PROCESS FOR IMPLEMENTING IN VITRO SPERMATOGENESIS AND ASSOCIATED DEVICE

Abstract

The present invention relates to a process for in vitro spermatogenesis from male germinal tissue comprising conducting maturation of testicular tissue comprising germ cells in a bioreactor which is made of a biomaterial and comprises at least one cavity wherein the germinal tissue is placed, and recovering elongated spermatids and/or spermatozoa.

Claims

1. Method for in vitro spermatogenesis from male germinal tissue comprising conducting maturation of testicular tissue comprising germ cells in a bioreactor which is made of a biomaterial and comprises at least one cavity wherein the germinal tissue is placed, and recovering elongated spermatids and/or spermatozoa.

2. Method according to claim 1, wherein germinal tissue, preferably testicular tissue, comprises at least one seminiferous tubule or fragments of at least one seminiferous tubule, preferably several seminiferous tubules or fragments of several seminiferous tubules.

3. Method according to claim 2, wherein the germinal tissue, preferably testicular tissue, comprises fragments from 2 to 50, 3 to 40, 4 to 30, 5 to 20 seminiferous tubules.

4. Method according to claim 2, wherein the tubules and/or fragments are obtained through mechanical separation or enzymatic separation of seminiferous tubules.

5. Method according to claim 2, wherein the fragments of seminiferous tubules have a size comprised between about 1 mm and about 5 mm.

6. Method according to claim 1, wherein the germinal tissue, preferably testicular tissue, comprises germ cells, Sertoli cells and peritubular cells, and optionally Leydig cells.

7. Method according to claim 1, wherein cells selected from the group consisting of germ cells, Sertoli cells, peritubular cells and mixtures thereof, are added to the germinal tissue, preferably testicular tissue.

8. Method according to claim 1, wherein the volume of the cavity or the volume of germinal tissue, preferably testicular tissue, is from about 1 to about 100 mm.sup.3, preferably from about 0.5 to about 150 mm.sup.3, for example from about 1 to about 30 mm.sup.3.

9. Method according to claim 1, wherein the germinal tissue, preferably testicular tissue, is from: a healthy prepubertal or postpubertal patient about to undergo a gonado-toxic treatment or surgery, for example cancer-therapy; a postpubertal patient who does not produce spermatozoa for example due to genetic or acquired non-obstrusive azoospermia, bilateral chryptorchidism during childhood or severe sickle cell disease; a prepubertal patient having bilateral chryptorchidism or severe sickle cell disease; endangered species; horses, camel, dromedary or pets; and livestock.

10. Method according to claim 1, wherein the biomaterial is in the physical hydrogel state and preferably comprises a polysaccharide, preferably a natural polysaccharide, or structural protein, preferably collagen, or a mixture thereof.

11. Method according to claim 10, wherein the natural polysaccharide is chitosan, hyaluronic acid, alginate, pectine or a modified natural polysaccharide such as carboxymethylcellulose (CMC), alone or in mixture, preferably chitosan or alginate, alone or in mixture, preferably chitosan.

12. Method according to claim 1, comprising the following steps: a) Providing a sample of germinal tissue; b) Providing a bioreactor which is made of a biomaterial and comprises at least one cavity; c) Introducing said germinal tissue into the at least one cavity of said bioreactor; optionally sealing the bioreactor d) Placing said bioreactor containing said germinal tissue in a tank comprising a culture medium; e) Conducting maturation of the germinal tissue until elongated spermatids and/or spermatozoa are obtained; and f) Recovering elongated spermatids and/or spermatozoa from the bioreactor.

13. Method according to claim 1, wherein the bioreactor is formed around the germinal tissue.

14. Method according to claim 1, wherein the bioreactor is a hollow fiber of a biomaterial, comprising a channel wherein the germinal tissue is placed.

15. Method according to claim 1, wherein the bioreactor is made of an hydrogel.

16. Method according to claim 1, in which the bioreactor is placed in or at the contact of a culture medium, wherein: the culture medium comprises one or more of growth factors, hormones, testosterone, vitamins, antibiotics, metabolites, etc., alone or in mixture; or the culture medium comprises one or more of growth factors, hormones, vitamins, antibiotics metabolites, etc., alone or in mixture and testosterone is added to the culture medium in the course of the process.

17. Method according to claim 1, wherein the biomaterial is such that it enables air, carbon dioxide, and the elements of the culture medium into which the bioreactor is placed for maturation, to diffuse through it.

18. Process of in vitro fertilization comprising the following steps: a) Preparation of elongated spermatids and/or spermatozoa according to the process of claim 1; or providing elongated spermatids and/or spermatozoa by the process according to claim 1; b) Fertilization of an oocyte with the elongated spermatids and/or spermatozoa obtained.

19. Kit for implementing in vitro spermatogenesis from male germinal tissue comprising a bioreactor which is made of a biomaterial and comprises at least one cavity wherein germinal tissue is placed.

