According to survival data of mammalian cells at elevated temperatures, there are no surviving cells starting at about 48°C even though the cells are heated for less than 30 seconds (Fig. 1). Higher temperatures (about 58-60°C) sharply shorten the critical exposure time required to kill the cells by heating to less than 2-3 seconds. Such a short time to achieve cell death at 58-60°C is attributed to the fast and irreversible denaturation of cell proteins.
According to our experimental data, even at 48-50°C cell's protein denaturation does not take place. The heating rather destroys the selective permeability of the nuclear pore complexes (NPCs), so the pore becomes non-selective. Usually any intracellular cargo with a nuclear localization signal (NLS) exposed is destined for quick and efficient transport through the pore and to reside within the nucleoplasm. Our experiments reveal that a Red Fluorescent Protein (RFP) fused to NLS (RFP-NLS) irreversibly leaves nuclei after short-term (2-3 seconds) heating of the cells at 48-50°C (see Images) (Fig. 2, right). All cells with "leaking" nuclei round up and detach from the substrate within a couple of hours (see Video 1). These results suggest that all unwanted cells in a culture dish could be "erased" by selective heating them to 48-50°C with following washing them out with fresh medium (after waiting 2-3 hours) (Fig. 3).
We have developed an improved technology (“CellEraser™”) for selective hyperthermic damage of target cells, specifically for irreversible damage (ablation) of target cells within a cell population, that overcome the limitations of previously known methods and devices. CellEraser™ is specially designed for selective heating of cells within cell culture in order to ablate them ("erase") from the culture dish. The content of the unwanted cells (proteins, DNA, etc.) will not leak out to the culture medium. The cells outside of the heating area are not damaged (stay intact) and continue growing.
Next time when you are going to drink too hot tea or coffee (50°C or higher) think about what will happen with epithelial cells in your esophagus. In recent years, the idea has emerged that drinking very hot beverages like coffee and tea could contribute to esophageal cancer. The theory has been that hot liquids such as coffee or tea could destroy the inner lining of the esophagus, requiring the cells to continually regenerate. During this process, there is a greater chance that something can go wrong and turn normal cells into cancer cells. Seeing is believing - just look at Figure 2-3. Cells with damaged/leaky nuclear envelopes never survive. 45°C will induce same effect but exposure time should be 5-10 minutes. Drinking of hot beverages is related to an esophageal cancer called squamous cell carcinoma.
CellEraser™ is very compact, safe and user-friendly microscope attachment (CellEraser™ is patented and protected under US patent laws). It is fed by low voltage of 5-10 V. Specific absorption rate (SAR) of W-band millimeter waves produced by CellEraser™ is very low i.e much lower than SAR of Millimeter wave scanners that are used for a whole-body imaging at airports security checkpoints). Millimeter wavelength radiation is a subset of the microwave radio frequency spectrum. Even at its high-energy end, it is still more than 3 orders of magnitude lower in energy than its nearest radiotoxic neighbor (ultraviolet) in the electromagnetic spectrum. As such, millimeter wave radiation is non-ionizing and incapable of causing cancers by radiolytic DNA bond cleavage.
The instrument is easy calibrated to any desired temperatures (see CellEraser™ calibration). Currently there are no CellEraser™ analogs on the market. Our competitors use lasers-based equipment (Laser Capture Microdissection and LEAP) or very complicate and expensive robotic setup (AVISO CellCelector™). Please, contact us with any details regarding CellEraser™ price and ordering.
Technology
The following examples clarify possible applications of the described method and device for selective hyperthermic damage of target cells.
EXAMPLE 1
Selection of desirable single cell clones in biotechnology applications
In some applications cells are seeded at low density to isolate single cell colonies for cell line development. Usually several cells are placed per well in a cell culture plate (manually via delimited dilution or automatically by flow cytometry sorting) to allow formation of separated colonies. If cells are single cell sorted or seeded to get single colonies, man cell lines show poor survival of single cell clones and slow initial outgrow rate. Better cell survival and growth rate are achieved with placement of several or more cells per well as they condition the culture medium. As the end result, only one single-cell colony that possesses some useful properties (fluorescent protein knock-in, the knock-out of a surface antigen, etc.) needs to be isolated. Therefore all unwanted colonies that helped the desired colony to survive should be eliminated. Overheating by millimeter wave radiation offers a safe and effective way to do so. The elimination is based on a feature distinguishing the desired cells that are kept alive (a specific morphology, a fluorescent signal coming from genetically engineered fluorescent proteins, a surface marker that could be revealed by fluorescently-labeled antibodies, etc.). The method of the current invention allows eliminating all unwanted colonies and keeping alive only a specific single cell colony that exhibits a desired phenotype.
EXAMPLE 2
Generation of tumor cell cultures originated from cancer patient tumor biopsy.
