Technical

Cell fragmentation, cell fragmentation technology refers to the use of external force to destroy the cell membrane and cell wall, so that the cell contents including the target product components are released. When a certain protein is isolated and purified by cell fragmentation, the protein must first be released from the tissue or cell and maintained in its original natural state without losing its activity. Therefore, appropriate methods should be used to disrupt tissue and cells. Different organisms or tissues in different parts of the same organism have different difficulty in breaking cells and different methods used. For example, the cell membranes of animal organs are fragile and easy to break. Plants and microorganisms have stronger cell membranes. Cell wall composed of cellulose and hemicellulose.

1. Introduction

2. Principles and Techniques

3. Hole effect

4. Impact effect

5. Clipping effect

6.  Application introduction

 6.1.▪ Cell disruption (E. coli, yeast, bacteria, etc.)

 6.2.▪ Maintain Cell Viability

 6.3.▪ Bacteria

 6.4.▪ Yeast

 6.5.▪ Fungus

 6.6▪ Plant cells

7.  Cell disruption techniques and common methods

 7.1.  Mechanical

 7.2. Non-mechanical method

▪ Introduction

Cell disruption technology refers to the use of external force to destroy the cell membrane and cell wall, so that the cell contents including the target product components are released. It is the separation and purification of non-secreted biochemical substances synthesized in cells (product) basis. Combined with major advances in recombinant DNA technology and tissue culture technology, proteins can be produced on a large scale.

▪ Principles and Techniques

Sonication utilizes a 15-25kHz ultrasonic probe emitted by an ultrasonic oscillator to process cell suspensions. There are different types of ultrasonic oscillators, the most commonly used is the electroacoustic type. It is composed of a generator and a transducer. The generator can generate high-frequency current. The function of the transducer is to convert electromagnetic oscillations into mechanical vibrations. Ultrasonic oscillators can be divided into two types: slot type and probe directly inserted into the medium.

The high-pressure homogenizer is composed of a high-pressure plunger pump and a special-structured homogenizer valve. The material to be processed enters the adjustable pressure under the high-pressure condition caused by the plunger. In large and small valve groups, the depressurized material is ejected from the adjustable flow restriction slit at a very high flow rate (1000 m/s, up to 1500 m/s), and hits the impact ring of one of the valve assemblies .

hole effect

The material compressed by the plunger accumulates extremely high energy. When it passes through the adjustable current limiting gap, the pressure is instantly lost, resulting in the release of high energy and the explosion of the cavity, resulting in strong crushing of the material. refinement. (mainly used for homogenization)

Bump effect

The material passing through the adjustable flow restriction gap hits the impact ring made of special material at the above-mentioned extremely high speed, causing the material to be crushed. (mainly used for cell disruption)

Clip effect

Shearing effect occurs when high-speed material passes through the channel in the pump chamber and the slit in the valve port. The material treated by the three effects can be uniformly refined to a particle size of 0.03μm-2μm. (mainly used in emulsification)

▪ App introduction

Cell disruption (E. coli, yeast, bacteria, etc.)

Using Escherichia coli, yeast and other strains for fermentation, and then extracting intracellular enzymes, proteins and other active products after wall breaking is an important technology in bioengineering. An important step in these processes is cell disruption.

Maintain cell viability

The most important parameters for maintaining cell viability are temperature and time. When the material is inside the high-pressure homogenizing valve for about 0.01 seconds, the temperature will rise to about 10 under the action of high pressure. ~15°C.

After passing through the homogenizing valve and under the action of a high-efficiency heat exchanger, the temperature will drop rapidly in 2~3 seconds, so the material is only under the condition of relatively high temperature. Hold for up to 3 seconds.

Bacteria

The cell wall of almost all bacteria is composed of peptidoglycan, which is an insoluble glycan chain cross-linked by short peptides A mesh structure that surrounds the cells and gives them shape and strength. Short peptides generally consist of four or five amino acids, such as L-alanine-D-glutamine-L-lysine-D-alanine. Moreover, D-amino acid and diaminopimelic acid are often present in short peptides. The main resistance to breaking bacteria comes from the network structure of peptidoglycan. The density and strength of the network structure depend on the number of peptide bonds existing on the glycan chain and the degree of cross-linking, if the degree of cross-linking is large.

Yeast

The innermost layer of the yeast cell wall is composed of fine fibers of glucan, which constitute the rigid skeleton of the cell wall, giving the cell a certain shape, and covering the fine fibers It is a layer of glycoproteins, and the outermost layer is mannan, which is covalently linked by 1,6-phosphodiester bonds to form a network structure. Inside this layer, there are mannan-enzyme complexes, which may or may not be covalently attached to the network. As with bacterial cell walls, the resistance to disrupting yeast cell walls is primarily determined by how tightly the wall structure is cross-linked and how thick it is.

Fungus

There are mainly three kinds of polymers in the cell wall of mold, glucan (mainly connected by β-1,3 glycosidic bonds, some are connected by β-1,6 glycosidic bonds) , chitin (in the form of microfibrils), and glycoproteins. The outermost layer is a mixture of alpha- and beta-glucans, the second layer is a network of glycoproteins, glucans are combined with glycoproteins, the third layer is mainly protein, and the innermost layer is mainly chitin , the microfibrils of chitin are embedded in the protein structure. Like the cell walls of yeast and bacteria, the strength of the fungal cell wall is related to the network structure of the polymer, and not only that, it also contains the fibrous structure of chitin or cellulose, so the strength is increased.

