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Cell disruption

Cell disruption, also known as cell lysis, is the process of breaking down the outer cell membrane and wall to release intracellular components such as proteins, nucleic acids, organelles, and metabolites for subsequent analysis, purification, or utilization in biotechnological processes.^[1] This technique is essential in fields like molecular biology, diagnostics, and industrial biotechnology, where it facilitates the extraction of valuable biomolecules from microorganisms, plant cells, or animal tissues.^[1] The importance of cell disruption lies in its role as a critical step in downstream processing, enabling efficient recovery of intracellular products while minimizing damage to sensitive molecules.^[2] Methods are categorized into mechanical/physical approaches, which apply shear forces or pressure—such as high-pressure homogenization (operating at 15–150 MPa) and bead milling (using 0.25–0.5 mm beads)—and non-mechanical methods, including thermal treatments (e.g., freezing-thawing cycles or heating above 90°C), chemical lysis (e.g., detergents or alkaline solutions at pH 11.5–12.5), and enzymatic digestion (e.g., lysozyme for bacterial peptidoglycan).^[1] Emerging microscale techniques, developed over the past 25 years in microfluidics, incorporate electrical fields (2–300 V DC/AC), acoustic waves, optical lasers, or nanoscale mechanical structures to achieve high-throughput lysis with reduced sample volumes.^[1] Applications span protein purification, DNA/RNA extraction for genomics, pathogen detection in diagnostics, and biofuel production from microalgae, with the global cell lysis market valued at USD 4.02 billion in 2025 and projected to reach USD 6.05 billion by 2030.^[3] Selection of disruption method depends on cell type—rigid walls in yeast or bacteria often require intensive mechanical or chemical approaches—while gentle methods like enzymatic lysis preserve bioactivity for therapeutic proteins.^[2] Historical advancements include partial lysis via patch clamping in 1984 and microfluidic innovations in the late 1990s, enhancing precision and scalability.^[1]

Overview

Definition and Purpose

Cell disruption, also referred to as cell lysis, is the controlled rupture of cellular boundaries—such as the plasma membrane in animal cells or the rigid cell wall and membrane in microorganisms and plant cells—to liberate intracellular contents including proteins, enzymes, nucleic acids (DNA and RNA), and metabolites, while avoiding excessive degradation or denaturation of these biomolecules.^[1] This process ensures that the released components remain viable for subsequent analysis or utilization, distinguishing it from uncontrolled cell death mechanisms.^[4] The primary purpose of cell disruption lies in its role as a critical step in biotechnological downstream processing, where it facilitates the isolation and recovery of high-value biomolecules for applications in research, pharmaceutical production, diagnostics, and food industries.^[5] By enabling access to intracellular products, it supports goals such as maximizing extraction yields—often aiming for near-complete release of target molecules—and minimizing issues like protein denaturation, contamination from host cell debris, or loss of bioactivity, which are essential for maintaining product quality and efficiency in large-scale operations.^[6] Cell disruption primarily targets microorganisms such as bacteria (prokaryotes) and yeast (fungi), as well as plant and animal cells, each presenting unique challenges due to structural differences.^[7] Prokaryotic cells like bacteria and fungal cells like yeast feature rigid peptidoglycan or β-glucan-based cell walls that resist lysis, necessitating robust methods to achieve effective release without compromising biomolecule integrity, whereas animal cells, lacking cell walls, are generally easier to disrupt but require care to prevent over-lysis.^[4] Plant cells, with their cellulose walls, similarly demand targeted approaches to overcome mechanical barriers.^[4] The basic workflow of cell disruption begins with the preparation of a cell suspension in an appropriate buffer to maintain physiological conditions and protect sensitive components, followed by the application of a disruption technique to break open the cells, and concludes with separation of the resulting lysate (containing released biomolecules) from insoluble debris via centrifugation or filtration.^[8] This sequence ensures efficient recovery while allowing for downstream purification steps.

Historical Background

Cell disruption techniques originated with rudimentary manual methods, such as grinding tissues using a mortar and pestle, which date back to ancient practices and were adapted for biochemical tissue homogenization in the early 20th century.^[9] These tools applied shear forces through mechanical pressure to break down cellular structures, enabling the extraction of intracellular components from small sample volumes, though labor-intensive and limited to batch processing of 0.1–500 mL.^[10] By the mid-20th century, advancements addressed the need for more efficient lysis, particularly for microorganisms. In the 1950s, significant progress came with the invention of the French press by Charles Stacy French, a biophysicist at the Carnegie Institution, who developed the device to disrupt plant and bacterial cells under high pressure (up to 40,000 psi) by forcing suspensions through a narrow orifice, causing shear-induced lysis while preserving organelles.^[11] French's innovation, detailed in his 1955 collaboration with H.W. Milner, facilitated studies on photosynthetic bacteria and algae, marking a shift toward controlled, high-yield disruption for cytological research.^[12] This batch method influenced subsequent pressure-based techniques, emphasizing minimal heat generation to protect sensitive biomolecules. The 1960s and 1970s saw the introduction of ultrasonication, leveraging cavitation from high-frequency sound waves (around 20 kHz) to fragment cells, as demonstrated in early studies on yeast and bacterial lysis.^[13] Concurrently, bead beating emerged in the late 1970s, pioneered by BioSpec Products, which commercialized shaking systems with glass or ceramic beads to generate impact and shear forces, effectively lysing tough microbial walls in small volumes (0.2–50 mL).^[14] Cryogenic methods also advanced during this period, with liquid nitrogen cryo-impacting described by Smucker and Pfister in 1975, involving freezing cells in liquid nitrogen followed by mechanical impacting in a ball mill, achieving over 90% disruption in vegetative bacteria and spores with reduced enzyme denaturation compared to some mechanical alternatives.^[15] Nitrogen decompression, an earlier cryogenic approach pioneered by Fraser in 1951 using pressures around 1,000 psi to dissolve gas in cells followed by sudden release causing cavitation and bursting, achieved up to 75% disruption in bacteria such as E. coli.^[16] From the 1980s, high-pressure homogenization gained commercial traction for industrial-scale applications, building on earlier pressure concepts like the 1951 nitrogen decompression method by Fraser but evolving into continuous-flow systems (e.g., Gaulin models at 10,000–20,000 psi) that processed larger volumes for microbial lysis.^[16] Bead mills further developed for scale-up, enhancing throughput in bioprocessing. The 1990s biotechnology boom, driven by recombinant protein production, spurred refinements in these methods, including optimized pressure profiles and hybrid approaches to improve yields from inclusion bodies in engineered bacteria, as explored in key studies on E. coli disruption.^[17] French's foundational work on plant cells continued to inform adaptations for eukaryotic systems amid this expansion.^[18] In the late 1990s, microfluidic innovations emerged, enabling precise, high-throughput cell lysis using electrical, acoustic, or mechanical forces in microscale devices, paving the way for miniaturized and automated biotechnological processes as of the early 21st century.^[1]

