RU2815539C1 - Method for creating cell model of premature ovarian failure in wistar rats - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to experimental medicine, namely to a method for creating models of diseases, and can be used to create a cell model of premature ovarian failure (POF). Culture of granulosa cells of Wistar rat ovary after five stages of subculturing is treated with preparation of cyclophosphamide with provision of working concentration in growth medium of 0.1 mg/ml with subsequent incubation for 6 hours.

EFFECT: method provides 100% simulation efficiency, creation of a model in a short time, high manufacturability and environmental safety of POF simulation, allows studying efficacy of various substances as a drug for treating POF on its basis, improving personification of treatment.

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Description

The invention relates to experimental medicine, namely a method for creating disease models, and can be used to create a cellular model of premature ovarian failure (POF). POI is characterized by accelerated loss of follicles during the fertile period and premature cessation of ovarian function. According to the European Society of Human Reproduction and Embryology (ESHRE) Guidelines, the diagnosis of POI is based on the presence of oligomenorrhea or amenorrhea for at least 4 months in combination with an increase in follicle-stimulating hormone (FSH) levels > 25 IU /l, confirmed twice (not during ovulation), with an interval of more than 4 weeks (Webber L. et al., ESHRE guideline. Management of women with premature ovarian insufficiency. Hum Reprod. 2016; 31(5):926-37. https://doi.org/10.1093/humrep/dew027. Reed D. Primary ovarian insufficiency: a primary care overview. Proc UCLA Heal, 2019. 23). This syndrome is characterized by adverse long-term consequences for a woman's health, including an increased risk of coronary heart disease, osteoporosis, dementia, dyslipidemia and many other chronic diseases (Dhanushi Fernando W. et al. Premature ovarian insufficiency and infertility. Aust J Gen Pract. 2023; 52( l-2):32-38. doi:10.31128/AJGP-08-22-6531). In addition, there are psychological consequences of loss of fertility, which can provoke the development of depression and cause a significant decrease in quality of life.

In a recent large meta-analysis that included data from 31 studies, the prevalence of POI was 3.7%. At the same time, at the age of 20 years, POI occurs in 1 woman out of 10 thousand, and at 30 years - in 1 woman out of 1000 (Golezar S. et al. The global prevalence of primary ovarian insufficiency and early menopause: a meta-analysis. Climacteric 2019;22(4):403-11. https://doi.org/10.1080/13697137.2019.1574738).

POI is a polyetiological disease, and in a significant number of women the cause remains unknown. The only treatment method for POI is hormone replacement therapy, which is continued until the average age of natural menopause. The implementation of reproductive function is possible using methods of assisted reproductive technologies (donor oocytes).

Currently, there is no single approach or universal method for treating POI, given the extremely polymorphic clinical picture. Therefore, further study of the pathogenesis of POI with the development of alternative methods of pathogenetically based therapy is necessary. It is promising to study the effectiveness of various drugs on the course of the disease in the framework of preclinical trials using cell models.

A number of experimental models of POI in laboratory animals have been described in the literature, but in the available literature we have not found any reports on methods for modeling POI on a cell line.

The technical result of the invention is the creation of a cellular model of POI, ensuring 100% modeling efficiency, creating a model in a short time, high manufacturability and environmental safety of modeling a pathological condition, the ability to use the created model to test the medicinal effectiveness of chemicals for the purpose of their further use in medicine.

Cell cultures for modeling various pathologies attract the attention of researchers as promising potential systems for studying the mechanism of the disorder, as well as the possibility of testing various pharmacological drugs. In this case, an important advantage is the more appropriate use of experimental animals (the need to use multiple groups of animals is eliminated), the duration of the experiment (much shorter, since long exposure after administration to a laboratory animal is not required).

An additional advantage of this invention is that cell lines provide a more technologically advanced and environmentally friendly method for modeling a pathological condition, and the scale of cultivation ensures the production of cell lines in quantities sufficient for clinical trials and therapeutic use.

This technical result is achieved in a method for creating a cellular model of POF, in which a culture of Wistar rat ovarian granulosa cells after five stages of subcultivation is treated with the drug cyclophosphamide, ensuring a working concentration in the growth medium of 0.1 mg/ml, followed by incubation for 6 hours.

