WO2025247927A1 - Human fallopian tube organoids and uses thereof - Google Patents

Abstract

The present application relates to a mammal fallopian tube organoid within a cell-culture insert comprising a porous membrane supported by a frame, the cell-culture insert being accommodatable within a culture vessel to separate the culture vessel into two compartments providing an upper compartment and a lower compartment, and wherein the mammal fallopian tube organoid comprises at least ciliary cells and secretory cells, as well as uses and methods thereof.

Description

HUMAN FALLOPIAN TUBE ORGANOIDS AND USES THEREOF

[TECHNICAL FIELD]

[0001] The present disclosure relates to human fallopian tube organoids and cell-culture systems using human fallopian tube organoids, as well as methods and uses thereof. In particular, the present disclosure relates to human fallopian tube organoids within cell-culture inserts for gamete-cells culture and fertilization.

[BACKGROUND]

[0002] In humans, the fallopian tubes play a pivotal role in the processes essential for achieving pregnancy. Events such as gamete migration, interactions with tubal epithelium, oocyte fertilization, and embryo development occur within the fallopian tubes, which are anatomically divided into four segments: the intramural or intra-uterine segment, the isthmus, the ampulla, and the infundibulum or fimbriated end of the tube (Ng, et al., Hum Reprod Update 2018;24: 15-34). Following ovulation, where the cumulus-oocyte complex is captured in the infundibulum, and the ascent of spermatozoa along the tubal ampulla — site of fertilization — early embryo cleavages occur at the ampulla-isthmus junction and in the isthmus. Subsequently, processes such as cavitation, blastocyst expansion, and early embryo development occur primarily in the isthmus and the intramural segment (Croxatto, Reprod Biomed Online 2002;4: 160-169).

[0003] The tubal mucosa, recognized alongside cervical mucus, serves as one of two reservoirs for spermatozoa within the female reproductive tract (Holt et al., Mol Hum Reprod 2015 ;21 : 491-501). Its epithelium, through various functions performed by ciliated and secretory cells, facilitates sperm transport, nutrition, capacitation, and fertilization, as well as early embryo development and migration towards the uterus, up to the blastocyst stage (Croxatto, Reprod Biomed Online 2002;4: 160-169). The composition of tubal fluid, including proteins like lactoferrin, plays a crucial role in gamete interactions and embryo development (Gardner et al., Fertil Steril 1996;65: 349-353; Leese et al., Reproduction 2001 ; 121 : 339-346; Tay et al., Hum Reprod \ 997 \ 2: 2451-2456). Recent studies also highlightthe role of extracellular vesicles from tubal epithelium in sperm capacitation (Alminana et al., Theriogenology 2020;150: 59-69.; Alminana et al., BMC Genomics 2018; 19: 622.; Bathala et al., Mol Hum Reprod 2018;24: 143- 157.), suggesting a comprehensive understanding of tubal epithelium is essential for assessing the pre-implantation environment accurately. [0004] Despite being fundamental to reproductive function, fallopian tube research has been somewhat neglected since the advent of In Vitro Fertilisation (IVF). While IVF has addressed many cases of infertility, live birth rates from oocyte retrieval remain relatively low in Europe, ranging from 15% to 30% (Calhaz-Jorge, et al., Hum Reprod 2017;32: 1957-1973). Hence, there is a clear imperative to improve handling and culture conditions of gametes and preimplantation embryos during IVF, which remain suboptimal, through a deeper understanding of tubal biology.

[0005] The recent expansion of organoid models in medical research, facilitated by advancements in stem cell technology and 3D cultures, offers promising avenues for studying the female reproductive tract. More specifically, the development of the first human fallopian tube organoid models demonstrated the involvement of the Notch and Wnt signaling pathways in the growth and differentiation of these organoids (Kessler, et al., Nat Commun 2015;6: 8989). These fallopian tube organoid models have enabled studying various aspects including prolonged co-culture with pathogens, like Chlamydia trachomatis, and modeling ovarian serous carcinomas (Kessler et al., Nat Commun 2019;10: 1194; Yucer et al., Sci Rep 2017;7: 10741). Moreover, these organoids have shown physiological responses during hormonal modulation (Boretto et al., Development 2017;144: 1775-1786; Jung et al., Nat Commun 2017;8: 15680; Zhang et al., Nat Commun 2019;10: 5367).

[0006] However, at present, very few studies have looked at the development of female reproductive tract organoids for the study of reproductive functions. Organoid models emerge as leading tools for understanding the fallopian tube mechanism reproduction.

[0007] Therefore, there is a need to develop solutions for studying mammal reproductive functions, in particular in human.

[0008] There remains a need to develop novel solutions for providing a better understanding of the interactions between the tubal environment of a fallopian tube and the gametes and then the preimplantation embryos.

[0009] There remains a need to develop novel solutions for improving and/or maintaining the fertilizing capacity of mammal, in particular human, sperm and/or oocyte cells.

[0010] There remains a need to develop novel solutions for enhancing and/or maintaining the motility and vitality of mammal, in particular human, sperm cells.

[0011] There remains a need to develop novel solutions for improving the conditions for managing and culturing gametes and embryos during in vitro fertilization. [0012] There remains a need to develop novel solutions for improving the manipulation and culture of mammal, in particular human, gametes and preimplantation embryos during Assisted Reproductive Technique (ART).

[0013] There remains a need to develop novel solutions for improving in vitro fertilization, in particular for the fertilization capacity of mammal, in particular human, gametes, compared to current techniques.

[0014] There remains a need to develop novel solution for enhancing the quality of the preimplantation embryo.

[0015] There is also a need to develop novel solutions for producing extracellular vesicles from fallopian tube.

[0016] The present disclosure has for goal to satisfy all or part of those needs.

[SUMMARY]

[0017] According to one of its objects, the present disclosure relates to a mammal, in particular a human, fallopian tube organoid in (or within) a cell-culture insert comprising a porous membrane supported by a frame, the cell-culture insert being accommodatable within a culture vessel to separate the culture vessel into two compartments providing a upper compartment and a lower compartment, and wherein the mammal fallopian tube organoid comprises at least ciliary cells and secretory cells.

[0018] In some particular embodiments, the upper compartment and the lower compartment comprise a first cell-culture medium suitable for a mammal fallopian tube organoid.

[0019] As detailed in the example section, the inventors have surprisingly observed that it was possible to develop a reproductible model of human fallopian tube organoids within a cellculture insert successfully exhibiting different human fallopian tube cell types, a simple prismatic epithelium and an axonemal cilia structure. Specifically, this model of human fallopian tube organoids offers or enables easier access to the apical compartment of fallopian tube organoids. Such access to the apical compartment opens up new possibilities for utilizing fallopian tube organoids, particularly in studying gametes cells, the mechanisms of fertilization or preimplantation embryo development.

[0020] Subsequently, the inventors have observed that using this model of human fallopian tube organoids within a cell-culture insert as a gamete cell-culture system, significantly enhance the motility of sperm cells, as compared to the effectiveness of the current commercial medium for sperm fertilization, while maintaining a high vitality. This observation presents promising implications for ameliorate assisted reproductive technique, notably in vitro fertilization, by improving the competency of sperm cells and potentially favoring fertilization and preimplantation embryo development.

[0021] From the present examples, while the human fallopian tube organoids within a cell-culture inserts have shown to be promised for maintaining fertilizing sperm cells, it has been also observed that the retrieved apical supernatant of such developed human fallopian tube organoids within a cell-culture inserts exhibited good results in terms of sperm motility. Thus, the cell-culture system of the present disclosure may be used as bioreactor for producing extracellular vesicles that be used as additive components in conventional gamete and preimplantation embryo preparation and culture media during in vitro fertilization.

[0022] According to another of its objects, the present disclosure relates to a cellculture system for gamete cells, the cell-culture system comprising a mammal fallopian tube organoid comprising at least ciliary cells and secretory cells; a culture vessel; a first cell-culture medium suitable for a mammal fallopian tube organoid; and a cell-culture insert comprising a porous membrane supported by a frame, the cell culture insert being accommodatable within the culture vessel to separate the culture vessel into two compartments defined as an upper compartment and a lower compartment; and wherein the porous membrane supports, onto its upper surface, the mammal fallopian tube organoid.

[0023] In some embodiments, the first cell-culture medium can be comprised in the upper compartment and/or the lower compartment.

[0024] In some embodiments, the first cell-culture medium can be DMEM/Ham’s F12 or RPMI 1640.

[0025] In some particular embodiments, the first cell-culture medium can be DMEM/Ham’s F12.

[0026] In some embodiments, the first cell-culture medium can be supplemented with L-glutamine, a growth factor, Noggin, N-acetylcysteine, a ROCK inhibitor, a Wnt protein and Prostaglandin E2.

[0027] In some embodiments, the first cell-culture medium can be further supplemented with HEPES buffer, B27, N-acetylcysteine, Vitamin B3, and SB202190.

[0028] In some embodiments, the first cell-culture medium can be further supplemented with a differentiation fallopian tube cell factor, and a female steroid hormone.

[0029] In some embodiments, the first cell-culture medium can be further supplemented with Keratinocyte Growth Factor (KGF),WNT7a, estradiol and progesterone. [0030] In some embodiments, the porous membrane can be made of at least one material selected from polycarbonate, polyester (PET), collagen-coated polytetrafluoroethylene (PTFE) and a combination of collagen, fibronectin and laminin.

[0031] In some particular embodiments, the porous membrane can be a porous polycarbonate membrane.

[0032] In some embodiments, the porous membrane can have pores having an average pore size ranging from about 0.1 pm to about 50 pm,

[0033] In some particular embodiments, the porous membrane can have pores having an average pore size ranging from about 0.1 pm to about 7 pm.

[0034] In some particular embodiments, the porous membrane can have pores having a pore size of about 0.4 pm.

[0035] In some embodiments, the frame (4) of the cell-culture insert (2) can comprise a uniform wall, in particular a cylindric uniform wall, wherein the upper surface of the wall is open, and the lower surface of the wall is connected to the porous membrane (3).

[0036] In some embodiments, the mammal fallopian tube organoid originates from pluripotent stem cells (PSCs) or adult stem cells (ASCs).

[0037] In some particular embodiments, the mammal fallopian tube organoid originates from adult stem cells, in particular from adult stem cells isolated from isthmus and/or ampulla regions of one or more mammal fallopian tube tissue.

[0038] In some embodiments, the mammal fallopian tube organoid is a human fallopian tube organoid.

[0039] In some embodiments, the mammal fallopian tube organoid can be a non-human fallopian tube organoid.

