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WO2024160853A1 - Champs électriques pulsés en histologie 3d - Google Patents

Champs électriques pulsés en histologie 3d Download PDF

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Publication number
WO2024160853A1
WO2024160853A1 PCT/EP2024/052277 EP2024052277W WO2024160853A1 WO 2024160853 A1 WO2024160853 A1 WO 2024160853A1 EP 2024052277 W EP2024052277 W EP 2024052277W WO 2024160853 A1 WO2024160853 A1 WO 2024160853A1
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Prior art keywords
biological tissue
tissue
pef
solvent composition
optionally
Prior art date
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PCT/EP2024/052277
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English (en)
Inventor
Francesca CATTO
Stephan Fox
Robert AXELROD
Jana BUENDER
Mirko Meboldt
Alexander Mathys
Adriano Aguzzi
Leandro Silvano Guido BUCHMANN
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Eidgenoessische Technische Hochschule Zurich ETHZ
Zurich Universitaet Institut fuer Medizinische Virologie
Original Assignee
Eidgenoessische Technische Hochschule Zurich ETHZ
Zurich Universitaet Institut fuer Medizinische Virologie
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Publication of WO2024160853A1 publication Critical patent/WO2024160853A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the present invention is directed to a method for introducing charged compounds into three- dimensional biological tissues comprising the step of introducing at least one charged compound of interest into the biological tissue, wherein a composition for dissolving and removing lipids and/or a composition for introducing at least one charged compound of interest into the biological tissue is injected into the biological tissue and a pulsed electrical field (PEF) is applied to the biological tissue.
  • PEF pulsed electrical field
  • the invention further relates to a device for use in this method, and a use of this method or this device for histological staining.
  • Spatial 3D staining and imaging requires prior clearing, i.e. the increase in transparency (Rl, refractive index), of lipids (delipidation) from the 3D tissue sample. Clearing can be based on tissue treatment with organic solvent(s) (BABB, Dodt, H.U., et al., Nat Methods, 2007. 4(4): p. 331-6); 3DISCO, Erturk, A., et al., Nature Protocols, 2012. 7(11): p. 1983-1995; iDISCO, Reiner, N., et al., Cell, 2014. 154(4) : p. 896-910 ; Bard, F., et al., Nature Medicine, 2000. 6(8): p.
  • the CLARITY hydrogel-based clearing method is one of the most widely used methods for tissue clearing prior to staining.
  • a fixative solution comprising acrylamide and bisacrylamide, a polymerization initiator and paraformaldehyde (PFA) are incorporated into the tissue of interest, e.g. by soaking and/or by multiple microinjection, followed by repetitive lipid removal with detergents, for example sodium dodecyl sulfate (SDS), for delipidation and homogenization of RL
  • detergents for example sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • the hydrogel-treated tissue will be degassed and the hydrogel is polymerized to form a scaffold inside the tissue sample. This scaffold protects the sample's integrity and structure during the subsequent lipid removal by the detergents.
  • PFA fixates proteins and maintains their original position in the tissue matrix.
  • a detergent-mediated lipid extraction can be performed passively (diffusion only, Chung, K., et al., Nature, 2013. 497(7449): p. 332-337) or actively via electrophoresis (Lee, E., et al., Scientific Reports, 2016. 6(1): p. 1-15171).
  • tissue samples are washed, e.g. with PBS, and immersed in a histodenz-based solution (Sigma Aldrich) for refractive index (Rl) homogenization.
  • Passive CLARITY lipid extraction takes several weeks, whereas active CLARITY lipid extraction still requires one or more weeks for staining-sufficient transparency.
  • Electric fields for example formed by a capacitator consisting of two oppositely loaded electrodes, apply a Coulomb force to a charged element, for example charged molecules like surfactants, fatty acids, and charged staining agents.
  • a charged element for example charged molecules like surfactants, fatty acids, and charged staining agents.
  • PEF pulsed electric fields
  • PEF discharge electric fields in pulses with a specified energy force.
  • CEF constant electric fields
  • PEF can admit extreme voltages and electric currents at lower temperatures for the same time of process.
  • Pulse duration is typically between a few nano- and a few microseconds.
  • a pulse is typically characterized by pulse increase time, pulse -shape, pulse event, pulse amplitude, level and full width at half maximum (FWHM).
  • PEF can be implemented as batch or continuous processes.
  • PEF applications in medicine are presently limited to the use of PEF in therapy, i.e. of living tissues, usually for electroporation where living cells are pierced by PEF pulses due to the change in the membrane potential of the cells resulting in enhanced membrane permeability.
  • the increased permeability results in an improved transmembrane transport of proteins, nucleic acids, small molecules, etc.
  • Increasing the electric field or the pulse event causes cell disruption.
  • PEF are also applied to counteract biofouling for the transmission of chemotherapeutic drugs in cancer tissues, transdermal drug delivery, and bacterial washing of water and beverages.
  • tissue staining in biology and medical diagnosis is typically still two-dimensional. 3D tissue clearing and staining remains complicated and faces a number of obstacles relating to the limited diffusion of compounds within tissues, e.g. ineffective and long termed treatment, or relating to the negative influences of active diffusion enhancement techniques such as electrophoretic enhancement, e.g. loss of tissue structure, tissue component degradation, etc. Tissue samples for 3D staining are much larger than thin-sliced 2D samples. For staining 3D biological tissues the reagent or antibody concentration influences the 3D dye penetration.
  • Concentration gradient and temperature can be adjusted to aid diffusion.
  • the tissue can be made more permeable by detergents, pore size can be increased (Li, J., et al., Sci Rep, 2015. 5: p. 10640.) or smaller staining molecules can be selected. Whereas for 2D staining dye penetration is a minimal problem, 3D staining with tissues the size of a few micrometers to centimeters penetration depth is a substantial problem. In particular if the pore size of the tissue of interest is smaller than the staining molecule, e.g. an antibody, the tissue will form a pore network like a semi-permeable membrane resulting in flow blockage and surface dye accumulation.
