RU2415929C2 - Specialised medium for human amniotic fluid and fibroblast cell culture - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for oncogenetic analysis of amniotic fluid cells, as well as for cultivation of fibroblasts and other mesenchymal cells for laboratory purposes. Human amniotic fluid and fibroblasts are cultured in a specialised medium having a modified base. The base of the cell culture medium contains organic and inorganic salts, amino acids, vitamins, nucleosides.

EFFECT: effective replication of proliferative low-activity cells in vitro.

2 cl, 2 dwg, 7 tbl, 3 ex

Description

Application area

The invention relates mainly to the field of medical diagnostics, experimental medicine and cell biology. The invention can be used for cytogenetic analysis of amniotic fluid cells, as well as culturing mesenchymal cells for laboratory purposes.

State of the art

The widespread introduction of methods for prenatal diagnosis of genetic diseases is an urgent task in the preventive direction of modern medicine. Early detection of hereditary abnormalities in the fetus provides predictable results of pregnancy management, assessment of genetic risks, opens the possibility of their prevention and relief in newborns [1].

Conducting most of the procedures of cytogenetic and genetic diagnostics involves obtaining the embryo / fetus or trophoblast structures necessary in quantity and quality of cellular material. For example, a routine cytogenetic analysis or FISH hybridization of metaphase chromosomes requires a representative sample of proliferating cells. For this purpose, the cell material obtained by invasive methods is cultured in special media that ensure active reproduction of fetal cells in vitro [1, 2].

It is known that in the practice of prenatal diagnosis (in addition to chorionbiopsy and cordocentesis), the safest way to obtain a method for producing fetal cellular material during the period of 12-23 weeks of pregnancy is to obtain amniotic fluid cells [1, 3].

Amniocytes precipitated by centrifugation are propagated in the presence of cell growth stimulators in special media, block mitosis at the metaphase stage and carry out karyological / molecular cytogenetic analysis in situ or in suspension culture [3, 4].

The main problem of the technology of working with amniotic fluid cell culture is the creation of an adequate system for culturing primary floating cells having various sources of origin from fetal tissues (urethral, skin, intestinal, myeloid, etc.) [5]. It should be noted that cells floating in amniotic fluid are often limited in terms of viability and clonal growth in culture, which is an indispensable condition for obtaining high-quality material for analysis. Only 10–40% of all cells remain viable and have a proliferative potential for the formation of colonies up to 500 cells in size for 7–9 days when cultured in special media [6]. A number of nutrient media are presented on the global biopharmaceutical market to provide procedures for the efficient cultivation of fetal tissue cells for conducting prenatal genetic diagnostics [4]. However, only a small number of the products offered are truly effective and recognized in the practice of perinatology. The qualitative and quantitative composition of these products is a trade secret and are not disclosed in the press.

The domestic patent RU 2236457 “METHOD FOR PRODUCING A LIFFOCAR NUTRIENT MEDIUM FOR CULTIVATION OF HUMAN AND ANIMAL PERIPHERAL BLOOD LYMPHOCYTES” is known from the prior art. Obviously, the developed medium can also be used to multiply cord blood lymphocytes during prenatal diagnosis. At the same time, the cordocentesis procedure requires special staff qualifications, has increased risks associated with contamination, traumatic blood loss and premature miscarriage.

Patent US 5,879,937 "Cytokine-induced proliferation of amniotic t-cells", which describes a method for producing proliferating T lymphocytes by culturing amniotic fluid cells in a medium containing a complex of cytokines: IL-2, IL-4 and IL-7, is related to the present invention . The method allows cytogenetic analysis and the procedure of HLA typing of the fetus for transplant compatibility. However, the proposed method has limited use, due to the high cost of the reagents used, the complexity and instability of the selective cultivation of immature forms of lymphocytes. Closely related to the present invention is EP 2000/907644, PHARMACEUTIC, DIETETIC AND COSMETIC COMPOSITIONS BASED ON THIOCTIC ACID AND CYSTEINE, which describes the optimization of an amniotic fluid cell culture medium - Amniomed (EuroClone, Italy) by administering the composition amino acids with antioxidant properties (thiooctium and cysteine). The authors showed a 30 to 50% increase in the number of colonies of proliferating cells and a high proliferation rate of amniocytes. At the same time, the basic formulation of the tested media remains closed for component analysis of the composition.

