / ml to 6 pg / ml transferrin. In some embodiments, the cell culture medium comprises about 5 pg / ml transferrin. In some embodiments, the cell culture medium comprises 5 pg / ml transferrin.

[0164] In some embodiments, the cell culture medium (e.g. any of the cell culture mediums described herein) comprises selenium. In some embodiments, the cell culture medium comprises 3 pg / ml to 7 pg / ml selenium. In some embodiments, the cell culture medium comprises 4 pg / ml to 6 pg / ml selenium. In some embodiments, the cell culture medium comprises about 5 pg / ml selenium. In some embodiments, the cell culture medium comprises 5 pg / ml selenium.

[0165] In some embodiments, complete growth media for use in the methods described herein (e.g. culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte to form an oocyte-follicle somatic cell complex) comprises a mixture of aMEM Glutamax and F-12 Glutamax at a ratio of 1 :1 to 1 :0, supplemented with 5% FBS, 10 mIU / ml to 150 mIU / ml FSH, 5 pg / ml insulin, 5 pg / ml transferrin, 5 pg / ml selenium, and 50 ng / ml to 200 ng / ml GDF9.

[0166] In some embodiments, complete growth media for use in the methods described herein (e.g. culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte to form an oocyte-follicle somatic cell complex) comprises a mixture of aMEM Glutamax and F-12 Glutamax at a ratio of 1 :1 , supplemented with 5% FBS, 100 mIU / ml FSH, 5 pg / ml insulin, 5 pg / ml transferrin, 5 pg / ml selenium, and 100 ng / ml GDF9.

[0167] Culturing the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 The methods described herein comprise culturing the cells of the cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte in the presence of GDF9. In such embodiments, GDF9 may be provided in cell culture medium comprising GDF9. The GDF9 may be provided in the cell medium at an appropriate concentration for oocyte-follicle somatic cell complex to form.

[0168] In some embodiments, culturing the cells of the cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte in the presence of GDF9 comprises culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in cell culture medium comprising 50 ng / ml to 200 ng / ml GDF9. It will be appreciated that in such embodiments, the culture medium comprises GDF9 at a final concentration of 50 ng / ml to 200 ng / ml GDF9. In some embodiments, culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 comprises culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in cell culture medium comprising 75 ng / ml to 150 ng / ml GDF9. In some embodiments, culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 comprises culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in cell culture medium comprising about 100 ng / ml GDF9. In some embodiments, culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in the presence of GDF9 comprises culturing the cells of the cell suspension comprising follicle somatic cells and an oocyte in cell culture medium comprising 100 ng / ml GDF9.

[0169] In some embodiments, the cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte is cultured in the presence of GDF9 (e.g. under conditions which promote cell aggregation as described hereinabove) for an appropriate period of time for an oocyte-follicle somatic cell complex to form (e.g. 24, 36, 48 or 72 hours).

[0170] In some embodiments, the cell suspension comprising follicle somatic cells and an oocyte is cultured in the presence of GDF9 for 36 to 60 hours. In some embodiments, the cell suspension comprising follicle somatic cells and an oocyte is cultured in the presence of GDF9 for 40 to 56 hours. In some embodiments, the cell suspension comprising follicle somatic cells and an oocyte is cultured in the presence of GDF9 for 44 to 52 hours. In some embodiments, the cell suspension comprising follicle somatic cells and an oocyte is cultured in the presence of GDF9 for about 48 hours. In some embodiments, the cell suspension comprising follicle somatic cells and an oocyte is cultured in the presence of GDF9 for 48 hours.

[0171] Hydrogel Bead Culture

[0172] The methods described herein employ a hydrogel culture system. Hydrogels are three-dimensional, water-absorbing polymers with a gel-like consistency. The role of hydrogels in cell culture is reviewed e.g. in Caliari et al., Nat Methods. (2016) 13(5):405-14, which is hereby incorporated by reference in its entirety. Hydrogel beads are three-dimensional, water-absorbing polymers with a gel-like consistency and having a spherical shape. Hydrogel beads provide a controlled and supportive 3D matrix that enhances integrity and functionality of the artificial follicles produced by the methods described herein.

[0173] In some embodiments, the hydrogel beads comprise alginate. Alginate is a water-soluble polysaccharide found in brown algae. It is commonly used for cell culture and tissue engineering because of its biocompatibility, non-toxicity, and low immunogenicity. Alginate hydrogels and their use in 3D cell culture are reviewed e.g. in Andersen et al. Microarrays. (2015) 4(2):133-61 , which is hereby incorporated by reference in its entirety.

[0174] In some embodiments, the hydrogel beads comprise a basement membrane-derived preparation (also called a basement membrane extract). Such hydrogel beads may also be referred to herein as basement membrane-derived hydrogels. Basement membranes are thin sheets of extracellular matrix (ECM) that can provide a substrate for many cell types. The major molecular constituents of basement membranes are collagen IV, laminin-entactin / nidogen complexes and proteoglycans. The structure, assembly and cellular interactions of basement membrane proteins are reviewed e.g. in Paulsson. Crit Rev Biochem Mol Biol. (1992) 27(1-2):93-127, which is hereby incorporated by reference in its entirety. A ‘basement membrane-derived preparation’ as used herein refers to a preparation comprising one or more basement membrane constituent components. Accordingly, in some embodiments, the basement membrane- derived preparation comprises, or consists of, one or more of collagen, laminin, entactin and / or proteoglycan.

[0175] In some embodiments, the basement membrane-derived preparation comprises, or consists of, laminin and / or collagen. Accordingly, in some embodiments, the hydrogel beads comprise laminin. In some embodiments, the hydrogel beads comprise collagen e.g. collagen peptides). In some embodiments, the hydrogel beads comprise collagen IV.

[0176] Commercially available basement membrane-derived preparations are known in the art, for example ECM Gel Matrix (Catalog number:E1270; Sigma-Adrich) and Matrigel (Catalog number:354234; Corning, USA).

[0177] In some embodiments, the hydrogel beads comprise Matrigel. Matrigel (Catalog number:354234;

[0178] Corning, USA) is a basement membrane matrix preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcomas. The composition of Matrigel (also called “EHS matrix”) is variable, however the primary components are four major basement membrane ECM proteins: laminin (~60%), collagen IV (~30%), entactin (~8%) and the heparin sulfate proteoglycan perlecan (~2-3%).

[0179] Methods for producing hydrogel beads are known to the skilled person. Hydrogel beads for use in the methods described herein may be produced by mixing a hydrogel solution (e.g. a solution having a concentration of about 1 % w / v of the hydrogel) with suitable cell culture media on ice, followed by crosslinking. Salts including NaCI and CaCh may be used cross-link hydrogel beads.