In another application, the tumor biopsies are collected from cancer patients to establish the primary human tumor cell cultures. However, the biopsy samples are extremely heterogeneous due to the presence of other tissue cells like fibroblasts. In most cases these cells grow fast in cultures and easily can overgrow the desired tumor cells. The disclosed device may be used to distinguish the unwanted contaminating cells (via differences in morphology or using fluorescent labels) and to eliminate (ablate) them without damaging the neighboring tumor cells. The millimeter wave radiation hyperthermia ablation of fast-growing primary non-tumor cells (fibroblasts or others) may provide superior conditions for the growth of the desired tumor cells and may allow establishing more homogeneous culture of patient tumor cells.
EXAMPLE 3
Hyperthermia therapy studies
Hyperthermia therapy is a type of medical treatment in which body tissue is exposed to slightly higher temperatures to damage and kill cancer cells or to make cancer cells more sensitive to the effects of radiation and certain anti-cancer drugs. Hyperthermia may kill or weaken tumor cells, and is controlled to limit effects on healthy cells. In many cases hyperthermia is performed via application of radiofrequency (RF) waves to damage tumor tissues. Cancerous cells are not inherently more susceptible to the effects of heat. When compared in in vitro studies, normal cells and cancer cells show the same responses to heat. However, the cancer cells might be selectively sensibilized to hyperthermia treatment with special drugs. The disclosed device and method may provide an experimental tool for high-content screening of compounds that selectively sensibilize cancer cells to hyperthermia therapy. For example, the cancer cells might be mixed with normal cells to yield a mixed cell culture. The tested compound is added to the culture media and after a pre-incubation period the millimeter wave radiation are applied to small area (1 mm in diameter) of the culture to increase the temperature to sub-lethal level (42-43°C). If the tested compound makes cancer cells significantly less able to tolerate the added heat stress than the healthy cells, the heating will lead to cancer cells dying but normal cells surviving. The found hit compounds could be used to increase the effect of radiofrequency ablation in cancer patients.
Recently published paper showed that S-phase cancer cells heated to 45.5°C for 30 minutes become senescent i.e. undergo permanent cell cycle arrest.
Unfortunately there are too many wrong hypotheses about mechanisms of cells damage after application of high temperatures. For example authors of this paper states:
"Hyperthermia >43°C is capable of inducing cell death both directly, by inducing apoptosis, and indirectly, by protein denaturation or DNA damage (1). Hyperthermia has also been shown to adversely affect the fluidity and stability of cellular membranes, the function of transmembrane transport proteins, and cell surface receptor expression (1). Notably, tumor cells are more sensitive to sudden increases in temperature than normal cells, making hyperthermia an attractive therapeutic tool (2)."
But we showed that even higher temperature (50°C) if applied for short time (several seconds) will not induce any protein denaturation or DNA damage.
DNA damage via double-strand breaks happens as a result of exposure to ionizing radiation and chemical compounds. Most anticancer therapies induce multiple DSBs to kill cancer cells.
Fig. 1. Visualization of intact and DSB-containing chromatin domains in U2OS cells. (A) Intact chromatin domains labeled with Cy3–dUTP (red). (B) Cells with telomeres labeled with TRF1–mCherry (red). (C) Cells with centromeres labeled with CENP-B–GFP (green). (D) Cells expressing 53BP1–GFP (green), exposed to 5 Gy of γ-radiation and fixed 30 minutes later. (E) Cells treated as in D, additionally stained for γH2AX (red). The inset shows the intensity profile of a single confocal scan measured along the white bar. Scale bar, 5 μm (Chromatin mobility is increased at sites of DNA double-strand breaks P. M. Krawczyk, T. Borovski, J. Stap, T. Cijsouw, R. ten Cate, J. P. Medema, R. Kanaar, N. A. P. Franken, J. A. Aten)
TP53BP1 is a DNA damage checkpoint protein that interacts with HDAC4 protein to mediate the DNA damage response. p53-binding protein 1 (53BP1) bound to damaged chromatin carries out several functions. First, 53BP1 recruits additional DNA double-strand break (DSB) signalling and repair proteins to the site of DNA damage. Second, 53BP1 promotes ataxia-telangiectasia mutated (ATM)-dependent checkpoint signalling, especially at low levels of DNA damage. Third, it is a key player in DSB repair pathway choice. Fourth, 53BP1 promotes the synapsis of distal DNA ends during non-homologous end-joining (NHEJ)
Etoposide forms a ternary complex with DNA and the topoisomerase II enzyme (which aids in DNA unwinding), prevents re-ligation of the DNA strands, and by doing so causes DNA strands to break (visible as bright foci in nuclei). Cancer cells rely on this enzyme more than healthy cells, since they divide more rapidly. Therefore, this causes errors in DNA synthesis and promotes apoptosis of the cancer cell..