  • the staining molecule e.g. an antibody
  • the dye concentration, temperature, solvent viscosity, tissue porosity, and dye molecule size can be varied. Due to instability issues most dyes will limit the temperature to at most 37 to 40 °C. Staining solutions are generally water-based, in particular for protein and antibody dyes, and low-viscosity solvents often have a tendency to osmotically dehydrate tissues.
  • tissue penetration include sonification by ultrasound, for example for penetrating drugs through the skin, which promotes cell to cell exchange as well as drainage and adsorption of lipids.
  • the technical problem underlying the present invention is the provision of improved methods for the removal and/or introduction of charged compounds in isolated three-dimensional histological samples.
  • it is an objective to (i) decrease the removal and introduction times for charged compounds, optionally cell lipids as well as targeting and/or staining agents, to (ii) improve tissue/cell integrity and stability as well as to (iii) maintain the original overall cell status (cell/tissue structure, cell component distribution, no or little degradation or undesired delocalization of relevant cell components) of histological samples.
  • the objectives are solved by a method for introducing charged compounds into three- dimensional biological tissues, wherein a composition comprising a charged compound for dissolving and removing lipids or a composition for introducing at least one charged compound of interest into the biological tissue is injected into the biological tissue, and a pulsed electrical field (PEF) is applied to the biological tissue.
  • a pulsed electrical field PEF
  • a composition comprising a charged compound for dissolving and removing lipids may be a composition comprising a charged detergent(s) and suitable solvent(s) for dissolving cellular lipids within the biological tissue.
  • Detergent(s) and solvent(s) dissolve the lipids upon injection of the composition and disperse and diffuse actively within the biological tissue due to the pulsed movement caused by the PEF. With the charged detergent(s) most of the tissue lipids will be charged (e.g. fatty acids).
  • compositions for introducing at least one charged compound of interest into the biological tissue can be a composition comprising, for example, a charged research or diagnostic agent such as an antibody.
  • a charged research or diagnostic agent such as an antibody.
  • the charged compound Upon injection into the biological tissue, the charged compound will disperse and diffuse actively within the biological tissue due to the pulsed movement caused by the PEF.
  • the composition comprising a charged compound for dissolving and removing lipids can be used for clearing the lipids from the biological tissue
  • the composition comprising a charged compound of interest e.g. a targeting antibody, can be used for staining tissue components in the cleared biological tissue.
  • this aspect of the present invention comprises a step for dissolving and removing lipids and/or a step for introducing a charged compound of interest as long as at least one of the steps, optionally both steps, involve(s) injection of the respective composition into the biological tissue and subsequent PEF treatment for dispersion.
  • the method of the invention is one comprising the steps of (i) providing an isolated three-dimensional biological tissue of interest;
  • introducing a physiologically acceptable solvent composition to the biological tissue by one or more injections into the biological tissue wherein the solvent composition comprises at least one charged compound suitable for dissolving lipids within the biological tissue; at least partially immersing the biological tissue in a suitable or the solvent composition and applying a pulsed electrical field (PEF) to the biological tissue suitable for effecting movement of charged compounds and reagents within the biological tissue; removing the solvent composition and dissolved lipids from the outside surface of the biological tissue; and optionally repeating the introduction and removal of the solvent composition for the removal of lipids from the biological tissue;
  • PEF pulsed electrical field
  • introducing a physiologically acceptable solvent composition to the biological tissue by one or more injections into the biological tissue wherein the solvent composition comprises at least one charged compound of interest for introduction into the biological tissue; at least partially immersing the biological tissue in a suitable or the solvent composition and applying a pulsed electrical field (PEF) to the biological tissue suitable for effecting movement of the charged compounds of interest within the biological tissue; wherein the method comprises step (i) and at least one of steps (iib) or (iii).
  • the method of the present invention is generally suitable for introducing charged compounds into three-dimensional biological tissues, in particular for introducing charged and optionally target-specific compounds for staining isolated three-dimensional tissues of interest within the inner tissue, i.e. below the tissue surface and throughout the tissue.
  • isolated tissue as used herein, is meant to indicate that the tissue no longer forms part of a living body, e.g. an animal or human.
  • the three-dimensional tissue can have any size that still allows for tissue lipid removal and introduction of compounds of interest, e.g. by needle injection.
  • the three-dimensional biological tissue has cross sectional dimensions of at least 100, 200 or 250 pm, and at most 2, 3 or 5 cm.
  • physiologically acceptable solvent composition is meant to indicate that the composition's components and solvent(s) do not degrade, react with, or form artefacts with the tissue, do not essentially alter the natural biological components and structure of the biological tissue to the extent that the introduction, diffusion and interaction with compounds of interest is impaired.
  • the lipids within the tissue can be at least partially removed not to hinder the movement of compounds of interest within the tissue.
  • the lipid removal can be achieved conventionally by adding a physiologically acceptable solvent composition to the biological tissue, wherein the solvent composition is suitable for dissolving lipids within the biological tissue, and the solvent composition stays in contact with the biological tissue for diffusion (A) of the solvent composition into the tissue and (B) of dissolved lipids to the solvent composition outside the biological tissue; removing the solvent composition and dissolved lipids from the outside surface of the biological tissue; and optionally repeating the addition and removal of the solvent composition for the removal of lipids from the biological tissue.
  • the time for sufficient lipid removal depends on natural diffusion, temperature, the nature of the composition and the frequency and number of additions and removals of the solvent composition.
  • the physiologically acceptable solvent composition comprises detergents that assist the dissolution of the lipids in the biological tissue.
  • a physiologically acceptable solvent composition is introduced to the biological tissue by one or more injections into the biological tissue, wherein the solvent composition comprises at least one charged reagent suitable for dissolving lipids within the biological tissue; the biological tissue is at least partially immersed in a suitable solvent composition and a pulsed electrical field (PEF) is applied to the biological tissue suitable for effecting movement of charged compounds and reagents within the biological tissue; the solvent composition and dissolved lipids are removed from the outside surface of the biological tissue; and optionally the solvent composition is introduced and removed repeatedly for the removal of lipids from the biological tissue.