[0040] In some embodiments, the cell-culture insert is a Transwell insert.

[0041] According to another of its objects, the present disclosure relates to an in vitro method for improving and/or maintaining the fertilizing capacity of mammal sperm cells, the method comprising at least the steps of:

(a) providing mammal sperm cells previously obtained from a male mammal subject,

(b) providing a mammal fallopian tube organoid as described herein, or a cell-culture system as described herein,

(c) introducing the mammal sperm cells, with a second cell-culture medium, within the upper compartment of the mammal fallopian tube organoid or of the cell-culture system, and

(d) incubating the mammal sperm cells under suitable conditions for mammal sperm cells. [0042] In some embodiments, the second suitable cell-culture medium at step (b) can be suitable for maintaining and/or improving the fertilizing capacity of mammal sperm cells.

[0043] In some embodiments, the second suitable cell-culture medium at step (b) can be suitable for fertilization.

[0044] In some embodiments, the second cell-culture medium can be identical to the first cell-culture medium.

[0045] In some embodiments, the second cell-culture medium can be a Minimum Essential Medium (MEM).

[0046] In some embodiments, before proceeding to step (c), the first cell-culture medium can be removed from the upper compartment and replaced with the second suitable cellculture medium.

[0047] In some embodiments, the mammal sperm cells at step (d) can be incubated for a time of about 12 hours to about 7 days.

[0048] In some particular embodiments, the mammal sperm cells at step (d) can be incubated for a time of about 3 days to about 5 days.

[0049] In some embodiments, the mammal sperm cells can be human sperm cells. In some embodiments, the mammal sperm cells can be non-human sperm cells.

[0050] According to another of its objects, the present disclosure relates to an in vitro fertilization method, the method comprising at least the steps of:

(a) providing at least one oocyte previously obtained from a female mammal subject and providing sperm cells previously obtained from a male mammal subject,

(b) introducing said oocyte and said sperm cells, with a second cell-culture medium suitable for fertilization, within a mammal fallopian tube organoid as described herein, or a cell-culture system as described herein,

(c) incubating the oocyte and the sperm cells under conditions suitable for the occurrence of fertilization, thereby obtaining a preimplantation embryo, and

(d) optionally, culturing the preimplantation embryo to a desired development stage before use or conservation.

[0051] According to another of its objects, the present disclosure relates to the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein for improving and/or maintaining the fertilizing capacity of mammal gamete cells.

[0052] In some embodiments, the mammal gamete cells are sperm cells and/or oocytes. [0053] According to another of its objects, the present disclosure relates to the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein for the development of a preimplantation embryo to a desired development stage.

[0054] According to another of its objects, the present disclosure relates to the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein for in vitro fertilization.

[0055] According to another of its objects, the present disclosure relates to the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein as a bioreactor for producing extracellular vesicles (EVs).

[0056] According to another of its objects, the present disclosure relates to a cellculture kit-of-part for improving and/or maintaining the fertilizing capacity of mammal gamete cell, the kit comprising at least:

(i) a mammal fallopian tube organoid within a cell-culture insert as described herein,

(ii) one or more mammal gamete cells,

(iii) a first cell-culture medium suitable for mammal fallopian tube organoid, and

(iv) a second cell-culture medium suitable for fertilizing mammal gamete cells.

[DESCRIPTION OF THE FIGURES]

[0057] FIGURE 1 depicts images of a patient’ fallopian tube tissue before cell culture. Left: Image of an ex-vivo human fallopian tube retrieved from patients; the ampulla and the isthmus are physically separated before mechanical and enzymatic treatments. The dot arrows show the segments treated by standard histological procedure. Scale bar = 3 cm. In the middle: Hemalun-eosine staining of the fallopian ampulla (top). Scale bar = 200 pm. Hemalun-eosine staining of the fallopian isthmus (bottom). Scale bar = 200 pm. Right: Scanning Electron Microscopy (SEM) of patient’s tissue from ampulla (top) and isthmus (bottom).

[0058] FIGURE 2 shows an illustration of a cell-culture insert (top image) and of a mammal fallopian tube organoid within a cell-culture insert of the present invention (bottom illustration). The bottom illustration shows a mammal fallopian tube organoid (1) within a cellculture insert (2) comprising a porous membrane (3) supported by a frame (4), the cell-culture insert (2) being accommodatable within a culture vessel (5) to separate the culture vessel into two compartments providing an upper compartment (6) and a lower compartment (7). Gamete cells (8) being easily drop-off and pick-up in the upper compartment (6).

[0059] FIGURE 3 shows human fallopian tube ampulla (HFTA) and human fallopian tube isthmus (HFTI) organoids cultures derived directly from patients’ cells. Figure 3A shows organoids culture of HFTA. Left: At passage n°0, day 5 (left). Scale bar = 40 pm. Centre: At passage n°l, day 24. Scale bar = 200 pm. Right: at passage n°l, day 24. Scale bar = 90 pm. Figure 3B shows organoids culture of HFTI. Left: At passage n°0, day 5 (left). Scale bar = 40 pm. Centre: At passage n°l, day 24. Scale bar = 200 pm. Right: at passage n°l, day 24. Scale bar = 90 pm.

[0060] FIGURE 4 depicts a boxplot showing organoids’ axis length at passage n°l, day 24 of to human fallopian tube ampulla (HFTA) and human fallopian tube isthmus (HFTI) organoids. Great axis of organoids was measured with an adjustment on number of organoid by well (linear regression). Abscissa (from left to right): Human fallopian tube isthmus (HFTI) organoids and Human fallopian tube ampulla. Ordinate: Organoid size in micrometer.

[0061] FIGURE 5 depicts electron microscopy images of Human Fallopian Tube Ampulla (HFTA) and Human Fallopian Tube Isthmus (HFTI) organoids. Figure 5A shows electron microscopy of a 24 days-grown HFTA organoids. Left: SEM image. Scale bar = 20 pm. Centre: TEM image; C = ciliated cell; S = secretory cell. Scale bar = 5 pm. Right: TEM image showing axonema structure of cilae. Scale bar = 2 pm. Figure 5B shows electron microscopy of a 24 days-grown HFTI organoids. Left: SEM image. Scale bar = 10 pm. Centre: TEM image; C = ciliated cell; S = secretory cell. Scale bar = 5 pm. Right: TEM image showing axonema structure of cilae. Scale bar = 2 pm.

[0062] FIGURE 6 illustrates the relative gene expression of eleven targeted genes, related to ciliary, secretory and others functions which characterize human fallopian tubes, in HFT patient tissue, HFT undifferentiated organoids, HFT differentiated organoids in Matrigeln and HFT organoids on Transwell®. Abscissa: Boxes for HFT ampulla organoid and HFT isthmus organoid, from left to right, (a) Human fallopian tube patient tissue, (b) Human fallopian tube undifferentiated organoids, (c) Human fallopian tube differentiated 3D organoids on Matrigel, (d) Human fallopian tube differentiated organoids on Transwell®. Ordinate: Relative gene expression of ARMC4 (Figure 6A), DNAI1 (Figure 6B), FOXJ1 (Figure 6C), SLC12A2 (Figure 6D), PAX8 (Figure 6E), LRRC6 (Figure 6F), OVGP1 (Figure 6G), ESRI (Figure 6H), LGR5 (Figure 61), PGRB (Figure 6J), and K167 (Figure 6K).

[0063] FIGURE 7 illustrates the sperm vitality and motility of sperm cells in culture within the following organoids or media: (1) Medium for human sperm fertilization (Universal IVF Medium, CooperSurgical®), (2): A differentiated medium supplemented with KGF, WNT7a, estradiol E2 and progesterone P4 used for HFT organoid culture, (3) a minimal medium MEM® (Gibsco), (4) The apical compartment of human colon organoids on Transwell inserts , (5) The apical compartment of HFT isthmus organoids on Transwell inserts, (6) The apical compartment of HFT ampulla organoids on Transwell inserts, (7) in suspension in tube within retrieved apical supernatants of HFT isthmus organoids after differentiation, and (8) in suspension in tube within retrieved apical supernatants of HFT ampulla organoids after differentiation . Figure 7A: Abscissa: Time of the sperm cell incubation in the different organoids or media ((1) to (8)), expressed in hours at 0, 48 h and 96 h. Ordinate: Sperm vitality expressed in percentage. Top of figure 7A represents a histogram at 48h and 96 h of the sperm vitality (%) relative to TO. Figure 7B: Abscissa: Time of the sperm cell incubation in the different organoids or media ((1) to (8)), expressed in hours at TO, 48h and 96h. Ordinate: Total sperm motility expressed in percentage. Top of figure 7B represents a histogram at 48h and 96h of total sperm motility (%) relative to TO. Figure 7C: Abscissa: Time of the sperm cell incubation in the different organoids or media ((1) to (8)), expressed in hours at TO, 48h and 96h. Ordinate: Progressive sperm motility expressed in percentage. Top of figure 7C represents a histogram at 48h and 96 h of progressive sperm motility (%) relative to TO. Bars are mean ± SEM.

[0064] FIGURE 8 illustrates the percentage of acrosome reacted spermatozoa (sperm cell) after induction with lonomycin (A23187) without EVs incubation (control) and after 6 hours of incubation with EVs from 10K and 100K centrifugation of segments of HFT tissue (left) or incubation with EVs from 10K and 100K centrifugation of segments of HFT organoids of the present invention (right). Segments of HFT: 1ST - Isthmus; AMP - Ampulla. Experiments were replicated 10 times (n = 10) using spermatozoa from different donors. *p < 0.05; ** p < 0.01

[DETAILED DESCRIPTION]

Definitions

[0065] The terms used in this specification generally have their ordinary meanings in the art. Certain terms are discussed below, or elsewhere in the present disclosure, to provide additional guidance in describing the products and methods of the presently disclosed subject matter.

[0066] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form.

[0067] The following definitions apply in the context of the present disclosure. [0068] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, “a cell” is understood to represent one or more cells. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0069] Furthermore, the term “and/or”, where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a sentence such as “A and/or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone).

[0070] Furthermore, the term "optionally, where used herein, is used to denote that an element or component place after the term optionally in the sentence is discretionary and may or may not be present within the described object or composition or combination. Thus, for instance, the term “optionally” as used in a sentence such as “A and, optionally B” herein is intended to denote that A is mandatory and B is discretionary and may or may not be present.

