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micromachines
Review
A Review on Macroscale and Microscale Cell
Lysis Methods
**Mohammed Shehadul Islam, Aditya Aryasomayajula and Ponnambalam Ravi Selvaganapathy ***
Department of Mechanical Engineering, McMaster University, Hamilton, ON L8S 4L7, Canada; [email protected] (M.S.I.); [email protected] (A.A.) ***** Correspondence: [email protected]; Tel.: +1-905-525-9140 (ext. 27435) Academic Editors: Aaron T. Ohta and Wenqi Hu Received: 21 January 2017; Accepted: 3 March 2017; Published: 8 March 2017
Abstract: The lysis of cells in order to extract the nucleic acids or proteins inside it is a crucial unit operation in biomolecular analysis. This paper presents a critical evaluation of the various methods that are available both in the macro and micro scale for cell lysis. Various types of cells, the structure of their membranes are discussed initially. Then, various methods that are currently used to lyse cells in the macroscale are discussed and compared. Subsequently, popular methods for micro scale cell lysis and different microfluidic devices used are detailed with their advantages and disadvantages. Finally, a comparison of different techniques used in microfluidics platform has been presented which will be helpful to select method for a particular application.
Keywords: cell lysis; cell lysis methods; microfluidics; electrical lysis; mechanical lysis; thermal lysis
1. Introduction Cell lysis or cellular disruption is a method in which the outer boundary or cell membrane is broken down or destroyed in order to release inter-cellular materials such as DNA, RNA, protein or organelles from a cell. Cell lysis is an important unit operation for molecular diagnostics of pathogens, immunoassays for point of care diagnostics, down streaming processes such as protein purification for studying protein function and structure, cancer diagnostics, drug screening, mRNA transcriptome determination and analysis of the composition of specific proteins, lipids, and nucleic acids individually or as complexes. Based on the application, cell lysis can be classified as complete or partial. Partial cell lysis is performed in techniques such as patch clamping, which is used for drug testing and studying intracellular ionic currents [ 1 ]. In this technique, a glass micropipette is inserted into the cell, rupturing the cell membrane partially. Complete cell lysis is the full disintegration of cell membrane for analyzing DNA, RNA and subcellular components [ 2 ]. Different methods have been developed in order to lyse the cell. The nature of lysis method chosen is influenced by the ease of purification steps, the target molecules for analysis, and quality of final products [ 3 ]. Laboratory and industrial scale cell lysis methods have been developed and used for many years now. There are a few companies that have also developed equipment (e.g., sonicators and homogenizers) and chemicals (reagents, enzymes and detergents) to lyse cells, which are commercially available. The global market for cell lysis is estimated at 2.35 billion dollars in 2016 and is expected to reach 3.84 billion dollars by 2021 [ 4 ]. In past 25 years, conventional laboratory-based, manually-operated bioanalytical processes have been miniaturized and automated by exploiting the advances in microfabrication in the microelectronic industry [ 5 ] leading to emergence of a new field known as Microfluidics. Microfluidic technology involves the handling and manipulation of tiny volumes of fluids (nanoliter to picoliter) in the
micrometer scale and offers various advantages which include low reagent volume, high surface to volume ratio, low cost and easy handling of small volumes of fluids which are suited for cell analysis. Microfluidic devices have shown great promise in cell lysis and in general cell analysis due to the similar operating size scale [ 6 ]. Various researchers have developed microfluidic devices to lyse cells [ 7 ]. Researchers have also developed single cell lysis techniques for single cell analysis [ 8 ]. This paper reviews several methods of cell lysis techniques that have been used in both macro and micro scale. Finally, a competitive analysis has been performed, which might be helpful to select a process to lyse cell depending on the application and motivation of lysis.
2. Overview of Cell Lysis Cells are the fundamental unit of all living organisms. Similar to the human body, cells also have a set of organs known as organelles, which are responsible for the cell’s ability to perform various kinds of functions. Additionally, the genetic information for the development and functioning of any organism is encoded in DNA or RNA sequences that are located inside the cell. The cell has an outer boundary called cell membrane, which encloses all the contents. The cell membrane serves as a barrier and regulates the transport of material between the inside and outside of the cell. The cell membrane must be disrupted or destroyed in order to access the DNA from inside the cell for molecular diagnosis, such as to identify pathogens [ 9 ]. A schematic representation of the cell lysis procedure is shown in Figure 1 where a detergent is used to disrupt the membrane chemically. Detergents react with cell membrane forming pores on the surface of membrane resulting in release of intracellular components such as DNA, RNA, proteins, etc.