Fundamentals of Cell Structure

Prokaryotic Cells

Prokaryotic cells, encompassing bacteria and archaea, are characterized by a distinct cell envelope that imparts mechanical strength and influences the efficacy of disruption processes. In bacteria, the envelope composition differs markedly between Gram-positive and Gram-negative species. Gram-positive bacteria possess a thick peptidoglycan layer that constitutes 60-90% of the cell wall dry weight, forming a robust network through extensive cross-linking of glycan strands and peptide bridges. Gram-negative bacteria, in contrast, feature a thinner peptidoglycan layer (approximately 2-7 nm thick) sandwiched between the inner cytoplasmic membrane and an outer membrane rich in lipopolysaccharides, which contribute to permeability barriers and structural integrity. Archaea often exhibit a proteinaceous surface layer, known as the S-layer, which assembles into a crystalline lattice and frequently serves as the primary or sole non-membrane component of the cell envelope, enhancing resistance to environmental stresses. This S-layer provides additional mechanical protection, particularly in extremophilic species. The rigidity of prokaryotic envelopes presents significant challenges for cell disruption, primarily due to the cross-linked peptidoglycan in bacteria, which resists deformation and requires substantial shear or hydrolytic forces to breach. In archaea, the S-layer further complicates access to intracellular contents by forming a tightly packed, porous yet protective barrier that maintains cell shape and integrity. Disruption of prokaryotic cells targets the release of key intracellular components, including plasmids for genetic engineering, ribosomes for translational studies, and metabolic enzymes for biochemical assays. A prominent example is Escherichia coli, widely used as a host for recombinant protein production, where lysis enables extraction of these over-expressed proteins alongside native cellular machinery. Prokaryotes' small dimensions, typically ranging from 0.5 to 5 μm in length for most bacteria (though some reach 10 μm), combined with their tough envelopes, render them less susceptible to disruption than animal cells, which lack a cell wall and can often be lysed under milder osmotic or chemical conditions.

Eukaryotic Cells

Eukaryotic cells exhibit greater structural diversity than bacterial cells, which typically feature a uniform peptidoglycan-based cell wall, necessitating tailored disruption strategies that account for varied extracellular barriers and intracellular compartments.^[19] This diversity spans fungi, plants, and animals, influencing the mechanical and biochemical challenges in cell lysis while emphasizing the need to minimize damage to delicate internal structures. Fungal cell walls, such as those in yeast like Saccharomyces cerevisiae, are primarily composed of chitin and β-glucans, including branched β-(1,3)-glucans that form a rigid scaffold linked to chitin via β-(1,4) bonds, providing structural integrity and resistance to enzymatic or mechanical breakdown.^[20]^[21] In contrast, plant cell walls consist mainly of cellulose microfibrils embedded in a matrix of hemicelluloses, such as xyloglucans and arabinoxylans, which contribute to tensile strength and flexibility but complicate disruption due to their multilayered architecture.^[22]^[23] Animal cells, including mammalian ones, lack a cell wall entirely, relying solely on a phospholipid-based plasma membrane for protection, which renders them more susceptible to lysis but requires careful handling to prevent unintended membrane fragmentation.^[24]^[25] A key consideration in eukaryotic cell disruption is the preservation of organelles, such as mitochondria, nuclei, and chloroplasts, to prevent secondary damage like membrane rupture or loss of enzymatic activity.^[26] These membrane-bound structures, which house critical metabolic pathways, demand isotonic conditions during processing to maintain integrity, as demonstrated in cell fractionation techniques where sucrose buffers protect mitochondrial and chloroplast function.^[27] Eukaryotic cells are generally larger, ranging from 10 to 100 μm in diameter, with plant cells often containing prominent central vacuoles that store osmotic regulators, enabling hypotonic swelling as a viable preliminary step but heightening vulnerability to mechanical shear that could shear-sensitive organelles like nuclei.^[19]^[28] This size and compartmentalization contrast with the smaller, simpler prokaryotic architecture, underscoring the need for balanced forces in eukaryotic lysis. In practical contexts, such as enzyme production from yeast like S. cerevisiae, the chitin-β-glucan wall necessitates strategies that target these components without excessive fragmentation, while mammalian cells used for antibody extraction benefit from gentler approaches to safeguard fragile plasma membranes and intracellular proteins.^[29]^[30] These examples highlight how eukaryotic structural variations dictate disruption parameters to optimize intracellular release while preserving functional components.

