The present disclosure relates generally to evaluation of biological tissues, and more particularly, to ex vivo analysis of resected live tissue samples, such as primary or metastatic solid tumors.
Potential treatment options for solid tumors continues to expand, but effective models to evaluate these new options prior to undertaking clinical trials remains limited. While mouse models have been developed in some cases, non-human models continue to be limited in terms of accuracy and application. Indeed, non-human models are non-existent for many cancers. Moreover, a greater understanding of the tumor microenvironment (TME), including dynamic interactions between cells in this heterogenous tissue, can help guide the development of new treatments as well as inform the prescription of existing treatment options. For example, immunotherapy has had been successful in the treatment of solid tumors, but some cancers are resistant to such treatments, with many individuals failing to respond to therapies that are otherwise effective for other similarly-diagnosed patients. Yet, such information about the fine-grained organization of cells and their functional state or interactions cannot be readily obtained by dispersed cell methods and standard sequencing techniques. Rather, a tumor model that accurately and faithfully recapitulates the heterogeneity of the human TME, inclusive of 3-D structure, stromal components, and immune populations is needed.
While patient-derived xenografts (PDX), patient-derived tumor organoids (PDO), and organ-on-chips have presented some progress over standard 2-D cell culture in modeling these complex interactions, they fail to provide the necessary prerequisites outlined above. For example, PDXs are cancer models where tissue resected from a human patient is implanted into a nonhuman, such as a mouse. As such, PDXs have stroma comprised of murine cells, lack a competent immune system, and represent a departure from human cell signaling and interactions. PDOs are generated by plating resected human tumor tissue in a basement membrane extract with a growth medium, but this model does not contain immune cells, blood vessels, or the necessary stromal cells. In organ-on-chips, different cell types can be added to the same model to observe interactions between tumor and healthy cells. However, cells from multiple patients are typically used to create the microenvironment, and the quantities of cells in the chip may not match normal physiological proportions. These models can suffer from genomic instability and evolution, and otherwise fail to imitate the tumor and its microenvironment with physiologic ratios and orientation of stromal and immune cells.
Accordingly, a need exists for a tumor model that accurately reflects the TME and can be used to test or customize treatment options. A further need exists for a model that is readily amenable to interrogation, such that resistance can be understood and rational combinations of treatment strategies can be ascertained. Indeed, mechanistic interrogation will move past the unrealistic “black and white” readouts currently available and allow understanding of how to best combine therapies for a desired effect. Embodiments of the disclosed subject matter may address one or more of the above-noted problems and disadvantages, among other things.
Embodiments of the disclosed subject matter provide systems, methods, and devices for ex vivo analysis of resected tissue samples, which can be used to provide an improved tumor model. A thin portion of tissue (e.g., a surface portion of the mesothelium) resected from a patient is mounted on a sample platform, which supports the tissue in a flow of perfusate within a perfusion chamber to allow exchange of nutrients, oxygen, water, and/or other chemicals (e.g., drugs or hormones) between the tissue and perfusate. Drugs can be added to the perfusate flow, for example, via an infusion port or valve within a fluid circuit connected to the perfusion chamber. The sample platform is designed to be removable from the perfusion chamber, for example, for analysis of the tissue (e.g., by imaging or other investigation technique), for treatment (e.g., partial or full immersion in a chemotherapeutic agent), or for any other purpose. After removal, the sample platform can be returned to the perfusion chamber for continued viability of the tissue ex vivo.
In some embodiments, when imaging with a microscope, the sample platform can be installed on an imaging platform holder, which holds the tissue on a viewing stage of the microscope and can easily reposition the tissue (e.g., by moving the tissue up or down) with respect to focal point of the microscope. In some embodiments, the sample platform can be installed on an exposure platform holder, which positions the tissue (or a portion thereof) within a fluid (e.g., stain for imaging, drug for treatment, etc.). In some embodiments, even though the sample platform is removed from the perfusion chamber, the sample platform, the imaging platform holder, and/or the exposure platform holder can include features that supply the tissue with perfusate and/or oxygen in order to maintain tissue viability outside the perfusion chamber.
In one or more embodiments, an ex vivo tissue analysis method comprises mounting a portion of live tissue resected from a patient on a sample platform. The method further comprises, using the sample platform, positioning the resected tissue portion within a perfusion chamber. The method also comprises flowing perfusate through the perfusion chamber and into contact with the resected tissue portion such that diffusion of oxygen occurs between the perfusate and the resected tissue portion. During the flowing, the resected tissue portion maintains a competent immune system.
In one or more embodiments, a system for ex vivo tissue analysis comprises a perfusion chamber and a sample platform. The perfusion chamber has an inlet, an outlet, and internal volume between the inlet and outlet. The sample platform has a tissue mount section and a chamber mount section coupled to the tissue mount section. The tissue mount section is constructed for mounting of a resected tissue portion thereon. The chamber mount section is constructed to releasably support the sample platform with respect to the perfusion chamber such that the resected tissue portion is positioned within the internal volume of the perfusion chamber. The tissue mount section of the sample platform has an opening that exposes a backside of the mounted resected tissue portion, such that both a frontside and a backside of the mounted resected tissue portion are exposed to perfusate within the perfusion chamber.
Any of the various innovations of this disclosure can be used in combination or separately. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
Embodiments will hereinafter be described with reference to the accompanying drawings, which have not necessarily been drawn to scale. Where applicable, some elements may be simplified or otherwise not illustrated in order to assist in the illustration and description of underlying features. Throughout the figures, like reference numerals denote like elements.
For purposes of this description, certain aspects, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods and systems should not be construed as being limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another. The methods and systems are not limited to any specific aspect or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present, or problems be solved. The technologies from any embodiment or example can be combined with the technologies described in any one or more of the other embodiments or examples. In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the disclosed technology.
Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods. Additionally, the description sometimes uses terms like “provide” or “achieve” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.
The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods, as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited. Whenever “substantially,” “approximately,” “about,” or similar language is explicitly used in combination with a specific value, variations up to and including 10% of that value are intended, unless explicitly stated otherwise.
Directions and other relative references may be used to facilitate discussion of the drawings and principles herein, but are not intended to be limiting. For example, certain terms may be used such as “inside,” “outside,”, “top,” “bottom,” “interior,” “exterior,” “left,” right,” “front,” “back,” “rear,” and the like. Such terms are used, where applicable, to provide some clarity of description when dealing with relative relationships, particularly with respect to the illustrated embodiments. Such terms are not, however, intended to imply absolute relationships, positions, and/or orientations. For example, with respect to an object, an “upper” part can become a “lower” part simply by turning the object over. Nevertheless, it is still the same part and the object remains the same.
As used herein, “comprising” means “including,” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.
Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and/or perform equally well. Nor does it mean that the alternatives are listed in a preferred order, unless stated otherwise. Unless stated otherwise, any of the groups defined below can be substituted or unsubstituted.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the appended claims.
Disclosed herein are exemplary systems, methods, and devices for ex vivo analysis of resected tissue samples, which can be used to provide an improved human tumor model. A thin portion of tissue (e.g., a surface portion of the mesothelium) can be resected from a patient and mounted on a sample platform. The sample platform can then be installed in a perfusion chamber and can support the tissue portion in a flow of perfusate to allow exchange of nutrients, oxygen, waste, water, and/or other substances (e.g., drugs or hormones). Drugs can be added to the perfusate flow, for example, via an infusion port or valve within a fluid circuit connected to the perfusion chamber.
The sample platform can be removed from the perfusion chamber for example, for analysis of the tissue (e.g., by microscopic imaging or other investigation technique), for experimental treatment (e.g., partial or full immersion in a chemotherapeutic agent), or for any other purpose. In some embodiments, when imaging with a microscope, the sample platform can be installed on an imaging platform holder, which holds the tissue on a viewing stage of the microscope and can easily reposition the tissue (e.g., by moving the tissue up or down with respect to the viewing stage), for example, to align different portions of the tissue with a focal point of the microscope. In some embodiments, the sample platform can be installed on an exposure platform holder, which positions the tissue (or a portion thereof) within a fluid (e.g., stain for imaging, drug for treatment, etc.). After removal, the sample platform can be returned to the perfusion chamber.
Unlike conventional tumor models, embodiments of the disclosed subject matter allow the tissue sample to maintain a competent immune system while preserving its tumor microenvironment (TME), which may offer more effective analysis of existing and new cancer treatment options and/or tailoring of a treatment to match the patient. Moreover, the configuration of the system (e.g., perfusion chamber, fluid circuit, sample platform, exposure platform holder, and/or imaging platform holder) can allow exposure of the tissue to treatment(s) (e.g., drugs, peripheral blood mononuclear cells (PBMCs), or other substances) while maintaining viability thereof. The system is also readily amenable to complex imaging, for example, to allow for analysis of a tumor's response to individual treatments on a cellular level.