[0071] The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0072] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “including,” “consisting of,” and “consisting essentially of’ aspects and embodiments. The words “include” and “comprise,” or variations such as “includes,” “including,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of’ implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of’ implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the characteristic(s) of the stated elements. It is understood that the different embodiments of the disclosure using the term “comprising”, “including” or equivalents cover the embodiments where this term is replaced with “consisting of’ or “consisting essentially of’. [0073] Within the disclosure, the term “significantly” used with respect to a change intends to mean that the observed change is noticeable and/or it has a statistic meaning.

[0074] As used herein, the term “mammal” refers to a vertebrate animal of the class Mammalia within the phylum Chordata. Mammals are characterized by the presence of milkproducing mammary glands. Examples of mammals can include, without limitation, mouse, dog, cat, cow, sheep, pig, rabbit, chimpanzee or human. In some embodiments, a mammal is a nonhuman mammal. In some preferred embodiments , a mammal is a human.

[0075] As used herein, the term “organoid(s)” refers to an in vitro population of cells that mimics the structural and functional characteristics of a specific organ or tissue found in vivo. This population of cells typically comprises self-organizing cells derived from stem cells, such as from induced stem cells, embryonic stem cells or adult stem cell, and cultivated under controlled conditions to obtain the physiological behavior of the target organ or tissue. An organoid should satisfy at least one of the following criteria: containing a plurality of cell types of the target organ or tissue, and in vitro exhibiting one or more of the specific functions of the target organ or tissue. A mammal fallopian tube organoid as described herein can consist of a mammal fallopian tube tissue organoid. A mammal fallopian tube organoid as described herein can consist of a mammal fallopian tube epithelium organoid. In the present disclosure, a mammal fallopian tube organoid refers to an in vitro population of cells, in particular human cells, that mimics the structural and functional characteristics of a mammal fallopian tube epithelium. A mammal fallopian tube organoid as described herein can be obtained from stem cells, optionally adult stem cells, that self-organized and differentiated under controlled conditions within a culture medium.

[0076] As used, herein the term “accommodatable” refers to the capability of a system, device or structure, to be adjusted, configured, or modified to accommodate varying conditions, requirements, or preferences without necessitating significant structural changes or redesign. In particular, a cell-culture insert can be removed, adjusted or manipulated, and then replaced within a culture vessel.

[0077] As used herein, the term “adult stem cell” refers to a multipotent or unipotent cell found in various differentiated tissues and organs of mammal adult organism. Adult stem cells are capable of self-renewal and differentiation into one or more cell types within the same tissue or organ lineage. In the present disclosure, an adult stem cell originates from a fallopian tube tissue of a mammal adult organism, in particular from a human adult. Generally, adult stem cells are isolated and purified from a tissue before being cultured in vitro. [0078] As used herein, the term “pluripotent stem cells (PSCs)” refers to a type of undifferentiated cell having the capacity to differentiate into cell types of all three germ layers, namely ectoderm, endoderm, and mesoderm. A pluripotent stem cell can be an embryonic stem cell or an induced pluripotent stem cell. An embryonic stem cell originates from the inner cell mass of blastocysts. An induced pluripotent stem cell is artificially derived from a somatic non- pluripotent cell, such as an adult somatic cell, by a reprogramming process inducing a "forced" expression of certain genes. By introducing specific genetic factors, such as Oct4, Sox2, Klf4, and c-Myc, into somatic non-pluripotent cells, they can be induced to revert to a pluripotent state similar to embryonic stem cells and then be differentiated into cell types of all three germ layers.

Fallopian tube organoids within a cell-culture insert

[0079] The present disclosure relates to a mammal fallopian tube organoid within a cellculture insert.

[0080] As used herein, the term “fallopian tube organoid” (FTO) refers to a fallopian tube organoid within a cell-culture insert, unless otherwise described. Thus, “fallopian tube organoid” and “fallopian tube organoid within a cell-culture insert” can be used interchangeably.

[0081] In some embodiments, a mammal fallopian tube organoid can be a human fallopian tube organoid. In some embodiments, a mammal fallopian tube organoid can be a nonhuman fallopian tube organoid.

[0082] A mammal fallopian tube organoid as described herein comprises at least ciliary cells and secretory cells.

[0083] In some embodiments, the liver organoids disclosed here may present secretory and ciliary related gene relative expression, featured by expression of different biomarkers including ADM, MMP7, ELAFIN, SerpineA5, K167, IL8, ARMC4, DNAI1, LRC6 or Claudin 2.

[0084] A mammal fallopian tube organoid within a cell-culture insert as described herein can be prepared following the protocol described in the example section.

[0085] A mammal fallopian tube organoid as disclosed herein can be obtained from the differentiation of at least one pluripotent stem cell or adult stem cell.

[0086] In some embodiments, a mammal fallopian tube organoid as disclosed herein can originate from a plurality of adult stem cells. In particular, an adult stem cell is isolated from a fallopian tube tissue, preferably from isthmus or ampulla region, of an adult mammal organism, preferably an adult human. [0087] The process for obtaining a mammal fallopian tube organoid as disclosed herein implements a first cell-culture medium that can be suitable for growing and differentiating the pluripotent stem cell or adult stem cell into a fallopian tube organoid.

[0088] In some embodiments, the first cell-culture medium can be suitable for maintaining the viability of a fallopian tube organoid.

[0089] A skilled person will understand from common general knowledge the types of culture media that might be used for as the basal medium in the first cell culture medium to be used in the cell-culture insert or the cell-culture system or the methods as disclosed herein.

[0090] The cell culture media to be used in the different compartments of the cell-culture insert disclosed may be identical or different. Identical cell culture media may have the same basic components but may be supplemented with different supplements such as nutrients, cytokines, growth factors, hormones. Different cell culture media have different basic components. They may be supplemented with different or identical supplements. Different cell culture media may be used at different steps of the methods disclosed herein.

[0091] Potentially suitable cell culture media are available commercially, and include, but are not limited to, Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham’s F12, Advanced DMEM/Ham’s F12, Iscove’s Modified Dulbecco’s Media (IMDM), Ham's F-10, Ham’s F-12, Medium 199, RPMI 1640 Media, and Fallopian tube cell culture media.

[0092] In some embodiments, the first cell-culture medium can be MEM, DMEM, DMEM/Ham’s F12 or RPMI 1640.

[0093] A first cell-culture medium can be supplemented with free amino acids, a growth factor, a ROCK inhibitor, cytokines, and hormones suitable for the development of a fallopian tube organoid.

[0094] In some embodiments, a free amino acids can be selected from L-glutamine, L- arginine, L-lysine, L-leucine, L-methionine, L-histidine, L-cysteine, N-acetylcysteine and a combination thereof.

[0095] In some embodiments, a free amino acids can be L-glutamine and N- acetylcysteine.

[0096] In some embodiments, a growth factor can be selected from EGF, FGF-2, and/or VEGF. In some embodiments, a growth factor can be EGF.

[0097] In some embodiments, a cytokine can be selected from Noggin and/or a Wnt protein (R-spondin and/or Wnt3a). [0098] In some embodiments, a ROCK inhibitor can be Y-27632 and/or SB431542. In some embodiments, a ROCK inhibitor can be SB431542.

[0099] In some embodiments, a first cell-culture medium can be supplemented with free amino acids, a growth factor, Noggin, a ROCK inhibitor, R-spondin, Wnt3a, and Prostaglandin E2.

[00100] In some embodiments, a first cell-culture medium can be supplemented with L- glutamine, a growth factor, Noggin, a ROCK inhibitor, R-spondin, Wnt3a, and Prostaglandin E2.

[00101] In some embodiments, a first cell-culture medium can be supplemented with L- glutamine, EGF, Noggin, a ROCK inhibitor, R-spondin, Wnt3a, and Prostaglandin E2.

[00102] In some embodiments, a first cell-culture medium can be further supplemented with HEPES buffer, B27, N-acetylcysteine, Vitamin B3 (Nicotinamide), N2 and/or SB202190.

[00103] In some embodiments, a first cell-culture medium can be further supplemented with a penicillin/streptomycin solution.

[00104] In some embodiments, a first cell-culture medium can be supplemented with a differentiation fallopian tube cell factor and a female steroid hormone.

[00105] A differentiation fallopian tube cell factor can be selected from Keratinocyte Growth Factor (KGF) and/or WNT7a.

[00106] A female steroid hormone can be selected from estrogen (estradiol), and/or progesterone.

[00107] In some embodiments, a first cell-culture medium can be supplemented with Keratinocyte Growth Factor (KGF),WNT7a, estradiol and progesterone.

[00108] Illustratively, a first cell-culture medium as described herein can be a DMEM/F12 media supplemented with L-glutamine, HEPES buffer, N2, B27 minus vitamin A, a penicillin/streptomycin solution, EGF, Noggin, Wnt3a, N-acetylcysteine, Y-27632, A83-01, R-Spondin, nicotinamide, SB202190, Prostaglandin E2 (PGE2), KGF, WNT7a, estradiol and progesterone.

[00109] In some embodiments, a first cell-culture medium can be further supplemented with glucose.

Cell-culture insert

[00110] The fallopian tube organoid as described herein is specifically developed within a cell-culture insert. Cell-culture inserts are well known in the art and a skilled person in the art can select the appropriate one for implementing the present disclosure. [00111] A cell-culture insert as implemented herein comprises a porous membrane supported by a frame.

[00112] The frame of the cell-culture insert can comprise at least one side wall, in particular a uniform cylindrical side wall, wherein the upper surface of the side wall is open, and the lower surface of the side wall is connected to the porous membrane.

[00113] The cell-culture insert as described herein can be accommodatable within a culture vessel to separate the culture vessel into two compartments providing an upper compartment and a lower compartment.

[00114] The culture vessel comprises side walls and a solid base. In some embodiments, the side walls are in cylindrical. In some embodiments, the side walls can be a uniform cylindrical side wall.

[00115] The cell-culture insert is arranged within the culture vessel to form a gap, for containing a cell culture medium within the lower compartment (7), between the cell-culture insert and the side walls of the culture vessel which gap extents around the entire rim of the cellculture insert.

[00116] The cell-culture insert and the culture vessel are of similar shape, but the cross- sectional area of the cell-culture insert is smaller than that of the culture vessel, so that the cellculture insert may sit within the culture vessel, leaving the gap therebetween. In some embodiments, the gap can be of constant size around the cell-culture insert.

[00117] In some embodiments, the cell-culture insert can be a Transwell insert.

[00118] In some embodiments, the upper compartment and the lower compartment of the culture vessel as described herein can contain a first cell-culture medium as described herein.