[0180] In some embodiments, the hydrogel beads (i.e. the first or second hydrogel beads for use in the methods described herein) have a volume of 3 pl to 10 pl. In some embodiments, the hydrogel beads have a volume of 4 pl to 8 pl. In some embodiments, the hydrogel beads have a volume of 4 pl to 6 pl. In some embodiments, the hydrogel beads have a volume of about 5 pl. In some embodiments, the hydrogel beads have a volume of 5 pl. In some embodiments, a hydrogel bead (e.g. a 5 pl hydrogel bead) for use in the present methods may be crosslinked in a solution of CaCh and NaCI, for 2 to 5 minutes (e.g. about 3 minutes). In some embodiments, a hydrogel bead (e.g. a 5 pl hydrogel bead) for use in the present methods may be crosslinked in a solution of 50 mM CaCh and 140 mM NaCI, for 2 to 5 minutes (e.g. about 3 minutes).

[0181] The skilled person is able to select appropriate cell culture media for producing the first and second hydrogel beads. For example, the cell culture medium may be a cell culture medium suitable for the culture of mammalian cells as described hereinabove. In some embodiments, the cell culture medium may be complete growth medium as described hereinabove.

[0182] In some embodiments, the hydrogel beads comprise complete growth media, wherein the complete growth media comprises a mixture of aMEM Glutamax and F-12 Glutamax at a ratio of 1 :1 to 1 :0, supplemented with 5% FBS, 10 mIU / ml to 150 mIU / ml FSH, 5 pg / ml insulin, 5 pg / ml transferrin, 5 pg / ml selenium, and 50 ng / ml to 200 ng / ml GDF9. This growth medium can also be used in both the first hydrogel bead and second hydrogel bead follicle cultures.

[0183] In some such embodiments, the hydrogel beads comprise complete growth media, wherein the complete growth media comprises a mixture of aMEM Glutamax and F-12 Glutamax at a ratio of 1 :1 , supplemented with 5% FBS, 100 mIU / ml FSH, 5 pg / ml insulin, 5 pg / ml transferrin, 5 pg / ml selenium, and 100 ng / ml GDF9.

[0184] In some embodiments, the methods of producing antral follicles described herein employ a two-step hydrogel culture system, using first hydrogel beads for initial follicle formation, followed by second hydrogel beads for further maturation. Accordingly, in some embodiments, the methods comprise culturing the oocyte-follicle somatic cell complex in a first hydrogel bead to form an artificial follicle, and further comprise culturing the artificial follicle in a second hydrogel bead to form an antral stage follicle. This sequential culture system mimics the dynamic changes in the follicular environment during natural development, promoting optimal growth and maturation of the oocytes.

[0185] In such embodiments, the hydrogel concentration in the first hydrogel bead may be greater ( / .e. more concentrated) than the hydrogel concentration in the second hydrogel bead. For example, the concentration of the alginate and / or the basement membrane-derived preparation in the first hydrogel bead may be greater than the concentration of the alginate and / or the basement membrane-derived preparation in the second hydrogel bead, respectively. In some embodiments, the concentration of the basement membrane-derived preparation in the first hydrogel bead is greater than the concentration of the basement membrane-derived preparation in the second hydrogel bead.

[0186] First hydrogel bead culture

[0187] The methods described herein comprise culturing the oocyte-follicle somatic cell complex in a first hydrogel bead to produce an artificial follicle. In other words, an oocyte-follicle somatic cell complex produced by culturing a cell suspension comprising follicle somatic cells (e.g. GCs) and an oocyte is encapsulated in the first hydrogel bead and cultured to form an artificial follicle. In some embodiments, the methods described herein comprise culturing the oocyte-follicle somatic cell complex in a first hydrogel bead to produce an artificial follicle, wherein the artificial follicle is a primary follicle, secondary follicle or antral follicle. In some embodiments, the methods described herein comprise culturing the oocyte-follicle somatic cell complex in a first hydrogel bead to produce a secondary follicle.

[0188] In some embodiments, the first hydrogel bead comprises alginate. In some embodiments, the first hydrogel bead comprises 0.25% w / vto 1% w / v alginate. In some embodiments, the first hydrogel bead comprises about 0.25% w / v alginate. In some embodiments, the first hydrogel bead comprises 0.25% w / v alginate.

[0189] In some embodiments, the first hydrogel bead is a hydrogel comprising 1 mg / ml to 4 mg / ml of a basement membrane-derived preparation. In some embodiments, the first hydrogel bead is a hydrogel comprising 2 mg / ml of a basement membrane-derived preparation.

[0190] In some embodiments, the first hydrogel bead is a hydrogel comprising 1 mg / ml to 4 mg / ml of a basement membrane-derived preparation and 0.25% w / v to 1% w / v alginate. In some embodiments, the first hydrogel bead is a hydrogel comprising 1 mg / ml to 4 mg / ml of a basement membrane-derived preparation and about 0.25% w / v alginate. In some embodiments, the first hydrogel bead is a hydrogel comprising about 2 mg / ml of a basement membrane-derived preparation and 0.25% w / v to 1% w / v alginate. In some embodiments, the first hydrogel bead is a hydrogel comprising about 2 mg / ml of a basement membrane-derived preparation and about 0.25% w / v alginate.

[0191] In some embodiments, the first hydrogel bead is a hydrogel comprising 1 mg / ml to 4 mg / ml of Matrigel. In some embodiments, the first hydrogel bead is a hydrogel comprising 2 mg / ml of Matrigel.

[0192] In some embodiments, the first hydrogel bead is a hydrogel comprising 1 mg / ml to 4 mg / ml of Matrigel and 0.25% w / vto 1% w / v alginate. In some embodiments, the first hydrogel bead is a hydrogel comprising 1 mg / ml to 4 mg / ml of Matrigel and about 0.25% w / v alginate. In some embodiments, the first hydrogel bead is a hydrogel comprising about 2 mg / ml of Matrigel and 0.25% w / vto 1% w / v alginate. In some embodiments, the first hydrogel bead is a hydrogel comprising about 2 mg / ml of Matrigel and about 0.25% w / v alginate. In such embodiments, the first hydrogel bead may also be referred to herein as an ‘Alginate-Matrigel (high)’ bead.