At least a perfusion chamber 104 can be disposed within the interior volume of the environmental control apparatus 102, although other components of system 100 may also be disposed therein. The perfusion chamber 104 has at least one inlet 112, through which perfusate can be introduced into an internal volume 106 thereof, and at least one outlet 114, through which perfusate can be extracted from the internal volume 106. The perfusate can comprise blood plasma or culture medium mixed with blood plasma. Optionally, the plasma (with or without culture medium) can be supplemented with one or more drugs, hormones, and/or nutrients (e.g., amino acids or amino acid precursors, glutathione, dextrose, antibiotics, and/or insulin). In some embodiments, the blood plasma is taken from the same patient as the tissue sample (e.g., autologous plasma).
In some embodiments, the inlet 112 may be misaligned with respect to the outlet 114, so as to encourage adequate mixing of perfusate within internal volume 106 of the perfusion chamber 104. For example, the inlet 112 may be at a height (as measured from a bottom of the perfusion chamber) greater than that of the outlet 114, or vice versa. In another example, one or both of the inlet 112 and outlet 114 can be offset from a radial direction of the perfusion chamber 104 in a plane perpendicular to the direction of gravity. The perfusion chamber 104 can optionally include additional passive structures or active structures designed to further agitate the perfusate within internal volume 106 of perfusion chamber 104 to encourage adequate mixing. Control system 122 can be operatively connected to the one or more active structures to effect control thereof. Exemplary active structures can include, but are not limited to, a magnetic stirrer bar 116 that spins within the internal volume 106 upon application of an externally applied magnetic field. Alternatively, in some embodiments, the perfusion chamber 104 is provided without any active structures for perfusate flow agitation. Exemplary passive structures can include, but are not limited to, baffles on bottom, side, or top internal surfaces of the internal volume 106, configuration or arrangement of inlet 112 or outlet 114, and orientation of perfusate flow (from inlet 112 to outlet 114) within the perfusion chamber. For example, inlet 112 can include a nozzle or other structure designed to instigate turbulent flow of perfusate within the internal volume 106 to enhance mixing. In another example, multiple inlets 112 and/or multiple outlets 114 can be arranged around a circumference of the perfusion chamber 104. Alternatively, in some embodiments, the perfusion chamber 104 is provided without any additional passive structures for perfusate flow agitation.
In some embodiments, an oxygen-generating biomaterial (OGB) can be disposed within, or at least in contact with, perfusate in the perfusion chamber. The OGB can be configured to release oxygen into the perfusate by diffusion of entrapped, absorbed oxygen or by chemical generation of oxygen. OGB materials can include, but are not limited to, sodium percarbonate (SPO), calcium peroxide (CaO2), magnesium peroxide (MgO2), and hydrogen peroxide (H2O2) (which may be combined with catalase or other antioxidant) contained in a supporting structure formed from hydrogel, ethyl cellulose, or a biocompatible polymeric material (e.g., polydimethylsiloxane (PDMS), poly(lactic-co-glycolic acid) (PLGA), etc.). For example, calcium peroxide powder can be mixed with PDMS and loaded into a cylindrical silicone mold (e.g., having a depth of 1 mm) for curing. After curing, the PDMS-CaO2 disk can be removed from the mold and disposed at a bottom of the perfusion chamber 104 aligned with, or at least proximal to, sample 110.
The OGB structure may continuously release O2 into the perfusate, which may supplement the oxygen infusion by the gas exchange unit 124, described below. Alternatively, the OGB can form a part of the perfusion chamber 104 (e.g., coating of a surface of the chamber), or be coupled to or integrated with other structures within the perfusion chamber (e.g., as part of stirrer bar 116 or sample platform 108). When multiple samples 110 are provided in the perfusion chamber 104, each sample may have its own separate OGB structure.
An external fluid circuit 118, composed of one or more fluid conduits (e.g., glass, metal, or polymer tubing), connects the inlet 112 and outlet 114 together so as to recirculate exiting perfusate back to the perfusion chamber 104. One or more pumps 120 (e.g., a peristaltic pump) is provided to move perfusate through the fluid circuit 118, and thereby cause flow of perfusate through internal volume 106 of the perfusion chamber 104. Alternatively, the external fluid circuit 118 may have a non-recirculation configuration, for example, where a first conduit network of the fluid circuit connects inlet 112 to a source of fresh perfusate and a second conduit network of the fluid circuit connects outlet 114 to a receptacle (e.g., for waste collection or recycling) or drain (e.g., for waste disposal). In such a configuration, each fluid conduit network may have a respective pump 120. Control system 122 can be operatively connected to the one or more pumps 120 to effect control thereof.
A gas exchange unit 124 (also referred to as a gas exchanger) can be connected to the external fluid circuit 118. The gas exchange unit 124 can be configured to infuse perfusate flowing therethrough with gaseous oxygen from a source 126 (in addition to or in place of OGB structures) and to remove gaseous carbon dioxide from the perfusate. For example, source 126 can be an oxygen gas canister or oxygen supply line, either of which may be located external to the environmental control apparatus 102. In some embodiments, source 126 provides humidified oxygen to help compensate for any fluid loss during the flowing of perfusate. A sensor 128 can monitor the perfusate flowing through the gas exchange unit 124 (or monitor perfusate within internal volume 106) to provide feedback for regulation of gas exchange, for example, by sending a signal to control system 122. In some embodiments, the gas exchange unit 124 can comprise an oxygenator, which has a perfusate flow path therein separated from an air flow path therein by a membrane. For example, the oxygenator can have a size of 1000 cm2 or less, preferably 100 cm2 or less. The inlet end of the oxygenator air flow path can be connected to the source of oxygen while the outlet end of the oxygenator air flow path can be connected to an outlet valve. The control system 122 can be operatively coupled to the outlet valve to effect control thereof, for example, to cause venting of CO2 gas (e.g., external to the environmental control apparatus 102) to achieve a predetermined pH as measured by sensor 128. Alternatively or additionally, gas exchange unit 124 can comprise a gas mixer that blends together gases from an oxygen source (e.g., source 126) and a CO2 source (not shown) to supply a dynamic, customized gas mixture to the perfusate. For example, the control system 122 can control the gas mixer to change the gas mixture composition in order to manipulate the levels of CO2 dissolved in the perfusate and thereby adjust the pH of the perfusate to be within a predetermined range or to maintain the pH at a predetermined value.
An infusion device 132 can be connected to the external fluid circuit 118 by way of connection 130. For example, connection 130 can comprise a valve (e.g., a multi-position valve), a union (e.g., a Y-junction), an injection or infusion port, or any other fluidic structure that allows the combination of the infused substance and the perfusate (or a portion thereof) in fluid circuit 118. The infusion device 132 can be used to continuously or periodically inject a substance (e.g., drug, hormone, fluid, cells such as PBMCs, or any other chemical or substance) into the flowing perfusate. For example, the infusion device 132 can comprise a syringe pump or an infusion pump. In some embodiments, the infusion device 132 can continuously (substantially continuous while perfusate is flowing, except for brief periods of downtime for setup, replacement, or repair) provide insulin and/or sterilized water to the fluid circuit. Alternatively or additionally, the infusion device 132 can provide a single injection or provide periodic injections of one or more drugs, for example, to test a particular treatment option for cancer, and/or cells, for example, PBMCs isolated from the patient. The control system 122 can be operatively coupled to the infusion device 132 to effect control thereof, for example, to cause control a rate and/or timing of infusion. The control system 122 also may be operatively coupled to the connection 130, for example, to configure a multi-position valve to allow infusion by infusion device 132.
In some embodiments, a sampling device 136 can also be connected to the external fluid circuit 118 by way of connection 134. For example, connection 134 can comprise a valve (e.g., a multi-position valve), a union (e.g., a Y-junction), a withdrawal port, or any other fluidic structure that allows removal of a portion of the perfusate in fluid circuit 118. The sampling device 136 can be used to continuously or periodically withdrawal a portion of the perfusate from fluid circuit 118, for example, for interrogation by sensor 138 or for testing outside of system 100. For example, perfusate may be sampled periodically by sampling device 136 to test physiological parameters such as pH, oxygen, and metabolite levels using sensor 138. Alternatively or additionally, the perfusate can be sampled continuously or periodically to ascertain biomarker levels therein, for example, to assess response of the tissue (or a tumor therein) to a drug in the perfusate or to previously performed treatment outside of the perfusion chamber. The control system 122 can use feedback from sensor 138 (e.g., via one or more signals) to control gas exchange unit 124, infusion device 132, or a perfusate supply (not shown) to take corrective action, or to notify a user to take corrective action.