Mechanical Disruption Methods

Bead Beating

Bead beating is a mechanical cell disruption technique that relies on the high-speed agitation of a cell suspension intermixed with small abrasive beads, which collide with cells to generate shearing and impact forces that rupture cell walls and membranes. The beads, typically composed of glass, ceramic, or zirconia and ranging in diameter from 0.1 to 2 mm, are selected based on cell type, with smaller beads (0.1-0.5 mm) suited for bacteria and larger ones (0.5-2 mm) for yeast or tougher structures. This abrasive grinding action ensures efficient lysis without relying on chemical or enzymatic agents, making it particularly effective for releasing intracellular contents like proteins and nucleic acids.^[1]^[31] Equipment for bead beating includes specialized bead mills and bead beaters, such as the BeadBeater or Mini-BeadBeater devices, which accommodate sample volumes from microliters to larger batches and operate via vigorous shaking or vortexing. Key operational parameters encompass a bead load filling 20-50% of the vial volume (often around 50% for optimal contact), agitation speeds of at least 2000 rpm (up to 4800 rpm in some models), and disruption cycles lasting 1-3 minutes, sometimes repeated with cooling intervals to manage heat buildup. These settings can be adjusted for sample type, with heavier zirconia beads enhancing disruption efficiency by approximately 50% compared to glass due to increased impact force.^[31]^[32] The method is especially suitable for lysis-resistant cells, including Gram-positive bacteria (e.g., Bacillus subtilis), yeast, fungi, and spores, where it achieves high disruption yields of 80-95% after multiple passes or optimized cycles. For instance, in purple non-sulfur bacteria, bead beating at 2000 rpm for 30 seconds in three cycles yielded 92.1% protein extraction efficiency, demonstrating its reliability for biochemical recovery from robust microbial sources. Unlike continuous-flow methods like high-pressure homogenization, bead beating operates in batch mode, allowing precise control for small-scale laboratory applications while remaining adaptable to industrial milling setups.^[33]^[31] Bead beating offers advantages in scalability, from high-throughput 96-well formats to larger production volumes, and its mechanical nature preserves sample integrity without reagent contamination when using disposable components. However, the process generates significant frictional heat during agitation, necessitating cooling systems or ice baths to prevent thermal degradation of sensitive biomolecules like proteins or RNA. Prolonged exposure beyond 3-5 minutes can lead to protein denaturation or incomplete recovery due to over-shearing, particularly in heat-sensitive samples, though these limitations are mitigated by short cycles and proper parameter tuning.^[1]^[31]

Ultrasonication

Ultrasonication is a mechanical cell disruption technique that utilizes high-frequency sound waves, typically ranging from 20 to 40 kHz, to induce acoustic cavitation within a liquid suspension of cells. These sound waves, generated by an ultrasonic transducer, propagate through the medium and create alternating high- and low-pressure cycles, leading to the formation, growth, and violent implosion of microscopic gas bubbles. The collapse of these cavitation bubbles generates intense localized shear forces, shock waves, and microjets that exert mechanical stress on cell walls and membranes, effectively lysing the cells and releasing intracellular contents.^[34]^[35] Additionally, the extreme conditions during bubble collapse—reaching temperatures up to 5000 K and pressures exceeding 1000 atm—can produce free radicals, such as hydroxyl radicals, which contribute to oxidative damage and further aid in cell rupture, though this chemical effect is secondary to the physical forces.^[34] The primary equipment for ultrasonication includes laboratory-scale ultrasonic processors equipped with titanium probes (horn-type sonicators) that directly immerse into the sample for efficient energy transfer, or indirect bath sonicators where samples are placed in sealed containers within an ultrasonic bath. Operational parameters are critical for optimizing disruption while minimizing damage: probe amplitude is adjustable from 20% to 100% of maximum output, treatment times vary from 30 seconds to 5 minutes depending on sample volume and cell type, and pulsed modes (e.g., 10-30 seconds on/off cycles) are employed to dissipate heat and prevent thermal degradation. Cooling strategies, such as immersion in an ice bath, are routinely integrated to maintain sample temperatures below 10°C during processing.^[36]^[35] This method proves particularly suitable for small-volume samples (up to a few milliliters) and softer cell types, including bacteria like Escherichia coli, yeast such as Saccharomyces cerevisiae, and microalgal or animal tissue suspensions, where disruption efficiencies of 70-90% can be achieved with proper parameter tuning. It performs well across a range of biomass concentrations due to its independence from cell density, making it versatile for laboratory applications in biotechnology and biochemistry. However, scalability to industrial levels remains challenging, as energy distribution becomes uneven in larger volumes, often resulting in lower and more inconsistent lysis rates.^[34]^[36] Ultrasonication offers distinct advantages, including rapid processing times and a non-contact approach that avoids contamination from grinding media, enabling efficient extraction of proteins, lipids, and other biomolecules without chemical additives. Despite these benefits, notable limitations include the generation of excessive heat, which can denature heat-sensitive enzymes or proteins if not controlled, and the formation of free radicals that may oxidize and degrade nucleic acids or delicate metabolites, often requiring the inclusion of antioxidants like dithiothreitol in the lysis buffer. Heat management in ultrasonication aligns with strategies used in bead beating, such as pulsed operation and external cooling, to preserve biomolecular integrity.^[35]^[36]

High-Pressure Homogenization

High-pressure homogenization is a mechanical cell disruption technique that involves forcing a cell suspension through a narrow orifice or valve under extreme pressure, generating intense shear forces, turbulence, and cavitation that rupture cell walls and membranes. In this process, cells suspended in a liquid medium are pumped at pressures typically ranging from 500 to 2000 bar, leading to explosive decompression upon release from the restricted space, which induces mechanical stress and causes intracellular contents to be liberated. The primary mechanisms include hydraulic shear from the high-velocity flow and inertial forces arising from rapid acceleration and deceleration of the cell contents, with studies showing that at around 560 bar, yeast cells experience wall tensions up to 8 N/m, sufficient for breakage.^[37]^[38]^[37] Common equipment for high-pressure homogenization includes the French press, a batch device invented by Charles Stacey French in the 1940s that manually builds pressure in a cell before releasing it through a valve, and continuous-flow systems like the Microfluidizer, which uses fixed-geometry interaction chambers to achieve consistent shear rates. Operational parameters such as pressure levels (often 1000-1500 bar for microbial cells), number of passes (typically 1-5 to optimize disruption without excessive heating), and flow rates (up to 10 L/min in industrial-scale units) are adjusted based on cell type and desired yield. For instance, Microfluidizer processors can achieve over 99% rupture of Escherichia coli in a single pass at suitable pressures, highlighting their efficiency for uniform processing.^[39]^[40]^[41] This method is versatile and particularly effective for disrupting prokaryotic cells like bacteria (especially Gram-negative strains, achieving 90-99% efficiency), as well as eukaryotic cells such as yeast and algae, where multiple passes at higher pressures (up to 2000 bar) can yield 95% lysis for resilient yeast strains. It excels in large-scale bioprocessing due to its ability to handle viscous or concentrated suspensions without additives, making it suitable for extracting proteins, lipids, or pigments from microbial biomass.^[42]^[43]^[44] Key advantages include scalability for industrial applications through continuous operation, which supports high throughput and reproducibility, and minimal thermal damage when controlled properly, preserving sensitive biomolecules better than some alternative mechanical methods. However, limitations encompass high energy consumption due to the powerful pumps required, potential for equipment clogging with highly viscous or fibrous samples, and the need for pre-treatment to avoid blockages in certain biomass types. Maintenance costs can also be elevated owing to wear on valves and orifices from abrasive particles.^[38]^[45]^[1]