Figure 1. Cell lysis using detergent to open the cell membrane and release the intracellular components. Reproduced with permission from Genomics education program.
2.1. Classification of Cell Types Cells are of two types: eukaryotic (such as mammalian cells) and prokaryotic (such as bacteria). The main difference between these two types is in their structure and organization. Figure 2 illustrates the difference between mammalian cells and bacteria. Mammalian cells have a boundary called cytoplasmic membrane that encloses the contents of the cell. In the case of bacteria, there are multiple layers enclosing the cell content and the innermost and outermost of them are called the plasma membrane and cell wall, respectively. Depending on the type of bacteria, the number of these layers varies. In the case of gram-positive bacteria, the plasma membrane is surrounded by another membrane known as cell wall or the peptidoglycan layer, whereas gram-negative bacteria, such as E. coli, consist of a cytoplasmic membrane, cell wall and an outer membrane. The composition of these cell layers such as structure and properties, have been extensively reviewed [ 10 – 12 ].
2.1.2. Cell Wall
Osmotic pressure is developed inside the cell due to the concentration difference of solutes across the membrane. For E. coli, this pressure is estimated around 2 atm [ 15 ]. To withstand these pressures, bacteria contains a cell wall or peptidoglycan layer, which also contributes to the shape and rigidity of the cell. This layer consists of two sugar derivatives named N-acetylglucosamine and N-acetylmuramic acid as well as a small group of amino acids consisting of L-alanine, D-alanine and D-glutamic acid. The basic structure of this peptidoglycan layer is a thin sheet where the aforementioned sugar derivatives are connected to each other by glycosidic bond forming a glycan chain. These chains are cross-linked by amino acid and the whole structure gives the cell rigidity in all directions. The strength of this structure depends on the frequency of chains and their cross linking. In gram-positive bacteria, peptidoglycan layer makes up 50%–80% of the cell envelope and 10% of this layer is associated with teichoic acid which provides a greater structural resistance to breakage [ 16 ]. In contrast, 10%–20% of the cell envelope of gram-negative bacteria is composed of a 1.2 to 2.0 nm thick peptidoglycan layer [ 16 ].
2.1.3. Outer Membrane
In addition to the peptidoglycan layer, there is another layer in the gram-negative bacteria known as the outer membrane. This layer is made of lipopolysaccharide which contains polysaccharides, lipids and proteins. It isolates the peptidoglycan layer from the outer environment and increases the structural firmness of the bacteria. The outer membrane is not permeable to enzymes. While the focus of the paper is the disruption of the cell boundary, this brief discussion regarding types of cells and their bounding structures is critical in selecting the appropriate methods and materials for lysis. In the next section, the different cell lysis techniques are explained.
3. Classification of Cell Lysis Methods
A number of methods, as depicted in Figure 5 , have been established to lyse cells in the macro and micro scale and these methods can be categorized mainly as mechanical and non-mechanical techniques.
Figure 5. Classification of cell lysis methods.
3.1. Mechanical Lysis
In mechanical lysis, cell membrane is physically broken down by using shear force. This method is the most popular and is available commercially because of a combination of high throughput and higher lysing efficiency. Different types of mechanical lysis techniques are discussed below.
3.1.1. High Pressure Homogenizer High Pressure Homogenizer (HPH) is one of the most widely used equipment for large scale microbial disruption. In this method, cells in media are forced through an orifice valve using high pressure. Disruption of the membrane occurs due to high shear force at the orifice when the cell is subjected to compression while entering the orifice and expansion upon discharge. Figure 6 shows an example of a commercially available HPH system. In this system, two storage tanks are employed which alternate the feed and allow for multiple passes of the homogenate. A positive displacement pump is used to draw the cell suspension. Depending on the types of the cells, 15–150 MPa is required [ 3 , 17 ].
Figure 6. Example of a high pressure homogenizer system. Reproduced with permission from [ 18 ].
Sauer et al. [ 19 ] proposed a model to relate the amount of protein released by homogenizer to the applied pressure for E. coli. ln
Rm Rm − R
= KNPa^ (1)
where R is protein released, Rm is the maximum protein available for release, P is pressure in MPa, N is the number of passes, K is the rate constant and a is the pressure exponent. Since the release of protein is independent of biomass concentration, higher concentration of cell can be disrupted at the same time. However, generation of heat is a problem in this method. Cooling systems can be used to minimize the heat generated. Augenstein et al. [ 20 ] reported the degradation of some enzymes during homogenization due to the high pressure. A combination of lysis methods, for example chemical treatment along with homogenization, has shown better results [ 18 ].