The method is illustrated in Fig. 1, 2, where:

In fig. 1 - graphical confirmation of the resulting signal change in response to the introduction of follicle-stimulating hormone (FSH), indicating that the cells under study are ovarian granulosa cells.

In fig. 2 - graphical confirmation of the emerging dysfunction of complex I of the electron transport chain associated with the action of cyclophosphamide.

The inventive method is based on experimental studies involving 9 female Wistar rats.

The method is carried out, for example, as follows.

In a sexually mature Wistar rat, the ovary is collected by laparotomy, the resulting fragment is washed three times with a sterile isotonic NaCl solution, the tissue is mechanically crushed, washed with Versene solution, followed by trypsinization. Next, the trypsin solution is removed, and the fragments are washed three times with DMEM solution and collagenase is added (0.2% collagenase solution; 10 ml of solution per 1 g of tissue), after which collagenase is neutralized by adding fetal bovine serum, and the tissue is crushed until a homogeneous suspension is obtained. The resulting suspension was washed three times with DMEM solution and suspended in complete growth medium. 5 stages of subcultivation of the culture of granulosa cells of Wistar rats are carried out, subcultured into sterilized round coverslips and treated with the drug cyclophosphamide to ensure a working concentration in the growth medium of 0.1 mg/ml, followed by incubation for 6 hours, treated with the drug cyclophosphamide to ensure a working concentration of growth medium 0.1 mg/ml, followed by incubation for 6 hours. The magnitude and mechanism of maintaining mitochondrial membrane potential are assessed.

It is known that the drug cyclophosphamide causes the induction of chromosomal aberrations, exchange of sister chromatids, single-strand breaks and the formation of double cross-links of DNA, which subsequently leads to increased cell apoptosis (Kalich-Philosoph L. et al. (2013) Cyclophosphamide triggers follicle activation and burnout; AS101 prevents follicle loss and preserves fertility. Sci Transl Med 5: 185ra62). Our results demonstrated a decrease in the fluorescence intensity of TMRM (tetramethylrhodamine methyl ester), which indicates a sharp drop in the value of the mitochondrial membrane potential (Fig. 2). At the same time, in contrast to the control, in model cells there was practically no depolarization of mitochondria in response to the introduction of rotenone, which indicates emerging dysfunction of complex I (NADH-dehydrogenase complex) of the electron transport chain associated with the action of cyclophosphamide.

The inventive method can be easily reproduced in experimental laboratories. All experiments and preparations were carried out under sterile conditions.

An example of the method.

The culture of Wistar rat granulosa cells was used as a cell line. In a mature Wistar rat (age - 4 months, quarantine - 2 weeks) weighing 258 g, the right ovary was collected under isoflurane anesthesia: laparotomy, isolation of the right uterine horn, visualization of the right ovary, ligation and intersection of the neurovascular bundle were performed. The resulting fragment was washed three times with a sterile isotonic NaCl solution. Then the ovary was mechanically crushed and washed with Versene solution, followed by trypsinization (0.25% trypsin solution; 10 ml of solution per 1 g of tissue) at a temperature of 37°C for 45 minutes. Then the trypsin solution was removed, the tissue fragments were washed three times with DMEM and exposed to collagenase (0.2% collagenase solution; 10 ml of solution per 1 g of tissue) at 37°C for 45 minutes. The action of the enzyme was stopped by adding 2 ml of fetal bovine serum (FBS), after which the tissue fragments were crushed by pipetting to obtain a homogeneous suspension. Cells were washed three times with DMEM followed by suspension in complete growth medium (DMEM with 1 g/l glucose, 10% FBS, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine, 100 U/ml penicillin, 100 μg/ ml streptomycin). Cell cultivation was carried out in a CO2 incubator at a temperature of 37°C and a CO2 content of 5% until the confluency of the adhesion culture monolayer was achieved at least 70%. To model premature ovarian failure, cells obtained after five stages of subculturing were used. To perform reseeding, the culture was washed twice with Versene solution (0.1 ml per 1 cm ² of the surface of the culture bottle), followed by treatment with a trypsin solution (0.25%, 0.1 ml per 1 cm ² of the surface of the culture bottle) and kept at a temperature of 37° With periodic rocking. After complete detachment of the monolayer, the enzyme was inactivated by adding complete growth medium (0.1 ml per 1 cm ² of the surface of the culture flask), separated by centrifugation, followed by decantation of the supernatant, and the cells were washed twice with complete growth medium. The number of viable cells in the resulting suspension was monitored by staining with trypan blue (working concentration 0.2%) and counting in a Goryaev chamber. The initial concentration in the culture flask during subculture was 104 cells per 1 ^cm2 .

