FR3101356A1 - In vitro methods of ovarian tissue culture - Google Patents

Abstract

The invention relates to an in vitro or ex vivo method of preserving germinal ovarian tissue or ovarian germ cells, or of folliculogenesis from said germinal ovarian tissue or cells, comprising culturing the germinal ovarian tissue or ovarian germ cells in a bioreactor whose walls are composed of a biomaterial in the form of a hydrogel.

Description

In vitro methods for culturing ovarian tissue

The invention relates to in vitro methods for culturing ovarian tissue, which enable tissue preservation and folliculogenesis .

Technology background

The ovaries are the female sexual glands which measure on average 3 x 1.8 cm ² in women. These glands have a role in the secretion of sex steroids (estrogens, progesterone and androgens) and also have a role in reproduction through folliculogenesis and oocyte maturation. These functions are exercised cyclically during puberty, until menopause. The ovary is mainly composed of the cortex which contains the follicles and which is the seat of folliculogenesis, and of the medulla where the vessels and supporting tissues are located. The stock of primordial follicles is formed during intrauterine life. It reaches its maximum at the ^7th month of intrauterine life. Then the number of follicles contained in the ovarian cortex decreases with age. At birth, each ovary contains an average of 500,000 follicles, or a total of one million follicles, but this estimate varies between individuals. This number drops to 100,000 at age 20, 10,000 at age 40, and 100 at age 45.

Folliculogenesis is the development of the follicle from the primordial stage to the tertiary stage in the ovarian cortex. It can only take place completely from puberty. The primordial follicle transforms into a primary, secondary and then tertiary follicle. Less than 0.1% of primordial follicles reach the tertiary follicle stage.

Figure 15 shows the different stages of follicle development (see also Yilmaz Guzel and Ozgur Oktem, Mol Reprod Dev.2017;84:544–559).

In women, about 450 oocytes are ovulated in a lifetime. However, certain therapeutic treatments with a gonadotoxic effect, such as chemotherapy or radiotherapy, can lead to impairment of female reproductive potential. To deal with this premature ovarian failure, preservation techniques exist.

Ovarian freezing, which is one of the fertility preservation methods, is the only method that can be offered to prepubescent girls and women requiring emergency treatment. It consists of surgically removing an ovary and then freezing the cortex until the request for use by the patient for a parental project. The ovarian cortex is then thawed and grafted. However, the protocols for self-preservation and then for transplantation of ovarian tissue in women still need to be improved. In addition, in patients suffering from malignant pathologies, the major drawback of using this cryopreserved ovarian tissue is the risk of reintroduction of malignant cells linked to the autograft.

An alternative technique to autograft is the in vitro folliculogenesis technique. However, today this technique is not yet developed, and at present no team has succeeded in obtaining mature fertilizable oocytes.

Complete in vitro folliculogenesis indeed requires a long culture time, approximately three months to begin at the primordial follicle stage and end at the mature follicle stage containing a mature fertilizable oocyte.

There is therefore still a need for a technique of in vitro preservation and in vitro folliculogenesis which would make it possible, on the one hand, to preserve the ovarian tissue in vitro without freezing, and on the other hand to obtain mature oocytes, in particular from reserve follicles.

The inventors propose to culture and confine ovarian tissue in a structure, advantageously three-dimensional, consisting of a biocompatible material having cavities. The ovarian tissue contains the follicular stock necessary for regular ovulation. Culture in this biocompatible structure allows the primordial and primary follicles to be cultured with the ovarian medulla. Ovarian tissue is secreted growth factors, cytokines and paracrine mediators essential for folliculogenesis. This culture allows the development of medullary tissue and follicular growth which occurs by outgrowth on the surface of the ovary.

One aspect of the invention relates to an in vitro or ex vivo method for preserving germinal ovarian tissue or germinal ovarian cells, comprising culturing the germinal ovarian tissue or germinal ovarian cells in a bioreactor whose walls are composed of a biomaterial in the form of a hydrogel, the culture of the ovarian tissue being conducted preferably from 1 month to 6 months.

According to another aspect, the invention provides an in vitro or ex vivo method of folliculogenesis from ovarian germinal tissue or ovarian germinal cells, comprising maturing the ovarian germinal tissue or ovarian germinal cells in a bioreactor whose walls are composed of a biomaterial in the form of a hydrogel.

The biomaterial in the form of a hydrogel allows exchanges, in particular of nutrients and gaseous exchanges with the culture medium in which the bioreactor is placed.

In this culture system structured in three dimensions, the size of the structure and the cavities as well as the geometry vary according to the technical needs: size of the ovarian tissue, stage of folliculogenesis. The bioreactor is therefore a hydrogel which imitates the extra cellular matrix of the tissue to be matured, it is rich in water, elastic and permeable.

This bioreactor makes tissue culture possible for a long period (at least 2 or 3 months) by allowing paracrine exchanges within the ovarian tissue which remains confined in the cavity or cavities of the bioreactor. Paracrine exchanges are protected in the compartments when changing the culture medium.

Legends of figures

: photograph of a compartmentalized structure being closed

: photograph of a compartmentalized structure according to the invention closed at each end by a suture thread

: photograph of a macroscopic examination of the structure and of fragment B of the fetal ovary (F1) after stopping the culture at D90.