Cryogenic Methods

Cryogenic methods involve freezing cells at extremely low temperatures, typically using liquid nitrogen at -196°C, to embrittle cellular structures and facilitate mechanical disruption. This approach leverages the formation of ice crystals within cells, which expand and rupture membranes upon freezing, making subsequent fracturing more efficient. These techniques are particularly valuable in biotechnology for extracting intracellular components from samples sensitive to heat or shear forces.^[46] The primary mechanism begins with rapid freezing of cell suspensions or tissues in liquid nitrogen, where water inside the cells forms sharp ice crystals that pierce and disrupt lipid bilayers. This is followed by mechanical pulverization or decompression to complete the lysis. For instance, in cryopulverization, the frozen material is ground while maintaining cryogenic conditions to prevent thawing and enzyme degradation. In nitrogen decompression, cells are first equilibrated under high-pressure nitrogen gas (often chilled), allowing gas dissolution into the cells; rapid release then causes explosive bubble formation that shears membranes, akin to cavitation but gentler than ambient-pressure methods.^[31]^[47]^[48] Specific techniques include cryopulverization via mortar and pestle grinding of frozen pellets or automated cryogenic mills, where samples are frozen for 5-10 minutes before processing at controlled speeds to achieve uniform particle sizes. Nitrogen decompression uses specialized chambers where samples are pressurized to 600-2200 psig for 5-30 minutes, followed by instantaneous release. Equipment for these includes cryogenic mills like the CryoGrinder™ or BioPulverizer for grinding, and stainless-steel vessels such as the Parr Model 4635 for decompression, handling sample volumes from 0.5 mL to 5 L.^[46]^[31]^[47] These methods are best suited for plant tissues, fungi, and heat-sensitive samples like mammalian cells or those requiring intact organelles, achieving disruption yields of 85-95% while preserving thermolabile compounds such as enzymes and RNA. For example, cryogenic grinding effectively lyses tough plant cell walls composed of cellulose, outperforming room-temperature methods in protein release.^[30]^[46]^[48] Advantages include minimal heat generation, which protects labile biomolecules, and the ability to process heterogeneous tissues without chemical additives. Limitations encompass labor-intensive manual grinding, challenges in scaling for industrial use, and the need for safe handling of liquid nitrogen to avoid hazards like asphyxiation. Additionally, nitrogen decompression requires pretreatment for cell walls tougher than those in mammalian cells.^[31]^[47]^[46]

Non-Mechanical Disruption Methods

Enzymatic Lysis

Enzymatic lysis involves the use of specific hydrolase enzymes to selectively degrade components of the cell wall, facilitating osmotic rupture and release of intracellular contents without mechanical force.^[49] This method targets the structural polysaccharides and proteins that provide rigidity to microbial and plant cell walls, making it particularly suitable for organisms with well-defined extracellular matrices.^[1] The primary mechanism relies on enzymes that hydrolyze key bonds in cell wall polymers. For prokaryotic cells, lysozyme (EC 3.2.1.17) cleaves the β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major structural component of bacterial walls; this is highly effective against Gram-positive bacteria but requires pretreatment for Gram-negative species.^[50] In Gram-negative bacteria, ethylenediaminetetraacetic acid (EDTA) is often added to chelate divalent cations, destabilizing the outer lipopolysaccharide layer and enhancing lysozyme access.^[51] For eukaryotic fungi like yeast, zymolyase (a mixture including β-1,3-glucanase) degrades the β-glucan layer, weakening the rigid cell wall and promoting spheroplast formation followed by lysis in hypotonic conditions.^[49] In plant cells, cellulase (EC 3.2.1.4) hydrolyzes β-1,4-glucan chains in cellulose microfibrils, often combined with pectinases to disrupt the middle lamella, enabling protoplast isolation.^[52] Typical protocols involve resuspending cells in an isotonic or hypotonic buffer (e.g., phosphate-buffered saline at pH 7.0-8.0) with enzyme concentrations of 0.1-1 mg/mL for zymolyase or cellulase, and 1-10 mg/mL for lysozyme, followed by incubation at 25-37°C for 30-60 minutes with gentle agitation.^[53] For Gram-negative bacteria, 1-5 mM EDTA is included in the lysis buffer to permeabilize the outer membrane prior to lysozyme addition.^[54] Post-incubation, cells are subjected to osmotic shock or mild centrifugation to collect the lysate, with enzyme activity monitored via turbidity reduction (e.g., OD600 decrease).^[1] This approach is well-suited for prokaryotes, fungi, and plants where cell wall composition is known, achieving 70-90% lysis efficiency in optimized conditions for sensitive applications like protein or enzyme extraction.^[49] It is species-specific, excelling with peptidoglycan-rich Gram-positive bacteria (e.g., Bacillus subtilis) or β-glucan-containing yeasts (e.g., Saccharomyces cerevisiae), but less effective against Gram-negative bacteria without EDTA or certain algae with resistant walls.^[1] Key advantages include its gentleness, which preserves the native activity of intracellular enzymes and biomolecules (e.g., maintaining >80% enzymatic functionality post-lysis), and high specificity that minimizes contamination from wall debris.^[55] It operates under low-energy, ambient conditions, avoiding heat-induced denaturation.^[56] However, limitations encompass high enzyme costs (e.g., lysozyme at $100-500/kg), prolonged incubation times that may allow microbial contamination, and incomplete lysis (10-30% unlysed cells) in heterogeneous or resistant strains, necessitating protocol customization.^[49]