Russian analogues of media for culturing amniotic fluid cells have not yet been presented on the domestic market of biopharmaceuticals, which determines the need for the procurement of appropriate products from Western manufacturers and their high cost.

The aim of the invention is to develop a method for culturing amniotic fluid cells in a domestic production of Amniocar and determine the growth properties of this medium in relation to cells of mesenchymal origin using an example of a secondary culture of human fibroblasts.

EFFECT: method for culturing amniotic fluid cells in an Amniocar medium allows for the implementation of prenatal diagnosis of genetic diseases using amniotic fluid cells and for efficient cultivation of human fibroblasts in vitro.

The implementation of the invention

The claimed technical result is achieved due to the fact that the incubation of amniotic fluid cells is carried out in a specialized environment "Amniocar", which provides effective attachment and proliferation of cellular material in the monolayer. A method of cultivating cells of amniotic fluid and human fibroblasts in a specialized environment that provides effective attachment and proliferation of cellular material in a monolayer, characterized in that the cells are incubated in ventilated culture bottles in an atmosphere of 5% CO ₂ and incubated for 7-11 days. In addition, a complete change of medium, if necessary, is carried out 72 hours after the start of incubation. The specialized Amniocar medium for the cultivation of cells of amniotic fluid and other cells of mesenchymal origin, including glucose, organic and inorganic salts, amino acids, characterized in that it contains a modified base and cell growth stimulators that ensure efficient proliferation of proliferatively inactive cells. Ventilated culture bottles (CO ₂ -incubators - thermostatically controlled chambers) are necessary to create a gas atmosphere corresponding to exhaled air (5% -CO ₂ ). This percentage of carbon dioxide in the gas phase ensures the maintenance of the buffer capacity and physiological pH values (7.0-7.5) for most nutrient media containing bicarbonate buffer as the main acidity regulator.

The essence of the invention lies in the development of a method for effective in vitro cultivation of cells of primary amniocytes and human fibroblasts using a special medium “Amniocar” developed by the Applicant.

It was shown that the use of the Amniocar medium allows one to increase the proliferative activity level of cells of the secondary culture of fibroblast-like and epithelial-like amniocytes in comparison with the classical DMEM medium containing 10% fetal calf serum (ETF) by 4 and 3 times, respectively. It has been established that the Amniocar medium provides the number of mitoses at the level of the metaphases analyzed in the range from 2.5 to 6.0% during the cultivation of primary amniocyte cells. This indicator is 6 times higher than the corresponding metaphase level when using DMEM medium with 10% ETS and 2.4 times higher than the corresponding indicator for the classical Cheng medium containing specific stimulators of amniocyte growth in vitro [7]. The high efficiency of Amniocar medium in the process of culturing human skin fibroblasts has been demonstrated. The cell yield obtained using this medium is 4-6 times higher than the number of fibroblasts grown on classical DMEM medium with 10% ETS. Thus, the Amniocar medium can be effectively used for culturing amniotic fluid cells during prenatal genetic diagnostics and for rapid accumulation of fibroblast biomass during in vitro cultivation.

The use of an optimized composition of basic components in the composition of the Amniokar environment

It is known that the first effective medium for culturing amniotic fluid cells, proposed by Cheng [7], contained as a basis the well-proven composition of DMEM and F12 media in a 1: 1 ratio. At the same time, the specificity of the cultivation of amniocytes, taking into account the special requirements for maintaining buffer and antioxidant properties, as well as providing easily absorbable nutrients and vitamins, requires optimization of the basic composition of the nutrient medium.

The applicants proposed the basic composition of the components of the Amniocar medium, which ensures the rapid growth of cultures of proliferatively inactive cells (including amniotic fluid cells and fibroblasts).

The qualitative and quantitative composition of the components of the basis of the Amniocar medium is presented in table 1.

