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WO2023060199A1 - Méthode et système intégré pour l'élimination sélective de cellules défectueuses et de fluides œdémateux à partir du poumon - Google Patents

Méthode et système intégré pour l'élimination sélective de cellules défectueuses et de fluides œdémateux à partir du poumon Download PDF

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Publication number
WO2023060199A1
WO2023060199A1 PCT/US2022/077701 US2022077701W WO2023060199A1 WO 2023060199 A1 WO2023060199 A1 WO 2023060199A1 US 2022077701 W US2022077701 W US 2022077701W WO 2023060199 A1 WO2023060199 A1 WO 2023060199A1
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WIPO (PCT)
Prior art keywords
target tissue
cells
acoustic waves
low intensity
tissue
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Ceased
Application number
PCT/US2022/077701
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English (en)
Inventor
Jinho Kim
Gordana Vunjak-Novakovic
John D. O'NEILL
Meghan Pinezich
Seyed Mohammad MIR
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Columbia University in the City of New York
Stevens Institute of Technology
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Columbia University in the City of New York
Stevens Institute of Technology
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Publication of WO2023060199A1 publication Critical patent/WO2023060199A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B2017/00969—Surgical instruments, devices or methods used for transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0004—Applications of ultrasound therapy
    • A61N2007/0017—Wound healing