U2OS sells expressing 53BP1–GFP (green) were exposed to etoposide. DSBs are visible as bright foci inside of cell nuclei.
U2OS cells expressing TP53BP1-GFP and RFP:2A:RFP-NLS plasmid were heated to 50°C (region on right side of grid line) during 2-3 seconds. RFP-NLS was leaked out of nuclei because of damage of nuclear pore complex (NPC). However, foci of TP53BP1-GFP were not observed in heated area.
Thus, our experimental data clearly shows that cells after short-term hyperthermia irreversible lose a control of molecular transport across the nuclear envelope. In eukaryotic cells this transport is solely controlled by the nuclear pore complex (NPC).
Nuclear pore. Left: Side view. 1. Nuclear envelope. 2. Outer ring. 3. Spokes. 4. Basket. 5. Filaments. (Drawing is based on electron microscopy images). Right: nucleoporins are arrangement (read the paper).
NPCs perforate the nuclear envelope and act as the main transport gate, therefore have a fundamental role in cellular homeostasis. The NPC is a modular, donut-shaped assembly of ~30 different proteins (nucleoporins or NUPs), arranged in multiples of eight around a central axis that is aligned with the main transport channel. The NPC provides two types of nucleocytoplasmic transport: passive diffusion of small molecules and active chaperon-mediated translocation of large molecules. Single molecule studies have revealed that cargoes up to 29 kDa can smoothly diffuse through the pore, while transport of cargoes larger than 61 kDa is prohibited. Efficient passage through the complex requires several protein factors. Karyopherins, which may act as importins or exportins are part of the Importin-β super-family which all share a similar three-dimensional structure.We hypothesizes that hyperthermia (50°C) applied to cells for a few seconds can irreversibly damages protein complex that build NPC and the damaged NPC just can't control protein transport between nucleus and cytoplasm. As a result proteins and other molecules equilibrate between cytoplasm and nucleus by free diffusion during tens seconds. Cells that lose a control on main transport gate undergo rapid apoptosis and detach from substrate (see Video 1). Particular mechanisms of NPC damage is still unknown (need special research). Similar damage also happens at mild hyperthermia (45°C) but cell exposure time is longer (tens seconds). The percentage of damaged NPCs is also unknown i.e. we can only approximate that undamaged NPCs (if any) can not support equilibrium between cytoplasm and nucleoplasm . It is known that NPC renewal or repair is possible in healthy cells but it looks like that sudden failure of function in big number of NPCs leads to imminent cell death . An understanding of mechanisms of temperature-induced NPC damage can open a new page in hyperthermia therapy of cancer.
More data and information about CellEraser is available in our recently published paper
Novel millimeter-wave-based method for in situ cell isolation and other applications Barney Boyce & Natalia Samsonova Scientific Reports Volume 8, Article number: 14755 (2018)
Figure 2. Left: cells before heating to 50°C; right: cells immediately after heating to 50°C (RFP-NLS protein distributed equally between cytoplasm and nuclei) (40x).
Video 1. 2 hours time-lapse observation of cells (shown in image 1) after heating to 50°C. Cells rounded and start to detach from substrate.
Figure 3. Heated region after wash out (20x). Cells that were outside of the heated area are not damaged (RFP-NLS is still nuclear and continue growing (see Movie 3 in Images)
GFP-tubulin expressing cells were heated to 50C during 2-3 seconds: monomers of GFP-tubulin normally never enters cell nucleus but after the heating they rapidly enter nuclei and then never exit
All mentioned above mechanisms of cell damage at ≥50°C are wrong i.e. there are no cell membrane collapse, protein denaturation, mitochondrial dysfunction, halt in enzyme function, no DNA damage.
Understanding the biological factors that help or hinder thermal ablation has tremendous importance for enhancing or augmenting outcome.
CellEraser™ is the only tool that uses high power millimeter wave (W band) radiation for thermal fixation ("thermofixation") of small areas of cells (array) in live cell cultures.
High-power energy beam instantly heats cells to high temperature (>70°C) that reduces and denatures all cellular proteins and permeabilizes cell membranes. Thermofixed cells remain firmly attached to a substrate and all cell proteins become detectable with Western Blot (WB) antibodies that recognize proteins with linearized epitopes. This method allows precise quantitative analysis of protein expression level between fixed spots with WB antibodies i.e. to perform quantitative in situ Western assay within live cell culture.Thermofixation is a simple and fast alternative to conventional chemical fixation: fixation and permeabilization steps happen simultaneously and take just a few seconds; blocking buffer might be added to cells before thermofixation therefore blocking step starts immediately after ROI is fixed; a thermofixation does not induce autofluorescence in fixed cells as it usually happened in chemically-fixed cells (see Fixation-induced autofluorescence) and therefore it significantly improve signal-to-noise ratio in fixed ROI.