Cultivation of fibroblast-like and epithelioid cells of secondary amniocytes in the Amniocar medium

Amniotic fluid cells represent many heterogeneous in origin and cultural properties of amniocyte populations [5, 6]. Therefore, a comparative analysis of the proliferative activity of cells cannot be reliably evaluated using limited-volume samples of amniotic fluid. Monoclonal cells of amniotic fluid of fibroblast-like and epithelial-like morphology, which were selected and propagated as a result of 2 passages on the Amniomax-C100 commercial medium (Invitrogen) [4], were used to carry out an objective assessment of the effectiveness of cultivation in the Amniocar medium.

Example 1

Test environments:

1. "Amniocar" - a medium for the cultivation of amniotic fluid cells - development of gr. biotechnology MGNTs RAMS (LLC NPP PanEko)

2. Control environment: DMEM + 10% ETS.

Experiment Conditions

50 thousand cells of secondary cultures of fibroblast-like amniocytes (Amn-P) and epithelioid amniocytes (Amn-K) were inoculated in 5 ml of test media and incubated at 37 ° C, 5% CO ₂ for 7 days in ventilated SPL culture bottles, 25 cm ² . The change of medium was carried out 4 days after the start of incubation. At the end of the incubation, the grown cultures were removed from the plastic surface and quantitative indicators of cell growth were determined (PI, T2).

Proliferation Index (PI): PI = C2 / C1,

where C1 is the total number of cells or the concentration of inoculum (cells / ml), C2 is the total cell yield at the end of the incubation time, t or its concentration (cells / ml).

The doubling time of the experimental and control cultures was calculated by the formula T2 = t × ln2 / ln (PI) [8],

where T2 is the doubling time (hour), t is the total incubation time of the culture (hour), PI is the proliferation index.

The data obtained for cultures of two morphological types are presented in tables 2 and 3.

Thus, the analysis shows a significant positive effect of the composition of the Amniocar medium on cell proliferation of the main morphological types of secondary amniocyte cultures.

Cultivation of Amniocyte Primary Culture Cells in Amniocar Medium A targeted test of the efficiency of culturing amniotic fluid cells in Amniocar medium was carried out using primary cell material obtained from amniotic fluid samples using the ″ flask ″ cultivation method [3].

Example 2

Test environments:

1. "Amniocar" - a medium for the cultivation of amniotic fluid cells - development of gr. biotechnology MGNTs RAMS (LLC NPP PanEko).

2. Wednesday Cheng [5].

3. Control medium: DMEM + 10% ETS.

Experiment Conditions

A 30 ml sample of amniotic fluid (17 weeks pregnant) was divided into 3 10 ml aliquots into conical plastic tubes and centrifuged (200g, 10 min). 100 thousand cells of the primary amniocyte culture, in a volume of 1 ml of the supernatant suspension, were inoculated in 5 ml of each test medium and incubated using the Flask method at 37 ° C, 5% CO ₂ for 11 days in ventilated SPL culture vials, 25 cm ² . A complete change of medium was carried out 72 hours after the start of incubation. At the end of the incubation, cultures were treated with colchicine (1 μg / ml, for 1 hour), followed by removal of the cells with Trypsin / EDTA solution (0.05%) from the plastic surface. Quantitative indicators of cell growth were determined by directly counting the density of cell suspensions in cultures using a Goryaev camera. To determine the mitotic index, the resulting cell suspensions were precipitated by centrifugation (200g, 10 min). Then the cells were treated with a hypotonic solution, fixed and metaphase chromosome preparations were prepared according to the standard technique with Giemsa staining [3]. The mitotic index (MI) was determined as the ratio of the number of metaphase plates per the total number of 1000 nuclear elements, expressed as a percentage:

MI = N _MP / 1000 × 100%

The efficiency of the cultivation process in Amniocar medium was evaluated by proliferative activity, mitotic index, and the quality of the microscopic picture of metaphase chromosome plates.

The morphology and cellular composition of the main types of colonies of amniocytes developing in the Amniocar medium are presented in Fig. 1, where A is a colony of epithelial cells (actively proliferating); B - Colony of cells of the epithelial type (slowly proliferating); B - Colony of fibroblast-like cells of type 1 (actively proliferating); D - Colony of fibroblast-like cells type 2 (moderately proliferating).

A comparative analysis of the proliferative activity of amniocytes in the environments of the tested variants is presented in Table 4.

