Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of co-culturing cells
Reexamination Certificate
2001-05-22
2002-07-30
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of co-culturing cells
C435S404000, C435S408000
Reexamination Certificate
active
06426219
ABSTRACT:
This invention pertains to compositions and methods for mammalian cell culture in vitro, for example the culture of mammalian oocytes or embryos.
Embryo Cell Culture Media
Various cell culture media have been used to support the growth of mammalian cells in vitro. Many of these media are quite satisfactory in supporting the growth of certain cell types. Other cell types, however, have proven more difficult to support in vitro. There is a continuing need for improved media to support the growth of such cell types.
There is a particular need for improved media to support mammalian oocytes and embryos. A high percentage of embryos that are fertilized or transferred in vitro cease development-prematurely. The consequences are felt at both the economic and the human levels.
In the livestock industry, the value of in vitro-fertilized (IVF) embryos from genetically superior stock can be very high. Significant economic savings will result from methods that reduce the high rate of loss of bovine embryos.
Many forms of fertility treatments for humans involve the in vitro fertilization or transfer of oocytes or embryos. The success rates of human fertility treatments are not high. The low success rates impose substantial economic and emotional costs. Even incremental improvements in the success rate can be of substantial benefit. One of the many causes of the low overall success rate is the frequent failure of embryos to grow and develop properly in vitro. Improved media to better support embryo growth can not only enhance the success rate of fertility treatments, but ironically can also reduce the rate of multiple pregnancies resulting from the treatments. Because the overall success rate of current methods is low, practitioners often implant multiple embryos to increase the likelihood of pregnancy. Implanting multiple embryos increases the likelihood of multiple pregnancies as well. If each individual embryo were more likely to survive, then the perceived need to implant multiple embryos simultaneously would decline, and the rate of multiple pregnancies would decrease.
The development of bovine embryos has been supported by media containing inorganic salts, amino acids, carbohydrates, growth factors, and antioxidants. Development has been enhanced by co-culturing embryos with reproductive tissue cells and complex media containing sera and macromolecules. However, embryotoxic substances in these co-culture systems can interfere with the development of embryos. For example, it has been reported that ammonium and purines in the culture system have an embryotoxic effect on the pre-implantation development of bovine and murine embryos. Thus despite some improvements, the overall rate of successful development of embryos in vitro remains low. There is a continuing, unfilled need for improved media for the culture or co-culture of oocytes and embryos.
In vitro fertilization (IVF) and embryo transfer (ET) techniques for bovine oocytes and embryos have been used for both commercial and research purposes. Similar techniques have been used in infertility treatments for humans. Although several researchers have achieved high IVF success rates (~90%), only a small proportion (~20%) of in vitro fertilized zygotes develop to the morula and blastocyst stages. A large number of inseminated zygotes cultured in vitro cease development at the 8- to 16-cell stage, a time corresponding to embryonic genome activation. Relatively few morulae or blastocysts derived from IVF are suitable for ET.
Papers disclosing in vitro culture and co-culture systems for oocytes and embryos include the following: J. Lim et al., “Roles of Growth Factors in the Development of Bovine Embryos Fertilized in vitro and Cultured Singly in a Defined Medium,” Reprod. Fertil. Dev., 8:1199-1205 (1996); W. Eyestone et al., “Co-culture of Early Cattle Embryos to the Blastocyst Stage with Oviductal Tissue or in Conditioned Medium,” J. Reprod. Fert. 85:715-720 (1989); J. Thibodeaux et al., “Role of Platelet-Derived Growth Factor in Development of in vitro Matured and in vitro Fertilized Bovine Embryos,” J. Reprod. Fert. 98:61-66 (1993); J. Thibodeaux et al., “Stimulation of Development of In Vitro-Matured and In Vitro-Fertilized Bovine Embryos by Platelets,” J. Anim. Sci. 71:1910-1916 (1993); J. Lim et al., “A Serum-Free Medium for Use in a Cumulus Cell Co-Culture System for Bovine Embryos Derived from In Vitro Maturation and In Vitro Fertilization,” Theriogenology 45:1081-1089 (1996); J. Lim et al., “Intracytoplasmic Glutathione Concentration and the Role of &bgr;-Mercaptoethanol in Preimplantation Development of Bovine Embryos,” Theriogenology 46:429-439 (1996); J. Lim et al., “A Continuous Flow, Perifusion Culture System for 8- to 16-Cell Bovine Embryos Derived from In Vitro Culture,” Theriogenology 46:1441-1450 (1996); and J. Lim et al., “Perifusion Culture System for Bovine Embryos: Improvement of Embryo Development by Use of Bovine Oviduct Epithelial Cells, and Antioxidant and Polyvinyl Alcohol,” Reprod. Fertil. Dev., 9:411-418 (1997).
