The cell
The cell structure
Cells are the fundamental units of biological activity, a term coined by Robert Hooke in 1665, derived from the Latin word "cella," meaning storeroom or chamber There are two main types of cells: prokaryotic and eukaryotic Prokaryotic cells have a simple structure, while eukaryotic cells, which include those found in plants and animals, possess a more complex architecture This article focuses specifically on the structure of eukaryotic cells, particularly cardiomyocyte-like and neuron-like cells.
Eukaryotic cells range in size from 1 to 100 micrometers and are protected by a plasma membrane that separates them from the external environment This membrane is rich in biomolecules, including proteins that form ion channels to facilitate the movement of substances in and out of the cell Inside, various subcellular components collaborate to sustain cellular functions, with the nucleus housing genetic information that regulates gene expression—the process of synthesizing functional proteins from genes Additionally, the nucleolus within the nucleus is responsible for ribosome synthesis, which can be found either free in the cytoplasm or attached to the rough endoplasmic reticulum, where they contribute to protein production.
The smooth endoplasmic reticulum is crucial for lipid metabolism and synthesis In muscle cells, the specialized sarcoplasmic reticulum stores and releases calcium, facilitating excitation and contraction Additionally, proteins synthesized in the endoplasmic reticulum are transported to membranes or lysosomes through vesicles via the Golgi apparatus, where they play vital roles in cellular functions.
F IGURE 2.1: Structure of the eukaryotic cell contains many structural subunits:
1) Nucleolus, 2) Nucleus, 3) Ribosome, 4) Vesicle, 5) Rough endoplasmic reticu- lum, 6) Golgi apparatus, 7) Cytoskeleton, 8) Smooth endoplasmic reticulum, 9) Mitochodria, 10) Vacuoles, 11) Cytoplasm, 12) Lysosomes, 13) Centriole. biomolecules, including macromolecules such as proteins, nucleic acids, or small molecules, are disassembled by hydrolytic enzymes This pro- cess is similar to the activity of digestion [22].
Lysosomes play a crucial role in recycling processes within the cell, alongside their function in molecular degradation To preserve cell shape and size, all cells contain a dynamic structure known as the cytoskeleton, which is essential for rapid cell growth The cytoskeleton is composed of three primary types of filaments: microfilaments, microtubules, and intermediate filaments Microfilaments, also referred to as actin filaments, are constructed from actin proteins and serve as tracks for the movement of myosin molecules.
In muscle cells, the interaction between myosin and actin is essential for contraction in cardiomyocytes Microtubules function as tracks for organelle movement, while their interactions with microfilaments are vital for neurite outgrowth and guidance Intermediate filaments, along with microfilaments, help maintain cell shape and facilitate cell-cell connections The centriole, composed of two bundles of microtubules, plays a key role in establishing cell polarity Mitochondria generate energy for the cell by converting it into adenosine triphosphate (ATP), earning them the title of the cell's "powerhouse."
Membrane potential
Ion channels, as detailed in subsection 2.1.1, are embedded in cell membranes and play a crucial role in regulating ion flow The movement of ions in and out of the cell creates a difference in electrical potential between the inside and outside of the cell, known as membrane potential.
Signals within cells are conveyed by the opening and closing of ion channels in the membrane, leading to localized changes in membrane potential This ion movement creates an asymmetric distribution, establishing ion gradients across the cell membrane Cells leverage these ion gradients to communicate with their environment, particularly in electrogenic cells like neurons and muscle cells, where changes in membrane potential initiate action potentials for signal transmission.
The resting membrane potential occurs when the influx and efflux of ions are balanced, maintaining the cell's resting state This equilibrium is sustained by the movement of ions, particularly sodium (Na+), potassium (K+), and chloride (Cl–), which are distributed both inside and outside the cell The concentrations of these key ions are detailed in Table 2.1 The cell membrane's selective permeability significantly influences this ion distribution.
The outflow of K+ ions is the primary mechanism for maintaining the resting membrane potential, as it is more significant than the movement of other ions The equilibrium potential is defined as the voltage at which the net ion flow across the membrane reaches zero.
T ABLE 2.1: Approximate concentrations of intracellular and extrallular ions in mammalian cells (Note: these are concentrations of free ions).
The equilibrium potential of an ion, such as potassium (K+), can be determined using the Nernst equation, which takes into account the ion's charge and its concentration gradient across the membrane.
E eq,K + = RT zF a ln[K + ] out
The equilibrium potential for potassium ions (K+) is determined by the Nernst equation, which incorporates the universal gas constant (R), absolute temperature (T), the number of elementary charges (z), the Faraday constant (F), and the concentrations of K+ inside ([K+] in) and outside ([K+] out) the cell.
The resting membrane potential in real cells is influenced by multiple ions, primarily potassium (K+), chloride (Cl-), and sodium (Na+), rather than solely K+ The Goldman-Hodgkin-Katz equation accounts for the contributions of these three ions, providing a more accurate estimation of the membrane potential by considering the strongest ionic currents across the membrane.
F a lnP K [K + ] out +P Na [Na + ] out +P Cl [Cl − ] in
The relative permeabilities of ions are represented as P K for potassium (K+), P Na for sodium (Na+), and P Cl for chloride (Cl−), with the equation P K [K+] in + P Na [Na+] in + P Cl [Cl−] out Typically, permeability values are expressed relative to P K At the resting membrane potential, most cells exhibit a higher permeability for K+ compared to Na+ and Cl− The charge (z) is implicitly included in the individual ion terms.
K + A sketch of various ion channels embedded in the plasma membrane is shown in Figure 2.2.
Action potentials and signal propagation
The electrical potential difference across the membrane arises from varying ion concentrations, leading to rapid fluctuations in membrane potential and subsequent changes in voltage.
The action potential, crucial for neuronal signaling, consists of three main phases, as illustrated in Figure 2.3 The initial phase, known as depolarization, involves a rapid shift in membrane potential from its resting state to the equilibrium potential of ions This occurs when a stimulus opens ion channels, resulting in an influx of Na+ ions and a significant increase in membrane potential, shifting it from approximately -70 mV at rest to around 40 mV during depolarization.