[0193] In some embodiments, the oocyte-follicle somatic cell complex is cultured in a first hydrogel bead (e.g. in complete growth medium as described herein) for an appropriate period of time for an artificial follicle to form (e.g. 24, 36, 48 or 72 hours). In some embodiments, the oocyte-follicle somatic cell complex is cultured in a first hydrogel bead for 36 to 60 hours. In some embodiments, the oocyte-follicle somatic cell complex is cultured in a first hydrogel bead for 40 to 56 hours. In some embodiments, the oocyte-follicle somatic cell complex is cultured in a first hydrogel bead for 44 to 52 hours. In some embodiments, the oocyte-follicle somatic cell complex is cultured in a first hydrogel bead for about 48 hours. In some embodiments, the oocyte-follicle somatic cell complex is cultured in a first hydrogel bead for 48 hours. The skilled person is able to select appropriate cell culture media for culturing the oocyte-follicle somatic cell complex in the first hydrogel bead to form an artificial follicle. For example, the cell culture medium may be a cell culture medium suitable for the culture of mammalian cells as described hereinabove. In some embodiments, the cell culture medium may be complete growth medium as described hereinabove.

[0194] Second hydrogel bead culture

[0195] In some embodiments, the methods comprise further culturing the artificial follicle in a second hydrogel bead to form an antral stage follicle. In other words, the artificial follicle produced during the first hydrogel bead culture step is encapsulated in the second hydrogel bead and cultured to form an antral stage follicle. In some embodiments, the antral stage follicle is a graafian follicle. The second hydrogel bead may also be referred to herein as an ‘Alginate-Matrigel (low)’ bead.

[0196] Methods comprising culturing the artificial follicle in a second hydrogel bead to form an antral stage follicle may comprise the step of extracting the follicles from the first hydrogel bead. Methods of extracting or isolating cells or cellular aggregations from hydrogel beads are known to the skilled person. For example, cells or cellular aggregations may be extracted from a hydrogel bead using a cell recovery solution (e.g. Catalog Number:354253, Corning EU) or dissolution enzyme such as dispase or alginate lyase.

[0197] Accordingly, in some embodiments the method further comprises the step of extracting the follicles from the first hydrogel bead. In some embodiments the method further comprises the step of extracting the follicles from the first hydrogel bead using a dissolution enzyme. In some embodiments the method further comprises the step of extracting the follicles from the first hydrogel bead using alginate lyase.

[0198] In some embodiments, the second hydrogel bead comprises alginate. In some embodiments, the second hydrogel bead comprises 0.25% w / vto 1% w / v alginate. In some embodiments, the second hydrogel bead comprises about 0.25% w / v alginate. In some embodiments, the second hydrogel bead comprises 0.25% w / v alginate.

[0199] In some embodiments, the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation. In some embodiments, the second hydrogel bead is a hydrogel comprising 0.1 mg / ml of a basement membrane-derived preparation.

[0200] In some embodiments, the second hydrogel bead is a hydrogel comprising 0 mg / ml to 1 mg / ml of a basement membrane-derived preparation and 0.25% w / v to 1% w / v alginate. In some embodiments, the second hydrogel bead is a hydrogel comprising 0 mg / ml to 1 mg / ml of a basement membrane-derived preparation and about 0.25% w / v alginate. In some embodiments, the second hydrogel bead is a hydrogel comprising about 0.1 mg / ml of a basement membrane-derived preparation and 0.25% w / v to 1% w / v alginate. In some embodiments, the second hydrogel bead is a hydrogel comprising about 0.1 mg / ml of a basement membrane-derived preparation and about 0.25% w / v alginate. In such embodiments, when the second hydrogel bead comprises 0 mg / ml of a basement membrane-derived preparation, it may also be referred to as an ‘alginate bead’. In some embodiments, the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of Matrigel. In some embodiments, the second hydrogel bead is a hydrogel comprising 0.1 mg / ml of Matrigel.

[0201] In some embodiments, the second hydrogel bead is a hydrogel comprising 0 mg / ml to 1 mg / ml of Matrigel and 0.25% w / v to 1 % w / v alginate. In some embodiments, the second hydrogel bead is a hydrogel comprising 0 mg / ml to 1 mg / ml of Matrigel and about 0.25% w / v alginate. In some embodiments, the second hydrogel bead is a hydrogel comprising about 0.1 mg / ml of Matrigel and 0.25% w / v to 1 % w / v alginate. In some embodiments, the second hydrogel bead is a hydrogel comprising about 0.1 mg / ml of Matrigel and about 0.25% w / v alginate. In such embodiments, when the second hydrogel bead comprises 0 mg / ml of Matrigel, it may also be referred to as an ‘alginate bead’.

[0202] In some embodiments, the artificial follicle is cultured in a second hydrogel bead for an appropriate period of time for an antral stage follicle (e.g. a graafian follicle) to form (e.g. 72, 96, 120, 144 or 168 hours). In some embodiments, the artificial follicle is cultured in a second hydrogel bead for 4 to 7 days. In some embodiments, the artificial follicle is cultured in a second hydrogel bead for at least 5 days. As used herein, at least 5 days refers to any suitable period of time of 5 days or longer, for example 5 days, 5.5 days ( / .e. 5 days and about 12 hours) 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days or 10 days. In some embodiments, the artificial follicle is cultured in a second hydrogel bead for 5 to 6 days. In some embodiments, the artificial follicle is cultured in a second hydrogel bead for about 5 days or about 6 days. In some embodiments, the artificial follicle is cultured in a second hydrogel bead for 5 days or 6 days.

[0203] The skilled person is able to select appropriate cell culture media for culturing the artificial follicle in the second hydrogel bead to form an antral stage follicle. For example, the cell culture medium may be a cell culture medium suitable for the culture of mammalian cells as described hereinabove. In some embodiments, the cell culture medium may be complete growth medium as described hereinabove.

[0204] Further culture

[0205] The artificial follicle produced by the methods described herein may be further matured to obtain a mature oocyte. In some embodiments, the method further comprises culturing an artificial follicle produced by the methods described herein (e.g. an antral follicle produced by the methods described herein) to produce a graafian follicle (also called a mature ovarian follicle). It will be appreciated that a mature oocyte is produced during maturation of an antral (or graafian) follicle. Accordingly, methods of producing a graafian follicle described herein refer to methods of producing a follicle comprising a mature oocyte.

[0206] Methods for culturing follicles to produce a graafian follicle, including suitable culture conditions ( / .e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), culture periods etc. are well known to the skilled person. For example, in vitro culture of human follicles and oocytes is reviewed in Yang et al., Front Endocrinol (Lausanne). 2020 Aug 11 ;11 :548, which is hereby incorporated by reference in its entirety. In some embodiments, any of the methods described herein further comprise culturing the antral follicle (e.g. a graafian follicle) from step (iv) to obtain a mature oocyte.