: graph showing the evaluation of the follicular density of the fresh tissue, at D45 of culture and at D90 of culture in the two groups: control tissue (group A) and tissue in bioreactor (group B).

: photographs of fetal ovary sections (F1) stained with HPS (hematoxyline-phloxine-saffron). On the photo plate, the left column shows the control tissue that was cultured in group A and the right column shows the tissue that was cultured in group B for the duration of the culture.

: graph showing the evaluation of the cell proliferation of the fresh tissue, at D45 of culture and at D90 of culture in the two groups: control tissue (group A) and tissue in bioreactor (group B).

: Photographs of sections of fetal ovary (F1) marked with Ki67, objective 10. The cells marked in brown are positive while the blue cells are negative. The left column shows the tissues of group A and the right column shows the tissue of group B, and this throughout the duration of the culture.

: photographs of sections of fetal ovary (F1) marked with Ki67, objective 40. The cells marked in brown are positive while the blue cells are negative. The left column shows the tissues of group A and the right column shows the tissue of group B, and this throughout the duration of the culture.

: photographs of sections of fetal ovary labeled with Ki67, tissue group A at D90 of culture, objective 40. Presence of acellular zones. Loss of cell cohesion.

: photographs of sections of fetal ovary labeled with Ki67, group B tissue at D90 of culture, objective 40. Presence of crystalline inclusion without loss of cellular cohesion.

: Photograph of a group B human fetal ovary section (F2) stained with HPS, D90 culture after 10 days of activin A, objective 40. The growing follicles are outlined in white.

: photograph of a group B human fetal ovary section (F2) stained with HPS after 90 days of culture including 10 days of activin A from D80 to D90; objective 40. Presence of a secondary follicle contoured in white.

: photograph of a group B human fetal ovary section (F2) stained with HPS after 90 days of culture including 10 days of activin A from D80 to D90; objective 100. Presence of granulosa cells and thecal cells.

: photograph of a group A human fetal ovary section (F2) stained with HPS after 90 days of culture including 10 days of activin A from D80 to D90; objective *40. Presence of primordial and primary follicles. Absence of growing follicle.

: Chronology of follicular growth and maturation in the human ovary, modified from Gougeon, 1986, Hum Reprod. 1986;1:81-87.

Detailed description of the invention

Cultured ovarian tissue

The method of the invention culture an ovarian biopsy hereinafter referred to as "ovarian tissue". This is preferably fresh, but it can also be ovarian tissue preserved by freezing. Preferably, it is ovarian tissue from a human being, but it can also be ovarian tissue from other non-human mammals, for example sheep, goats, cattle, horses (in particular for the selection of race horses ), camelids, and threatened or endangered species. Ovarian tissue can be obtained from a healthy prepubertal human subject. In a particular embodiment, the ovarian tissue is from a pubescent patient about to undergo gonadotoxic treatment, such as chemotherapy or surgery.

Advantageously the realization of this invention does not require a large quantity of ovarian tissue. This ovarian tissue may in particular be i) a fragment of ovary which comprises all or part of the cortex and all or part of the medulla, ii) an entire ovary, or iii) a tissue filtrate comprising primordial follicles associated with cells of the ovarian medulla, which can be obtained by dissociation and filtration of an ovarian tissue.

Preferably, an ovary is used which is cut into a fragment comprising the cortical (site of the follicular reserve) and the medulla, preferably over a thickness of 2 to 5 mm, more preferably of 3 to 5 mm. In this cutting plan, the tissue surface is on average 10 x 5mm. In other words, a preferred fragment has a sagittal surface of approximately 2 x 5 mm, for a longitudinal surface of approximately 10 x 5 mm.

In general, the folliculogenesis process is preferably carried out until at least one tertiary follicle has formed. A tertiary follicle can typically be produced by the method described, after a maturation time typically comprised between 10 days and 3 months in women, for example approximately 2 months.

You can also continue until at least one follicle containing a mature oocyte is formed, which typically occurs after about 3 months, or even 4 months.

The bioreactor

The bioreactor is composed of a biomaterial forming at least one cavity in which the ovarian tissue is placed or confined.

According to the present invention, it is to be understood that the bioreactor is i) pre-constituted and comprises one or more cavities in which the tissue is introduced or ii) formed around the tissue, the tissue thus being included in a cavity which is formed around it during the formation of the bioreactor. Typically, the cavity(ies) is/are filled or substantially filled with ovarian tissue. Typically, the tissue is confined within the cavity(ies).

According to one characteristic, the bioreactor is preferably closed or obstructed. According to the invention, the biomaterial is in the form of a hydrogel and preferably comprises a polysaccharide, or a mixture of polysaccharides, or a protein, preferably collagen, or a mixture of proteins, or even a mixture of polysaccharide(s) and protein(s). The biomaterial may in particular comprise polysaccharides, or modified polysaccharides or combinations of polysaccharides with collagen or other proteins. The biomaterial may also include other components, such as coagulants.

In a preferred embodiment, the polysaccharide is a chitosan, hyaluronic acid, an alginate, a pectin or a modified polysaccharide such as carboxymethylcellulose (CMC), alone or in a mixture.