The second phase of the action potential is repolarization, where the membrane potential returns to its resting state due to the opening of potassium channels that allow K+ ions to flow outward Following repolarization is the refractory period, a brief phase during which sodium channels are inactivated while some potassium channels remain open, continuing the outward flow of K+ ions Ultimately, Na+/K+ pumps restore the membrane to its resting condition.
F IGURE 2.3:A schematic shows various phases of a typical action potential.
Cardiac action potentials are distinct from those in other electrogenic cells, as they originate from specialized pacemaker cells within the heart A typical cardiac action potential consists of five phases, starting with the resting membrane potential (phase 4), where the membrane remains stable due to a balance between ion influx (sodium and calcium) and efflux (potassium) Upon receiving a stimulus, fast sodium channels open, resulting in a rapid influx of sodium ions and subsequent depolarization of the cell membrane.
As sodium channels close quickly, potassium channels open, allowing potassium ions to flow out of the cell and making the membrane potential slightly more negative This sudden depolarization activates calcium and potassium channels, facilitating calcium influx and potassium efflux The simultaneous opening of these channels creates a balance between calcium entry and potassium exit, resulting in a plateau phase.
The action potential of a cardiomyocyte consists of several phases: depolarization, first repolarization, plateau, second repolarization, and resting membrane potential During phase 2, calcium channels close while potassium channels remain open, resulting in potassium efflux that facilitates full repolarization of the cell in phase 3 Ultimately, the potassium channels close, allowing the membrane potential to return to its resting state in phase 4.
Pacemaker cells are specialized cardiac cells located in the sinoatrial node of the heart that can generate action potentials spontaneously, without external stimuli These cells play a crucial role in regulating the heart's beating rate At rest, pacemaker cells gradually increase the membrane potential by allowing sodium ions to flow into the cell Once the membrane potential reaches a certain threshold, an action potential is triggered, initiating the heartbeat.
When the membrane potential reaches -40 mV, calcium channels open, allowing calcium ions to flow into the cell and trigger depolarization This is followed by the opening of potassium channels, which leads to potassium ions exiting the cell and repolarizing it After repolarization, sodium leak channels can create instability in the resting potential, resulting in the generation of the next action potential The inherent instability of pacemaker cells facilitates spontaneous and rapid depolarizations that occur regularly These fast depolarizations regulate the contractile cardiomyocytes through a process known as overdrive suppression, where the cells with the highest depolarization frequency dictate the heart's overall rhythm.
Gap junctions play a crucial role in cardiac function by connecting pacemaker cells to neighboring cells, enabling local depolarization This connectivity allows pacemaker cells to regulate contractions in cardiomyocytes effectively.
Signal propagation in cardiac cells
Gap junctions are specialized channels that connect the plasma membranes of adjacent cells, facilitating communication through the passage of molecules, ions, and electrical impulses These channels are formed by proteins known as connexons, each composed of six connexin proteins When cells experience depolarization beyond a specific threshold, it triggers the rapid propagation of action potentials through these gap junctions, enabling swift signal transmission, particularly in cardiomyocyte-like cells such as HL-1 cells.
E XCITATION - CONTRACTION COUPLING OF CARDIAC CELLS In cardiac cells, the excitation-contraction coupling is a key process of turning elec- trical excitations into mechanical contractions This process is initiated by
Excitation-contraction coupling in cardiac cells begins with local membrane depolarization, which triggers calcium-induced calcium release from the sarcoplasmic reticulum (SR) During the action potential, voltage-gated L-type calcium channels in the T-tubule membrane open, allowing calcium ions to flow inward and increase intracellular calcium concentration This influx binds to ryanodine receptors (RyR) on the SR, facilitating further calcium release The elevated intracellular calcium enables binding to troponin C, leading to a conformational change in tropomyosin that uncovers the myosin binding site on actin, ultimately generating force as myosin interacts with actin.
At the conclusion of the contraction cycle, calcium is released from troponin-C, allowing the muscle to relax This calcium is then either expelled from the cell through the sodium-calcium exchanger or reabsorbed into the sarcoplasmic reticulum by the SERCA pump.
The contraction process in individual cardiomyocytes involves the formation of gap junctions when these cells are cultured as a confluent layer This setup allows for effective signal propagation across the cell layer through these gap junctions.
TRPV channels as temperature sensors
Temperature sensing is crucial for organisms to adapt to environmental changes, primarily through ion channels in cellular membranes These channels, known as thermal-transient receptor potential (TRPV) channels, are sensitive to heat The temperature dependence of TRPV channels can be estimated using the Q10 value, which measures the change in reaction rate with a 10°C increase in temperature.
R 1 ) T 2 10 −T 1 , (2.3) where R 1 and R 2 are the rate of the reaction at temperature T 1 and T 2 , respectively.
The thermal TRPV channels are characterized for having greater ther- mal sensitivity with Q 10 values much higher than 2 (6 < Q 10 < 30)
The TRPV family of thermal-sensing ion channels includes TRPV1, TRPV2, TRPV3, and TRPV4, all characterized by a structure similar to the TRP ion channel superfamily, consisting of six transmembrane domains that form cation-permeable channels These channels open in response to rising temperatures, allowing ion influx TRPV1 channels, activated by capsaicin, have a temperature threshold of 43 °C and respond rapidly within milliseconds, though their sensitivity decreases with prolonged exposure to constant temperatures TRPV2 channels activate at noxious temperatures up to 53 °C, while TRPV3 channels respond to lower temperatures, ranging from 23 to 39 °C Lastly, TRPV4 channels activate at temperatures starting from 22 °C but become desensitized between 24 and 36 °C.
