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WO2025247927A1 - Organoïdes humains de trompe de fallope et leurs utilisations - Google Patents

Organoïdes humains de trompe de fallope et leurs utilisations

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Publication number
WO2025247927A1
WO2025247927A1 PCT/EP2025/064686 EP2025064686W WO2025247927A1 WO 2025247927 A1 WO2025247927 A1 WO 2025247927A1 EP 2025064686 W EP2025064686 W EP 2025064686W WO 2025247927 A1 WO2025247927 A1 WO 2025247927A1
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WIPO (PCT)
Prior art keywords
cell
mammal
fallopian tube
organoid
culture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/064686
Other languages
English (en)
Inventor
Nicolas GATIMEL
Nathalie Vergnolle
Guillaume Perez
David SAGNAT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Toulouse
Universite de Montpellier
Ecole Nationale Veterinaire de Toulouse ENVT
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Universite de Toulouse
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Toulouse
Universite de Montpellier
Ecole Nationale Veterinaire de Toulouse ENVT
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Universite de Toulouse
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, Centre Hospitalier Universitaire de Toulouse, Universite de Montpellier, Ecole Nationale Veterinaire de Toulouse ENVT, Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement, Universite de Toulouse filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of WO2025247927A1 publication Critical patent/WO2025247927A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract

Definitions

  • 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.
  • the present disclosure relates to human fallopian tube organoids within cell-culture inserts for gamete-cells culture and fertilization.
  • 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).
  • the tubal mucosa 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).
  • 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).
  • 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.
  • the upper compartment and the lower compartment comprise a first cell-culture medium suitable for a mammal fallopian tube organoid.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first cell-culture medium can be comprised in the upper compartment and/or the lower compartment.
  • the first cell-culture medium can be DMEM/Ham’s F12 or RPMI 1640.
  • the first cell-culture medium can be DMEM/Ham’s F12.
  • 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.
  • the first cell-culture medium can be further supplemented with HEPES buffer, B27, N-acetylcysteine, Vitamin B3, and SB202190.
  • the first cell-culture medium can be further supplemented with a differentiation fallopian tube cell factor, and a female steroid hormone.
  • the first cell-culture medium can be further supplemented with Keratinocyte Growth Factor (KGF),WNT7a, estradiol and progesterone.
  • KGF Keratinocyte Growth Factor
  • WNT7a Keratinocyte Growth Factor
  • estradiol estradiol
  • progesterone a Keratinocyte Growth Factor
  • 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.
  • the porous membrane can be a porous polycarbonate membrane.
  • the porous membrane can have pores having an average pore size ranging from about 0.1 pm to about 50 pm,
  • the porous membrane can have pores having an average pore size ranging from about 0.1 pm to about 7 pm.
  • the porous membrane can have pores having a pore size of about 0.4 pm.
  • 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).
  • the mammal fallopian tube organoid originates from pluripotent stem cells (PSCs) or adult stem cells (ASCs).
  • PSCs pluripotent stem cells
  • ASCs adult stem cells
  • 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.
  • the mammal fallopian tube organoid is a human fallopian tube organoid.
  • the mammal fallopian tube organoid can be a non-human fallopian tube organoid.
  • the cell-culture insert is a Transwell insert.
  • 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:
  • the second suitable cell-culture medium at step (b) can be suitable for maintaining and/or improving the fertilizing capacity of mammal sperm cells.
  • the second suitable cell-culture medium at step (b) can be suitable for fertilization.
  • the second cell-culture medium can be identical to the first cell-culture medium.
  • the second cell-culture medium can be a Minimum Essential Medium (MEM).
  • MEM Minimum Essential Medium
  • the first cell-culture medium before proceeding to step (c), can be removed from the upper compartment and replaced with the second suitable cellculture medium.
  • the mammal sperm cells at step (d) can be incubated for a time of about 12 hours to about 7 days.
  • the mammal sperm cells at step (d) can be incubated for a time of about 3 days to about 5 days.
  • the mammal sperm cells can be human sperm cells. In some embodiments, the mammal sperm cells can be non-human sperm cells.
  • the present disclosure relates to an in vitro fertilization method, the method comprising at least the steps of:
  • 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.
  • the mammal gamete cells are sperm cells and/or oocytes.
  • 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.
  • 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.
  • 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).
  • 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).
  • EVs extracellular vesicles
  • 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:
  • FIGURE 1 depicts images of a patient’ fallopian tube tissue before cell culture.
  • 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).
  • FIGURE 3 shows human fallopian tube ampulla (HFTA) and human fallopian tube isthmus (HFTI) organoids cultures derived directly from patients’ cells.
  • 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.
  • HFTA human fallopian tube ampulla
  • HFTI human fallopian tube isthmus
  • Abscissa from left to right
  • Ordinate Organoid size in micrometer.
  • FIGURE 5 depicts electron microscopy images of Human Fallopian Tube Ampulla (HFTA) and Human Fallopian Tube Isthmus (HFTI) organoids.
  • 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®.
  • 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 .
  • (1) Medium for human sperm fertilization Universal IVF Medium, CooperSurgical®
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a cell-culture insert can be removed, adjusted or manipulated, and then replaced within a culture vessel.
  • 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.
  • an adult stem cell originates from a fallopian tube tissue of a mammal adult organism, in particular from a human adult.
  • adult stem cells are isolated and purified from a tissue before being cultured in vitro.
  • pluripotent stem cells 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.
  • somatic non-pluripotent cells 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.
  • specific genetic factors such as Oct4, Sox2, Klf4, and c-Myc
  • the present disclosure relates to a mammal fallopian tube organoid within a cellculture insert.
  • fallopian tube organoid refers to a fallopian tube organoid within a cell-culture insert, unless otherwise described.
  • fallopian tube organoid and “fallopian tube organoid within a cell-culture insert” can be used interchangeably.
  • 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.
  • a mammal fallopian tube organoid as described herein comprises at least ciliary cells and secretory cells.
  • 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.
  • a mammal fallopian tube organoid within a cell-culture insert as described herein can be prepared following the protocol described in the example section.
  • 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.
  • a mammal fallopian tube organoid as disclosed herein can originate from a plurality of adult stem cells.
  • 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.
  • 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.
  • the first cell-culture medium can be suitable for maintaining the viability of a fallopian tube organoid.