Preferably, the biomaterial is a hydrogel comprising from 1.5% to 6% chitosan and from 94 to 98.5% water.

The greater the degree of acetylation of chitosan, the more flexible the hydrogel, which possibly makes it possible to adapt the mechanical properties of the bioreactor. To guarantee stability throughout the culture process, it is preferable to select a degree of acetylation between 0 and 30%, preferably between 0 and 15%. Preferably, the biomaterial is a chitosan hydrogel having a degree of acetylation less than or equal to 5% and a molar mass of the order of 300,000 g/mol, or more, for example from 300,000 g/mol to 600,000 g /mol. The measurement of the mass and number average molar mass is carried out by steric exclusion chromatography coupled with a measurement of refractive index and a measurement of multi-angle light scattering, as detailed in the literature (for example Sorlier, et al, Biomacromolecules, 2001, 2(3), 765-772).

Hydrogels are macromolecular networks, with a high water content, capable of at least partially reproducing the extracellular matrix of hydrated native tissues. According to the invention, the term "hydrogel" designates a viscoelastic mass comprising at least 70%, preferably at least 80% or 90% by weight of water, for example between 94 and 99% by weight of water, for example from 96 to 98.5% by weight of water. The hydrogel of the present invention is a chemical hydrogel (the interactions responsible for the inter-chain crosslinking are of the covalent bond type) or a physical hydrogel (the interactions responsible for the cross-links are of the physical type, for example hydrogen bonds and/or or hydrophobic interactions, electrostatic interactions, physical cross-linking of crystallites or nanocrystallites). Preferably, the hydrogel of the present invention is a physical hydrogel, in order to avoid the toxicity of the covalent crosslinking agent. Preferably, the hydrogel of the invention does not contain any chemical (covalent) or physical crosslinking agent.

A high water content (preferably greater than 90%, more preferably greater than 95%, more preferably approximately 98%) makes it possible to mimic the natural hydration of the fabric.

Preferably, the total mass fraction of polysaccharide and/or collagen in the hydrogel biomaterial is from 0.1 to 20%, preferentially from 0.1 to 3%.

In a preferred embodiment, the hydrogel is formed by dissolving chitosan in an aqueous acid solution, for example hydrochloric acid, so as to obtain a percentage by mass of chitosan in the aqueous solution, preferably between 1 and 2.5%. After complete dissolution of the chitosan, an alcohol, for example 1,2-propanediol, is added to the solution obtained. The quantity by mass of alcohol introduced is advantageously identical to the quantity by mass of aqueous acid solution used. The solution is homogenized, for example, with stirring, then is poured into a mold of size and shape chosen beforehand according to the intended application for the hydrogel which is going to be formed. The gelation is then carried out at a temperature of between 30 and 70° C., advantageously of the order of 50° C., for example by positioning the mold in an oven. The gelation time depends on the concentration of chitosan in the starting solution, but also on the degree of acetylation and the molar mass of the chitosan, the gelation temperature, the quantity of solution placed in the mold and finally the ratio mold height/area. It is therefore clearly understood that at the end of the gelation, all of the polyelectrolyte solution introduced into the mold has gelled in mass due to evaporation of a significant fraction of water. This alcogel, which is unstable in a physiological medium, is then immersed in an aqueous coagulating bath (for example an aqueous solution of 1M sodium hydroxide) to neutralize the polymer and form a hydrogel. Interrupting the neutralization by taking the alcogel out of the coagulating bath during neutralization, then resuming the neutralization by a second immersion in the coagulating bath makes it possible to generate cavities between membranes, as described in the literature ( Ladet, et al, Nature, 2008, 452 (7183), pp.76-79) and patent application EP1855792). The biomaterial has permeability (by diffusion through its porosity) to the culture medium formulated for folliculogenesis, as well as for CO ₂ and oxygen. Very advantageously, the biomaterial indeed has microcavities and nanocavities containing water or culture medium, between the polymer chains. This porous structure allows air, CO ₂ and elements of the culture medium (such as hormones, vitamins, nutrients) in which the bioreactor is placed, to diffuse through the hydrogel to reach the ovarian tissue.

The thickness of the walls of the bioreactor, that is to say of the biomaterial constituting the bioreactor around the cavity, is preferably between 0.1 and 10 mm, preferably between 0.5 and 2 mm.

The bioreactor is made with the flexible biomaterial described below, and comprises at least one cavity in which the ovarian tissue is placed and preferably confined, i.e. separated from the culture medium by one or more permeable membranes.

The cavity or cavities are formed by an interrupted neutralization of the hydrogel to form a multimembrane object, or come from a mold whose shape makes it possible to generate the cavity.

The bioreactor can be pre-made or optionally formed around ovarian tissue. The initial bioreactor may for example have a cavity with a minimum volume of 1 mm ³ .

The method advantageously comprises at least one step of transferring the ovarian tissue or the ovarian cells in culture, to a bioreactor of larger size, allowing the growth of the ovarian tissue and/of the follicle(s). For example, for growth and folliculogenesis of human ovarian tissue, it is possible to use a bioreactor providing a cavity with a volume of approximately 350 mm ³ to 380 mm ³ .

In general, the tissue volume/cavity volume ratio is advantageously from 1/1 to 1/350, preferably 1/15.