[00119] In some embodiments, the porous membrane of the cell-culture insert can be made of at least one material selected from polycarbonate, polyester (PET), collagen-coated polytetrafluoroethylene (PTFE) and a combination of collagen, fibronectin and laminin.

[00120] In some embodiments, the porous membrane of the cell-culture insert can be made of polycarbonate.

[00121] In some embodiments, the porous membrane of the cell-culture insert can have pores having an average size ranging from about 0.1 pm to about 50 pm, or from about 0.1 pm to about 25 pm, or from about 0.1 pm to about 10 pm, or from about 0.1 pm to about 7 pm, or from about 0.1 pm to about 0.5 pm.

[00122] In some embodiments, the porous membrane of the cell-culture insert can have pores having an average size of about 1 pm, or about 2 pm, or about 3 pm, or about 4 pm, or about 5 pm, or about 6 pm, or about 7 pm, or about 8 pm. [00123] In some embodiments, the porous membrane of the cell-culture insert can have pores having an average size of about 0.1 pm, or about 0.2 pm, or about 0.3 pm, or about 0.4 pm, or about 0.5 pm, or about 0.6 pm, or about 0.7 pm, or about 0.8 pm, or about 0.9 pm, or about 1 pm.

[00124] In some embodiments, the porous membrane of the cell-culture insert can have pores having a size of about 0.4 pm.

[00125] In some embodiments, the porous membrane of the cell-culture insert can have a thickness ranging from about 5 pm to about 30 pm. In some embodiments, the porous membrane of the cell-culture insert can have a thickness ranging from about 7 pm to about 20 pm, or from about 8 pm to about 15 pm, or from about 9 pm to about 12 pm, or from about 9.5 pm to about 11 pm.

[00126] In some embodiments, the porous membrane of the cell-culture insert can have a thickness of about 10 pm.

[00127] In some embodiments, the porous membrane of the cell-culture insert can be made of polycarbonate, with a thickness of 10 pm, and comprising pores having a size of about 0.4 pm.

The cell-culture system

[00128] The present disclosure also relates to a cell-culture system for gamete cells.

[00129] The cell-culture system comprises:

- a mammal fallopian tube organoid (1) comprising at least ciliary cells and secretory cells,

- a culture vessel (5),

- a first cell-culture medium suitable for a mammal fallopian tube organoid, and

- a cell-culture insert (2) comprising a porous membrane (3) supported by a frame (4), the cell culture insert (2) being accommodatable within the culture vessel (5) to separate the culture vessel (5) into two compartments defined as an upper compartment (6) and a lower compartment (7); and wherein the porous membrane (3) supports, onto its upper surface, the mammal fallopian tube organoid (1).

[00130] In some embodiments, the first cell-culture medium can be contained in the upper compartment (6) and/or the lower compartment (7).

[00131] The mammal fallopian tube organoid, the culture vessel, the first cell-culture medium, and the cell-culture insert forming the cell-culture system are described thorough the present disclosure and are applicable for the cell-culture system of the present disclosure. Methods and used for improving and/or maintaining the fertilizing capacity of mammal sperm cells

[00132] The present disclosure also provided uses and methods implementing a mammal fallopian tube organoid as described herein or a cell-culture system as described herein.

[00133] In some embodiments, it is disclosed an in vitro method for improving and/or maintaining the fertilizing capacity of mammal sperm cells, the method comprising at least the steps of:

(a) providing mammal sperm cells previously obtained from a male mammal subject,

(b) providing or obtaining a mammal fallopian tube organoid within a cell-culture insert as described herein, or a cell-culture system as described herein,

(c) introducing the mammal sperm cells (8), with a second cell-culture medium, within the upper compartment (6) of the mammal fallopian tube organoid within a cell-culture insert; or within the upper compartment (6) of the cell-culture system, and

(d) incubating the mammal sperm cells under suitable conditions for mammal sperm cells.

[00134] In some embodiments, the in vitro method can be suitable for improving and/or maintaining the vitality and motility of sperm cells.

[00135] In some embodiments, a male mammal subject can be a human. In some embodiments, a male mammal subject can be a non-human.

[00136] In some embodiments, the mammal sperm cells can be human sperm cells. In some embodiments, the mammal sperm cells can be non-human sperm cells

[00137] In some embodiments, the second cell-culture medium can be suitable for maintaining and/or improving fertilizing mammal sperm cells. Otherwise said, in some embodiments, the second cell-culture medium can be suitable for maintaining and/or improving the fertilizing capacity of mammal sperm cells.

[00138] In some embodiments, the second cell-culture medium can be suitable for fertilization.

[00139] In some embodiments, the second cell-culture medium can be identical to the first cell-culture medium.

[00140] In some embodiments, the second cell-culture medium can be a Minimum Essential Medium (MEM). [00141] In some embodiments, the second cell-culture medium can be supplemented with growth factors, cytokines and/or hormones.

[00142] In some embodiments, before proceeding to step (c), the first cell-culture medium can be removed from the upper compartment and replaced with the second suitable cellculture medium within the upper compartment.

[00143] In some embodiments, the mammal sperm cells at step (d) can be incubated for a time of about 12 hours to about 7 days, or from about 2 days to about 6 days, or from about 3 days to about 5 days.

[00144] In some embodiments, the mammal sperm cells at step (d) can be incubated for a time of about 3 days, 4 days or 5 days.

[00145] In some embodiments, the mammal sperm cells are incubated under controlled conditions maintaining viability and motility of sperm cells preserving their fertilizing capacity. These conditions are well known in the art and thus can be adjusted by the skilled person in the art accordingly. For instance, parameters such as temperature, pH, duration, oxygen levels, and/or light exposure may require regulation to ensure optimal conditions. Generally, these conditions are adjusted to mimic the physiological environment of the cells in vivo.

[00146] In some embodiments, it is disclosed the use of a mammal fallopian tube organoid as described herein or the use of a cell-culture system as described herein for improving and/or maintaining fertilizing mammal gamete cells. Otherwise said, in some embodiments, it is disclosed the use of a mammal fallopian tube organoid as described herein or the use of a cellculture system as described herein for improving and/or maintaining the fertilizing capacity of mammal gamete cells.

[00147] In some embodiments, the mammal gamete cells are mammal sperm cells and/or mammal oocytes.

[00148] In some embodiments, the mammal sperm cells can be human sperm cells. In some embodiments, the mammal sperm cells can be non-human sperm cells.

[00149] In some embodiments, the mammal oocytes can be human oocytes. In some embodiments, the mammal oocytes can be non-human oocytes.

[00150] A mammal sperm cell can be characterized as “fertilizing” when the sperm cell typically has the capacity to interact and penetrate a female gamete cell, in particular an oocyte, to initiate the formation of an embryo. Typically, improving and/or maintaining the fertilizing capacity of mammal sperm cells induced that the sperm vitality, motility, capacitation and ability of sperm cells to make the acrosome reaction and to fertilize oocyte is improved and/or maintained. [00151] A mammal oocyte can be characterized as “fertilizing” when a sperm cell can penetrate the cell membrane of the oocyte and fuses with the oocyte's cytoplasm, to initiate the formation of an embryo.

In vitro fertilization methods and used

[00152] The present disclosure also provides in vitro fertilization methods and uses implementing a mammal fallopian tube organoid as described herein or a cell-culture system as described herein.

[00153] In some embodiments, it is disclosed the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein for in vitro fertilization.

[00154] The in vitro fertilization method comprises at least the steps of:

(a) providing at least one oocyte previously obtained from a female mammal subject and providing sperm cells previously obtained from a male mammal subject,

(b) introducing said oocyte and said sperm cells, with a second cell-culture medium suitable for fertilization, within a mammal fallopian tube organoid within a cell-culture insert as described herein, or a cell-culture system as described herein,

(c) incubating the oocyte and the sperm cells under conditions suitable for the occurrence of fertilization, thereby obtaining a preimplantation embryo.

[00155] The oocyte and the sperm cells are incubated under conditions, in particular chemical and physical conditions, suitable for the occurrence of fertilization. These conditions are well known in the art and thus can be adjusted by the skilled person in the art accordingly. For instance, parameters such as temperature, sperm concentration, culture media composition, pH, duration, oxygen levels, and/or light exposure may require regulation to ensure optimal conditions. Generally, these conditions are adjusted to mimic the physiological environment of the cells in vivo. Illustratively, the physical and chemical conditions suitable for the occurrence of fertilization are provided in Wai et al. Hum Reprod Update. 2016 Jan-Feb;22(l):2-22. Illustratively, the Revised guidelines for good practice in IVF laboratories (2015) provide recommendations in the organization and management of in vitro fertilization (De Los Santos et al. Hum Reprod. 2016 Apr;31(4):685-6.). In some embodiments, the method further comprises a step (d) of culturing the preimplantation embryo to a desired development stage before transfer into the uterus of a subject or conservation before the transfer. [00156] Typically, a preimplantation embryo is an embryo that has been created through in vitro fertilization (IVF) but has not yet been transferred into the uterus for further development.

[00157] In some embodiments, a desired development stage of a preimplantation embryo is a stage wherein the preimplantation embryo is suitable for transfer into the uterus of a subject. In some embodiments, a desired development stage of an embryo is the blastocyst stage.

[00158] In some embodiments, the preimplantation embryo can be conserved in a cryogenic environment before being transferred into the uterus of a subject.

[00159] In some embodiments, it is disclosed the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein for culturing a preimplantation embryo to a desired development stage.

[00160] Preimplantation embryos obtained with the methods of the present disclosure are not intended for industrial or commercial purposes.

Bioreactor

[00161] In some embodiments, it is disclosed the use of a mammal fallopian tube organoid as described herein or of a cell-culture system as described herein as a bioreactor for producing extracellular vesicles (EVs).

[00162] As used herein, a “bioreactor” is a culture cell system designed to mimic the physiological environment of one or more cell lines to support cell growth and to stimulate the release of extracellular vesicles from the cells.

[00163] Extracellular vesicles from fallopian tube organoid can be used as additive components in cell culture media for conventional in vitro gamete and preimplantation embryo development during in vitro fertilization (Li Y, et al. Hum Reprod Open. 2023 Feb 21 ;2023(2)).

[00164] In some embodiments, the extracellular vesicles can be proteins, RNAs, and/or lipids.

[00165] Technics to isolate extracellular vesicles from the luminal fluid generated by a fallopian tube organoid are well known by the skilled person. Illustratively, extracellular vesicles can be isolated using the ultracentrifugation method described previously in Lopera-Vasquez R, Hamdi M, Femandez-Fuertes B, Maillo V, Beltran-B renaP, Calle A, Redruello A, Lopez-Martin S, Gutierrez- Adan A, Yanez-M6 M. et al. Extracellular vesicles from BOEC in in vitro embryo development and quality. PLoS One 2016;2:e0148083.; Mazzarella R, Bastos NM, Bridi A, Del Collado M, Andrade GM, Pinzon J, Prado CM, Silva LA, Meirelles FV, Pugliesi G. et al. Changes in oviductal cells and small extracellular vesicles miRNAs in pregnant cows. Front Vet Sci 2021;8:639752. ).

Kit of parts

[00166] The present disclosure also relates to kit-of-parts for improving and/or maintaining the fertilizing capacity of mammal gamete cells, the kit comprising at least:

(i) a mammal fallopian tube organoid within a cell-culture insert as described herein,

(ii) one or more mammal gamete cells,

(iii) a first cell-culture medium suitable for mammal fallopian tube organoid, and

(iv) a second cell-culture medium suitable for mammal gamete cells.

[00167] In some embodiments, the kit-of-parts further comprises a set of instructions for improving and/or maintaining the fertilizing capacity of mammal gamete cells.

[00168] The present disclosure also relates to kit-of-parts for in vitro fertilization, the kit comprising at least:

(i) a mammal fallopian tube organoid within a cell-culture insert as described herein,

(ii) one or more mammal gamete cells,

(iii) a first cell-culture medium suitable for mammal fallopian tube organoid, and

(iv) a second cell-culture medium suitable for mammal gamete cells.

[00169] In some embodiments, the kit-of-parts further comprises a set of instructions for in vitro fertilization.

[00170] All definitions and embodiments provided in the present disclosure are intended to apply to all systems, methods, and uses described herein.

[00171] Without limiting the present disclosure, examples are described below for the purpose of illustration.

[EXAMPLES]

EXAMPLE 1

[00172] In the present examples, the development of a human fallopian tube organoid model is demonstrated, which has proven successful in its application as an in vitro culture system for improving and/or maintaining the vitality and motility of human spermatozoa.

PART 1: MA TERIALS AND METHODS

Fallopian Tube tissue collection

[00173] Fallopian tubes were collected from 10 patients undergoing bilateral salpingectomy by laparoscopy for contraceptive sterilization. Patients signed an inform consent for collecting tissue for research purposes. Patients were aged from 33 to 41 years. All of them have at least one child obtained by natural conception and have no previous significant medical and chirurgical history nor fertility treatment.

Human fallopian tube organoid culture

[00174] Fresh fallopian tubes tissues (N=10) (Figure 1) were kept for few minutes in Leibovitz medium (L-15 Sigma Aldrich) at room temperature (RT). A 15mm lengh section was cut from proximal region (isthmus) and from distal region (ampulla). A little part of these sections was kept for conventional histology and the other parts for further treatment. On the contralateral tube, mucosa from the same sections (ampulla and isthmus) was scratched for transcriptomic analysis and whole tissue kept for MEB/MET analysis for control conditions.

[00175] The rest of the section tissues was digested with a collagenase digestion A or I (2,5 mg/ml) +/- Dispase II (Roche l,2U/ml) at 37°C on a hot block with agitation for 40 minutes. After one washing 600G for 10 min, the tissues are subsequently digested by Trypsine (0.25%) / EDTA for 10 min at 37°c. Then, the cells are washed with PBS BSA 3% and filtrated on 70pM filter. The pellet was suspended in 1 ml PBS. Then cell count was performed after blue Tryptan staining. Fifteen thousand cells for 25 pL were embedded in hESC-qualified matrix (Matrigel, Coming) on ice and seeded in 48 wells plates (Greiner). The Matrigel was incubated for polymerization for 20 minutes at 37 °C, and was supplemented with 250 pl/well of complete medium A (advanced DMEM/F12, 2mM Glutamax (L-glutamine), 10 mM hepes, IX N2 (Invitrogen), IX B27 minus vitamin A (Life technologies), IX penicillin/streptomycin solution (Invitrogen), 50 ng/ml human EGF (Gibco), 25 ng/ml human noggin (Peprotech), 0.5 ng/ml human Wnt3a (R&D systems), 1.25 mM N-acetylcysteine (Sigma Aldrich), lOpM Y-27632 (Axon MedChem), 0.5pM A83-01 (Axon MedChem), 0.5 pg/ml human R-spondin (R&D systems), 10 mM nicotinamide (Sigma Aldrich), 10 pM SB202190 (Sigma Aldrich), 0.01 pM PGE2 (Sigma Aldrich)).

[00176] Medium was changed every other day with complete medium A and culture was maintained in a humidified incubator at 37°C under 5% CO2 and 95% air atmosphere for an average of 10 days.

[00177] After a cell passage at day 10, using 300pL of TrypLE (ThermoFisher Scientific) by well, pipetting and washing by centrifugation, cells was seeded for one part in 48-wells plate on Matrigel as the primary culture and for the other part in a thin layer of Matrigel (polycarbonate membrane) on Transwell device (Coming) (Figure 2).

[00178] After passage and seeding, organoids that were in 48-wells plate and in Transwell® were cultured in the proliferation medium A (whose composition is described above) during 10 days.

[00179] Then, differentiation was gradually induced adding 5ng/ml KGF (Peprotech), lOng/ml WNT7a (Peprotech), 0.2 ng/ml estradiol E2 (Sigma), and lOng/ml progesterone P4 (Sigma).

Immunofluorescence Labeling for organoid and Fallopian tissue

[00180] Fallopian Tube organoids (in 96 wells plate) were fixed with 4% formaldehyde (FA, Sigma Aldrich) in Hank’s Buffered Salt Solution (HBSS, Gibco) at 37°C for 20 min. Organoids were permeabilized with 1% triton X-100 (Sigma Aldrich) in HBSS at RT for 40 min and were incubated with a blocking buffer containing 1% triton X-100 (Sigma Aldrich) and 3% bovine serum albumin (BSA, Sigma Aldrich) 1 h at 37°C. Fallopian Tube organoids were incubated overnight at RT with primary antibodies and Ih at 37°C.

[00181] Next, organoids were incubated with secondary antibodies 1 h at 37°C and then stained for 20 min with DAPI (Invitrogen) and Phalloidin. Finally, slides were counted with Vectashield mounting medium (VectoLaboratories).

[00182] The percentage of cells with positive labelling for Ki67, PAX8 and Tub Detyr, relative to the number of DAPI nucleic, was calculated for isthmus and ampulla organoids.

[00183] Analytical parameters and threshold settings were the same for isthmus and ampulla in each patient. A paired analysis was performed to compare isthmus and ampulla within HFT organoids.

Primary Antibodies [00184] Mouse Monoclonal a-E-Cadherin (BD Biosciences, Cat. No. 610181, 1:200) [00185] Rabbit polyclonal a-detyrosinated tubulin (Abeam, Cat. No. 48389, 1:100) [00186] Rabbit polyclonal a-Pax8 (Proteintech, Cat. No. 10336-1-AP, 1: 100) [00187] Rabbit polyclonal a-Ki 67 (Cell Signaling, Cat. No. 9027, 1:100) [00188] Rat monoclonal a-CD49f (Biorad MCA699GA, 1 :400)

[00189] Mouse monoclonal a-acetyl-alpha tubulin, clone 6-1 IB (Sigma, Cat. No. 6-11- Bl, ref MABT868 1:100)

[00190] Rabbit monoclonal a-Era SP1 (Abeam abl6660 1:200).

Secondary antibody

[00191] Chicken a-Rat 488 (A-21470 Invitrogen 1:1000)

[00192] Chicken a-Rabbit 647 (pour PAX8, pour LGR6, pour Tubuline) (A-21443 Invitrogen 1 :500)

[00193] Donkey a-Mouse 568 (A10037 Invitrogen 1 :500).

Transmitted light microscopy for organoid observation and morphometry

[00194] The images of Fallopian Tube organoids were taken by inverted microscopy for brightfield observation (Nikon Eclipse) or by confocal microscopy with Opera Phenix. Harmony software was used to process and analyze images.

Transmission and Scanning Electronic Microscopy

[00195] Fallopian Tube organoids grown in a drop of Matrigel were fixed in 2 % glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4) for 4 h at 4°C and washed overnight in 0.2 M phosphate buffer. They were post-fixed in 1% osmium tetroxide in 250mM saccharose and 0.05 M phosphate buffer for 1 h in the dark at room temperature (RT). The samples were then dehydrated in a series of graded ethanol solutions, up to 100% ethanol. From then, organoids wells were separated in two for both techniques.

[00196] For SEM, bigger organoids were cut in two with a micro dissecting knife, still in 100% ethanol. They were then dried by critical point drying with a Leica EM CPD 300. The samples were coated with 6 nm Platinium on a Leica EM Med 020 before being examined on a FEI Quanta 250 FEG scanning electron microscope, at an accelerating voltage of 5 kV.

[00197] For TEM, after further dehydration in acetone and an acetone-resin mix, the organoids were embedded in Embed 812 (Electron Microscopy Sciences) according to current commercial protocols. Finally, the tissues were sliced into 70-nm thick sections (Ultracut Reichert Jung) and mounted on 100-mesh collodion-coated copper grids prior to staining with 3% uranyle acetate in 50% ethanol and Reynold’s lead citrate. Examinations were carried out on a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 80 kV.

RNA isolation and quantitative real-time PCR analysis

[00198] Human tissue samples were homogenized in blender Precellys ® (Bertin Technologies, France ) with CK14 beads in TRI reagent. Total RNAs from tissue or organoids were extracted by TRI reagent® (Euromedex, France) after removal Matrigel for organoids; samples were purified and traited by DNAse using the Direct-zol RNA kit (Zymo Research - Ozyme, France) according to manufacturer’s instructions. The quantity and quality of RNAs were assessed by Nanodrop (NanoPhotometer® P-330 Implen, Thermo Fisher Scientific). The desired amount of RNA (between 0.5 and 5 pg/ qsp 14 pL water) was taken and added to 4 pL reaction buffer and 2 pL enzyme (RT Life Technology Fermentas, ref.K1642). Reverse transcription was performed using GeneAmp® PCR System 9700. Quantitative PCR was performed with Fluidigm technology at GENTYANE facility (Clermont Ferrand, France). cDNA was studied with Fluidigm chips on BioMark TM HD system. Fluidigm is an automated real time qPCR integrating dynamic arrays of microfluidic circuits. The instrument uses an array of chips called dynamic arrays for qPCR, in which a typical chip format allows 9,216 PCR reactions (chip format 96.96; 96 samples x 96 assays) in a single qPCR analysis (Jang, et al., BMC Genomics 2011 ;12: 144. 2011). Other advantages ofFluidigm over standard qPCR include a greater number of reactions per plate, making it more cost-effective and less time-consuming. In addition, the protocol includes cDNA pre-amplification, enabling quantification of very small amounts of mRNA (Olwagen, et al., Sci Rep 2019;9: 6494, 2019). Sample preparation was recommended and carried by Gentyane facility (INRAE Clermont Ferrand, France). High- throughput qPCR with Fluidigm arrays on BioMark™ HD System. Sample preparation was recommended by the gentyane platform instructions. The concentration of the sample is 5 ng/pL of cDNA. The expression levels of genes were normalized to mean housekeeping genes (HPRT, GADPH and TBP genes). The primers were ordered at Eurogentec, PCR efficiency >90% and are shown in Table 1.

