CELL DISRUPTION

CELL DISRUPTION

Introduction

Biological products synthesized by fermentation or cell culture are either intracellular or extracellular. Intracellular products either occur in a soluble form in the cytoplasm or are produced as inclusion bodies (fine particles deposited within the cells). Examples of intracellular products include recombinant insulin and recombinant growth factors. A large number of recombinant products are found as inclusion bodies in order to accumulate in larger quantities within the cells. In order to obtain intracellular products the cells first have to be disrupted to release these into a liquid medium before further separation can be carried out. Certain biological products have to be extracted from tissues, an example being porcine insulin which is obtained from pig pancreas. In order to obtain such a tissue­ derived substance, the source tissue first needs to be homogenized or ground into a cellular suspension and the cells are then subjected to cell disruption to release the product into a solution. In the manufacturing process for intracellular products, the cells are usually first separated from the culture liquid medium. This is done in order to reduce the amount of impurity: particularly secreted extracellular substances and unutilized media components. In many cases the cell suspensions are thickened or concentrated by microfiltration or centrifugation in order to reduce the process volume.

Several factors must be considered.

Volume or sample size of cells to be disrupted

If only a few microliters of sample are available, care must be taken to minimize loss and to avoid cross-contamination.

Disruption of cells, when hundreds or even thousands of liters of material are being processed in a production environment, presents a different challenge. Throughput, efficiency, and reproducibility are key factors.

How many different samples need to be disrupted at one time?

Frequently when sample sizes are small, there are many samples. As sample sizes increase, fewer samples are usually processed. Issues are sample cross contamination, speed of processing, and equipment cleaning.

How easily are the cells disrupted?

As the difficulty of disruption increases (e.g. E. coli), more force is required to efficiently disrupt the cells. For even more difficult samples (e.g. yeast), there is a parallel increase in the processor power and cost. The most difficult samples (e.g. spores) require mechanical forces combined with chemical or enzymatic efforts, often with limited disruption efficiency.

What efficiency of disruption is required?

Over-disruption may impact the desired product. For example, if subcellular fractionation studies are undertaken, it is often more important to have intact subcellular components, while sacrificing disruption efficiency.

For production scale processes, the time to disrupt the cells and the reproducibility of the method become more important factors.

How stable is the molecule(s) or component that needs to be isolated?

In general, the cell disruption method is closely matched with the material that is desired from the cell studies. It is usually necessary to establish the minimum force of the disruption method that will yield the best product. Additionally, once the cells are disrupted, it is often essential to protect the desired product from normal biological processes (e.g. proteases) and from oxidation or other chemical events.

What purification methods will be used following cell disruption?

It is rare that a cell disruption process produces a directly usable material; in almost all cases, subsequent purification events are necessary. Thus, when the cells are disrupted, it is important to consider what components are present in the disruption media so that efficient purification is not impeded.

Is the sample being subjected to the method biohazardous?

Preparation of cell-free extracts of pathogens presents unique difficulties. Mechanical disruption techniques are not always applicable owing to potential biohazard problems associated with contamination of equipment and generation of aerosols.

Cells

Different types of cell need to be disrupted in the bio­industry:

·         Gram positive bacterial cells

·         Gram negative bacterial cells

·         Yeast cell

·         Mould cells

·         Cultured mammalian cells

·         Cultured plant cells

·         Ground tissue

Bacterial cells: The cell wall of Gram positive bacteria is thick and mainly composed of thick layer of peptidoglycan layer. While the plasma or cell membrane which is made up of phospholipids and proteins is relatively fragile. In certain cases polysaccharide capsules may be present outside the cell wall. The cell wall of gram positive bacteria is particularly susceptible to lysis by the antibacterial enzyme, lysozyme. Unlike gram positive bacteria, gram negative bacteria do not have distinct cell walls but instead have multi­layered envelops. The peptidoglycan layer is significantly thinner than in gram positive bacteria. An external layer composed of lipopolysaccharides and proteins is usually present. Another difference with gram positive bacteria is the presence of the periplasm layers which are two liquid filled gaps, one between the plasma membrane and the peptidoglycan layer and the other between the peptidoglycan layer and the external lipopolysaccharides. Periplasmic layers also exits in gram positive bacteria but these are significantly thinner than those in gram negative bacteria. The periplasm is important in bioprocessing since a large number of proteins, particularly recombinant proteins are secreted into it. An elegant way to recover the periplasmic proteins is by the use of osmotic shock. This technique is discussed below.

Yeast/Mould cells: Yeasts which are unicellular have thick cell walls, typically 0.1 to 0.2 microns in thickness. These are mainly composed of polysaccharides such as glucans, mannans and chitins. The plasma membrane in a yeast cell is composed of phospholipids and lipoproteins. Mould cells are largely similar to yeast cells in terms of cell wall and plasma membrane composition but are multicellular and filamentous.