Chemical Lysis

Chemical lysis involves the use of chemical agents to disrupt cell membranes and walls, releasing intracellular contents without mechanical force. These agents primarily target the lipid bilayer of cell membranes or the structural components of cell walls, such as peptidoglycan in bacteria, by altering molecular interactions that maintain cellular integrity. This method is particularly valuable in laboratory settings for its simplicity and ability to process samples in suspension, though it requires careful selection of agents to minimize damage to target biomolecules.^[1] The mechanisms of chemical lysis vary by agent type. Detergents, such as sodium dodecyl sulfate (SDS) and Triton X-100, solubilize lipid bilayers by inserting into the membrane and disrupting hydrophobic interactions between lipids and proteins; SDS, an ionic detergent, additionally denatures proteins through strong electrostatic binding, while Triton X-100, a non-ionic detergent, is milder and preserves protein structure better. Solvents like toluene and chloroform dissolve lipid components and break hydrophobic-hydrophilic balances in the membrane, leading to perforation and content leakage. Chelators such as EDTA weaken bacterial cell walls by sequestering divalent cations (e.g., Mg²⁺) that stabilize peptidoglycan cross-links and lipopolysaccharide layers in Gram-negative bacteria, enhancing membrane permeability.^[1]^[57]^[1]^[58] A typical protocol entails adding the chemical agent at concentrations of 0.1-1% (v/v) for detergents or 1-10 mM for chelators to a cell suspension, followed by incubation at room temperature or 4°C for 10-30 minutes with gentle mixing to allow uniform disruption. The mixture is then centrifuged at 10,000-16,000 × g for 5-10 minutes to separate the lysate supernatant from cellular debris. This process achieves 80-95% lysis efficiency for animal cells and certain bacterial strains, though optimization may be needed for robust cell types.^[1]^[59]^[60]^[1] Chemical lysis is highly suitable for fragile animal cells due to their thin plasma membranes but less effective alone for Gram-positive bacteria with thick peptidoglycan layers, where it may require complementary treatments for full efficacy. While effective, agents like SDS can denature enzymes and other sensitive proteins, limiting its use in applications requiring native biomolecule activity. Advantages include its reagent-based simplicity, scalability for small to medium volumes, and lack of need for specialized equipment, making it accessible for routine extractions. However, limitations arise from potential toxic residues in the lysate, necessitating downstream purification steps like dialysis or chromatography, and its unsuitability for preserving heat-labile or shear-sensitive biomolecules.^[1]^[57]^[1]^[1]

Physical Methods

Physical methods of cell disruption utilize environmental stresses, such as temperature fluctuations, osmotic imbalances, and rapid heating, to induce cell lysis without introducing chemical agents or enzymes. These approaches are valued for their simplicity and gentleness, making them suitable for lab-scale applications where preserving biomolecule integrity is crucial. Common techniques include freeze-thaw cycles, osmotic shock, and microwave heating, each exploiting physical forces to compromise membrane integrity.

Freeze-Thaw Cycles

Freeze-thaw lysis operates through repeated cycles of freezing and thawing, where ice crystal formation during the freezing phase mechanically punctures the cell membrane, and subsequent thawing induces osmotic excursions and lipid phase transitions that further destabilize the membrane.^[61]^[1] This process is distinct from cryogenic methods that incorporate mechanical grinding, as freeze-thaw relies purely on thermal cycling for disruption.^[1] The standard protocol involves subjecting cell suspensions to 3-5 cycles of freezing at -80°C for 30 minutes to several hours, followed by thawing at 37°C or room temperature until fully liquid.^[62]^[1] This method is particularly gentle and effective for animal cells and plant tissues, achieving 50-80% lysis efficiency, and is best suited for small-scale processing of soft, non-rigid cell types where downstream purity is prioritized over speed.^[1]^[63] Its primary advantages include low equipment costs and non-invasive application, requiring only standard laboratory freezers and incubators; however, it is limited by its time-intensive nature, variability in efficiency across cell types, and risk of uneven disruption due to incomplete ice formation.^[1]^[30]

Osmotic Shock

Osmotic shock disrupts cells by alternating exposure to hypertonic and hypotonic solutions, causing initial shrinkage followed by rapid water influx that swells and bursts the membrane due to increased internal pressure.^[64]^[1] A typical protocol entails suspending cells in a hypertonic solution such as 20% sucrose for 10-30 minutes to dehydrate them, followed by rapid dilution into hypotonic distilled water or buffer to induce swelling and lysis, often with gentle agitation.^[8]^[1] It is well-suited for fragile animal cells and plant protoplasts lacking robust walls, offering 50-80% efficiency in lab-scale extractions of intracellular contents from soft tissues.^[1]^[63] Advantages encompass its low cost, ease of implementation with basic buffers, and minimal invasiveness; limitations include slow kinetics, inconsistent results for walled cells, and potential for partial lysis leading to variable yields.^[1]^[30]

Microwave Heating

Microwave heating achieves cell disruption through rapid absorption of electromagnetic energy by water molecules, generating localized thermal expansion and pressure gradients that rupture the membrane.^[65]^[1] Protocols generally involve short bursts of microwave irradiation (e.g., 30-60 seconds at 500-800 W) in a controlled microwave oven, with intermittent cooling to prevent overheating, tailored to sample volume for uniform exposure.^[65]^[1] This technique is gentle for animal and plant cells in lab-scale settings, yielding 50-80% efficiency for soft tissues, though it excels more with aqueous suspensions.^[65]^[1] It offers advantages in speed and reduced equipment needs beyond a standard microwave; however, limitations include uneven heating, risk of thermal degradation of sensitive biomolecules, and variability in larger volumes.^[65]^[1]