Cell Models
HL-1 cells: A cardiomyocyte cell line
HL-1 cells, derived from the AT-1 mouse atrial cardiomyocyte tumor by Dr William Claycomb, are a valuable model for studying cardiac biology due to their ability to maintain contraction activity during multiple passages in culture These cells can be stored in liquid nitrogen and exhibit spontaneous contraction when forming a confluent monolayer, with approximately 30% of them acting as pacemaker cells Their unique characteristics make HL-1 cells suitable for various model systems aimed at investigating cellular functions in cardiomyocytes, particularly in relation to the effects of temperature on heart cells.
PC12 cells as a model for neurons
PC12 is a rat adrenal medulla-derived cell line established by Green and Tischler in 1976, widely utilized in neuronal studies due to its ease of culture and ability to be passaged multiple times These cells form small clumps and exhibit a significant response to nerve growth factor (NGF), which induces neurite formation and enhances microtubule assembly essential for neuronal extension NGF-treated PC12 cells undergo phenotypic changes, displaying characteristics typical of sympathetic neurons, and their elongating neurites connect to form neuronal networks The growth cones at the distal ends of these neurites, equipped with motile filopodia and lamellipodia, facilitate environmental exploration and neurite orientation, making NGF-treated PC12 cells an important model for neurite outgrowth research.
Devices and fabrication techniques
Microwire array
Photo-lithography, also known as optical lithography, is a critical micro-fabrication technique used to pattern structures on substrates This process involves transferring geometric designs from a photomask to the substrate through ultraviolet (UV) light exposure and photosensitive materials known as photoresists Photoresists, which consist of a base resin, a photoactive compound, and a solvent, can be categorized into positive and negative types based on their response to light exposure Positive photoresists dissolve in the developer after UV exposure, allowing for easy removal of exposed regions, while negative photoresists, like AZ nLOF2020, become less soluble and create cross-linking upon exposure, enabling the unexposed areas to be removed The photolithography process for fabricating microwire arrays can be outlined in a straightforward manner.
A thin oxide layer is formed on a silicon wafer by heating it to temperatures between 900 and 1150 °C in steam or humidified oxygen, which acts as an insulating layer Following this, a photoreist layer is applied to the substrate through spin coating, with its thickness varying based on the spin-coating speed and the viscosity of the photoresist used.
After spin coating, the wafer undergoes a soft bake at 115 °C, with the duration varying based on the thickness of the AZ nLOF2020 photoresist; for thinner layers, this lasts 90 seconds Following soft baking, the wafer is exposed to illumination, transferring mask structures onto the resist and inducing a chemical reaction that alters solubility in the exposed areas A post-exposure bake is then performed to finalize the chemical reaction and minimize residual resist after development The process concludes with the resist being developed in a solvent, resulting in the desired structure, as illustrated in Figure 2.8.
Microfluidic channels for cell culture
N EURONAL GUIDANCE AND ORIENTATION USING MICROFLUIDIC CHAN -
NELS Microfluidic devices have been shown to be a useful tool for study- ing cellular behavior Microfluidic platforms allow biologists deep in-
F IGURE 2.7:The schematic shows changes in characteristics of the negative and the positive photoresist after being exposed.
Microwire array chips, illustrated in Figure 2.8, demonstrate a precise structure after development and metal deposition, with scale bars indicating 100 µm These microfluidic devices are pivotal for studying cellular functionality, particularly in neurite guidance and polarization, by controlling the micro-environment within living cells Various microfluidic structures have been developed to facilitate localized physical and chemical stimulation on selected neurites, aiding research into neuronal regeneration and degeneration One notable design by Taylor et al features a microchannel structure that separates neurite outgrowth from neuronal cell bodies using micron-sized grooves and a physical barrier This device comprises two distinct cell culture chambers—a somatic and an axonal compartment—connected by microchannels measuring 10 µm in width and 3 µm in height, effectively preventing neuronal somata from entering the microchannels This innovative structure enables the quantification of neurite outgrowth properties, including growth length, growth rate, and retraction rate, by allowing neurites to grow straight along the microchannels.
Peyrin et al introduced a novel microfluidic structure designed for neurite orientation, featuring asymmetrical channels with wide inlets and narrow outlets This unique design functions as a directional filter, allowing neurites and axons to project selectively in one direction, similar to a "diode." The structure, characterized by a 15 µm inlet and a 3 µm outlet, significantly reduces the number of neurites reaching the opposite chamber, thereby creating a directional model for neural networks that was previously unattainable.
The microfluidic platform designed for neuron culture features two distinct compartments: the somal side, depicted in black, and the axonal side, shown in yellow, which are interconnected by microchannels This innovative setup allows neurites to extend from the somal side into the axonal side through these microchannels, facilitating advanced studies in neuronal behavior and connectivity.
The microfluidic platform designed for neurite polarization and orientation features culture chambers with two compartments linked by asymmetrical microchannels The specific dimensions of the microchannels' outlets and inlets play a crucial role in this setup.
Fabrication technologies for microfluidic channels 22
Soft lithography is a fabrication technique that utilizes elastomeric materials, primarily polydimethylsiloxane (PDMS), known for its low cost, biocompatibility, and optical transparency The PDMS prepolymer is composed of a polymer base and a curing agent, typically mixed in a 10:1 ratio The soft lithography process begins with the creation of a master mold, which is achieved by photo-patterning SU-8 microchannel structures on a silicon wafer.
8 is a negative photoresist widely utilized for fabricating high-aspect ratio three-dimensional patterns To create a PDMS microchannel, the PDMS prepolymer is poured onto a master mold and cured with heat, after which it is removed from the mold A biopsy puncher is then used to create holes in the PDMS for cell culture chambers For bonding the PDMS to a glass substrate or chip surface, oxygen plasma treatment is applied, which introduces polar functional groups, primarily silanol groups (SiOH), transforming the PDMS from hydrophobic to hydrophilic This treatment facilitates the formation of covalent bonds between the treated PDMS and the glass substrate Alternatively, silanization can be employed to bond PDMS to a polyimide chip's surface, establishing a covalent -Si-O-Si- bond.