3.1.2. Bead Mill Bead mill, also known as bead beating method, is a widely used laboratory scale mechanical cell lysis method. The cells are disrupted by agitating tiny beads made of glass, steel or ceramic which are mixed along with the cell suspension at high speeds. The beads collide with the cells breaking open the cell membrane and releasing the intracellular components by shear force. This process is influenced by many parameters such as bead diameter and density, cell concentration and speed of agitator. Smaller beads with a range of 0.25–0.5 mm are more effective and recommended for lysis [ 3 , 21 ]. Using this technique, several kinds of cells can be lysed for example yeast and bacteria [ 22 , 23 ]. Cell membrane can become totally disintegrated by this method confirming that the intracellular molecules are released. Thus, the efficiency of this method of lysing cells is very high. However, complete disintegration produces small cell debris and thereby separation and purification of sample becomes harder. In addition, heat generation occurs in this process due to the collision between beads and cells. This elevated heat may degrade proteins and RNA.
E. coli in large quantities (100 mg) in about 2 h. In their method, the E. coli are pretreated with lysozyme prior to passing through a heat exchange coil set at 70 ◦C to lyse the cells. They used peristaltic pump and two heating coils at constant temperature and avoided the use of centrifugation step which enabled them to develop a continuous and controllable flow through protocol for lysing the cells at high throughput and obtaining large quantities of plasmid DNA. Thermal lysis is an attractive method at the micro scale used in many microfluidic devices. The high surface to volume ratio in microfluidic devices helps in cell lysis by quickly dissipating the heat and rupturing the cell membranes effectively. These techniques are covered later in Section 5.
Cavitation Cavitation is a technique which is used for the formation and subsequent rupture of cavities or bubbles. These cavities can be formed by reducing the local pressure which can be done by increasing the velocity, ultrasonic vibration, etc. Subsequently, reduction of pressure causes the collapse of the cavity or bubble. This pressure fluctuation is of the order of 1000 MPa [ 3 ]. During the collapse of a bubble, a large amount of mechanical energy is released in the form of a shockwave that propagates through the media. Since this shock wave has high energy, it has been used to disintegrate the cell membrane. Ultrasonic and hydrodynamic methods have been used for generating cavitation used to disrupt cells. Ultrasonic Cavitation is a widely known laboratory based technique for disruption of the cells. Ultrasonic vibration (15–20 kHz) can be used to generate a sonic pressure wave [ 5 ]. It has been shown that disruption is independent of biomass concentration and proportional to power input. This technique also produces very small cell debris which might be a problem for subsequent processes. In addition, large amount of heat is generated which needs to be dissipated. Enzymes that come out from cell after Ultrasonic Cavitation have also been reported to be degraded [ 30 ]. To overcome the problems associated with ultrasonic cavitation, such as high power requirement and high energy to dissipate heat problem, hydrodynamic cavitation has been used to disrupt the cell membrane [ 31 ]. Hydrodynamic cavitation is produced by pumping the cell suspension through a constricted channel which results in an increase in velocity. Lee et al. [ 32 ] have demonstrated the use of hydrodynamic cavitation as an efficient method to disrupt the cell membrane of cells to extract the lipids. They report that the energy required for lipid extraction from cells using the hydrodynamic cavitation technique was 3 MJ/kg which is 10 times more efficient compared to sonication in terms of energy consumption. In another study by Capocellia et al. [ 33 ] the acoustic and hydrodynamic cavitation methods were compared for microbial cell disruption. Their simulation results show that the hydrodynamic cavitation is an order of magnitude more efficient than acoustic cavitation method for cell disruption.
Osmotic Shock When the concentration of salt surrounding a cell is suddenly changed such that there is a concentration difference between the inside and outside of the cell, the cell membrane becomes permeable to water due to osmosis. If the concentration of salt is lower in the surrounding solution, water enters the cell and the cell swells up and subsequently bursts. This technique is suitable for mammalian cell due to the fragile structure of membrane; however, periplasmic proteins may be released in the case of gram-negative bacteria [ 34 ]. Chen et al. [ 35 ] compared osmotic shock method and sonication for recovery of recombinant creatinase from E. coli. They found osmotic shock method resulted in a 60% creatinase recovery and 3.9 fold purification compared to sonication. They also observed that when the cells were pretreated with divalent cation (Ca2+^ or Mg2+) the efficiency of osmotic shock method could be improved to 75% and 4.5 fold purification. Another study by Byreddy et al. [ 36 ] showed that osmotic shock method resulted in the highest yield of lipids from Thraustochytrid strains when compared to grinding with liquid nitrogen, bead vortexing and sonication methods.