Plant cells

For the plant cell wall that has grown, it can be divided into two parts: primary wall and secondary wall. The primary wall is formed during cell growth. The secondary wall is the structure that forms inside the primary wall after the cell stops growing. The more popular primary cell wall structure is the "latitude and longitude" model proposed by Lampert et al. According to this model, cellulose microfibrils are deposited layer by layer in a direction parallel to the plane of the cell wall. The filaments are arranged in parallel, but in different directions at different levels, forming a certain angle with each other, forming an independent network, which constitutes the "warp" of the cell wall. The "latitude" in the model is a structural protein (hydroxyproline-rich protein), which is secreted by the cytoplasm, arranged perpendicular to the plane of the cell wall, and is cross-linked by isodityrosine to form a structural protein network, and the radial microfibril network and the zonal structural protein network are cross-linked with each other to form more complex network systems. Colloids such as hemicellulose and pectin fill the network, making the entire cell wall both rigid and elastic. In the secondary wall, the content of cellulose and hemicellulose is much higher than that in the primary wall, and the microfibrils of cellulose are arranged more tightly and regularly. Therefore, the formation of the secondary wall increases the rigidity of the cell wall, making the plant cells have high mechanical strength.

▪ Cell disruption techniques

The crushing method can be classified into two categories: mechanical method and non-mechanical method.

Mechanical method

High pressure homogenization method (homogenization)

A high-pressure homogenizer is a commonly used equipment. It consists of a positive displacenemt pump that can generate high pressure and a discharge valve. The discharge valve has a A narrow orifice whose size can be adjusted. The cell slurry enters the pump body through the non-return valve, and is forced to rush out at high speed in the small hole of the discharge valve under high pressure, and shoots to the impact ring. Due to the sudden decompression and high-speed impact, the cells are subjected to high liquid phase shear. Broken by force. In terms of operation mode, single pass through the homogenizer or multiple circulation passes can be adopted, and continuous operation is also possible. In order to control the temperature increase, the temperature can be adjusted with dry ice at the inlet, so that the outlet temperature can be adjusted at about 20°C. In industrial-scale cell disruption, for cells that are difficult to disrupt, such as yeast, and in high concentrations or in the stationary phase of growth.

Skaking Bead

Place equal volumes of small tissue samples and high-density ZircoBeads into a sealable 2ml screw-cap microtube, then add buffers and stabilizers to a volume of 1.5ml , use the 6500RPM soup shaker to vibrate up and down for 8 seconds at high speed, rest for 8 seconds, and then vibrate for 8 seconds. This method is currently the fastest and can process the most samples at a time. One machine can process up to 2400 samples a day. It is convenient for small and diverse people.

High-speed stirring bead grinding method (fine grinding)

Milling is a commonly used method in which the cell suspension is rapidly stirred with abrasives such as glass beads, quartz sand or alumina to break up the cells. In industrial-scale crushing, high-speed bead mills are often used.

Ultrasonication

Sonication utilizes a 15-25kHz ultrasonic probe emitted by an ultrasonic oscillator to process cell suspensions. There are different types of ultrasonic oscillators, the most commonly used is the electroacoustic type. It is composed of a generator and a transducer. The generator can generate high-frequency current. The function of the transducer is to convert electromagnetic oscillations into mechanical vibrations. Ultrasonic oscillators can be divided into two types: slot type and probe directly inserted into the medium.

non-mechanical

osmotic shock

Osmotic shock is a milder disruption method, placing cells in a solution of high osmotic pressure (such as a certain concentration of glycerol or sucrose solution), due to the osmotic pressure When the balance is reached, the medium is rapidly diluted, or the cells are transferred into water or buffer solution. Due to the sudden change of osmotic pressure, the extracellular water quickly penetrates into the cells. inside, causing the cells to expand rapidly and rupture.

Freezing and thawing

Freeze the cells at low temperature (about -15°C), then thaw them at room temperature, repeatedly to break the walls. Due to freezing, on the one hand, the hydrophobic bond structure of the cell membrane can be broken, thereby increasing the hydrophilic properties of the cell; on the other hand, the intracellular water crystallizes to form ice crystals, which causes the cell to expand and rupture. This method can be used for bacteria with fragile cell walls.

Enzyme lysis

Enzymolysis is to use enzymes that dissolve the cell wall to treat the bacterial cells, so that the cell wall is partially or completely destroyed, and then the cell membrane is destroyed by osmotic pressure shock and other methods to further increase the intracellular volume. permeability of the product. Lysozyme (lysozyme) is suitable for the decomposition of gram-positive bacteria cells, and when applied to gram-negative bacteria, it needs to be supplemented with EDTA to make it more effective on the cell wall. The cell wall of eukaryotic cells is different from that of prokaryotic cells and requires different enzymes.

chemical treatment

Chemical treatment can lyse cells or extract intracellular components. Commonly used chemical reagents such as acids, bases, surfactants and organic solvents.

Detergents

--------------------------------------------------------- -------------

If you want to know more, please call for details.

+86 574 8908 5816  +86 186 5743 5285

Or swipe the QR code below: