Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0608—Germ cells
  • C12N5/0609—Oocytes, oogonia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/05—Inorganic components
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/34—Sugars

Definitions

• the invention relates to culture media and more particularly to culture media for in vitro fertilization and embryo transfer.
• n vitro fertilization of human oocytes, cleavage of embryos and embryo transfer require that a culture medium be used to support the embryo for a period of up to three or four days during the various processes necessary for fertilization and early incubation before embryo transfer and reimplantatlon.
• the oocyte In the natural process of fertilization for a human oocyte the oocyte is supported within the mother within a fluid known as human tubal fluid andi t i s the object of the present invention to provide a culture medium as a synthetic human tubal fluid.
• the present invention we have devised a synthetic culture medium which is believed to approximate human tubal fluid but with desirable additional components and variations in the actual composition, including ratios of concentrations of sodium ions to potassium ions.
• the present invention may be said to reside in a culture medium for in vitro fertilization and embryo transfer comprising the following compounds in the following ranges of concentration;
• the ratio of sodium ion concentration to potassium ion concentration is in the range from 28 to 32.
• the ratio of concentrations of sodium ions to potassium ions is 29.3.
• the synthetic human tubal fluid may have the following composition
• the invention may be said to reside in a method of assisting with the in vitro fertilization of human oocytes including the step of handling the human oocytes in a culture medium, the culture medium being comprised of the compounds listed below in the range of compositions listed as follows;
• the ratio of sodium ion concentration to potassium ion concentration is in the range of from 28 to 32.
• the ratio concentration of sodium ions to potassium ions is 29.3.
• the method includes the step of handling the oocytes in a culture medium comprising;
• thei nvention may be said to reside in a culture medium for thei n vitro fertilization of human oocytesi ncluding sodium potassiumi ons wherein the ratio of sodium ions to potassium ions is i n the range of from 28 to 32.
• the ratio of sodium ions to potassium ions is 29.3.
• synthetic human tubal fluid culture medium is as given in Table 1 below (marked synthetic HTF).
• the medium may be prepared by using rainwater which has been distilled in glass six times.
• the bicarbonate-buffered medium is gassed for a minimum of five minutes with humidified 5% oxygen 5% carbon dioxide 90% nitrogen mixture and sterilized by passage through a 0.45-0.2 micrometre filter membrane (Millipore, Sydney, Australia or Amicon Sterilet Sydney, Australia) and then stored at 4°C for up to two weeks before use.
• a minimum of six hours or preferably the day before being used the bicarbonate buffered medium is gassed again for two to three minutes with the same gas mixture as above and the protein component is added.
• Tests have been carried out using both mouse embryo development in vitro and with initiation of human pregnancy in an endeavour to discover which components of the T6 medium and the synthetic human tubal fluid according to this invention might be responsible for observed differences in mouse embryo development in vitro and initiation of human pregnancies.
• the results show that for human pregnancy initiation almost three times as many pregnancies occurred when fertilization and culture were carried out using the synthetic human tubal fluid of the present invention over the T6 medium.
• the present invention therefore provides a synthetic human tubal fluid culture medium which is more than just a direct replication of naturally occurring human tubal fluid but has enhanced viability.
• Potassium chloride (Kcl) 4.69 mM
• the invention may be said to reside i n a culture medium for the in vitro fertilization of human oocytes i ncluding sodium potassium i ons wherein the ratio of sodium i ons to potassium ions i s in the range of from 28 to 32.
• the ratio of sodium ions to potassiumi ons is 29.3.
• synthetic human tubal fluid culture medium i s as given in Table 1 below (marked synthetic HTF).
• the medium may be prepared by using rainwater which has been distilled in glass six times.
• the bicarbonate-buffered medium is gassed for a minimum of five minutes with humidified 5% oxygen 5% carbon dioxide 90% nitrogen mixture and sterilized by passage through a 0.45-0.2 micrometre filter membrane (Millipore, Sydney, Australia or Amlcon Sterilet Sydney, Australia) and then stored at 4°C for up to two weeks before use.
• a minimum of six hours or preferably the day before being used the bicarbonate buffered medium is gassed again for two to three minutes with the same gas mixture as above and the protein component is added.
• Tests have been carried out using both mouse embryo development in vitro and with initiation of human pregnancy in an endeavour to discover which components of the T6 medium and the synthetic human tubal fluid according to this invention might be responsible for observed differences in mouse embryo development in vitro and initiation of human pregnancies.
• the results show that for human pregnancy initiation almost three times as many pregnancies occurred when fertilization and culture were carried out

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• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
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• Biotechnology (AREA)
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• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
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• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 1986
  • 1986-06-12 EP EP19860903165 patent/EP0230430A4/de not_active Withdrawn
  • 1986-06-12 WO PCT/AU1986/000170 patent/WO1986007377A1/en not_active Application Discontinuation
• 1987
  • 1987-02-10 DK DK066187A patent/DK66187A/da not_active Application Discontinuation

Non-Patent Citations (6)

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