Definitions

  • the disclosed subject matter relates to a method and integrated system for treatment of tissue by administration of biochemical agents and application of low intensity acoustic forces.
  • ARDS acute respiratory distress syndrome
  • a method of treating tissue includes applying a biochemical agent to a surface of a target tissue and applying low intensity focused acoustic waves from an acoustic energy source toward the target tissue.
  • the method selectively dissociates and detaches injured epithelial cells, removes of pathological tissue, and/or clears alveolar spaces filled with edematous fluids and apoptotic cell debris, while leaving intact healthy cells and minimal to no damage to surrounding tissue and cells.
  • the acoustic source is low intensity and has a frequency less than about 2 kHz.
  • the acoustic energy can be in the form of a pulse wave (LIP AW).
  • the output in the pulse wave may be between about 40 Hz, 100 Hz, or 1,000 Hz for example, but not limitation.
  • the acoustic waves can be externally applied to the target tissue.
  • the acoustic waves generated have a peak positive pressure at the target tissue of at about 3kPa and a frequency of about 2 kHz or less.
  • Pulsing low intensity focused acoustic waves comprises pulsing low intensity focused acoustic waves such that each pulse has a duration of 0.1- 100 ms or longer.
  • the biochemical agent for example is an enzyme, such as trypsin.
  • Other biochemical agents are also suitable.
  • surfactants or acid and base solutions may be suitable.
  • the target tissue can be an ex vivo organ, and detaching the cells of the target tissue may comprise moving the acoustic source across the surface of the organ to detach to create an ex vivo organ suitable for transplantation into a patient in need thereof.
  • the ex vivo organ is a lung from a lung donor.
  • the target tissue may be an airway of a subject.
  • the method and system described herein may be used to treat the airway of a subject’s lung, by removing edematous fluids.
  • the embodiments herein can be used to treat acute lung injury, for example, acute respiratory distress system.
  • a method of treating tissue including, applying a biochemical agent to a surface of a target tissue to form a portion of treated cells having cellular attachments at least partially disrupted by the application of the biochemical agent; and pulsing low intensity focused acoustic waves from an acoustic energy source toward the surface of the target tissue, such that the low intensity focused acoustic waves are pulsed at a frequency and amplitude that does not induce boiling or cavitation of the target tissue, thereby further disrupting and detaching only the portion of treated cells having cellular attachments at least partially disrupted by the application of the biochemical agent, leaving the untreated cells intact.
  • FIGURE 1 is a block diagram illustrating a method of disrupting and removing defective cells, tissues and edematous fluids from the lung with no to minimal damage to surrounding normal lung cells and tissue in accordance with an embodiment of the present technology.
  • FIGURE 2 is a simplified schematic view of an imaging-enable bioreactor platform in accordance with an embodiment of the present technology.
  • FIGURE 3 illustrates a procedure for de-epithelialization of tissue of a subject
  • FIGURE 4(a) is a photograph and FIGURE 4(b) is schematic diagram illustrating an imaging probe being used for visual inspection of in vitro-cultured tissue.
  • FIGURE 5 illustrates bright-field images of the interior of the tissue before carboxyfluorescein succinimidyl ester (CFSE) labeling of the epithelial layer.
  • CFSE carboxyfluorescein succinimidyl ester
  • FIGURE 6 illustrates fluorescence images of the interior of the tissue before CFSE labeling of the epithelial layer.
  • FIGURE 7 illustrates fluorescence images of native and (ii) de-epithelialized (Deepi) rat trachea lumen that was labelled with CFSE of the epithelium.
  • FIGURE 8 illustrates fluorescence images of de-epithelialized (De-epi) rat trachea lumen that was labelled with CFSE of the epithelium.
  • FIGURE 9 illustrates the H&E histologic analysis of native and de-epithelialized rat tracheas with 2% SDS.
  • FIGURE 10 illustrates immunostaining images of epithelial cell adhesion molecule (EpCAM) (left) and laminin (right) showing removal of the tracheal epithelium and preservation of ECM components within the rat trachea native and treated with 2% SDS.
  • EpCAM epithelial cell adhesion molecule
  • FIGURE 11 illustrates SEM images showing the luminal surface of the ex vivo rat tracheas native and de-epithelialized rat tracheas with 2% SDS.
  • FIGURE 12 illustrates Fluorescence images of CFSE-labelled MSCs being cultured on the tissue surface over the course of 7 days, day 1 (top) and day 7 (bottom).
  • the disclosed subject matter generally relates to methods and integrated systems for selectively dissociating and detaching injured epithelial cells, removing of pathological tissue, and/or clearing alveolar spaces filled with edematous fluids and apoptotic cell debris, with high precision such that healthy lung cells and lung tissue remain intact and undamaged.
  • a small volume of liquid bolus i.e., about 100 to 300 microliter, containing biochemical agent, such as an enzyme, is locally infused and deposited directly onto the surface of the target tissue of the lung, for example the airway.
  • biochemical agent such as an enzyme
  • Suitable biochemical agents include surfactants, acids and bases, or enzymes.
  • the suitable biochemical agents may include: sodium dodecyl sulfate, Triton X, sodium deoxycholate, CHAPS, peracetic acid, EDTA, trypsin, deoxyribunuclease (DNase), ribonuclease RNase).
  • the enzymatically treated cells are washed out from the lung through airway lavage that generates shear flow.
  • the clearing of cells from the distal lung regions can be challenging due to their small diameters of the airways, typically less than 500 micrometers, in the regions.
