System and method for predicting the effectiveness of a treatment

Abstract

Systems and methods are disclosed for predicting patient response to various drugs and / or drug combinations using ex vivo dosing and imaging methods. In one example, a method for determining the effectiveness of a treatment includes analyzing solid cell cultures over time, e.g., a first and second response of the solid cell cultures to each treatment can be compared to determine the treatment effectiveness of each treatment. Systems and methods are disclosed for applying treatments to cell cultures and analyzing the cell cultures and effectiveness.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 219,697, filed Jul. 8, 2021, entitled "Treatment Efficacy Prediction Systems and Methods", which is hereby incorporated in its entirety for all purposes. The described embodiments are generally related to systems and methods for determining the efficacy of treatments, such as anti - cancer agents and the like.

Background Art

[0002] A series of treatment agents, such as anti - cancer agents and the like, can be used to treat cancerous cells, such as cells associated with various types of tumors. Several factors, such as tumor type, progression, patient characteristics, characteristics of the anti - cancer agent, etc., can affect the efficacy of a given treatment. These and other factors may prevent the ability of healthcare providers to select the most suitable anti - cancer agent, such as the one with the highest efficacy. There is a risk that the anti - cancer treatment may result in only minimal efficacy and / or reduce the overall treatment of the patient. Similarly, other types of treatments for a particular disease can vary depending on the patient - specific factors. Therefore, there is a need for systems and techniques to facilitate the discovery of individualized biomarkers and the diagnosis and / or prediction related to the efficacy of specific medical treatments, such as treatments using anti - cancer agents and other treatment agents.

Summary of the Invention

[0003] Embodiments of the present invention are related to systems and methods for predicting treatment efficacy. In one example, a microfluidic chip is disclosed. The microfluidic chip comprises a body defining a channel and a cell culture chamber fluidly connected to the flow channel. The microfluidic chip further comprises a connecting portion attached to the body and defining an inlet and an outlet. The channel extends between the inlet and the outlet, defining a flow path in which the cell culture chamber is positioned. The microfluidic chip further comprises a gas permeable membrane covering the cell culture chamber. In another example, the channel is configured to deliver growth medium to a cell culture chamber. The cell culture chamber may have a substantially cylindrical shape. For example, the cell culture chamber may have a substantially cylindrical shape, preferably with a diameter of about 6.75 mm. In other cases, the diameter may be greater or smaller than 6.75 mm, depending on the specific application, for example, at least about 5.0 mm, at least about 3.0 mm, or other suitable diameters. In another example, the main body defines a cell culture chamber having a closed bottom and an open top. A gas permeable membrane may cover the open top of the cell culture chamber. Furthermore, the gas permeable membrane may be attached to the main body portion with an adhesive. In another example, the cell culture chamber may be a first cell culture chamber. Thus, the main body may further define a second cell culture chamber fluidly connected to a channel along a flow path between the first cell culture chamber and the inlet or outlet of the connecting portion. The first cell culture chamber may have a first volume, and the second cell culture chamber may have a second volume different from the first volume; however, this is not mandatory. Furthermore, the first cell culture chamber may have a first shape, and the second cell culture chamber may have a second shape different from the first shape; however, this is also not mandatory.

[0004] In another example, the main body has a multilayer structure. Thus, the multilayer structure comprises a first main body layer defining a cell culture chamber. The multilayer structure further comprises a second main body layer connected to the first main body layer and defining a channel that is fluidly connected to the cell culture chamber. The multilayer structure further comprises a third main body layer connected to the second main body layer on the opposite side of the first main body layer and defining an opening or pore on the cell culture chamber. In some cases, the connecting portion may be joined to the third main body layer. Thus, the third main body layer further defines a first lumen that is fluidly connected to the inlet of the connecting portion and extends to the flow channel of the second main body layer. The third main body portion may further define a second lumen that is fluidly connected to the outlet of the connecting portion and extends to the flow channel of the second main body layer.

[0005] In another example, the inlet and outlet define the barb of the tube. The barb may protrude from the outermost surface of the tip. Many material constructs are conceivable, but the body may include or be entirely formed from acrylic or silicone-based materials. In another example, a microfluidic device is disclosed. The microfluidic device may include a dosing bank having a plurality of reservoirs. Each of the plurality of reservoirs may be configured to hold a growth medium. The microfluidic device may further include a staging section or “stage” configured to arrange a plurality of microfluidic chips. The plurality of reservoirs may correspond to a plurality of microfluidic chips. In some cases, the plurality of reservoirs may be used with any given microfluidic chips. The microfluidic device may further include a pump that is fluidically connectable to the plurality of reservoirs and the plurality of microfluidic chips, for defining a fluid circuit between each corresponding pair of the plurality of reservoirs and the plurality of microfluidic chips. The pump may further induce circulation of the growth medium through the fluid circuit for each corresponding pair.

[0006] In another example, each reservoir of multiple reservoirs is fluidically isolated from one another. Thus, fluid circuits can be fluidly isolated from one another. The pump may be further configured to selectively induce the circulation of growth medium through the individual fluid circuits of multiple fluid circuits. The growth medium may contain treatment agents, such as anticancer drugs, and / or suspended cells. Thus, cells may be present in circulation with the growth medium, as may contain drugs, cells, and / or other agents. In some cases, the step of circulating cells can be used to create an immunotherapy model or to test cell therapy. Additionally or alternatively, the step of circulating other agents may be useful in characterizing the behavior or characteristics of cells in circulation.

[0007] In another example, multiple reservoirs may be exposed to the atmosphere. For instance, a reservoir may have a lid that can be opened to expose the reservoir to air for the purpose of loading a treatment. Once the treatment is loaded, the lid can be closed. Once the reservoir is closed with the lid, the microfluidic device can operate as described herein. Thus, multiple reservoirs may be configured to accept treatment agents or combinations of agents during the operating period of the pump. In another example, the drug bank may comprise a tray or other structure configured to hold multiple reservoirs in a substantially upright position. The device may further comprise tubing that fluidly connects a pump to each reservoir and each microfluidic tip housed in a staging section. Another example discloses a method for forming a solid culture. The method includes the step of isolating target cells from a patient sample. The method further includes the step of forming stained cells from the isolated cells by staining the isolated cells with a photoresponsive dye. The method further includes the step of encapsulating the stained cells within a hydrogel. In one example, the hydrogel may contain hyaluronic acid, collagen, and / or other elements configured to mimic the core components of the extracellular matrix of human tissue and / or disease-specific cell niches.