Mammalian cells: Mammalian cells do not possess the cell wall and are hence quite fragile i.e. easy to disrupt.

Plant cells: Plant cells on the other hand have very thick cell walls mainly composed of cellulose and other polysaccharides. Cell wall wherever present is the main barrier which needs to be disrupted to recover intracellular products. A range of mechanical methods can be used to disrupt the cell wall. Chemical methods when used for cell disruption are based on specific targeting of key cell wall components. For instance, lysozyme is used to disrupt the cell wall of gram positive bacteria since it degrades peptidoglycan which is a key cell wall constituent. In gram negative bacteria, the peptidoglycan layer is less susceptible to lysis by lysozyme since it is shielded by a layer composed of lipopolysaccharides and proteins.

Cell membranes: Cell membranes or plasma membranes are composed of phospholipids arranged in the form of a bi­layer with the hydrophilic groups of the phospholipids molecules facing outside (Figure below). The hydrophobic residues remain inside the cell membrane where they are shielded from the aqueous environment present both within and outside the cell. The plasma membrane can be easily destabilized by detergents, acid, alkali and organic solvents. The plasma membrane is also quite fragile when compared to the cell wall and can easily be disrupted using osmotic shock i.e. by suddenly changing the osmotic pressure across the membrane. This can be achieved simply by transferring the cell from isotonic medium to distilled water.

Figure 1: Plasma membrane

Cell disruption methods can be classified into two categories: Mechanical methods and Non-mechanical methods.

A. Mechanical Methods of Cell Disruption:  uses mechanical forces to disintegrate the cells like soli shearing, liquid shearing, pressure etc. Some methods are:

1.                  Disruption in bead mill

2.                  Disruption using a rotor­ stator mill

3.                  Homogenization

4.                  Disruption using French press

5.                  Disruption using ultrasonic vibrations

B. Non-mechanical Methods of Cell Disruption: includes physical, chemical and biological treatment of cells to disrupt the cells.

1.      Physical methods

a.       Disruption using osmotic shock

b.      Freeze-thaw method

2.      Chemical methods

a.       Detergents

b.      Solvents

c.       Acid and alkali methods

3.      Biological methods

a.       Enzymes e.g. lysozyme

b.      Phage

c.       Autolysis

The mechanical methods are targeted more towards cell wall disruption while the non-mechanical methods are mainly used for destabilizing the cell membrane.

A. Mechanical methods for cell disruption

1. Cell disruption using bead mill

Bead mill equipment consists of a tubular vessel made of metal or thick glass within which the cell suspension is placed along with small metal or glass beads. The tubular vessel is then rotated about its axis and as a result of this the beads start rolling away from the direction of the vessel rotation. At higher rotation speeds, some the beads move up along with the curved wall of the vessel and then cascade back on the mass of beads and cells below. The cell disruption takes place due to the grinding action of the rolling beads as well as the impact resulting from the cascading beads.

Figure 2: Cell disruption by Bead Milling

Bead milling can generate enormous amounts of heat. While processing thermolabile material, the milling can be carried out at low temperatures, i.e. by adding a little liquid nitrogen into the vessel. This is referred to as cryogenic bead milling. An alternative approach is to use glycol cooled equipment. A bead mill can be operated in a batch mode or in a continuous mode and is commonly used for disrupting yeast cells and for grinding animal tissue. Using a small scale unit operated in a continuous mode, a few kilograms of yeast cells can be disrupted per hour. Larger unit can handle hundreds of kilograms of cells per hour.

Disadvantages:

• Occasional problems with foaming and sample heating, especially for larger samples.
• Tough tissue samples such as skin or seeds are difficult to disrupt unless the sample is very small or has been pre-chopped into small pieces.

2. Cell disruption using rotor­-stator mill

A rotor­-stator mill device consists of a stationary block with a tapered cavity called the stator and a truncated cone shaped rotating object called the rotor. Typical rotation speeds are in the 10,000 to 50,000 rpm range. The cell suspension is fed into the tiny gap between the rotating rotor and the fixed stator. The feed is drawn in due to the rotation and expelled through the outlet due to centrifugal action. The high rate of shear generated in the space between the rotor and the stator as well as the turbulence thus generated are responsible for cell disruption. These mills are more commonly used for disruption of plant and animal tissues based material and are operated in the multi­pass mode, i.e. the disrupted material is sent back into the device for more complete disruption. The cell disruption caused within the rotor­-stator mill can be described using the equations discussed for a bead mill.