  • PEF pulsed electrical field
  • This lipid clearing method is particularly suitable for dispersing injected charged detergents within the biological tissue and for removing the dispersed lipids, most of which are charged, by PEF-assisted diffusion.
  • a charged compound(s) of interest can be introduced into the tissue.
  • a charged compound(s) of interest can be introduced into the tissue.
  • a physiologically acceptable solvent composition is introduced to the biological tissue by one or more injections into the biological tissue, wherein the solvent composition comprises at least one charged compound of interest, optionally a targeted staining agent, for introduction into the biological tissue;
  • the biological tissue is at least partially immersed in a suitable or the solvent composition and a pulsed electrical field (PEF) is applied to the biological tissue suitable for effecting movement of the charged compounds of interest within the biological tissue
  • PEF pulsed electrical field
  • the method of the invention requires that either the clearing step and/or the compound introduction step, optionally staining step, is injection- and PEF-assisted.
  • a method with conventional lipid clearing but injection/PEF-assisted compound introduction is a method of the invention, the same as an injection/PEF-assisted lipid clearing and conventional diffusive compound introduction. The method is most effective when both, an injection/PEF-assisted lipid clearing and an injection/PEF-assisted compound introduction are combined.
  • Biological tissues have the tendency to degrade and/or decompose at higher or lower temperatures than those in the original living organism.
  • the method of the present invention is optionally applied to mammalian tissues, and the method parameters are optionally adjusted so that the biological tissue is maintained during any one of steps (i) to (iii) at a temperature of at most 40, 42 or 45 °C and/or at least 15, 20 or 25 °C.
  • the temperature is maintained for 0.25 to 72 or 1 to 36 hours of treatment.
  • the biological tissue can be rotated during the exposure to a static pulsed electrical field (PEF), and/or the electrodes for the pulsed electrical field (PEF) can be rotated around the biological tissue, and/or the surrounding fluid can be static and/or be pumped through the PEF treatment chamber.
  • PEF pulsed electrical field
  • the pumping of the surrounding fluid through the PEF treatment chamber can involve the application of a milli-fluidics system for the staining process.
  • the circulation of a liquid around the tissue can be achieved by a pump system featuring, for example,
  • adjustable pressure settings from 0 to 2 bar, optionally 0 to 6 bar, optionally 0 to 15 bar to ensure optimal interaction without causing damage, and/or
  • the milli-fluid system pumping the fluid can be optimized for a range of tissue types, for example, tissues including epithelial, connective, muscle, nerve, and lymphoid tissues, optionally by implementing interchangeable nozzle heads for different sizes and tissue types, programmable fluidic pathways for even distribution over irregular or dense tissues, and/or integrated sensors for real-time monitoring and automatic adjustments.
  • tissue types for example, tissues including epithelial, connective, muscle, nerve, and lymphoid tissues
  • interchangeable nozzle heads for different sizes and tissue types
  • programmable fluidic pathways for even distribution over irregular or dense tissues
  • integrated sensors for real-time monitoring and automatic adjustments.
  • the pumping may optionally comprise a feedback mechanism within the milli-fluidic system, optionally including sensors to detect the effectiveness of staining at various depths in the tissue, automatic adjustments based on real-time data, and/or a user interface for real-time feedback and necessary manual adjustments.
  • a feedback mechanism within the milli-fluidic system, optionally including sensors to detect the effectiveness of staining at various depths in the tissue, automatic adjustments based on real-time data, and/or a user interface for real-time feedback and necessary manual adjustments.
  • the method of the invention may optionally employ a matrix of needles for injections, improving the speed of introducing compounds into the biological tissues.
  • This matrix may optionally allow for multiple simultaneous injections for efficient and uniform distribution, customizable needle configurations based on tissue size, type, and density, and/or reduced tissue damage and increased penetration due to uniform distribution.
  • a needle array for use in the present invention may be integrated into the device delivery unit, providing features such as, for example, an adjustable configuration mechanism to change needle spacing and pattern, advanced control systems to adjust the injection depth and volume for each needle, and/or a feedback system that monitors injection effectiveness and adjusts parameters in real time.
  • a needle array for use in the present invention may feature a needle diameter of 50 nm to 100 pm, optionally 50 nm to 1 mm, a needle distance of 100 urn to 1 mm, optionally 100 pm to 5 mm, optionally 100 pm to 20 mm, and/or a material that has electrically conductive or non-conductive properties.
  • the PEF for practicing the method of the invention is characterized by a number of parameters.
  • the biological tissue is, for example, exposed to an electrical field characterized by one or more parameters selected from the group consisting of
  • step (c) a PEF exposure time (s) of 0.01 to 1000 or 0.1 to 500, optionally 0.1 to 50 for step iii, or 1 to 100 for step (iib);
  • a specific energy input (kJ/kg) of 1 to 100,000, optionally 10 to 10,000 or 50 to 600 for step (III), or 800 to 8,000 for step (lib);
  • a conductivity of the solvent composition for injection and/or at least partially immersing the biological tissue of 0.1 to 30 mS/cm, optionally 1 to 18 mS/cm,
  • composition temperatures during PEF exposure of 15 to 45, 30 to 45, or 37 to 40 °C.
  • the method of the invention is universally applicable to all natural biological tissues, for example, an isolated three-dimensional biological tissue of interest selected from the group consisting of epithelial, connective, muscle, nervous, lymphoid, brain, heart, liver, kidney, breast, lymph node, spleen, skin, prostate, bone, uterus and lung tissues; biologically tissues suspected of cancer, neurological disorders, microbial, bacterial, parasitic, fungal, yeast or viral infections, optionally selected from the group consisting of carcinomas, sarcomas, leukemia, lymphomas, Alzheimer's disease, acute spinal cord injury, amyotrophic lateral sclerosis, brain tumors, meningitis, multiple sclerosis, muscular dystrophy, Parkinson's disease, encephalitis, , and transmissible spongiform encephalopathies.