[00199] It was chosen targeted genes related to FT secretory and ciliary activity, proliferation, differentiation, female steroid receptor, sternness, protease activity, inflammation, etc... : ARMC4, DNAI1, FOXJ1, LRRC6, OVGP1, SLC12A2, PAX2, PAX8, PGRB, ESRI, WT1, IGFBP4, PLTP, HSP90abl, GPX1, ADM, GAPDH, YWHAE, MYH9, TXN, SOD1, SOD2, GSTP1, VCP, ENO1, GPI, ADAMI 7, MMP9, MUC16, ANXA5, SLPI, TIMP2, MMP7, WNT3a, E cadherme, K167, EREG, CD24, CyclineDl, NOTCH, LGR5, CD44vl, CD44v6, Pl 10 a, Pl 10 b, BMI1, Fnzzled4, SOX9, ELAFIN, ELA2, MMP3, F10, F2RL3, PRSS1, PRSS2, SerpineA5, F2R, Areg, EGF, AXIN2, Beat, Epcam, claudine 2, IL18, IL33, IL8, TGFb, TNFa, ITGA5, CD166, F2RL1, SI, SLC26A3, chgA, PRSS3, IL18, Muc2, occludine, Lgr4, PTEN, ALDH1A1, TFF3, CDX1, P63, ATF6, TP53.

[00200] Table 1: Primers Colon organoids

[00201] Waste biological samples obtained from surgical resections of patients treated at the Toulouse University Hospital were collected after the patient gave their informed consent (CODECOH national agreement DC2015-2443, COLIC Collection). Samples were collected in healthy zones of the resections from 5 patients. Colon crypts were isolated and cultured as previously described in 48 wells plates (Aldebert, et al., 6th Edition edn, WHO 2021. World Health Organisation, Cambridge). Organoid cultures were expanded in hESC-qualified matrix (Matrigel, Coming) and then seeded on Transwell inserts (24-well plates, 0.4 pm pore size, Coming COSTAR, #3470). Colon organoids were collected and dissociated into single cells by incubation in pre-warmed TrypLE Express Enzyme (Gibco, 12605010) for 10 min at 37 °C in agitation (1000 rpm). After addition of 5 mL of Advanced DMEM/F12 (Invitrogen, 12634-010) plus 2 mM Glutamax (Invitrogen, A1286001), 10 mM hepes (Gibco, 15630-056) and 10% FBS (ThermoFisher), and centrifugation (400 rpm, 5 min, 4 °C), dissociated cells were resuspended in organoid culture medium.

[00202] Cells were counted and 4 x 105 of them in 200 pL of culture medium were seeded on individual transwells pre-coated with diluted Matrigel (1:40 v/v in PBS) (2 h at 37°C). Six hundred microliters of culture medium was added to the basolateral side and cells were incubated at 37°C in 5% CO2.

Sperm samples collection and preparation.

[00203] Semen samples were collected from men who have undergone a fertility checkup in the reproductive unit of University Hospital of Toulouse. Semen samples were collected by masturbation after 2-7 days of sexual abstinence and after liquefaction for 15 to 60 min. On the unselected fresh sperm, we carried out a sperm count, motility and vitality. Men who have normal conventional parameters on fresh semen samples were included. Sperm was prepared on density gradient of Puresperm (Nidacon) during 20 min at 300g and a washing in Universal medium (Origio) during 5 min at 400g. Patients signed an inform consent for collecting data and remaining wasted sperm samples for research purposes. GERMETHEQUE biobank (BB-0033- 00081), site of Toulouse, provided 5 samples of fresh prepared sperm samples and their associated data to realize this project. GERMETHEQUE obtained consent from each patient to use their samples (CPP 2.15.27). The GERMETHEQUE pilotage committee approved the study design the 11/03/2021. The Biobank has a declaration DC-2021-4820 and an authorization AC- 2019-3487. The request’s number made to Germetheque is the 20211009 and its contract is referenced under the number 22 277 C.

Sperm cells injection, “coculture”, and retrieving in organoid apical compartment

[00204] Before sperm injection, at the first day of coculture, organoid culture medium of the apical compartment in Transwell was removed and replaced after rinsing by simple minimum essential medium : MEM® (Gibco). In the basal compartment, organoid culture medium was maintained and changed every other day.

[00205] Sperm preparation (N=5) was gently drop off in the apical compartment of tubal organoid for 96 hours of incubation in order to obtain a sperm concentration of 5 Million/ml in 200 pL of organoid apical medium in Transwell or in 200 pL of control media.

[00206] The different conditions used for sperm cells culture were:

Control 1 : Sperm cells in a specific medium for human sperm fertilization (Universal IVF Medium, CooperSurgical®).

Control 2: Sperm cells a in a differentiated medium supplemented with KGF, WNT7a, estradiol E2 and progesterone P4 used for HFT organoid culture (as described in Human fallopian tube organoid culture).

Control 3 : Sperm cells in a minimal medium MEM® (Gibsco)

Control 4: Sperm cells in apical compartment of human colon organoids on Transwell inserts

Test 5: Sperm cells within apical compartment of HFT isthmus organoids on Transwell inserts

Test 6: Sperm cells within apical compartment of HFT ampulla organoids on Transwell inserts

Test 7: Sperm cells in suspension in tube within retrieved apical supernatants of HFT isthmus organoids after differentiation

Test 8: Sperm cells in suspension in tube within retrieved apical supernatants of HFT ampulla organoids after differentiation

Sperm analysis

[00207] Sperm vitality and motility was performed at 0, 48 and 96 hours of incubation of spermatozoa within organoids or media. Sperm vitality was performed on 200 spermatozoa by sample using Eosin staining (Sperm VitalStain™, Nidacon, Mblndal, Sweden) according to WHO 2021 guidelines (WHO 2021). Sperm motility was assessed using CASA (Computer Aid Semen Analysis). For CASA analysis, sperm motility was performed with the Sperm Class AnalyzerTM (SCA™, Microptic™ Automatic Diagnostic Systems SL, Barcelona, Spain) following the manufacturer’s guidelines. The software version used for the study is 6.6.45. A high-resolution digital camera able to capture 100 images per second (Basler, Vision- Technology) was connected to a microscope with phase contrast (Nikon, Eclipse Ci) to visualize the sample. Acquisition was done under lOx magnification. A 4pL sample of sperm was loaded into analysis chambers with a depth of 20 mm (Slides SCA Mot, Microptic™, Barcelona, Spain).

[00208] Analyses were performed, according to the manufacturer’s instructions to assess a minimum of 500 cells. Five to ten image acquisitions were performed for each sample. Results appear as mean for proportion of progressive, non-progressive and immotile sperm. Quality controls were run according to the manufacturer’s recommendations.

Statistical analysis

[00209] Statistical analyses were performed by using Prism software (GraphPad Software, La Jolla, California) and STATA software (StataCorp, College Station, Texas). Data sets were summarized with descriptive statistics. For comparison of organoids sizes in the different conditions, a linear regression was performed with adjustement on number of organoids by well. For quantitative immunocytochemistry labelling, a Wilcoxon test for paired series was performed. For global comparison of relative gene expression (-DelatCt) after RTqPCR and for sperm parameters (vitality, total sperm motility and progressive motility), means between the different groups were compared with a Friedman’s test for paired data. For pairwise comparisons, we used a Conover test (which a post-hoc test for Friedman) with Bonferroni correction. With the Conover test, a p-value < 0.025 indicates statistical significance.

PART 2: RESULTS

Tubal Adult Stem Cells (ASC) formed reproducible Human Fallopian Tube (HFT) organoids on Matrigel

[00210] The presented HFT organoids were obtained from adult stem cells (ASC) from human fallopian tube tissue collection of a total of 10 donor patients. A patient human fallopian tube tissue before cell culture is presented in Figure 1.

[00211] After one cell passage and 24 days of culture and differentiation of the ASC, 200 organoids in each condition, from ampulla and isthmus, were randomly observed and measured using transmitted light microscopy (Figures 3 A and 3B). At the end of the culture, organoids were no longer of spheroids forms, but are more elongated, with a thicker, darker, and more plicated epithelial wall.

[00212] After adjustment on number or organoid by well, it is observed that HFT organoids from ampulla were larger than HFT organoid from isthmus 347.0 pm [82-1419] vs 311 pm [64-762] respectively (p=0.028) (Figures 4).

HFT organoids shows a high level of differentiation specific to Fallopian tube epithelium with some differences compared to patient tissue

[00213] Specific fallopian tube differentiation of our HFT organoid model was confirmed by scanning and transmitted electronic microscopy (TEM) observations that exhibited ciliary and secretory cell in both ampulla and isthmus HFT organoids (Figures 5A and 5B).

[00214] TEM confirms the presence of simple prismatic epithelium in our organoids and the typical axonemal ciliae structure that includes nine peripheral microtubule doublets and a pair of central one. Our HFT organoids have been shown to respond to exogenous hormonal stimuli, demonstrated by a comparative transcriptome analysis between the non-differentiated and differentiated organoids. It is observed a significant better level of most of secretory and ciliary related gene relative expression in differentiated HFT organoids (ampulla and isthmus) compared to undifferentiated organoids.

[00215] The transcriptomic panel revealed that relative expression of most of the targeted genes regulating cell and adhesion, ciliated cell function and differentiation, and hormonal prostaglandin function express no significant differences in HFT organoids compared to naive in vivo patient tissue (data not shown). However, it was observed that the HFT organoid model exhibits a more proliferative profile compared to the patient tissue as shown by the relative gene expression of Ki67 (>2DelatCt; p<0.001).