PDMS is widely utilized in microfluidic devices, but its sponge-like structure can absorb molecules from solutions, leading to issues such as sample drying and altered osmolarity To address these challenges, there is a need to enhance PDMS microfluidic channels by incorporating alternative materials, such as SU-8, for improved performance.
F IGURE 2.11:a) The chemical reaction of oxygen plasma and b) silanization. rificial layer process is probably a promising substitute method This method will be described below.
The microchannel fabrication technique involves the deposition, photolithographic patterning, and selective etching of thin films to create microchannel structures This process begins with the deposition and patterning of a sacrificial layer, which defines the shape and size of the microchannels A mechanical layer is then deposited and patterned onto the sacrificial layer, followed by sacrificial etching to dissolve the first layer Various materials, including metal, polymer, and silicon dioxide, can be used for the sacrificial layer, which is fabricated through methods such as physical vapor deposition (PVD) and chemical vapor deposition (CVD) PVD is particularly utilized for depositing thin films in low-pressure environments, typically within a vacuum chamber.
F IGURE 2.12: The soft lithography process a) and b) are the lithophotogra- phy processes used to produce a SU-8 master mold, c) Pouring PDMS onto the master mold d) Releasing PDMS after curing.
The microchannel fabrication process involves several key steps: first, a sacrificial layer is deposited and patterned onto the substrate; next, a mechanical layer is applied and patterned on top of the sacrificial layer; finally, the sacrificial layer is etched away The deposition methods include evaporation and sputtering, where evaporation involves vaporizing source materials that condense on the substrate, while sputtering ejects atoms from a target material using energetic particles Physical Vapor Deposition (PVD) is primarily used for metal deposition, whereas Chemical Vapor Deposition (CVD) utilizes diffusive-convective mass transfer to deposit a variety of materials Although CVD offers versatility in material options, it often involves toxic gases and higher costs.
Photoresist serves as an alternative sacrificial layer material for microchannel fabrication, offering a streamlined approach that minimizes the number of patterning steps required Utilizing the photolithography process, photoresist is applied to the substrate, enabling the creation of high aspect ratio structures through the patterning of a thick photoresist layer.
Heat stimulation
Resistive heating
Resistive heating, or Joule heating, occurs when electrical energy is transformed into heat as an electric current flows through a conductor When an electric field is applied, it exerts a force on charge carriers, causing free electrons to accelerate against the electric field However, this acceleration is quickly diminished due to collisions with atoms and other charge carriers, leading to the conversion of kinetic energy into vibrational energy of the lattice atoms Consequently, this process raises the temperature of the conductor.
In a circuit featuring a resistor with resistance R and a battery providing a potential difference V, positive charges move counterclockwise through the circuit As these charges flow through the resistor, they lose energy due to collisions with atoms, resulting in the generation of internal energy The power (P) delivered to the resistor is directly proportional to the rate at which the charge loses energy.
P= IV, (2.4) whereIis the current in the circuit,Vis the potential difference of the bat- tery According to Ohm’s law, we have the potential difference
V = IR, in whichRis the electrical resistance of the wire The resistance of the wire is defined following the equation:
Whereρis the electrical resistivity of material,l is the length of the wire,and Ais the cross-sectional area of the wire.
If we substitute the potential differenceV = IR into the equation 2.4, we can express the power delivered to the resistor in other forms:
Heat transfer
This study explores the use of resistive heating in microwires as a precise heat source for localized stimulation of cellular networks To assess how thermal energy dissipates in relation to the supplied power, we employ finite element simulations alongside fluorescence lifetime imaging (FLIM) to measure local temperature changes The upcoming section will discuss the fundamental theories of heat transfer relevant to this system.
Heat transfer occurs through three primary mechanisms: conduction, convection, and radiation Conduction involves the transfer of heat through a medium due to a temperature gradient Convection occurs when heat is transferred between a surface and a moving fluid at different temperatures Lastly, radiation refers to the transfer of heat in the form of electromagnetic waves.
F IGURE 2.15: Three types of heat transfer: heat conduction, convection and radiation.
In this section, the three main heat transfer mechanisms will be re- viewed and a heat transfer equation will be presented.
Conduction is the process of energy transfer at the microscopic level, where higher energy particles collide with less energetic particles, facilitating energy movement within a substance In a metal with a temperature gradient, particles near the heat source vibrate more vigorously, transferring energy to neighboring particles through collisions This transfer of energy increases the temperature of particles located farther from the heat source, resulting in heat transfer that occurs from areas of higher temperature to areas of lower temperature.
The rate of heat conduction is influenced by the properties of the material, with metals being excellent thermal conductors due to the abundance of freely moving electrons in their structure Conversely, materials like glass and polymers exhibit poor thermal conductivity To assess a substance's ability to conduct heat, thermal conductivity is measured, where high thermal conductivity indicates effective heat conduction, and low thermal conductivity signifies suitability for thermal insulation.
Heat conduction occurs when heat flows from a region of higher temperature to one of lower temperature To quantify the heat transfer rate, Fourier's law of heat conduction is utilized, particularly in one-dimensional systems with a temperature distribution T(x) This law is represented by the equation q_x = -k (dT/dx), where q_x denotes the heat flux in watts per square meter (W/m²), k represents the thermal conductivity of the material in watts per meter per Kelvin (W m⁻¹ K⁻¹), and dT/dx indicates the temperature gradient in the x-direction The negative sign signifies that heat transfer occurs in the direction of decreasing temperature Fourier's one-dimensional equation can be expanded to accommodate three-dimensional heat transfer scenarios.
F IGURE 2.16: A convection mode of heat transfer with fluid moving over a heated surface.
The heat flux, as defined by equation 2.8, is characterized by both a specific direction and magnitude By understanding the boundary conditions, temperature gradients, materials, and heat conductivity, one can accurately evaluate the heat flux using this equation.
