COMPOSITION FOR IMPROVING THE CULTURE AND IMPLANTATION OF MAMMALIAN EMBRYOS, PREPARATION METHOD AND USE THEREOF

Abstract

A composition is for improving the culture and implantation of mammalian embryos. The composition includes one or more of the fractions of human plasma fractionation using the Cohn method wherein in the composition human serum albumin (HSA) is between 90% and 96% of the total proteins in the composition, alfa and beta globulins are between 3.5% and 9.99% of the total proteins in the composition, and gamma globulin is between 0.01% and 0.5% of the total proteins in the composition.

Claims

1. A composition for improving an implantation of mammalian embryos, comprising one or more of fractions of a human plasma fractionation using Cohn method, wherein in said composition human serum albumin (HSA) is between 90% and 96% of the total proteins in the composition, alfa and beta globulins are between 3.5% and 9.99% of the total proteins in the composition, and gamma globulin is between 0.01% and 0.5% of the total proteins in the composition.

2. The composition according to claim 1, characterized in that said fractions are selected from Fraction I, Fraction II+III, Fraction IV and Fraction V or supernatant of Fraction I, supernatant of Fraction II+III, supernatant of Fraction IV and supernatant of Fraction V of the Cohn method.

3. The composition according to claim 2, characterized in that said fractions are the supernatant of Fraction II+III and the Fraction V of the Cohn method.

4. The composition according to claim 3, characterized in that the supernatant of Fraction II+III of the Cohn method is further processed by dilution, clarification, diafiltration, nanofiltration, concentration by ultrafiltration, sterilisation, filling into vials and final lyophilisation of said vials, prior to submission of said vials to gamma irradiation, or a combination thereof.

5. The composition according to claim 3, characterized in that the Fraction V of the Cohn method is further processed by diafiltration, heat treatment, sterilisation, filling into vials and final pasteurization of said vials, prior to submission of said vials to quarantine, or a combination thereof.

6. The composition according to claim 3, characterized in that the supernatant of Fraction II+III and the Fraction V, as such or as further processed production intermediates or final products, are submitted to one or more production steps with capacity to eliminate pathogens.

7. The composition according to claim 1, characterized in that said HSA is at a final concentration of 50 mg/ml to 250 mg/ml.

8. The composition according to claim 1, characterized in that said HSA is at a final concentration of 100 mg/ml.

9. A method preparation of a embryo culture medium comprising the composition of claim 1, comprising: obtaining the composition of claim 1; by adding 10-50% (v/v) of said composition to an embryo culture medium used for a mammalian embryo culture.

10. The method for preparation according to claim 9, characterized in that the composition is present in the embryo culture medium at 20% (v/v).

11. A method of culturing mammalian embryos prior to their implantation, comprising culturing the embryos in a culture medium that comprises the composition according to claim 1.

Description

[0032] The present invention will be described below in reference to the figures, in which:

[0033] FIG. 1 is a graphic of the results showing the improved performance of composition III (composition of the present invention) for the culture of mammalian embryos at the bastocyst stage compared to composition I and composition II. The improvement of said culture was measured as the percentage of embryos that progress sequentially from “bleb” to “half hatcihng”, until they reach the complete “hatching”. “Arrested” refers to those embryos blocked at blastocyst, which did not develope further.

[0034] FIG. 2 is a graphic of the results of the adhesion of the hatched embryos to the culture plate surface in the culture using composition I and composition III.

[0035] FIG. 3 is an illustration of confocal microscopy images of the implantation of embryos in collagen hydrogels using using composition I and composition III.

[0036] Hereinafter, the present invention is described with reference to examples, which however are not intended to limit the present invention.

EXAMPLES

Example 1

Preparation and Characterization of the Composition of the Present Invention and Comparative Compositions.

[0037] Composition I (table I) differs from composition III in that it has a higher amount of albumin and a lower amount of alpha-1 globulin. On the other hand, composition II differs from composition III in that it has a lower amount of albumin and a lower amount of alpha-1 globulin.

[0038] The composition of the majority components of said compositions is shown in the table below:

TABLE-US-00001 TABLE I Composition of the majority components of compositions I, II and III (composition of the present invention). Composition III Composition I Composition II (present invention) n = 3 (x ± SD) n = 4 (x ± SD) n = 3 (x ± SD) Seroalbumin 198.1 ± 0.4 22.7 ± 1.8  107.6 ± 1.5  (g/L) Alpha-1  2.3 ± 0.2 1.3 ± 0.2 6.4 ± 0.4 globulin (g/L) Alpha-2 Not detected 2.7 ± 0.7 2.2 ± 0.2 globulin (g/L) Beta Not detected 2.2 ± 0.4 1.6 ± 0.3 globulin (g/L) Gamma Not detected 0.5 ± 0.1 0.5 ± 0.1 globulin (g/L) Seroalbumin  98.9 ± 0.1 77.1 ± 2.2  91.0 ± 0.5  (%) Alpha-1  1.1 ± 0.1 4.5 ± 0.3 5.4 ± 0.4 globulin (%) Alpha-2 Not detected 9.0 ± 1.4 1.8 ± 0.2 globulin (%) Beta Not detected 7.5 ± 0.7 1.3 ± 0.2 globulin (%) Gamma Not detected 1.6 ± 0.3 0.4 ± 0.1 globulin (%) Total protein 200.3 ± 0.3 29.5 ± 3.2  118.3 ± 1.1  (g/L)

EXAMPLE 2

Evaluation of the Development of Mouse Embryos in the Presence of the Composition of the Present Invention and Comparative Compositions (shown in FIG. 1).