Nitric Oxide
The nitric oxide (NO) molecule controls a wide range of biological activities, including programmed cell-death (apoptosis), activation of guanylyl cyclase, interaction with superoxide anions to form peroxynitrite, regulation of glycolysis by modification of glyceraldehyde-3-phosphate dehydrogenase activity, control of the mitochondrial transport electron chain, the citric acid cycle, DNA synthesis, binding to the iron-sulphur center of enzymes, stimulation of ADP-ribosylation of proteins, production of arachidonic acid metabolites such as prostaglandin E2 and 5-hydroxyeicosatetraenoic acid, sperm motility and viability, and capacitation and hyperactivation of spermatozoa in vitro.
NO plays an important role in the regulation of various aspects of cell metabolism. During human pregnancy, NO is produced in the placenta, the decidua, and the endometrium. It has been reported that NO synthesis increases during pregnancy but decreases toward the end of gestation. A peak in NO synthesis has also been reported in the rat uterus during pregnancy. See Novaro et al., “Nitric oxide synthase regulation during embryonic implantation,” Reprod. Fertil. Dev. 9:559-564 (1997). Early embryonic loss has been associated with local production of NO by decidual cells in the mouse uterus. See E. Haddad et al., “Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells,”
J Exp. Med.,
182, 1143-1151 (1995).
Papers discussing nitric oxide and its role in reproductive biology include the following: L. McDonald et al., “Nitric Oxide and Cyclic GMP Signaling,” PSEBM, 211:1-6 (1996); S. McCann et al., “The Role of Nitric Oxide in Reproduction,” PSEBM, 211:7-15 (1996); Z. Katu{haeck over (s)}ić et al., “Nitric Oxide Synthase: From Molecular Biology to Cerebrovascular Physiology,” NIPS 9:64-67 (1994); A. Jablonka-Shariff et al., “Hormonal Regulation of Nitric Oxide Synthases and Their Cell-Specific Expression during Follicular Development in the Rat Ovary,” Endocrinology 138:460-468 (1997); and M. Vega et al., “Expression of Nitric Oxide Synthase (NOS) in Human Corpus Luteum (hCL) and the Role of Nitric Oxide (NO) on Luteal Steroidogenesis” (abstract), Biol. Reprod., 54 (Suppl. 1):66 (1996).
A. Fukuda et al., “Production of nitric oxide from mouse embryo and effect of nitrite on mouse embryonic development in vitro,” Biol Reprod 54:173 (abstr) (1996) reported that NO may have a regulatory role in preimplantation embryonic development in the mouse.
Hemoglobin (Hb) is widely known as the iron-containing molecule in red blood cells responsible for the transport of oxygen and carbon dioxide. It has recently been recognized that hemoglobin also binds nitric oxide with high affinity. Hemoglobin has been used in some experimental systems as a “sink” for nitric oxide that diffuses outside the cell during a process being studied.
The Invention
We have discovered that nitric oxide adversely affects the development of certain cells in vitro, such as the pre-implementation developmen
Hansel William
Lim Jeong-Mook
Board of Supervisors of Louisiana State University and Agricultu
Runnels John H.
Witz Jean C.
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