[0207] In some embodiments, the method further comprises culturing an antral follicle (e.g. a graafian follicle) produced by the methods described herein in maturation medium. Accordingly, in some embodiments, the method further comprises culturing an antral follicle (e.g. a graafian follicle) produced by the methods described herein in maturation medium to obtain a mature oocyte.

[0208] In some embodiments, the maturation medium comprises human chorionic gonadotropin (hCG) (e.g. recombinant human chorionic gonadotropin; rhCG). hCG is a hormone produced by the placenta during pregnancy. rhCG is known to the skilled person (see e.g. Chang et al., Fertil Steril. (2001) 76(1):67-74, which is hereby incorporated by reference in its entirety). In some embodiments, the maturation medium comprises 0.5 to 5 lU / ml hCG. In some embodiments, the maturation medium comprises 1 to 3 lU / ml hCG. In some embodiments, the maturation medium comprises 1 to 2 lU / ml hCG. In some embodiments, the maturation medium comprises about 1.5 lU / ml hCG. In some embodiments, the maturation medium comprises 1.5 lU / ml hCG.

[0209] In some embodiments, the maturation medium comprises epidermal growth factor (EGF) (e.g. human EGF). EGF is a peptide which can be used in cell culture induce cell growth, proliferation and differentiation. In some embodiments, the maturation medium comprises 5 ng / ml to 15 ng / ml EGF. In some embodiments, the maturation medium comprises 7 ng / ml to 12 ng / ml EGF. In some embodiments, the maturation medium comprises about 10 ng / ml EGF. In some embodiments, the maturation medium comprises 10 ng / ml EGF.

[0210] In some embodiments, the maturation medium comprises follicle-stimulating hormone (FSH). In some embodiments, the maturation medium comprises 10 mIU / ml to 150 mIU / ml FSH. In some embodiments, the maturation medium comprises 50 mIU / ml to 120 mIU / ml FSH. In some embodiments, the maturation medium comprises about 100 mIU / ml FSH. In some embodiments, the maturation medium comprises 100 mIU / ml FSH.

[0211] In some embodiments, the maturation medium comprises fetal bovine serum (FBS). In some embodiments, the maturation medium comprises 3% to 15% FBS. In some embodiments, the maturation medium comprises about 10% FBS. In some embodiments, the maturation medium comprises 10% FBS.

[0212] In some embodiments, the maturation medium comprises aMEM Glutamax supplemented with: 3% to 15% FBS, 0.5 lU / ml to 5 lU / ml hCG, 5 ng / ml to 15 ng / ml EGF and 10 mlU / ml to 150 mIU / ml FSH. In some embodiments, the maturation medium comprises aMEM Glutamax supplemented with: 10% FBS, 1.5 lU / ml hCG, 10 ng / ml EGF and 100 mIU / ml FSH.

[0213] In some embodiments, the cell culture medium may be complete growth medium as described hereinabove. In some embodiments, the antral follicle is cultured (e.g. in maturation medium as described herein) for an appropriate period of time to obtain a mature oocyte (e.g. 14, 15, 16, 17 or 18 hours). In some embodiments, the antral follicle is cultured for 12 to 20 hours to obtain a mature oocyte. In some embodiments, step (v) comprises culturing the antral follicle for 12 to 20 hours. In some embodiments, the antral follicle is cultured for 14 to 18 hours to obtain a mature oocyte. In some embodiments, step (v) comprises culturing the antral follicle for 14 to 18 hours. In some embodiments, the antral follicle is cultured for about 16 hours to obtain a mature oocyte. In some embodiments, step (v) comprises culturing the antral follicle for about 16 hours. In some embodiments, the antral follicle is cultured for 16 hours to obtain a mature oocyte. In some embodiments, step (v) comprises culturing the antral follicle for 16 hours.

[0214] In some embodiments, the graafian follicle is cultured e.g. in maturation medium as described herein) for an appropriate period of time to obtain a mature oocyte (e.g. 14, 15, 16, 17 or 18 hours). In some embodiments, the graafian follicle is cultured for 12 to 20 hours to obtain a mature oocyte. In some embodiments, the graafian follicle is cultured for 14 to 18 hours to obtain a mature oocyte. In some embodiments, the graafian follicle is cultured for about 16 hours to obtain a mature oocyte. In some embodiments, the graafian follicle is cultured for 16 hours to obtain a mature oocyte.

[0215] It will be appreciated that the artificial follicles are cultured under suitable environmental conditions e.g. as described hereinabove. Conveniently, cultures of artificial follicles according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. Accordingly, in some embodiments artificial follicles are cultured at, or at about 37°C.

[0216] In some embodiments, the methods described herein comprise isolating a mature oocyte from a follicle. ‘Isolating a mature oocyte from a follicle’ as used herein refers to physically separating the oocyte from the follicle, and encompasses both ovulating an oocyte from a follicle and removing an oocyte from a follicle using needle aspiration.

[0217] In some embodiments, the methods described herein comprise isolating a mature oocyte from the graafian follicle. In some embodiments, the methods described herein comprise ovulating a mature oocyte from the graafian follicle. In some embodiments, the methods described herein comprise isolating a mature oocyte from the graafian follicle using needle aspiration.

[0218] In some embodiments, the isolated mature oocyte is subsequently used for in vitro fertilization. Methods of IVF are known to the skilled person and refer to a procedure where an egg is combined with sperm in vitro (i.e. outside the body).

[0219] Functional properties

[0220] Artificial follicles produced by methods described herein may be characterised by reference to certain functional properties.

[0221] In some embodiments, an artificial follicle produced by methods described herein may possess one or more of the following properties: having a three-dimensional structure, having a native follicle-like structure, and having the ability to support oocyte maturation.

[0222] In some embodiments, an oocyte grown in an artificial follicle produced by methods described herein may possess one or more of the following properties: exhibits normal extrusion of the polar body, exhibits normal spindle morphology, exhibits normal chromosome alignment at the metaphase plate, exhibits normal distribution of mitochondria, exhibits normal chromosome cohesion, exhibits a normal level of ATP, exhibits a normal fertilization rate, exhibits a normal post-fertilization development rate, exhibits a normal live birth rate, and exhibits a normal life birth rate post-IVF.

[0223] It will be appreciated that a given artificial follicle or oocyte may display more than one of the properties recited in the two preceding paragraphs.