3.2.2. Chemical Cell Disruption Chemical lysis methods use lysis buffers to disrupt the cell membrane. Lysis buffers break the cell membrane by changing the pH. Detergents can also be added to cell lysis buffers to solubilize the membrane proteins and to rupture the cell membrane to release its contents. Chemical lysis can be classified as alkaline lysis and detergent lysis.
Alkaline Lysis In alkaline lysis, OH−^ ions are the main component used for lysing cell membrane [ 37 ]. The lysis buffer consists of sodium hydroxide and sodium dodecyl sulphate (SDS). The OH−^ ion reacts with the cell membrane and breaks the fatty acid-glycerol ester bonds and subsequently makes the cell membrane permeable and the SDS solubilizes the proteins and the membrane. The pH range of 11.5–12.5 is preferable for cell lysis [ 3 , 38 ]. Although this method is suitable for all kinds of cells, this process is very slow and takes about 6 to 12 h. This method is mostly used for isolating plasmid DNA from bacteria [ 39 , 40 ].
Detergent Lysis Detergents also called surfactants have an ability to disrupt the hydrophobic-hydrophilic interactions. Since the cell membrane is a bi-lipid layer made of both hydrophobic and hydrophilic molecules, detergents can be used to disintegrate them. Detergents are capable of disrupting the lipid–lipid, lipid–protein and protein-protein interactions. Based on their charge carrying capacity, they can be divided into cationic, anionic and non-ionic detergents. Detergents are most widely used for lysing mammalian cells. For lysing bacterial cells, first the cell wall has to be broken down in order to access the cell membrane. Detergents are often used along with lysozymes for lysing bacteria (e.g., yeast). Table 2 lists all the detergents according to their charge and properties. Out of the three types of detergents, non-ionic detergents are mostly preferred as they cause the least amount of damage to proteins and enzymes. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), a zwitterionic detergent, is one of the most popular non-ionic detergents. Other non-ionic detergents include Triton-X and Tween series. Ionic detergent such as SDS is widely used for lysing cells because of its high affinity to bind to proteins and denature them quickly. It is used in gel electrophoresis and western blotting techniques. The hydrophilic part of an anionic detergent is mostly a sulphate or carboxylic group whereas for cationic detergent it is ammonium group. Apart from ionic and non-ionic detergents, chaotropic agents can also be used for cell lysis. These include urea, guanidine and Ethylenediaminetetraacetic acid (EDTA) which can break the structure of water and make it less hydrophilic and there by weakening the hydrophobic interactions. An additional purification step has to be in cooperated into the cell lysis protocol when using detergents [ 41 ].
Table 2. List of some detergents and their properties. A comprehensive list of detergents can be found here [ 42 ].
Detergent Charge Properties Sodium dodecyl sulphate (SDS) Anionic Strong lysis agent. Good for most cells. Not suitable forsensitive protein extraction. Triton X (100, 114) Non-ionic Mild lysis agent. Good for protein analysis. NP-40 Non-ionic Mild lysis agent. Good for isolating cytoplasmic proteinsbut not nuclear proteins. Tween (20, 80) Non-ionic Mild lysis agent. Good for cell lysis and protein isolation. Cetyltrimethylammonium bromide (CTAB) Cationic Generally used for isolating plant DNA. CHAPS, CHAPSO Zwitterionic Mild lysis agent. Good for protein isolation.
Table 3. Cont.