Applications and Considerations

Biotechnology Applications

Cell disruption plays a pivotal role in biotechnology by enabling the recovery of valuable intracellular products from microbial and mammalian cells, supporting large-scale production in pharmaceuticals and biofuels. In pharmaceutical manufacturing, it is essential for extracting recombinant human insulin from Escherichia coli inclusion bodies, where mechanical methods like high-pressure homogenization release the proinsulin precursor, achieving high yields critical for meeting global diabetes treatment demands.^[66] Similarly, in biofuel production, cell disruption facilitates enzyme recovery from recombinant E. coli, particularly cellulases used in biomass hydrolysis; optimized thermal and mechanical lysis protocols enhance enzyme release, improving process efficiency for bioethanol generation.^[67] For isolation of secondary metabolites from Streptomyces species, disruption techniques can be applied to access intracellular compounds, with combined mechanical and chemical methods used in some cases to ensure effective extraction while preserving bioactivity in downstream purification. In research settings, cell disruption is fundamental for isolating biomolecules in omics studies. For proteomics, gentle lysis methods release proteins from cells for comprehensive analysis, allowing high-resolution identification of cellular proteomes without significant degradation.^[6] In genomics, it enables efficient DNA and RNA extraction from diverse cell types, supporting sequencing and gene expression profiling essential for understanding genetic regulation.^[68] Metabolomics benefits from rapid disruption protocols that quench metabolism and extract intracellular metabolites, providing snapshots of biochemical pathways for biomarker discovery.^[69] Scaling cell disruption from laboratory batch processing to industrial continuous flow systems in bioreactors is crucial for economic viability, with targets often exceeding 90% recovery yields to offset costs in commercial operations. Batch methods suit small-scale research for precise control, while continuous homogenization in flow systems handles high volumes for sustained production, minimizing downtime and enhancing throughput in recombinant protein manufacturing.^[17] Notable case studies illustrate these applications: in vaccine production, cell disruption of mammalian cell cultures releases virus-like particles or viral antigens, as seen in influenza vaccine processes where lysis optimizes yield and purity for immunization efficacy.^[70] In food processing, lysis of brewer's yeast (Saccharomyces cerevisiae) from beer fermentation recovers nutrients and flavors, with autolysis and enzymatic methods valorizing spent yeast into protein-rich extracts for nutritional supplements.^[71] Recent advances include automated cell lysis systems, which improve throughput and reduce labor in high-volume biotechnological processes as of 2025.^[72]

Efficiency Factors

The efficiency of cell disruption is primarily evaluated through metrics such as yield, which quantifies the percentage of intracellular components like proteins or DNA released relative to the total available in the biomass. For instance, protein yield is often measured using assays like the modified Lowry method, where disruption efficiency can reach 40-43% of total biomass protein with methods like bead milling or NaOH-assisted sonication in purple non-sulfur bacteria.^[73] Factors influencing yield include cell density, typically ranging from 10^8 to 10^10 cells/mL for optimal processing, and the number of passes in mechanical systems like high-pressure homogenization, where multiple cycles can increase release from 67% to over 90%.^[74]^[75] Purity in disrupted samples is achieved by minimizing contamination from cellular debris and preventing unwanted protein modifications. Post-disruption centrifugation at 10,000-20,000 × g for 10-20 minutes effectively pellets unbroken cells and insoluble fragments, yielding a clearer supernatant for downstream applications.^[30] For sensitive proteins, shear forces in mechanical methods can induce aggregation, which is mitigated by maintaining samples on ice during processing to avoid denaturation.^[30] Enzymatic approaches generally provide higher purity due to their selectivity, reducing non-target release compared to mechanical techniques.^[74] Method selection hinges on matching the technique to the cell type and target biomolecule while balancing cost and energy demands. Rigid cell walls in bacteria or yeast favor mechanical methods like bead beating or homogenization for yields up to 95%, whereas mammalian or fragile cells benefit from enzymatic lysis using lysozyme or proteases to achieve gentle, high-purity extraction without excessive damage.^[2]^[74] Energy trade-offs are critical; ultrasonication consumes 0.1-0.5 kWh/L but offers rapid processing, while enzymatic methods require lower energy input (<0.34 kWh/kg biomass) yet incur higher costs from enzyme reagents.^[76]^[77] Troubleshooting common issues enhances overall efficiency, particularly in controlling heat and scaling operations. Excessive heat from methods like ultrasonication, which can exceed 60°C and denature proteins, is managed by processing in ice baths or cooling systems during short bursts.^[30] Scaling from laboratory volumes (mL) to industrial scales (m³) poses challenges, as lab methods like sonication are limited to <100 mL, necessitating robust alternatives like high-pressure homogenization that maintain yields above 80% at larger volumes but require optimization for uniform energy distribution.^[30]^[74] High yields from optimized disruption are especially vital in biotechnology applications like recombinant protein production.