Preferably, the bioreactor comprises a pre-formed tube.

The volume of the tubular channel is in agreement with the tissue volume to be cultured.

According to one embodiment, the tube is ligated on each side hermetically or partially to ensure its confinement and the assembly is placed in a culture medium.

According to another embodiment, the bioreactor is sealed by the formation of at least one hydrogel-based plug. For example, for a chitosan hydrogel, a chitosan solution with a chitosan concentration preferably higher than the concentration of the hydrogel (for example, the solution for the obturation can have a chitosan concentration of 3 to 3.5% w/ m for a hydrogel originating for example from an extruded solution of 2.5% m/m), can be introduced into the lumen of the tube at one or both ends and gelled by neutralization with a basic solution to form the stopper. This ligation or sealing prevents substantial leakage (i.e. leakage of cells outside the bioreactor), and also prevents possible effects that changes in culture medium could cause.

According to a preferred embodiment, the bioreactor has the shape of a hollow tube, preferably tied or obstructed, at least partially, at its ends, preferably with an outer diameter of approximately 1.5 mm to 3 cm, preferably 6mm to 2cm, or a spherical or hemispherical shape, preferably of an outside diameter of about 2mm to 10cm, preferably 6mm to 2cm.

Preferably, the hollow fiber is prepared by wet spinning under coagulation, as described for example in patent application WO2009/044053. The polysaccharide(s) and/or collagen fibers then preferably comprise, at least over their length, a coaxial membrane.

The method as described in patent application WO2009/044053 thus comprises a step (a) of preparing a spinnable solution of a coagulable macromolecular assembly. Characteristically, it then comprises: b) a step of extruding the solution of the coagulable macromolecular assembly through a cylindrical die, also called a capillary die; c) at least one partial coagulation cycle, comprising on the one hand, a coagulation step consisting in introducing the solution of the extruded coagulable macromolecular assembly into a coagulation bath containing a coagulation agent, the diffusion of which in said solution allows locally passing the macromolecular assembly to the coagulated state, under conditions making it possible to obtain a macrofiber whose section is partially coagulated, and, on the other hand, a coagulation interruption step following each coagulation step ; d) and a step for expelling the luminal solution and/or internal membranes.

By the expression “capillary die” is meant here a die which is not annular or which does not implement a central element for forming a central channel in the fiber by internal coagulation, in addition to the external coagulation.

The expression “spinnable solution” is understood to mean a solution of a macromolecular assembly whose characteristics, in particular rheological characteristics, make it capable of being extruded continuously. The expression “coagulable macromolecular assembly” means any macromolecule, in particular of the polymer or protein type, which can pass from a liquid phase to a solid phase by the action of a treatment agent, called a coagulation agent.

This process makes it possible to modify the internal diameter of the central channel, by controlling the interruption of coagulation during the last cycle, so that the core of the fiber remains in the form of a liquid solution, not coagulated. Thanks to this process, it is possible to obtain hollow fibers, mono or multi-membrane.

Alternatively, the bioreactor can have a shape of the capsule or sphere type, and can be obtained for example as described in patent application WO2006/092540.

By capsule is meant a closed volume, also called heart, delimited by at least one membrane acting as an envelope. Such capsules can be of any shape, for example cylindrical, parallelepipedic, spherical or ovoid. By plurimembrane capsule is meant a capsule delimited by at least two successive membranes. The capsules of the present invention can have any size. The constituent membranes, for their part, have an average thickness linked, in particular, to the dimension of the capsule and to the number of membranes.

The capsule or sphere can thus be formed from a succession of non-united membranes and made up of the same polysaccharide, in the form of a physical hydrogel. A process for producing it typically comprises the following successive steps consisting in: a) forming a physical hydrogel of an ionizable polysaccharide, in cationic or anionic form, in a mold of the shape adapted to the desired shape for the capsule, b) immersing the hydrogel in an alkaline bath in the case of a cationic polysaccharide, or acid in the case of an anionic polysaccharide, for a given immersion time, then leaving it to rest outside the bath for a given rest time, so as to obtain a first membrane, c) repeat step b), as many times as additional membranes desired, to form successive membranes from the outside towards the inside of the capsule.

The preservation and folliculogenesis process

The bioreactor is preferably closed, or at least partially closed, to promote tissue containment. Partial opening may nevertheless be possible, preferably to promote oxygenation. Then the bioreactor is placed in a culture medium for tissue maturation.

A culture medium is used. The culture medium in which the bioreactor is placed or immersed comprises one or more components among growth factors, hormones, vitamins, amino acids, antibiotics, antifungals, proteins such as albumin, alpha-globulin, beta-globulin, metabolites. Gonadotropins such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are typically added to the culture medium.

The process allows the preservation of the ovarian tissue for a sufficient time, without the need to resort to freezing, for example while waiting for a transplant of the preserved ovarian tissue.

A method is therefore also described here for grafting ovarian tissue to a subject (human or non-human mammal), in which the ovarian tissue is previously cultured and preserved by the method of the invention. In a particular embodiment, it is an auto-graft.