A tissue sample 110 is positioned within the flowing perfusate in the internal volume 106 of the perfusion chamber 104 by a sample platform 108. The sample platform 108 can include one or more structures (not shown) that releasably couple the sample platform 108 to the perfusion chamber 104. For example, the sample platform 108 can include a tissue mount section to which the tissue sample 110 is mounted, a chamber mount section, and one or more arms connecting the tissue mount section to the chamber mount section. The chamber mount section can be configured to interact with a top, bottom, side, or any other portion of the perfusion chamber 104 or surrounding structures to hold the tissue mount section (and the tissue sample 110 mounted thereon) in position within the flowing perfusate within the internal volume 106 of the perfusion chamber 104.
In some embodiments, the chamber mount section of sample platform 108 and a portion of the perfusion chamber 104 can comprise one or more cooperating features that align and/or retain (e.g., lock) the sample platform in a predetermined orientation with respect to the perfusion chamber 104 and/or contents thereof. For example, as shown in
The cooperating features of recesses and protrusions act to position the chamber mount section 162 and the attached tissue mount section in a fixed orientation with respect to the perfusion chamber, for example, with respect to a direction of perfusate flow in the perfusion chamber. Moreover, in some embodiments, the cooperating features can resist rotation of the sample platform, for example, due to forces of the perfusate flow on the tissue mount section or other portions of the sample platform. In some embodiments, the cooperating features can be keyed, such that only one orientation is possible for the sample platform installed to the perfusion chamber. For example, in some embodiments, the chamber mount section 162 can have a single protrusion 168, and the perfusion chamber portion 104a has a single recess 166. In another example, the chamber mount section 162 can instead have a pair of protrusions 168 that are not diametrically aligned (e.g., not disposed on a common axis 165 passing through a center of opening 164), and the perfusion chamber portion 104a can have a corresponding arrangement of recesses 166. Other configurations for such cooperating features to align and/or retain the sample platform are also possible according to one or more contemplated embodiments.
Referring to
In some embodiments, the periphery 110c of the tissue sample 110 can extend beyond edges of the tissue mount section of the sample platform 108. In such configurations, the tissue sample 110 may overhang and come into contact with sides 108c and/or a surface of the tissue mount section opposite the mounting surface 108a. The overhanging portion of the tissue sample 110 may be used to secure the sample to the tissue mount section without otherwise preventing perfusate access or obscuring visual inspection of the front surface 110a. The tissue sample 110 can be mounted on the sample platform 108 using mechanical attachment (e.g., suture wrapped around the tissue overhang and side 108c, a rubber band placed around the tissue overhang and side 108c, etc.) or a medical or biocompatible adhesive (e.g., between periphery 110c and mounting surface 108a).
In operation, tissue assembly 140, formed by the mounting of tissue sample 110 on sample platform 108, is inserted into perfusion chamber 104 such that tissue sample 110 is fully submerged within the perfusate within the internal volume 106, as shown in
When the tissue sample 110 is of a tumor (e.g., a primary cancerous tumor or a metastasis thereof), a drug under investigation for treatment of the tumor can be injected into the perfusate, for example, using infusion device 132 and fluid circuit connection 130. Alternatively, the tissue sample 110 may be of healthy tissue, and the drug administration may serve as an experimental control or to assess side effects. Again, the relatively thin thickness of the tissue sample 110, coupled with the exposure to perfusate of both front surface 110a and back surface 110b of the sample 110 by the sample platform 108, allows the drug to diffuse from the perfusate to most (and preferably all) cells of the tissue sample 110.
To investigate the effect of the drug on the tissue, the tissue assembly 140 can be removed from the perfusion chamber 104 for interrogation, for example, for microscopic imaging. Alternatively or additionally, the ability to interrogate the tissue sample 110 may be built into system 100, for example, by providing an objective lens of the microscope within the environmental control apparatus 102 at an appropriate location outside perfusion chamber 104, with a wall of the perfusion chamber 104 between the tissue sample 110 and the objective lens being transparent.
In some embodiments, the tissue assembly 140 can be removed from the perfusion chamber 104 and temporarily mounted to an imaging platform holder 142 for imaging of tissue sample 110 by a microscope (e.g., with objective lens 152), as shown in
In some embodiments, the tissue assembly can be adapted to maintain viability of the tissue sample over an extended period of time (e.g., ˜12 hours) outside of the perfusion chamber, for example, to allow for prolonged imaging, treatment, transport, or any other reason. For example,
In some embodiments, the tissue sample 110 can be removed from the perfusion chamber and disposed in contact with a fluid or otherwise subjected to a treatment outside of the perfusion chamber. For example, in some embodiments, part or all of the tissue sample 110 can be immersed in a fluid (e.g., stain for imaging or a chemotherapeutic agent). In such embodiments, the sample platform can be installed within an exposure platform holder, which positions the tissue sample (mounted on the sample platform) at a predetermined position with respect to the fluid. In some embodiments, the exposure platform holder can be adapted to maintain viability of the tissue sample during the immersion and/or treatment (e.g., 60-90 minutes). For example,
In some embodiments, the exposure platform holder can be adapted to maintain viability of the tissue sample 110 over a predetermined period (e.g., ˜60-90 minutes for a chemotherapeutic treatment regimen). For example, exposure platform holder 182 can include a fluid supply 190 supported by member 188 to deliver perfusate (e.g., plasma and/or culture media) to prevent the tissue sample 110 from drying out and/or to provide nutrients to the tissue sample 110. In some embodiments, the perfusate supplied to the tissue sample 110 can be oxygenated, for example, by dissolving oxygen in the perfusate within supply 190 or by providing an appropriate OGB structure within supply 190 or adjacent to tissue sample 110. The fluid supply 190 may be configured to deliver the perfusate to the back surface 110b of the tissue sample 110 as a substantially continuous or periodic supply of droplets 192.
Although the above discussion has focused on a single tissue sample 110 within a perfusion chamber 104 and a single perfusion chamber 104 within environmental control apparatus 102, embodiments of the disclosed subject matter are not limited thereto. Rather, any number of the above described components are possible according to one or more contemplated embodiments. Indeed, multiple tissue samples (with respective sample platforms) can be provided within a single perfusion chamber 104. For example, different tissue samples (e.g., healthy and cancerous tissue) can be provided in the same perfusion chamber 104 and subjected to the same investigation conditions (e.g., drug exposure, PBMCs, etc.) to compare efficacy and/or determine side effects. For example, in some embodiments, each perfusion chamber 104 can support four sample platforms 108 therein with respective tissue samples 110. In such embodiments, the four sample platforms 108 may be symmetrically arranged with respect to the perfusate flow through the perfusion chamber 104, for example, with two sample platforms 108 on opposite sides of a perfusate axis extending from the inlet 112 to the outlet 114.
Moreover, multiple perfusion chambers 104 (with respective fluid circuits 118) can be provided within environmental control apparatus 102 and may share one or more of the system 100 components, such as pump 120, control system 122, and/or oxygen source 126. For example, different perfusion chambers 104 can be provided to allow for parallel testing of multiple tissue samples. In some embodiments, two perfusion chambers 104 (e.g., each with four sample assemblies therein) can share a single pump 120. Other configurations are also possible according to one or more contemplated embodiments.
With reference to
A computing system may have additional features. For example, the computing environment 252 includes storage 266, one or more input devices 268, one or more output devices 270, and one or more communication connections 272. An interconnection mechanism (not shown) such as a bus, controller, or network interconnects the components of the computing environment 252. Typically, operating system software (not shown) provides an operating environment for other software executing in the computing environment 252, and coordinates activities of the components of the computing environment 252.
The tangible storage 266 may be removable or non-removable, and includes magnetic disks, magnetic tapes or cassettes, CD-ROMs, DVDs, or any other medium which can be used to store information in a non-transitory way, and which can be accessed within the computing environment 252. The storage 266 can store instructions for the software 264 implementing one or more innovations described herein.
The input device(s) 268 may be a touch input device such as a keyboard, mouse, pen, or trackball, a voice input device, a scanning device, or another device that provides input to the computing environment 252. The output device(s) 270 may be a display, printer, speaker, CD-writer, or another device that provides output from computing environment 252.