Convection heat transfer involves two key mechanisms: random molecular movement (diffusion) and fluid movement The fluid's motion, driven by a large number of molecules, plays a significant role in heat transfer This section discusses heat transfer when there is a temperature gradient between a heated surface and a moving fluid For instance, when fluid flows over a heated surface, its velocity changes from u_s at the fluid-surface interface to a finite value at the fluid's surface, particularly when the surface temperature (T_s) exceeds the ambient temperature (T_∞).
T ∞ represents the fluid's surface temperature, where convection heat transfer takes place from the heated surface to the fluid flow Convective heat transfer is categorized into two types: forced convection and natural convection Forced convection arises when the flow is driven by external forces, such as pumps or atmospheric conditions.
T ABLE 2.2: Typical values of the convective heat transfer coefficient (adapted from [60]).
Liquids 100-20000 winds Natural convection is induced by differences in density because of temperature variations in the fluid.
The convective heat transfer rate between a surface and a fluid is determined by Newton’s law of cooling, expressed as q = h(Ts - T∞), where q represents the convective heat flux in watts per square meter (W/m²), Ts is the temperature of the heated surface, and T∞ is the temperature of the fluid The convective heat transfer coefficient, h (W/m²K), plays a crucial role and varies based on boundary layer conditions, which are affected by the surface geometry, fluid motion, and various thermodynamic and transport properties of the fluid Typical values for the convective heat transfer coefficient can be found in Table 2.2.
In this work, the fluid on the chip’s surface is stilled Therefore, the contribution of convective heat transfer is the natural convection.
Radiation is the energy released through the thermal vibrations of molecules, transmitted as electromagnetic waves The amount of thermal radiation emitted is influenced by the surface area and the thermal energy of the material it encompasses.
However, in biological systems, thermal conduction and convection are the two dominant factors, which are caused by mass transport in the capillary system.
F IGURE 2.17:A schematic describes heat transfer in a control volume.
The heat transfer equation is essential for calculating heat dissipation in a system, illustrating the relationship between the heat generated by electrical current in a microwire, the temperature distribution within the wire, and its surroundings This analysis is based on the first law of thermodynamics, which in a closed system emphasizes the principle of conservation of energy.
The equation E int = E ch + E out illustrates the relationship between the internal heat generated by Joule heating (E int), the rate of change of internal heat energy (E ch), and the heat energy dissipated from the microwire through conduction (E out).
A volumetric heat release distributed through the volume isq v (W/m 3 ). This is the result of resistive heating Thus the heat generated within the volume V is:
The rate of change of internal heat energy is defined by
The outflow of heat energy (E_out) is directly proportional to the surface area (A), with an infinitesimal surface element represented as dA In this context, the unit normal vector (n̂) and the heat flux vector (q) are defined at the surface point The heat energy conducted through the area dA can be expressed mathematically, highlighting the relationship between density (ρ) and specific heat capacity (c) in the process.
(⃗q)(⃗ndA), (2.13) substitute the heat flux⃗qfrom equation 2.8 to the equation 2.13, the heat energy transferred out of area dAcan be written as follows:
(−k∇T)(⃗ndA) (2.14) Applying Gauss’s theorem,E out can be written as follows:
V ∇(−k∇T)dV, (2.15) substituteE int ,E out , andE ch to equation 2.10, the equation 2.10 can be written as follows: ˆ
Because the volumeV is arbitrary, the integrand can be taken out, so the equation 2.16 can be written: q v = ρc ∂T
∂t +∇(− k ∇ T ) (2.17) Finally, a general form of the heat transfer equation is obtained as follows:
By applying equation 2.18 with the known parameters of microwires, including their dimensions, materials, and boundary conditions, we can determine the temperature distribution This approach is effective primarily for simple geometries and symmetric boundary conditions In our study, we utilized the numerical method with COMSOL Multiphysics to analyze heat transfer in microwire arrays.
Optical detection
Calcium imaging
Intracellular calcium concentration is crucial for the regulation of cardiomyocyte function, as discussed in section 2.1.3 Monitoring changes in this calcium level provides insights into the cells' functional state To visualize calcium signals, this study employed calcium imaging, a fluorescence-based technique for remote calcium detection.
Calcium imaging involves staining living cells with a fluorescent dye to monitor changes in fluorescence intensity This technique utilizes a sensitive camera integrated with a microscope and standard fluorescent filters to capture the dynamic responses When the dye enters the cell, it binds to intracellular calcium (Ca 2+), resulting in alterations to the dye's fluorescent characteristics, such as intensity or wavelength.
Fluorescent dyes are typically impermeable to cell membranes, necessitating techniques for cellular loading such as microinjection, macroinjection, and ester loading Microinjection involves diluting calcium indicators in a cytosol-like solution and using a glass micropipette for injection; however, this method is limited to approximately 100 cells per hour, making it challenging for larger cell populations In contrast, macroinjection allows for the simultaneous loading of indicators into multiple cells, but both micro and macroinjection techniques are invasive and require specialized equipment and expertise.
This thesis explores the use of Fluo-4, a fluorescent calcium sensing dye, to investigate intracellular calcium concentration changes The dye is modified with acetoxymethyl (AM) ester groups, allowing it to cross cell membranes as an uncharged molecule Once inside the cell, esterases cleave the AM ester groups, resulting in a high concentration of the dye within the cell.
The dissociation constant (K d ) is a crucial factor in selecting a calcium indicator, as it reflects the indicator's calcium binding affinity Indicators with a K d below or near the target concentration provide reliable calcium sensitivity High-affinity calcium indicators produce intense fluorescence but can saturate at lower calcium levels, potentially buffering intracellular calcium In contrast, low-affinity dyes are ideal for measuring elevated calcium levels in subcellular organelles, offering rapid response times that enable high temporal resolution in monitoring calcium concentration changes.
In order to qualitatively measure the change in intracellular calcium concentration, a relative change of fluorescence signal R f can be calcu- lated as follows:
F base (2.19) where F is the measured fluorescent intensity of the calcium indicator,
The F base represents the baseline intensity of the calcium indicator prior to stimulation While the exact concentration of calcium cannot be directly measured, this ratio serves as an effective approximation of intracellular calcium levels.
