SU1727822A1 - Method for microsurgery with ovums and embryos - Google Patents

Abstract

Uses: biotechnology, mammalian embryo transplantation. SUMMARY OF THE INVENTION: Cattle embryos at the morula stage are obtained on the 7th day after seed replacement. Place them in a 1.5 M sucrose solution in Dulbecco's phosphate buffer. A sharpened micropipette makes a puncture of the shell and remove the contents of the cell. The bare embryo is transferred to an isotonic solution. The embryo is divided into two halves. Two parts of the embryo are transferred to a 1.5 M sucrose solution. Each of the parts of the embryo is placed in the extracellular membrane and transferred to physiological saline. 2 ill., 1 tab.

Description

-

fe

The invention relates to biotechnology, in particular to the microsurgery of biological objects having an extracellular membrane, and can be used in the transplantation of mammalian embryos.

The aim of the invention is to increase the safety of the biological object and increase the manufacturability of the operation.

The goal is achieved by the fact that a biological object is extracted from the extracellular envelope in a hypertonic solution of substances of 1.2–1.5 M penetrating through the cell membrane in a medium that provides viability.

A capillary is introduced through the envelope, the cellular mass of the biological object is drawn into it and removed from the envelope, microsurgery is performed with the cell mass in the solution, ensuring viability, then, if necessary, placed again in one or several extracellular envelopes in a hypertonic solution and transferred to isotonic saline.

The method of microsurgical operations is based on the fact that the extracellular membrane of a biological object, in particular, the transparent membrane (zone of pellucida) of the cells and embryos, does not have selective permeability, while the cytoplasmic membrane of cells, in particular of the cells and blastomeres embryos have selective permeability. This leads to the fact that when placed in hypertonic solutions that are impermeable through cytoplasm membranes, qualities such as sodium chloride, sucrose, by osmolarity of 1.2–1.5 M, the volume of the cell mass of the biological object decreases to 2.5-2.8

Yu

s

00

N5. GO

I

fold, and the size of the extracellular membrane does not change.

In Figure 1, by the example of a 2-cell embryo, the volumes of the intracellular mass of the embryo in an isotonic solution are displayed; 2 - the same, in a hypertonic solution with the osmolality of 1,2-.1,5-М. v

The choice of a concentration of 1.2-1.5 M is due to the fact that n $ and a lower concentration of eneium and. The volume of the cell mass is insignificant and it is difficult to remove it from the extracellular membrane, and a higher concentration of non-penetrating substances damage the cells (see table.).

The essence of the method is as follows.

A biological object that has an extracellular membrane that is in a physiological solution is transferred into a hypertonic solution of non-penetrating substances with an osmolarity of 1.2-1.5 M. It is kept in solution for 1 minute and then fixed in position with a microsuckle. A micro-needle or micronosuma makes a puncture or incision of the extracellular membrane, inserts a micropipette and removes a whole or part of the biological object from its own membrane. Transfer it to an isotonic solution; where the object takes the same shape and size. Perform the necessary operations and again transfer the whole or part of the object into the hypertonic solution, where they are reduced in volume. If necessary, place a part or whole biological object in an empty extracellular membrane.

Example. Cattle embryos at the morula stage are obtained on the 7th day after insemination. Embryos suitable for work are placed in a 1.5 M solution of sucrose in Dulbecco's phosphate-saline buffer. In this solution, the volume of the intracellular mass of the embryo is reduced by 2.8 times, and the size of the extracellular membrane does not change, and the space between the membrane and the cytoplasmic membrane increases. Microsurgery is performed under a Biolam R-2 microscope with the help of two

micromanipulator positioners, embryos are retained by micro-slips. A sharpened micropipette with a diameter comparable to the size of an intracellular

the masses in the hypertonic solution make a puncture of the membrane and extract the contents of the cell. At the same time, the gap of the extracellular membrane is insignificant, and the embryo itself is extracted from it without

damage. The bare embryo is transferred to an isotonic solution, where the embryo restores its original volume, and the embryo is separated by a glass needle into two halves. Then

the two parts of the embryo are transferred to a 1.5 M sucrose solution again where the size of the halves is reduced. One of the parts of the embryo is placed, through a hole in the extracellular membrane from which the embryo was extracted, and the second part of the embryo into the other membrane from which the contents have been previously extracted. After a microsurgery, the halves of the embryos in the extracellular

shell transferred to saline.

The experimental results show that the use of hypertonic solution when removing the extracellular membrane

allows you to manipulate embryos without the risk of damage and, therefore, increase the percentage of viable halves after separation of the embryos.

Claims (1)

Invention Formula A method of microsurgical operations with eggs and embryos, including removing them from the extracellular membrane and then returning to an empty membrane, characterized in that, in order to increase the safety of the biological object and improve the manufacturability of the operation; extraction of cells and embryos from the extracellular envelope and their return to the empty envelope are carried out in hypertonic solution of non-penetrating substances by the osmolarity of 1.2-1.5 M in the medium providing the viability of the objects.  Biological objects are placed in a hypertonic solution, incubated for 1 h, returned to physiological conditions and cultured outside the body.  Biological objects are cultivated outside the body in an isotonic solution. Zw /