The communication connection(s) 272 enable communication over a communication medium to another computing entity. The communication medium conveys information such as computer-executable instructions, audio or video input or output, or other data in a modulated data signal. A modulated data signal is a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media can use an electrical, optical, RF, or other carrier.
Any of the disclosed methods can be implemented as computer-executable instructions stored on one or more computer-readable storage media (e.g., one or more optical media discs, volatile memory components (such as DRAM or SRAM), or non-volatile memory components (such as flash memory or hard drives)) and executed on a computer (e.g., any commercially available computer, including smart phones or other mobile devices that include computing hardware). The term computer-readable storage media does not include communication connections, such as signals and carrier waves. Any of the computer-executable instructions for implementing the disclosed techniques as well as any data created and used during implementation of the disclosed embodiments can be stored on one or more computer-readable storage media. The computer-executable instructions can be part of, for example, a dedicated software application or a software application that is accessed or downloaded via a web browser or other software application (such as a remote computing application). Such software can be executed, for example, on a single local computer (e.g., any suitable commercially available computer) or in a network environment (e.g., via the Internet, a wide-area network, a local-area network, a client-server network (such as a cloud computing network), or other such network) using one or more network computers.
For clarity, only certain selected aspects of the software-based implementations are described. Other details that are well known in the art are omitted. For example, it should be understood that the disclosed technology is not limited to any specific computer language or program. For instance, aspects of the disclosed technology can be implemented by software written in C++, Java, Perl, any other suitable programming language. Likewise, the disclosed technology is not limited to any particular computer or type of hardware. Certain details of suitable computers and hardware are well known and need not be set forth in detail in this disclosure.
It should also be well understood that any functionality described herein can be performed, at least in part, by one or more hardware logic components, instead of software. For example, and without limitation, illustrative types of hardware logic components that can be used include Field-programmable Gate Arrays (FPGAs), Program-specific Integrated Circuits (ASICs), Program-specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), etc.
Furthermore, any of the software-based embodiments (comprising, for example, computer-executable instructions for causing a computer to perform any of the disclosed methods) can be uploaded, downloaded, or remotely accessed through a suitable communication means. Such suitable communication means include, for example, the Internet, the World Wide Web, an intranet, software applications, cable (including fiber optic cable), magnetic communications, electromagnetic communications (including RF, microwave, and infrared communications), electronic communications, or other such communication means. In any of the above described examples and embodiments, provision of a request (e.g., data request), indication (e.g., data signal), instruction (e.g., control signal), or any other communication between systems, components, devices, etc. can be by generation and transmission of an appropriate electrical signal by wired or wireless connections.
Referring to
The method 200 can proceed to process block 204, where the resected sample is prepared for mounting to the sample platform. The tissue resection may be transported from the surgical center to a preparation stage and disposed in a warmed bath of culture medium. For example, the preparation can include trimming a size of the resected sample (e.g., to fit a size or shape of the tissue mount section of the sample platform) and/or removing excess tissue (e.g., adipose tissue) from the sample. In embodiments, the prepared tissue sample may be relatively thin, for example, having a thickness less than 1 mm.
The method 200 can proceed to process block 206, where the resected sample is mounted to the sample platform, in particular, the tissue mount section thereof. The resected sample can be positioned over the tissue mount section with its native surface (e.g., mesothelial surface) facing away from the tissue mount section and such that regions of interest (e.g., a macroscopically visible tumor) are centered on the tissue mount section. A periphery of the sample can then be attached to the tissue mount section, for example, using a circumferential attachment member (e.g., a 1-0 or 0 silk suture, a rubber band, etc.). A time period between the resection of tissue (process block 202) and completion of the tissue mounting (process block 206) may be less than or equal to five minutes, and preferably less than two minutes.
The method 200 can proceed to process block 208, where the sample platform is inserted into the perfusion chamber, such that the mounted tissue sample is positioned within perfusate in the perfusion chamber. For example, the perfusion chamber can include one or more receptacles in a surface thereof that allow the tissue mount section to pass therethrough but prevent the chamber mount section of the sample platform from passing. The chamber mount section thus releasably supports the sample platform on the perfusion chamber, with the tissue sample being suspended within the perfusion chamber.
The method 200 can proceed to process block 210, where perfusate is flowed through the perfusion chamber and circulated from the perfusion chamber outlet back to the perfusion chamber inlet via an external fluid circuit. As noted above, the perfusate can comprise blood plasma (e.g., autologous plasma, obtained from the same patient as the tissue sample) or culture medium mixed with plasma. Optionally, the plasma can be supplemented with one or more drugs, hormones, and/or nutrients (e.g., amino acids or amino acid precursors, glutathione, dextrose, antibiotics, and/or insulin). The relatively-thin thickness of the tissue sample, coupled with the exposure to perfusate offered by the sample platform, allows nutrients and oxygen (e.g., supplied by a gas exchange unit, such as an oxygenator, a gas mixer, and/or an OGB structure within the perfusion chamber) to diffuse from the perfusate to cells of the tissue sample while waste and carbon dioxide diffuse from the cells into the perfusate, thereby supporting the viability of the tissue sample ex vivo.
The method 200 can proceed to decision block 212, where it is determined if the tissue sample should be exposed to a substance. For example, the drug could be a potential treatment option for a cancer of the patient. The tissue sample thus serves as a model to assess efficacy of the drug for the patient's cancer. The determination at decision block 212 can be based on timing (e.g., for periodic drug exposure or waiting a period after introduction of the sample to the perfusion chamber to allow it to equilibrate), a status of the tissue sample (e.g., whether the tissue sample is to serve as an experimental control), or any other criteria. If perfusate-based drug exposure is desired at decision block 212, the method 200 can proceed to process block 214, where a drug is added to the perfusate. The method 200 can then return to process block 210 for perfusate circulation, such that the drug is transported to the tissue sample in the perfusion chamber via the flowing perfusate for interaction with cells of the sample.
Alternatively or additionally, decision block 212 can include determining whether the tissue sample should be exposed to PBMCs. For example, PBMCs can be isolated from whole blood of the patient (i.e., the same patient from which the tissue sample has been resected), for example, using density centrifugation, using cell preparation tubes (CPTs), using SepMate tubes, or by any other methodology. If exposure to isolated PBMCs is desired at decision block 212, the method can proceed to process block 214 where the isolated PBMCs can be introduced to the tissue sample via addition to the flowing perfusate. In such an example, the isolated PBMCs may be tagged (e.g., using a nanoparticle tracking dye) prior to introduction to the tissue sample so as to aid in differentiation from immune cells that reside in the TME of the sample. The introduction of PBMCs may be part of a cancer treatment that increase a number or ability of circulating immune cells to target the tumor. The method can then return to process block 210 for perfusate circulation, such that the PBMCs are transported to the tissue sample in the perfusion chamber via the flowing perfusate for interaction with cells of the sample.
In some embodiments, after process block 214, the continuing to circulate perfusate of process block 210 can include continuously or periodically sampling the perfusate (e.g., for subsequent testing) or continuously or periodically detecting a substance within the flowing perfusate. For example, perfusate can be periodically sampled and tested to determine levels of one or more biomarkers therein. For example, in some embodiments, levels of transforming growth factor beta (TGF-β) in the flowing perfusate can be measured over time to assess response of the tissue sample, in particular a tumor therein, to an administered drug or treatment.
Otherwise, the method 200 proceeds to decision block 216, where it is determined if the tissue sample should be removed from the perfusion, for example, for imaging or treatment. For example, the tissue sample could be imaged after being exposed to the drug and/or PBMCs to assess the effect on various cells of the tissue. The determination of imaging at decision block 216 can be based on timing (e.g., waiting a period of time after initial drug exposure), a status of the tissue sample (e.g., whether the tissue sample is to serve as an experimental control), or any other criteria. If imaging is desired at decision block 216, the sample platform can be removed from the perfusion chamber and the method 200 can proceed to process block 218. If instead treatment is desired at decision block 216, the sample platform can be removed from the perfusion chamber and the method 200 can proceed to process block 224.
Alternatively or additionally, decision block 216 can involve determining if the tissue sample should be permanently removed, for example, for an analysis that involves tissue dissociation. For example, in some embodiments, the sample platform can be removed from the perfusion chamber, and the tissue sample removed from the sample platform. The tissue sample can then be analyzed, for example, by flow cytometry, multi-omics profiling methods, etc.
If temporary or permanent removal of the sample platform from the perfusate chamber is not desired at decision block 216, the method 200 can return to process block 210, where the circulation of perfusate continues.