  • an isolated three-dimensional biological tissue of interest selected from the group consisting of epithelial, connective, muscle, nervous, lymphoid, brain, heart, liver, kidney, breast, lymph node,
  • the initial distribution of the physiologically acceptable solvent composition for dissolving and removing tissue lipids or the physiologically acceptable solvent composition for introducing at least one charged compound of interest into the biological tissue depends on the one or more injections into the biological tissue.
  • the composition's distribution within the tissue will depend on the injection volume, density, depth and the 3D injection distribution.
  • the one or more injections are optionally characterized by
  • a further optional embodiment for practicing the present invention but also an independent aspect is an injection system for injecting fluid, e.g. solvent compositions into biological tissues.
  • This system is characterized by a modular injection system comprising a spindle unit and a controlling unit that regulates the injection speed, position, depth and amount.
  • three-dimensional control of the injection points can be achieved by the flexible movement of a remodeled Prusa i3 MK2S 3D printer (Prusa research, Prague, Czechia).
  • a unique spindle and holder can be installed to allow the connection of a disposable syringe and precise control of the plunger position to an accuracy of 1 pl.
  • This arrangement and set up allows for precisely injecting a given volume of fluid at any point in the biological tissue.
  • the viscosity of the injected fluid can be varied and adopted to the particular injection fluid and optionally ranges 0.5 to 10 or 1 to 5 mPas when measured at a shear rate of 5 s 1 at 25°C.
  • This injection system can be used for practicing the present invention, i.e. with PEF- assisted dispersion, and it can also be used for injecting fluids of interest without subsequent active PEF-dispersion.
  • physiologically acceptable solvent composition for dissolving and removing lipids from the biological tissue is aqueous, physiologically acceptable, i.e. not harmful to the tissue or the purpose of the method, and dissolves tissue lipids.
  • physiologically acceptable solvent composition for dissolving and removing lipids can comprise (i) physiological acceptable buffer compounds, optionally selected from the group consisting of phosphate, Tris, Tricine, phosphate-buffered saline (PBS);
  • hydrogel-forming compounds or combinations thereof optionally selected from the group consisting of acrylamide and bisacrylamide, polyethylene glycol (PEG), hyperbranched polyglycerols
  • polymerization initiators for hydrogel-forming compounds optionally selected from the group consisting of azo initiators (e.g. VA-044), thermal initiators, light initiators, radical initiators;
  • protein-stabilizing and/or fixating compounds optionally selected from the group consisting of paraformaldehyde (PFA), formaldehyde, formalin;
  • detergents optionally selected from the group consisting of sodium dodecyl sulfate (SDS), , Tween 20, Triton X-100, urea, lipase, DMSO, xylene, dichloromethane (DCM) , dibenzylether, Tween 80, deoxycholate, cholate, sarkosyl, Decylmaltoside (DM), n-dodecyl-0- D-maltoside (DDM), digitonin, Fos-cholines, HEGA-10, CHAPS, n-Nonyl P-D-l-thiomalto- pyranoside (NTM), Cymal-5, N,N-Dimethyl-n-dodecylamine N-oxide (LDAO), Octyltetraglykol (C8E4), Octaethylene glycol monododecyl ether (C12E8), Octyl-beta-Glucoside (OG);
  • SDS sodium do
  • tissue pore forming and pore widening compounds optionally selected from the group consisting of boric acid, sodium citrate, tris-tricine;
  • reactive index matching compounds or compositions optionally selected from the group consisting of Histodenz, glycerol, fructose, sucrose, sorbitol, xylitol, polyethylene glycol, iohexol, iodixanol, diatrizoic acid, antipyrine, N-methylnicotinamide, nicotinamide, N-methyl- d-glucamine, DMSO, TDE, phosphoric acid, ethers esters, methyl salicylate, benzyl alcoholbenzyl benzoate (1:2, BABB), dibenzyl ether (DBE), diphenyl ether (DPE), ethyl-3-phenylprop- 2-enoate (ECi) PEGMMA500.
  • reactive index matching compounds or compositions optionally selected from the group consisting of Histodenz, glycerol, fructose, sucrose, sorbitol, xylitol, polyethylene glycol, iohex
  • Exemplary but non-limiting physiologically acceptable solvent compositions for dissolving and removing lipids from biological tissues are selected from the group consisting of: SDS, Triton- X100, and Tween20; TritonXIOO, Tween 20, methanol, DCM, and DBE; SDS, TritonXIOO, Tween- 20, and Lipase; SDS, methanol, DCM, and DBE; SDS, Triton X, Tween20, methanol, DCM, and DBE; SDS, TritonX, urea, Tween 20, methanol, DCM, and DBE; and SDS, TritonX, Tween20, DCM, and DBE.
  • the charged compound(s) of interest in the physiologically acceptable solvent composition for introduction of these by injection and subsequent PEF exposure are soluble in the aqueous composition and can be selected, for example, from the group consisting of charged compounds for targeting, binding and/or detecting cellular components, optionally binding and/or detecting nucleotide-, amino acid-, or sugar- based cellular components.
  • the charged compounds can be selected from lectins, nucleotide sequences, nucleotide sequence derivatives, antibodies, antibody fragments, antibody derivatives, and antibody like binding proteins.
  • Antibodies, antibody fragments, antibody derivatives and antibody-like binding proteins for practicing the present invention may be selected from Fab fragments, Fab2 fragments, miniantibodies (also called small immune proteins), tandem scFv-scFv fusions as well as scFv fusions with suitable domains (e.g. with the Fc portion of an immunoglobulin), polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, Fv- fragments, Fab-fragments and Fab2-fragments and antibody-like binding proteins, e.g. affilines, anticalines and aptamers.