  • 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.
  • DMEM Dulbecco's Modified Eagle Media
  • MEM Minimal Essential Medium
  • KO-DMEM Knockout-DMEM
  • G-MEM Glasgow Minimal Essential Medium
  • BME Basal Medium Eagle
  • 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
  • Fallopian tube cell culture media 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
  • the first cell-culture medium can be MEM, DMEM, DMEM/Ham’s F12 or RPMI 1640.
  • 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.
  • 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.
  • a free amino acids can be L-glutamine and N- acetylcysteine.
  • a growth factor can be selected from EGF, FGF-2, and/or VEGF. In some embodiments, a growth factor can be EGF.
  • a cytokine can be selected from Noggin and/or a Wnt protein (R-spondin and/or Wnt3a).
  • a ROCK inhibitor can be Y-27632 and/or SB431542. In some embodiments, a ROCK inhibitor can be SB431542.
  • 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.
  • a first cell-culture medium can be supplemented with L- glutamine, a growth factor, Noggin, a ROCK inhibitor, R-spondin, Wnt3a, and Prostaglandin E2.
  • a first cell-culture medium can be supplemented with L- glutamine, EGF, Noggin, a ROCK inhibitor, R-spondin, Wnt3a, and Prostaglandin E2.
  • a first cell-culture medium can be further supplemented with HEPES buffer, B27, N-acetylcysteine, Vitamin B3 (Nicotinamide), N2 and/or SB202190.
  • a first cell-culture medium can be further supplemented with a penicillin/streptomycin solution.
  • a first cell-culture medium can be supplemented with a differentiation fallopian tube cell factor and a female steroid hormone.
  • a differentiation fallopian tube cell factor can be selected from Keratinocyte Growth Factor (KGF) and/or WNT7a.
  • KGF Keratinocyte Growth Factor
  • WNT7a WNT7a
  • a female steroid hormone can be selected from estrogen (estradiol), and/or progesterone.
  • a first cell-culture medium can be supplemented with Keratinocyte Growth Factor (KGF),WNT7a, estradiol and progesterone.
  • KGF Keratinocyte Growth Factor
  • WNT7a Keratinocyte Growth Factor
  • estradiol estradiol
  • progesterone Keratinocyte Growth Factor
  • 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.
  • PGE2 Prostaglandin E2
  • a first cell-culture medium can be further supplemented with glucose.
  • 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.
  • the culture vessel comprises side walls and a solid base.
  • the side walls are in cylindrical.
  • the side walls can be a uniform cylindrical side wall.
  • 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.
  • the gap can be of constant size around the cell-culture insert.
  • the cell-culture insert can be a Transwell insert.
  • the upper compartment and the lower compartment of the culture vessel as described herein can contain a first cell-culture medium as described herein.
  • 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.
  • PET polyester
  • PTFE collagen-coated polytetrafluoroethylene
  • the porous membrane of the cell-culture insert can be made of polycarbonate.
  • 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.
  • the porous membrane of the cell-culture insert can have pores having a size of about 0.4 pm.
  • 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.
  • the porous membrane of the cell-culture insert can have a thickness of about 10 pm.
  • 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 The cell-culture system
  • the cell-culture system comprises:
  • a mammal fallopian tube organoid (1) comprising at least ciliary cells and secretory cells
  • 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).
  • the first cell-culture medium can be contained in the upper compartment (6) and/or the lower compartment (7).
  • 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
  • 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.
  • an in vitro method for improving and/or maintaining the fertilizing capacity of mammal sperm cells comprising at least the steps of:
  • a male mammal subject can be a human. In some embodiments, a male mammal subject can be a non-human.
  • the mammal sperm cells can be human sperm cells. In some embodiments, the mammal sperm cells can be non-human sperm cells
  • 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.
  • the second cell-culture medium can be suitable for fertilization.
  • the second cell-culture medium can be identical to the first cell-culture medium.
  • the second cell-culture medium can be a Minimum Essential Medium (MEM).
  • MEM Minimum Essential Medium
  • the second cell-culture medium can be supplemented with growth factors, cytokines and/or hormones.
  • the first cell-culture medium before proceeding to step (c), can be removed from the upper compartment and replaced with the second suitable cellculture medium within the upper compartment.
  • 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.
  • the mammal sperm cells at step (d) can be incubated for a time of about 3 days, 4 days or 5 days.
  • 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.
  • 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 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.
  • the mammal gamete cells are mammal sperm cells and/or mammal oocytes.
  • the mammal sperm cells can be human sperm cells. In some embodiments, the mammal sperm cells can be non-human sperm cells.
  • the mammal oocytes can be human oocytes. In some embodiments, the mammal oocytes can be non-human oocytes.
  • 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.
  • 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.
  • 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.
  • the in vitro fertilization method comprises at least the steps of:
  • the oocyte and the sperm cells are incubated under conditions, in particular chemical and physical conditions, suitable for the occurrence of fertilization.
  • conditions are well known in the art and thus can be adjusted by the skilled person in the art accordingly.
  • parameters such as temperature, sperm concentration, culture media composition, pH, duration, oxygen levels, and/or light exposure may require regulation to ensure optimal conditions.
  • these conditions are adjusted to mimic the physiological environment of the cells in vivo.
  • 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.
  • 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.
  • 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.
  • 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.