To conduct research, cells were removed from the culture flask using the described method using trypsin, after which they were seeded onto pre-sterilized round coverslips and cultured until a confluency of at least 70% was achieved. In order to identify the culture, a functional cell analysis was performed based on the activation of calcium signaling in ovarian granulosa cells using FSH. To do this, cells on coverslips were washed three times from the growth medium using Hanks' balanced salt solution (HBSS), followed by incubation in a 5 μM solution of the ratiometric calcium-sensitive probe Fura-2 AM for 45 minutes at 37°C with the addition of 0.005% Pluronic F127 followed by three washes with HBSS. Fluorescence studies were carried out on a wide-field fluorescence microscope with a fluorite immersion objective (×20) using two excitation wavelengths - 380 nm and 340 nm for the free and calcium-bound forms of the Fura-2 probe. The ratio of fluorescence intensity upon excitation at wavelengths of 340 nm and 380 nm (ratio 340/380) was used as an analytical signal. The measurement protocol included recording the baseline level of the analytical signal followed by the administration of a follicle-stimulating hormone drug (Fig. 1).

A change in the nature of the signal in response to the introduction of FSH indicates that the cells under study are ovarian granulosa cells.

To model POI, cells on cover glasses were treated with a cyclophosphamide preparation providing a working concentration in the growth medium of 0.1 mg/ml, followed by incubation for 6 hours and assessment of the magnitude and mechanism of maintaining mitochondrial membrane potential. To do this, cells on coverslips were washed three times from the growth medium using Hanks' balanced salt solution (HBSS), followed by incubation in a 25 nM solution of the mitochondrial targeting fluorescent probe tetramethylrhodamine (TMRM) for 45 minutes at 37°C. Fluorescence studies were carried out on a laser scanning confocal microscope using an excitation wavelength of 561 nm and recording fluorescence intensity in the range of 565-650 nm. The measurement protocol included recording the baseline signal level, the signal after the addition of the F1-F0-ATP synthase inhibitor oligomycin A (2 μg/ml), the complex I inhibitor rotenone (2 μM), and the mitochondrial uncoupler FCCP (2 μM). The magnitude of the mitochondrial membrane potential was assessed by the amplitude of the change in fluorescence intensity between the baseline level and the level after the addition of FCCP. The mechanism of maintaining mitochondrial membrane potential was assessed by the nature of the signal change after supplementation. Ovarian granulosa cells that were not treated with cyclophosphamide served as a control.

In comparison with the control, cells treated with the drug are characterized by an almost 3-fold decrease in the intensity of TMRM fluorescence, which indicates a sharp drop in the mitochondrial membrane potential. Moreover, in contrast to the control, in model cells there is practically no depolarization of mitochondria in response to the introduction of rotenone (Fig. 2), which indicates the emerging dysfunction of complex I of the electron transport chain associated with the action of cyclophosphamide.

The inventive method ensures 100% modeling efficiency, creation of a model in a short time, high manufacturability and environmental safety of modeling a pathological condition, makes it possible to further study the effectiveness of various chemicals as a drug for the treatment of this syndrome, improve the personalization of the treatment of the disease, and use this cellular model in fundamental and applied scientific research.

Claims (1)

A method for creating a cellular model of premature ovarian failure, which consists in treating a culture of Wistar rat ovarian granulosa cells after five stages of subcultivation with the drug cyclophosphamide, ensuring a working concentration in the growth medium of 0.1 mg/ml, followed by incubation for 6 hours.