When imaging is desired, after the sample platform is removed from the perfusion chamber, the tissue sample is prepared for imaging at process block 218. For example, the preparation can include one or more staining procedures (e.g., hematoxylin and eosin (H&E), immunohistochemical CD3, immunohistochemical CD20, immunohistochemical CD68, antigen Ki-67, etc.). During the preparation of process block 218, the tissue sample is not removed from the sample platform. Rather, in some embodiments, process block 218 may employ an exposure platform holder to perform the staining of the tissue sample, for example, using the platform holder of
The method 200 can proceed to process block 222, where the tissue sample supported by the imaging platform holder is imaged. For example, the imaging system can comprise a microscope system, such as a confocal microscope, and the imaging platform holder can be constructed to rest on a horizontal sample stage of the microscope. The imaging system can be configured to perform any type of known interrogation technique or imaging modality, such as, but not limited to, confocal microscopy, fluorescence microscopy, two-photon or multiphoton microscopy, time-lapse microscopy (e.g., for live cell imaging), phase contrast microscopy, holotomography, etc.
Once imaging is completed, the method 200 can proceed to decision block 228, where it is determined if the tissue sample should be returned to the perfusion chamber for continued viability and/or testing. The determination at decision block 228 can be based on timing (e.g., a predetermined lifetime of the sample or experimentally determined maximum lifetime of the sample), a status of the tissue sample (e.g., whether imaging reveals the sample is no longer viable), or any other criteria. If return is desired at decision block 228, the sample platform can be removed from the imaging platform holder, and the method 200 can return to process block 208, where the sample holder is reinstalled in the perfusion chamber.
When treatment is desired, after the sample platform is removed from the perfusion chamber at decision block 216, the method 200 can proceed to process block 224, where the tissue sample can be temporarily mounted on an exposure platform holder, for example, the exposure platform holder of
Once treatment is completed, the method 200 can proceed to decision block 228, where it is determined if the tissue sample should be returned to the perfusion chamber for continued viability and/or testing. The determination at decision block 228 can be based on timing (e.g., a predetermined lifetime of the sample or experimentally determined maximum lifetime of the sample), a status of the tissue sample, or any other criteria. If return is desired at decision block 228, the sample platform can be removed from the exposure platform holder, and the method 200 can return to process block 208, where the sample holder is reinstalled in the perfusion chamber.
Otherwise, the tissue sample can be discarded at 228 when it is determined that return is not desired. In some embodiments, the sample platform may be a disposable component, in which case the tissue sample and sample platform can be disposed of as a unit. In other embodiments, the sample platform may be a reusable component, in which case the tissue sample is dismounted from the tissue mount section and discarded. The sample platform can then be sterilized (e.g., via autoclave) for use with a subsequent tissue sample at 206.
Alternatively or additionally, after imaging in process block 222 or treatment in process block 226, it can be determined at decision block 228 to refrain from returning the tissue to the perfusion chamber in order to perform further analysis. For example, the tissue can be removed from the sample platform, subject to a chemical fixation process (e.g., using formaldehyde or glutaraldehyde), and then embedded within optimal cutting temperature (OCT) compound for subsequent frozen tissue sectioning (e.g., using a microtome-cryostat). The tissue sections can then be stained for complex imaging, such as immunohistochemistry (IHC) imaging (e.g., multiplex immunofluorescence imaging). Other analyses are also possible according to one or more contemplated embodiments.
Although
In some embodiments, the chamber mount section has an opening 326 that extends through flange 314 and cylindrical bottom member 312. In other embodiments, the chamber mount section may be substantially solid, for example, with flange 314 as a solid disk and/or bottom member 312 as a solid cylinder. The tissue mount section is connected to the chamber mount section by a pair of arms 306a, 306b, thereby spacing the tissue mount section from the chamber mount section along an axial direction of the sample platform 300. The arms 306a, 306b can be positioned on opposite sides of through-hole 328, thereby forming an open region 316 therebetween which communicates with the open through-hole 328 and opening 326.
Upper ends of arms 306a, 306b connect to lower portion 324 of the tissue mount section, while lower ends of arms 306a, 306b connect to the cylindrical bottom member 312. In some embodiments, each arm can have a respective support portion 308a, 308b at the lower ends and connected to the cylindrical bottom member 312. The support portions 308a, 308b may improve the rigidity and/or reliability of arms 306a, 306b. Each arm 306a, 306b can also include one or more through-holes 310 in a region proximal to the tissue mount section. Each through-hole 310 can extend through a thickness of the respective arm 306a, 306b to communicate with open region 316, thereby improving perfusate access to the backside of the tissue sample. In some embodiments, the through-holes 310 can be replaced by a single large hole (e.g., a rectangular window), a plurality of smaller holes (e.g., an array or mesh), or any other configuration of opening or openings.
In some embodiments, flange 314 can include one or more surface features (e.g., protrusion or recess) designed to cooperate with corresponding surface features of the holder (e.g., top circular disk 609 of lid 608) in order to lock, or at least releasably position, the sample platform in a predetermined orientation within the perfusion chamber. For example, in some embodiments, the predetermined orientation of the sample platform 300 is such that an upper portion of arms 306a, 306b (e.g., having through-holes 310) and/or open region 316 are substantially parallel to a direction of the perfusate flow through the perfusion chamber (e.g., in a top down plan view).
In a fabricated example, the flange 314 had an outer diameter of 18.6 mm, the upper portion 318 had an outer diameter of 9.56 mm, the inner diameter of the through-hole 310 was 6.68 mm, the groove 322 was indented from the upper portion outer diameter by 0.5 mm, the arms 306a, 306b were 16.75 mm in length along the axial direction and 4.78 mm in width, and each arm 306 had four through-holes 310 of 1 mm diameter. Other dimensions are also possible according to one or more contemplated embodiments. For example, a diameter and/or thickness of upper portion 318, a diameter of lower portion 324, and/or a width of groove 322 (e.g., in a direction extending between the upper portion 318 and the lower portion 324) can be increased from the above-noted exemplary dimensions to assist in attaching the tissue sample to the sample platform. For example, in some embodiments, the diameter of upper portion 318 can be made larger than that of the lower portion 324, such that the top annular member 302 is mushroom-shaped in side view.
In some embodiments, a jig can be used to hold and stabilize the sample platform during the mounting of the tissue sample thereto. For example,
In use, the jig 400 is first inserted into a circular dish, with portions of walls 402 in contact with a circumferential wall 414. The sample platform 300 is then placed within open region 404 at a radially innermost end of arms 408a, 408b. The chamber mount section of the sample platform 300 is aligned with the grooves 412a, 412b of the arms 408a, 408b, and the sample platform 300 is slid radially outward to engage with the grooves 412a, 412b, as shown in
As noted above, in some embodiments, the mechanical attachment of the tissue sample to the tissue mount section may be by way of an annular flexible member that applies a radial compressive force to the periphery of the tissue sample, for example, a rubber band.
In use, a tissue sample is draped over the top annular member 302 of the sample platform 300, and then the bottom end 514 of the applicator is positioned over and into contact with the tissue sample (e.g., as suggested by the configuration shown in
The perfusion chamber 600 can also include a lid 608 (also referred to as a platform holder) having an internal volume 614 defined by a substantially cylindrical wall 612 extending from a circumference of a top circular disk 609. The cylindrical wall 612 has an inner diameter greater than an outer diameter of cylindrical wall 618, such that lid 608 can fit over a top end of the dish 616 to enclose internal volume 622. The top circular disk 609 of the lid 608 includes four recesses 610a-610d extending therethrough and communicating with internal volume 614. Recesses 610a-610d are designed to receive respective sample platforms therein, in particular, allowing the tissue mount section to pass therethrough. However, a diameter of each recess 610 is smaller than a diameter of flange 314 of the chamber mount section, such that the sample platform 300 is releasably coupled to the lid 608 while suspending the rest of the sample platform 300, including the mounted tissue sample, within the internal volume 622, as shown in
In some embodiments, the lid 608 and the dish 616 can comprise one or more cooperating features that align and/or retain (e.g., lock) the lid 608 (and the sample holders supported thereby) in a predetermined orientation with respect to the dish 616 and/or contents thereof. For example, similar to the configuration illustrated in
In some embodiments, the perfusion chamber 600 can also include a cover 602 having an internal volume 606 defined by a substantially cylindrical wall 604 extending from a circumference of a top circular disk 605. The cover 602 can be designed to fit over the lid 608, thereby protecting open ends of the chamber support sections of sample platforms 300 inserted into recesses 610a-610d of the lid 608. In some embodiments, the cover 602, lid 608, or both can include one or more spacers that hold the top circular disk 605 spaced away from the flange 314 of each sample platform 300 retained in recesses 610a-610d of lid 608, for example, to prevent adhesion of the cover 602 to the sample platforms, the lid, or both. Alternatively or additionally, the spacer can be a separate member (e.g., an O-ring) disposed between cover 602 and lid 608.