[0008] In another example, the method may further include the step of culturing the dissociated cells in a hydrogel. The culturing step may further include forming a two-dimensional cell culture of the dissociated cells. The culturing step may further include forming a three-dimensional cell culture of the dissociated cells. The culturing step may further include forming a cell culture consisting of a single population of dissociated cells. The culturing step may further include forming a cell culture from multiple cell types. In another example, the dissociated cells include cancer cells, as well as normal / non-transformed cells, stromal cells, or immune cells. Thus, the culturing step further includes forming a co-culture consisting of cancer cells, normal / non-transformed cells, stromal cells, and / or immune cells. The dissociated cells can be isolated from patient-derived tissue or tumor samples.

[0009] In another example, the method may further include the step of forming spheroids or organoids. In some cases, the method may include the step of culturing the spheroids or organoids in a hydrogel. The spheroids or organoids may include cancer cells, normal / untransformed cells, stromal cells, or immune cells. In some cases, the culturing step further includes forming a co-culture consisting of cancer cells, normal / untransformed cells, stromal cells, and / or immune cells. Isolated cells may be isolated from patient-derived tissue or tumor samples. In another example, the method further includes the step of processing the patient sample using digestive enzyme-based operations, hemolytic solutions, or selective operations to isolate target cells from the patient sample. Processing may allow the isolation of many different cell types, such as many different cell types that remain viable, from the patient tissue or tumor sample. Thus, different cells, including several cells, can be co-cultured in either a dissociated cell culture or a spheroid / organoid, attached to cancer cells, normal / non-transformed cells, immune cells, and / or stromal cells.

[0010] In another example, a photoresponsive dye may be configured to allow tracking of target cells via fluorescence microscopy. The photoresponsive dye may be configured to stain the mitochondria of target cells to track viable cells. The photoresponsive dye may be configured to stain the nuclei of target cells to track dead cells. In another example, the step of forming stained cells may further include staining isolated cells with a first photoresponsive dye. The first photoresponsive dye may be configured to stain the mitochondria of target cells in order to track viable cells. The step of forming stained cells may further include staining isolated cells with a second photoresponsive dye, the second photoresponsive dye may be configured to stain the nuclei of target cells in order to track dead cells. In another example, a photoresponsive dye may be configured to cause a change in color in stained cells when the stained cells transition from living to dead cells. Target cells may, without limitation, be tumor cells, including breast cancer, colorectal cancer, lung cancer, kidney cancer, pancreatic cancer, ovarian cancer, brain cancer, or stomach cancer. In another example, the patient sample may include tissue slices, surgical excisions, and / or xenografts. The patient sample may also include biopsy samples, such as core needle biopsy samples in specific circumstances. Thus, the method may further include the step of culturing the tissue slices, cores, surgical excisions, and / or xenografts in a hydrogel.

[0011] In another example, a method for loading onto a microfluidic chip is disclosed. The method includes the step of positioning and configuring a solid cell culture within the cell culture chamber of the microfluidic chip. The microfluidic chip comprises a body defining the cell culture chamber and a channel traversing the cell culture chamber and extending between the inlet and outlet of the microfluidic chip. The method further includes the step of positioning a gas permeable membrane over the cell culture chamber while the inlet and outlet remain exposed for connection to the circulating system.

[0012] In another example, the placement step may further include attaching a gas permeable membrane to the main body and covering the cell culture chamber. The placement and configuration step of the solid cell culture may further include dropping a certain amount of the solid cell culture (e.g., cell culture in a hydrogel) into the cell culture chamber using a pipette. In another example, a solid cell culture may be the first solid cell culture, and a cell culture chamber may be the first cell culture chamber. Thus, the method may further include the step of arranging and configuring a second solid cell culture within a second cell culture chamber of a microfluidic chip. The second cell culture chamber may be defined by a body and may be fluidly connected to a channel between an inlet and an outlet. The first cell culture chamber may have a first volume, and the second cell culture chamber may have a second volume different from the first volume. The first cell culture chamber may have a first shape, and the second cell culture chamber may have a second shape different from the first shape.

[0013] In another example, a method for operating a microfluidic chip is disclosed. The method includes the step of fluidically connecting the microfluidic chip to a microfluidic device in order to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump. The microfluidic chip contains a solid cell culture, and the reservoir contains a growth medium. The method further includes the step of causing a flow of growth medium through the circuit so that the solid cell culture in the microfluidic chip is exposed to the growth medium and an exposed cell culture is formed. As used herein, “solid cell culture” means cells in a hydrogel material. “Exposed cell culture” means a situation in which the solid cell culture is exposed to a medium (e.g., growth medium) while the cell culture itself remains solid (e.g., growth medium flows along a hydrogel containing cells). As used further herein, “liquid cell culture” means cells exclusively in a liquid medium. For example, a liquid cell culture may include immune cells introduced into a growth medium / serum and then circulated using the systems and techniques described herein. The method includes the step of analyzing the response of the solid cell culture to the growth medium.

[0014] In another example, the growth medium contains a treatment agent. In some cases, the fluid coupling step further includes fluidically coupling a first tubing section to the inlet of a microfluidic device. The first tubing section may be connected to a second tubing section which is fluidically coupled to a reservoir, but the first and second tubing sections define a common tube. The fluid coupling step may further include fluidically coupling a third tubing section to the outlet and reservoir of the microfluidic device in order to define a fluid circuit. The microfluidic device may define a flow path through which fluid passes. In another example, the microfluidic chip may comprise a cell culture chamber holding a hydrogel containing target cells. The channel may traverse and / or pass near the cell culture chamber. Thus, the method may further include causing a flow of growth medium along the channel while the hydrogel confines the target cells to prevent them from escaping from the cell culture chamber. In another example, the first tubing section may be fluidically connected to a pump. The second tubing section may be fluidically connected to a reservoir. The first and second tubing sections may be parts of the same tube. The microfluidic device may further include a third tubing section connected to a reservoir and a microfluidic tip to complete the circuit. The circuit may be a closed circuit.

[0015] In another example, the method further includes the step of fluidically connecting a second microfluidic chip to a microfluidic device in order to define a second fluid circuit between a second microfluidic chip, a second reservoir, and a pump; the second microfluidic chip contains a second solid cell culture, and the second reservoir contains a second growth medium. The method may further include the step of causing a second flow of the second growth medium through the second circuit so that the second solid cell culture on the second microfluidic chip is exposed to the second growth medium. The method may further include the step of analyzing the response of the second solid cell culture to the second growth medium.

