WO2023060199A1 - Method and integrated system for selective removal of defective cells and edematous fluids from the lung - Google Patents

Abstract

A method of treating tissue, the method comprising: applying a biochemical agent to a surface of a target tissue, and pulsing low intensity focused acoustic waves from an acoustic energy source toward 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 selectively removing cells of the target tissue by the low intensity acoustic waves, while leaving healthy cells of the target tissue substantially intact.

Description

METHOD AND INTEGRATED SYSTEM FOR SELECTIVE REMOVAL OF DEFECTIVE CELLS AND EDEMATOUS FLUIDS FROM THE LUNG

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 63,252,926, filed October 6, 2021, which is incorporated by reference in its entirety herein..

GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under grant EB027062 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[0003] 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.

BACKGROUND

[0004] Nearly 80% of lungs offered by organ donors are unacceptable for transplantation due to their poor quality and diminished function mainly caused by airway tissue injury established within the respiratory tract. Typical methods for repair of injured donor lungs involve complete removal of entire cellular components from the lung by filling the airspace and blood vessels with biochemical reagents (e.g., decellularization agents) that disrupt the cells. The lungs are then washed rigorously to remove the cell debris and the residual decellularization agents using saline solution. While this approach is effective for complete removal of all cellular components from the lungs, the harsh process of solution instillation and extraction with strong decellularization reagents can cause severe damages to the delicate lung tissue. [0005] Also, patients with acute lung injury or severe pneumonia frequently develop pulmonary edema in which alveolar spaces fill with excess fluid and debris, cause by fluid leakage from the capillaries, build-up of apoptotic cell debris, and immune cell infiltration. Together, these events contribute to significantly impaired gas exchange and ultimately, lead to acute respiratory distress syndrome (ARDS). Currently there is no effective way to resolve pulmonary edema and the resulting ARDS. This unmet clinical need has been exacerbated by COVID-19 pandemic, as ARDS is the main contributor to high morbidity and mortality in COVID-19 patients.

[0006] Thus, there remains a need for a method and system for clearing alveolar spaces filled with edematous fluids and apoptotic cell debris, as well as dissociating and detaching injured epithelial cells, and/or removing of pathological tissue with precision such that healthy cells and tissue remain intact and undamaged.

SUMMARY OF THE DISCLOSED SUBJECT MATTER

[0007] According to one aspect of the disclosed subject matter, a method of treating tissue is provided. The method 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.

[0008] 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. [0009] The acoustic waves can be externally applied to the target tissue. In some embodiments, 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.

[0010] The biochemical agent for example is an enzyme, such as trypsin. Other biochemical agents are also suitable. For example, in addition to enzymatic biochemical agents, surfactants or acid and base solutions may be suitable.

[0011 ] 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. For example, the ex vivo organ is a lung from a lung donor. In other applications, however, the target tissue may be an airway of a subject. In this regard, the method and system described herein may be used to treat the airway of a subject’s lung, by removing edematous fluids. Thus, the embodiments herein can be used to treat acute lung injury, for example, acute respiratory distress system.

[0012] In a further aspect, a method of treating tissue is provided 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

[0014] FIGURE 2 is a simplified schematic view of an imaging-enable bioreactor platform in accordance with an embodiment of the present technology.

[0015] FIGURE 3 illustrates a procedure for de-epithelialization of tissue of a subject,

[0016] 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.

[0017] FIGURE 5 illustrates bright-field images of the interior of the tissue before carboxyfluorescein succinimidyl ester (CFSE) labeling of the epithelial layer.

[0018] FIGURE 6 illustrates fluorescence images of the interior of the tissue before CFSE labeling of the epithelial layer.

[0019] FIGURE 7 illustrates fluorescence images of native and (ii) de-epithelialized (Deepi) rat trachea lumen that was labelled with CFSE of the epithelium. [0020] FIGURE 8 illustrates fluorescence images of de-epithelialized (De-epi) rat trachea lumen that was labelled with CFSE of the epithelium.

[0021] FIGURE 9 illustrates the H&E histologic analysis of native and de-epithelialized rat tracheas with 2% SDS.

[0022] 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.

[0023] FIGURE 11 illustrates SEM images showing the luminal surface of the ex vivo rat tracheas native and de-epithelialized rat tracheas with 2% SDS.

[0024] 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).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0025] 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. In several embodiments, for example, 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. The deposition of biochemical agent bolus allows for continued normal ventilation of the lungs even during the enzyme treatment. The region of airways that receives the enzyme bolus can finely be controlled by modulating the enzyme delivery deposition, such as volume and instillation speed, to target only pathological tissue. Theoretical analysis and mathematical models can be used to understand the interactions between the enzyme molecule and target cells, and also to predict the mechanisms of enzyme film deposition onto the surface of the tissue. Suitable biochemical agents include surfactants, acids and bases, or enzymes. For the purpose of illustration, the suitable biochemical agents may include: sodium dodecyl sulfate, Triton X, sodium deoxycholate, CHAPS, peracetic acid, EDTA, trypsin, deoxyribunuclease (DNase), ribonuclease RNase).

[0026] 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.