[0224] Where the functional properties are compared (e.g. where the oocytes grown in an artificial follicle produced by methods of the present disclosure are compared with other oocytes that are competent to develop into a mature oocyte), comparisons are performed under equivalent conditions (e.g. using concentrations and / or quantity of the relevant agents).

[0225] A given artificial follicle or oocyte may be evaluated for the properties recited in the preceding paragraphs using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in nonhuman animals. In some embodiments, the assays may be e.g. ex vivo assays, i.e. performed using cells / tissue / an organ obtained from a subject. Such assays may be utilised to screen for artificial follicles or oocytes with a desired functional property. Such assays may comprise e.g. using antibodies against mitochondrial membrane proteins, tubulins, and histones to determine normal distribution and / or morphology of mitochondria, spindles and / or chromosomes.

[0226] In some embodiments, markers for cell components are used to determine normal distribution and / or morphology of mitochondria, spindles and / or chromosomes. In some embodiments, MitoTracker (catalog number: M22426, ThermoFisher) is used to as a mitochondrial marker. In some embodiments, SiR- Tubulin (e.g. catalog number: SC002, Spirochrome) is used to determine spindle morphology. In some embodiments, Hoechst stain (e.g. catalog number: H1399, ThermoFisher) is used as a chromosome marker. Methods of Rejuvenating an oocyte

[0227] Any of the methods of producing an artificial follicle described herein may be used in methods of rejuvenating an oocyte. Accordingly, disclosed herein are methods of rejuvenating an oocyte. The methods may be performed in vitro.

[0228] As used herein, the term ‘rejuvenating’ in the context of an oocyte refers to the process of promoting the development, growth and maturation of the oocyte, such that the oocyte has potential to develop into a viable embryo.

[0229] Accordingly, in vitro methods of rejuvenating an oocyte are provided, comprising the steps of:

[0230] (i) providing a cell suspension comprising an aged oocyte and follicle somatic cells (e.g. GCs),

[0231] (ii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex,

[0232] (iii) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle, and

[0233] (iv) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle.

[0234] In some embodiments, the aged oocyte may be an oocyte derived from a human female over the age of 20, 25, 30, 35 or 40 years. In some embodiments, the aged oocyte may be an oocyte derived from a human female over the age of 35 years. In some embodiments, the subject from which the oocyte is derived is a geriatric subject.

[0235] Phenotypes that can be used to determine the competence of the oocyte to develop into a mature oocyte may comprise but are not limited to: normal extrusion of the polar body, morphology of the spindles, alignment of the chromosomes, distribution of mitochondria, chromosome cohesion and level of ATP as described herein above.

[0236] Therapeutic applications

[0237] Methods of producing artificial follicles described herein may find therapeutic applications in treating infertility or may be used to improve fertility treatments. In other words, the methods of producing an artificial follicle described herein may be used in methods of treating infertility. The methods may be especially useful for treating subjects with a diminished ovarian reserve or poor oocyte quality.

[0238] The term ‘infertility’ as used herein refers to a condition where a person with a uterus is unable to get pregnant. In other words, it is a disease of the female reproductive system that is characterized by inability or reduced ability of the oocyte to be fertilized by male reproductive cells or the inability or reduced ability of the fertilized oocyte to grow and develop into a viable embryo. The methods of treating infertility described herein may also be referred to as methods of improving fertility. The term ‘fertile’ as used herein refers to the ability of the oocyte to be fertilized by male reproductive cells or the ability of the fertilized oocyte to grow and develop into a viable embryo.

[0239] Provided herein is a method of treating infertility or improving fertility, comprising obtaining an oocyte from a subject and generating an artificial follicle using any of the methods described herein.

[0240] In some embodiments, the method further comprises implanting the artificial follicle to treat infertility or improve fertility. As used herein, ‘implanting’ refers to inserting the artificial follicle into the body of a subject. For example, in some embodiments the artificial follicle is implanted into the body of a subject at a pelvic site such as the ovary, ovarian fossa, or broad ligament.

[0241] In some preferred embodiments, the method comprises implanting the artificial follicle into the same subject as the subject from which oocyte was obtained prior to production of the artificial follicle (also known as autogeneic). In other words, in some embodiments, the method further comprises implanting the artificial follicle into a genetically identical subject to the subject from which oocyte was obtained prior to production of the artificial follicle.

[0242] In such embodiments, the follicle somatic cells (e.g. GCs) are preferably allogeneic with respect to the oocyte. In other words, the follicle somatic cells may preferably be obtained from a subject who is genetically non-identical to the subject from which the oocyte is derived. It will be appreciated that in some such embodiments, follicle somatic cells may be “off the shelf’. In some embodiments, the follicle somatic cells are cells of a cell line.

[0243] Subjects

[0244] A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals ( / .e. veterinary use).

[0245] The subject may be a subject in need of such intervention (e.g. a subject who is infertile). The subject is preferably mammalian. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). The subject may be a female mammalian subject. The subject may be a non-human mammal, but is more preferably human. The subject may be female. The subject may be a patient.

[0246] A subject may have (e.g. may have been diagnosed with) a disease or condition described herein (e.g. infertility), may be suspected of having such a disease / condition, or may be at risk of developing such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease / condition. In some embodiments, the subject is a human female over the age of 20 years. As used herein, the term ‘over the age of 20’ refers to any time after the subject reaches 20 years of age ( / .e. the subject may be age 20 years and 1 day). In some embodiments, the subject is a human female over the age of 25, 30, 35 or 40 years. In some embodiments, the subject is a human female over the age of 30 years. In some embodiments, the subject is a human female over the age of 35 years.

[0247] In some embodiments, the subject is a ‘geriatric’ subject. As used herein, a ‘geriatric subject’ refers to a human female subject over the age of 35 years.

[0248] In some embodiments, the subject has a condition characterised by premature oocyte aging. Conditions characterised by premature oocyte aging may be associated with high serum FSH and are reviewed e.g. in Sukur et al., J Turk Ger Gynecol Assoc. (2014) 8; 15(3): 190-6, which is hereby incorporated by reference in its entirety. In some embodiments, the aged oocyte is an oocyte derived from a subject with Premature Ovarian Aging (POA) (also known as Early ovarian ageing (EOA)).

[0249] Kits

[0250] The present disclosure also provides kits of parts.

[0251] In some embodiments, the kit of parts may comprise follicle somatic cells (e.g. GCs) according to the present disclosure, and optionally materials for producing the artificial follicle. In some embodiments, the kit of parts may comprise a system for producing an artificial follicle according to the present disclosure. In some embodiments, the kit of parts may comprise a solid support which facilitates aggregation of the follicle somatic cells and oocyte. In some embodiments, the kit of parts may comprise components for production of hydrogel beads.