  • Application of low intensity focused acoustic therapy to selected lung regions induces mechanical agitation sufficient to detach and remove only the enzymatically disrupted cells, while maintaining intact the healthy cells leaving them undamaged or only minimally damaged from the treatment.
  • This targeted approach combines externally applied low intensity focused acoustic waves with localized airway lavage to clear edematous fluid from the airway with no to minimal tissue damage.
  • the application of low intensity focused acoustic energy is advantageous over application of high intensity acoustic waves, which often results in boiling the cells and tissue, leaving the method less precise and damaging to surrounding healthy cells and tissue.
  • the disclosed method can reduce the duration of mechanical ventilation and increases the chances of survival among patient with severe edema and ARDS.
  • the present method can treat acute lung injury and in particular ARDS.
  • the method can improve lungs offered by organ donors to an acceptable condition for transplantation into a patient in need of a lung transplant.
  • the enzymatic and low intensity focused acoustic treatment can be used to decellularize a tissue mass to form a scaffold that can later be used for regenerative medicine and/or other applications.
  • the low intensity focused acoustic system is automated to ensure homogenous application of acoustic energy throughout the desired tissue regions for the purpose of acoustic cell disruption and/or acoustics enabled removal of edematous fluid.
  • the acoustic source is an acoustic transducer that emits low levels of acoustic energy, i.e., sound waves, toward desired target tissue region.
  • the acoustic transducer modulates both acoustic intensity and region of treatment to reduce off-target tissue damage.
  • the acoustic transducer can be operatively engaged to a computer-controlled motorized system combined with 3D scanning technology to allow superior precision and control of the movement of the acoustic transducer over the surface of the lung.
  • the disrupted cells and/or tissue is selective and precise such enabling the treatment to result in no to minimal damage to healthy cells and tissue. This precision cannot be attained through application of high intensity focused applications.
  • the acoustic transducer in some embodiments, is operatively engaged to a function generator and, optionally, an amplifier.
  • the acoustic source is low intensity, and has a frequency less than about 2 kHz (2000 Hz). For example, 0.2-2 kHz, and/or values in-between.
  • the acoustic energy can be output in the mode of a pulse wave, for example, is low intensity pulsed acoustic wave (LIP AW).
  • LIP AW low intensity pulsed acoustic wave
  • output in the mode of pulse wave 40 Hz, 100 Hz and 1,000 Hz
  • acoustic wave of intensity is 0.03 W/cm 2 (or 30 mW/cm 2 ), a pulse ratio 1:2 with the acoustic wave frequency of 40 Hz.
  • the low intensity acoustic energy is applied in a way to avoid unstable cavitation and necrosis typical of high intensity focused ultrasound (HIFU).
  • the low intensity acoustic system can emit a pulsing protocol to mechanically disrupt target lung tissue and cells.
  • an acoustic transducer can propagates millisecond-long bursts of non-linear low intensity waves toward the target region of the lung tissue. This results in agitation of tissue and cells at the target region that dissociates and detaches defective tissue and cells selectively.
  • a function generator can initiate a pulsing protocol to generate acoustic waves with peak amplitudes of about 3 kPa at the target tissue.
  • the wave amplitudes may be 2.9 kPa, 2 kPa, 1 kPa, 0.1 kPa, 0.01 kPa, 0.001 kPa, and/or any values between 3 kPa and 0.001 kPa.
  • FIGURE l is a block diagram illustrating a method 100 of disrupting and detaching defective cells in accordance with an embodiment of the present technology.
  • the method 100 is implemented with suitable low intensity focused acoustic systems (e.g., LIP AW) and can be performed on a selected tissue in vivo or ex vivo.
  • the method 100 can include administration of a microliter volume of biochemical agent solution 101 to the surface of the target airway tissue.
  • the administration of the biochemical agent solution can result in forming a portion of treated cells having their cellular connections at least partially disrupted.
  • the method 100 further can include 102 pulsing low intensity focused acoustic energy from an acoustic source toward a target tissue.
  • the low intensity acoustic energy can be pulsed in accordance with the pulsing protocols described above to mechanically disrupt defective tissue after administration of bolus enzyme solution to the surface of the tissue.
  • the method 100 continues by disrupting cells of the tissue with the low intensity acoustic energy 103 to at least substantially detach a volume of defective cells and tissue.
  • the defective cells include the portion of cells treated by the biochemical agent solution that have been partially disrupted.
  • the application of the low intensity acoustic energy further disrupts the treated cells, and leaves healthy cells and tissue intact such that the area surrounding the treatment region remains with minimal to no damage.
  • the disruption and detachment of most or all defective cells within the tissue region can occur within minutes.
  • Evaluation of the airway tissue following cell and/or edematous fluid removal is achieved non-destructively in real time by using a micro-optical imaging probe that can be inserted directly into the local airway and obtain both bright-field and fluorescent images within the lung.
  • This system can be used to monitor functional properties of the airway epithelium including, but not limited to: mucociliary clearance, tight junction integrity, and cell viability and metabolism.
  • This optical based approach eliminates the need for tissue biopsy that is destructive and can potentially cause complications including bleeding and infection.
  • An innovative imaging-enabled rat trachea bioreactor that can allow real-time monitoring of the internal space of the trachea at the cellular level during long-term ex vivo culture.