[0027] In one aspect the disclosed method can reduce the duration of mechanical ventilation and increases the chances of survival among patient with severe edema and ARDS. Thus, the present method can treat acute lung injury and in particular ARDS. In another aspect, the method can improve lungs offered by organ donors to an acceptable condition for transplantation into a patient in need of a lung transplant. In certain embodiments, 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.

[0028] In one aspect, 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. In one embodiment, the acoustic source is an acoustic transducer that emits low levels of acoustic energy, i.e., sound waves, toward desired target tissue region. In one embodiment, 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. In this regard, 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.

[0029] 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). In some embodiments, output in the mode of pulse wave (40 Hz, 100 Hz and 1,000 Hz) and delivered at a much lower intensity (<1 W/cm²) than traditional high intensity focused ultrasound (HIFU) that typically generates over 1000 W/cm². In one embodiment, acoustic wave of intensity is 0.03 W/cm² (or 30 mW/cm²), 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).

[0030] In various embodiments, the low intensity acoustic system can emit a pulsing protocol to mechanically disrupt target lung tissue and cells. In one embodiment, 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. In certain embodiments, a function generator can initiate a pulsing protocol to generate acoustic waves with peak amplitudes of about 3 kPa at the target tissue. For example, 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.

[0031] 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. As shown in FIG. 1, 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. In some embodiments, 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.

[0032] 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.

EXAMPLE

[0033] 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.

[0034] To study differentiation and therapeutic functionality of airway stem cells, various in vitro platforms have been created and used. Traditionally, 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. The 2D cultured cell monolayer enabled fundamental studies related to cell signaling pathways, cellular responses, and cell differentiation. Notably, lung-on-a-chip (LOC) devices with 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. For instance, airway epithelium and endothelium were cultured on the apical and basal sides of a porous membrane, respectively, to mimic breathing airway, allowing in vitro drug screening and disease modeling. However, these in vitro platforms are incapable of recapitulating the complex three-dimensional (3D) architecture and dynamic cellmatrix interactions found in vivo, resulting in considerable differences between 2D cultured cells and in vivo cells in terms of morphology, proliferation, and differentiation.

[0035] 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. For example, 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. When 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. 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. Using this platform, we demonstrated selective removal of the epithelial layer from the trachea lumen without disrupting the underlying tissue layers and extracellular matrix (ECM) components, onto which exogeneous airway cells or stem cells could be implanted to restore functional epithelium. Further, we created a micro-imaging device that can be inserted into the inner space of the in vitro-cultured trachea to allow in situ visual monitoring of the cell replacement and cultivation. Notably, the de-epithelialized rat tracheas supported survival of exogenous cells, such as mesenchymal stem cells (MSCs) that were topically seeded onto the denuded tracheal lumen. We envision that the imaging-enabled bioreactor platform and tissue manipulation methodology established in this study could enable creation of humanized airway tissues by combining with human airway stem cells, allowing in vitro study of airway diseases and expediting development of therapeutics and intervention modalities.

[0037] Results. 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. Using this system, 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. Disrupted cells can be removed from the tissue surface with phosphate buffered saline (PBS) washing solution. 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 stress to the seeded cells.

[0038] De-epithelialization of ex vivo rat trachea. To selectively remove the tracheal epithelium without disrupting the rest of the underlying airway tissue (FIG. 3), we deposited a thin layer of 2% or 4% SDS detergent topically onto the tracheal lumen by instilling a small volume (50 pL) of the detergent solution. Instillation of a small volume of liquid (e.g., aqueous solution) through the respiratory tract can generate a thin layer of the liquid onto the airway lumen. Following disruption of the epithelium through detergent exposure, 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. In particular, 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) can be seeded onto the denuded tracheal lumen to reconstitute the epithelium within the bioreactor.

[0039] In situ visualization of the trachea lumen. We used our custom-built in situ airway imaging device to inspect the luminal surface of the rat trachea tissue during in vitro cell removal. We used a 1951 USAF (U.S. Air Force) test target to evaluate the resolution of the images obtained using the device. Obtained images showed high resolution and good quality with approximately 5pm of the smallest features resolvable. To visualize the trachea lumen, the imaging probe was directly inserted into the trachea through a cannula connected to the trachea (Figs 4(a)-(b)). Both bright-field images (FIG. 5) and fluorescence images (FIG. 6) of the local luminal surface were obtained, respectively, by using white light and 488-nm laser for illumination prior to carboxyfluorescein succinimidyl ester (CFSE) labeling of the epithelial layer. While no fluorescence signal was observed before labeling, a discernible signal (i.e., green light) was observed when the epithelium was labeled with CFSE (FIG. 7). Notably, following de- epithelialization, the intensity of the fluorescent signal substantially decreased, indicating clearance of the epithelium from the tracheal lumen (FIG. 8). This result highlights the utility of our imaging approach in in situ and minimally invasive monitoring of the epithelium removal.

