RU2630658C1 - Method of cultivation of amniotic liquid cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method of cultivation of amniotic liquid cells comprising adding amniotic liquid of the nutrient medium to the cells and incubating in vials, several changes of the nutrient medium followed by treatment of cells with colchicine, detaching the cells from the bottom of the vial, hypotonic treatment and treatment with a fixative, where "Amniomax" or "Amniocar" nutrient medium are used as a nutrient medium in the ratio of the initial hallmark: medium, equal to 1.4:1, the first change of the medium is carried out 5 days from the beginning of cultivation with the pretreatment of cells with Hank's solution at a ratio of 2.3:1, the subsequent changes of the medium are performed every 2 days, then in 6-8 days from the beginning of cultivation trypsinization is performed with 0.05% trypsin-EDTA solution for 2-3 minutes at 37°C, the first trypsinization is carried out in one of the vials of the cell culture with the most active growth, the next day trypsinization is carried out in the second vial with a medium rise, a day later - in the third vial with minimal growth, treatment of cells is carried out with colchicine with a concentration of 1 µg/ml for 2.5 hours at 37°C in the presence of 5% CO₂, the detachment of the cells from the bottom of the vial is carried out with 0.05% of trypsin-EDTA solution for 2-3 minutes at 37°C, hypotonic treatment of the cells is carried out with an aqueous solution of embryonic calf serum (2:1) for 20 minutes at 37°C, and fixation of cellsis carried out with a solution containing ethanol and glacial acetic acid at a ratio of 3:1.

EFFECT: acceleration of the method and increasing the yield of cultured cells.

1 dwg, 1 tbl, 2 ex

Description

A method of culturing amniotic fluid cells.

The present invention relates to the field of medical diagnostics and relates to the cultivation of fetal cells from amniotic fluid, which can then be used for diagnostic or scientific purposes, in particular, to identify quantitative and structural abnormalities of chromosomes in the fetus.

Chromosomal diseases occupy a significant place in the structure of hereditary and congenital human pathology. The average number of patients with chromosomal diseases in Russia is about 400 thousand people. Given the severe course, disability, and sometimes the early death of patients with chromosomal disorders, the prevention of these diseases is of paramount importance. As you know, a change in the normal number or structure of chromosomes is one of the many causes of impaired normal prenatal development. It is estimated that the frequency of chromosomal abnormalities in children with multiple congenital malformations (MVD) reaches 50%. In numerous studies of domestic and foreign authors, it is shown that 60-75% of spontaneously aborted embryos have chromosomal abnormalities. At the same time, about 0.6% of newborns have chromosomal diseases [1]. In view of this, prenatal cytogenetic diagnostics, which can prevent chromosomal diseases by eliminating fetuses with a violation of the karyotype, is of great importance.

It is known that the safest method of prenatal cytogenetic diagnosis by the method of obtaining the material is amniocentesis [2]. Amniotic fluid cells precipitated by centrifugation are propagated in the presence of cell growth stimulants in special nutrient media, mitosis is blocked at the metaphase stage, chromosome preparations are prepared after treatment of cells with a hypotonic solution and fixation, and then cytogenetic analysis is performed. It should be noted that the percentage of living cells in samples of amniotic fluid (AF) is only 10-20% [3].

The main disadvantages of this method are the significant duration of cultivation (14-21 days), the receipt of an insufficient amount of material for a complete analysis, the presence of artifact (due to cultivation) mosaicism, the risk of infection of the culture, the high cost of reagents and consumables.

A known method of cultivating AF cells and human fibroblasts in an Amniocar medium, which provides effective attachment and proliferation of cellular material in a monolayer, characterized in that the incubation of primary amniocytes is carried out in ventilated culture bottles in an atmosphere of 5% CO ₂ and incubated for 11 days with the implementation of a complete change of medium 72 hours after the start of incubation [4].

The disadvantages of this method are:

1. An early change of medium (72 hours after the start of incubation), leading to the loss of cells that did not have time to attach, as well as to the additional expense of an expensive nutrient medium.

2. The absence before the first change of medium of the stage of processing the culture with Hanks solution, necessary for the maximum removal of dead, non-adherent cells.

3. The lack of trypsinization of the culture to obtain a uniform monolayer of cells with a synchronized cell cycle. Colonies of primary amniocytes growing in a vial without trypsinization are not synchronized, which does not allow us to obtain a sufficient number of metaphase plates for analysis in the future.

