SU1451164A1 - Method of in vitro assessment of cellular populations - Google Patents

Abstract

This invention relates to biology, in particular to the study of cultured cell cultures. The aim of the invention is to simplify the method and simultaneously evaluate the duration of the stages of the cell cycle and the distribution of population cells to these stages. The invention provides estimates of the duration of the premitotic and synthetic phase of the mitotic cycle and the distribution of population cells in these phases. Assessments are obtained when studying a cell culture grown in a single matrix. Monolayer-cultured cells are pulsed incubated with a radio-active DNA precursor. A culture mattress is placed in a fractionation apparatus and connected with nutrient tubes, a fraction collector (CF) and a cell collector for incubation with a fluorescent dye (CC). Cells entering mitosis are fractionated from the population by periodic shaking. Each time, a fraction of the fraction obtained is pumped into the CF and the VAC is fed into filters for the subsequent determination of radioactivity. The second half is poured into the QC, stained and pumped through the chamber for flow counting of the fluorescent microscope. Fresh nutrient is pumped into the mattress and after a certain time the whole procedure is repeated. The intensity of DNA synthesis (IC) is determined by the ratio of the radioactivity of each fraction to the number of cells of this fraction. As a result, the duration of the synthetic phase and then the duration of the premitotic phase are determined by HC and the time of preparation of the fractions. The cell distribution in the fractions obtained during each phase is estimated by the number of cells. 2 ill., 4 tab. sl O5 4

Description

This invention relates to biology, in particular, to the study of cultured cell cultures.

The purpose of the invention is to simplify the method and simultaneously evaluate the duration of the stages of the cell cycle and the distribution of the cells of the population among these stages.

An increase in the information content and simplification of the method is achieved by periodically fractionating the studied cell population, extracting as a result of shaking, nitotic cells in the form of fractions, in which the cell concentration and radioactivity are determined, the results of these measurements are calculated cells and specific radioactivity from time, by which the duration of prenytotic Gj, synthetic S, and presynthetic 0 stages of the cell cycle, cell distribution fraction by these stages and the distribution of cells throughout these stages.

The invention is illustrated by the following examples.

Example 1. Use two

cultures of fibroblasti-like cells of the FECh-L22 and CHECh-L23 lines. Cells were subcultured for 2 passages. Cell subcultures are carried out using the standard method using standard nutrient media. 3,410 cells / mattress are seeded into experimental matrices. Take 1 mat with the FECH-L22 culture and 1 mattress with the FECH-L23 culture. The stage of prefractional cultivation is 23 hours. 1 hour before the start of the fractionation, the nutrient medium in the experimental bottles is replaced with a medium of the same composition containing H-thymidine (5 mCi / ml). Cell cultures were incubated in this medium for 60 minutes at 37 ° C, removed from non-included DNA precursor with three volumes of fresh nutrient medium, and 45 ml nutrient medium was added again. As a medium for fractionation, a medium of standard composition is used. The fractionation step is carried out in a one-hour cycle. The culture mode is 50 minutes, the fractionation mode is 10 minutes. During the experiment, 29 cell fractions were collected.

0

five

The results are shown in table 1 and 2.

Figure 1 and 2 presents the M- and S-profiles of cultures FEC-L23 FAC-L22, respectively.

The results of calculations of intermediate values for estimating the parameters of the duration of the stages of the cell cycle are shown in Table 3 and are indicated by arrows in Figures 1 and 2. Data for estimating the parameters of the distribution of the growth fraction cells by herds and m of the cell cycle and for determining the distribution of culture cells in each. th stage of the cycle are presented in table.1 and 2 (column 9).

The values of the kinetic parameters for the studied cultures are given in Table 4.

Example 2. Calculation of initial values.

The initial data for calculating the kinetic parameters are the characteristics of the mitotic profile (M profile), which is a curve describing the change in the number of cells in a population entering mitosis depending on the time of the fraction, and the S profile, which describes the change in intensity incorporation of a radioactive DNA precursor (specific radioactivity) from the time the fraction was obtained. Table 1 shows the data for the construction of M- and S-profiles. To carry out calculations of the parameters of the duration of the stages of the cell cycle, the quantitative yield of cells and the specific specific rad and inactivity data are transformed according to the moving average method. The M- and S-profile curves are plotted according to the data in columns 7 and 8 (Table 1).