A microscopic picture of a group of metaphase plates of amniocytes proliferating in the Amniocar medium is presented in FIG. 2.

A comparative analysis of the mitotic index for the tested media options is presented in Table 5.

Thus, the Amniocar medium demonstrates the best characteristics among the tested media variants and meets the requirements for the media providing a high level of proliferative activity of a heterogeneous cell population.

Cultivation of skin and lung fibroblast cells in Amniocar medium

Fibroblasts are the main cellular and forming element of connective tissue of mesenchymal origin. Fibroblast cells can be easily isolated from human skin and can be used both for research and for a number of biochemical and genetic tests for clinical diagnostic purposes [9].

Example 3

Test environments:

1. "Amniocar" - a medium for the cultivation of amniotic fluid cells - development of gr. biotechnology MGNTs RAMS (LLC NPP PanEko).

2. Control medium: DMEM + 10% ETS.

50 thousand cells of secondary cultures of adult human skin fibroblasts HSF (passage 15) and alveolar fibroblasts of the human embryo A-Fh (passage 5) were inoculated in 5 ml of test media and incubated at 37 ° C, 5% CO ₂ for 7 days in ventilated culture bottles SPL, 25 cm ² . At the end of the incubation, the grown cultures were removed from the plastic surface and quantitative indicators of cell growth were determined (PI, T2: see Example 1). The data obtained for cultures of two morphological types are presented in Tables 6 and 7. The data presented indicate an increase in the proliferative properties of fibroblasts of cultures of both types in the Amniocar medium by 4-5 times compared with the classical medium DMEM + 10% ETS.

The reproducibility of the test results allows the use of secondary fibroblast cultures as a test object for testing the quality of the media used for the cultivation of amniocytes. In this case, as a quantitative criterion for the proliferative properties of the medium can be an indicator of the relative proliferation index (IPR):

IPR = N _M / N _DMEM ,

where N _M is the number of cells taken from the bottle with the test medium;

N _DMEM - the number of cells taken from a bottle with a control medium DMEM + 10% ETS.

The IPR index for Amniocar medium in a series of 10 independent experiments ranged from 4.5 ± 0.3 for A-Fh cell culture to 5.1 ± 0.4 for HSF cell culture. Thus, the data presented in Example 3 indicate:

1) the suitability of the Amniocar medium for the efficient proliferation of fibroblasts;

2) the possibility of using a fibroblast culture as a test object when testing media for culturing amniotic fluid cells.

Information sources

1. B.C. Baranov. Soros Educational Journal, No. 10, 32-36, 998.

2. V.V. Chestkov. Medical Genetics, vol. 4, No. 1, 20-22, 2005.

3. B.C. Baranov, T.V. Kuznetsova. Cytogenetics of human embryonic development, Ed. N-L, St. Petersburg, 2007.

4. S. Kuligowski et al, Focus, Vol. 21, No. 2, 35-37, 1999.

5. A-R. Prusa, M. Hengstschlager, Med. Sci. Monitor, Vol. 8, No. 11, RA253-257, 2002.

6. C. M. Gosden, Br. Med. Bull., Vol. 39, 348-354, 1983.

7. Z. H. Chang et al, Proc. Natl. Acad. Sci. USA, Vol. 79, 4795-4799, 1982.

8. M.K. Jr. Patterson, Measurement of growth and viability of cells in culture. In: W. B. Jacoby, I. H. Pastin, eds., Methods in Enzymology, New York: Academic Press Inc., Vol. 58, 141-152, 1979.

9. B. Fowler et al, Pediatric Research, Vol.41, No. 1, 145-151, 1997.

A method of cultivating cells of amniotic fluid and human fibroblasts in a specialized environment and a specialized environment "amniocar" for its implementation

table 2 Comparative analysis of the proliferative properties of fibroblast-like amniocytes in test media Test environment The total cell yield (thousand) IP / T2 % Control Amniocar 2956 43.4 / 31.0 394 Control: DMEM + 10% EMF 750 15.0 / 43.0 one hundred

Table 3 Comparative analysis of the proliferative properties of epithelial-like amniocytes in test media Test environment The total cell yield (thousand) IP / T2 % Control Amniocar 1688 33.8 / 33.1 303 Control: DMEM + 10% EMF 558 11.2 / 48.3 one hundred

