METHOD FOR DEVELOPING CELL CULTURE FROM EGG CELLS OF CAUCASIAN HONEY BEE (APIS MELLIFERA CAUCASICA)

Abstract

The invention relates to the fields of biotechnology and cell culture development, and specifically concerns a cell culture development method for culturing and multiplying cells obtained from eggs of the Caucasian honey bee (Apis mellifera caucasica) in vitro.

Description

CAUCASIAN HONEY BEE (APIS MELLIFERA CAUCASICA) EGGS A METHOD FOR DEVELOPING CELL CULTURE FROM CELLS TECHNICAL FIELD The invention relates to the fields of biotechnology and cell culture development, in particular. cells obtained from eggs of the Caucasian honey bee (Apis mellifera caucasica) a cell culture for culturing and multiplying cells in vitro It is related to the development method. PREVIOUS TECHNIQUE Honey bees are important for the continuity of ecosystems and agricultural production. living organisms, and the study of their physiological and cellular characteristics, exposure important in terms of understanding the effects of the biological and chemical agents they are exposed to. It carries, in particular, viral agents such as Deformed Wing Virus (DWV) that interact with cells. This allows for the investigation of the interaction, infection, and replication processes of pesticides and In vitro for evaluating the direct cellular effects of environmental stressors Cell cultures are used. In studies on culturing honeybee cells under in vitro conditions Although primary cell cultures have been established, the viability of the cells and limitations in terms of maintaining its proliferation for a long period This situation involves virus replication and pesticides in honeybee-derived cells. a sustainable and reproducible in vitro testing system for studies such as toxicity It restricts its use as such. The effect of starting material on the continuity of honey bee cell cultures preparation, ensuring sterile conditions, cells from the starting material decomposition, culture medium composition, culture temperature, pH value, and passage interval Parameters such as these are influential. Differences in these parameters affect the cells. different effects on the vitality, proliferation and sustainability of culture in the cultural environment It can produce results. 1 THE PURPOSE OF THE INVENTION The aim of the invention is to produce eggs from the Caucasian honey bee (Apis mellifera caucasica). This allows the obtained cells to be cultured and multiplied under in vitro conditions. The goal is to develop a method for developing cell cultures. Another aim of the invention is to enzymatically process cells obtained from eggs. isolation, culture of isolated cells supplemented with bee hemolymph extract placed in an environment and incubated under temperature and pH conditions suitable for bee physiology. This involves supporting the viability and proliferation of cells. Another aim of the invention is to allow the resulting cell culture to be analyzed at specific intervals. It is through the transmission of information that the continuity of culture is ensured. Another objective of the invention is to enable the resulting cell culture to undergo DWV replication. This is an in vitro cell that can be used in pesticide toxicity studies and related research. It is about creating culture. DETAILED DESCRIPTION OF THE INVENTION The invention was obtained from eggs of the Caucasian honey bee (Apis mellifera caucasica). It is a method for culturing isolated cells in an in vitro environment. The method collecting eggs in a sterile manner, performing the sterilization process, cells from eggs were enzymatically separated using a trypsin / EDTA solution. separation, centrifugation of the separated cells, and the resulting The cells are placed in a culture medium containing bee hemolymph extract, and the cells are stored at 34°C. Incubation at a certain temperature and in an environment containing 5% CO₂ stimulated cell proliferation. microscopic monitoring and passage of the culture at specific intervals It includes. Within the scope of the invention, Caucasian honey was used as the starting material for cell culture. Bee eggs are used. The eggs are used in cell culture. They are collected under sterile conditions to prevent contamination. Egg collection and subsequent cell culture processes are performed in a sterile laminar flow cabinet. This is carried out within it. Thus, during the preparation of the cell culture... This ensures that the access of environmental microorganisms to the culture is limited. The collected eggs are sterilized before being cultured. It is kept in place. 70% ethanol and UV light are used in the sterilization process. The sterilization process takes 20 minutes. This process sterilizes the eggs. 2 Maintaining the sterility of the culture before the cell separation stage. It is being prepared as follows. Separation of cells from eggs after sterilization process. This is carried out using a 0.05% trypsin / EDTA solution for cell separation. Eggs are washed with a 0.05% trypsin / EDTA solution for 5 minutes. It undergoes enzymatic decomposition. The use of trypsin / EDTA solution. the cells in the egg material can be separated from each other and transferred to culture. The cell suspension is obtained. Material containing cells obtained after enzymatic decomposition process. It is subjected to centrifugation. The centrifugation process takes place at a speed of 1500 rpm for 5 minutes. This is carried out. Thus, the cells obtained after enzymatic decomposition are processed. It is separated in order to be transferred to the cultural environment. The cells obtained after centrifugation are transferred to a culture medium. The culture medium used in this invention is primarily L-15 medium. The L-15 medium in question contains 10% FBS and 5% bee hemolymph extract. This is supported. A combination of L-glutamine and antibiotics is also used in the culture medium. It is located. FBS promotes the viability and proliferation of cells in culture medium. It is used in the support of honey bee hemolymph extract, culture medium. It ensures that the cells are supported in a way that meets their needs. L- Glutamine is one of the nutritional components of cell culture medium. The antibiotic combination, on the other hand, prevents microbial contamination during cell culture. It is used for the purpose of limiting [the use of] L-15 medium, 10% FBS. Culture containing a combination of 5% bee hemolymph extract, L-glutamine, and antibiotics. the environment, the viability of isolated Caucasian honeybee cells under in vitro conditions and It creates a culture medium in which proliferation is supported. The cells taken into the culture medium are placed in an incubator. The incubation process... The culture is carried out at a constant temperature of 34°C and in a medium containing 5% CO₂. The pH of the environment is regulated to 6.6. In this way, the cells are treated with Caucasian honey. at temperature and pH conditions determined according to the physiological conditions of the bee is being held. The condition of the cells in the culture medium during incubation is observed microscopically. is being monitored. Microscopic examination is performed at 200x magnification and 3 The morphological characteristics of the cells and their proliferation within the culture are monitored. This By monitoring, the development of cells in the culture medium is tracked. The cells that proliferate in culture are passaged at intervals of 3-4 days. Passaging is performed in this way... The continued growth of cells present in the existing culture in the new culture medium This ensures that the eggs of Caucasian honeybees are initially produced. the continuation of the proliferation of the obtained cells in the culture medium and the cells The aim is to ensure the continuity of the culture. The bee hemolymph extract used in the invention is a culture medium for Caucasian honey. in a way that supports the nourishment and multiplication of cells obtained from the bee It is one of the components created. The ratio of bee hemolymph extract in the culture medium. It is determined as 5%. FBS, on the other hand, is present at a rate of 10% in the culture medium. This The viability of cells under culture conditions with two components L-15 medium and It creates an environment that supports its proliferation. The 0.05% trypsin / EDTA solution used within the scope of this invention is for eggs. It enables the separation of cells from the material. Enzymatic separation 5 With a time limit of only minutes, it is suitable for transferring cells to the culture medium. The cell suspension is obtained. Within the scope of the invention, the temperature is 34°C and the pH value of the culture medium is 6.6. These temperature and pH conditions are determined for in vitro culture of the cells. It creates the physiological conditions in which the child is kept in the incubation environment. The incubation environment also... It contains 5% CO₂. The resulting cell culture allows us to observe the responses of the cells to external factors in vitro. This allows for the examination of pesticides in a controlled environment. In this context, cell culture is used to analyze pesticides. It can be used in the study of cellular toxicity effects. Pesticide toxicity. In their studies, cellular changes occurred after cultured cells were exposed to pesticides. Changes can be evaluated through the cultural system. The subject of the invention is the method. Cell cultures obtained with DWV were also used in studies conducted with DWV. It can be used. For this purpose, the DWV reference sample is used in the testing phase. and the sensitivity of the resulting cell culture to DWV can be evaluated. Thus, regarding DWV replication of cells obtained from Caucasian honey bees... It is made possible to use it in vitro for research purposes. Consequently, the invention involves sterilizing Caucasian honeybee eggs. obtaining, enzymatic decomposition with 0.05% trypsin / EDTA solution, decomposed Centrifuging the cells at 1500 rpm for 5 minutes, L-15 medium, 10% FBS, 5% purified water. 4 culture medium containing hemolymph extract, L-glutamine and antibiotic combination the cells were collected, incubated at 34°C and 5% CO₂ under pH 6.6 conditions. its proliferation is monitored microscopically at 200x magnification and the culture is followed for 3-4 weeks. In vitro testing of Caucasian honey bee cells through daily passage. The development of its culture is ensured.