The procedure for estimating the kinetic parameters (Fig. 1) is as follows.

From the curve describing the M-profile, it is clear that it is represented by three peaks. The first peak has a maximum at 2 hours and a minimum at a point corresponding to 3 hours of fractionation. The second peak has a maximum coming at 5 hours and two minima at points 3 and 10 hours. The third peak has a maximum at 16 hours and two minima at points 10 and 22 hours.

The S-profile curve is represented by two peaks, which are described

maximums in mums at points with a maximum at a point real minimum

point

Itoll

31

8 I and mini- and 16 h and max - 26 h with corresponding points 16 and

29 h. To calculate the Td, Td, and Tq parameters, it is necessary to determine the beginning and the end of the exit of cells that are in GJ, S, and G, periods of the cell cycle (which corresponds to the first, second, and third peaks of the mitotic profile). To determine the time intervals corresponding to the entry of Gj, S, and G subpopulations of cells into mitosis, the curves of M- and S-n-profiles were approximated by piecewise linear functions. In general, this procedure consists in describing each peak of the M and S profiles by two linear functions:

 t

t

but .

but .

(12)

where is the regression coefficient characterizing the upstream part of the peak; t is the time during which

variable y increases f - regression coefficient, the difference between the descending part of the peak}

t is the time during which the variable y decreases. Equating the values of y and y to zero, we find the points of beginning and ending of the peak. In Figure 1, we consider the second peak of the M-profile curve (interval between 3 and 10 hours of fractionation). The upstream part of the peak begins at the point corresponding to 3 hours and lasts for the next two hours, is described by an expression that is a transformation of equation (1) according to the example (Table 1, column 7, 3-5 fractions):

for 100.5 ts - 183.3.

(3)

The downstream part of the peak occurs in 5-10 hours of fractionation and is described by the following expression (based on Equation 2 and the above example):

y -62.8 t + 611.1,

(four)

ts start of cell diy cell release

S is in mitosis, defined by M-profile}

- the end of the release of cells of the S-stage of the cell cycle into mitosis, determined by the M-profile. Equating y and y to zero we get estimates

1.82

(five)

 iMIjLl 9 73rd and -62, 8

(6)

Similarly, other intermediate values necessary for determining the duration Q (Td, Tg, TO,) of the cell cycle stages are calculated. Below are the conventions and definitions of these values M-profile:

tq - the beginning of the release of G cells

Stage 5 in mitosis

t

Cg stage

 Q,

-ending the release of G cells into mitosis;

-the beginning of the release of G cells

0

five

stages in mitosis;

t-Q is the termination of the release of G cells, stages in mitosisJ profile;

the beginning of the release of S-stage cells into mitosis;

t, - the end of the exit of cells of the S stage into mitosis;

S - the beginning of the release of cells into mitosis.

past the second cycle

Calculations t

L - "

by s-profile

40

performed using: equations (1) and (2). Having obtained estimates of nine quantities (tC, t, t ;, t4. T,,, t, t j t), the duration of the cell cycle periods 45 is determined using the following formulas.

Example 3. Evaluation of the parameters of the duration of the stages of the cell cycle.

Duration G st.adii

50

t ,,).

(7)

(AT)

G stages, tg are not determined.

The first peak of the M-profile has

only the downstream part (Fig. 1). AT

In this case, the beginning of the period G is taken as the beginning of the fractionation, i.e. zero point.

Duration S stage

2

(T) (9)

-

L

+ ts

2

(ten)

t t - t

S

- the beginning of S stage}

- end of S stage.

(eleven)

de td

GRN

 s Duration G

gr,

T, tLi-tl

2

stage (1). (12)

.

t: - t

Q,

; i3)

(14)

Where

t

T

M1

-the beginning of G, period;

-end G ;, stage. Calculated intermediate values

values are given in table 3.

