Centrifugation 101: How Scientists Separate the Inner World of Cells

Before we learn about centrifugation, we need to understand cell fractionation.

• It is the process of splitting open a cell into its individual organelles by keeping its function intact.
• This is especially useful when you want to extract an intracellular protein or biological product (protein is produced in the cell and is not secreted outside).
• It has a wide range of applications, especially in the biopharmaceutical industry, where you will have to extract your protein of interest from inside your cell.
• To maintain the integrity of the organelles, you will need to do the fractionation under isotonic conditions.
• Cell fractionation is typically done using a homogenizer. This is followed by centrifugation to sediment the individual organelles based on their density.
  • Types of homogenizers-
    • Mechanical homogenizers- this includes blenders, mortar & pestles that use sheer force to disrupt the tissues. It is usually for tougher tissues and larger cells.
    • Sonication– it uses ultrasound waves to create changes in pressure that disrupt the cells. The high-frequency sound waves cause cavitation bubbles in the liquid medium. When these collapse, they produce shock waves that lyse the cells. This procedure is typically done on ice in order to prevent overheating.
    • Osmotic lysis– it uses osmotic pressure to lyse cells like RBCs. Cells are placed in a hypotonic solution, and when there is an influx of water, the cells swell up and burst.
      

Centrifugation

• It helps us separate mixtures based on centrifugal force.
• The separation can be based on size, viscosity, density, and shape
• One can purify cells, cell organelles, viruses, proteins, and nucleic acids
• Working principle– it sediments particles suspended in a liquid medium using gravitational force. Gravity causes the solid particles to settle to the bottom and separate from the liquid.
• Major parts of a centrifuge–
  •  Motor– this creates the spin. It houses the centrifuge tubes with the liquid solution to be centrifuged.
  • The rotor assembly comprises a shaft and a rotor. The rotor is attached to the motor through the shaft. The rotor converts electrical energy to mechanical energy. Unit of rotation- relative centrifugal force. They can be fixed at an angle (fixed angle rotors), horizontal (swinging bucket), or vertical
  • Containers– they can be bottles, bags, test tubes, or cuvettes.
  • A control panel to set temperature and rpm (rotations per minute
• Types of centrifugation
  • Differential centrifugation is based on size, shape, and density. Particles with different sizes and densities will sediment at different speeds. Larger and denser particles will sediment quickly.

• Density gradient centrifugation– separation is based on buoyant density or sedimentation rate. Preformed density gradients include CsCl for isolating DNA and plasmids, NaBr and NaL for fractionating lipoproteins, and dextran for whole cells. they can be of two types-
  • Rate zonal sedimentation is based on mass and size.
  • Isopycnic– sedimentation is exclusively based on density
• Ultracentrifugation– runs at extremely high speeds, like 100000g. It can separate microscopic organelles from more prominent organelles with high purity.
• Major applications
  •  Used in the dairy industry to separate cream from milk
  •  Used in water treatment to sediment solid wastes and get relatively pure water
  • Used in the biopharma industry for producing biological products and drugs