RU2084523C1 - Method of preparing the nutrient medium for human blood lymphocytes culturing - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: nutrient medium has medium RPMI-1640 with HEPES-buffer, cattle serum blood, 3% L-glutamine solution with antibiotics and phytohemagglutinin. The obtained dissolved mixture is sterilized by filtration through diaphragms (pore diameter is 0.22 mcm), dosed in small bottle and subjected for lyophilic drying. Invention provides culturing the human blood lymphocytes at mitotic index = 30, not less, preparing the medium that is stable for prolonged period. Medium is used for diagnostic studies of chromosomal anomalies. EFFECT: improved method of preparing, enhanced quality of medium, simplified using. 2 cl

Description

 The proposed technical solution relates to biotechnology and relates to a method of obtaining a nutrient medium for the cultivation of human blood lymphocytes in diagnostic studies of chromosomal abnormalities.

 Diagnostic kits for the study of human chromosomes produced by the firms Difco (USA), Gibco (Great Britain) are known, the main disadvantage of which is the need to dose the individual reagents that are part of the kit before use, which requires aseptic conditions and a significant investment of time, as well as the scarcity of these components.

 Most often in medical practice, when studying chromosomal abnormalities in humans, blood lymphocytes are cultured in nutrient media, which are prepared each time before the introduction of a blood sample, mixing the starting components according to the prescriptions of well-known foreign companies [1] In addition to characteristic for diagnostic kits, the main disadvantage of this method of obtaining media is the irreproducibility of the results of chromosome analysis. Nutrient media of standard manufacture for the cultivation of human blood cells [2] and animals [3] are described in analogues, however, without introducing into their composition mitogen, which is very labile in a liquid medium. These media are not suitable for the cultivation of blood lymphocytes in order to isolate the chromosome preparation and subsequent diagnostic studies.

 The objective of the invention is the creation of a finished form of a standard composition of a nutrient medium that ensures the cultivation of human blood lymphocytes with a mitotic index of at least 30, stable for a long period, convenient for use in medical practice in any conditions, including and field.

To solve this problem, a nutrient medium for the cultivation of human blood lymphocytes, which includes a nutrient medium RPM1-1640 with HEPES buffer, serum of cattle (cattle), a solution of a 3% mass fraction of L-glutamine with antibiotics, according to the invention , at the stage of mixing the starting components, phytohemagglutinin is additionally introduced until they are completely dissolved, the resulting dissolved mixture is sterilized by filtration through membranes with a pore diameter of 0.22 mmk, metered into vials and freeze-dried in the following ratio tion of the initial components:
Culture medium RPM1-1640 with HEPES 3.0 3.5 ml (3.027 ml)
A solution of a 3% mass fraction of L-glutamine with antibiotics 0.06 0.08 ml (0.072 ml)
Phytohemagglutinin 0.04 0.15 mg
Serum of cattle 0.3 0.9 ml (0.865 ml)
When using 1 dose of dry sterile medium obtained from 4 ml of a dissolved mixture, 4 ml of sterile deionized water (resistivity 10 18 MΩ) and 0.4 ml of human blood are introduced into a vial with medium and, using the vial as an fermenter, lymphocytes are cultured at ( 37 ± 0,1) ^o C for 72 hours. The nature of the growth of blood lymphocytes standard suspension.

In 1 l of the solution, a 3% mass fraction of L-glutamine with antibiotics contains 30.0 g of L-glutamine, 2.5 g of kanamycin sulfate, 5.0 g of streptamycin sulfate, 5 • 10 ⁶ units. activity of benzylpencillin sodium salt.

 Since the cultivation of lymphocytes is stimulated by the labile PHA mitogen, which at optimal concentrations promotes the division of lymphocytes, and in elevated ones inhibits growth and causes destruction of lymphocytes, before the preparation of the medium, mitotic activity of PHA is controlled by introducing it into samples of the medium at various concentrations from 0.01 up to 0.04 μg / ml and determination of the mitotic index for blood samples of the same person.