Claims

1. In vitro cells from eggs of Caucasian honey bee (Apis mellifera caucasica) It is a method of developing culture, and its characteristic feature is; - Collection of Caucasian honeybee eggs under sterile conditions and subjected to sterilization process, - Enzymatic decomposition of sterilized eggs with trypsin / EDTA solution the separation of cells by subjecting them to a process, - centrifuging the material containing the separated cells, - After centrifugation, the obtained cells were placed in L-15 medium, FBS, and bee balm. culture containing hemolymph extract, L-glutamine and antibiotic combination being brought into the environment - Incubation of cells transferred to culture medium, - microscopic monitoring of cell proliferation, - involves the steps of passage of cells that proliferate in culture It is characteristic.

2. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method characterized by the sterilization process using 70% ethanol and UV light. It is characterized by being performed for a period of 20 minutes using [method / technique].

3. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method, and its characteristic feature is that it contains 0.05% in the enzymatic decomposition process. It is characterized by the use of a trypsin / EDTA solution for a period of 5 minutes.

4. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method, characterized by centrifugation at 1500 rpm for 5 minutes. It is characterized by being carried out over a period of time.

5. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method, characterized by its culture medium being L-15 medium, 10% FBS, 5% It contains a combination of bee hemolymph extract, L-glutamine and antibiotics. It is characteristic.

6. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method, characterized by the fact that the pH value of the culture medium is 6.

6. It is characteristic. 6 7. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method characterized by the cells being kept at a temperature of 34°C and containing 5% CO₂. It is characterized by incubation in a suitable environment.

8. In vitro cell culture from Caucasian honeybee eggs according to Claim 1. It is a development method, and its characteristic feature is that the cells multiplying in culture are 3-4 days old. It is characterized by its passages that are spaced intermittently. 7