Figure 3: Cell disruption using rotor-stator mill

3. Homogenization

Liquid based homogenization is the most widely used cell disruption technique for small volumes and cultured cells. Cells are lysed by forcing the cell or tissue suspension through a narrow space known as clearance space (0.001 mm- 0.006 mm), thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Dounce homogenizer consists of a round glass pestle that is manually driven into a glass tube. A Potter-Elvehjem homogenizer consists of a manually or mechanically driven Teflon pestle shaped to fit a rounded or conical vessel. The number of strokes and the speed at which the strokes are administered influences the effectiveness of Dounce and Potter-Elvehjem homogenization methods. Both homogenizers can be obtained in a variety of sizes to accommodate a range of volumes.

Figure 4: Cell disruption using homogenizer

4. Cell disruption using French press

A French press is a device commonly used for small ­scale recovery of intracellular proteins and DNA from bacterial and plant cells. The device consists of a cylinder fitted with a plunger which is connected to a hydraulic press. The cell suspension is placed within the cylinder and pressurized using the plunger. The cylinder is provided with an orifice through which the suspension emerges at very high velocity in the form of a fine jet. The cell disruption takes place primarily due to the high shear rates and differential pressure. The internal FRENCH Pressure Cell pressure increases as the pressure developed by the Laboratory Press increases. The intracellular pressure increases as well. As the sample is dispensed through the sample outlet tube, the external pressure on the cell wall drops rapidly toward atmospheric pressure. The pressure within the cell drops as well but not as quickly as the pressure external to the cell. This pressure differential causes the cell wall membrane to burst, releasing the intra-cellular contents. A French press is frequently provided with an impact plate, where the jet impinges causing further cell disruption. Typical volumes handled by such devices range from a few milliliters to a few hundred milliliters. Typical operating pressure ranges from 10,000 to 50,000 psi.

Advantages:

—  This technique results in more uniform and complete disruption

—  Cells do not require pre-treating.

—  Easy to use

Figure 5: Cell disruption using French press

5. Cell disruption using ultrasonic vibrations

Ultrasonic vibrations (i.e. having frequency greater than 18 kHz) can be used to disrupt cells. The cells are subjected to ultrasonic vibrations by introducing an ultrasonic vibration emitting tip into the cell suspension (Figure below). Ultrasound emitting tips of various sizes are available and these are selected based on the volume of sample being processed. The ultrasonic vibration could be emitted continuously or in the form of short pulses. A frequency of 25 kHz is commonly used for cell disruption. The duration of ultrasound needed depends on the cell type, the sample size and the cell concentration. These high frequency vibrations cause cavitations, i.e. the formation of tiny bubbles within the liquid medium. When these bubbles reach resonance size, they collapse releasing mechanical energy in the form of shock waves equivalent to several thousand atmospheres of pressure. The shock waves disrupt cells present in suspension. For bacterial cells such as E. coli, 30 to 60 seconds may be sufficient for small samples. For yeast cells, this duration could be anything from 2 to 10 minutes.

Ultrasonic vibration is frequently used in conjunction with chemical cell disruption methods. In such cases the barriers around the cells are first weakened by exposing them to small amounts of enzymes or detergents. Using this approach, the amount of energy needed for cell disruption is significantly reduced.

Disadvantages:

• Heat generated by the ultrasound process must be dissipated.
• High noise levels (most systems require hearing protection and sonic enclosures)
• Yield variability
• Free radicals are generated that can react with other molecules.

Figure 6: Cell disruption by sonication

6. Mortar and Pestle

It is a manual grinding method, most commonly used to disrupt plant cells. In this method, tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of tensile strength of the cellulose and other polysaccharides comprising the cell wall, this method is the fastest and most efficient way to access plant protein and DNA.

B. Non-mechanical methods of cell disruption

B.1. Physical methods:

1. Cell disruption by osmotic shock

Osmotic pressure results from a difference in solute concentration across a semi permeable membrane.

—  Hypertonic – Shrinkage

—  Isotonic – Equilibrium

—  Hypotonic – turbid,  burst

Cell membranes are semi permeable and suddenly transferring a cell from an isotonic medium to distilled water (which is hypotonic) would result is a rapid influx of water into the cell. This would then result in the rapid expansion in cell volume followed by its rupture, e.g. if red blood cells are suddenly introduced into water, these hemolyse, i.e. disrupt thereby releasing hemoglobin. Osmotic shock is mainly used to lyse mammalian cells. With bacterial and fungal cells, the cell walls need to be weakened before the application of an osmotic shock.

2. Freeze-Thaw method

The freeze-thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient release recombinant proteins located in the cytoplasm of bacteria and are recommended for the lysis of mammalian cells in some protocols.