  • the charged compounds may be selected from any research-, clinically and diagnostically relevant antibody, antibody fragment or antibody derivative, or other targetspecific charged compound, selected from the group consisting of: (Cancer:) anti-BRAF, anti- BRD4, anti-CD molecules (example CD45, CD1, CD5, CD20, CD38 etc), anti- BCL6 / B-cell lymphoma 6 protein, anti-beta catenin, anti- cytokeratins (example: cytokeratins 8, cytokeratin 18 etc), anti-E-Cadherins, anti- Ep-Cam Epithelial Antigen, anti-Cyclin DI, anti-c-Fos, anti-EGFR, anti-40 kd PAN Carcinoma, anti- melanoma Black / Pmell7, anti-prostate specific antigen, a nti-Ki67, anti- BAF47, anti-pancreatic lipase, anti- T Cell Leukemia/Lymphomal, anti
  • Neurodegeneration anti-Beta Amyloids, anti-alpha-Synuclein, anti-claudins, anti-GFAP, anti-LAMP, anti-GPCRs, anti-NeuN, anti-Neurotensin Receptors, anti-Neurofilament Protein, anti-NECAB2, anti-Phospho-GSK-3beta, anti-somatostatin, anti- Synaptophysin.
  • the charged compound of interest for introduction into the biological tissue can be dissolved in a physiologically acceptable aqueous solvent composition, for example, compositions selected from the group consisting of: tris-tricine buffer (TTB) and low melting agarose (LMA); PBS and LMA; Tween-20, PBS (PBST20) and LMA; PBS, Heparin (PBSH) and LMA; PBST, heparin (PBSTH) and LMA; TTB, PBS, PBST20, PBSTH; and may optionally comprise further components, e.g. citrate, formic acid.
  • TTB tris-tricine buffer
  • LMA low melting agarose
  • PBS and LMA Tween-20, PBS (PBST20) and LMA
  • PBS Heparin
  • PBSH Heparin
  • PBSTH heparin
  • TTB PBS, PBST20, PBSTH
  • further components e.g. citrate, formic acid.
  • the present invention relates to a device for practicing the method of the invention, i.e. a device combining at least means for presenting a three-dimensional tissue of interest immersed in a solution, means for injecting a solution into the tissue, and one or more electrodes for applying a PEF to the tissue.
  • the device for the introduction or removal of charged compounds from isolated biological tissue comprises: a) a delivery unit configured to inject a solvent composition into biological tissue; b) a tissue processing unit including at least one electrode configured to apply a pulsed electric field (PEF) to the biological tissue for the dispersion of charged compounds within the biological tissue; c) a controller for controlling the delivery of the solvent composition into the biological tissue and/or for the processing of the biological tissue with the electrical field such that a temperature of the biological tissue during exposure to the electrical field remains below a temperature threshold.
  • PEF pulsed electric field
  • the delivery unit is configured to provide a three-dimensional injection pattern of the injected solvent composition within the biological tissue.
  • the biological tissue is contained at least partially immersed in a solvent composition.
  • the tissue processing unit of the device may comprise at least two, three, four or more electrodes.
  • the device may further comprise means for adding and removing a solvent composition from the biological tissue.
  • the device may optionally further comprise means for controlling the temperature of the biological tissue lower than 45, 42 or 40 °C.
  • the device may further comprise means for rotating the biological tissue and/or the electrodes for the pulsed electrical field (PEF).
  • PEF pulsed electrical field
  • the present invention is directed to the use of a method according to the invention or a device of the invention as taught herein for histological staining, optionally for the histological staining of isolated tissues suspected of comprising mutated, infected or cancer cell.
  • the method is used for identifying cancer cells in an isolated tissue of interest.
  • Another aspect of the invention pertains to a method for the identification of a disease, for example, forming part of a diagnostic method, optionally for identifying cancer, a neurological disease or an infection, comprising the steps of
  • tissue sample of interest from a mammal suspected of suffering from a disease, wherein the disease alters said tissue, optionally from a mammal suffering from cancer, a neurological disease or an infection;
  • physiologically acceptable solvent compositions are injected three- dimensionally into said tissue
  • the solvent composition for the removal of lipids comprises at least one charged reagent suitable for dissolving lipids within the biological tissue
  • the solvent composition for introducing detectable disease-specific marker compounds comprises at least one charged detectable disease-specific marker compound
  • PEF pulsed electrical field
  • the motor of the rotating chamber was powered by a 9 V battery.
  • the PC was connected by way of a driver module to the motor, (c) a continuous PEF treatment chamber, allowing the continuous passage of a fluid.
  • the pulse repetition frequency of the bipolar pulse was set to 40 Hz.
  • the pulse repetition frequency of the bipolar pulse was set to 35 Hz and for the square wave pulse to 1.5 Hz.
  • the pulses shown here were applied in the static treatment chamber setup.
  • Fig. 4 are three graphs showing time-temperature profiles and specific energy input for three PEF treatments of mouse tissue,
  • Fig. 5 shows pictures of cross sections of the imaged mouse tissue (brain halves) labelled with fluorescent anti-histone H3 antibodies
  • Bipolar exponential decay pulses were used for all three treatments.
  • CLAHE Contrast Limited Adaptive Histogram Equalization
  • Rbulk bulk resistor
  • CDL double layer capacitor
  • WD Warburg element
  • Fig. 7 shows a typical time-temperature profile for a 134 V cm' 1 , 7.5 ps, 40 Hz, 75001240 kJ kg’ 1 .
  • the gap between 148 min and 157 min reflects the manual buffer change.
  • the dashed line represents the upper temperature limit, above which the tissue is expected to suffer damages.
  • Fig. 9 shows and compares different staining (lectin for blood vessel detection) procedures of murine tissue samples: (a) injection only using 5 injection points, (b) passive diffusion during 16 h, (c) PEF treatment (exponential decay pulses, 909 V cm' 1 ), and (c) the combination of an injection step and a PEF treatment.
  • Fig. 10 shows a representative image of a digital section obtained from a whole cleared cerebellum and brain stem of a mouse brain stained with anti-histone-3 antibodies diluted in 0.1% LMA in IX PBS with a 1:500 ratio.
  • the injection spots spaced 0.5 mm apart from each other in x, y and 0.3 mm apart in z. The injected amount was 10 pl per spot.