The Ca2+ measurements in this thesis were captured using imaging software that employed a rolling average filter with a width of 4 frames and a sequential subtraction of 4 frames to enhance grey-scale resolution This live processing allowed for the analysis of Ca2+ imaging through temporal averaging of fluorescent intensity.
F av , (2.20) whereF norm is the normalized fluorescent intensity,F av is the temporal average of the fluorescence intensity, and F is the measured fluorescent intensity of the indicator.
Fluorescence lifetime imaging (FLIM)
This study utilized fluorescence lifetime imaging microscopy to effectively measure temperature dissipation on the microwire array The following section provides a concise overview of the fundamental principles underlying this measurement technique.
FLIM measurement is a technique for assessing temperature by analyzing the lifetime of a fluorescent indicator excited by brief laser pulses When a molecule absorbs a photon, it transitions to an excited state and eventually returns to its ground state by emitting a photon Utilizing fast detectors, the timing of photon arrivals and decay is captured in relation to the laser pulses and beam position This data reveals spatial distribution and fluorescent decay times, ultimately presenting results in an array where each point reflects the corresponding fluorescent decay time.
[63] is an exponential function given as:
The fluorescent intensity over time is described by the equation I(t) = I0 exp(−τt), where I(t) represents the intensity at time t, I0 is the initial intensity after a pulse, and τ denotes the fluorescent lifetime This equation allows for the determination of the fluorescent lifetime, which can be calibrated against temperature using a predefined set of values obtained through a curve fitting algorithm Consequently, temperature can be inferred from lifetime measurements Fluorescence Lifetime Imaging Microscopy (FLIM) is widely utilized as a noninvasive technique for temperature assessment, as it is independent of the concentration and absorption of the fluorescent indicator However, the FLIM method has limitations, as the properties of fluorescent indicators may change in response to environmental factors like pH variations, and quenching effects can occur when imaging at micrometer distances from the sample.
In this work, the temperature measured from FLIM method is com- pared with the numerical method to have a reliable temperature distribu- tion on the microwire.
SIGNAL PROPAGATION IN HL-1 CELL
This chapter was reproduced from the unpublished work “K.M Dang,
P Rinklin, D Afanasenkau, G Westmeyer, T Schürholze, S Wiegand and
B Wolfrum, Chip-based heat stimulation for modulating signal propaga- tion in HL-1 cell networks.” in submission.
Preamble
This study explores how localized temperature gradients affect signal propagation in cardiac cell networks These gradients are produced by resistive heating through microwire arrays on a chip that supports a confluent cell network The research demonstrates that heating locally accelerates the velocity of the propagating Ca²⁺ wave within the network Additionally, it examines the relocation of the pacemaker cell and the deformation of the Ca²⁺ wavefront resulting from increased local temperatures.
Introduction
Endotherms maintain a healthy body temperature within a narrow range, as deviations can lead to metabolic and functional issues in cardiac activity The contraction of cardiac muscle relies on intracellular Ca 2+ dynamics, where the release of Ca 2+ from the sarcoplasmic reticulum increases cytosolic Ca 2+ concentration, triggering a Ca 2+ transient that facilitates muscle contraction Once the Ca 2+ concentration decreases, the cardiac muscle cell relaxes This Ca 2+ signaling is dependent on key molecular components such as ryanodine receptors, voltage-gated Ca 2+ channels, and Ca 2+ pumps, all of which are sensitive to temperature fluctuations.
Assessing the impact of local temperature variations at the cellular level remains challenging, as most studies on temperature effects on Ca 2+ transients in cardiac myocytes focus on ambient temperature control, potentially obscuring local influences While localized heating using focused infrared lasers can excite action potentials in muscle and nerve cells, this method is complex and not suitable for long-term or high-throughput applications Alternatively, the use of electromagnetically excited nanoparticles for localized heating has been reported, but this approach often leads to global temperature increases due to heat dissipation across a large area, along with the added risk of cell damage from nanoparticle toxicity.
Recent advancements in microsystems have opened new avenues for spatially and temporally controlled stimuli and cellular activity recording Notable applications include microchip devices for electrical stimulation, single-vesicle release measurement, and the investigation of signal propagation in cardiac myocyte networks The introduction of microwire array chips enables the creation of thermal lesions in cell cultures to analyze network connectivity This study utilizes microwire arrays to induce localized hyperthermia, generating precise temperature gradients to examine dynamic intracellular Ca²⁺ signal changes in HL-1 cardiomyocyte-like cells, which are derived from mouse cardiomyocyte tumors The HL-1 cells exhibit spontaneous action potentials and contractions upon confluency Our findings indicate that localized heating significantly affects Ca²⁺ signal propagation, influencing speed, direction, and the positioning of the pacemaker cell.
Materials and methods
Fabrication of microwire array chips
At the Helmholtz Nanoelectronic Facility, I fabricated advanced microwire array chips consisting of 17 microwires, utilizing cutting-edge microfabrication technology These chips were designed to apply resistive heating, inducing thermal stress on cellular networks The fabrication process began with a 4-inch diameter, 500 µm thick thermally oxidized silicon wafer as the substrate A double layer resist (LOR3B and AZ nLOF 2020) was spin-coated onto the wafer, followed by photolithographic patterning of the microwire array, which featured wires 2.5 µm wide and spaced 30 µm apart After exposure and development, a metal stack of 10 nm Ti/300 nm Au/10 nm Ti was deposited via electron-beam vapor deposition, with titanium layers enhancing gold adhesion To passivate the chip for cell-culture applications, a polyimide layer (PI2545) was spin-coated, and a subsequent photolithography step was performed to expose the outer contact pads for electrical connections.
