Introduction to the principle of stem cell centrifuge separation

Cell separation is the process of separating biological tissues into single cell dispersions by physical, biological, and chemical methods. There are many methods for stem cell separation, and centrifugation is one of the commonly used methods.

Centrifugal technology is to use the centrifugal force generated by the high-speed rotation of the object to cause the suspended particles placed in the rotating body to settle or float, so that some particles can be concentrated or separated from other particles. The suspended particles herein often refer to cells, organelles, viruses, and biological macromolecules which are in a suspended state. When the centrifuge rotor rotates at a high speed, when the density of the suspended particles is greater than the density of the surrounding medium, the particles move away from the axial direction and sedimentation occurs; if the density of the particles is lower than the density of the surrounding medium, the particles move toward the axial direction to float. According to the principle of centrifugation, the centrifugation technique can be further divided into differential centrifugation, density gradient centrifugation and sedimentation equilibrium centrifugation. In the cell separation technique, differential centrifugation and density gradient centrifugation are mainly used.

1. Differential centrifugation The size and shape of cells of different types and functions are different. Differential centrifugation is to gradually precipitate the cell particles of different sizes in a heterogeneous mixture by gradually increasing the relative centrifugal force. Thereby achieving the purpose of separation.

2. Density gradient centrifugation
A continuous or discontinuous density gradient is formed in the centrifuge tube with a certain medium, and the cell suspension or homogenate is placed on the top of the medium, and the cells are separated and separated by the action of gravity or centrifugal force field. Commonly used media for density gradient centrifugation are barium chloride, sucrose and polysucrose. The medium for separating living cells requires: 1 to produce a density gradient, and when the density is high, the viscosity is not high; 2PH neutral or easy to adjust to neutral; 3 when the concentration is large, the osmotic pressure is not large; 4 is non-toxic to cells.

1) The speed zone is centrifuged. Before centrifugation, the centrifuge tube is filled with density gradient medium such as sucrose, glycerin, CsCl or Percoll. The sample to be separated is placed on top of the gradient solution, at the bottom of the centrifuge tube or in the middle of the gradient layer, and centrifuged together with the gradient solution to utilize the particles in the gradient solution. The difference in the sedimentation velocity or the floating velocity is such that particles having different sedimentation velocities are in a gradient layer of different densities to achieve separation from each other. This method can separate biological samples such as various cells, viruses, chromosomes, lipoproteins, DNA and RNA.

2) Prefabricated gradient equal density centrifugation. It is required to prepare a density gradient medium with a thick bottom and a thin tube top before centrifugation. The commonly used medium is sucrose, CsCl, Cs2SO4, etc. The sample to be separated is generally laid on the top of the gradient liquid, if it needs to be immersed in the middle of the gradient liquid or at the bottom of the tube. The sample liquid density needs to be adjusted. After centrifugation, the sample particles of different densities reach a gradient layer equal to their own density, i.e., reach a position of equal density to obtain separation.

3) Self-forming gradient equal density centrifugation. Some density media will self-form gradient after centrifugation. For example, Percoll can form a gradient quickly. CsCl, Cs2SO4 and triiodoformyl glucosamine can also produce a stable gradient after long-term centrifugation. The sample that needs to be separated by centrifugation can be uniformly mixed with the gradient medium. After the start of centrifugation, the gradient medium gradually forms a dense density gradient at the bottom of the tube due to the centrifugal force, and at the same time, the original mixed sample particles can also be restarted. The distribution, reaching a gradient layer equal to its own density, reaches a position of equal density and is separated.

3. Settlement equilibrium centrifugation
Separation according to the difference in buoyancy density of the separated substances, the initial density of the medium used is approximately equal to the density of the separated material, and the medium forms a density gradient during centrifugation, and the separated material settles or floats to reach the medium region with the same density. And form a zone.

The most critical instrument for centrifugation is the centrifuge. It is generally believed that a centrifuge with a rotational speed of 10 to 25 Kr/min is called a high-speed centrifuge; a rotational speed exceeding 25 Kr/min and a centrifugal force greater than 89 Kg is called an ultracentrifuge. At present, the maximum speed of the ultracentrifuge can reach 100Kr/min, and the centrifugal force exceeds 500Kg. The number of revolutions of the centrifuge used for the separation of stem cells is about 5Kr/min.