[0039] For determining the efficiency of the embryo culture, different protein formulations were prepared using as a reference the final concentration of HSA. KSOMAA (Millipore; USA) was used as a base medium with bovine serum albumin (BSA, 5 mg/ml) for bringing the mouse embryo culture E0.5 to the blastocyst stage (E3.5). Upon arrival at the blastocyst stage some embryos remained as a control in KSOMAA (BSA 5 mg/ml) and the rest of the embryos were transferred to a new KSOMAA medium without BSA and supplemented with: [0040] Group I: composition I based on a fraction of the human plasma fractionation using the Cohn method comprising more than 96% of HSA, less than 3.5% of alfa and beta globulins and less than 0.5% of gamma globulins. The fraction should be added at a final concentration of 22.5 mg/ml of HSA. [0041] Group II: composition II based on a fraction of the human plasma fractionation using the Cohn method comprising between 70% and 90% of HSA, between 9.5% and 28% of alfa and beta globulins and between 0.5% and 2% of gamma globulins. The fraction should be added at a final concentration of 5 mg/ml of HSA. [0042] Group III (composition III, which is the composition of the present invention): a fraction of the human plasma fractionation using the Cohn method comprising between 90% and 96% of HSA, between 3.5% and 9.99% of alfa and beta globulins and between 0.01% and 0.5% of gamma globulins. The fraction should be added at a final concentration of 22.5 mg/ml of HSA.

[0043] Embryos at the blastocyst stage (E3.5) were left in culture in the incubator until day E6.5 to allow complete hatching. Their morphology was recorded daily through photographs. At the end of the incubation period (E6.5), the stage of development of the embryos in culture was evaluated.

[0044] As shown in FIG. 1, the highest percentage of embryos with complete hatching success were those cultured in the presence of the composition of Group III which is the composition of the present invention. Specifically, 62% of embryos in the presence of the composition of Group III managed to hatch, while in the presence of the composition of Group I 46.8% did. Embryos cultured in the presence of BSA (5 mg/ml) or in the presence of the composition of Group II hatched with an efficiency of 53.9% and 37.2%, respectively. The distribution of embryos at different stages of development shows that embryos grown in the presence of the composition of Group III have two preferential stages, either they are still in blastocyst, or most progress directly to complete hatching. Under these conditions, few embryos were at intermediate steps, for example they were not detected at bleb stage. These results suggest that the composition of the present invention allows progress in the development of viable embryos more efficiently than under the other conditions.

EXAMPLE 3

Evaluation of the Adhesion of the Embryos Hatched in the Culture (shown in FIG. 2).

[0045] In this example, hatched mouse embryos are able to adhere to the culture plate and proliferate if they are grown under optimal conditions. The adhesion involves an outgrowth of the embryo trophoblast attaching to the glass or plastic surface, which cannot be re-attached by flushing with a pippete. In FIG. 2, measurements of the adhesion capacity of blastocysts cultured from stage E3.5 to E7.5 in the presence of the composition of Group I were compared to embryos cultured with the composition of Group III. The results showed a surprising increase in the adhesion of hatched blastocysts when they were cultured in the presence of the composition of Group III. Specifically, 73.3% of the embryos cultured in the composition of Group III adhered to the plate, while, at most, 1.3% of embryos were able to adhere in the presence of the composition of Group I. Under the other two conditions only a marginal or zero percentage managed to adhere to the plate.

EXAMPLE 4

Implantation of Embryos in Collagen Hydrogels (shown in FIG. 3).

[0046] In preliminary studies, mouse embryos were embedded at the blastocyst stage within collagen hydrogels to allow their implantation (US 20150305774 A1). Confocal microscopy allows the observation of the transgenic fluorescent embryo in three dimensions within the implantation matrices. The collagen fibers can be observed by the reflection technique, and appear as a destructured mesh of white fibers, distributed in a relatively homogeneous way. As shown in FIG. 3, embryos cultured in the presence of the composition of Group III induce in certain areas in contact with the surface of the embryo, a type of dramatic remodeling in the fibers. This type of matrix remodeling suggests that the embryo is implanted in the matrix by strongly adhering and exerting force on it as documented during in vitro cell culture (Legant et al., Measurement of mechanical tractions exerted by cells in three-dimensional matrices, Nat Methods, 2010;7(12):969-71; Lesman et al., Cell tri-culture for cardiac vascularization, Methods Mol Biol, 2014;1181:131-7). However, the control population of embryos cultured in the presence of the composition of Group I and transferred to the collagen matrix failed to implant and the embryos do not progress. Since collagen polymerization occurs during the first hour after embryo embedding, these results discard the possibility that the process of deformation of the matrix is produced by heterogeneities in the polymerization of collagen when embryos are inserted. On the contrary, said deformations happen gradually over time, so their presence suggests the active mechanical action of the embryos. It is also surprising that supplementation by the composition of the present invention not only improves the implantation of the embryo but also extends its life until at least day E9.5.