3.1. Creation of rho0 Cell Cultures

To obtain rho0-human cells, the method of M. King and G. Attardi was used [30].

The algorithm for creating rho0-human cells is as follows:

1. At the first stage, the possibility of creating rho0-cells was tested. For this purpose, a normal culture of monocytic origin, THP-1, was cultured in a growth medium in two variants:

With the addition of uridine and ethidium bromide;

With the addition of ethidium bromide only.

The growth medium contained pyruvate and glucose at the concentration indicated for the complete DMEM medium. The necessary conditions for the cultivation of THP-1 for the creation of rho0-cells were determined:

Time of cultivation;

Concentration of additives used in the medium.

2. At the second stage, the rho0 cell line was created. THP-1 was placed in a growth medium with the addition of uridine and ethidium bromide. With the use of ethidium bromide, the mitochondrial genome was blocked, and complete mitochondrial dysfunction occurred. Afterwards the cultured cell line was placed on medium with only uridine (but without ethidium bromide). In the process of culturing on this medium, the cell line was to lose all non-functioning mitochondria and to become an mtDNA-less cell culture (rho0). It should be noted that it took us 18 weeks to create a rho0 culture of monocytic origin. Thirty-six passages were conducted (two passages per week). If a stable, rho0 cell line was obtained using the culture conditions described above, then an analysis was performed to confirm that the mitochondria in the cell line were absent. To achieve this aim, the analysis of the number of mitochondrial genome copies in rho0 culture was carried out (Figure 1).

Number of copies of mtDNA in the culture of THP-1 cells (reference matrix-mutation m.12315G>A).

In the process of creating rho0 cells and cybrid cultures, the following solutions and media were used (Table 1).