CS257098B1 - Process for preparing water for embryo, cell and tissue culture - Google Patents

Abstract

The subject of the solution is the preparation of water for media intended for the cultivation of cells, tissues and embryos. The procedure consists in treating raw water containing inorganic and organic impurities with an oxidizing agent, for example potassium permanganate and concentrated sulfuric acid or ozone and a precipitating agent with compounds containing heavy metals, for example carbonates, sulfates or phosphates of alkali metals, the resulting precipitates are removed, for example by sedimentation, and then the water thus treated is subjected to at least one distillation. The water thus treated can be used in basic research, in human and veterinary medicine, in agriculture for the purposes of diagnostics, treatment of infertility and in the biology of reproduction of farm animals.

Description

The invention provides the preparation of water for media intended for the cultivation of cells, tissues and embryos so as not to contain substances adversely affecting the culture result, in particular heavy metal compounds and certain organic substances.

Conventional redistilled and / or deionized water does not meet the requirements for preparing growth media for culturing cells, tissues, and in particular oocytes and early embryos. Cell growth in culture media prepared from such water results in abnormalities or no cell division at all. The water purification methods used to date are mainly based on repeated distillation or a combination of distillation with deoinization by means of ion exchangers.

The main disadvantage of the methods used so far is the energy intensity due to up to five times distillation and further imperfect removal of harmful substances, which during the distillation partially pass into the distillate and are not all captured even by ion exchangers. This problem is usually solved by selecting the source of the starting water and leads to abnormalities and high costs of water purification for cultivation.

The drawbacks of the known processes for preparing water for cultivation are overcome by the process according to the invention. It is based on the treatment of raw water containing inorganic and organic impurities with an oxidizing agent such as potassium permanganate and concentrated sulfuric acid or ozone and a precipitating agent with heavy metal compounds such as alkali metal carbonates, sulphates or phosphates, the resulting precipitates are removed, for example, by sedimentation, and then the water thus treated is subjected to at least one distillation.

The process according to the invention ensures the elimination of all substances that jeopardize the success of the cultivation, with minimal energy and material costs. With the water purified according to the invention, completely standard cultivation results were achieved, both of the murine eggs in the medium modification itself and in the fertilization of human eggs and their further cultivation according to Pilka L. et al .: Delivery after intrauterine transfer of embryos obtained by fertilization. culturing oocytes in vitro (Cs. gynecol. 50, 452-459, 1985). After the transfer of embryos grown in the medium prepared from the water purified according to the invention, in 1986 there have been seven pregnancies in infertile women.

The preparation for the culture media is explained in more detail in the following examples.

Example 1

0.05% potassium permanganate and 0.05% concentrated sulfuric acid were added to the drinking water. The water was boiled for 30 min and 0.1% anhydrous sodium carbonate and 0.05% sodium tertiary phosphate were added hot. After cooling slowly, the weld containing inorganic impurities and permanganate reduction products was removed by sedimentation in decantation and the water was distilled twice at atmospheric pressure in a glass apparatus.

Example 1 a d 2

To the drinking water were added 0.05 anhydrous sodium sulfate, 0.05% anhydrous sodium carbonate and 0.05 sodium tertiary phosphate. Thereafter, ozone-enriched air was introduced from the ozonator for 10 min. The water was boiled and after cooling, a precipitate containing inorganic impurities was removed by sedimentation and decantation, followed by two distillations at atmospheric pressure in a glass apparatus.

The quality of the water prepared according to the invention was compared with the quality of the water pre-treated with cation exchanger and 4x distilled. In the cultivation of early mouse embryos in their own modification of the culture medium (Happy and Lawn), the in vitro from the two-cell embryos developed the microscopic structure of the morulae and blastocysts. Folia Morph. (Prague) 22, 86-95, 1984) using the water according to the invention, 100% of the cultivations were successful (development from the cell-to-mula-to-blastocyst stage).

The same medium, but prepared from water pre-treated with cation exchanger and 4x distilled, was used in the comparative sample. Under otherwise the same conditions, 60% of cultivations were successful (2-cell to morule to blastocyst stage), 20% partially successful (2-cell to 4-8 stage, or part of embryo from 2-cell stage (morule to blastocyst stage) and 20% unsuccessful (embryos did not develop). From this it can be concluded that the absolute standard of water quality for cultivation has been achieved. ·

The culture water according to the invention can be used for the preparation of growth media for cell, tissue and embryo cultivation and for fertilization of eggs in basic research, in human and veterinary medicine and in agriculture for diagnostics, infertility treatment and livestock reproduction biology. It can be stored for a long time in the refrigerator or after sterilization even at normal temperature.

Claims (1)

SUBJECT OF THE INVENTION A process for the preparation of embryo, cell and tissue culture water, characterized in that raw water containing inorganic and organic impurities is treated with an oxidizing agent such as potassium permanganate and concentrated sulfuric acid or ozone and a precipitating agent with heavy metal containing compounds such as alkali metal carbonates, sulphates or phosphates, the resulting precipitates are removed, for example, by sedimentation, and then the water so treated is subjected to at least one distillation.