  • the preimplantation embryo can be conserved in a cryogenic environment before being transferred into the uterus of a subject.
  • Preimplantation embryos obtained with the methods of the present disclosure are not intended for industrial or commercial purposes.
  • 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).
  • 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.
  • 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)).
  • the extracellular vesicles can be proteins, RNAs, and/or lipids.
  • 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.
  • 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:
  • the kit-of-parts further comprises a set of instructions for improving and/or maintaining the fertilizing capacity of mammal gamete cells.
  • kits-of-parts for in vitro fertilization comprising at least:
  • the kit-of-parts further comprises a set of instructions for in vitro fertilization.
  • 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.
  • 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.
  • 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).
  • 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.
  • FFA formaldehyde
  • HBSS Hank’s Buffered Salt Solution
  • BSA bovine serum albumin
  • 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).
  • 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)
  • 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.
  • RNA 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).
  • RNA between 0.5 and 5 pg/ qsp 14 pL water
  • 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).
  • chip format 96.96 9,216 PCR reactions
  • Other advantages ofFluidigm over standard qPCR include a greater number of reactions per plate, making it more cost-effective and less time-consuming.
  • 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).
  • 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, PRSS
  • Waste biological samples obtained from surgical resections of patients treated at the Jardin 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.
  • Semen samples were collected from men who have undergone a fertility checkup in the reproductive unit of University Hospital of Jardin. 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.
  • Puresperm Nidacon
  • Origio Universal medium
  • GERMETHEQUE biobank (BB-0033- 00081), site of Jardin, 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.
  • organoid culture medium of the apical compartment in Transwell was removed and replaced after rinsing by simple minimum essential medium : MEM® (Gibco).
  • MEM® simple minimum essential medium
  • organoid culture medium was maintained and changed every other day.
  • 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 Malem cells within apical compartment of HFT isthmus organoids on Transwell inserts
  • Test 6 Malem cells within apical compartment of HFT ampulla organoids on Transwell inserts
  • Test 7 Malem 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
  • a high-resolution digital camera able to capture 100 images per second 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, MicropticTM, Barcelona, Spain).
  • ASC Tubal Adult Stem Cells
  • HFT Human Fallopian Tube
  • 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.
  • organoids in each condition were randomly observed and measured using transmitted light microscopy ( Figures 3 A and 3B).
  • organoids were no longer of spheroids forms, but are more elongated, with a thicker, darker, and more plicated epithelial wall.
  • HFT organoids shows a high level of differentiation specific to Fallopian tube epithelium with some differences compared to patient tissue
  • 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.
  • 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).
  • 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.
  • genes linked to ciliate function (ARMC4, DNAI1, LRC6) were overexpressed in the tissue compared with the HFT organoids.
  • 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 exhibit good features for sperm survival, total sperm motility and mainly progressive sperm motility
  • FIG. 7C Progressive sperm motility values at 0 h, 48 h and 96 h of incubation time under the different conditions are shown in Figure 7C.
  • 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).
  • HFT isthmus organoids No significant difference is observed between HFT isthmus organoids and HFT ampulla organoids concerning sperm vitality, total sperm motility nor progressive sperm motility.
  • HFT Human Fallopian Tube
  • MEM minimal medium
  • CooperSurgical Universal FVF Medium. CooperSurgical
  • 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.
  • 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.
  • 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.
  • EVs extracellular vesicles
  • HFT Human Fallopian Tube
  • 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.
  • EVs extracellular vesicles
  • 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).
  • PBS filtered phosphate-buffered saline
  • 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).
  • 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.
  • 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.
  • FITC fluorescein-isothiocyanate
  • PNA peanut agglutinin
  • 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).
  • ART Assisted Reproductive Technology
  • IVF in vitro fertilization
  • Kessler M Hoffmann K, Brinkmann V, Thieck O, Jackisch S, Toelle B, Berger H, Mollenkopf HJ, Mangier M, Sehouli J et al.

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Abstract

La présente invention concerne un organoïde de trompe de Fallope de mammifère à l'intérieur d'un insert de culture cellulaire comprenant une membrane poreuse soutenue par un cadre, l'insert de culture cellulaire pouvant être logé à l'intérieur d'un récipient de culture afin de séparer le récipient de culture en deux compartiments, à savoir un compartiment supérieur et un compartiment inférieur, et l'organoïde de trompe de Fallope de mammifère comprenant au moins des cellules ciliaires et des cellules sécrétoires. L'invention concerne également des utilisations et des procédés associés.
PCT/EP2025/064686 2024-05-28 2025-05-27 Organoïdes humains de trompe de fallope et leurs utilisations Pending WO2025247927A1 (fr)

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WO2018176001A2 (fr) * 2017-03-24 2018-09-27 Cedars-Sinai Medical Center Procédés et compositions pour la production d'épithélium de trompes de fallope
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