Estimated T parameters (

LQ, ig, Iq

are listed in the table

Example 4: Evaluation of Parameters Characterizing the Distribution of Growth Cell Cells by Stages of the Cell Cycle

Nc ,, N5, Nq,

).

To determine the number of cells in each stage of the cell cycle, the calculated estimates of the duration of each stage of the cell cycle are calculated. The counting of the cells that were in each stage of the cycle is carried out by summing the number of cells obtained in the fractions over the period of the corresponding stage of the cell cycle (T, Td, Tq,). For the FECh-L22 culture, the duration of the Gj stage was (1.1-6.4 h (Table 4). Summed up the number of cells obtained in the first six hourly fractions and added the cells obtained in 0.4 hours of the seventh fraction, we get the G2 stage in mitosis included 1465.6x10 cells (Table 2, column 9). In a similar way, estimates of the number of cells from other stages of the cell cycle (Ng and N, 5) are calculated. Thus, the structure of the growth fraction of the studied population

. consists of 34.7% N, 44.1% N5,

U511646

13% NQ and 8.2% of cells that have passed the second cycle.

Example 5. Evaluation of parameters reflecting the distribution of cells of a population at each stage of a cell

cycle (Rc, 5,

In general, these parameters are calculated by the formula for the linear regression coefficient

. (t ..- t) (logy; - logy)

1 el

 (t, - 1) 2

(15)

0

t; y

five

Q

where K is the number of the last fraction of cells of the corresponding stage of the cell cycle, the time of obtaining the fractions of cells of the corresponding stage of the cell cycle, the number of cells of the corresponding stage of the cell cycle, obtained by the time t. For example (Table 2, column 9), for an FEC-L22 culture t; TC, 1.1 h, t.Tc 6.4 h. The number of cells during this time increased from y {0 208, to the Criminal Code of 1465.6x10 cells. The value of A c calculated by formula (15) is 0.0012.

Thus, using the proposed method, in one experiment 5, three groups of parameters were obtained. These parameters characterize the duration of the stages of the cell cycle, the distribution of population cells by these stages and the distribution of cells within each stage of the cycle.

Claims (1)

Formula of gain A method for estimating the kinetics of cell populations in vitro by pulsed incubation with a radioactive DNA precursor with a subsequent assessment of kinetic parametrob, characterized in that, in order to simplify the method and simultaneously evaluate the duration of cell cycle stages and the distribution of cell populations by these stages, the studied cell population is periodically fractionated, the mitotic cells are removed by shaking in the form of fractions, in which the cell concentration and radioactivity are determined, by to these of  U511648 measures the specific radioactive S and presynthetic G, potency, plotting changes in cell cycle stages, distribution of cell numbers and specific radio cell growth of the growth fraction in G, activity from time to fractionation-S and G, the stages and distribution of a kennel, which determine the length over the G., S and G. stages Premitotic G, Sieu-Cell cycle. Table 1 eleven 1451164 12 Continued table. 2 Culture Parameters h kZ Zl LlIiIl EZAn n lZ PHEH-L22 1.11 9.7 5.16 15.6 7.83 20.98 2.62 17.25 15.95 PHEC-L23 - 3.64 1.82 9.73 10.12 21.7 0 , 68 15.48 16.32  T a b l and c a 4 5, 3 (1.1-6.4) 2.2 (0-2.2) 12.5 (3.9-16.4) 11.3 (1.3-12.6) 5.5 (12.04-18.5) 6.3 (12.8-19.0) 1465.6 (34.7%) 793.2 (23.5%) 1864.5 (44.1% 1295.7 (38.4%) 550.9 (13.0%) 1014.3 (39, Note. In parentheses, the start time of the end cell cycle stages. Parameters h I fUI30 ffT} Ulf / Oor) V (i tfDHH / dffi Th u lr  yitniiHDdcb og moszi osui:) 3hTii / Q u lr gu4JOHffniU) ivoiigvd X  OS)) J;  a Th ts 5G , l