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Physical Cell Disruption Technologies for Intracellular Compound ...
Sep 24, 2024 · This review focuses on the physical disruption techniques in extracting intracellular compounds, a critical step that significantly impacts yield and purity.
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Cell Disruption and Membrane Preparation
### Workflow for Cell Disruption: Preparation, Disruption, Separation
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(PDF) Mechanical/Physical Methods of Cell Disruption and Tissue ...
Aug 10, 2025 · This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available ...
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The Evolution of Homogenizers | Lab Manager
Throughout the early years, however, homogenization was limited to the ancient technology of the mortar and pestle, or to the destructive power of the Waring ...
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[PDF] An Efficient, Easily Constructed Cell Homogenizing Press - NCBI
254-261. FRENCH, C. S., AND H. W. MILNER. 1955. Disintegration of bacteria and small particles by high-pressure extrusion, p. 64-67. In S. P. Colowick and N ...
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A device for facilitating the use of the French press - ResearchGate
Aug 7, 2025 · The French pressure cell for disruption of microbial cells was invented by Stacey French in the 1950s (French and Milner, 1955). Since that ...Missing: Charles | Show results with:Charles
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Cell Disruption by Ultrasound - Science
Cell Disruption by Ultrasound: Streaming and other activity around sonically induced bubbles is a cause of damage to living cells.
[13]
The Beadbeater and the Evolution of Mechanical Cell Disruption ...
Dec 17, 2019 · Beadbeating technology was developed and originally commercialized by BioSpec Products in the late 1970's -- over time becoming the ...
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Liquid Nitrogen Cryo-Impacting: a New Concept for Cell Disruption
1975, p. 445449. Copyright ©D 1975 American Society for Microbiology ... decompression, shearing, or chemical extrac- tion ... SMUCKER AND PFISTER. 4. FIG. 1.
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Chapter 2 Methods of cell breakage: assessing their suitability and ...
... French press (Milner et al. 1950) Hughes press (Hughes 1951). Pressure homogenisation, originally introduced by Fraser in 1951, has also featured in the ...
[16]
Bacterial cell disruption: A key unit operation in the recovery of ...
The need for microbial cell disruption has hindered the large scale production of commercial biotechnological products of intracellular derivation.
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Charles Stacy French | Biographical Memoirs: Volume 88
Stacy French was one of those inventors. His prestigious inventions include the “French pressure cell” for breaking of cells; the first auto-. Page 64 Share ...
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1.2 Cellular Size - Cell & Molecular Biology
Prokaryotic cells are 0.1 to 5.0 μm, while eukaryotic cells range from 10 to 100 μm. Animal cells are typically 10-100 μm.Missing: osmotic mechanical shear risks
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On the evolution of fungal and yeast cell walls - PMC
The fibrous component of yeast cell walls usually consists of β-glucan and/or chitin. ... β(1–3)glucan in the Saccharomyces cerevisiae cell wall. Eukaryot Cell.
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The Fungal Cell Wall: Structure, Biosynthesis, and Function
In most fungal species the inner cell wall consists of a core of covalently attached branched β-(1,3) glucan with 3 to 4% interchain and chitin (9, 10). β-(1,3 ...
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Visualizing chemical functionality in plant cell walls
Nov 30, 2017 · Cellulose, hemicellulose, and lignin entangle to form a complex matrix. One challenge for efficient utilization of cellulose, hemicellulose, and ...Missing: disruption | Show results with:disruption
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The plant cell wall—dynamic, strong, and adaptable—is a natural ...
Cell walls certainly fit this definition as they can undergo subtle or dramatic changes in structure, assume many shapes, and perform many functions.
[23]
Cell Disruption - an overview | ScienceDirect Topics
Cell disruption is crucial for the release of intracellular components such as nucleic acids, metabolites or proteins.
[24]
Overview of Cell Lysis and Protein Extraction | Thermo Fisher Scientific
In animal cells, the plasma membrane is the only barrier separating cell ... The lack of an extracellular wall in animal cells makes them relatively easy to lyse.
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Cell fractionation: Techniques and applications - Abcam
Cell fractionation requires maintaining isotonic conditions to preserve the integrity of organelles3. In this process, isotonic buffers like sucrose or mannitol ...
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Fractionation of Cells - Molecular Biology of the Cell - NCBI Bookshelf
Experiments on mitochondria and chloroplasts purified by centrifugation, for example, demonstrated the central function of these organelles in converting energy ...
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The difference between prokaryotic and eukaryotic cells
Dec 11, 2017 · Prokaryotes lack a nucleus and are smaller (1-2µm), while eukaryotes have a nucleus, are larger (10-100µm) and have more complex internal ...<|separator|>
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Yeast (Saccharomyces cerevisiae): evaluation of cellular disruption ...
Jun 11, 2019 · Cell rupture of the yeast can provide a significant increase in protein content and improvement in digestibility since the cell wall is ruptured ...
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Traditional Methods of Cell Lysis for Protein Extraction
Several methods are commonly used to physically lyse cells to extract proteins, including mechanical disruption, liquid homogenization, high frequency sound ...
[30]
Cell Disrupters: A Review - BioSpec Products
Cell disruption occurs by the crushing action of the beads as they collide with the cells. Compared to ultrasonic and high-pressure methods of cell disruption, ...
[31]
Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a ...
The mechanism of lysis by bead-beating has been attributed to high shear rates between beads and strong periodic vortical flow fields (13).
[32]
Evaluation of cell disruption methods for protein and coenzyme Q10 ...
Mar 6, 2024 · These cellular disruption techniques can be broadly classified into mechanical treatment (such as bead milling and homogenization), physical ...
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On the mechanism of microbial cell disruption in high-pressure ...
In this study we examine the mechanism of homogenisation by considering a rigid, thin-walled, liquid-filled sphere (i.e., an idealised rigid cell) moving with ...
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Mechanical Cell Disruption Technologies for the Extraction of Dyes ...
Mar 9, 2021 · Ultrasonication is a technique whereby cavitation is created by introducing ultrasonic waves into a liquid medium via a resonance rod. It occurs ...
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French Pressure Cell Disruptor
The device was invented by an American scientist, Charles Stacey French, at the Rockefeller Institute, and is named for him. The conditions of cell disruption ...
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Equipment for Cell Disruption and Cell Lysis - Microfluidics
Cell disruption is the process of breaking cells to recover and maintain the intracellular content integrity with high efficiency.
[40]
[PDF] High Cell Disruption Rates and Protein Yield Utilizing a ...
Microfluidizer processors enable extremely effective cell disruption (often >99% rupture for E.coli in only one pass), with high protein recovery. Protein ...<|control11|><|separator|>