[0016] In another example, the growth medium and the second growth medium contain different treatment agents, such as different anticancer drugs or cells. The method may further include a step of determining the effectiveness of the treatment by comparing the response of the first solid cell culture with the response of the second solid cell culture. The first and second circuits may be fluidically isolated from each other. Thus, the pump may be configured to control the first flow and the second flow independently. In another example, the method may further include a step of fluidically disconnecting the microfluidic chip from the microfluidic device prior to the step of analysis. Thus, following the step of analysis, the method may further include a step of fluidically connecting the microfluidic chip to the microfluidic device in order to define a fluid circuit between the microfluidic chip, a reservoir, and a pump. The method may further include a step of inducing another flow of growth medium (e.g., the same growth medium or a different growth medium) through the circuit so that the solid cell culture on the microfluidic chip is exposed to the growth medium. Following the step of inducing the other flow, the method may further include a step of analyzing the subsequent response of the solid cell culture to the growth medium.

[0017] In another example, the method may further include a step of determining the effectiveness of the treatment by comparing the response of the solid cell culture to the growth medium with the subsequent response of the solid cell culture to the growth medium. The method may further include a step of analyzing the response of the solid cell culture to the growth medium to determine the quantity of a first cell population. The method may further include a step of analyzing the subsequent response of the solid cell culture to the growth medium to determine the quantity of a second cell population. As described herein, the method may further include a step of analyzing further subsequent responses of the solid cell culture, and a step of determining the quantity of a third cell population, a fourth cell population, etc., over several days or other suitable intervals. The method may further include a step of comparing the quantity of the first cell population with the quantity of the second cell population to determine a change in the quantity of cell populations that suggests the effectiveness of the treatment. Thus, the method may further include a step of analyzing the response of a solid cell culture to a growth medium in order to determine the location of a first cell population, and the step of analyzing the subsequent response of the solid cell culture to the growth medium may include determining the location of a second cell population. As described herein, the method may further include a step of analyzing further responses of the solid cell culture, and a step of determining the location of a third cell population, a fourth cell population, etc., over several days or at other suitable intervals. The method may further include a step of comparing the location of the first cell population with the location of the second population to determine a change in the location of the cell population that suggests the effectiveness of the treatment.

[0018] In another example, the analysis step may include performing fluorescence microscopy operations on the solid cell culture on the microfluidic chip. In some cases, the analysis step may include collecting three-dimensional images of the solid cell culture on the microfluidic chip. The analysis step may further include collecting two-dimensional images of the z-stack of the solid cell culture on the microfluidic chip. The analysis step may further include analyzing multiple responses of the solid cell culture to the growth medium over time. The analysis may be performed daily. In another example, the analytical step may include performing confocal microscopy operations on the solid cell culture on the microfluidic chip. Additionally or alternatively, the analytical step may include performing bright-field microscopy operations on the solid cell culture on the microfluidic chip. Additionally or alternatively, the analytical step may include performing lattice light sheet microscopy operations on the solid cell culture on the microfluidic chip.

[0019] In another example, the analysis step involves using one or more processing elements of a computer to execute instructions on a non-transient computer-readable medium in order to determine the effectiveness of treating growth medium treatments on solid cell cultures.

[0020] Another example discloses a method for analyzing solid cell cultures over time. The method includes determining a first response of a solid cell culture to a growth medium containing a treatment agent. The solid cell culture is held in a cell culture chamber of a microfluidic chip. The method further includes determining a second response of a solid cell culture to a growth medium containing a treatment agent. The method further includes comparing the first and second responses to determine the effectiveness of the treatment. In some cases, as described herein, multiple responses of solid cell cultures may be compared to determine the effectiveness of the treatment, for example, comparing three, four, or more responses for each solid cell culture / chip for a given application. Thus, while an example of comparing first and second responses is presented for illustrative purposes, it is recognized that the computer vision and imaging techniques described herein can be applied to compare and analyze any number of responses over any suitable period of time. In another example, either or both of the first or second response may include at least one of the following: the color of the solid cell culture, the pixel intensity of the image of the solid cell culture, the shape of the solid cell culture, the size of the solid cell culture, the position of cells in the solid cell culture, or the number of cells in the solid cell culture. The effectiveness of the treatment may represent the viability of cells in the solid cell culture in response to the treatment agent. In another example, the step of determining the first response includes taking an image of the solid cell culture. From this, the method may further include determining cell viability from the image and predicting a patient response to the treatment agent based on the cell viability. The method may further include determining cell proliferation from the image and predicting a patient response to the treatment agent based on the cell proliferation. The method may further include determining cell location from the image and predicting a patient response to the treatment agent based on the cell location.

[0021] In another example, the image may be a first image. From this, the step of determining the second response may include taking a second image of the solid cell culture. The method may further include comparing the first image and the second image (and / or additional images) to determine the cell migration distance of the cells of the solid cell culture over time. The method may further include predicting a patient response to the treatment agent using the cell migration distance. In another example, the method may further include comparing the first image and the second image to determine the cell migration speed of the cells of the solid cell culture over time. Next, the method may further include predicting a patient response to the treatment agent using the cell migration speed. In another example, the method may further include comparing the first image and the second image to determine the migration distances of a plurality of cells that define a subset of the solid cell culture over time. Next, the method may further include predicting a patient response to the treatment agent using the migration distances.

[0022] In another example, the method may further include comparing the first image and the second image to determine the migration speeds of a plurality of cells over time. Next, the method may further include predicting a patient response to the treatment agent using the migration speeds. In another example, in relation to determining migration distance and / or migration speed, a subset of cells may include the most invasive cells, accounting for 5% of the multiple cells. In other cases, the multiple cells may include the most invasive cells, accounting for 2% of the multiple cells. In other cases, the multiple cells may include the most invasive cells, accounting for 1% of the multiple cells. Additionally or alternatively, the multiple cells may include a subset of cells expressing a specific biomarker.

[0023] In another example, the method may further include the step of comparing a first image with a second image to determine the cell having the maximum migration vector in a solid cell culture over time. The method may then further include the step of using the maximum migration vector to predict the patient's response to a treatment agent. In another example, the method may further include the step of comparing a first image with a second image to determine the cell having the highest migration speed in a solid cell culture over time. The method may then further include the step of using the highest migration speed to predict the patient's response to a treatment agent.

