Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/16—Physical preservation processes
  • A01N1/162—Temperature processes, e.g. following predefined temperature changes over time

Definitions

• the present invention relates to methods of preserving functionality of an organ, preserving fertility of a patient undergoing a treatment expected to cause sterility and assuring a supply of viable gametes for future use and, more particularly, to a methods which rely upon removal of an organ, cryopreservation of the removed organ and reintroduction of the cryopreserved organ into a recipient in such a way that the vasculature of the recipient supplies blood to the introduced organ or a portion thereof.
• cryopreservation of large-volume samples introduces heat transfer problems.
• macroscopic samples there is a large thermal gradient from the surface of the sample to the interior.
• the survival of cells in monolyer was higher than isolated cells using the determined optimal cooling rate of 0.2°C/min. This indicates that the attached cells were more tolerant of slow cooling injury (Armitage et al., 1996).
• the need for a slow cooling rate is further illustrated by the cooling rate at which a Wood frog survives freezing (less than 0.1°C/minute; Schmid, 1982).
• the sheep ovary is similar to the human ovary in that it has a dense fibrous stroma and relatively high density of primordial follicles in the ovarian cortex.
• a method of preserving functionality of an organ includes: (a) removing at least a whole organ including vasculature from a donor; (b) cryo-preserving the whole organ including vasculature; (c) allowing a period of time to elapse; (d) thawing the whole organ including vasculature so that at least a portion of cells contained therein resume biological activity; and (e) introducing the whole organ including vasculature into a recipient so that a blood vessel present in the vasculature of the whole organ including vasculature is supplied with blood by a vasculature system belonging to the recipient.
• the phrase "whole organ including vasculature” refers to those blood vessels responsible for local supply of blood to the removed organ.
• a method of preserving fertility of a patient undergoing a treatment expected to cause sterility includes; (a) removing at least one whole gonadal organ including vasculature from the patient; (b) cryo-preserving the at least one whole gonadal organ including vasculature; (c) conducting the treatment and waiting for an effect thereof to subside; (d) thawing the at least one whole gonadal organ including vasculature so that at least a portion of cells contained therein resume biological activity; and (e) introducing at least one whole gonadal organ including vasculature into the patient so that a blood vessel present in the gonadal organ is supplied with blood by a vasculature system belonging to the patient.
• a method of assuring a supply of viable gametes for future use includes;(a) removing at least one whole gonadal organ including vasculature from a patient; (b) cryo-preserving the at least one whole gonadal organ including vasculature; (c) waiting until viable gametes are desired by the patient; (d) thawing the gonadal organ so that at least a portion of cells contained therein resume biological activity; and (e) introducing the at least one whole gonadal organ including vasculature the patient so that the at least one whole gonadal organ including vasculature is supplied with blood by a vasculature system belonging to the recipient.
• a single individual serves as both the donor and the recipient.
• the whole organ including vasculature has a capacity to fulfill at least one function selected from the group consisting of oogenesis, spermatogenesis, supply of a hormone, supply of a growth factor, metabolism of a precursor into an end-product, elimination of waste and regulation of a physiologic process.
• the method further includes facilitating angiogenesis of the whole organ including vasculature.
• the organ is a gonadal organ selected from the group consisting of an ovary and a testicle.
• the period of time is selected from the group consisting of: (i) sufficient time for resolution of an acute medical condition of the donor; (ii) sufficient time for a change in a physiologic state of the recipient; (iii) sufficient time for identifying a recipient unlikely to reject tissue from the donor.
• the single individual is a cancer patient attempting to avoid sterility.
• the single individual is a woman wishing to alleviate menopausal symptoms by implantation of a previously removed ovary including vasculature after onset of menopause.
• the single individual is a woman wishing to achieve altered fertility status by implantation of a previously removed whole ovary including vasculature.
• the at least one whole gonadal organ including vasculature is selected from the group consisting of an ovary and a testicle.
• the method of further includes installing at least one prosthetic testicle in a scrotum of the patient. According to still further features in the described preferred embodiments the method of further includes administering sex hormones to the patient.
• the present invention successfully addresses the shortcomings of the presently known configurations by providing methods of preserving functionality of an organ, preserving fertility of a patient undergoing a treatment expected to cause sterility and assuring a supply of viable gametes for future use.
• FIG. 1 is a cartoon of a device used for freezing of a whole organ suitable for use in the context of the present invention ;
• FIG. 2 is a flow diagram illustrating essential steps in methods according to the present invention
• FIGs. 3a-d are photomicrographs of oocytes (panels a and b) and enbryos (panels c and d) from experimental (panels a and c) and control (panels b and d) ovaries; and
• FIG. 4 is a graph illustrating estradiol and progesterone levels as a function of time post transplantation.