B.2. Chemical methods:

1. Cell disruption using detergents

Detergent-based cell lysis is an alternative to physical disruption of cell membranes. Detergents disrupt the structure of cell membranes by solubilizing their phospholipids disrupting lipid:lipid, lipid:protein and protein:protein interactions. These chemicals are mainly used to rupture mammalian cells. For disrupting bacterial cells, detergents have to be used in conjunction with lysozyme. With fungal cells (i.e. yeast and mould) the cell walls have to be similarly weakened before detergents can act. Detergents are classified into three categories: cationic, anionic and non­ionic. Non­ionic detergents are preferred in bioprocessing since they cause the least amount of damage to sensitive biological molecules such as proteins and DNA. Commonly used non­ionic detergents include CHAPS, a zwitterionic detergent, the Triton­ X series and the Tween series. However, it must be noted that a large number of proteins denature or precipitate in presence of detergents. Also, the detergent needs to be subsequently removed from the product and this usually involves an additional purification/polishing step in the process. Hence the use of detergents is avoided where possible. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function. SDS, an ionic detergent that binds to and denatures proteins, is used extensively for studies assessing protein levels by gel electrophoresis and western blotting.

2. Cell disruption using organic solvents

Organic solvents like acetone mainly act on the cell membrane by solubilizing its phospholipids and by denaturing its proteins. Some solvents like toluene are known to disrupt fungal cell walls. Others are ether, phenylethly alcohol, DMSO, benzene, Methanol, chloroform etc. Chemical permeabilization can also be achieved with antibiotics, sufactants, chaotropic agents and chelates.

EDTA (Ethylenediaminetetraacetic acid): It is an organic solvent used as chelating agent. It binds divalent cations of Ca²⁺ and Mg²⁺ and stabilizes the structure of outer membrane. Once these cations are removed from the EDTA, the LPS are removed increasing permeability areas of the outer walls.

The limitations of using organic solvents are similar to those with detergents, i.e. the need to remove these from products and the denaturation of proteins. However, organic solvents on account of their volatility are easier to remove than detergents.

3. Acid/Alkali treatment:

It is the easiest and least expensive method available in general lab. The method is fast, reliable and relatively clean way to isolates DNA from cells. It can be used for both laboratory and industrial scale. It is most common for plasmid DNA isolation however prolonged exposure of disrupted cells at high pH may denature the proteins. NaOH is used in nucleic acid extraction which solubilizes the phospholipid and protein components of the cell membrane.

B.3. Biological methods:

1. Cell disruption using enzymes

The use of enzymatic methods to remove cell walls is well-established for preparing cells for disruption, or for preparation of protoplasts (cells without cell walls) for other uses such as introducing cloned DNA or subcellular organelle isolation. The enzymes are generally commercially available and, in most cases, were originally isolated from biological sources (e.g. snail gut for yeast or lysozyme from hen egg white). The enzymes commonly used include lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase etc.

Lysozyme (an egg based enzyme) lyses bacterial cell walls, mainly those of the gram positive type. Lysozyme on its own cannot disrupt bacterial cells since it does not lyse the cell membrane. The combination of lysozyme and a detergent is frequently used since this takes care of both the barriers. Lysozyme is also used in combination with osmotic shock or mechanical cell disruption methods. The main limitation of using lysozyme is its high cost. Other problems include the need for removing lysozyme from the product and the presence of other enzymes such as proteases in lysozyme samples.

Disadvantages include:

• Not always reproducible.
• Not usually applicable to large scale as large scale applications of enzymatic methods tend to be costly and irreproducible.
• The enzyme must be removed (or inactivated) to allow cell growth or permit isolation of the desired material.

In addition to potential problems with the enzyme stability, the susceptibility of the cells to the enzyme can be dependent on the state of the cells. For example, yeast cells grown to maximum density (stationary phase) possess cell walls that are notoriously difficult to remove whereas midlog growth phase cells are much more susceptible to enzymatic removal of the cell wall.

2. Phage

Viruses that lyse bacterial cells-lytic phage are commonly used for cell disruption. Most bacteriophages accomplish lysis with a tandem, late transcriptional, two gene products: A holin: a small membrane protein that oligomerizes in the membrane to form non-specific lesions or ‘holes’ and a specific endolysin.

At a ‘programmed’ time, the holes cause a permeabilization of the membrane that facilitates the action of the active endolysins, murein hydrolases that degrade the bacterial cell wall. As endolysins coding genes do not harbor secretory signal sequence they accumulate in a fully folded and active state in the cytoplasm during the vegetative cycle until they reach the peptidoglycan, hydrolyse it and lyse the cells.

Limitations:

·         Phage is specific to its host so it is difficult to choose phage for different host cells. E.g. Coliphage in E. coli.

·         DNA isolation is not applicable