4. The low quality of the obtained metaphase plates, not satisfying the necessary criteria for analysis [5].

Closest to the claimed method - the prototype - is a method of culturing amniotic fluid cells [6], which consists in the following. Amniocentesis AF samples in an amount of 10-20 ml are collected in sterile vials, poured into 2-3 sterile centrifuge tubes and centrifuged for 10 min (1000 rpm). About 1.5 ml of AF are left in a test tube with a cell pellet, which is resuspended in fetal calf serum (ETF), 3-5 ml of culture medium consisting of 80% RPMI-1640 medium (NLP PanEco, Russia), Ham's are added F-12 (Merck, Germany) and 20% ETS. Antibiotics are added to the culture, vials with cells are placed in a CO ₂ incubator at + 37 ° C for 72 hours, then the culture mixture is changed. Subsequent changes in the culture mixture produced every 3-5 days. At the end of the 2nd - beginning of the 3rd week of cultivation, the first fixation of the cells is carried out, while the cell readiness is evaluated under an inverted microscope. Before fixation, a nutrient change is carried out in 24 hours. Then colchicine is introduced into the vial and placed for 4-5 hours in a thermostat at + 37 ° C, the medium is drained and 2-3 ml of trypsin-versene solution (1: 1) is added. After 2 minutes, this solution, along with part of the detached cells, was poured into a centrifuge tube with 2 ml of cold Eagle medium (NLP PanEco, Russia) to inhibit the action of trypsin. After repeating this procedure, cold medium is added to the cell suspension to a volume of 10-15 ml and the tubes are centrifuged for 5 min (1000 rpm), the supernatant is removed, leaving about 1 ml of liquid above the precipitate. After hypotonic treatment for 40 min at 37 ° C, the tubes are centrifuged for 5 min, the supernatant is removed, 7-10 ml of fixative I is poured into the cell suspension (methanol, glacial acetic acid 2: 1) and left for 15 min at + 4 ° C . Fixer I is changed twice to fixative II (methanol, glacial acetic acid 3: 1). After removal of the supernatant, the resuspended cell pellet was digged onto clean chilled glass slides and air dried. The duration of AF cell cultivation is 14 or more days.

The disadvantages of the prototype are:

1. An early change of medium (72 hours after the start of incubation), leading to the loss of cells that did not have time to attach, as well as to the additional expense of an expensive nutrient medium.

2. The absence before the first change of medium of the stage of processing the culture with Hanks solution, necessary for the maximum removal of dead, non-adherent cells.

3. The lack of trypsinization of the culture necessary to obtain a uniform monolayer of cells with a synchronized cell cycle.

4. Too long (4-5 hours) treatment of cells with colchicine, which can lead to insufficient metaphases with long chromosomes on the preparations necessary for a detailed structural analysis of the fetal karyotype.

5. Treatment with colchicine in a thermostat without maintaining the level of carbon dioxide necessary for normal cell growth (5%), which leads to the death of some cells.

6. The use of a mixture of methanol and ethyl alcohol for cell fixation, which poses a threat to the health of personnel, since methanol is a poison.

The technical result of the invention is to accelerate the method and increase the yield of cultured cells.

The technical result is achieved by the proposed method, which consists in the following.

Amniocentesis AF samples (21 ml) are collected in sterile vials and delivered to the laboratory. AF is divided into three equal aliquots, placed in test tubes, centrifuged, the supernatant is removed, and the precipitate is resuspended in AF. Then, Amniomax or Amniocar culture medium is added in the ratio of the initial sample: medium equal to 1.4: 1, and incubated in SPL culture bottles at 37 ° C, 5% CO ₂ . The first change of medium is carried out after 5 days from the start of cultivation with pre-treatment of the cells with Hanks solution in a ratio of 2.3: 1, subsequent changes of the medium are carried out every 2 days. After 6-8 days from the start of cultivation, to obtain a synchronized cell culture, trypsinization with a 0.05% trypsin-EDTA solution (NLP PanEco, Russia) is carried out at a temperature of 37 ° C for 2-3 minutes. The change of the nutrient medium is carried out 1-2 days after trypsinization, another 18-19 hours - fixation of the material (upon reaching the cell monolayer of sufficient density). To block mitosis at the metaphase stage, the cultures are treated with colchicine solution (final concentration 1 μg / ml) for 2.5 hours at 37 ° C, 5% CO ₂ . Then carry out hypotension of the cells by treatment with an aqueous solution of fetal calf serum (2: 1) for 20 min at 37 ° C. After this, the cells are fixed with a mixture of 95% ethanol and glacial acetic acid (3: 1), preparations are prepared, they are treated with a ready-made 0.25% trypsin solution and stained with Giemsa stain according to the standard procedure [2].

