Cell separation is the process by which biological tissues are separated into single-cell dispersion systems by physical, biological and chemical methods。Stem cellsThere are many methods of separation, and centrifugal separation is a common method. Centrifugal technology creates a powerful centrifugal power when objects are rotated at high speed, making them rotate...
Cell separation is the process by which biological tissues are separated into single-cell dispersion systems by physical, biological and chemical methods. There are many methods of stem cell separation, and centrifugal separation is a common method。
Centrifugal technology is the use of a high-speed rotation of objects to produce a powerful centrifugal force, which allows suspended particles placed in a rotating body to sink or float, thereby enabling some particles to concentrate or separate from others. Suspended particles are often used to refer to cells, cellular devices, viruses and biomolecular molecules manufactured in suspended states. When the centrifuge rotor rotates at high speed, when the suspended particle density is greater than the density of the surrounding medium, the particles move away from the axis and sink; if the particle density is lower than the density of the surrounding medium, the particles float in the direction of the axis. According to the centrifugal principle, centrifuge technology can be divided into differential centrifugal methods, density gradient centrifugal methods and sediment equilibrium centrifugal methods. In the case of cell separation techniques, differential centrifugal and density gradient centrifugal methods are used。
1. The differential centrifugal method
The sizes and shapes of cells of different types and functions are different, and the differential centrifugal method is to achieve separation by gradually increasing the relative centrifugal power and dissipating the particles of cells of different sizes in an unequally mixed fluid。
2. Centrifugal method of density gradients
Using a medium to form a continuous or non-continuous density gradient within the centrifuge, cell-mixed fluid or slurry is placed at the top of the medium, and the cell is layered and separated by the operation of gravity or centrifuge field. The media commonly used for density gradients are caesium chloride, sugar cane and polycane sugar. The medium for the separation of active cells requires: 1 to produce density gradients with low viscosity when density is high; 2ph is neutral or is easily neutral; 3 is low permeability at large concentrations; 4 is not toxic to cells。
1) speed zone centrifuge method. Before the centrifugal, the centrifugal tube is filled with a density gradient medium such as sugar, glycerine, cscl, percoll, to be separated from the sample at the top of the gradient fluid, at the bottom of the cavity or in the middle of the gradient layer, centrifugal with the gradient fluid, using the different sedimentation or drift rates of the particles in the gradient, so that the particles with different sedimentation rates are within the gradient layers at different density levels for purposes of separation. The act allows the separation of biological samples such as cells, viruses, chromosomes, lipid proteins, dna and rna。
2) prefabricated gradients equal to density centrifugal method. The pre-formulation of thin, pre-centrifugal, tube-dependant gradient mediums with sugar, cscl, cs2so4, etc., is required, and the samples to be separated are typically laid on the top of the gradient, and the liquid density of the samples is regulated if it is to be held between the gradient fluids or the bottom of the tube. After centrifugalization, the particles of the sample of different density reach a gradient layer equal to their own density, i. E. At the same density and are separated。
3) the density centrifugal method. Some density media are self-contained gradients after centrifugalization, e. G. Percoll, which can rapidly form gradients, and cscl, cs2so4 and triodide glucoseamines can produce stable gradients after long periods of centrifugalization. Samples requiring centrifuge separation can be evenly mixed with the gradient medium, and, after the centrifuge begins, the gradient medium has the thinest density gradient due to the operation of centrifugal forces that gradually form a tube base thick, while the original mixture of sample particles can be redirected to the gradient layer equal to its own density, i. E. To the equivalent density position。
3. Deposition balance centrifugal method
Separating on the basis of the floating density difference of the separated substance, the initial density of the medium used is approximately equal to the density of the separated substance. The medium forms a density gradient during the centrifugal process, and is deposited or floated up to an area of the medium corresponding to its density。
The most critical instrument for centrifuge technology is the centrifuge. It is generally accepted that centrifuges at 10-25 kr/min are referred to as high-speed centrifuges; at more than 25 kr/min, the centrifuge is referred to as ultra-velocity centrifuges with greater than 89 kg. The current maximum speed of the hypervelocity centrifuge can be up to 100 kr/min with a centrifugal power exceeding 500 kg. The number of centrifuges used for stem cell separation is about 5kr/min.