[00216] When comparing the differences in gene expression between the starting patient tissue and the HFT organoids from which they are derived : genes that are more than 4-fold more expressed (>2 delta Ct difference) in HFT organoids than in patient tissue in both isthmus and ampulla are ADM, MMP7, ELAFIN, SerpineA5, Ki67, IL8, Claudin 2 ; those expressed more than 4-fold less in organoids are OVGP1, PGRB, WT1, ESRI, F10, PRSS3, LGR5, TP53, IL15, IL33, ITGA5. For the ampulla specifically, genes linked to ciliate function (ARMC4, DNAI1, LRC6) were overexpressed in the tissue compared with the HFT organoids.

[00217] HFT organoids on Transwell® exhibit features close to HFT 3D organoids on Matrigel (HFT differentiated organoids) as it is observed no significant difference of relative gene expression between HFT organoids on Transwell® and HFT differentiated organoids on Matrigel.

HFT organoids from proximal (isthmus) and distal (ampulla) ASC didn’t show significant different features

[00218] There was no significant difference in confocal microscopy between isthmus and ampulla for high labelling of Ki67, PAX8 and Tub Detyr; Ki67: 19.0% ±12.4 vs 17.1% ±6.7; PAX8: 17.8% ±8.0 vs 17.6% ±6.7; TubDetyr%: 5.3% ±2.8 vs 4.5% ±2.4 in HFT organoid of isthmus and ampulla respectively. Also, transcriptomic analysis doesn’t evidence significant difference between organoids from the proximal and distal portion of tissue.

HFT organoids exhibit good features for sperm survival, total sperm motility and mainly progressive sperm motility

[00219] Vitality and motility of spermatozoa was tested in HFT ampulla organoid models and HFT isthmus organoid models on Transwell®. Using Transwell device, it was developed a cell culture system that enable a direct access to the apical compartment in order to drop off and remove gametes in the apical compartment easily.

[00220] Sperm vitality values at 0 h, 48 h and 96 h of incubation time under the different conditions are shown in Figure 7A. As expected, sperm vitality decreases over time but remain at relatively elevated level (around 50%) even after 96 hours in HFT organoids (isthmus and ampulla), Universal Medium Origio and culture medium for organoids. At 96h, the highest vitality values are obtained under the following conditions: HFT organoids (ampulla and isthmus) and universal medium Origio without significant difference between these four conditions. There was no significant difference with the fifth ranked condition supernatant of HFT organoid from ampulla (p>0,05) (Figure 7A).

[00221] Total sperm motility values at 0 h, 48 h and 96 h of incubation time under the different conditions are shown in Figure 7B. At 48 and 96h, the highest total sperm motility values are obtained when spermatozoa are in apical compartment of HFT organoids compare to all other conditions. At 96h, the difference with the third ranked Universal medium Origio condition is not significant (p>0.05) (Figure 7B).

[00222] Progressive sperm motility values at 0 h, 48 h and 96 h of incubation time under the different conditions are shown in Figure 7C. We demonstrated a superiority of HFT organoid apical compartment for progressive sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm fertilization media). At 48 hours of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 30.6% ±17.3, isthmus 29.3% ±14.8) than in commercial fertilization media (15.3% ±14.6) (p<0.05) and compared with all other conditions (Figure 7C, Table 2). The difference between HFT organoid and apical supernatant of organoids was not significant at 48h (p=0.84). At 96 hours progressive sperm motility was almost nil (<1%) in all conditions except for spermatozoa within HFT organoids (p<0.05): 11.5% ±14.7 and 12.9% ±16.9 in ampulla and isthmus organoids, respectively (Figure 7C, Table 2).

[00223] No significant difference is observed between HFT isthmus organoids and HFT ampulla organoids concerning sperm vitality, total sperm motility nor progressive sperm motility.

Table 2: Sperm vitality after eosin staining (%), Total sperm motility (%), and progressive sperm motility (%) at 48 and 96 hours of incubation within Human Fallopian Tube (HFT) organoids ampulla and isthmus, within retrieved apical supernatants of HFT organoids ampulla and isthmus, within colon organoids, and within different control media : a minimal medium (MEM), a specific medium for sperm fertilization (Universal FVF Medium. CooperSurgical), and the medium for HFT organoid culture in basal compartment. Values are mean ± SD. Significant differences appear with letter in exponent (p<0.025).

For vitality, a=0.015; b,c,d=0.001; e=0.0002; f=0.013; g=0.0021; h=0.0076.

For progressive motility, a,b,c,d,e,f,i,j <0.0001; g=0.0056; h=0.0015; k=0.0085; 1=0.0024.

For total motility, a=0.0014; b=0.0169; c=0.035.

PART 3: CONCLUSIONS

[00224] In conclusion, the present invention has succeeded in developing a reproducible model of HFT organoids which exhibits good features of differentiation after exposure to female sexual steroids.

[00225] It has been developed a culture system that gives easy access to the apical compartments of HFT organoids, and it has been demonstrated that the HFT organoid model in permeable support (Transwell®) can improve a level of motility for spermatozoa, up to the 96th hour of observation, which is higher than in various control conditions.

[00226] Consequently, it has been developed an HFT organoid model allowing to observe one of the functions of the tubal epithelium in the reproductive process.

[00227] Finally, based on the present invention, human fallopian tube organoids on permeable support can provide a faithful human cellular model to investigate complex interactions between the tubal epithelium and gametes, and pre-implantation embryos. These HFT organoid models offer considerable promise for future research in reproductive medicine. The present invention also opens the way for further work to study the behaviour of organoids with oocytes and embryos, as well as in the pre-implantation embryo development for having improvements in the handling and culture of gametes and human embryos during ART, and so improve medical care for infertile couples.

EXAMPLE 2: [00228] In the present examples, it has been explored the molecular mechanisms underlying the role of the tubal epithelium in the acquisition of sperm fertilizing ability, through the role of extracellular vesicles (EVs) from tubal tissue and from Human Fallopian Tube (HFT) organoids on sperm acrosome reaction.

[00229] To this end, it was conducted co-culture experiments of EVs from human tissue and from the HFT organoid model of the present invention with human spermatozoa from fertile donor. The acrosome reaction is a key event during fertilization and must occur at a precise location and timing within the Fallopian tube.

PART 1: MATERIALSAND METHODS

[00230] Human Fallopian tubes were cultured in Transwell inserts like in “Human fallopian tube organoid culture” in Part 1 of Example 1 above. Once the epithelial cells were differentiated, apical supernatants were collected daily over a period of 10 consecutive days, with one medium change per day. At the end of this 10 days-collect, the pooled apical supernatants were sequentially centrifuged (i) first at 500 * g for 5 minutes and then (ii) at 2500 x g for 20 minutes, to remove cellular contaminants. Subsequently, to isolate extracellular vesicles (EVs), the supernatant underwent differential ultracentrifugation at 10,000 x g for 30 minutes and then at 100,000 x g for 2 hours. Pellets obtained from each ultracentrifugation step (10K and 100K pellets) were retained and resuspended in filtered phosphate-buffered saline (PBS). The presence of EVs was confirmed by use of nanoparticle tracking analysis (NT A) and western blotting.

[00231] Functional assays were conducted after coculture of human spermatozoa at a concentration of 2 million cells per mL with EVs at a ratio of 500 EVs per spermatozoon (sperm cell).

[00232] After semen liquefaction for 30 minutes at 37°C, sperm was isolated using a density gradient and then incubated for 5-6 hours to allow capacitation. The acrosome status was assessed before and after the acrosome induction on addition of 10 mM ionomycin (Sigma- Aldrich, St-Quentin-Fallavier, France) with a minimum count of 100 sperm cells per patient. To do so, acrosomal content was labeled with fluorescein-isothiocyanate (FITC)-conjugated peanut agglutinin (PNA)-FITC (Sigma-Aldrich, St-Quentin-Fallavier, France) (25 mg/mL), which is lost after acrosome reaction. Groups were compared using the Kruskal-Wallis test.

PART 2: RESULTS

[00233] The results are presented in Figure 8. [00234] In the fertile control group without EV exposure, calcium ionophore induced acrosome reaction in 39.0% ± 2.6 SEM of sperm cells. This reaction was significantly enhanced when spermatozoa were incubated with EVs derived from segments isthmus (1ST) or ampulla (AMP) of Human Fallopian Tube (HFT) tissue and organoids.

[00235] Specifically, EVs from HFT tissue (100K pellet) increased acrosome reaction to 55.7% ± 4.2 SEM (p=0.048) and 58.0% ± 2.9 SEM (p=0.006) for EVs from the 100K 1ST and 100K AMP fractions, respectively.

[00236] Similarly, EVs from HFT organoids significantly enhanced the reaction to 55.6% ± 3.8 SEM (p=0.048), 55.40% ± 4.70 SEM (p=0.048), and 56.8% ± 3.9 SEM (p=0.016) for EVs from the 10K 1ST, 10K AMP and 100K AMP fractions, respectively.

[00237] These findings demonstrate that extracellular vesicles (EVs) from both HFT tissue and organoids (10K and 100K pellets) significantly promote ionophore-induced acrosome reaction in human sperm.

PART 3: CONCLUSIONS

[00238] The results demonstrate that extracellular vesicles (EVs) isolated from both fallopian tube tissues and organoids significantly enhance ionomycin-induced acrosome reaction in human sperm. This suggests that EVs play a crucial role in promoting sperm fertilizing potential, highlighting the importance of tubal epithelial-sperm communication mediated by EVs.

[00239] Furthermore, the use of the fallopian tube organoid culture system of the present invention as a bioreactor for EV production presents a promising tool for improving sperm function in Assisted Reproductive Technology (ART) settings, such as in vitro fertilization (IVF).

[00240]

[REFERENCES]

Aldebert E, Quaranta M, Sebert M, Bonnet D, Kirzin S, Portier G, Duffas JP, Chabot S, Lluel P, WHO laboratory manual for the examination and processing of human semen. 6th Edition edn, WHO 2021. World Health Organisation, Cambridge. Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients. Front Cell Dev Biol 2020;8: 363.

Alminana C, Bauersachs S. Extracellular vesicles: Multi-signal messengers in the gametes/embryo-oviduct cross-talk. Theriogenology 2020; 150: 59-69.

Alminana C, Tsikis G, Labas V, Uzbekov R, da Silveira JC, Bauersachs S, Mermillod P. Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo. BMC Genomics 2018;19: 622.