  • airway stem cells have been cultured and assessed in a static two-dimensional (2D) environment of a petri dish or transwell insert that can provide controllable cell culture conditions.
  • 2D cultured cell monolayer enabled fundamental studies related to cell signaling pathways, cellular responses, and cell differentiation.
  • LOC lung-on-a-chip
  • lung-mimetic designs have been developed that can allow co-culturing of different lung cells in an environment where fluids (e.g., air and culture media) are dynamically manipulated.
  • Decellularized allogeneic or xenogeneic tissue grafts have been used to provide in vzvo-like microenvironments to the airway epithelial cells or stem cells during cell culture.
  • isolated rat or mouse tracheas with their endogenous cellular components completely removed via repeated freezing and thawing or chemical treatments were seeded with airway cells.
  • the cell-seeded decellularized airway tissues were implanted subcutaneously into immunodeficient host animal functional airway epithelial layer was regenerated on the luminal surface of the tissues.
  • the in vivo cultured tissue specific scaffolds allowed study of stem cell differentiation and confirmed regenerative capacity of the airway stem cells.
  • tissue culture approach Because cell-seeded tissue scaffolds are embedded in the host body, however, major limitations of this tissue culture approach include lack of ability for real-time manipulation of the cell culture conditions and monitoring of the cellular responses. In particular, creation of the air-liquid interface and timedependent supply of growth factors or cytokines that are essential for stem cell differentiation are difficult to achieve in the in vzvo-cultured tissue scaffolds. Further, different from in vzfro-cultured models, microscopic assessments of the cultured cells are only possible after removing the celltissue constructs from the host after completion of each experiment. [0036] Here, we report an imaging-enabled rat lung bioreactor system that allows longterm in vitro cultivation of isolated rat trachea and direct visualization of the tracheal lumen at the cellular level.
  • Imaging-enabled bioreactor for in vitro cultivation of isolated rat trachea We constructed an imaging-enabled bioreactor system that enabled selective removal of the epithelium and long-term culture of the tracheal tissue (FIG. 2). The bioreactor was designed and constructed in a way that the luminal surface of the trachea can be treated using different solutions (e.g., decellularization solution, washing solution, cells, culture medium) while the entire trachea is submerged in a cell culture medium to maintain the viability of the trachea tissue during planned experiments.
  • solutions e.g., decellularization solution, washing solution, cells, culture medium
  • isolated rat trachea can be de-epithelialized by introducing decellularization reagents (i.e., sodium dodecyl sulfate detergent; SDS) with specified volumes and concentrations directly into the inner space of the trachea.
  • decellularization reagents i.e., sodium dodecyl sulfate detergent; SDS
  • SDS sodium dodecyl sulfate detergent
  • Disrupted cells can be removed from the tissue surface with phosphate buffered saline (PBS) washing solution.
  • PBS phosphate buffered saline
  • the airway imaging device integrated into the bioreactor enables direct in situ visual inspection of the trachea lumen in both bright-field and fluorescence modes.
  • the trachea could be supplied with a gas flow (e.g., air) via the ventilation port connected to the bioreactor to create air-liquid interface within the trachea or to provide in vivo-like flow shear
  • the lysed cells were then cleared from the trachea by washing with PBS buffer while the entire trachea was vibrated mechanically using our custom-built shaker.
  • the trachea was oscillated at 20 Hz with a vertical displacement of approximately 0.3 mm
  • Exogeneous cells e.g., airway epithelial or stem cells
  • the de-epithelialization method preserved laminin (green), collagen I, elastin, and smooth muscles of the trachea tissue. Furthermore, immunostaining of the de-epithelialized tissues with endothelial cell marker (cluster of differentiation 31; CD31) confirmed that the blood vessels of the tissue remained intact (Fig.
  • CFSE-labelled mesenchymal stem cells were introduced and topically deposited onto the inner space of ex vivo rat tracheas with their epithelium removed via 4% SDS. Immediately after cell seeding, the micro-imaging probe was inserted into the trachea to confirm the deposition of the cells onto the deepithelialized tracheal lumen.
  • the bioreactor containing the cell-seeded trachea was then transferred to an incubator and connected to the perfusion pumps for extended in vitro culture (i.e., 1, 4, and 7 days).
  • the fluorescent images of the MSCs cultured on the deepithelialized tracheal lumen showed that the density of the cells covering the surface gradually increased over time (i.e., day 1 : 15.6 ⁇ 6.1 cells. mm-2, day 4: 94.6 ⁇ 15.1 cells. mm-2, day 7: 145 ⁇ 12.1 cells. mm-2; FIG. 12).
  • the average circularity of the seeded cells which is a normalized ratio of area to perimeter of the cells, was calculated to quantitatively evaluate attachment and engraftment of the cells on the surface.

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Abstract

L'invention concerne une méthode de traitement d'un tissu, la méthode consistant à : appliquer un agent biochimique sur une surface d'un tissu cible, et faire pulser des ondes acoustiques focalisées de faible intensité à partir d'une source d'énergie acoustique vers le tissu cible, de telle sorte que les ondes acoustiques focalisées de faible intensité sont pulsées à une fréquence et à une amplitude qui n'induisent pas d'ébullition ou de cavitation du tissu cible, permettant ainsi d'éliminer de manière sélective des cellules du tissu cible par les ondes acoustiques de faible intensité, tout en laissant les cellules saines du tissu cible sensiblement intactes.
PCT/US2022/077701 2021-10-06 2022-10-06 Méthode et système intégré pour l'élimination sélective de cellules défectueuses et de fluides œdémateux à partir du poumon Ceased WO2023060199A1 (fr)

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