  • Table 1 Composition of the solutions used for the spatial imaging workflow.
  • PFA paraformaldehyde
  • SDS sodium dodecyl sulfate
  • VA-044 2,2-azobis[2-(2-imidazolin-2- yl)propane]dihydrochloride).
  • mice were either non-transgenic (C57BL/6J, The Jackson Laboratory, Bar Harbour ME, US) or transgenic (B6.Cg-Tg(APPswe,PSENldE9)85Dbo/Mmjax, The Jackson Laboratory, Bar Harbour ME, US).
  • the age of the mice used in the trials ranged from 6 to 24 months.
  • the human lymph node used as a model tissue for clearing was extracted from a patient undergoing an autopsy in strict compliance with the rules and regulations of the Ethics Committee of the canton of Zurich.
  • the pulsed electric field treatment chamber was connected to a RUP6-15CL pulse generator (GBS Elektronik GmbH, Radeberg, Germany).
  • the pulse generator was coupled to an external trigger (15 MHz FG300, Yokogawa Electric, Musashino, Tokyo, Japan) to set the pulse duration and pulse repetition frequency.
  • the treatment time t t solely considers the exposure time of fluid or tissue elements to the electrical pulses and is calculated by multiplying the residence time t r by the pulse repetition frequency f and the pulse length T P (Eq. 1).
  • tt t r - f - T p Eq. 1
  • the bipolar exponential decay pulses were characterized using a current monitor (Model 110, Pearson Electronics Inc., Palo Alto, CA, USA) and a voltage probe (P6015A, Tektronix Inc., Beaverton, OR, USA).
  • the signals from the voltage probe and current monitor were visualized by an oscilloscope (Wave Surfer 10, Teledyne LeCroy GmbH, Heidelberg, Germany).
  • the temperature increase (ohmic heating) in the treatment chamber was quantified using a fiber optic sensor (TS4, Weidmann Technologies GmbH, Dresden, Germany).
  • the static treatment chamber (shell and meshed cage) was 3D printed (Prusa research, model i3 MK3S, Prague, Czechia), using polyethylene terephthalate glycol.
  • the maximum total treatment chamber volume without the cage and without the sample (Vmax) was 10 mL.
  • the parallel plate stainless steel electrodes had a height of 4.5 cm, a width of 3.0 cm and an electrode distance of 1.1 cm.
  • the temperature sensor could not be fixed to a specific position.
  • the shell of the treatment chamber and the connecting parts were 3D printed (Crealty, Ender 3 V2, Shenzhen, China), using polylactic acid.
  • the cage was 3D printed (Prusa Research, SL1, Prague, Czechia) from resin tough (Prusa research, Prague, Czechia).
  • the maximum total treatment chamber volume without the cage and without the sample (Vmax) was 12 mL.
  • the parallel plate stainless steel electrodes had a height of 4.3 cm, a width of 1.2 cm and an electrode distance of 1.9 cm. A small hole was drilled in the corner of the shell to hold the temperature sensor in place.
  • a high-geared DC motor (Comvec, N20, Taucha, Germany), a DC motor driver with a pulse width modulation control (Sinotech Mixic Electronics, MX1508, Guangdong, China), a single-board microcontroller (Arduino, Uno Rev3, Turin, Italy), and a 9 V battery were used.
  • the motor was operated in the stop-and-go mode with short impulses.
  • the speed of rotation was set by the respective delay time.
  • the delay time was set to 300 ms (6 rpm) and for the human tissue, to 600 ms (13 rpm).
  • a static and a rotating treatment chamber are shown in Fig. 1 (a) and (b), respectively.
  • the underlying protocol used for the clearing and staining pipeline was the Clear Lipid- exchanged Acrylamide-hybridized Rigid Imaging/lmmunostaining/lnsitu hybridization-compatible Tissue-hYdrogel (CLARITY) method (Chung et al., Nature 497, 332-337).
  • CLARITY Clear Lipid- exchanged Acrylamide-hybridized Rigid Imaging/lmmunostaining/lnsitu hybridization-compatible Tissue-hYdrogel
  • mice were deeply anaesthetized with ketamine and xylazine. Chilled PBS and the hydrogel premix were used for the transcardial perfusion of the mice.
  • the mouse brains were then extracted and incubated in the hydrogel premix at 4°C. After 24 h, nitrogen was used to degas and purge the tissue hydrogel. The temperature was increased to 37°C for 2.5 h to initiate the hydrogel polymerization. The polymerized hydrogel was removed from the gel and placed into the clearing solution until further use.
  • the human lymph node was immersed in the hydrogel premix overnight at 4°C.
  • the steps after the fixation in the hydrogel premix were the same as for the mouse tissue: (i) degassed and purged with nitrogen, (ii) hydrogel polymerization at 37°C for 2.5 h, (iii) removal from gel and storage in clearing solution.
  • the mouse brain is a relatively loose tissue, especially in comparison to human lymph nodes. Therefore, active and passive clearing result in a cleared tissue. Active clearing in a chamber filled with clearing solution and an applied DC voltage of 10-60 V reached a cleared tissue within 2 days. The temperature of the clearing solution was kept between 37°C and 50°C. During the whole clearing process, fresh clearing solution was used to replace spent liquid. Passive clearing, where the samples were immersed in a plastic tube, reached a cleared sample within 20 days at 39°C. The clearing solution was replaced regularly, and an incubator shaker was used continuously.
  • the lymph node (m 54 mg) was immersed in 9 mL of clearing solution and treated by PEF in the rotating treatment geometry.
  • the clearing solution was manually exchanged every 2-4 h.
  • a bipolar exponential decay pulse was applied to reduce electrochemical effects at the stainless steel electrodes and to further increase the randomness of the migration direction (see Fig. 3).
  • the pulse length for one pulse was 7.5 ps, according to the definition, where the pulse length is defined as the time needed to decrease the voltage by 37% of its peak value (Raso et al., Innov. Food Sci. Emerg. Technol. 37, 312-321.).