Table 4 Comparative analysis of proliferative properties of primary amniocytes in test media Test environment The total cell yield (thousand) % Control Amniocar 387 516 Wednesday Cheng 268 357 Control: DMEM + 10% EMF 75 one hundred

Table 5 Comparative analysis of the mitotic index of primary amniocytes in test media Test environment MI (%) Amniocar 5.3 Wednesday Cheng 2.2 Control: DMEM + 10% EMF 0.88

Table 6 Comparative analysis of the proliferative properties of HSF skin fibroblasts in test media Test environment The total cell yield (thousand) IP / T2 % Control Amniocar 960 19.2 / 39.4 485 Control: DMEM + 10% EMF 198 3.96 / 84.6 one hundred

Table 7 Comparative analysis of proliferative properties of alveolar fibroblasts of the A-Fh embryo on test media Test environment The total cell yield (thousand) IP / T2 % Control Amniocar 1450 29.0 / 34.6 469 Control: DMEM + 10% EMF 309 6.2 / 64.0 one hundred

Claims (2)

1. The basis of the medium for the cultivation of cells of amniotic fluid and human fibroblasts, containing, mg / l:
Inorganic salts NaCl 6200 KCl 400 MgSO ₄ 73.00 Na ₂ HPO ₄ 150.00 NaH ₂ PO ₄ 54.00 NaHCO ₃ 2960.0 Ca (NO ₃ ) ₂ · 4H ₂ O 50.00 CaCl ₂ one hundred FeSO ₄ · 7H ₂ O 0.04 CuSO ₄ · 5H ₂ O 0.001 ZnSO ₄ · 7H ₂ O 0.20 Carbohydrates D-glucose 4000 Organic acids and salt Hypoxanthine Na 2.00 Lipoic acid 0.2 Pyruvate sodium 110 Sodium Acetate 3H ₂ O 2.00 Putrescin 2НСl 0,03 Amino Alcohols 2-aminoethanol 0.80 Amino acids L-Alanine 10.00 L-Arginine Hcl 137 L-Glycine thirty L-histidine 25.00 L-Isoleucine 88.00 L-leucine 88.00 L-Lysine Hcl 108 L-Methionine 25.00 L-Phenylalanine 55.00 L-Serine 35.00 L-Threonine 65.00 L-Tryptophan 16.00 L-Valine 69.00 L-Asparagine 16.00 L-Aspartic Acid 6.00 L-Glutamine 7.00 L-hydroxyproline 5.00 L-proline 9.00 L-Ornithine 0.40 L-Taurine 0.30 L-cystine 45.30 L-tyrosine 59.00 Peptides L-Alanyl-Glutamine 330.0 Glutathione 0.80 Vitamins Vitamin C 3.00 D-Ca pantothenate 3.00 Choline chloride 5.00 i-Inositol 20.00 Niacinamide 4.00 Pyridoxal Hcl 1.00 Pyridoxine Hcl 2,50 Riboflavin 0.30 Thiamine Hcl 3.00 P-aminobenzoic acid 0.25 B ₁₂ 0.30 Folic acid 3.00 Biotin 0.10 α-tocopherol phosphate Na ₂ 0.002 Menadione bisulfate Na ₂ 0,030 Ergocalciferol 0,020 Vitamin A Acetate 0,020 Nucleosides Ribonucleosides Adenosine 5.00 Cytidine 5.00 Guanosine 5.00 Thymidine 0.45 Uridine 5.00 Deoxyribonucleosides 2'-deoxyadenosine 5.00 2'-Deoxyadenosine · H ₂ O 5.40 2'-Deoxycytidine Hcl 5.50 2'-deoxyguanosine 5.00 2'-Deoxyguanosine · H ₂ O 5.40 5-methylcytosine 0.0045 2'-deoxythymidine 5.00 2. A method of cultivating cells of amniotic fluid and human fibroblasts in a specialized medium based on claim 1, characterized in that the cells are incubated in ventilated culture bottles in an atmosphere of 5% CO ₂ and incubated for 7-11 days with a complete change medium 72 hours after the start of incubation.

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