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Applications of High and Ultra High Pressure Homogenization for ...
However, HPH was firstly employed as an useful method for cell disruption and recovery of intracellular bio-products (Keshavarz Moore et al., 1990; Shirgaonkar ...<|separator|>
[42]
Cell disruption methodologies - Dyhydromatics
Different types of cells, e.g., mammalian, bacterial, yeast, plant cells and algal cells have unique characteristics and therefore different cell disruption ...
[43]
High Pressure Homogenisation for the Cell Disruption of Algae
High pressure homogenisation (HPH) offers the best solution to meet this requirement, and is the most widely used method. A 2015 study on green alga cell ...Missing: homogenization | Show results with:homogenization
[44]
Detailed Analysis of High Pressure Homogenizer for Cell Disruption
Ability to achieve high levels of disruption: High pressure homogenizers can achieve a high level of cell disruption due to the intense shear forces generated ...
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Mechanical Disruption Methods: Grinding - OPS Diagnostics
This cryogenic grinding makes the sample brittle and fracture easily, but it also preserves analytes that are heat labile or which may rapidly degrade upon ...
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[PDF] Cell Disruption by Nitrogen Decompression
The principle of the method is quite simple. Large quantities of nitrogen are first dissolved in the cell under high pressure within a suitable pressure vessel.Missing: workflow | Show results with:workflow<|control11|><|separator|>
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https://pubmed.ncbi.nlm.nih.gov/21041386/
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Lysozyme Lysis of Gram-Negative Bacteria without Production of ...
Summary: Cells of Gram-negative bacteria undergo lysis when treated with lysozyme in the presence of ethylenediaminetetraacetic acid (EDTA) and tris buffer, ...Missing: protocol | Show results with:protocol
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Separating Inner and Outer Membranes of Escherichia coli by EDTA ...
Mar 20, 2023 · Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria.
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A fully automatable enzymatic method for DNA extraction from plant ...
Nov 3, 2005 · We describe here a new method for the lysis of plant tissues using a powerful cocktail of enzymes isolated from Trichoderma longibrachiatum.Discussion · Methods · Enzymatic Cocktail
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https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/protein-lysis-and-extraction/lysing-enzymes
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Cell Disruption | Bio-Rad
Enzymatic lysis, Suspension of cells in iso-osmotic solutions containing enzymes that digest the cell wall (for example, cellulase and pectinase for plant cells ...
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Current techniques for single-cell lysis - PMC - NIH
However, when used after first treating the cell with a weak detergent such as digitonin, sonication can lyse cells within 3 s, allowing efficient separation of ...
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Molecular Sieve Mechanism of Selective Release of Cytoplasmic ...
In this procedure, bacterial cells are preincubated in a hyperosmotic sucrose solution supplemented with EDTA to permeabilize their outer membrane and then ...
[58]
Preparation and Extraction of Insoluble (Inclusion-Body) Proteins ...
The usual detergent concentration is between 0.5% to 5%. Triton X-100 will ... cell lysis. If unbroken cells are present in the low-speed pellets, they ...
[59]
Detergent Induction of HEK 293A Cell Membrane Permeability ... - NIH
A fixed number of cells (e.g., 5 million cells) were suspended in ECS and incubated with various concentrations of detergents for 15 min. The suspension was ...Introduction · Experimental Methods · Results
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Freeze/thaw-induced destabilization of the plasma membrane and ...
Freeze/thaw causes plasma membrane destabilization via osmotic excursions and lipid phase transitions. Cold acclimation increases tolerance to these excursions ...
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scFTD-seq: freeze-thaw lysis based, portable approach toward ...
In the next sections, we show that our freeze-thaw lysis based approach yields efficient cell lysis and mRNA capture, and high-quality sequencing libraries ...
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Cell lysis techniques | Abcam
Introduction to cell lysis for cellular analysis. Cell lysis or cellular disruption is the process that helps destroy or break the cell membrane or outer ...
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Osmotic Shock - an overview | ScienceDirect Topics
Osmotic shock is the technique of weakening cells caused by lysis, which is brought about by increased internal pressure as water rapidly enters cells.
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Cell disruption methods for improving lipid extraction efficiency in ...
Feb 8, 2015 · The advantages of microwave treatment lies in the fact that the high internal temperature and pressure gradient acting on the cell wall created ...
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Downstream processing of recombinant human insulin and its ... - NIH
Jul 27, 2021 · Here, a comprehensive review of downstream processing of recombinant human insulin/analogue production from E. coli inclusion bodies is presented.
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Optimization of cell culture and cell disruption processes to enhance ...
Jan 17, 2019 · The effect of pH, temperature and time on cell disruption and the resulting FnCel5A yield was demonstrated in this study.
[67]
DNA Extraction and Polymerase Chain Reaction - PMC - NIH
Cell lysis can be done using nonionic detergent (sodium dodecyl sulfate), Tris–Cl, and Ethylene diamine tetraacetic acid (EDTA), and this step is followed by ...
[68]
Methodological approaches to help unravel the intracellular ...
Jul 11, 2013 · Rapid, efficient, and gentle cell disruption is a key prerequisite for metabolite extraction in metabolome analyses.
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Downstream processing of cell culture-derived virus particles - NIH
Here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy.
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Spent Brewer's Yeast Lysis Enables a Best Out of Waste Approach ...
Exposing cells to ultrasounds (≥20 kHz frequency) is often used as a yeast cell lysis method. The mechanism of cell disruption through ultrasonication is based ...
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Evaluation of cell disruption methods for protein and coenzyme Q10 ...
Mar 7, 2024 · Bead milling is often regarded as one of the most efficient cellular disruption and extraction techniques (D'Hondt et al., 2017; Hu and Bassi, ...Missing: definition | Show results with:definition
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Cell Disruption - an overview | ScienceDirect Topics
The methods involved in cell disruption can be categorized into two types - mechanical and non-mechanical. The non-mechanical methods can be further sub- ...
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https://pmc.ncbi.nlm.nih.gov/articles/PMC10976975/
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Ultrasound for microalgal cell disruption and product extraction
Ultrasonic frequency, intensity, and duration affect algal cell disruption. Acoustic cavitation, heat, pressure and free radicals are the major mechanisms.Missing: microjets | Show results with:microjets
[75]
Energy consumption and water-soluble protein release by cell wall ...
Enzymatic treatment required low energy input (<0.34 kWh.kg−1biomass), but it only released ±35% protein (w/w). Pulsed Electric Field (PEF) was neither energy- ...