[0024] In another example, one or both of the steps of determining a first response or a second response include determining the characteristics of a single cell among a group of cells. The single cell among the group of cells may have characteristics that can be used to predict the response of the group of cells (for example, a cell with the longest moving vector may be a predictive biomarker). Thus, the step of determining a first response includes determining the characteristics of a single cell in a first period. The step of determining a second response includes determining the characteristics of a single cell in a second period following the first period. The method then further includes predicting the patient response to the treatment agent based on a comparison of the measured characteristics of the single cell in the first period and the second period and / or additional periods, as described herein. In another example, either or both of the first or second image include a weighted cytometry of a single cell or multiple cells. The method may then further include a step of predicting the patient's response to a treatment based on the weighted cytometry. In some cases, the first image includes a first weighted cytometry, and the second image includes a second weighted cytometry. The method then further includes a step of predicting the patient's response to a treatment based on a comparison of the first weighted cytometry and the second weighted cytometry.

[0025] In another example, one or both of the first or second image include information relating to the radius or diameter of the spheroid or organoid. The method may further include a step of predicting the patient's response to the treatment based on the radius or diameter of the spheroid or organoid. In some cases, the first image includes the first radius or first diameter of the spheroid or organoid, and the second image includes the second radius or second diameter of the spheroid or organoid. The method then further includes a step of predicting the patient's response to the treatment based on a comparison of the first radius or first diameter with the second radius or second diameter. In some cases, dissociated / single cells may be analyzed in a similar manner. For example, dissociated / single cells may be tracked or measured, or measured separately, according to substantially any relevant metrics as described herein. As an illustrative example, the count and / or location of dissociated / single cells resulting in spheroids, migration distance, migration speed, etc., may be determined and analyzed according to the techniques described herein for determining the effectiveness of a treatment. In another example, one or both of the first or second image include information relating to one or more of the length, width, or height of the surgical excision, tissue slice, and / or xenograft. The method further includes predicting the patient's response to the treatment based on the length, width, or height of the surgical excision, tissue slice, and / or xenograft. In some cases, the first image includes a first length, first width, or first height of the surgical excision, tissue slice, and / or xenograft, and the second image includes a second length, second width, or second height of the surgical excision, tissue slice, and / or xenograft. The method then further includes predicting the patient's response to the treatment based on a comparison of the first length, first width, or first height with the second length, second width, or second height.

[0026] In another example, the comparison step further includes using one or more processing elements of a computer to execute instructions on a non-temporary computer-readable medium in order to determine the effectiveness of the treatment.

[0027] Further embodiments and models will become apparent from reference to the drawings and from consideration of the following description, in addition to the exemplary embodiments and models described above. [Brief explanation of the drawing]

[0028] [Figure 1] This figure shows a functional diagram of the treatment effectiveness prediction system. [Figure 2] This figure shows a flow diagram for determining the effectiveness of a treatment. [Figure 3] This figure shows an example of a patient sample. [Figure 4] This figure shows a user interface containing information related to stained cells. [Figure 5] This figure shows a chart illustrating the relative concentration and size distribution of living and dead cells. [Figure 6] This figure shows a 2D microscope image of an example of a spheroid. [Figure 7A] This figure shows the first representation of an ovarian tumor, suggesting viable cells. [Figure 7B] This figure shows a second representation of the ovarian tumor in Figure 7A, suggesting dead cells. [Figure 7C] This figure shows a third visible representation of the ovarian tumor in Figure 7A, which includes a complex of living and dead cells. [Figure 8A] This figure shows an exploded view of an example of a microfluidic chip. [Figure 8B] Figure 8A shows a cross-sectional view of a microfluidic chip. [Figure 8C] This figure shows a cross-sectional view of a flow restrictor used in conjunction with a microfluidic chip within a fluid circuit. [Figure 9A] This figure shows the operation of the microfluidic chip in Figure 8A. [Figure 9B] This figure shows another operation of the microfluidic chip shown in Figure 8A. [Figure 9C] This figure shows another operation of the microfluidic chip shown in Figure 8A. [Figure 9D] This figure shows another operation of the microfluidic chip shown in Figure 8A. [Figure 9E] This figure shows another operation of the microfluidic chip shown in Figure 8A. [Figure 10] This figure shows a schematic top view of the microfluidic device used with the microfluidic chip shown in Figure 8A. [Figure 11] This is an isometric view of the microfluidic device used for drug delivery. [Figure 12] This figure shows examples of spheroids from isolated and stained tumor cells after drug administration. [Figure 13A] This figure shows a chart representing the initial number of cells at the first time point. [Figure 13B] This figure shows another chart representing the second number of cells at the second time point. [Figure 14A] This figure shows a chart representing the three-dimensional position of the number of cells at the first point in time. [Figure 14B]This figure shows another chart representing the three-dimensional position of the number of cells at the second time point. [Figure 15] This figure shows a chart representing a quiver plot, which suggests the three-dimensional migration of cells. [Figure 16] This figure shows an example of a report that includes visualization of different medication procedures performed on a given patient sample. [Figure 17] This figure shows a chart representing the distribution of cancer cell survival rates based on one or more of the drug administration procedures described herein. [Figure 18] This figure shows a flow diagram for forming a cell culture. [Figure 19] This figure shows a flow diagram for loading a microfluidic chip. [Figure 20] This figure shows a flow diagram for administering and imaging cultures in a microfluidic chip. [Figure 21] This figure shows a flow diagram for determining the effectiveness of a treatment. [Figure 22] This is a diagram showing a functional block diagram of a system including computing devices. [Modes for carrying out the invention]

[0029] The following description includes sample systems, methods, and apparatus that embody various elements of this disclosure. However, the disclosure described herein should be understood to be applicable in various forms in addition to those described herein. This disclosure relates to systems and methods for predicting patient responses to various drugs and / or drug combinations using ex vivo administration and imaging techniques. In some examples, the systems and methods may be applied to ex vivo monitoring of oncology, disease progression, e.g., cancer progression, testing of treatment agents, e.g., anticancer drugs, patient stratification, and other testing of the effectiveness of medical treatments. The systems and methods enable the discovery, identification, or validation of therapeutic, diagnostic, and / or predictive biomarkers for the purpose of drug development and treatment decision-making.

[0030] In one embodiment, an organ-on-a-chip and a computer vision system are used to analyze biomarkers for predicting a patient's response to a treatment agent, such as an anticancer drug. In one example of the implementation, tumor and / or healthy tissue samples may be cultured in a hydrogel or other environment and placed in a cell culture chamber of a microfluidic chip. The tumor and / or healthy tissue sample may be divided into multiple segments, e.g., fixed volumes, for culture in multiple chips or individual cell culture chambers. The sample is labeled or stained for tracking, for example, via a photoresponsive dye, such as a fluorescent dye. As described herein, stained cells may be tracked by microscopy and processed and analyzed by various computer-implemented techniques, such as the computer vision techniques disclosed herein. Fluorescent dyes may selectively stain viable cells. Other fluorescent dyes selectively stain dead cells. Some dyes may stain all cells, regardless of whether they are viable or dead. In some cases, dyes may be used to stain cells expressing a particular biomarker or target, such as a particular protein. DNA or RNA may also be stained using dyes based on the techniques disclosed herein.

[0031] A microfluidic chip, including a cell culture chamber, may be configured (e.g., fluidically connected) with a microfluidic device or other system or pump to introduce growth medium, treatment agents, and other media to the cell culture on the chip. For example, the device may contain various treatment agents held in a reservoir along with the growth medium. The media are circulated from the cell culture chamber to the microfluidic chip using a pump, such as a peristaltic pump or pneumatic pump. A solid cell culture containing target cells and a hydrogel may be deposited within the microfluidic chip. The solid cell culture may be exposed to the circulating media (which may contain treatment agents) to define the solid cell culture exposed within the microfluidic chip. The cell culture chamber of a microfluidic chip with a solid cell culture may be imaged using various techniques, such as microscopy (including confocal microscopy), to track viable and dead cells over time. Imaging may be based on cell identification resulting from cell staining or labeling. For example, the viability / death status of cells may be tracked using fluorescent dyes or other tracking dyes. Three-dimensional (3D) images, or stacks of two-dimensional (2D) images acquired layer by layer, are collected for analysis using computer vision tools.

[0032] Computer vision tools, which can be run on one or more computing devices (for example, via software running one or more algorithms or machine learning models), can track cellular features (e.g., shape, size, location (x, y, z), and color) within a given image. Cells can be stained using selectively activated dyes so that the number of viable and dead cells in the image can be determined. For example, computer vision can determine the location of a particular cell by analyzing pixel information, such as hue and intensity (e.g., viable cells may have a first color and dead cells may have a second color), and the locations and numbers of different cells can be tracked over time by analyzing subsequent images or image frames. One or more images taken at different points in time can be used to monitor the progression of malignant cells (e.g., cancer cells) ex vivo, and can also be used to predict a patient's possible response to a given drug. For example, when comparing a first image frame containing viable cells at a first location, such as a first pixel, it can be compared to a second image frame taken at a second time point, which contains viable cells at a different location. In this case, the system can estimate that the cells have moved or shifted from the first location to the second location. The distance can then be determined by analyzing the difference in pixel locations within the images. Various metrics, including cell viability over time, distance, and speed of cell migration, can be derived from such images. Such assessments, used alone or in combination, can be used to predict patient outcomes for a given drug.

[0033] Multiple microfluidic chips can be analyzed in parallel with determining the effectiveness of a series of anti-cancer treatments. This allows for comparisons between images to predict patient responses for microfluidic chips exposed to no drug (baseline) or various single and / or combination drugs, such as chemotherapy. By comparing various drugs and / or drug combinations, the techniques described herein can be used to discover therapeutic, diagnostic, and predictive biomarkers (which can aid in the treatment of cancer patients and the development of new therapeutic agents).

[0034] Herein, we will refer to the accompanying drawings, which are useful in illustrating the various characteristics of this disclosure. The following description is provided for illustrative and explanatory purposes. Furthermore, the description is not intended to limit the embodiments of the invention to those disclosed herein. Accordingly, the following teachings, as well as modifications and alterations commensurate with the skills and knowledge of the relevant art, are within the scope of the embodiments of the invention.

[0035] Referring to Figure 1, a functional diagram of the treatment efficacy prediction system 100 is shown. The treatment efficacy prediction system 100 may be broadly configured to facilitate ex vivo monitoring of disease progression, such as cancer progression, testing of treatment agents, and patient stratification. Broadly, the system 100 may comprise modules, devices, assemblies, subassemblies, etc. (described in relation to examples in Figures 3 to 17) that operate concurrently with each other to evaluate the efficacy of selected treatment agents, combination agents, and / or other treatments used when treating a particular patient. It is understood that any of the modules, devices, assemblies, subassemblies, etc. may be introduced separately from each other as described herein. Treatment agents and other agents may be more or less effective based on an individualized context consisting of the treatment regimen, patient characteristics, cancer type, and progression or stage, in addition to a series of other complex and often interrelated factors. Conventional treatment regimens may lack the ability to adequately assess these factors, which can lead to less-than-optimal treatment delivery. In conventional systems, various treatment agents are often administered and monitored in vivo, which can hinder the patient's treatment and overall health outcome. For example, using conventional approaches, it may be impractical, or even impossible, to determine whether a particular cancer treatment is effective without administering the treatment to the patient over a long period and evaluating the patient. Furthermore, the patient may have adverse or undesirable outcomes from a particular cancer treatment. Evaluating the effectiveness of a treatment can also be extremely time-consuming for clinicians, potentially leading to a deterioration of the patient's overall health during that period.

[0036] System 100, functionally represented in Figure 1, may mitigate these and other obstacles in the context of the proposed treatment agents by enabling ex vivo monitoring of cancer progression. System 100 may enable a clinician or other healthcare provider to evaluate a spectrum of different treatment agents on one or more patient samples, e.g., samples containing cancerous tumors. System 100 may enable the creation of an ex vivo environment that replicates in vivo conditions for patient samples. For example, target cells, e.g., any of the cells described herein, may be cultured and encapsulated in a hydrogel to provide structural stability for the cells, mechanical properties to mimic the physiological environment within human organs, and clues to the microenvironment. More generally, a modular environment may be provided, including a set of cell culture conditions (including conditions in which human serum is present to provide conditions that can be similar to those in the human body). Over a period of time, e.g., several days, the clinician can monitor the progression and response of each tissue sample for each treatment agent. As described in more detail below, the treatment agent with the most appropriate response or most suitable efficacy may be identified for administration to the patient for treatment purposes.

[0037] To facilitate the above, Figure 1 provides an overview of the system 100, which includes a culture module 104, a drug delivery module 108, and an analysis module 112. The culture module 104 may generally include various mechanical components, instruments, solutions, and devices used to prepare patient samples for evaluation and drug delivery. While many functions are discussed and described herein, the culture module 104 may be broadly configured to isolate target cells from patient samples, form stained cells from the isolated cells, and encapsulate the stained cells in a medium. Target cells may be derived from patient-derived tissue or tumor samples. Target cells may be derived from tissue slices, cores, surgical resections, and / or xenografts. Sample target cells may, non-limitingly, include cells associated with malignant tissue, such as cells derived from breast cancer, colorectal cancer, lung cancer, kidney cancer, pancreatic cancer, ovarian cancer, brain cancer, or gastric cancer. The cell culture module 104 can isolate target cells and stain the cells with photoresponsive dyes, such as fluorescent dyes. For example, staining with fluorescent dyes can stain or tag an entire cell population, stain individual cell populations, or stain individual components of a given cell population (e.g., staining mitochondria or individual nuclei), enabling cell tracking via fluorescence microscopy or other procedures. To facilitate the reproduction of core components of human tissue, stained cells can be encapsulated in a medium, such as a hydrogel, and thus form a solid cell culture within the culture module 104. Stained cells can be encapsulated in a medium, such as a hydrogel, or formed as spheroids or organoids, and thus form a solid cell culture.