In a fabricated example, the lid 608 had an outer diameter of 61.49 mm and a length of 9 mm, the wall 612 of the lid flared radially outward by about 2n (e.g., forming an angle of 92° with top disk 609), a thickness of top disk 609 was 2 mm, each recess 610a-610d had a diameter of 15.9 mm, the dish 616 had an outer diameter of 56.71 mm and a length of 19.35 mm, the wall 618 of the dish flared radially outward by about 2° (e.g., forming an angle of 92° with bottom disk 620), a bottom of outlet 626 was 2 mm above the bottom disk 620, and a bottom of inlet 624 was 5 mm above the bottom disk 620. Other dimensions are also possible according to one or more contemplated embodiments.
The second member 814 has a substantially cylindrical wall 816 extending from a circumference of a top circular disk 815. A substantially cylindrical shaft 834 extends from a bottom surface of the top circular disk 815. A central threaded opening 818 extends through both the top circular disk 815 and the cylindrical shaft 834. The cylindrical wall 816 has an inner diameter greater than an outer diameter of cylindrical wall 822, such that second member 814 can fit over a top end of the dish 820 to enclose internal volume 828, with a bottom end of cylindrical wall 816 resting on stops 824 of the dish 820.
The first member 806 similarly has a top circular disk 807 and a substantially cylindrical shaft 832 extending from a bottom surface of the top circular disk 807. A central opening 812 extends through both the top circular disk 807 and the cylindrical shaft 832. The cylindrical shaft 832 has a threaded circumferential surface that corresponds to threads of central opening 818 of the second member 814. The shaft 832 of the first member 806 is thus coupled with opening 818 of the second member 814 and rotated therein to move the first member 806 axially with respect the second member 814 and the dish 820. The first member 806 may optionally include a knurled circumferential edge 808 to facilitate rotation by a user. By rotating the first member 806 with respect to the second member 814, the tissue sample can be moved axially to or from the bottom disk 826 of dish 820, for example, to move a different portion of the tissue sample into focus for imaging.
Central opening 812 is designed to receive a sample platform 300 therein, in particular, allowing the tissue mount section (e.g., top annular member 302) to pass therethrough. However, a diameter of opening 812 is smaller than a diameter of flange 314 of the chamber mount section, such that the sample platform 300 is releasably coupled to the first member 806 while suspending the rest of the sample platform 300, including the mounted tissue sample, within the internal volume 828, as shown in
In some embodiments, the height of the gap 830a, 830b is greater than a thickness of the flange 314. Alternatively or additionally, a distance between facing ends of the securing arms 810a, 810b may be greater than an outer diameter of flange 314. Accordingly, the securing arms 810a, 810b alone may not adequately retain the sample platform 300 to the imaging platform holder 800, for example, where fluid within dish 820 generates an axial force due to buoyancy of the sample as the first member advances toward the dish 820. Locking member 802 can thus be provided between the securing arms 810a, 810b and the exposed surface of the flange 314 and form a transition fit (e.g., tight fit, similar fit, or fixed fit) or an interference fit (e.g., press fit) that secures the flange 314, and thereby the sample platform 300, to the first member 806, as shown in
Multiple solid tumors commonly manifest as metastatic disease to the mesothelial surface (peritoneum, pleura, liver capsule). Mesothelium is thin, translucent and is commonly resected both with open and laparoscopic/thoracoscopic surgery as part of both staging and standard treatment algorithms. The thin tumors of mesothelial surfaces enable oxygen diffusion and facilitates real-time or recent (e.g., within a few minutes) imaging interrogations. Accordingly, tumor-bearing mesothelium was suspended in the perfusate using the above-described platforms, which were designed to maximize gas exchange and facilitate imaging.
The perfusate was composed of patient blood-type matched human fresh frozen plasma (FFP), Dulbecco's Modified Eagle Media (DMEM) with GlutaMAX (ThermoFisher Scientific), glutathione (1 mM), insulin (Novolin R 100 units/mL), 5% dextrose (0.25 mL per 30 mL) and penicillin-streptomycin (Pen-Strep). The perfusate was prepared in a sterile fashion inside a biosafety cabinet. A one-to-one ratio of DMEM to FFP was utilized, with addition of 0.5 mL of insulin (Novolin R 100 units/mL) and Pen-Strep for a total volume of 30 mL. Alternatively, this volume permits plasma to be donated by the individual patient from whom tumor is used (as opposed to the blood bank), which can facilitate biomarker and correlative science discovery, and is acceptable from a drug utilization perspective. In some configurations, the volume can be reduced further by decreasing sizes of the perfusion chamber, the sample platform, and/or associated fluid circuit (e.g., diameter, length, and/or shape of tubing between components of the system), such that a minimum total volume of perfusate for the system is 12-15 mL. In such configurations, the perfusate may be 100% autologous plasma (e.g., without culture media, although potentially supplemented with one or more drugs, hormones, and/or nutrients).
Humidified oxygen was used with the system in order to minimize evaporate losses (and the accompanying electrolyte derangements), and pH was controlled with the CO2 pressure control valve on the venting outlet of the oxygenator to let off excess carbon dioxide. In alternative configurations, the perfusion chamber can also be provided with OGB structures to generate oxygen within the perfusate. In further alternative configurations, a humidified low-flow gas blender, such as MCQ Gas Blender 100 (sold by MCQ industries in Rome, Italy) can be used to supply a dynamic, customized mixture of oxygen and CO2 gases.
To compensate for evaporative loss of water, sterile water and insulin were continuously infused at a rate of 0.25-0.75 mL/hour using a metered syringe injector. Although continuous infusion of insulin was used, any drug/hormone/growth factor/cells/etc. can be added to the system in a similar manner. To ensure maintenance of physiological parameters, point of care testing was conducted/carried out at interval timepoints with an iStat analyzer (sold by Abbott of Chicago, Illinois) to assess pH, oxygen, and metabolite levels, and appropriate adjustments were made, as needed.
Tissues introduced to the system were kept viable for up to four days. Every 24 hours, a complete perfusate exchange was performed. Experimental drugs were introduced into the perfusate as indicated. Tissue samples taken immediately upon extirpation from the patients were preserved in 10% neutral buffered formalin and served as a control (Day 0) for a given experiment. Throughout the four-day experiment, sample platforms were removed from the perfusion chamber at various endpoints to demonstrate viability and drug effects.
Sample platforms containing tumor-bearing and adjacent normal mesothelium tissues were assembled. The maintenance and preservation of the normal tissue can be important to prevent any uncertain effects on the adjacent tumor. Moreover, the potential side of effects of new agents could be estimated using normal mesothelial platforms run in parallel to tumor-bearing mesothelium. Peritoneum, liver capsule and pleura tissues were assembled and perfused using the system of
Using standard histologic evaluation (H&E), cellular preservation (95%) of samples at 96 hours was demonstrated as shown in
With the goal of establishing a system that can evaluate immunomodulatory agents, it was sought to demonstrate that native immune cells can respond to stimulants in the system of
Using the setup of
Any meaningful translational platform should be readily amenable to interrogation to understand why some cells within a tumor respond while others are ostensibly resistant. The design of the system of
For example,
With the application of a leucocyte marker (CD45), the relationship between tumor and immune cells becomes apparent. In particular, the GIST demonstrates “cold” tumor morphology with immune cells essentially excluded or surrounding nests of tumor cell. In contrast, the gastric tumor in
Finally, immune cells can be tracked and monitored in the system of
Given a functional immune system, the use of full-length antibodies for cell recognition may result in unintended consequences. Accordingly, in some embodiments, fluorescently-labelled antigen-binding fragments (Fab fragments) and nanobodies can be used for various immune subpopulations instead of or in addition to full-length antibodies.
The system of
In
The system of
In another example, the system of
In any of the above discussed examples, tissue tested in system 700 was similarly prepared. For example, a specific tissue was procured directly from the operating room at the beginning of each operation and transported to the ex-vivo laboratory in pre-warmed Dulbecco's Modified Eagle Media (DMEM). Upon arrival to a sterile biosafety hood, the tissue was transferred into a larger cell-culture dish containing DMEM equilibrated to 37° ° C. using a heating plate, in order to prevent temperature fluctuations and warm ischemia to the tissue. Following visual inspection, excess adipose tissue was stripped carefully and mounted loosely over the small ring (e.g., top annular member 302) of the sample platform, with the mesothelial surface facing outward. Macroscopically visible tumor was positioned over the center of the platform before securing it in place with a 1-0 or 0 silk suture. The duration of the tissue preparation and mounting to the sample platform was limited to an average of five minutes. The sample platform was immediately transferred into the system 700, with the tissue side facing down within a sterile incubator (Thermo Heracell VIOS 160i CO2 incubator) at 37° C. and 5% CO2 for the duration of experimentation.
