Cell Line Culture

Pet cloning refers to the process of asexual reproduction by using nuclear transplantation (i.e. transplanting the same genetic material as the original PET) to create a new life. An important part of this process is cell line culture. Generally speaking, cell line culture usually includes two main processes: somatic cell line establishment and somatic cell nuclear transfer.

The main process of donor cell line: complete and healthy active cells are necessary for the successful culture of the donor cell line. This means that some of the raw materials we need for cloning must be somatic cells of cloned organisms and must be active. When the pet just died, the somatic cells are still active. However, over time, the activity will gradually decrease, so it is necessary to sample and complete cell preservation and culture as soon as possible. Under appropriate conditions, active cells can be extracted from the skin and muscle tissue of the remains. After obtaining tissue samples from pets, healthy cells are obtained and cultured. Then, several cells in the best condition will be selected as gene donor cells.

The steps of somatic cell nuclear transfer: 1. extract a small piece of tissue from the pet; 2. select a cell as the donor cell after cell culture and establishment; 3. transfer the nucleus of the donor cell containing genetic material to the egg cell with the nucleus removed; 4. use microcurrent stimulation to combine the two, and then promote the division and reproduction of the new cell; 5. develop into an embryo; 5. when the embryo develops to a certain extent, implant it into an animal's uterus to make the animal pregnant. An animal with the same gene as the nuclear donor will be born.

In addition to the two methods described above, in the process of cell line culture, technicians also need to use the following important technologies and pay attention to relevant precautions:

Standard Cell Culture Protocol

Cell culture, also known as cell cloning technology, refers to extracting cells from animals or plants and then conducting scientific research in an artificial environment. Using cell culture technology, a cell can be transformed into a simple single cell or several differentiated multi cells after a large number of cultures. For either the whole bioengineering technology or biological cloning technology, cell culture is an essential process. The technological process itself is the large-scale cloning of cells.

There are several basic cell culture protocols in all cell culture laboratories. It is important to be familiar with and understand operating standards.

Aseptic Technology

The consistent operation of aseptic technology can help ensure the sterility of all media and dishes, reduce the exposure of cells to contaminants and keep the culture healthy, feasible, and pure. Strict aseptic technology is an essential prerequisite for successful cell culture to protect the culture from microbial contamination and cell cross-contamination.

Primary Cell Isolation

After correctly collecting the pet tissue sample, the primary cell separation operation needs to be carried out. There are many different methods to prepare samples to obtain the best cell separation effect. The method chosen depends on your starting sample and may involve removing certain elements from the sample or creating only a single-cell suspension. When isolating cells from intact tissue, mechanical force or proteolytic enzymes must first be used to destroy the extracellular matrix that holds cells together.

Cell Subculture

Subculture refers to the dilution of cells that have reached a high degree of fusion so that they can be continuously cultured. Adherent cells should be subcultured at 80-90% confluence, and suspended cells should be subcultured when the cells are caked and turbid. It is very important to record the cell transmission algebra of each cell line. This helps to monitor protocell viability and plan experiments before it reaches senescence. It is important to always treat cells gently, because severe or severe treatment may cause cell damage or death. 

Cryopreservation And Resuscitation

Cell lines are valuable resources, so it is very important to preserve stocks for long-term storage. Cryopreservation refers to the process of cooling and preserving cells at very low temperatures to maintain their viability. The exact freezing conditions depend on the cell line used. It is important to examine cell line-specific conditions, otherwise, frozen seed cells may not produce living cells during resuscitation and reculture. 

Detection Of Mycoplasma Infection

The main problem in cell culture is mycoplasma infection. This bacterial infection will change cell behavior and metabolism, and have adverse effects on cells. It is important to perform mycoplasma tests regularly, especially for continuous cell lines. It is better to test mycoplasma before freezing the cells, when new cell lines are received, and the protocell seeds are recovered and cultured.

BioVenic has advanced cell experiment equipment and provides an aseptic environment. It can professionally cultivate your pet cells while maintaining good cell health, providing the best nuclear donor cells for the next process. BioVenic strives to contribute to the rapid breeding and gene preservation of excellent pet animals, and provides you the best dog cloning and cat cloning services.

References 

1. Schmitz J, et al. Heterogeneity Studies of Mammalian Cells for Bioproduction: From Tools to Application. Trends Biotechnol. 2019 Jun;37(6):645-660.
2. Ravi M, et al. The culture conditions and outputs from breast cancer cell line in vitro experiments. Exp Cell Res. 2019 Oct 15;383(2):111548.
3. Prior N, et al. Liver organoids: from basic research to therapeutic applications. Gut. 2019 Dec;68(12):2228-2237.