[0038] The drug delivery module 108 may generally comprise various mechanical components, instruments, solutions, and devices used to administer treatment agents to cell cultures and collect data on the culture's response to the treatment agents. Thus, the drug delivery module 108 may include a microfluidic chip configured to accept solid cell cultures (e.g., cells and hydrogels). Growth media may be introduced into circulation along with the solid cell cultures to define the exposed solid cell cultures. The microfluidic chip may hold cells within the hydrogel and enable circulation of the treatment agent along with the growth medium in a gas-permeable environment (e.g., providing oxygen to the cells). A pump in the microfluidic device of the drug delivery module 108 may cause circulation of the treatment agent and growth medium through the chip. Multiple chips (each fluidically connected within a separate closed circuit) may allow the device to circulate different treatment agents to each chip, enabling treatment within a cell culture chamber to evaluate the effectiveness of different treatments. The chips may be analyzed at selected intervals, for example, daily (1, 2, 3, 4, 5, or more days), and the response to the treatment agent may be determined. As an example, fluorescence microscopy may be used to determine the concentrations of viable and dead cells in a given culture using a photoresponsive dye, as described in more detail below. More generally, any suitable camera or imaging device may be used, as described herein. Images may be taken over time and presented in two-dimensional and / or three-dimensional formats to provide a sufficient dataset for analysis of the effectiveness of the treatment.

[0039] The analysis module 112 may generally include various computer vision systems (e.g., computer system 2200 in Figure 22), or image analysis systems, computer-implemented technologies, and analysis devices that can be operated to determine the effectiveness of one or more treatment agents administered to cells. Photoresponsive dyes may be used to represent the concentration of dead cells, viable cells, and the transition of cells from viable to dead states. The concentration of viable or dead cells over time may, for example, provide evidence of the survival of cancer cells throughout the process of administering a particular treatment agent. The characteristics of the captured images (including, but not limited to, cell characteristics, such as the color of a solid or liquid cell culture, the pixel intensity of an image of a solid or liquid cell culture, the shape of a solid or liquid cell culture, the size of a solid or liquid cell culture, the position of cells in a solid or liquid cell culture, or the number of cells in a solid or liquid cell culture) may be further analyzed to predict the effectiveness of the treatment. In some cases, two-dimensional or three-dimensional images of cell cultures may be generated at multiple different time points throughout the process of ex vivo administration. By using photoresponsive dyes, images can contain individualized information for each cell, including cell location and type. By taking multiple images over time, the images can be compared to determine various characteristics, including cell migration, cell migration speed, and cell migration distance. The images may be further analyzed to determine, among other features, the maximum migration speed and / or maximum migration vector. Thus, a collective image of the ex vivo response of cells to a treatment agent may suggest the in vivo response to the treatment agent and can therefore be used to predict the effectiveness of the treatment.

[0040] Figure 2 shows a flow diagram 200 for determining the effectiveness of the treatment. The flow diagram 200 can perform one or more of the operations of the system 100 described above with respect to Figure 1. Accordingly, the operations described with respect to the flow diagram 200 can be performed by one or more of the culture module 104, the drug delivery module 108, and / or the analysis module 112.

[0041] In operation 204, patient samples are collected. Patient samples may be delivered to the laboratory, including samples containing tumors of various cancer types. In operation 208, tumor samples may be processed using digestive enzyme-based cell isolation kits, hemolytic solutions, and other selection steps to isolate viable cells while removing blood and contaminants. The output may be a viable mixed cell population containing various cells derived from the primary tumor, including cancer cells, normal / non-transformed cells, stromal cells, and immune cells.

[0042] In operation 212, cells may be stained using viable and / or dead cell labeling dyes. Additionally or alternatively, cell-specific / biomarker-targeting dyes may be used. Such dyes may be photoresponsive dyes that stain or mark cells. For example, dyes may be selected specifically for their ability to track cells without requiring cell fixation. In one example, MitoView® 633 may be used to track viable cells by staining the mitochondria of the cells. This dye appears red when imaged using fluorescence microscopy or other imaging techniques (including confocal and lattice-sheet microscopy). The red color may disappear after cell death. In another example, NucView® 488 may be used to track dead cells by staining the nuclei of the cells. The dye may have a green color when imaged using fluorescence microscopy or other fluorescence imaging techniques; however, substantially any color is usable, and the colors described above are presented as illustrative examples. Green may appear when cells die. Additionally or alternatively, both dyes may be used to stain cells simultaneously to more accurately track their transition from survival to death. Red and green are recognized above for illustrative purposes. More generally, the photoresponsive dyes described herein may have a range of different colors, depending in part on the fluorescent dye used. Thus, various other colors, color intensities, etc., may be used to stain cells and perform one or more analytical operations, such as any of the analytical techniques described herein.

[0043] In operation 216, spheroids may be formed. For example, an ultra-low adhesion (ULA) plate may be used to form spheroids. As shown in operation 220, cells or spheroids may be embedded in a hydrogel. The hydrogel may contain hyaluronic acid and collagen to mimic the core components of the extracellular matrix of human tissue and / or disease-specific cell niches. Spheroids may generally contain target cells and / or stained cells that have spheroid shape (including, but not limited to, spheroid size, shape, or other characteristics, which can facilitate the determination of the cell's response to the treatment agent by allowing tracking of spheroid features over time).

[0044] In operation 224, spheroids, dissociated cell cultures (e.g., those without spheroid structures), or other cell cultures including hydrogels and stained cells may be placed in a microfluidic chip (e.g., microfluidic chip 800 in Figures 8A and 8B). For example, a microfluidic chip may comprise one or more cell culture chambers. Solid cell cultures may be placed in the first cell culture chamber using a pipette or other instrument. Flow channels are fluidically connected to one or more cell culture chambers. Flow channels may allow the microfluidic chip to be fluidly connected to a circulating system, as described herein with reference to Figures 10 and 11. The microfluidic chip may be configured to allow circulation through the flow channels and to facilitate interaction between the circulating flow and the hydrogel and cells in the chip. In operation 228, the hydrogel and cells are in the microfluidic chip and covered by a gas permeable membrane. For example, to seal the chip, a gas permeable membrane may be placed on the cell culture chamber and adhered to the body of the chip. Once the gas permeable membrane is placed on the chip, the cell culture chamber is covered. This allows the growth medium to flow into the chip without leakage or overflow, using tubes connected to the barbs on the chip's inlet and outlet.