[0252] In some embodiments, the kit of parts further comprises reagents, buffers and / or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0253] The manufacture of kits of parts according to the present disclosure preferably follows standard procedures which are known to the person skilled in the art.

[0254] ***

[0255] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0256] The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described. Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0257] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0258] As used herein, a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A ‘polypeptide’ is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids. Reference herein to peptides, polypeptides and proteins also includes glycopeptides / glycopolypeptides / glycoproteins, lipopeptides / lipopolypeptides / lipoproteins, nucleopeptides / nucleopolypeptides / nucleoproteins, etc.

[0259] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0260] Values may be expressed herein as ‘about’ a particular value. Similarly, ranges may be expressed herein as from ‘about’ a particular value, and / or to ‘about’ another particular value. The term ‘about’ in relation to a numerical value is optional, and means for example + / - 10%. By way of illustration, reference e.g. to ‘about 10%’ is to be construed as 9% to 11%. In instances herein where ‘about’ is recited, the value it precedes is also specifically contemplated. By way of illustration, reference e.g. to ‘about 10%’ also specifically contemplates 10%.

[0261] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0262] Methods described herein may preferably be performed in vitro or ex vivo. The term ‘in vitro' is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms. Accordingly, in such embodiments the methods described herein are not a method performed on the human or animal body.

[0263] Brief Description of the Figures

[0264] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures. Figure 1. Schematic Illustration of the Novel Method to Produce Artificial Follicles to Support Oocyte Development. This figure provides a step-by-step visual representation of the process involved in creating artificial follicles from follicle somatic cell suspensions. It outlines the preparation of the AggreWell plate, the isolation and suspension of follicle somatic cells (FSCs), the transfer and culture of the FSC suspension, the formation of oocyte-FSC cell complexes, and the exchange of Alginate-Matrigel (high) hydrogel beads to Alginate-Matrigel (low) hydrogel beads. The final result is the development of functional antral stage artificial follicles capable of supporting oocyte growth and maturation.

[0265] Figure 2. Artificial follicles produced by the present methods. (A) The use of the AggreWell™400 plate, which features inverse pyramidal microwells with 400 microns in diameter, facilitates the production of oocyte-FSC (follicle somatic cell) complexes from FSC single cell suspensions. (B) Oocyte-FSC complexes collected from the AggreWell plate after 2 days of in vitro culture. (C) An example of an artificial follicle (left) after 2 days of 3D culture in an Alginate-Matrigel (high) hydrogel bead. This artificial follicle can develop into an antral stage follicle (right) after an additional 5 days of culture in an Alginate- Matrigel (low) hydrogel bead. (D) The final antral stage follicle diameter shows no significant difference between the artificial follicle and intact follicle after in vitro culture.

[0266] Figure 3: Comparison of Oocyte Maturation and Quality Between Artificial and Intact Follicles. (A) An example of an oocyte grown in an artificial follicle stained with mitochondrial (MitoTracker), spindle (SiR-Tubulin), and chromosome (Hoechst) markers. (B) Comparison of oocyte maturation rates between those grown in artificial follicles and those grown in intact follicles. (C) Normal spindle morphology rate in oocytes grown in artificial follicles compared to those in intact follicles. (D) Chromosome alignment rate in oocytes grown in artificial follicles versus intact follicles.

[0267] Examples

[0268] Example 1 : Production of functional Artificial Follicles

[0269] 1.1 Production of Artificial Follicles

[0270] 1.1.1 Preparation of AggreWell™ Plate:

[0271] 500 pl of anti-adherence rinsing solution was added to each well of an AggreWell™ Plate. The plate was centrifuged at 2,000 xg for 5 minutes and incubated for 30 minutes at room temperature. The rinsing solution was aspirated and each well was washed with 1 ml of PBS. After aspirating the PBS, 500 pl of complete growth medium with 100 ng / ml Recombinant Human GDF9 (1 :1 mixture of aMEM Glutamax and F-12 Glutamax, supplemented with 5% FBS, 100 mlU / ml follicle-stimulating hormone, 5 pg / ml insulin, 5 pg / ml transferrin, and 5 pg / ml selenium and 100 ng / ml GDF9 (R&D Systems, Catalog number: 8266- G9)) was added to each well.

[0272] 1.1.2 Preparation of Granulosa Cell (GC) Single Cell Suspension:

[0273] Ovaries were dissected from a 2-3 month-old mouse and follicles were isolated. 0.25% Trypsin was used to dissociate and disaggregate the follicles into a single cell suspension. The cells were transferred to a 1 ,5-ml tube and centrifuged at 200 x g for 5-10 minutes. The cell pellet was resuspended in 100 pl of prewarmed complete growth medium.

[0274] 1.1.3 Production of oocyte-GC complexes:

[0275] One hundred microlitres of the cell suspension from step 2 was transferred into one well of the prepared AggreWell™ plates. The plate was mixed well and centrifuged at 500 x g for 5 minutes. The plate was cultured at 37°C in 5% CO2 in air for 2 days. The oocytes and GCs formed oocyte-GC complexes spontaneously (Figure 2A and Figure 2B).

[0276] 1.1.4 Formation of artificial follicles:

[0277] An Alginate-Matrigel (high) hydrogel bead was prepared by mixing alginate stock solution (1% w / v) with cell culture media (final concentration of 0.25% w / v) and Matrigel (final concentration of 2 mg / ml) on ice. A 5 pl Alginate-Matrigel (high) hydrogel bead was crosslinked in a solution of 50 mM CaCh and 140 mM NaCI for 3 minutes.

[0278] After 2 days, the oocyte-GC complexes were collected from the AggreWell™ plate. One oocyte-GC complex was cultured in an Alginate-Matrigel (high) hydrogel bead for 2 days using complete growth medium. Artificial follicles formed spontaneously within these 2 days (Figure 2C).

[0279] 1.1.5 Exchange to Alginate-Matrigel (Low) Hydrogel Bead:

[0280] Follicles were extracted from the Alginate-Matrigel (high) hydrogel bead using 10 lU / mL of alginate lyase. The follicles were encapsulated into an Alginate-Matrigel (low) hydrogel bead (Alginate 0.25% w / v, Matrigel 0.1 mg / ml) and cultured for another 5-6 days. The artificial follicles developed into antral stage follicles (Figure 2C).