In any of the above discussed examples, tissue from system 700 was subject to similar histopathologic evaluations. For example, the tissues were preserved in the 10% neutral buffered formalin, then through the tissue processor and embedded in paraffin and cut at 5-micron sections for hematoxylin and eosin (H&E) slides. Slides were prepared for H&E staining and immunohistochemistry, respectively to detect CD3 (T cells), CD68 (macrophages), Ki-67 and MIBI (mitotic activity). CD3 and CD68 were predilute antibodies as purchased, while Ki-67 and (MIB-1) were run at 1:200 dilution. All stains were done on an automated immunostainer (Ventana Benchmark Ultra). Viability on H&E was defined as preservation of normal tissue architecture, intact nuclei, and normal population of immune cells (qualitative and quantitative). Each sample was compared against day 0 control samples. Ki67 or stains were performed to assess proliferative activity of the tumor cells. Samples stained with Ki67 or MIB1 on day 4 were compared to day 0 samples to confirm no change in proliferative activity.
In any of the above discussed examples, tissue from system 700 was subject to similar immune cell activation testing. For example, in assessment of interferon-γ production, sample platforms were removed from the system 700 at 24 hour intervals for 4 days and placed in 2 mL of perfusate media and stimulated with recombinant human Interleukin-2 (IL-2) (1:1000 dilution), 50 ng/ml of phorbol 12-myristate 13-acetate (PMA), and 1 μg/mL of ionomycin. The tissue samples were allowed to incubate overnight. The following day the supernatant of each sample was harvested and stored at −80° ° C. Following collection of all samples, the supernatants were thawed and used for interferon-γ enzyme-linked immunosorbent assay (ELISA).
In preparation, flat-bottom 96-well plates were pretreated with 1 μg/ml of a human IFN-γ capture antibody, at 4° C. Immediately preceding the assay, the plates were blocked with 150 μl of 5% BSA in PBS for 30 min at room temperature. After removing the blocking solution, 50 μl of both the sample supernatants and the interferon-γ standard dilutions were added. Afterwards, 50 μl of 0.5 μg/ml biotinylated anti-human-IFN-γ detection antibody (50 μl/well, diluted in 5% bovine serum albumin (BSA) in phosphate buffer solution (PBS)) were added to each well. The plate was incubated for 60 minutes at room temperature, followed by four washes with ELISA buffer (PBS containing 0.05% Tween 20). 125 ng/ml streptavidin-horseradish peroxidase (150 μl/well, diluted in PBS with 5% BSA) was added and the plate was incubated again for 45 min at room temperature. The plate was then washed eight times with ELISA buffer and developed with TMB substrate (100 μl/well) in the dark. The reaction was stopped with 100 μl/well of 0.1 M H2SO4. Each ELISA plate was measured using a SpectraMax 190 microplate reader (Molecular Devices) and the associated software SoftMax Pro 6.2.2 (Molecular Devices).
In assessment of IL-2 production, sample platforms were removed from the system 700 at 24-hour increments for 4 days and placed in 2 mL of media and stimulated with Macrophage Colony Stimulating Factor (MCSF, 50 ng/mL) and lipopolysaccharide (LPS, 100 ng/ml). Macrophage function was assessed by cytokine bead capture of soluble IL-12p70 using the Legendplex Human Macrophage/Microglia kit. (BioLegend, San Diego, CA). The media was harvested and centrifuged at 6000×g for 60 seconds to separate debris from the supernatant. 25 μl of each respective sample supernatant was then added to a 96 well V-bottom plate followed by 25 μl of the assay buffer and 25 μl of the capture beads. The plate was then incubated for 2 hours at room temperature on a shaker to allow cytokine-bead binding. The plate was then centrifuged at 250×g for 5 minutes, inverted and flicked to remove the supernatant leaving the beads. The beads were then washed, followed by a second centrifugation at 250×g for 5 minutes and flicking of the wash buffer. 25 μl of the biotinylated detection antibody was then added to each well and incubated at room temperature, in the dark on a shaker, at 800 rpm. 25 μl of strepavidinphycoerythrin (SAPE) was added and the plate was placed on a shaker for 30 minutes. The plate was washed and flicked, and the beads were suspended in 150 μl of wash buffer. Samples were analyzed by flow cytometry with a BD FACSCanto I (BD Biosciences, San Jose, CA). A 575 nm phycoerythrin (PE) channel was used as the reporter channel and a 660 nm allophycocyanin (APC) channel was used as the classification channel. A linear mode forward and side scatter were used. Standard samples were first run to generate a standard curve followed by the experimental samples. Sample cytokine concentrations were determined using the Legendplex Data Analysis Software (BioLegend, San Diego, CA).
In any of the above discussed examples, tissue from system 700 was subject to similar RNA sequencing. For example, tumor bearing peritoneum samples were collected on day 0 and day 4 to assess for conservation of RNA expression. Selection of gene panels was made from commercially available methods that had been previously empirically validated. RNA was extracted from formalin fixed, paraffin embedded samples of tumor and 10 ng of RNA from each sample was used.
In any of the above discussed examples, tissue from system 700 was subject to similar tumor imaging techniques. For example, for live imaging, sample platforms were removed from the system 700 at indicated time points and transferred to a 24 well plate containing 1 ml perfusate supplemented with 0.5 μg fluorophore-conjugated antibodies (CD45; CD44; CD117) and incubated for 3 hours at 37° C. Sample platforms 300 were first secured in the first member 806 of imaging platform holder 800. The first member 806 was then screwed into second member 814 that fit on dish 820 with a coverglass 836 on its bottom. Following live imaging, the sample platforms 300 were retrieved from the imaging platform holder 800 and returned to the perfusion circuit to be reimaged at a later timepoint.
Image acquisition was performed on an inverted Leica SP8 setup using multiphoton excitation at 870 nm together with a 25×/0.95 W VISIR lens. For the timeseries, images were collected at 2-minute intervals for indicated time periods. Images were analyzed using ImageJ software. Following live imaging, tissue was preserved in a fixative solution containing 1% paraformaldehyde (diluted 1:4) at 4° C. for 24 hours, washed thoroughly with PBS, and then incubated in a 30% sucrose solution for 3 days. After incubation, specimens were removed from the sample platform 300 and mounted vertically in an embedding frame (HistoMold, 6×8 mm) with the perimeter of the tissue situated perpendicular to the base of the mold. The specimen was frozen with Optimum Cutting Temperature Compound (Tissue-Tek) on dry ice. The tissue was then sliced into 30 μm sections and placed on polarized microslides. The sections were incubated in a blocking solution containing 1% BSA, 0.3% Triton-X 100, and 0.05% NaN3 for 24 hours. After blocking, samples were then stained in primary antibody solutions for 48 hours, and secondary antibody solutions, if needed, for an additional 24 hours. Once staining was completed, samples were mounted with Fluoromount-G (Invitrogen) and secured with a cover glass. Images were acquired with an inverted Leica SP8 microscope equipped with a white light laser, using a 40× objective lens. Images were then processed with the Imaris software.
In view of the above described implementations of the disclosed subject matter, this application discloses the additional examples enumerated below. It should be noted that one feature of an example in isolation or more than one feature of the example taken in combination and, optionally, in combination with one or more features of one or more further examples are further examples also falling within the disclosure of this application.
Clause 1. An ex vivo tissue analysis method, comprising:
Clause 2. The ex vivo tissue analysis method of any example herein, particularly Clause 1, wherein the flowing the perfusate includes circulating the perfusate from an outlet of the perfusion chamber to an inlet of the perfusion chamber.
Clause 3. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-2, wherein during the flowing, perfusate within the perfusion chamber is agitated or mixed by passive structures within the perfusion chamber, by active structures within the perfusion chamber, or both.
Clause 4. The ex vivo tissue analysis method of any example herein, particularly Clause 3, wherein the active structures include a stirrer bar that is rotated by an external magnetic field.
Clause 5. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 3-4, wherein the passive structures include one or more baffles within the perfusion chamber, configuration of inlet and/or outlet ports within the perfusion chamber, arrangement of inlet and/or outlet ports within the perfusion chamber, orientation of perfusate flow within the perfusion chamber, or any combination of the foregoing.