Culture of HL-1 cells on the microwire array chip 40
HL-1 cells were cultured in T25 flasks at 37 °C with 5% CO2 using Clay-comb Medium (51800C, Sigma), enriched with 10% FBS (ThermoFisher Scientific), 100 µg/mL penicillin-streptomycin (ThermoFisher Scientific), 0.1 mmol norepinephrine (Sigma), and L-glutamine.
Cells were cultured in a humidified incubator with 2 mmol of a solution from ThermoFischer Scientific until they reached confluency and began beating Prior to cell plating, microwire chips were sterilized under UV light for one hour The chips were then treated with a mixture of fibronectin (5 µg/mL, Sigma) and gelatin (0.2 mg/mL, Sigma) for about 60 minutes Following this, 500 µL of the cell suspension was seeded onto the chips and incubated under standard conditions of 37 °C and 5% CO2.
Thermal stimulation and Ca 2+ imaging
The power supply utilized custom LabView software to deliver electrical stimulation to HL-1 cells, applying 0.7 W of power to a central microwire for 30 seconds Thermal stimulation was administered in a normal growth medium, with a 1-minute resting period between each stimulation pulse to allow for cell recovery To assess the relationship between local heating and cell activity, the HL-1 cells were stained with a calcium-sensitive dye.
The study utilized 4 μmol of Fluo-4 AM (Thermofisher) for 15-30 minutes, followed by a medium exchange and transfer to the measurement setup During thermal stimulation, the Ca²⁺ fluorescence signal was captured using a high-speed, low-light emCCD camera (Hamamatsu C9100-13) To investigate the impact of localized heating on the Ca²⁺ signal, the analysis focused on the fluorescence indicator dye, Fluo-4, which is known to have a decreasing dissociation constant with increasing temperature This temperature effect can alter both the intensity and waveform of the fluorescence signal To mitigate these influences, the Ca²⁺ signal was evaluated relative to the basal fluorescence, and signal propagation was assessed through the temporal correlation of signals, ensuring that the temperature-induced changes in the indicator's properties were minimal during the experiments.
Cross-correlation analysis
To analyze Ca 2+ imaging activity, a cross-correlation analysis was conducted after down-scaling images to 32 x 32 pixels for improved processing speed The pixel intensity was normalized by calculating the normalized fluorescent intensity, as outlined in equation 2.20 Active pixels were distinguished from inactive ones based on their root-mean-square (RMS) intensity changes, with the threshold set at the minimum RMS value plus half the standard deviation of all RMS values Additionally, the intensity traces of all pixels were smoothed using a Gaussian filter for the cross-correlation analysis.
Microwire arrays are utilized for localized thermal stimulation, as demonstrated by an encapsulated chip measuring 24 mm x 24 mm, which contains 17 parallel microwires When a current passes through these wires, it accelerates the speed of Ca 2+ signals in the heated area An optical image shows HL-1 cells cultured on the chip's surface, and the temporal propagation of calcium signals is analyzed by calculating the average intensity trace of all pixels Each pixel is cross-correlated with this reference trace to determine relative signal delays, which are then visualized in a color map Scale bars in the images correspond to 100 µm.
Results and discussions
Effect of localized heat stimulation on Ca 2+ signal
This study investigates the impact of localized heat stimulation on the propagation of Ca 2+ signals in HL-1 cell cultures, utilizing microwire arrays for heat generation through electrical power The temperature profile, as illustrated in Figure 3.2, rapidly stabilizes within approximately 100 ms after applying 0.7 W of power, with a notable sharp gradient observed near the microwire on the chip's surface.
We assessed the influence of temperature on cardiac cell activity through optical recordings during heat stimulation To visualize the propagation of Ca2+ signals within the cardiac cell network, we utilized a sensitive fluorescence dye, Fluo-4, AM Our Ca2+ imaging videos captured both spontaneous activity and responses to thermal stimulation.
The Comsol simulation illustrated in Figure 3.2 reveals the temperature distribution on the chip's surface caused by a microwire array operating at 0.7 W The temperature at the top of the stimulating wire stabilizes rapidly, reaching 32 °C within 100 ms, as shown in the time-dependence graph The temperature profile across the chip surface remains consistent at 10, 20, and 30 seconds due to this quick equilibration, with the center position of the stimulating microwire marked at an x-position of 0 mm Additionally, Figure 3.3 displays representative frames of the fluorescent signal captured both with and without thermal stimulation, highlighting the effects of the thermal process.
To assess the impact of heat on Ca 2+ signal propagation, a cross-correlation analysis of local fluorescence intensity from Ca 2+ imaging videos was conducted, as illustrated in Figure 3.4a The images represent time delays in Ca 2+ signal occurrences across different positions within the cell network, with color gradients indicating the signal's direction from blue to red The experiments involved recording Ca 2+ imaging videos for one minute without stimulation (left column) and for thirty seconds with thermal stimulation (right column) The results, depicted in Figure 3.4a, clearly demonstrate the alterations in Ca 2+ wave propagation induced by heat stimulation.
We assessed signal propagation velocity by calculating the spatial extension divided by the difference between maximum and minimum delay values within a circular area This method intentionally overlooked variations in signal propagation direction While absolute velocities and relative changes in velocity were considered, the focus remained on the overall propagation characteristics.
The Ca2+ imaging sequence reveals distinct signal propagation patterns in the sample, with spontaneous signals moving from the top left to the bottom right corner without thermal stimulation In contrast, when thermal stimulation is applied, the signal travels more rapidly along the heated area, demonstrating the influence of temperature on signal dynamics.
(Figure 3.4c) vary significantly between different cell culture experiments, we observe an increase of Ca 2+ wave propagation velocity upon heating in approximately 87 % of all the samples investigated (n= 15).
The findings offer a qualitative insight into the impact of heat stimulation on HL-1 cells; however, they lack a quantitative assessment of local propagation velocity in heated versus non-heated areas To evaluate how the stimulus affects the local propagation velocity of Ca 2+ signals, we conducted an analysis of correlation plots from various stimulation experiments, as illustrated in Figure 3.5.