[0045] In operation 232, drug administration may be initiated. Each microfluidic device may include a reservoir connected to a corresponding microfluidic tip via tubing. In one example, a peristaltic pump is used to collect growth medium into a 15 mL conical tube or reservoir and circulate the growth medium to the tip's inlet. The growth medium may then pass through a flow channel and flow out through the tip's outlet. The growth medium then returns to the reservoir using another line of tubing. To ensure sterility against bacterial and / or microbial contamination, a hermetic seal placed on the reservoir lid, as well as filters placed in the tip's inlet and / or outlet, may be present.

[0046] By installing multiple chips / cell culture chambers in parallel, various chemical agents, including chemotherapy, can be tested concurrently. In one example, six chips / reservoirs may be connected using peristaltic pumps or other pumps (e.g., Figures 10 and 11). One chip may be a control in which only growth medium is circulated to dissociated cell cultures in a circular chamber. Various drugs and combinations, including olaparib, AC-T, and AC-carbotaxol, can be circulated to other chips via their corresponding cell culture chambers. In the case of sequential combination therapies such as AC-T and AC-carbotaxol, a clearing process is provided to administer AC first, remove the drug from the cell culture chamber, and add fresh medium containing taxol or carbotaxol for the next round of dosing.

[0047] In operation 236, imaging may be initiated. Imaging may be periodic, for example, at selected intervals, or as appropriate depending on the application, such as daily or every 12 hours. In this example, imaging may occur daily. For this reason, the medium is removed from the microfluidic tip daily. The microfluidic tip can be detached from the microfluidic device so that it can be transported to the microscope for imaging. In one example, a confocal microscope is used to perform three-dimensional fluorescence imaging. Each tip has the length and width of a standard microscope slide to ensure fit. The tip is placed in the slide holder of the confocal microscope, and the laser settings are selected to match the excitation wavelength and the detection wavelength of the staining dye of each cell.

[0048] In operation 240, a two-dimensional image of the chip is acquired. For example, a confocal microscope may acquire layers of images layer by layer from the bottom to the top of the chamber. In one case, a step size of 5 micrometers may be used. In other cases, other step sizes, such as 10 micrometers or larger, may be appropriate. A chamber containing cells may be imaged in its entirety to capture the location of all cells. For example, a circular chamber may be imaged daily. In other cases, imaging may be performed at longer intervals (e.g., on day 1 and day 5) or shorter intervals (e.g., at hour 1 and hour 12). Between each imaging of the chip, the chip may be reconnected to a microfluidic device to deliver additional treatment agents to the chip. Subsequent imaging of the chip may then indicate the progression and response of the tumor to the treatment agent over time.

[0049] In operation 244, two-dimensional images can be merged together. For example, raw microscope images can be aligned by their z-position. Once the images are aligned from bottom to top, each layer can be merged along the z-axis. Cells are detected on each two-dimensional image and assigned x, y, and z positions. From this, three-dimensional images and other visible representations of the cells can be generated to determine the effectiveness of the treatment, as reflected in operation 248. For example, once the z-stack is complete, some cells may appear on two or more stacks due to confocal microscopy capturing the same cell in two or more layers. When a cell has the same x and y positions and appears two or more times in adjacent layers along the z-axis, the brightest pixel may be identified as the cell's true z-position and be the only pixel displayed in the final three-dimensional reconstruction. The final three-dimensional reconstruction contains all detected cells with x, y, and z coordinates. In one example, such overlapping cells can be identified through machine learning techniques, such as K nearest neighbor (KNN), and other techniques. This process can achieve the removal of duplicate cells traversing multiple Z positions within the z-stack. In some cases, such machine learning techniques can also be configured to match multiple stained mitochondria to the same cell. Deep learning and other relevant techniques of the computer vision systems described herein can also be used. Thus, if a cell has multiple mitochondria, the techniques described herein can be used to offset the multiple mitochondria to obtain a more accurate cell count.

[0050] More broadly, process 200 may include further analytical operations 252 related to the analysis of two-dimensional and three-dimensional images and the cellular response to the treatment agent. Analytical operations 252 may be performed using computer vision and related systems described herein, such as those described in more detail below with respect to Figures 13A to 16. In some cases, the operations may include the step of executing instructions on a non-transient computer-readable medium using one or more processing elements of a computer, such as the computing system 2200 in Figure 22. For example, computer vision tools configured to identify the color of each cell based on a photoresponsive dye or fluorescent dye are disclosed herein. Since each cell is treated as a unique object, the color of each cell may also change (for example, all red blood cells may change to blue, or a subset may change to blue based on their position or size).

[0051] In some cases, the array may be configured to store cell-specific data, such as the coordinates and color of each cell, for analysis across multiple time points. Data at a first time point may suggest a first response of the cell culture to the treatment agent (e.g., at the first period). Data at a second time point may suggest a second response of the cell culture to the treatment agent (e.g., as a second period following the first period). For example, cells with red fluorescence derived from MitoView® 633 may be counted on the first day (e.g., day 1) to determine the number of viable cells, and then compared with images of the same sample at a later date (e.g., day 5) to determine how many cells died between the first day and the later date. Cellular properties, such as the average z-position of cells, may be calculated at both the first and later dates to determine, for example, the average upward or downward cell migration between the first and later dates. Various other cellular metrics may be analyzed to determine changes over time when cells are exposed to a drug or combination of drugs. In another example, a computer vision analysis system configured to determine the behavior of each cell at the single-cell level over time is disclosed herein. The computer vision analysis system may be further configured to analyze the behavior at the single-cell level in order to distinguish cancer cells from immune or other cells. In some cases, the computer vision analysis system may be further configured to identify and differentiate between subtypes of cancer cells.

[0052] To facilitate the above, deep learning can be used to make predictions about cells and groups of cells, including the use of neural networks. For example, deep learning can enable the classification of cells by type (e.g., cancer cells, stromal cells, or immune cells). More generally, machine learning can also be used to support one or more of the analytical functions described herein. Further machine learning methods, such as k-means and support vector machines (SVMs), can be used to classify single cells and groups of cells. High-resolution and high-magnification microscopy can be used to distinguish cells by their size and shape and / or various other cellular characteristics. As one example, the KNN algorithm is used to match a single cell between the first day and a later day (e.g., day 1 and day 5). From this, the movement vector of each cell can then be visualized using a quiver plot (e.g., Figure 15 herein). The sum of the vectors may represent various trends, such as the total amount of cell movement in the sample. A subset of the vectors (e.g., the top 10%) or individual vectors can be used to help predict the patient's response to a given treatment.