[0281] 1.1.6 Results and Conclusions

[0282] By applying the aforementioned protocol, artificial follicles were successfully produced from cell suspensions comprising GCs and oocytes. The created artificial follicles exhibit a native-like architecture with the oocyte centrally positioned and GCs uniformly surrounding it. No significant difference was found in antral stage follicle diameter between the artificial follicle and intact follicle after in vitro culture (Figure 2D).

[0283] 1 .2 Artificial follicles support oocyte growth and maturation

[0284] The functionality of these artificial follicles was also assessed. Over the culture period, the artificial follicles demonstrated the ability to grow and progress to the antral stage, which is a critical phase in follicular development. Notably, the oocytes within these antral stage artificial follicles exhibited normal maturation processes, including the extrusion of the polar body, an essential indicator of oocyte developmental competence.

[0285] Artificial follicles were stained with mitochondrial (MitoTracker), spindle (SiR-Tubulin), and chromosome (Hoechst) markers to assess functionality (Figure 3A). The oocytes grown in these artificial follicles exhibit normal spindle morphology and chromosome alignment at the metaphase plate, which are indicators of high-quality eggs (Figure 3A, 3C and 3D). No significant differences in oocyte maturation rates were observed between those grown in artificial follicles and those grown in intact follicles (Figure 3B). The ability of these artificial follicles to facilitate oocyte maturation and maintain oocyte quality underscores their potential utility in IVF.

[0286] In conclusion, this innovative method has demonstrated effectiveness in creating functional follicles from cell suspensions comprising follicle somatic cells and oocytes in vitro. These artificial follicles not only replicate the structural and functional characteristics of intact natural follicles but also support oocyte growth and maturation. The successful development of antral stage follicles and the maturation of oocytes highlight the potential of this method in reproductive research and clinical applications. This method holds significant promise for advancing assisted reproductive technologies (ART). It offers a novel approach to follicle generation that can be utilized in clinical settings, providing new avenues for treating infertility, particularly in cases involving aged oocytes or compromised ovarian function. The ability to produce functional artificial follicles using patient’s own oocyte and readily available GC suspensions could revolutionize fertility treatments and enhance the success rates of ART procedures.

[0287] The entire process is designed to be efficient and scalable, making it suitable for large-scale production of artificial follicles. The scalability of this method allows for its use in diverse settings, from small-scale laboratory research to large-scale clinical applications. It also facilitates the development of high- throughput screening methods for assessing the effects of various treatments on oocyte and follicle quality. Furthermore, this technique could be instrumental in research focused on understanding folliculogenesis and oocyte maturation, providing a reliable in vitro model for high- resolution observations of ovarian biology and developing new therapeutic interventions.

[0288] In summary, these results indicate that this innovative method of creating artificial follicles is a powerful tool that could have far-reaching implications for both basic research and clinical practice in the field of reproductive medicine.

[0289] Numbered statements

[0290] The following numbered paragraphs describe particular aspects and embodiments of the present disclosure:

[0291] 1 . A method for producing an artificial follicle, comprising:

[0292] (i) providing a cell suspension comprising granulosa cells and an oocyte,

[0293] (ii) culturing the cells of the cell suspension from step (i) to form an oocyte-granulosa cell complex, and

[0294] (iii) culturing the oocyte-granulosa cell complex from step (ii) in a first hydrogel bead to form an artificial follicle.

[0295] 2. The method according to statement 1 , wherein the culturing in step (ii) is performed under conditions promoting cell aggregation.

[0296] 3. The method according to statement 1 or statement 2, wherein step (ii) comprises culturing the cells of the cell suspension in the presence of a solid support that promotes cell aggregation, optionally wherein the solid support is an inverse pyramidal microwell plate.

[0297] 4. The method according to any one of statements 1 to 3, wherein the culturing in step (ii) is performed in the presence of Growth Differentiation Factor 9 (GDF9).

[0298] 5. The method according to any one of statements 1 to 4, wherein the first hydrogel bead is a hydrogel comprising 1 to 4 mg / ml of a basement membrane-derived preparation.

[0299] 6. The method according to any one of statements 1 to 5, wherein step (iii) comprises culturing the cells of the cell suspension for 36 to 60 hours.

[0300] 7. The method according to statement 6, wherein step (iii) comprises culturing the cells of the cell suspension for about 48 hours.

[0301] 8. The method according to any one of statements 1 to 7, wherein the method further comprises:

[0302] (iv) culturing the artificial follicle from step (iii) in a second hydrogel bead to form an antral follicle.

[0303] 9. The method according to statement 8, wherein the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation.

[0304] 10 The method according to statement 8 or statement 9, wherein step (iv) comprises culturing the artificial follicle for 5 or 6 days.

[0305] 11 . The method according to any one of statements 8 to 10, wherein the method further comprises:

[0306] (v) culturing the antral follicle from step (iv) to produce a graafian follicle. 12. The method according to statement 11 , wherein the method further comprises:

[0307] (vi) isolating a mature oocyte from the graafian follicle from step (v).

[0308] 13. The method according to any one of statements 1 to 12, wherein the oocyte is obtained from, or has been obtained from, a female mammalian subject.

[0309] 14. The method according to statement 13, wherein the subject is a human.

[0310] 15. The method according to statement 13 or statement 14, wherein the subject is over 35 years of age.

[0311] 16. An artificial follicle produced by the method according to any one of statements 1 to 15.

[0312] 17. An in vitro method of rejuvenating an oocyte comprising:

[0313] (i) providing a cell suspension comprising granulosa cells and an aged oocyte,

[0314] (ii) culturing the cells of the cell suspension from step (i) to form an oocyte-granulosa cell complex,

[0315] (iii) culturing the oocyte-granulosa cell complex from step (ii) in a first hydrogel bead to form an artificial follicle, and

[0316] (iv) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle.

[0317] 18. The method according to statement 17, wherein the culturing in step (ii) is performed under conditions promoting cell aggregation.

[0318] 19. The method according to statement 17 or statement 18, wherein step (ii) comprises culturing the cells of the cell suspension in the presence of a solid support that promotes cell aggregation, optionally wherein the solid support is an inverse pyramidal microwell plate.

[0319] 20. The method according to any one of statements 17 to 19, wherein the culturing in step (ii) is performed in the presence of Growth Differentiation Factor 9 (GDF9).

[0320] 21 . The method according to any one of statements 17 to 20, wherein the first hydrogel bead is a hydrogel comprising 1-4 mg / ml of a basement membrane-derived preparation.

[0321] 22. The method according to any one of statements 17 to 21 , wherein step (iii) comprises culturing the cells of the cell suspension for 36 to 60 hours.