Since the thermofixation is very fast process, it allows to resolve very fast cellular processes like EGFR autophosphorylation after ligand (EGF) binding to EGFR receptor. WB assay can not resolve this process i.e. phospho-EGFR band appeared after 1 minute and the density only slightly increased after 2-5 minutes. Thermofixation can resolve the process within 1 minute after addition of EGF (added at room temperature to slow down the phosphorylation reaction). The thermofixed spots are simultaneously stained with beta-actin WB Ab (ab170325) and anti-EGFR (phospho Y1068). The phosphorylation already started after 10s after EGF adding and practically completed after 30 seconds.
When we presented CellEraser™ on Cell Bio 2022 we got many questions about "thermofixation" application. One question was: Why adherent cells heated to > 70ºC via millimeter wave radiation still firmly attached to substrate?
We have a hypothesis that explain this phenomena.
Indeed, all cell proteins and extracellular matrix (ECM) proteins are denatured at this temperature and ECM/substrate ECM/integrin links should be destroyed. Therefore, thermofixed cells should detach from substrate. On the contrary, we observe that the spot of thermofixed cells is firmly attached to substrate and can survive multiple washout steps (similar to chemically fixed cells). Chemical fixation cross-links cell proteins with ECM proteins.
Millimeter wave radiation heats only water and never heat plastic or glass. Extracellular matrix (ECM) proteins (collagen, fibronectin, and laminin) interacts with substrate surface (glass or plastic) via different types of forces that involves multiple electrostatic, hydrophobic, hydrogen bonding, and van der Waals interactions. ECM should be in native conformation to provide it absorption to surfaces. Denatured ECM most likely will lose their absorption properties.
We heat culture cells via internal heating - glass and plastic are transparent to millimeter waves and remain at room temperature when cells instantly heated to 70ºC. Therefore, ECM proteins contact with cold substrate surface (18-20ºC) during cells heating to 70ºC. This surface actually a good heat sink that not allows ECM and may cell proteins integrins that link cells to ECM to be heated to 70ºC and denatured. As a result, all proteins in "thermofixed" cells are denatured but cells still firmly attached to substrate surface.
We think that "thermofixation" phenomena is possible only when cells are rapidly heated via internal heating. Conventional heating for cell culture dishes happens differently - the thermal control strategy is based upon a thin-film coating of indium-tin oxide (ITO), a transparent, electrically conductive layer applied to the lower surface of the coverslip. Such approach is used Delta T or VAHEAT technologies. They applies an electrical current to the coated underside of the coverslip, thereby heating the entire dish to the desired temperature. Usually cell cultures are heated to 37ºC.
For example, VAHEAT technology allows fast and precise temperature adjustment with heating rates up to 100°C/s. Thus, VAHEAT allows to heat adherent cells to 100°C during 1-2 seconds. Question is why VAHEAT technology doesn't allow to do a thermofixation of adherent cells?
We explain it simply: they heat a substrate (glass or plastic) to 70ºC. Therefore, at some point the cells seat on "frying pan". Hot surface (70ºC) denatures ECM proteins and they lost their adherence properties to a substrate. Cells detached from substrate. An illustration of this hypothesis is in below figure. We don't have any information from VAHEAT users about any attempts of rapid heating of adherent cell culture to 70-80ºC. Maybe they can get cell thermofixation in VAHEAT chamber.
Title. Double click here.
Novel method to pick up single colony of cells (based on CellEraser)
Test on Collagen Type I fims
Holes in collagen film melted at 45°C during 2 s. Plumes of melted gel converted to sole moved up by convection are still visible on right images.
Test on HEK293 colonies growing on Collagen Type I (Corning #354236) film. CellEraser melted collagen under colony and detach it. Convection moves it up for short distance but after heating stopped (2s) the colony fell down because of gravity force. It was collected by collecting entire medium from well. Other colonies around are firmly attached to gel.
Our new method we are not going to compete with laser-based or mechanical/pipette-based methods (like A-picK™ system that designed to pick up individual cells from cell culture.These systems cannot pickup entire cell colonies and they can't work with iPS and ESC cells (too high shear stress).
CellEraser™ might be an effective and not expensive alternative to CellCelector Flex (Sartorius) that cost now $450,000. CellEraser price is 20 times less and customer will a bundle of different application like killing of unwanted colonies after single cell sorting, isolation of wanted cells in cultureware, thermofixation of small regions in live culture, etc. As far as we know customers that bought (or going to buy) CellCelector Flex are going to use it mostly for picking up individual iPS or ESC colonies. Because these cells are very fragile and too sensitive to excess of shear stress. The detachment of entire single colonies via gel melting should be an ideal method for labs that want to pickup tens single colonies per day. One of the big advantages of our method is that the collection is 100% contact-free and contamination-free (no need to open a cultureware lid, it can be performed even in T25 flasks).