A folliculogenesis activator, such as activin A or PI3-kinase activator 740YP (Tocris), or the agents MHY1485 or bpV (HOpic), can be added to the culture medium when in vitro folliculogenesis is desired . The activators can be used alone or in a mixture (for example a mixture of 740YP and bpV).

According to a particular example, the bioreactor containing the tissue is placed in a well or a tank containing the culture medium. The bioreactor is immersed in the culture medium or preferably at the interface between the medium and the ambient air, with an oxygen concentration which can typically be 20%.

In another embodiment, when the bioreactor is immersed in the culture medium, air bubbling is implemented in order to have sufficient oxygenation for the culture and/or the folliculogenesis.

Preferably, the bioreactor is placed at the interface between the air and the culture medium. This can be achieved by any method known to those skilled in the art and for example by adding to the reservoir comprising the culture medium a support, preferably a perforated support and the like, such as a grid, at the interface between air and said culture medium. This advantageously improves the oxygenation and consequently the ripening and the efficiency of the method according to the invention. As mentioned above, advantageously, the bioreactor has a permeability allowing exchanges between the culture medium and the biomaterial comprising the ovarian tissue, allowing contact between the ovarian tissue and the culture medium.

The method of the present invention is carried out under known temperature conditions by a person skilled in the art to allow culture and/or folliculogenesis of ovarian tissue. The ideal temperature for growing human ovarian tissue is 37°C. This temperature will be adapted according to the species, for example it is preferably 39°C in ewes (physiological conditions).

The follicles or oocytes obtained are recovered by opening the bioreactor, typically by puncture using a fine needle or by microdissection. The follicles which may be present in the culture medium are also advantageously recovered.

In one embodiment, a solution or gellable (eg, coagulable, cross-linkable, etc.) material is placed around the ovarian tissue. Then the material is left to harden or gel. The quantity of material formed around the tissue and its nature are adapted to allow confinement of the tissue. The culture of the ovarian tissue can also be facilitated by an additional solid structure (network type, grid, walls, container such as a Petri dish, tube, etc.) which can be used at the time of hardening or even be maintained to help preserve the structure of the bioreactor during the culture.

Follicles or oocytes can be cryopreserved pending a fertilization project.

Another object of the invention is therefore an in vitro fertilization process, comprising the preparation of at least one oocyte by means of the process as described here, and the fertilization of the oocyte by spermatozoa.

The examples and figures illustrate the invention without limiting its scope.

Example 1: Production of the hydrogel as a hollow tube

The first step consisted in dissolving chitosan (lyophilisates or flakes or powder) by immersing it in water and adding acetic acid in sufficient quantity, according to a molar ratio n _{Acetic acid} / n _glucosamine between 1 and 1, 1, (to protonate the NH ₂ functions of chitosan). This dissolution process was continued for 16 hours under mechanical stirring. The resulting solution was then centrifuged to remove air bubbles.

The next step consisted in gelling this solution in contact with an aqueous solution of sodium hydroxide at a concentration of 1M (gelling bath, volume: 5L). Tube formation was performed using a Nordson Ultimus pressurized fluid dispensing device. A syringe was filled with chitosan solution. The outlet of this syringe, or die, consists of interlocking tubes to control the final diameter of the extrudate. A plunger was placed above the solution in the syringe, with a pressure of 0.35 bar for 1 second. The viscous chitosan solution was thus extruded (polymer concentration: 2.5% mass/mass). The extrudate was immersed in the gelling bath. On contact with the sodium hydroxide solution, the chitosan solution externally gels the extrudate to form a tube containing the non-neutralized solution, with an external diameter conditioned by the die outlet diameter. After extrusion in the gelation bath, the chitosan extrudate was cut to separate it from the die and allowed to gel for 2 minutes with 200 rpm agitation. Then the rotational speed was increased to 600 rpm for 30 seconds.

Stopping gelling to form a tube was accomplished by removing the gelling extrudate from the sodium hydroxide solution. Then, the tube was washed in distilled water. Until this stage the tube contains a chitosan solution in its lumen. The ends of the tube were then cut off and the tube cleaned by removing the solution and gel residue with forceps. The last steps consist in rinsing the tube abundantly in distilled water to eliminate the sodium hydroxide until a neutral pH of the washing waters, then in sterilizing the tubular hydrogels by autoclave, at 120°C for 21 minutes.

Example 2: Culture of human fetal ovarian tissue in a closed hollow tube over a period of 3 months

1 Materials and methods

1.1 Choice of model:

Human fetal ovaries have the advantage of containing only primordial follicles which implies the absence of growing follicles. The selected samples had not undergone any gonadotoxic treatment. They came from fetuses from 17 to 24 weeks of amenorrhea.

1.2 Sample processing:

After being collected, the two ovaries were transported in an adequate transport medium (Leibovitz L15, Eurobio, Courtabeouf, France) to the laboratory for culturing. The time between stopping ovarian vascularization and culturing varied from 12 to 24 hours. In the laboratory, the ovary was weighed and then dissected into five fragments of 20 mg +/- 2 mg. A fragment was directly fixed in 4% formaldehyde (Sigma-Aldrich); this allows the study of the tissue in the fresh state on days 0 (D0). The other four fragments were cultured, two directly in the culture medium in a well (group A tissues) and two in the structure of the hydrogel-based bioreactor (group B tissues) which was then completely immersed in the culture centre. The advantage of using fragments from the same ovary is to be able to compare the effects of the different parameters tested on the same sample.

1.3 Culture medium:

The culture medium used was developed and tested on sheep ovaries. The composition as well as the frequency of medium changes were identical for all the samples. It is composed of alpha MEM medium, streptomycin, penicillin, amphotericin B, synthetic serum (Irvine), ascorbic acid (Sigma-Aldrich), insulin, transferrin, selenium and FSH.

1.4 Bioreactor structure:

These hydrogel structures take the form of a flexible tube of 6mm external diameter and 4 mm internal diameter. The fragment was inserted inside, then the tube was closed at each end with flexible suture threads (Figures 1 and 2).

1.5 Cultivation:

The culture started on the day of the dissection (D0) and continued for 90 days with a change of medium every 2 days.

1.5.1 Crop stops:

A first stop was made at 45 days to evaluate the effects of the mid-culture structure on the 2 ovaries studied. A fragment of group A and a fragment of group B from each ovary were arrested for macroscopic (appearance, weight) and microscopic study after fixation in 4% formaldehyde (Sigma-Aldrich). The culture medium from the day of the stop was frozen at -20°C. Group B fragments had to be extracted from the bioreactor. This was therefore cut using a scalpel, its appearance was noted (Figure 3).

A second stop was made at 90 days of culture; a fragment from group A and a fragment from group B from each ovary were arrested for macroscopic (appearance, weight) and microscopic study after fixation in 4% formaldehyde (Sigma-Aldrich). The culture medium from the day of the stop was frozen at -20°C.

1.5.2 Tissue activation at 80 days of culture:

At 80 days of culture, the ovary of the F1 fetus (fragment A and fragment B) was cultured in the culture medium without addition of activin A until D90. The ovary of the F2 fetus (fragment A and fragment B) was cultured in the culture medium with the addition of activin A at a concentration of 100 ng/mL for 10 days, i.e. until D90.

1.6 Histology:

All fragments, regardless of group, were dehydrated in 4% formaldehyde (Sigma-Aldrich) for 24 hours. They were then embedded in a paraffin block in order to proceed with microtome sectioning. Each fragment was cut into sections 4 µm thick. 80 sections were made; 20 were intended for histological study and 60 for immunohistochemical study.

For the histological study, we used Hematoxyline/Phloxine/Saffron (HPS; Sigma-Aldrich) staining. The reading was carried out using an optical microscope. The follicles were classified according to the classification of Gougeon, supra . Measurement of follicular diameters, follicular surfaces, vessel surfaces was performed using ImageJ software (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih .gov/ij/, 1997-2018.).

1.7 Immunolabelling:

Immunological labeling makes it possible to highlight protein targets expressed in the cells. In our study, we carried out two markings: a marking of Ki67 (Cell Signaling Technology) which is a protein expressed by proliferating cells and a marking of cleaved caspase 3 (Cell Signaling Technology) which is expressed by cells in apoptosis. .

2. Results

2.1 Histology:

2.1.1 Follicular density:

At each stop, follicular density was assessed by counting on HPS-stained slides. On the fresh tissue, there were on average 514.77 follicles per mm². After 45 days of culture of the group A fragments, the average follicular density was 366.17 follicles per mm² and after 90 days, the average follicular density was 276.34 follicles per mm². After 45 days of culture of the group B fragments, the average follicular density was 255.09 follicles per mm² and after 90 days, the average follicular density was 219.37 follicles per mm².

The average follicular density decreased significantly between the fresh tissue and the tissues of the two groups cultured at D44 and D90. This follicular density was lower in group B. However, the follicular density remained stable throughout the culture (Figure 4).

2.1.2 Appearance of the fabric:

In this study we observed tissues that retain their cellular cohesion with few cellular alterations. Primordial follicles remain intact. It is important to note the absence of growing follicles in the two groups up to D90 of culture in the absence of activin A (Figure 5).

2.2 Immunolabelling:

2.2.1 Study of cell proliferation: labeling of the Ki67 antigen

At each culture stop, immunological labeling of Ki67 was performed on the tissue sections to assess cell proliferation (Figures 7 and 8).

On fresh tissue, there was an average of 566.6 proliferating cells/mm². After 45 days of culture of the group A fragments, there were 106.17 cells/mm² proliferating and after 90 days of culture of the group A fragments, there were 84.34 cells/mm² proliferating. After 45 days of culture of the group B fragments, there were 155.37 cells/mm² proliferating and after 90 days of culture of the group B fragments, there were 158.82 cells/mm² proliferating (Figure 6).

We observed a significant decrease in the number of proliferating cells of the fragments of groups A and B compared to the initial fresh tissue. The number of proliferating cells within the A fragments decreased during the culture while that of the B fragments remained constant until the end of the culture. The bioreactor allows the maintenance of cell proliferation throughout the duration of the tissue culture.

It is interesting to note that at 90 days of culture, the fragments of group A presented acellular zones with loss of cohesion of neighboring cells, a sign of cellular alteration (Figure 9).

Whereas at 90 days of culture, the fragments of group B presented clear zones which are crystalline inclusions, of undetermined origin for the moment, there was therefore no loss of cellular cohesion (Figure 10) .

