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. A method for in vitro spermatogenesis from male testicular tissue comprising: (a) conducting maturation of testicular tissue in a bioreactor which is made of a biomaterial and comprises at least one cavity wherein the testicular tissue is confined, said bioreactor being placed in or in the contact of a culture medium until elongated spermatids and/or spermatozoa are produced; and (b) recovering said elongated spermatids and/or spermatozoa, wherein said cavity is closed during the maturation process, wherein the bioreactor has a permeability that enables sufficient gas exchange and metabolite circulation between the culture medium and the cavity, and the biomaterial being a hydrogel that enables air, carbon dioxide, and the elements of the culture medium into which the bioreactor is placed for maturation to diffuse through it; and wherein the testicular tissue comprises germ cells, Sertoli cells and peritubular cells, where the germ cells are in close association with Sertoli cells.

2. The method according to claim 1, wherein the testicular tissue comprises at least one seminiferous tubule or fragments of at least one seminiferous tubule.

3. The method according to claim 2, wherein the testicular tissue comprises fragments from 2 to 50, 3 to 40, 4 to 30, or 5 to 20 seminiferous tubules.

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

5. The method according to claim 2, wherein the fragments of seminiferous tubules have a length between about 1 mm and about 5 mm.

6. The method according to claim 1, wherein the testicular tissue further comprises Leydig cells.

7. The 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 testicular tissue.

8. The method according to claim 1, wherein the volume of the cavity or the volume of testicular tissue is from about 1 to about 100 mm.sup.3, from about 0.5 to about 150 mm.sup.3, or from about 1 to about 30 mm.sup.3.

9. The method according to claim 1, wherein the testicular tissue is obtained from a subject selected from the group consisting of: a healthy prepubertal or postpubertal patient about to undergo a gonado-toxic treatment or surgery; a postpubertal patient who does not produce spermatozoa 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; a horse, a camel, a dromedary or a pet; and livestock.

10. The method according to claim 1, wherein the biomaterial comprises collagen.

11. The method according to claim 1, wherein the biomaterial comprises a natural polysaccharide selected from the group consisting of chitosan, hyaluronic acid, alginate, pectin and a modified natural polysaccharide, and wherein said natural polysaccharide is used alone or in a mixture.

12. The method according to claim 1, further comprising: i) providing a sample of testicular tissue comprising germ cells, Sertoli cells and peritubular cells, where the germ cells are in close association with Sertoli cells; ii) providing a bioreactor which is made of a biomaterial and comprises at least one cavity, said bioreactor having a permeability that enables sufficient gas exchanges and metabolite circulation between the culture medium and the cavity comprising the testicular tissue, and the biomaterial being a hydrogel that enables air, carbon dioxide, and the elements of the culture medium into which the bioreactor is placed for maturation to diffuse through it; iii) introducing said testicular tissue into the at least one cavity of said bioreactor and sealing the bioreactor; and iv) placing said bioreactor containing said testicular tissue in a tank comprising a culture medium.

13. The method according to claim 1, wherein the bioreactor is formed around the testicular tissue during the formation of the bioreactor.

14. The method according to claim 1, wherein the bioreactor is a hollow fiber of a biomaterial comprising a channel wherein the testicular tissue is confined.

15. The method according to claim 1 wherein: the culture medium comprises a material selected from the group consisting of growth factors, hormones, testosterone, vitamins, antibiotics, metabolites, and mixtures thereof the culture medium comprises a material selected from the group consisting of growth factors, hormones, vitamins, antibiotics, metabolites, and mixtures thereof.

16. The method according to claim 1 wherein: the culture medium comprises a material selected from the group consisting of growth factors, hormones, vitamins, antibiotics, metabolites, and mixtures thereof.

17. The method according to claim 1, wherein the thickness of the biomaterial is from about 0.1 to 10 mm.

18. The method according to claim 1, wherein testosterone is added in the culture medium during the course of the process.

19. The method according to claim 1, wherein the biomaterial is chitosan.

20. The method according to claim 1, wherein the bioreactor is a hollow fiber of chitosan comprising a channel wherein the testicular tissue is confined.

21. Process of in vitro fertilization comprising: a) preparing 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; and b) fertilizing of an oocyte with the elongated spermatids and/or spermatozoa obtained.

Description

(1) The present invention will now be described by means of examples.

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

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

(4) FIGS. 5 and 6 represent testis cross sections of a 8-day and a 60-day-old rat respectively.

(5) FIGS. 11, 12 and 13 represent germ cells of 1.5 year-old cynomologus monkeys after 54 days of culture.

(6) FIGS. 14 and 15 represent germ cells of transsexual man after 34 days of culture.

PATIENTS

(7) 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).

(8) Preparation of Seminiferous Tubules

(9) 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).

(10) Example General Preparation of a Bioreactor:

(11) 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 3 mm. 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).

(12) Preparation of the Bioreactor Used for the Examples:

(13) 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.

(14) 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).

(15) Spermatogenesis

(16) 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.

(17) Histological Studies

(18) 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.

(19) The results of the process implemented are presented in the figures.

(20) Results on 20-Days-Old Rats

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

(22) 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).

(23) 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.

(24) Results on 8-Days-Old Rats

(25) 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.

(26) After 61 days of culture, cells are obtained after spreading the crushed cultured seminiferous tubules.

(27) 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.

(28) 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).

(29) 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).

(30) 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.

(31) 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.

(32) Results on 1.5-Year-Old Cynomolgus Monkeys

(33) 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.

(34) FIG. 11, shows the sperm flagella (F).

(35) 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.

(36) 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.

(37) Results on a Transsexual Man

(38) 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.

(39) After 34 days of culture, cells were obtained after spreading the crushed cultured seminiferous tubules.

(40) FIG. 14 shows flagella (F) of spermatozoa (shown by the arrows).

(41) At a higher magnification, FIG. 15 shows the head (H) and the flagellum (F) of a spermatozoon.