• the present invention is of methods of preserving functionality of an organ, preserving fertility of a patient undergoing a treatment expected to cause sterility and assuring a supply of viable gametes for future use which can be used to remove an organ, cryopreserve the removed organ and reintrodue the cryopreserved organ into a recipient. Specifically, the present invention assures that the vasculature of the recipient supplies blood to the introduced organ or a portion thereof.
• a freezing device 20 Multi-Gradient-Directional (MGD) IMT technologies; Israel) according to earlier teachings of Arav (United States Patent 5,873,254) was employed to freeze removed whole organs.
• device 20 includes a plurality of temperature domains (e.g. 22, 24, 26 and 28) within 270 mm copper blocks 36 .
• the cooling rate may be st to, for example, 0.03°C/min by varying velocity 32 at which tube 30 containing the organ passes through the gradient 34. Seeding is typically performed at the tip of the tube 30 and ice inte ⁇ hase is propagated according to the freezing point of the solution.
• Figure 2 illustrates a method 40 of preserving functionality of an organ.
• Method 40 includes removing 42 at least a whole organ including vasculature from a donor.
• Method 40 further includes cryo-preserving 44 the whole organ including vasculature and allowing 46 a period of time to elapse.
• Method 40 further includes thawing 48 the whole organ including vasculature so that at least a portion of cells contained therein resume biological activity.
• Method 40 further includes introducing 50 the whole organ including vasculature into a recipient so that a blood vessel present in the vasculature of the whole organ including vasculature is supplied with blood by a vasculature system belonging to the recipient.
• a single individual serves as both the donor and the recipient.
• the single individual may be, for example, a cancer patient attempting to avoid sterility. Alternately, the single individual may be a woman wishing to alleviate menopausal symptoms by implantation of a previously removed ovary including vasculature after onset of menopause.
• the single individual is a woman wishing to achieve altered fertility status by implantation of a at least one previously removed whole ovary including vasculature.
• a woman may elect to have one or both ovaries remove when she is relatively young, and then have one or both ovaries re-implanted at a later time when she wishes to conceive.
• the whole organ including vasculature may be introduced to fulfill at least one function including, but not limited to, oogenesis, spermatogenesis, supply of a hormone, supply of a growth factor, metabolism of a precursor into an end-product, elimination of waste or regulation of a physiologic process.
• the organ is a gonadal organ i.e. an ovary or a testicle.
• method 40 further includes facilitating angiogenesis of the whole organ including vasculature.
• Allowing a period of time to elapse may be for a variety of reasons, such as, for example, allowing sufficient time for resolution 52 of an acute medical condition of the donor, or allowing sufficient time for a change 54 in a physiologic state of the recipient, or allowing sufficient time for identifying 56 a recipient unlikely to reject tissue from the donor.
• the invention is further embodied by method 40 of preserving fertility of a patient undergoing a treatment expected to cause sterility.
• Method 40 includes removing 42 at least one whole gonadal organ including vasculature from the patient.
• Method 40 further includes cryo-preserving 44 the at least one whole gonadal organ including vasculature.
• Method 40 further includes conducting 45 the treatment and waiting for an effect thereof to subside.
• Method 40 further includes thawing 48 the at least one whole gonadal organ including vasculature so that at least a portion of cells contained therein resume biological activity; and introducing 50 the at least one whole gonadal organ including vasculature into the patient so that a blood vessel present in the gonadal organ is supplied with blood by a vasculature system belonging to the patient.
• the at least one whole gonadal organ including vasculature is either an ovary or a testicle depending upon the sex of the patient.
• method 40 preferably further includes installing 58 at least one prosthetic testicle in a scrotum of the patient. This step is performed to prevent damage to the patient's self image. Whether the patient is male or female, method 40 further includes administering 60 sex hormones to the patient if both gonadal organs are removed so that secondary sex characteristics are not affected.
• the present invention is further embodied by method 40 of assuring a supply of viable gametes for future use.
• Method 40 includes removing 42 at least one whole gonadal organ including vasculature from a patient.
• Cryo-preserving 44 the at least one whole gonadal organ including vasculature and waiting until viable gametes are desired 47 by the patient.
• Method 40 further includes thawing 48 the gonadal organ so that at least a portion of cells contained therein resume biological activity and introducing 50 the at least one whole gonadal organ including vasculature into the patient so that the at least one whole gonadal organ including vasculature is supplied with blood by a vasculature system belonging to the recipient.
• Arav G ⁇ T/d where ⁇ T is the temperature difference and d is the distance over which it is maintained.
• Organs e.g. ovaries
• Belzer UW solution (ViaSpan®, Do Pont Pharma, USA) +10% dimethyl sulfoxide( DMSO; Sigma Chemicals, St. Louis, USA) by insertion of a catheter into a blood vessel of the organ.
• the apparatus includes 4 temperature domains within 270 mm copper blocks.