Clause 6. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-5, wherein the resected live tissue comprises part of a solid tumor.
Clause 7. The ex vivo tissue analysis method of any example herein, particularly Clause 6, wherein the solid tumor is a metastasis of a primary cancerous tumor.
Clause 8. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 6-7, wherein the resected live tissue comprises a heterogenous human tumor microenvironment (TME) including 3-D tissue structure, stromal components, and immune populations.
Clause 9. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-8, wherein the resected live tissue comprises a surface portion of a mesothelium of the patient.
Clause 10. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-9, wherein the perfusate comprises blood plasma or culture medium combined with blood plasma.
Clause 11. The ex vivo tissue analysis method of any example herein, particularly Clause 10, wherein the blood plasma is from the patient (e.g., autologous plasma).
Clause 12. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-11, further comprising:
Clause 13. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-12, wherein, during the flowing, the perfusate is periodically or continuously infused with a drug, a hormone, water, or any combination of the foregoing.
Clause 14. The ex vivo tissue analysis method of any example herein, particularly Clause 13, wherein the hormone comprises insulin.
Clause 15. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-14, further comprising, during or before the flowing, introducing a drug for the resected tissue portion into the perfusate.
Clause 16. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-15, further comprising:
Clause 17. The ex vivo tissue analysis method of any example herein, particularly Clause 16, wherein (e) comprises immersing a first surface of the resected tissue portion in a drug (e.g., chemotherapeutic agent) for a predetermined period of time, the first surface being exposed from the sample platform.
Clause 18. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-17, further comprising introducing oxygen to the perfusate by:
Clause 19. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-18, further comprising introducing carbon dioxide to the perfusate so as to change or maintain a pH of the perfusate during the flowing.
Clause 20. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-19, wherein the mounting comprises securing a periphery of the resected tissue portion to a circumferential portion of the sample platform.
Clause 21. The ex vivo tissue analysis method of any example herein, particularly Clause 20, wherein the securing is by way of a suture.
Clause 22. The ex vivo tissue analysis method of any example herein, particularly Clause 20, wherein the securing is by way of an annular flexible member that applies a radial compressive force to the periphery of the resected tissue portion.
Clause 23. The ex vivo tissue analysis method of any example herein, particularly Clause 22, wherein the securing includes:
Clause 24. The ex vivo tissue analysis method of any example herein, particularly Clause 23, wherein the annular flexible member comprises a rubber band, and the advancing comprises rolling or sliding the rubber band along the outer circumferential surface of the applicator.
Clause 25. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-24, wherein:
Clause 26. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-25, further comprising resecting the portion of live tissue from the patient.
Clause 27. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-26, further comprising, after the flowing the perfusate:
Clause 28. The ex vivo tissue analysis method of any example herein, particularly Clause 27, further comprising, after the imaging, returning the resected tissue portion back to the perfusion chamber, and continuing the flowing of the perfusate through the perfusion chamber.
Clause 29. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 27-28, wherein the positioning the sample platform comprises:
Clause 30. The ex vivo tissue analysis method of any example herein, particularly Clause 29, wherein the adjusting an axial position of the imaging platform holder comprises rotating a threaded male shaft within a threaded female recess.
Clause 31. The ex vivo tissue analysis method of any example herein, particularly any one of Clauses 1-30, further comprising:
Clause 32. The ex vivo tissue analysis method of any example herein, particularly Clause 31, wherein the ring gasket is formed of an oxygen-generating polymer, and the providing the ring gasket and the filling at least part of the well are effective to create an oxygen microenvironment for the resected tissue portion while outside of the perfusion chamber.
Clause 33. A system for ex vivo tissue analysis, comprising:
Clause 34. The system for ex vivo tissue analysis of any example herein, particularly Clause 33, wherein:
Clause 35. The system for ex vivo tissue analysis of any example herein, particularly Clause 34, wherein two arms extend between and connect the annular platform to the circular base, the arms being on opposite sides of the opening in the annular platform from each other, and each arm has one or more through-holes or openings in a portion proximal to the annular platform.
Clause 36. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 34-35, wherein the annular platform includes a circumferential groove configured to receive an attachment member for securing a periphery of the mounted resected tissue portion to the annular platform.
Clause 37. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 34-36, wherein:
Clause 38. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-37, wherein the inlet of the perfusion chamber is at a height with respect to a bottom surface of the perfusion chamber that is different from that of the outlet of the perfusion chamber.
Clause 39. The system for ex vivo tissue analysis of any example herein, particularly Clause 38, wherein the inlet is higher than the outlet.
Clause 40. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-39, further comprising an oxygen-generating biomaterial (OGB) disposed within the perfusion chamber, the OGB being constructed to release oxygen into perfusate within the internal volume of the perfusion chamber.
Clause 41. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-40, further comprising a pump that moves perfusate through the internal volume of the perfusion chamber via flow into the inlet and out of the outlet.
Clause 42. The system for ex vivo tissue analysis of any example herein, particularly Clause 41, further comprising a fluid circuit with fluid conduits connecting the outlet of the perfusion chamber to the inlet of the perfusion chamber, wherein the pump is constructed to circulate perfusate through the fluid conduits and to flow the perfusate through the internal volume of the perfusion chamber.
Clause 43. The system for ex vivo tissue analysis of any example herein, particularly Clause 33-42, further comprising:
Clause 44. The system for ex vivo tissue analysis of any example herein, particularly Clause 43, wherein the gas exchanger comprises an oxygenator.
Clause 45. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 42-44, wherein the fluid circuit comprises a sampling port or valve, through which a portion or all of the perfusate is removed from the fluid circuit.
Clause 46. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 42-45, wherein the fluid circuit comprises an infusion port or valve, through which a drug, a hormone, cells, fluid, or any combination of the foregoing is introduced into the fluid conduits.
Clause 47. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-46, further comprising an incubator constructed to maintain a predetermined temperature for components therein, at least the perfusion chamber being disposed within the incubator.
Clause 48. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-47, further comprising a passive structure that agitates or mixes perfusate within the perfusion chamber, an active structure that agitates or mixes perfusate within the perfusion chamber, or any combination of the foregoing.
Clause 49. The system for ex vivo tissue analysis of any example herein, particularly Clause 48, wherein the active structure comprises a stirrer bar within the internal volume of the perfusion chamber that is rotated by an externally applied magnetic field.
Clause 50. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 48-49, wherein the passive structure comprises baffles within the perfusion chamber, arrangement of inlet and/or outlet ports within the perfusion chamber, orientation of perfusate flow within the perfusion chamber, or any combination of the foregoing.
Clause 51. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-50, further comprising a controller coupled to and controlling operation of the pump, the gas exchanger, the CO2 pressure control valve, the sampling port or valve, the infusion port or valve, the incubator, or any combination of the foregoing.
Clause 52. The system for ex vivo tissue analysis of any example herein, particularly Clause 51, wherein the controller comprises:
Clause 53. The system for ex vivo tissue analysis of any example herein, particularly any one of Clauses 33-52, further comprising an imaging platform holder constructed to receive the sample platform therein for microscopic imaging of the mounted tissue portion.
Clause 54. The system for ex vivo tissue analysis of any example herein, particularly Clause 53, wherein the imaging platform holder comprises:
For components of the disclosed systems that come into contact with the tissue sample (either directly or indirectly via fluid), such components can be made of any suitable biocompatible material. If reusability of components is desired, the components can be made of a biocompatible material that is also autoclavable for sterilization. For example, embodiments of the disclosed sample holder can be formed of a composite resin (e.g., a dental resin including bisphenol A-glycidyl methacrylate), medical grade stainless steel, glass, or a polymer (e.g., a fluoropolymer such as polytetrafluoroethylene (PTFE)).
Although some of the embodiments described above refer to “imaging,” the production of an actual image is not strictly necessary. Indeed, the mentions of “imaging” are intended to include the acquisition of data where an image may not be produced. Accordingly, the use of the term “imaging” herein should not be understood as limiting.
Any of the features illustrated or described with respect to
In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be taken as limiting the scope of the disclosed technology. Rather, the scope is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.
This application claims the benefit of U.S. Provisional Application No. 62/988,783, entitled “Systems, Methods, and Devices for Ex Vivo Analysis of Resected Tissues Samples,” filed Mar. 12, 2020, which is incorporated by reference herein in its entirety.
This invention was made with Government support under project number ZIA BC 011759 by the National Institutes of Health (NIH). The Government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2021/021525 | 3/9/2021 | WO |
Number | Date | Country | |
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62988783 | Mar 2020 | US |