Description

[0094] The present invention will now be described by means of examples.

[0095] FIGS. 1, 2, 9 and 10 represent germ cells of 20-days old rats after 39 days of culture.

[0096] FIGS. 3, 4, 5, 6, 7 and 8 represent germ cells of 8-days old rats after 61 days of culture.

[0097] FIGS. 5 and 6 represent testis cross sections of a 8-day and a 60-day-old rat respectively.

[0098] FIGS. 11, 12 and 13 represent germ cells of 1.5 year-old cynomologus monkeys after 54 days of culture.

[0099] FIGS. 14 and 15 represent germ cells of transsexual man after 34 days of culture.

[0100] Patients

[0101] The process according to the invention was implemented on 8 or 20-days-old male Sprague-Dawley rats, on two 1.5-year-old cynomolgus monkey and on a transsexual man. After anesthesia, rats were killed by decapitation and their testes were quickly removed. Testes of the cynomolgus monkeys and testes of the transsexual man were obtained by surgery. The testes were immersed in Ham's F-12/Dulbecco's Modified Eagle's medium (F12/DMEM, 1:1).

[0102] Preparation of Seminiferous Tubules

[0103] The tunica albuginea of testes was mechanically removed, and seminiferous tubules were isolated by digestion at 33 C. in F12/DMEM (1:1) containing collagenase, 2 mg/ml lima bean trypsin inhibitor, and 10 mg/ml DNase for 10 min under gentle agitation. Seminiferous tubules were harvested by low-speed centrifugation, washed twice with F12/DMEM (1:1).

[0104] Example General Preparation of a Bioreactor:

[0105] A solution of a biomaterial, for example chitosan, was prepared in deionized water. This can be operated in a closed reactor with mechanical stirring. After polymer dissolution, the solution is placed in a syringe and the bubbles can be removed by centrifugation (5000 g, 10 min). The solution was extruded using a syringe pump, the syringe is connected to an extrusion cone with extrusion hole diameter of 3mm. The extrusion is operated in a coagulation bath (NaOH aqueous solution with concentration 1M) inducing the formation of a physical hydrogel, preferably a physical chitosan hydrogel. An external membrane with tubular form is obtained by NaOH radial diffusion from the periphery to the center of the cylindrical extrudate. Depending on the coagulation time (ex: 2 minutes), the thickness of the coagulated hydrogel tube can be adapted to the desired value (ex: 1 mm). After a given coagulation time, the tubular hydrogel still containing a polymer, for example chitosan solution is poured in a large volume of deionized water in order to stop gelation. The non-gelated internal solution can be removed by the introduction of a water or air flux inside the tube thus creating the lumen of the tube, with a length of 1 to 100 cm. The bioreactors can then be cut from this preformed tube at the desired length (about 3 cm). The bioreactors can then be washed in a distilled water baths, and sterilized in water by autoclave treatment (121 C., 20 minutes).

[0106] Preparation of the Bioreactor Used for the Examples:

[0107] The bioreactors are mono-membrane hollow fibers of chitosan obtained by the process described in WO2009044053. The bioreactor is based on chitosan from squid pen chitin (Mahtani chitosan, Veraval, India; Mahtani batch indexes 114) with acetylation degree of 4% (as determined by Hirai method as described in WO2009044053) with mean molecular mass Mw of 550 kg/mol (as determined by Size exclusion chromatography coupled with refraction index measurement and multi angle light scattering as described in WO2009044053), the interior of the channel obtained has a volume of 20 to 50 mm.sup.3.

[0108] An acetate chitosan solution, with polymer concentration of 2% w/w in deionized water is prepared with stoichiometric amount of acetic acid with respect to the amine moieties of chitosan. This can be operated in a closed reactor with mechanical stirring. After polymer dissolution, the solution is placed in a syringe and the bubbles can be removed by centrifugation (5000 g, 10 min).

[0109] Spermatogenesis

[0110] 20 to 50 mm.sup.3 of the seminiferous tubules were introduced into chitosan tubes. The chitosan tubes were then sealed at both ends and then deposited in a conventional culture well containing approximately 8 ml of culture medium. The medium was changed every two days. The culture medium consisted of 15 mM Hepes-buffered F12/DMEM supplemented with antibiotics, 1.2 g/L NaHCO.sub.3, 10 g/ml insulin, 10 g/ml transferrin, 10.sup.4 M vitamin C, 10 g/ml vitamin E, 3.310.sup.7 M retinoic acid, 3.310-7 M retinol, 10.sup.3 M pyruvate (all from Sigma), 10-.sup.7M testosterone, and 50 ng/ml porcine FSH. For 8 days old rats, 1.5 year-old cynomolgus monkeys and the transsexual man, testosterone was added to the culture medium after several days of culture.