• the cooling rate was set to 0.1°C/min by varying the speed the tube passes through the temperature gradient. Seeding was performed at the tip of the test tube and ice inte ⁇ hase was propagated according to the freezing point of the solution.
• In-vitro follicular assays Follicles were aspirated using a 22 gauge needle connected to a 5ml syringe and thin ovarian cortex slices were cut. The granulose cells and follicular viability was assessed using FDA (fluorescein diacetate)/DAPI (4,6-diamidino-2-phenylindole) fluorescent live/dead staining (Molecular Probes, Leiden, the Netherlands and Merk, Germany).
• Thawing of cryopreserved organs Organs were first immersed in a high temperature (66-68 °C ) water bath for 10 to 20 seconds followed by immersion in a 38-40°C water bath for 10 to 120 seconds. Ovaries were then perfused with 0.5M sucrose and 150iu heparin to remove cryoprotectants and prevent blood clotting.
• Aspirated oocytes were then put into TCM- 199 maturation medium supplemented with 10 % (V/N) heat-inactivated fetal calf serum (FCS) (Bio-lab, Jerusalem, Israel), 0.2mmol Na pyruvate l "1 , 5 ⁇ g gentamicin l "1 , lO ⁇ g ovine LH ml "1 (NIADDK-NIH-26, AFP5551B, Bethesda, MD), l ⁇ g ovine FSH ml -1 (NIADDK-NIH-20, AFP7028D, Bethesda, MD) and l ⁇ g estradiol ml "1 and incubated in a 38.5°C in a humidified atmosphere of 5 % CO2 in air.
• FCS heat-inactivated fetal calf serum
• oocytes were denuded from cumulus cells in the maturation wells and placed for 5 min. in the ionomycin medium: 10 ml TCM-199, supplemented with 10 % (V/V) heat-inactivated FCS, 0.2mmol Na pyruvate l "1 , 5 ⁇ g gentamicin l "1 , and 5 ⁇ mol of ionomycin l "1 .
• Oocytes were transferred to 6-dimethylaminopurine (6-DMAP) medium (10 ml of TCM 199 supplemented with 2mmol of 6-DMAP l "1 ) for 4.5h.
• 6-DMAP 6-dimethylaminopurine
• oocytes were then washed three times in a cleavage medium (Sydney INF Cleavage Medium, Cook, Australia) and transferred into 50 ⁇ l cleavage drops under mineral oil for another three days.
• Example 1 In vitro assay of viability of sheep ovaries after thawing
• the cooling rate was set to 0.1°C/min by varying the speed of the tube through the temperature gradient. Seeding was performed at the tip of the test tube and ice interface was propagated according to the freezing point of the solution. 15 minutes after reaching the final temperature the test tube was thawed. When -196°C was used as the final temperature, ovaries were stored in L ⁇ for 2 days to one week before being thawed.
• Frozen ovaries were thawed as described hereinabove. The frozen-thawed whole ovaries were then washed in buffer medium at 38°C. Follicles were aspirated with a 22G needle connected to a 5ml syringe in order to obtain granulosa cells. Thin slices were cut from the ovarian cortex. The granulose cells and primordial and small antral follicules were evaluated for viability using fluorescent microscopy as described by Oktay et al. (Fertil. and Steril .
• Granulosa cells that were evaluated after freezing and thawing showed no statistically significant differences from controls (Table 2).
• the survival of small follicles (small antral and primordial follicules) showed no statistically significant differences from controls (Table 2).
• Means were calculated using the General Linear Model procedure of JMP (SAS Institute, 1994) and differences between treatments examined by analysis of variance. Significance was PO.05.
• Example 1 In order to verify the results of Example 1 in an in vivo system, eleven-month-old Assaf sheep were subjected to oophorectomy under general anaesthesia via longitudinal laparotomy. The uterus and ovaries were exposed and a dissection of the right ovarian artery and vein was performed. The ovary was perfused in vivo with UW + 10% DMSO for 3 min. It was then excised and inserted into a freezing tube containing UW + 10% DMSO.
• Slow freezing was performed by cooling from 0 to -6°C over 2 min, seeding was done before entering -6°C and cooling to -14°C, -30°C and thawing after 15 minutes or to -30°C and then direct plunging into LN and then thawing.
• the cooling rate of the three different procedures was 0.1 °C/min. After 15 min at the final temperature thawing was performed by plunging the test tube into a 66°C water bath for 20 s and then into a 40°C water bath for 2 min.
• Ovarian vascular transplantation was performed by re-anastomosing the ovarian artery and vein with 10 zero sutures (Ethilon, Johnson and Johnson). After the arterial anastomosis was completed, blood flow was verified by observing blood pulse in the ovary and venous return through the ovarian vein.

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• 2002
  • 2002-08-28 AU AU2002330726A patent/AU2002330726A1/en not_active Abandoned
  • 2002-08-28 EP EP02767822A patent/EP1545200A4/de not_active Withdrawn

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