  • the time between the onset of the pulses was 64 ps.
  • the pulse 99 repetition frequency of the bipolar pulse was set to 40 Hz.
  • PEF treatment pauses i.e., overnight
  • the sample was stored in the clearing solution at 37°C. Tissue staining of mouse tissue
  • the injection apparatus consisted of a modified 3D printer (Prusa research, model i3 MK2S, Prague, Czechia).
  • the movable nozzle (x- and z direction) of the 3D printer was replaced by a spindle machine to regulate the injection speed of a 1 mL syringe equipped with a G26 needle (ONCE Medical, Muang Samutsakhon, Thailand).
  • Half or parts of a mouse brain or human lymph nodes were fixed to a movable (y-direction) bottom plate.
  • the injection solution was composed of a low melting point agarose in TTB or PBS (concentration of agarose: 0.1%) and antibodies. Low melting point agarose reduces the backflow of the staining molecules during injection.
  • the commands for the injection system were written in GCode, which was created as a text file, following the same logic as a 3D printer.
  • the injection method facilitated precise control of various injection points. Different injection patterns were used to analyze the effects of injection amount and placement. Testing required writing codes for a wide range of injection patterns, from single-line 1x5x1 injections (X, Y, and Z) to patterns intended to cover the entire sample. The injection sites spaced 0.5 or 1 mm apart. Depending on the sample size, different injection patterns were used for staining the samples; these ranged approximately from 4x4x4 to 10x10x10 injection sites. Users can first choose the injection pattern they wish to create. The user can then define the number of steps in each direction and the step size for each axis. The result should be a cube that fits the sample to be injected.
  • a method of automatic injection of dye solution followed by application of a pulsed electric field (PEF) to distribute the molecules was applied.
  • a mixture of low melting point agarose, TTB, and fluorescent staining molecules was injected in a 8x6x5 (x, y, z) matrix (Fig. 5 and Fig.9).
  • the injection locations were 1 mm apart and each injection channel was filled with 20 pL of solution.
  • the needles used was a 26G.
  • a whole cleared cerebellum and brain stem of a mouse brain were injected with an anti-histone-3 antibody diluted in 0.1% LMA in IX PBS with a 1:500 ratio.
  • the sample was 5x4x3 mm in x,y,z and the used injection pattern was 10x8x6 mm.
  • the injection spots spaced 0.5 mm apart from each other in x, y and 0.3 mm apart in z.
  • the injected amount was 10 pl per spot.
  • the mixture of low melting point agarose, PBS, and antibody was injected in an 8x8x8 (x, y, z) matrix.
  • the injection locations were 0.5 mm apart and each injection channel was filled with 10 pL of solution.
  • the used needles were a 33G. This procedure was done for both primary and secondary antibody staining with extensive washing in PBST following both steps. After staining and washing, the lymph node samples were subjected to total clarification with organic solvents and evaluated for staining efficacy.
  • a mouse tissue sample was immersed in TTB.
  • the volume of TTB corresponded to 4.5 mL, in the rotating setup, the volume was measured to be 9 mL.
  • a bipolar exponential decay pulse was applied to the sample (see Fig. 3a, b).
  • the pulse length for one pulse was 18.1 ps
  • the time between the onset of the pulses was 64 ps
  • the pulse repetition frequency of the bipolar pulse was set to 35 Hz.
  • Tissue imaging Unbound antibodies were removed, i.e., washed out, by placing the stained tissue in fresh TTB for 1 h.
  • the sample was immersed in Rl matching solution overnight at 4°C.
  • mice All samples were imaged with mesoSPIM (Voigt et al., 2019, Nat. Methods 16, 1105-1108).
  • mesoSPIM mesoSPIM
  • the excitation laser was set to 647 nm with a 488-561-640 nm triple block filter.
  • human tissue unstained, autofluorescence
  • the excitation laser was set to 488 nm.
  • the lateral resolution of the imaging was 3.26 x 3.26 pm and the axial resolution was 3.00 pm. Impedance spectroscopy of mouse tissue
  • the impedance Z (Q) generalizes the concept of the resistance by additionally describing the storage of electrical energy.
  • the impedance therefore not only describes the resistance R (Q), but also the inductive reactance XL (Q) and the capacitive reactance XC (Q) (Eq. 2).
  • Electrochemical impedance spectroscopy is a powerful tool to characterize a material or a device by monitoring the frequency response. AC is applied for a range of frequencies. The changes in amplitude and phase are measured and the resulting data plotted in Bode and/or Nyquist plots.
  • Software was set automatically and ranged from 1 mV to 10 V. Each sample was measured three
  • the mechanical injection method of the present invention effectively stained mouse brain tissue and dense human tissue, thereby reducing the sample preparation time compared with classical processes such as passive diffusion.
  • the multiple dot matrix injection method alone could not perform complete coverage and staining of whole mouse hemisphere and lymph nodes.
  • full staining by only injection was achieved for a smaller mouse sample, such as the cerebellum and brain stem part (Fig. 5, Fig. 9, Fig 10). It was demonstrated that, under certain conditions (e.g. better tissue porosity or lower tissue density or smaller sample size), injection staining by itself, can yield acceptable results (Fig.
  • the electric current increased from 9.0 A to 12.2 A (Ssl: static chamber, 790 V cm' 1 , 18.1 ps, 15 Hz, 375 ⁇ 12 kJ kg -1 ), from 5.2 A to 6.7 A (Ss2: static chamber, 405 V cm' 1 , 18.1 ps, 35 Hz, 596 ⁇ 19 kJ kg 1 ), and from 4.2 A to 4.6 A (Sri: rotating chamber, 203 V cm 29.9 ps, 35 Hz, 301 ⁇ 10 kJ kg 1 ).
  • the energy inputs resulting from the applied pulses during the time of process increased the temperature of the fluid by around 16°C (see Fig. 4).
  • a preheating phase can be implemented to avoid the time-loss until higher temperatures are reached (pre-heating phase) and/or implementing a continuous system from a preheated fluid pool, allowing the application of higher energy inputs and thus, a faster time of process (see Fig. 1, Fig. 4).