[0322] 23. The method according to statement 22, wherein step (iii) comprises culturing the cells of the cell suspension for about 48 hours. 24. The method according to any one of statements 17 to 23, wherein the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation.

[0323] 25. The method according to any one of statements 17 to 24, wherein step (iv) comprises culturing the artificial follicle for 5 or 6 days.

[0324] 26. The method according to any one of statements 17 to 25, wherein the method further comprises:

[0325] (v) culturing the antral follicle from step (iv) to produce a graafian follicle.

[0326] 27. The method according to statement 26, wherein the method further comprises:

[0327] (vi) isolating a mature oocyte from the graafian follicle from step (v).

[0328] 28. The method according to any one of statements 17 to 27, wherein the aged oocyte is obtained from, or has been obtained from, a female mammalian subject.

[0329] 29. The method according to statement 28, wherein the subject is a human.

[0330] 30. The method according to statements 28 or 29, wherein the subject is over 35 years of age.

[0331] 31 . A chimeric follicle comprising an oocyte and somatic cells, wherein the somatic cells are obtained or obtainable from a granulosa cell suspension.

[0332] 32. A method of treating infertility or improving fertility, comprising:

[0333] (i) obtaining an oocyte from a subject,

[0334] (ii) producing a cell suspension comprising the oocyte of step (i) and granulosa cells,

[0335] (iii) culturing the cells of the cell suspension from step (i) to form an oocyte-granulosa cell complex,

[0336] (iv) culturing the oocyte-granulosa cell complex from step (ii) in a first hydrogel bead to form an artificial follicle,

[0337] (v) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle; and

[0338] (vi) a) implanting the follicle obtained in stage (v) to treat infertility or improve fertility, or b) culturing the antral follicle to produce a graafian follicle and isolating a mature oocyte from the graafian follicle. References

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Claims

Claims:1 . A method for producing an artificial follicle, comprising:(i) providing a cell suspension comprising follicle somatic cells and an oocyte,(ii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex, and(iii) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle.

2. The method according to claim 1 , wherein the culturing in step (ii) is performed under conditions promoting cell aggregation.

3. The method according to claim 1 or claim 2, wherein step (ii) comprises culturing the cells of the cell suspension in the presence of a solid support that promotes cell aggregation, optionally wherein the solid support is a round-bottomed microwell plate or an inverse pyramidal microwell plate.

4. The method according to any one of claims 1 to 3, wherein the culturing in step (ii) is performed in the presence of Growth Differentiation Factor 9 (GDF9).

5. The method according to any one of claims 1 to 4, wherein the first hydrogel bead is a hydrogel comprising 1 to 4 mg / ml of a basement membrane-derived preparation.

6. The method according to any one of claims 1 to 5, wherein step (iii) comprises culturing the cells of the cell suspension for 36 to 60 hours.

7. The method according to claim 6, wherein step (iii) comprises culturing the cells of the cell suspension for about 48 hours.

8. The method according to any one of claims 1 to 7, wherein the method further comprises:(iv) culturing the artificial follicle from step (iii) in a second hydrogel bead to form an antral follicle.

9. The method according to claim 8, wherein the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation.10 The method according to claim 8 or claim 9, wherein step (iv) comprises culturing the artificial follicle for 5 or 6 days.11 . The method according to any one of claims 8 to 10, wherein the method further comprises:(v) culturing the antral follicle from step (iv) to produce a graafian follicle.

12. The method according to claim 11 , wherein the method further comprises:(vi) isolating a mature oocyte from the graafian follicle from step (v).

13. The method according to any one of claims 1 to 12, wherein the oocyte is obtained from, or has been obtained from, a female mammalian subject.

14. The method according to claim 13, wherein the subject is a human.

15. The method according to claim 13 or claim 14, wherein the subject is over 35 years of age.

16. An artificial follicle produced by the method according to any one of claims 1 to 15.

17. An in vitro method of rejuvenating an oocyte comprising:(i) providing a cell suspension comprising follicle somatic cells and an aged oocyte,(ii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex,(iii) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle, and(iv) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle.

18. The method according to claim 17, wherein the culturing in step (ii) is performed under conditions promoting cell aggregation.

19. The method according to claim 17 or claim 18, wherein step (ii) comprises culturing the cells of the cell suspension in the presence of a solid support that promotes cell aggregation, optionally wherein the solid support is a round-bottomed microwell plate or an inverse pyramidal microwell plate.

20. The method according to any one of claims 17 to 19, wherein the culturing in step (ii) is performed in the presence of Growth Differentiation Factor 9 (GDF9).21 . The method according to any one of claims 17 to 20, wherein the first hydrogel bead is a hydrogel comprising 1-4 mg / ml of a basement membrane-derived preparation.

22. The method according to any one of claims 17 to 21 , wherein step (iii) comprises culturing the cells of the cell suspension for 36 to 60 hours.

23. The method according to claim 22, wherein step (iii) comprises culturing the cells of the cell suspension for about 48 hours.

24. The method according to any one of claims 17 to 23, wherein the second hydrogel bead is a hydrogel comprising 0 to 1 mg / ml of a basement membrane-derived preparation.

25. The method according to any one of claims 17 to 24, wherein step (iv) comprises culturing the artificial follicle for 5 or 6 days.

26. The method according to any one of claims 17 to 25, wherein the method further comprises:(v) culturing the antral follicle from step (iv) to produce a graafian follicle.

27. The method according to claim 26, wherein the method further comprises:(vi) isolating a mature oocyte from the graafian follicle from step (v).

28. The method according to any one of claims 17 to 27, wherein the aged oocyte is obtained from, or has been obtained from, a female mammalian subject.

29. The method according to claim 28, wherein the subject is a human.

30. The method according to claims 28 or 29, wherein the subject is over 35 years of age.31 . An artificial follicle comprising an oocyte and somatic cells, wherein the somatic cells are obtained or obtainable from a follicle somatic cell suspension.

32. A method of treating infertility or improving fertility, comprising:(i) obtaining an oocyte from a subject,(ii) producing a cell suspension comprising the oocyte of step (i) and follicle somatic cells,(iii) culturing the cells of the cell suspension from step (i) to form an oocyte-follicle somatic cell complex,(iv) culturing the oocyte-follicle somatic cell complex from step (ii) in a first hydrogel bead to form an artificial follicle,(v) culturing the artificial follicle of step (iii) in a second hydrogel bead to form an antral stage follicle; and(vi) a) implanting the follicle obtained in stage (v) to treat infertility or improve fertility, or b) culturing the antral follicle to produce a graafian follicle and isolating a mature oocyte from the graafian follicle.

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