In our experiments, we recorded Ca 2+ imaging videos from the same cells during repetitive stimulation and recovery phases The reversible change in Ca 2+ propagation patterns was evident when comparing the recovery phase patterns to those observed during stimulation Notably, the time delay of the signals across different experiments highlights the distinct dynamics of Ca 2+ signaling.
Figure 3.4 illustrates the direction and velocity of signal propagation in cell cultures, comparing conditions with and without heat stimulation Panel (a) presents data from three independent experiments, highlighting the differences in signal propagation Panel (b) summarizes the global signal velocity across all experiments under both conditions Panel (c) shows the normalized velocity, calculated as the ratio of signal velocities with and without heat stimulation for the same sample, with the dashed blue line indicating the threshold for equal velocities.
The reversible effects of localized heating on cardiac myocytes are illustrated through cross-correlation analysis of Ca²⁺ imaging videos The time delay of the propagating action potential is depicted using a color-coded scale, with the x- and y-axes representing the dimensions of the observed sample section Thermal stimulation was applied to a central wire for 30 seconds, followed by a 1-minute recovery period, resulting in localized changes in the Ca²⁺ signal behavior of the cardiac myocyte cells Each image corresponds to the same position within the sample, highlighting the effects of the thermal treatment.
The speed of Ca 2+ signal propagation in HL-1 cells is illustrated through two plots: one comparing delay time and signal progression with and without heat stimulation, represented by red and blue traces, respectively Additionally, a graph depicts the correlation between power levels and the velocity of the Ca 2+ signal, highlighting changes in signal speed as power supplies are activated and deactivated.
Figure 3.7 illustrates the signal delay recorded at a heated wire (red) and at a location 300 µm away (blue), demonstrating that signal propagation accelerates during thermal stimulation To analyze the propagation velocity in high-temperature zones, we assessed the delay time of the Ca²⁺ signal in both heated and non-heated conditions As shown in Figure 3.6a, the delay time was measured against the distance of signal propagation, comparing the diagonal frame without thermal stimulation (blue trace) to the y-axis in the thermally activated frame (red trace).
The data analysis employed a linear function to determine the propagation speed of the Ca 2+ signal, revealing that the slope without thermal stimulation is steeper than that with stimulation Quantitative results indicate that during heat activation, propagation speeds exceed 35 µm/ms, while without heating, speeds range from 16 to 20 µm/ms This slight increase in signal velocity during the recovery phase is attributed to a minor global temperature rise from repeated heat stimulation Notably, the propagation is affected near the heated region, resulting in a deformation of the signal wavefront A comparison of propagation speeds in different temperature regions shows a difference of approximately 10 µm/ms, indicating that microscopic heating can induce local variations in Ca 2+ signal propagation speed within a connected network.
Effect of localized heat stimulation on the pacemaker
Thermal stimulation alters the propagation direction of the Ca 2+ wave, potentially due to two main effects: a local change in propagation velocity and a shift in the pacemaker position within the cell layer As illustrated in Figure 3.8, without thermal stimulation, the intrinsic Ca 2+ signal originates from the top left corner and moves to the bottom right However, upon applying thermal stimulation, the signal origin shifts along the microwire, changing the propagation direction towards the y-axis, as shown in Figure 3.8b This shift in signal origin towards the heated area was observed in approximately 27% of experiments (n=15) This mechanism may result from local modulation of cellular kinetics, influencing the beating pattern, similar to effects seen during electrical stimulation.
[95, 100] a cell beating faster than the intrinsic pacemaker, can “hijack” the network and become the new pacemaker cell.
F IGURE 3.8:The effect of heat on the origin of the Ca 2+ signals a) The intrinsic
The propagation of the Ca 2+ signal occurs from the top left corner to the bottom right corner, while localized thermal activation alters this signal's pathway, directing it along the heated wire from top to bottom.
An increase of the beating frequency has been previously observed for global temperature stimulation [101] To assess if, correspondingly, the
The direction of Ca 2+ signaling is significantly influenced by local heat activation, as evidenced by our comparison of spontaneous signal propagation angles with those observed during local heat stimulation Results indicate that Ca 2+ signal directions shift towards the heated wire, aligning closely at 90° To better understand the conditions necessary for pacemaker relocation during heat stimulation, further experiments on geometrically defined cell-network structures are essential This research could ultimately clarify the mechanisms behind heat-controlled pacemaker activation.
Conclusions and outlook
Microwire arrays serve as efficient instruments for delivering localized thermal stimulation to cultured cells As illustrated in Figure 3.10, this process can lead to two distinct phenomena when using an individual microwire for localized heat application.
The distribution of propagation angles for spontaneous signals (a, n=15) and heat-stimulated signals (b, n=15) shows a notable change in the percentage of signals aligned with the heated wire Specifically, the percentage of signals within the angle range of 60° to 90° increases significantly from 20% to 47% following stimulation.
The Ca 2+ wave initiates in the upper left corner and travels to the bottom right of the cell network Our findings suggest that a localized increase in temperature can lead to two distinct effects within the cellular environment The first effect, referred to as Effect A, involves the disruption of the network's normal functioning.
The Ca 2+ wave pattern in heated areas is influenced by localized changes in propagation speed, leading to the thermal activation of an additional pacemaker This combination results in a disturbance of the signal wavefront and a relocation of the pacemaker within the heated region.
Heat stimulation in structured networks or tissues can enhance research on pacemaker selection and localized Ca 2+ effects By utilizing nanofabrication technologies, we can modify the geometry of heating zones and restrict cell growth to designated areas, such as within microfluidic compartments.
F IGURE 3.10: Schematic illustrating two possible effects of local heating to the
The dynamics of Ca 2+ in cardiac cells are influenced by localized temperature changes, leading to two key effects: first, the Ca 2+ wave experiences disruptions due to alterations in signal velocity in the heated region; second, a distinct pacemaker may be triggered in the same area These two phenomena can occur concurrently, affecting overall cardiac function.
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