Methods (^) Technique UsedEquipment and Advantages Disadvantages
Non-mechanical
Physical
Thermal lysis
- Independent of the cell type
- Easy to implement
- Expensive
- Damage to proteins and intracellular components
Cavitation
- Independent of cell type
- Large scale integration possible
- Operates at a lower temperature and energy level - Expensive technology - Can cause damage to sensitive proteins - Difficult to purify sample from debris
Osmotic shock -^ Can be used for extractingsensitive intracellular products -^ Not suitable for all cell types
Chemical
Alkaline lysis
- Suitable for extraction of sensitive intracellular components (proteins, enzymes, DNA)
- Suitable for all kinds of cells
Detergent lysis -^ Suitable for protein release
- Not suitable for isolating sensitive enzyme and proteins-Expensive reagents
- Removal of chemical reagent from sample after lysis is difficult
- Lower efficiency as complete lysis is not possible Biological
Enzymatic lysis
- Can be very specific for cell types
- Suitable for extracting proteins
- Complete lysis not possible
- Expensive reagents
- Has to be used in combination of detergents for bacteria. 4. Microfabricated Platforms for Cell Lysis Microfluidics is one of the emerging platforms for cell lysis on a micro scale. Microfluidics is the manipulation and handling of small volumes (nano- to picoliters) of liquid in microchannels. Due to the micro scale operation regime, microfluidics is well suited for application where the sample or sample volume is small. This lowers the cost of the analysis due to low consumption of reagents [ 46 ]. Microfluidics also enables integration of different modules (or operations) into one device. For example, cells can be lysed and the intracellular products can directly be post processed (PCR or DNA isolation for diagnostics) inside the same device [ 47 , 48 ]. Although there have been a number of reviews on cell lysis in the past 10 years [ 7 , 8 , 49 ], some of the recent developments in the field have not been reviewed. This review will focus on the recent developments from 2014 onwards and will briefly cover the developments from before, which have been extensively surveyed. Some of the macro scale techniques have been implemented in microfabricated devices for cell lysis. Techniques such as electrical lysis methods are applicable only in the micro scale. Microfluidic lysis technology can be broadly classified into six types. They include mechanical lysis, thermal lysis, chemical lysis, optical lysis, acoustic lysis and electrical lysis.
4.1. Mechanical Lysis Mechanical lysis in microfluidics involves physically disrupting the cell membrane using shear or frictional forces and compressive stresses. Berasaluce et al. [ 50 ] developed a miniaturized bead beating based method to lyse large cell volumes. Zirconium/silica beads were placed inside a cell lysis chamber along with a permanent magnet and actuation of an external magnetic field caused the motion of the beads inside the chamber. Figure 7 shows the various components and device assembled for cell lysis. Staphylococcus epidermidis cells were used in this study and they studied the effect of bead size, volume, flow rate and surfactant (Tween-20) on lysing efficiency. They found the optimum parameters achieved a 43% higher yield efficiency at a flow rate of 60 μL/min compared to off chip bead beating system.
ΐ
( a ) ( b )
Figure 7. Miniaturized bead beading cell lysis system: ( a ) various components: (1) inlet; (2) outlet; (3) stirring magnet; (4) zirconia/silica beads; (5) bead weir; (6) rotating magnet; and (7) electric motor coupling; and ( b ) image of the device for lysis. Reproduced with permission from [ 50 ].
Pham et al. [ 51 ] have recently used nanotechnology to fabricate black silicon nano pillars to lyse erythrocytes in about 3 min. They fabricated these nanopillar with ~12 nm tip diameter and 600 nm tall on silicon substrate using reactive ion etching technology. The authors showed that the interaction of erythrocytes cultured on nanopillar arrays causes stress induced cell deformation, rupture and lysis in about 3 min. Figure 8 shows the interaction of erythrocytes with the nanostructures. Mechanical lysis has been demonstrated by using nano-scale barb [ 52 ]. When cells are forced through small opening, high shear forces cause rupture of the cell membrane. Similar principle has been used here where “nanoknives” were fabricated in the wall of microchannels by using modified deep reactive ion etching (DRIE). Distance between these sharp edges was 0.35 μm and width of the channel was 3 μm. The lysis section of this device consisted of an array of these “nanoknives” patterned on a microchannel as shown in Figure 9 b. Human promyelocytic leukemia cells (HL-60) were used to pass through this section at sufficient velocity. The addition of this “nanoknives” pattern increased the amount of lysis. This device was used to extract protein from inside the cell. It has been estimated that as much as 99% of the cell was lysed but, only 6% protein was released. Alternatively, mechanical impingement through collision has also been used to lyse in the microscale [ 53 – 55 ]. Cells were suspended in solution with glass beads and placed on the microfluidic compact disc (CD) device, which was then set to rotate at a very high velocity. The centrifugal force generated by the rotation, causes collision and friction between cells and beads, which results in cell lysis. Various kinds of cells including mammalian, bacteria and yeast have been lysed using this technique. Though the efficiency of the mechanical lysis is very high, these disruption methods have some drawbacks in microscale application. Fabrication of these devices is complex as well as expensive and collecting the target materials from a complex mixture is very difficult.