The proposed method allows to reduce the duration of the cultivation of AF cells to 8-11 days (14 or more days in the prototype), increase the yield of cultured cells, reduce the likelihood of culture contamination and the likelihood of artifact (culture-related) mosaicism, fix cells without using methanol, which is poison . Comparative analysis of the proposed method in comparison with the prototype are presented in table 1.

The defining differences of the proposed method in comparison with the prototype are:

1. Amniomax or Amniocar nutrient medium is added to the AF sample in the ratio of the initial sample: medium equal to 1.4: 1, which allows to increase the yield of cultured cells by increasing the proliferation efficiency of proliferatively inactive cells.

2. The first change of the nutrient medium is carried out after 5 days from the start of cultivation with pretreatment of the cells with Hanks solution, which allows to increase the yield of target cells due to the maximum removal of dead, non-adherent cells, and subsequent changes of the medium are carried out every 2 days, which ensures active cell growth .

3. The trypsinization necessary to synchronize the cell culture is carried out with a 0.05% trypsin-EDTA solution after 6-8 days from the start of cultivation at a temperature of 37 ° C for 2-3 minutes, which allows to achieve separation of almost all colonies from the vial, but while maintaining the maximum number of viable cells.

Mostly the first trypsinization is carried out in one of the vials on the cell culture with the most active growth, the next day trypsinization is carried out in the second vial (with average growth), another day later in the third (with the minimum of three vials growth), which ensures time diversity the process of fixation of cells cultured in three bottles, and also allows you to avoid during trypsinization and fixation of failures that affect more than one bottle at a time.

4. To block mitosis at the metaphase stage of the culture, colchicine with a final concentration of 1 μg / ml for 2.5 hours at 37 ° C, 5% CO _{2 is} predominantly used, which allows one to obtain a sufficient number of metaphase plates for analysis and to obtain chromosomes sufficient for analysis lengths.

5. Ethanol, which, unlike methanol, is not toxic, is predominantly used for cell fixation.

The invention is illustrated by the following examples of specific performance.

Example 1

Amniotic fluid (20 weeks of gestation) was taken in the amount of 21 ml in the operating room, placed in a sterile plastic culture bottle SPL (25 cm ² , vented lid with filter), transported to the laboratory. The amniotic fluid was divided into three equal aliquots (7 ml each) in 15 ml sterile plastic conical tubes. It was centrifuged for 7 min at 1.5 thousand rpm, the supernatant was removed. 2 ml of amniotic fluid was left in a test tube with a cell pellet, which was resuspended, 5 ml of Amniomax culture medium (Gibco® AmnioMAX ™ C-100 Complete Medium, Invitrogen, USA) was added and incubated in SPL culture vials for 5 days at 37 ° C, 5% CO ₂ . After removing the medium from the vials, the cells were washed with 3 ml of Hanks solution (treatment was performed twice). Then 5 ml of culture medium was added to each vial. Further, control of cell culture growth was performed daily. The number and density of colonies in each vial were recorded in a laboratory journal. Trypsinization was carried out with a ready-made 0.05% trypsin-EDTA solution in the first bottle after 6 days from the start of cultivation, in the second after 7 days, in the third after 8 days. For this, after the nutrient medium was drained, a warm (37 ° C) trypsin-EDTA solution (3 ml) was added to the vial, held for 20 s, and the trypsin-EDTA solution was drained. The treatment was performed twice, then the vial was kept for 30 s, shaken vigorously, the cell separation from the bottom of the vial was monitored under an inverted microscope. The total processing time of the cells with trypsin-EDTA was 2-3 minutes. After separation of the cells, culture medium (5 ml) was added to the vial. Then, the medium was changed in all bottles one day after trypsinization, and fixation was performed after another 19 hours. Cell cultures were treated with colchicine solution (final concentration 1 μg / ml) for 2.5 hours at 37 ° C, 5% CO ₂ . Then the medium was drained from the vial into a centrifuge tube. The culture vial was treated with a warm (37 ° C) 0.05% trypsin-EDTA solution (3 ml), the vial was shaken, and the cell separation from the bottom of the vial was monitored under a microscope. A trypsin-EDTA solution with separated cells was poured into the same centrifuge tube. Processing was performed twice. The total cell treatment time with a 0.05% trypsin-EDTA solution was 2-3 minutes. After centrifugation and removal of the supernatant, 10 ml of a warm (37 ° C) hypotonic solution (water, fetal calf serum 2: 1) was added to the tube to fill the cells with water and better chromosome spreading during preparation, suspended, kept for 20 min at 37 ° C.

Then 5 drops of fixative were added, suspended, centrifuged, supernatant was removed, 7 ml of fixative (ethanol, acetic acid 3: 1) was added to fix the cells, suspended, the tube was kept for 20 min at 4 ° C. It was centrifuged, the supernatant was removed, 10 ml of fixative was added, suspended, and kept for 40 min at 4 ° C. Centrifuged, the supernatant was removed, leaving 1 ml with a precipitate. The fixative was added up to 2-2.5 ml (depending on the precipitate), suspended. The suspension was unearthed on fat-free, cold, wet glass slides. The resulting preparations were dried for 20 min under a stream of warm air, and stained according to the standard technique [2]. The material necessary for analysis was obtained from the first bottle after 8 days from the start of cultivation, from the second bottle after 9 days, from the third after 10 days.

The mitotic index was determined as the ratio of the number of metaphase plates per total number of 1000 nuclei, expressed as a percentage. The mitotic index was 6.6%.

We analyzed 50 metaphase plates to exclude mosaicism, since a marker chromosome was detected in one metaphase. Fetal karyotype: 46, XY.

In FIG. 1 presents a microscopic picture of a group of metaphase plates that are of high quality and satisfy the necessary criteria for analysis.

Example 2

Amniotic fluid (16 weeks of gestation) was taken in an amount of 21 ml in the operating room, placed in a sterile plastic culture bottle SPL, and transported to the laboratory. The amniotic fluid was divided into three equal aliquots (7 ml), placed in sterile plastic conical tubes with a volume of 15 ml, centrifuged for 7 min at 1.5 thousand rpm, the supernatant was removed. 2 ml of amniotic fluid was left in a test tube with a cell pellet, which was resuspended, 5 ml of Amniocar culture medium (NLP PanEco, Russia) was added and incubated in SPL culture vials for 5 days at 37 ° C, 5% CO ₂ . After removing the medium from the vials, the cells were washed with 3 ml of Hanks solution (treatment was performed twice). Then 5 ml of culture medium was added to each vial. Further, control of cell culture growth was performed daily. The number and density of colonies in each vial were recorded in a laboratory journal. Trypsinization was carried out with a prepared solution of trypsin-EDTA (0.05%) in the first vial after 7 days from the start of cultivation, in the second after 8 days, in the third after 9 days. For this, after the nutrient medium was drained, a warm (37 ° C) trypsin-EDTA solution (3 ml) was added to the vial, held for 20 s, and the trypsin-EDTA solution was drained. The treatment was performed twice, then the vial was kept for 30 s, shaken vigorously, the cell separation from the bottom of the vial was monitored under an inverted microscope. The total processing time of the cells with trypsin-EDTA was 2-3 minutes. After cell separation, culture medium (5 ml) was added to the vial. The medium was changed in the first and second vials two days after trypsinization, in the third - one day after trypsinization, material was fixed after 18 hours. The cultures were treated with colchicine solution (final concentration 1 μg / ml) for 2.5 hours at 37 ° C, 5% CO ₂ . Then the medium was drained from the vial into a centrifuge tube. The culture vial was treated with a warm (37 ° C) 0.05% trypsin-EDTA solution (3 ml), the vial was shaken, and the cell separation from the bottom of the vial was monitored under a microscope. A trypsin-EDTA solution with separated cells was poured into the same centrifuge tube. Processing was performed twice. The total processing time of the cells with a 0.05%) trypsin-EDTA solution was 2-3 minutes. After centrifugation and removal of the supernatant, 10 ml of a warm (37 ° C) hypotonic solution (water, fetal calf serum 2: 1) was added to the tube to fill the cells with water and better chromosome spreading during preparation, suspended, kept for 20 min at 37 ° C.

Then 5 drops of fixative were added, suspended, centrifuged, supernatant was removed, 7 ml of fixative (ethanol, acetic acid 3: 1) was added to fix the cells, suspended, the tube was kept for 20 min at 4 ° C. It was centrifuged, the supernatant was removed, 10 ml of fixative was added, suspended, and kept for 40 min at 4 ° C. Centrifuged, the supernatant was removed, leaving 1 ml with a precipitate. The fixative was added up to 2-2.5 ml (depending on the precipitate), suspended. The suspension was unearthed on fat-free, cold, wet glass slides. The resulting preparations were dried for 20 min under a stream of warm air, and stained according to the standard technique [2]. The material necessary for analysis was obtained from the first vial 10 days after the start of cultivation, from the second and third vials after 11 days. The mitotic index was 4.4%.

We studied 26 metaphase plates, used an additional type of staining - C-staining for analysis, since a polymorphic variant (9ph) was found in the analysis of G-stained chromosomes. C-staining was carried out according to the standard method [2]. Fetal karyotype: 46, XX, 9ph.

The proposed method allows to obtain from each vial within 8-11 days a large amount (2-2.5 ml) of a suspension of cultured fetal cells from amniotic fluid, which is sufficient to prepare 24-35 drugs (2-10 drugs are usually used for analysis). Preparations made from suspensions have a high mitotic index (4-6.9). The quantity and quality of metaphases is sufficient to conduct a complete cytogenetic study (in case of suspicion of mosaicism, analysis of up to 100 metaphase plates, if necessary, the use of additional diagnostic methods). Reducing cultivation time saves costly reagents, supplies and working time. In addition, the optimized method allows to minimize the likelihood of artifact pathology and contamination of crops by reducing the cultivation period and a single trypsinization in the culture.

Information sources

1. B.C. Baranov, T.V. Kuznetsova, T.E. Ivashchenko; under the editorship of B.C. Baranova and E.K. Aylamazyan Modern algorithms for prenatal diagnosis of hereditary diseases: guidelines. SPb Publishing House NL., 2009. 80 p.

2. Baranov B.C., Kuznetsova T.V. Cytogenetics of human embryonic development. SPb Publishing House NL, 2007. 640 p.

3. Nelson M.M. Amniotic fluid cell culture and chromosomes studies. In: Antenatal Diagnosis of Genetic Disease. Edinburg-London: Williams a. Wilkins. 1973. P. 69-81.

4. Patent RU 2415929 C2, op. 04/10/20115. Antonenko V.G., Lilp I.G. Recommendations for ensuring the quality and reliability of cytogenetic studies. European standards for cytogenetic studies of constitutive and acquired chromosomal abnormalities. Moscow. 2007.34 s.

6. Nazarenko S.A., Vasilyeva E.O. Test system of external quality control of cytogenetic studies in medical and genetic services: a manual for doctors. Tomsk: "Printing Manufactory." 2003.34 p.

Claims (1)

A method of culturing amniotic fluid cells, including adding a nutrient medium to the cells of the amniotic fluid and incubating in vials, several changes of the nutrient medium followed by treatment of the cells with colchicine, detaching the cells from the bottom of the vial, hypotonic treatment and fixative treatment, characterized in that the nutrient medium is used Amniomax or Amniocar medium in the ratio of the initial sample: medium equal to 1.4: 1, the first change of medium is carried out after 5 days from the beginning of cultivated I pre-treat the cells with Hanks' solution in a ratio of 2.3: 1, subsequent medium changes are carried out every 2 days, then after 6-8 days from the start of cultivation trypsinization is carried out using a 0.05% trypsin-EDTA solution for 2-3 minutes at 37 ° C, while the first trypsinization is carried out in one of the vials on the cell culture with the most active growth, the next day trypsinization is performed in the second vial with medium growth, another day later in the third vial with minimal growth, the cells are treated with colchicine with conc by centration of 1 μg / ml for 2.5 hours at 37 ° C in the presence of 5% CO ₂ , cells are detached from the bottom of the vial with a 0.05% trypsin-EDTA solution for 2-3 minutes at 37 ° C, hypotonic treatment of cells carry out an aqueous solution of fetal calf serum (2: 1) for 20 minutes at 37 ° C, and the fixation of the cells is carried out with a solution containing ethanol and glacial acetic acid in a ratio of 3: 1.

Images