Cellular culture medium, particularly for in vitro...

Details Cellular culture medium, particularly for in vitro... Cellular culture medium, particularly for in vitro...

2001-07-24

2003-08-26

Falk, Anne-Marie (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of culturing cells in suspension

C435S366000, C435S325000, C435S405000, C435S385000

Reexamination Certificate

active

06610543

ABSTRACT:

BACKGROUND OF HE INVENTION
1. Field of the Invention
The present invention has for its object compositions useful as cellular culture media, particularly for follicles in the course of development or for the maturation of cells of the male germinal line, as well as for the in vitro fertilization and development of embryos, and more particularly mammal embryos, particularly human embryos, if desired frozen.
2. Description of the Related Art
After maturation of the De Graaf follicle, ovulation permits the oocyte to pass into the Fallopian tube there possibly to be fertilized. The resulting embryo stays there 3 to 5 days for development and reaches the stages of morula, and then blastocyte.
In the tubes, the gametes, the embryo, the morula, then the blastocyte are surrounded by tube fluid. The composition of this latter is complex: it associates fundamental components of the internal medium of the mother with those of the follicle liquid, and it does not seem to undergo great qualitative changes in the course of the first days of the first phase of the post-ovulatory phase. Reaching the blastocyte stage, the embryo passes into the uterus whose mucosa have thus reached a stage of development favorable for the implantation and nidation of the egg.
The passage of the embryo into the uterus does not take place until one and the other have arrived at the level of physiological development compatible with the implantation and nidation: the blastocyte stage for the embryo, the endometrium near its secrotory phase for the uterus.
In the case of medically assisted conception, these physiological conditions suggest a single and constant culture medium, and not sequential ones as actually exists, associating all the elements necessary for the development, the in vitro fertilization, and if desired subsequent to ICSI (intra cytoplasmic sperm injection, or intracytoplasmic injection of spermatozoa), until transfer of the embryo.
SUMMARY OF THE INVENTION
The present invention results from the discovery by the inventors of constant compositions usable as well for follicle cultures as for cells of the male germinal line, and also for fertilization of the oocyte and the development of the embryo, until th blastocyte stage, and this with comparable yields, or even greater yields than those obtained with compositions now on the market.
The present invention has for its object compositions for the in vitro culture of cells, characterized in that they comprise at least two growth factors (hereinafter called GF) such as recombinant human growth factors, preferably in association with at least one compound of the corticoid family involved in the production of energy in mammals, and more particularly of the glucocorticoid family, such as hydrocortisone or a derivative of the same family, preferably in water soluble form, and, as the case may be, in association with at least one key co-enzyme of energy metabolism, such as NAD/NADH and NADP/NADPH.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
More particularly, the invention has for its object the above-mentioned compositions for the in vitro culture of follicles in the course of development for maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germinal line, or for the in vitro fertilization of oocytes by spermatozoa, or for the culture of embryos, if desired after thawing the follicles, male cells or embryos.
Preferably, the grown factors contained in the compositions of he invention belong to five families of cytokines. The hormones and the growth factors are present in equilibrium concentrations, compatible with physiological conditions. The compositions of the invention bring to the growing and differentiating cells the regulatory molecules which they must have. Thus, these compositions are adapted to permit the maturation of immature human oocytes cultivated in the presence of follicle cells, as well as the maturation of the cells of the mal germinal line. They can also ensure the development of the human embryo to the blastocyte stage. However, this stag has a higher percentage of implantation in the uterus than when the transfer of the embryo takes place at a less advanced stage, particularly at the stages of two to about eight cells.
The invention more particularly has for its object the above mentioned compositions, comprising at least two growth factors, selected from:
the hepatic growth factor, also called HGF (hepatocyte growth factor),
the transformation growth factor &agr;, also called TGF&agr; (transforming growth factor),
the factor for stimulating colonies of granulocytes and macrophages, also called GM-CSF (granulocyte-macrophage colony stimulating factor),
the epidermal growth factor, also called EGF and/or HB-EGF (heparin-binding epidermal growth factor),
the growth and differentiation factors, also called GDF, such as GDF-9,
the insulin-like growth factors, such as IGF-1 and/or IGF-2.
Preferably, the compositions of the invention contain at least three growth factors selected from those listed above.
Particularly preferred compositions contain at least IGF-1 and/or IGF-2.
Also preferably, the above-mentioned compositions of the invention comprise all the above-mentioned growth factors.
Preferably, the concentrations of the growth and differentiation factors in the above-mentioned compositions of the invention are nanomolecular, and preferably comprise between about 0.25 &mgr;g/L and about 60 &mgr;g/L, particularly between about 0.5 &mgr;g/L and about 50 &mgr;g/L.
Preferably, the concentrations of the growth and differentiation factors contained in the compositions of the invention are such that:
the concentration of EGF is about 40 &mgr;g/L to about 60 &mgr;g/L, particularly about 50 &mgr;g/L,
the concentration of TGF&agr; is about 20 &mgr;g/L to about 30 &mgr;g/l, particularly about 25 &mgr;g/L,
the concentration of HGF is about 40 &mgr;g/L to about 60 &mgr;g/L, particularly about 50 &mgr;g/L,
the concentration of GM-CSF is about 1.25 &mgr;g/L to about 1.75 &mgr;g/L, particularly about 1.5 &mgr;g/L,
the concentration of GDF-9 is about 4 &mgr;g/L to about 6 &mgr;g/L, particularly about 5 &mgr;g/L,
the concentration of IGF-1 is about 12.5 &mgr;g/L to about 17.5 &mgr;g/L, particularly about 15 &mgr;g/L,
the concentration of IGF-2 is about 12.5 &mgr;g/L to about 17.5 &mgr;g/L, particularly about 15 &mgr;g/L.
The invention also has for its object the above-mentioned compositions for in vitro culture, comprising a compound of the family of corticoids such as those defined above, and preferably hydrocortisone.
Preferably, the above-mentioned hydrocortisone is present in the form of a hydrosoluble salt, particularly in the form of hydrocortisone hemisuccinate. Preferably, the concentration of the hydrocortisone salt in the above-mentioned compositions, is comprised between about 5×10
−7
M and about 10
−6
M, and is particularly about 7×10
−7
M, or about 350 &mgr;g/L for hydrocortisone hemisuccinate.


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patent: 2001/0033835 (2001-10-01), Daley et al.
patent: WO 96/12793 (1996-05-01), None
Lim et al., Roles of growth factors in the development of bovine embryos fertilized in vitro and cultured singly in a defined medium, 1996, Reprod. Fertil. Dev., vol. 8, pp. 1199-1205.*
Invitrogen/Tech-On-Line/, www.lifetech.com. TPN (triphosphopyridine nucleotide-Na).*
D.T. Loo et al. “Differentiation of Serum-Free Mouse Embryo Cells Into Astrocytes Is Accompanied by Induction of Glutamine Synthetase Activity”, Journal of Neuroscience Research, 42, 1995, pp. 184-191.
Shyamal K. Roy, “Epidermal Growth Factor and Transforming Growth Factor—&bgr; Modulation of Follicle-Stimulating Hormone-Induced Deoxyribonucleic Acid Synthesis in Hamster Preantral and Early Antral Follicles”, Biology of Reproduction, 48, 1993, pp. 552-557.

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