What are the influencing factors of cell separation by velocity sedimentation?

1.        Cell sedimentation coefficient

Since the resolution depends on the ratio of the zone width to the radial path, the larger the sedimentation coefficient is, the larger the distance between the cell zones is. The smaller the sedimentation coefficient distribution of the same cell line is, the narrower the cell zone is. The higher the resolution, the easier it is to separate. When separating cells with small difference in sedimentation coefficient, the width of the initial cell suspension should be kept as small as possible, the amount of sample loading should be small, and the gradient column should be long, that is, separated by long centrifuge tube to compensate for the settlement coefficient. The adverse effects of the rate.

2.        Gradient density range

The cell density must be greater than the zui density of the gradient medium, using as much as possible the gradient of zui and the gradient of zui low viscosity, in order to complete the separation in a short time.

3.        Gradient volume

The number of isolated cells differs from that of the separation device. The volume of the gradient varies widely, ie the gradient volume is limited by the rotor used and the recovery method, typically ranging from 5 ml to 1600 ml. Unlike iso-density centrifugation, speed separation in very few gradient media is difficult.

4.        Cell loading

As a result of cell interaction or cell-to-cell association is a function of cell concentration. There are two problems. One is how many cells are added to the gradient to obtain the desired resolution; the other is how large the concentration of the cell suspension does not cause cell aggregation and can recover the number of cells and cell purity suitable for the study. Due to the large differences in cells from various tissue sources, the number of contaminated cells and the resolution of gradient media, it is difficult to develop a universally applicable method, generally based on experience or according to literature reporting methods, the theoretical methods discussed above. There are certain limitations in practical applications.

5.        Centrifugal speed and time

The choice of zuijia separation centrifugal speed is affected by several factors, including cell size and density, medium viscosity and density range. In any event, the applied centrifugal force is sufficient to allow the cells to move in the medium and migrate to a range of distances in due course. If the centrifugation speed is too low, the centrifugation time is too long. For example, when the cardiomyocytes are separated in a high-viscosity medium, the centrifugation time is too long, which may cause the cardiomyocytes to be deprived of oxygen and die. If the centrifugation speed is too high, the small cells will quickly pass through the gradient and even reach the bottom of the tube in a short time, re-mixing with the large cells. Therefore, in speed centrifugation, centrifugation speed and time are critical. In order to reduce the instability of unstable cells or the loss of activity of certain factors, the separation is completed as quickly as possible using a higher rotational speed. If the larger cell settling rate is too long, a highly viscous gradient medium can be used to settle at a suitable rate from the love gradient.

6.        stable

The isolated cells were mostly carried out at room temperature (20 to 25 ° C ), and in a few cases at 4 °C .