  • the pulsed electric field (bipolar or unipolar) can be continuously or incrementally rotated to increase the randomness of the distribution.
  • the mouse tissue samples were imaged.
  • Image processing approaches were tested to account for overexposed regions, thereby increasing the image quality (see Fig. 5).
  • computer vision can be used for the image processing.
  • Ssl was treated for 25 min. Less dense tissue regions were well stained, while more dense regions, in particular the hippocampus, remained unstained (see Fig.5a). Blurriness (before image processing) might be related to local damages triggered by the high voltages (Servant, A., et al., Journal of Materials Chemistry B, 2013. 1(36): p. 4593-4600). The voltage was therefore lowered for Ss2.
  • the pulse repetition frequency was increased from 15 Hz to 35 Hz to (i) ensure a similar timetemperature profile and, in addition to prolonging the time of process, (ii) to increase the ps PEF treatment time (Eq. 1) from 0.82 s to 4.56 s.
  • This parameter combination resulted in clearly distinguishable stained regions (white spots), including dense parts, such as the hippocampus (Fig. 5b).
  • the electrode distance was increased from 1.1 cm to 1.9 cm, thus decreasing the electric field from 405 V cm 1 to 203 V cm 1 for a similar voltage.
  • the ps PEF treatment time and the electric field intensity had the larger effects on the migration speed and randomness of the antibodies. Due to the altered treatment chamber, the pulse shape changed slightly, thereby increasing the psPEF treatment time (Eq. 1) from 4.56 s to 7.53 s.
  • Electrochemical impedance spectrometry (EIS) measurements showed a slight increase of the magnitude of impedance at higher frequencies after PEF treatments in comparison to the control (Fig. 6). However, this increase was detected for all applied treatments, including purely thermal treated samples. Based on these results, the observed differences might arise from minimal changes of the hydrogel structure or altered compound properties.
  • the hydrogel-embedded mouse tissue was modelled fairly accurately by a simplified Randles circuit, consisting of a bulk resistor Rbuik and a barrier response containing a double layer capacitor CDL and a Warburg element WD (HaGerstrom, H., et al., Journal of pharmaceutical sciences, 2003. 92(9): p. 1869- 1881) (Fig. 6b, c).
  • Rbuik reflects the resistance of the hydrogel (plateau in the Bode plot at high frequencies) and the barrier response represents the processes at the electrode/hydrogel interface, i.e., participation in electrolysis reactions or depositing at the interface (HaGerstrom, H., et al., Journal of pharmaceutical sciences, 2003. 92(9): p. 1869-1881; Lvovich, V. F., John Wiley & Sons, 2012. p. 1-112).
  • the Warburg element is connected in series with a charge transfer resistor RCT, the latter representing the resistance one electron has to overcome to be transferred to another particle (Lvovich, V. F., John Wiley & Sons, 2012. p. 1- 112).
  • the PEF clearing method was similar to the PEF staining approach. However, due to the higher conductivity of the clearing solution (14.1 mS cm 1 ) in comparison to TTB (1.4 mS cm' 1 ), the PEF parameters had to be adjusted to minimize ohmic heating effects, leading to tissue damage. Mainly, the electric field intensity and exponential decay pulse length were reduced by approximately a factor of 2 and 4, respectively. The temperature plateaued between 38°C and 39°C (Fig. 7).
  • a preheating phase can be implemented to avoid the time-loss until higher temperatures are reached (pre-heating phase) and/or a continuous system can be implemented from a preheated fluid pool, allowing the application of higher energy inputs and, thus, a faster time of process (see Fig. 1).
  • the pulsed electric field (bipolar or unipolar) can be continuously or incrementally rotated to increase the randomness of the distribution.
  • a continuous system replaces spent fluid, thereby enabling a constant temperature plateau throughout the treatment and potentially allowing a higher pulse repetition frequency, which will reduce the required time of process.
  • a treatment time Eq.
  • the applied parameters resulted in a cleared lymph node, showing a homogeneous autofluorescence throughout the tissue (Fig. 8).
  • the energy input at approximately 75001240 kJ kg’ 1 targeting the electrophoretic mobility of surfactants, was rather high, especially in comparison with more traditional PEF applications in the food and biobased industry (around 200 kJ kg 1 or lower targeting electropermeabilization) (Toepfl et al., Food Rev. Int. 22, 405-423, 2006.).
  • this treatment resulted in a cleared human lymph node in a relatively short treatment time span, especially in comparison to other methods (e.g., passive clearing).
  • other PEF parameter combinations from the expected working range, surfactants, solutions, and chemicals can be used to achieve similar results.
  • a rapid staining protocol with charged staining compounds e.g., lectin and anti-histone H3 antibodies
  • a rapid clearing protocol for, but not limited to, a dense human tissue was developed by implementing an injection device and pulsed electric field (PEF) technology.
  • charged staining compounds e.g., lectin and anti-histone H3 antibodies

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Abstract

La présente invention concerne un procédé d'introduction de composés chargés dans des tissus biologiques tridimensionnels comprenant l'étape d'introduction d'au moins un composé d'intérêt chargé dans le tissu biologique, une composition pour dissoudre et éliminer des lipides et/ou une composition pour introduire au moins un composé d'intérêt chargé dans le tissu biologique étant injectée dans le tissu biologique et un champ électrique pulsé (PEF) étant appliqué au tissu biologique. L'invention concerne en outre un dispositif destiné à être utilisé dans ce procédé, et une utilisation de ce procédé ou de ce dispositif pour la coloration histologique.
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Citations (3)

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WO2000002621A1 (fr) * 1998-07-13 2000-01-20 Genetronics, Inc. Therapie genique par champ electrique pulse visant la peau et les muscles
WO2018005511A1 (fr) * 2016-06-27 2018-01-04 Gala Therapeutics, Inc. Générateur et cathéter pourvu d'une électrode et procédé pour traiter une voie pulmonaire
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