2.2.2 Immunolabelling of cell apoptosis: cleaved caspase 3

We did not observe an increase in cell apoptosis during culture in both groups.

2.3 Follicular growth in the bioreactor

2.3.1 Presentation of the protocol used

We cultured two ovaries in vitro until D80 in the basic culture medium without activator. For each ovary we had a control tissue cultured in a well (group A) and a tissue cultured in the bioreactor (group B).

Then we added activin A for one of the two ovaries for the control tissue and the tissue cultured in a bioreactor; and for the other ovary no activator for the control tissue and the tissue cultured in a bioreactor.

Stopping the culture on D90 either after 10 days of additional culture with activin A or without activator.

Results obtained

2.3.2.1 Tissues cultured in the absence of activator:

- Control tissue (group A) at D90:

The ovarian tissue is opaque white in color. In histology, ovarian follicles are only primordial and primary follicles.

- Tissue in bioreactor structure (group B) at D90:

Ovarian tissue is pearly white in color, similar in appearance to fresh tissue. In histology, ovarian follicles are only primordial and primary follicles. Absence of growing follicles.

2.3.2.2 Tissues cultured in the presence of activator:

- Control tissue (group A) at D90:

The ovarian tissue is opaque white in color. In histology, ovarian follicles are only primordial and primary follicles. The average diameter of the follicles is 18.05 µm ± 7 (Photograph 12).

- Tissue in bioreactor (group B) at D90:

The ovarian tissue is pearly white in color, weighing 21 mg or 1 mg more compared to D0. In histology, presence of 3 secondary follicles with an average diameter of 545.64 µm ± 280.76 (Figures 11-13).

3. Conclusion:

On microscopic examination, 3 secondary follicles are observed in the fragment cultured within the bioreactor in the presence of activin A. This initiation of folliculogenesis within the ovarian tissue highlights the functionality of the tissue after three months of culture within of this structure and the capacity to support this initiation.

This bioreactor allows tissue survival for three months as well as in vitro folliculogenesis. It also makes it possible to maintain tissue activity verified by the presence of constant cell proliferation and the capacity for folliculogenesis after 80 days of culture.

Example 3: Preservation and Survival of Ewe Ovarian Tissue

1. MATERIALS AND METHODS

1.1 Collection of ovarian tissue

The ovaries were taken from prepubertal ewes from the slaughterhouses of Corbas (Lyon, France), after approval by the responsible veterinarian. Only one ovary per ewe was recovered. They were collected then immersed in Leibovitz L-15® medium (Eurobio, Courtaboeuf, France) and transported to the laboratory at 10°C in less than 40 minutes.

1.2 Sample processing

Each ovary was then dissected using a scalpel in a Petri dish under sterile conditions. The cortex was isolated, then cut into fragments of identical size. Each fragment was weighed before culturing.

1.3 Cultivation

1.3.1 Cultivation system

The fragment of ovarian tissue was introduced into a chitosan hydrogel tube with an external diameter of 6 mm, ie a lumen with a diameter of 4 mm, under sterile conditions under a laminar flow hood. Each end of the tube was closed with a surgical suture. The average fragment weight was 150 mg. It was cultured in a tube 3 cm in length reduced to 2 cm after suturing. The control fragment is cultured in wells, without culture support.

1.3.2 Cultivation conditions

The ovarian tissue fragments were cultured in an incubator at 37° C. under 5% CO ₂ . Tissues within the hydrogel-based bioreactor were cultured in flasks (test tissues). The control tissues were cultured in wells without bioreactor and in flasks without bioreactor (control tissues). For the same ovary, several test tissues and control tissues were carried out. A test tissue and a control tissue from the same ovary are studied at defined times for histological and immunohistochemical studies.

1.3.3 Culture medium

The culture medium is composed of alpha MEM supplemented with L-glutamine, insulin, transferrin and selenium, FSH, SSS, ascorbic acid and antibiotics.

1.4 Histological study

When the culture was stopped, the tissues were weighed and measured. The general aspect was noted (flexibility of the fabric, aspect, color). The tissues were then fixed in 4% formaldehyde (VWR, Strasbourg, France). Then they were dehydrated and embedded in paraffin to be cut into 4 µm sections. For each fragment, 120 cuts were made. For the histological study, 20 stages were made, spaced 16 µm apart with two sections per stage and stained with HPS. The other 20 levels are intended for the functional study of the tissue by immunohistochemistry.

The criteria for ovarian tissue alteration were the presence of tissue detachments, pyknotic nuclei, tissue disintegration.