Bathala P, Fereshteh Z, Li K, Al-Dossary AA, Galileo DS, Martin-DeLeon PA. Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility. Mol Hum Reprod 2018;24: 143-157.

Boretto M, Cox B, Noben M, Hendriks N, Fassbender A, Roose H, Amant F, Timmerman D, Tomassetti C, Vanhie A et al. Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability. Development 2017;144: 1775-1786.

Calhaz- Jorge C, De Geyter C, Kupka MS, De Mouzon J, Erb K, Mocanu E, Motrenko T, Scaravelli G, Wyns C, Goossens V. Assisted reproductive technology in Europe, 2013: results generated from European registers by ESHRE. Hum Reprod 2017;32: 1957-1973.

Croxatto HB. Physiology of gamete and embryo transport through the fallopian tube. Reprod Biomed Online 2002;4: 160-169.

De los Santos MJ, Apter S, Coticchio G, Debrock S, Lundin K, Plancha CE, Prados F, Rienzi L, Verheyen G, Woodward B, Vermeulen N. ESHRE Guideline Group on Good Practice in IVF Labs; Revised guidelines for good practice in IVF laboratories (2015). Hum Reprod. 2016 Apr;31(4):685-6. doi: 10.1093/humrep/dew016. Epub 2016 Feb 17. PMID: 26908842.

Gardner DK, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril 1996;65: 349-353.

Holt WV, Fazeli A. Do sperm possess a molecular passport? Mechanistic insights into sperm selection in the female reproductive tract. Mol Hum Reprod 2015;21 : 491-501. Jang JS, Simon VA, Feddersen RM, Rakhshan F, Schultz DA, Zschunke MA, Lingle WL, Kolbert CP, Jen J. Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays. BMC Genomics 2011;12: 144.

Jung D, Xiong J, Ye M, Qin X, Li L, Cheng S, Luo M, Peng J, Dong J, Tang F et al. In vitro differentiation of human embryonic stem cells into ovarian follicle-like cells. Nat Commun 2017;8: 15680.

Kessler M, Hoffmann K, Brinkmann V, Thieck O, Jackisch S, Toelle B, Berger H, Mollenkopf HJ, Mangier M, Sehouli J et al. The Notch and Wnt pathways regulate sternness and differentiation in human fallopian tube organoids. Nat Commun 2015;6: 8989.

Kessler M, Hoffmann K, Fritsche K, Brinkmann V, Mollenkopf H J, Thieck O, Teixeira da Costa AR, Braicu El, Sehouli J, Mangier M et al. Chronic Chlamydia infection in human organoids increases sternness and promotes age-dependent CpG methylation. Nat Commun 2019;10: 1194.

Leese HJ, Tay JI, Reischl J, Downing SJ. Formation of Fallopian tubal fluid: role of a neglected epithelium. Reproduction 2001 ;121 : 339-346.

Li Y, Liu C, Guo N, Cai L, Wang M, Zhu L, Li F, Jin L, Sui C. Extracellular vesicles from human Fallopian tubal fluid benefit embryo development in vitro. Hum Reprod Open. 2023 Feb 21;2023(2):hoad006. doi: 10.1093/hropen/hoad006. PMID: 36895886; PMCID: PMC9991590.

Ng KYB, Mingels R, Morgan H, Mackion N, Cheong Y. In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in human conception: a systematic review. Hum Reprod Update 2018;24: 15-34.

Olwagen CP, Adrian PV, Madhi SA. Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing. Sci Rep 2019;9: 6494.

Tay JI, Rutherford AJ, Killick SR, Maguiness SD, Partridge RJ, Leese HJ. Human tubal fluid: production, nutrient composition and response to adrenergic agents. Hum Reprod 1997;12: 2451-2456.

Wale PL, Gardner DK. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update. 2016 Jan-Feb;22(l):2-22. doi: 10.1093/humupd/dmv034. Epub 2015 Jul 22. PMID: 26207016.

Yucer N, Holzapfel M, Jenkins Vogel T, Lenaeus L, Ornelas L, Laury A, Sareen D, Barrett R, Karlan BY, Svendsen CN. Directed Differentiation of Human Induced Pluripotent Stem Cells into Fallopian Tube Epithelium. Sci Rep 2017;7: 10741. Zhang S, Dolgalev I, Zhang T, Ran H, Levine DA, Neel BG. Both fallopian tube and ovarian surface epithelium are cells-of-origin for high-grade serous ovarian carcinoma. Nat Commun 2019,10: 5367.

Claims

[CLAIMS]

1. A mammal fallopian tube organoid (1) within a cell-culture insert (2) comprising a porous membrane (3) supported by a frame (4), the cell-culture insert (2) being accommodatable within a culture vessel (5) to separate the culture vessel into two compartments providing an upper compartment (6) and a lower compartment (7), and wherein the mammal fallopian tube organoid comprises at least ciliary cells and secretory cells.

2. The mammal fallopian tube organoid (1) within a cell-culture insert (2) according to claim 1, wherein the upper compartment (6) and the lower compartment (7) contain a first cellculture medium suitable for a mammal fallopian tube organoid.

3. A cell-culture system for gamete cells, the cell-culture system comprising a mammal fallopian tube organoid (1) comprising at least ciliary cells and secretory cells, a culture vessel (5), a first cell-culture medium suitable for a mammal fallopian tube organoid, and a cellculture insert (2) comprising a porous membrane (3) supported by a frame (4), the cell culture insert being accommodatable within the culture vessel (5) to separate the culture vessel (5) into two compartments defined as an upper compartment (6) and a lower compartment (7); and wherein the porous membrane (3) supports onto its upper surface the mammal fallopian tube organoid (1).

4. The cell-culture system according to claim 3, wherein the first cell-culture medium is contained in the upper compartment (6) and/or the lower compartment (7).

5. The mammal fallopian tube organoid according to claim 2, or the cell-culture system according to claim 3 or 4, wherein the first cell-culture medium is DMEM/Ham’s Fl 2 or RPMI 1640.

6. The mammal fallopian tube organoid according to claim 2 or 5, or the cell-culture system according to any one of claims 3 to 5, wherein the first cell-culture medium is DMEM/Ham’s F12.

7. The mammal fallopian tube organoid according to claim 2, 5 or 6, or the cell-culture system according to any one of claims 3 to 6, wherein the first cell-culture medium is supplemented with free amino acids, a growth factor, Noggin, a ROCK inhibitor, a Wnt protein, and Prostaglandin E2.

8. The mammal fallopian tube organoid according to claim 7, or the cell-culture system according to claim 7, wherein the first cell-culture medium is further supplemented with HEPES buffer, B27, Vitamin B3 (Nicotinamide), and SB202190.

9. The mammal fallopian tube organoid according to claims 2, 5 to 8, or the cell-culture system according to any one of claims 3 to 8, wherein the first cell-culture medium is supplemented with a differentiation fallopian tube cell factor and a female steroid hormone.

10. The mammal fallopian tube organoid according to claims 1, 2, 5 to 9, or the cellculture system according to any one of claims 3 to 9, wherein the porous membrane is made of at least one material selected from polycarbonate, polyester (PET), collagen-coated polytetrafluoroethylene (PTFE) and a combination of collagen, fibronectin and laminin, preferably the porous membrane is a polycarbonate membrane.

11. The mammal fallopian tube organoid according to claims 1, 2, 5 to 10, or the cellculture system according to any one of claims 3 to 10, wherein the mammal fallopian tube organoid originates from pluripotent stem cells (PSCs) or adult stem cells (ASCs), optionally the mammal fallopian tube organoid originates from adult stem cells, in particular from adult stem cells isolated from isthmus or ampulla regions of a fallopian tube tissue.

12. An in vitro method for improving and/or maintaining the fertilizing capacity of mammal sperm cells, the method comprising at least the steps of:

(a) providing mammal sperm cells previously obtained from a male mammal subject,

(b) providing a mammal fallopian tube organoid within a cell-culture insert according to any one of claims 1, 2 and 5-9, or a cell-culture system according to any one of claims 3 to 9,

(c) introducing the mammal sperm cells (8), with a second cell-culture medium, within the upper compartment (6) of the mammal fallopian tube organoid within a cell-culture insert or of the cell-culture system, and

(d) incubating the mammal sperm cells under suitable conditions for mammal sperm cells.

13. The method according to claim 12, wherein the second suitable cell-culture medium at step (b) is suitable for maintaining and/or improving the fertilizing capacity of mammal sperm cells, in particular the second suitable cell-culture medium is suitable for fertilization.

14. An in vitro fertilization method, the method comprising at least the steps of:

(a) providing at least one oocyte previously obtained from a female mammal subject and providing sperm cells previously obtained from a male mammal subject,

(b) introducing said oocyte and said sperm cells, with a second cell-culture medium suitable for fertilization, within a mammal fallopian tube organoid according to any one of claims 1, 2 and 5-11, or a cell-culture system according to any one of claims 3 to 11,

(c) incubating the oocyte and the sperm cells under conditions suitable for the occurrence of fertilization, thereby obtaining a preimplantation embryo, and (d) optionally, culturing the preimplantation embryo to a desired development stage before transfer into the uterus of a subject or conservation.

15. Use of a mammal fallopian tube organoid according to any one of claims 1, 2 and 5-11 or of a cell-culture system according to any one of claims 3 to 11, for improving and/or maintaining the fertilizing capacity of mammal gamete cells.

16. Use of a mammal fallopian tube organoid according to any one of claims 1, 2 and 5-11 or of a cell-culture system according to any one of claims 3 to 11, for culturing a preimplantation embryo to a desired development stage.

17. Use of a mammal fallopian tube organoid according to any one of claims 1, 2 and 5-11 or of a cell-culture system according to any one of claims 3 to 11, for in vitro fertilization.

18. Use of a mammal fallopian tube organoid according to any one of claims 1, 2 and 5-11 or of a cell-culture system according to any one of claims 3 to 11, as a bioreactor for producing extracellular vesicles (EVs).

19. A kit-of-parts for improving and/or maintaining the fertilizing capacity of mammal gamete cells, the kit comprising at least:

(i) a mammal fallopian tube organoid within a cell-culture insert according to any one of claims 1, 2 and 5-11,

(ii) one or more mammal gamete cells,

(iii) a first cell-culture medium suitable for mammal fallopian tube organoid, and

(iv) a second cell-culture medium suitable for the fertilizing capacity of mammal gamete cells.

20. The kit of parts of claim 19, wherein the kit further comprises a set of instructions for improving and/or maintaining the fertilizing capacity of mammal gamete cells.