 The quantitative content of the components of the environment less than the specified boundary provides a mitotic index of less than 30. The quantitative content of the components of the environment greater than those specified in the proposed composition leads to the destruction of lymphocytes.

 The smallest amount of PHA, which provides at least 30 mitotic plates per 1000 culture cells, is optimal for this series of medium.

 The proposed technical solution is illustrated by the following examples.

 Example 1

1260 ml of RPM1-1640 nutrient medium containing 5.8 g of HEPES buffer are loaded into the reactor, 125 ml of sterile liquid cattle serum, 30 ml of a 3% solution of L-glutamine containing benzylpenicillin sodium salt are added at a concentration of 90 u / ml , kanamycin sulfate 0.045 mg / µ, streptomycin sulfate 0.09 mg / ml and 17 mg PHA dissolved in (15 ± 1) ml of sterile deionized water. The contents of the reactor are mixed for 15 to 20 minutes until they are completely dissolved and subjected to sterilizing filtration through membranes with a pore diameter of 0.22 μm with simultaneous dosing of (4.0 ± 0.1) ml into vials, cooled in a sublimation chamber to a temperature of minus 45 ^o C for 10 12 hours and freeze-dried for 48 hours. Receive 410 -420 bottles of dry nutrient medium suitable for the cultivation of human blood lymphocytes.

 Example 2

 1260 ml of RPM1-1640 nutrient medium containing 7.5 g of HEPES buffer are loaded into the reactor, 375 ml of sterile liquid blood serum of cattle, 30 ml of a 3% solution of L-glutamine containing antibiotics in concentration are added (see example 1) and 60 mg of PHA dissolved in (15 ± 1) ml of sterile deionized water. Further see example 1.

 Example 3

The suitability of the cultivation medium is monitored by introducing aseptically 4 ml of sterile deionized water and 0.4 ml of freshly collected peripheral blood into vials of dry medium. Cultivate at (37 ± 1) ^o C for 72 h, after which the cell division is stopped at the metaphase stage by adding 0.22 ml of colchicine solution with a concentration of 0.00004 mg / ml to the culture suspension. Lymphocyte cells are isolated by centrifugation. The number of metaphase plates is determined by applying a sample to a glass slide. After drying and staining with Giemsa-Romanovsky dye, the mitotic index is determined, which should be 30 or more.

 An analysis of experimental data shows that the proposed medium, in contrast to the known analogues, instead of fetal serum, contains more affordable and cheaper cattle blood serum produced according to FS 42 246 BC - 87, made from domestic components of RPM1-1640 medium (FS42-365 BC • 91) does not contain heparin. The proposed environment is convenient for use in medical practice in any conditions, including and field.

Literature
The Catalog for Cell Cultue and Cell Biology, p. 77-80, Cibco BRL.

 2. USSR author's certificate N1678830, class C 12 N 5/18, C 12 P 21/08, 1989.

 3. USSR author's certificate N1576562, class C 12 N 5/00, 1988.0

Claims (1)

1. A method of obtaining a nutrient medium for the cultivation of human blood lymphocytes by mixing a standard nutrient medium with blood serum, antibiotics and PHA, characterized in that the medium additionally contains L-glutamine, from standard nutrient media RPMI-1640 on Hepes buffer, from blood serum cattle, after which the resulting mixture is sterilized, dosed and freeze-dried in the following ratio of the starting components:
Culture medium RPMI-1640 3.0 3.5 ml
Hepes buffer 14 18 mg
A solution of a 3% mass fraction of L-glutamine with antibiotics 0.06 0.08 ml
Phytohemagglutinin 0.04 0.15 mg
Cattle blood serum 0.3 0.9 ml
2. The method according to claim 1, characterized in that the medium is sterilized by filtration through membranes with a pore diameter of 0.22 μm, and dosing is carried out in 4 ml.