[0040] Selective removal of tracheal epithelium with preserved tissue ECM. Histological evaluation of de-epithelialized tracheas showed complete removal of the epithelium across the luminal surface of de-epithelialized tracheas treated with 2% and 4% detergent solutions. In particular, high magnification of H&E images confirmed the removal of the pseudostratified columnar epithelium from the trachea mucosa and preservation of other cells in the submucosa, cartilaginous, and adventitia layers (FIG. 9). In addition, the structure pattern of the tissue layers and endogenous cells, such as cartilage and chondrocytes, underneath the basement membrane was well preserved following deepithelialization.

[0041] Further, we confirmed the preservation of ECM components of the de- epithelialized tracheas via immunofluorescence staining. Immunostaining of the trachea tissues by epithelial cell adhesion molecule (EpCAM) and 4’,6-diamidino-2-phenylindole (DAPI) revealed removal of the epithelial layer as no EpCAM (green) and DAPI (blue) signals were detected at the lumen of the trachea (Fig. 10). De-epithelialized tracheas treated with 4% SDS showed a reduction in the DAPI signal throughout the tissue, suggesting potential disruptions occurred to endogenous subepithelial cells due to increased detergent concentration. Nevertheless, 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.

4C).

[0042] Scanning electron microscopy (SEM) imaging of the tracheal lumen. We then investigated topological changes in the luminal surfaces of de-epithelialized trachea via SEM imaging where the images were obtained. See FIG. 11. The native trachea showed that the luminal surface was densely populated by different epithelial cells, predominantly multiciliated and goblet cells. On the other hand, the SEM images of de-epithelialized tracheas with 2% and 4% SDS showed absence of the tracheal epithelium. Notably, in both deepithelialized trachea lumen surfaces, a thin membrane layer, which is most likely the basement membrane, and mesh network of airway ECM were clearly visible. Structural disruption of the basement membrane and tissue ECM was more prominent in the tracheas treated with 4% SDS compared with that of 2% SDS as the porosity of the remaining ECM structure increased with the concentration of the SDS. Notably, use of mechanical vibration during the airway washing facilitated detachment of the epithelium as SDS-disrupted epithelium remained attached onto the lumen surface when no vibration was applied to the tissue (Fig. 4S). This result clearly indicated that oscillation energy provided to the trachea in the presence of the shear flow promoted disruption and detachment of the detergent- lysed cells from the airway tissue ECM.

[0043] We also investigated whether the sodium dodecyl sulfate (SDS)-treated tracheas can support attachment, growth, and proliferation of exogenous cells seeded on the lumen surface without generating a cytotoxic environment. To do this, CFSE-labelled mesenchymal stem cells (MSCs) 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). Further, 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. Analysis showed that cell circularity decreased over time, confirming active binding and incorporation of the cells onto the de-epithelialized tracheal lumen (i.e., day 1 : 0.90 ± 0.07, day 4: 0.22 ± 0.04, day 7: 0.25 ± 0.08; Fig. S5). Notably, SEM imaging showed that seeded MSCs quickly initiated binding onto de-epithelialized lumen at day 1 as multiple protrusions extending from the cell membrane to the tissue surface were observed. Collectively, the results indicated that the de-epithelialize trachea lumen provided a suitable microenvironment that allowed engraftment, survival, and proliferation of topically seeded exogenous cells.

Claims

CLAIMS What is claimed is:

1. A method of treating tissue, the method comprising: applying a biochemical agent to a surface of a target tissue, and pulsing low intensity focused acoustic waves from an acoustic energy source toward 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 selectively removing cells of the target tissue by the low intensity acoustic waves, while leaving healthy cells of the target tissue substantially intact.

2. The method of claim 1 wherein the acoustic waves are externally applied to the target tissue.

3. The method of claim 1, wherein the acoustic energy source is an acoustic transducer.

4. The method of claim 1, wherein 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.

5. The method of claim 1 wherein 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.

6. The method of any one of claims 1-5 wherein the target tissue is part of an ex vivo organ, and wherein detaching the cells of the target tissue comprises 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.

7. The method of claim 6, wherein the ex vivo organ is a lung from a lung donor.

8. The method of claim 1, wherein the target tissue is an airway of a subject’s lung, and the removed cells is edematous fluids.

9. The method of claim 8, wherein the method is a treatment for acute lung injury.

10. They method of claim 9, wherein the acute lung injury is acute respiratory distress syndrome.

11. A method of treating tissue, the method comprising: 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; 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.

12. The method of claim 11, further comprising, washing the target tissue to remove the portion of treated cells detached by the pulsing of low intensity focused acoustic waves.

13. The method of claim 11 wherein the acoustic waves are externally applied to the target tissue.

14. The method of claim 11, wherein the acoustic source is an acoustic transducer.

15. The method of claim 11, wherein 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.

16. The method of claim 11 wherein 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.

17. The method of any one of claims 11-15 wherein the target tissue is part of an ex vivo organ, and wherein detaching the cells of the target tissue comprises 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.

18. The method of claim 16, wherein the ex vivo organ is a lung from a lung donor.

19. The method of claim 11, wherein the target tissue is an airway of a subject’s lung, and the removed cells is edematous fluids.

20. The method of claim 18, wherein the method is a treatment for acute lung injury.

21. They method of claim 19, wherein the acute lung injury is acute respiratory distress syndrome.

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