1.5 Functionality of fabric

- Labeling of cleaved caspase 3

The primary antibody is a cleaved anti-caspase 3 rabbit monoclonal antibody (9664S; Cell Signaling Technology, Beverly, MA) which is a specific marker of apoptosis. The protocol used is that of the provider. The secondary antibody is an anti-rabbit IgG conjugated to FITC (Cell Signaling Technology). Tissue sections are deparaffinized in baths of methylcyclohexane (Sigma-Aldrich) then absolute ethanol (Sigma-Aldrich). After rinsing with clear water, the ovarian fragments are unmasked by microwave heating while immersed in citrate buffer (Merck, Darmstadt, Germany) at pH=6. This is followed by a step of blocking the non-specific binding sites and then incubating the cleaved anti-caspase 3 primary antibody (1:200 dilution) overnight at 4°C in a humid chamber. The slides are then rinsed with PBS and again incubated for 1h30 with the secondary antibody at 4°C protected from light (1:500 dilution). The tissues are counterstained with propidium iodide (Sigma-Aldrich) which stains the nuclei. The slides are mounted with an aqueous Fluoprep mounting medium (bioMerieux, Marcy l'étoile, France) and stored at 4°C away from light while awaiting reading by two observers under a fluorescence microscope (Axiovert 200M, Carl Zeiss, Oberkochen, Germany).

- Marking of DNA breaks by Tunel method

The ApopTag® fluorescein in situ apoptosis detection kit (Millipore, US) is used to detect cells in apoptosis by an indirect TUNEL method. We used an anti-digoxigenin antibody which is conjugated with a Fluorescein reporter molecule. DNA strand breaks are detected by labeling free 3'OH regions with modified nucleotides. Indeed, during DNA fragmentation, these new 3'OH regions are generated. This kit therefore allows us to detect both: single-stranded and double-stranded DNA breaks. There are many biochemical and morphological differences between apoptotic and necrotic cells, so one can focus only on apoptotic cells with this kit by specifically identifying DNA cleavage and cell-bound chromatin condensation. apoptotic. To reveal our results we use a fluorescence microscope with a FITC filter (Axiovert 200M, Carl Zeiss, Oberkochen, Germany).

2. RESULTS

Each step, from ovary recovery to culture, is controlled and reproducible.

We observed an absence of alteration of the ovarian tissue after 3 months of culture in our culture system in 7 ewes compared to the cultured control tissue.

The control tissues show a particularly significant DNA fragmentation, especially at the level of the medulla.

Regarding the tissues cultured in the structure of the hydrogel-based bioreactor, it can be noted that in the majority of cases the epithelium does not show any degradation of the DNA since it does not show fluorescence. Blood vessels and follicles are also only minimally affected.

Claims (13)

In vitro or ex vivo method for preserving ovarian germinal tissue or ovarian germinal cells, comprising culturing the ovarian germinal tissue or ovarian germinal cells in a bioreactor whose walls are composed of a biomaterial in the form of a hydrogel which allows exchanges of at least nutrients and gaseous exchanges with the culture medium in which the bioreactor is placed, the culture of the ovarian tissue being carried out preferably from 1 month to 6 months. Method according to claim 1, the culture medium in which the bioreactor is placed comprising one or more components among growth factors, hormones, vitamins, amino acids, antibiotics, antifungals, proteins such as albumin, alpha-globulin, beta-globulin , metabolites. An in vitro or ex vivo method of folliculogenesis from germinal ovarian tissue or germinal ovarian cells, comprising maturing the germinal ovarian tissue or germinal ovarian cells in a bioreactor whose walls are composed of a biomaterial in the form of a hydrogel which allows exchanges of at least nutrients and gaseous exchanges with the culture medium in which the bioreactor is placed. Method according to claim 3, the culture medium in which the bioreactor is placed comprising at least one folliculogenesis activator, preferably activin A. Process according to one of Claims 1 to 4, in which the bioreactor is closed or at least partially closed. Process according to one of Claims 1 to 5, in which the ovarian tissue is i) an ovary fragment comprising cortex and medulla, ii) an entire ovary, or iii) a tissue filtrate comprising primordial follicles associated to ovarian medulla cells, obtainable by dissociation and filtration of ovarian tissue. Process according to one of Claims 1 to 6, in which the bioreactor has the shape of a hollow tube, preferably tied or obstructed, at least partially, at its ends, preferably with an outside diameter of approximately 1.5 mm to 3 cm, preferably 6 mm to 2 cm, or a spherical or hemispherical shape, preferably of an outside diameter of about 2 mm to 10 cm, preferably 6 mm to 2 cm. Process according to one of Claims 1 to 7, in which the walls of the bioreactor have a thickness of 0.1 to 10 mm, preferably 0.5 to 2 mm. Process according to one of Claims 1 to 8, in which the biomaterial in the form of a hydrogel comprises a polysaccharide, a mixture of polysaccharides, or a protein or a mixture of proteins, preferably collagen, or else a mixture of polysaccharides ( s) and protein(s). Process according to Claim 9, in which the polysaccharide is a chitosan, hyaluronic acid, an alginate, a pectin or a modified polysaccharide such as carboxymethylcellulose (CMC), alone or in a mixture. A method according to claim 10, wherein the biomaterial is a hydrogel comprising 1.5 to 6% chitosan and 94 to 98.5% water. Process according to claim 9 or 10, in which the biomaterial is a chitosan hydrogel having a degree of acetylation less than or equal to 30%, preferably less than or equal to 15%, or even more preferably less than or equal to 5%, and a molar mass greater than 300,000 g/mol, preferably of the order of 300,000 to 600,000 g/mol. Process according to one of Claims 1 to 12, comprising at least one step of transferring ovarian tissue or ovarian cells in culture to a larger bioreactor, allowing the growth of the ovarian tissue and/or of the follicle(s).