[0111] Histological Studies

[0112] At selected days of culture, seminiferous tubules were extruded from the chitosan tubes, were crushed between two microscopic glass slides. Then the nuclei were stained by Harris's hematoxylin solution.

[0113] The results of the process implemented are presented in the figures.

[0114] Results on 20-Days-Old Rats

[0115] In 20-days-old rats, at the beginning of the culture, the most differenciated germ cells were Pachytene spermatocytes (stage X).

[0116] At different days of culture, germ cells were observed. Cells had a similar appearance to their appearance in vivo. Round spermatids (step 1-4 of spermiogenesis) and elongated spermatids (step 9 of spermiogenesis) were visualized on day 11 of culture. After 39 days of culture a cluster of elongated spermatids with their flagella was observed (FIG. 1). It is also observed heads (H in FIG. 2) and flagella (F in FIG. 2) of elongated spermatids (ES) (step 15-17 of spermiogenesis) at a higher magnification (FIG. 2).

[0117] A large cell cluster was observed at day 39 (FIGS. 9 and 10): early meiotic cells, Preleptotene (Prl) and Leptotene (Lept) spermatocytes were still abundant and a new wave of round spermatids (RS) with young Pachytene spermatocytes was present.

[0118] Results on 8-Days-Old Rats

[0119] By analogy with prebubertal boys who have only spermatogonia in their testes, semiferous tubule cultures from 8 days old rats were performed. Indeed, 8-days-old rats have only spermatogonia in their testes.

[0120] After 61 days of culture, cells are obtained after spreading the crushed cultured seminiferous tubules.

[0121] FIG. 3 shows a portion of a seminiferous epithelium with cell associations from the basement (B) of the tubule to the lumen (L). A cluster of elongated spermatids/spermatozoa (ES/SZ in FIG. 3) with their flagella in the lumen of the cultured seminiferous tubules were observed.

[0122] The organization of cells within the bounds of the seminiferous epithelium is seen in FIG. 4. Sertoli cell nuclei (SCn) and/or spermatogonia nuclei (Gn), Pachytene spermatocytes (PS), round spermatids (RS) and ES/SZ were identified at successively higher levels within the epithelium from the basement (B) to the lumen (L) of the seminiferous tubule. Note that no Preleptotene spermatocyte was present. Heads (H) and flagella (F) of ES/SZ were observed (FIG. 4). These results show a growth of cultured seminiferous tubules during the 61 days of culture compared to in vivo 8-day-old rat seminiferous tubules which have no lumen (FIG. 5 shows a cross section of a 8-day-old rat testis). FIG. 5 shows Sertolli cell (SC), the basement of the seminiferous tubules (B) and the cluster of spermatogonia (G).

[0123] The size of the 61 days cultured seminiferous tubules (FIG. 4) was slightly smaller than the size of in vivo 60-day-old rat seminiferous tubules (FIG. 6 shows a cross section of a 60-day-old rat testis with the basement (B) and lumen (L) of the seminiferous tubules, Spermatogona (G) Pachytene spermatocytes (PS), round spermatids (RS) and spermatozoa (SZ).

[0124] FIG. 7 shows an isolated spermatozoon (SZ) (step 19 of spermiogenesis). At a higher magnification (FIG. 8) it was observed the spermatozoon head (H), the cytoplasmic lobe (CL) and the flagellum (F) of the SZ.

[0125] These results show that the process according to the invention enables to carry out spermatogenesis from a germinal tissue, i.e. from spermatogonia to elongated spermatids or spermatozoa.

[0126] Results on 1.5-Year-Old Cynomolqus Monkeys

[0127] By analogy with the boys who have only spermatogonia in their testes, seminiferous tubules of two 1.5 year-old cynomolgus monkeys were cultured. Indeed, 1.5 year-old monkeys have only spermatogonia in their testes. After 54 days of culture, cells were obtained after spreading the crushed cultured seminiferous tubules. FIG. 11, shows the sperm flagella (F). FIG. 12 shows the head (H) and the flagellum (F) of an ES at a higher magnification. A Pachytene spermatocyte (PS) is besides the ES. FIG. 13: The presence of young spermatocytes (shown by the arrows) indicates that a new wave of meiotic germ cells was ready to differentiate into spermatozoa.

[0128] Results on a Transsexual Man

[0129] Seminiferous tubules of a transsexual man were cultured. After a hormonal treatment that inhibited his spermatogenesis, this man had only spermatogonia, scarce preleptotene spermatocytes and Sertoli cells in his seminiferous tubules. This patient is close to a young boy who has only spermatogonia in his testes. After 34 days of culture, cells were obtained after spreading the crushed cultured seminiferous tubules. FIG. 14 shows flagella (F) of spermatozoa (shown by the arrows). At a higher magnification, FIG. 15 shows the head (H) and the flagellum (F) of a spermatozoon.