[0053] A combination of metrics extracted from image data can be used to predict patient responses to various treatments, including single agents and drug combinations. In its simplest form, a threshold is set to classify treatment responses using a single metric, such as cell viability, total cell migration vector, or maximum single cell migration vector. Simple classification indices, receiver operating characteristic (ROC) curves, and logistic regression analysis are examples of methods for relating a single metric to patient responses in this invention.

[0054] Metrics output by computer vision processes can be used as input characteristics for making various predictions. For example, a decision tree can be used to predict patient outcomes using a complex decision-making process that correlates multiple patient response characteristics. In some cases, a decision tree can be used to perform complex metric calculations on multiple inputs to optimize predictive accuracy. Training data can be used to correlate inputs with patient responses and to determine which inputs have the greatest effect on predicting patient outcomes. A decision tree can have multiple knots and branches to facilitate the prediction of patient responses. The final knot (prediction) of the tree may be the predicted likelihood of the patient's response, the optimal predicted treatment, or both. Furthermore, a decision tree can be optimized to maximize sensitivity and specificity on the patient population. A decision tree can also be optimized to maximize positive and negative predictive values. For example, for each patient, the decision tree may output a predicted response (e.g., full response, partial response, or no response) for each proposed treatment.

[0055] With reference to Figures 3 to 17, examples of implementations of System 100 and Process 200 of Figure 1 are presented herein. For example, mechanical components, equipment, solutions, devices, user interfaces, charts, computing devices, etc., are described for illustrative purposes and may be used to perform one or more of the functions or techniques described above with respect to Figures 1 and 2. Accordingly, the examples in Figures 3 to 17 are not intended to limit this disclosure, but rather to illustrate various characteristics and functions of this disclosure. For this reason, other mechanical components, equipment, solutions, devices, user interfaces, charts, computing devices, etc., are considered herein for the implementation of the above functions and techniques within System 100 and Process 200.

[0056] Referring to Figure 3, an example of a patient sample 304 is shown in comparison with the measurement device 308. The patient sample 304 may be a patient-derived tumor sample obtained during the period of operation 204 described above with respect to Figure 2. The patient sample 304 may include cells of malignant tissue or related cells, such as cells of cancer, particularly breast cancer, colorectal cancer, lung cancer, kidney cancer, pancreatic cancer, ovarian cancer, brain cancer, or gastric cancer. The patient sample 304 may include tissue slices, cores, surgical resections, xenografts, and / or core needle biopsies. More broadly, the patient sample 304 is shown as substantially any patient-derived sample collected with respect to the culture module 104 described above. Thus, the patient sample 304 may include target cells that can be placed and configured within the culture for administration and analysis by the drug delivery module 108 and the analysis module 112. Therefore, the patient sample 304 may include dissociated or isolated cells. The patient sample 304 shown in Figure 3 may represent the patient sample before forming a solid cell culture or otherwise modifying the patient sample. Therefore, sample 304 may further contain cancer cells, normal / non-transformed cells, stromal cells, or immune cells. Sample 304 may also contain blood and other contaminants, which may be selected or filtered, or removed using, among several possibilities, digestive enzyme-based operations, hemolytic solutions, or selective operations, as described herein.

[0057] Referring to Figure 4, the user interface 400 is shown, including information associated with cells tagged using tracking dyes. The user interface 400 may be a visual representation associated with the stained cells. In this example, target cells may be stained with a first photoresponsive dye to facilitate tracking of viable cell counts and a second photoresponsive dye to facilitate tracking of dead cell counts. The user interface 400 may allow visualization of the presence of the first photoresponsive dye for viable cell tracking and the second photoresponsive dye for dead cell tracking.

[0058] As shown in Figure 4, the user interface 400 includes a display area 402. The display area 402 may represent at least a portion of cells derived from operation 212. For example, the display area 402 may represent a portion of dissociated cells (analyzed to facilitate the measurement of viable and dead cells). Thus, for illustrative purposes, the display area 402 is shown including viable cells 406 and dead cells 410. Viable cells 406 may have a first color or be associated with it, and dead cells 410 may have a second color or be associated with it. Viable cells 406 and dead cells 410 are represented within the display area 402 based on magnification 414 (which may be modified by user or computer implementation technology).

[0059] The representative first and second colors of viable cells 406 and dead cells 410 can enable analytical determination of various cell characteristics, respectively. For example, viable cells 406 and dead cells 410 having different colors can enable the measurement or determination of the number of cells per unit volume. The number of viable cells 406 and dead cells 410 per unit volume can be compared to determine the relative concentration of viable cells to dead cells. In the example in Figure 4, the display area 402 may have a control panel 452 stacked on top of it or associated with it. The control panel 452 can display current measurements of viable cell concentration 456 and dead cell concentration 470. For example, if the concentration of viable cells 406 is determined in comparison with dead cells 410, it may result in a viable cell concentration 456 with a value of 90% and a dead cell concentration 470 with a value of 10%. The user interface 400 is recognized as showing information related to the quantity of cells isolated from the patient tumor sample.

[0060] For example, referring to Figure 5, Chart 500 is shown, including additional information corresponding to the viable cells 406 and dead cells 410 shown in Figure 4. For example, Chart 500 may represent the relative concentration or number of viable / dead cells with respect to cell size. As shown in Figure 5, Chart 500 includes a cell size axis 504, a cell quantity axis 508, a viable cell distribution 512, a dead cell distribution 516, an average viable cell size 506a, and an average dead cell size 506b. The cell size axis 504 may represent the approximate size of a given target cell. In Figure 5, the size is expressed in micrometers; however, other suitable units may also be used. The cell quantity axis 508 may include a measurement of the number of cells in a given sample or portion thereof.

[0061] The viable cell distribution 512 and the dead cell distribution 516 may represent the number of cells for each cell size. In some cases, the distributions 512 and 516 may be histograms or other representations showing the number of cells for a given range of cell sizes. The mean viable cell count 506a may represent the average size of viable cells represented by the viable cell distribution 512. The mean dead cell count 506b may represent the average size of dead cells represented by the dead cell distribution 516. An example of culture 600 is shown with reference to Figure 6. Culture 600 may represent a solid cell culture formed via any of the spheroid formation procedures 216 and hydrogel embedding procedures 220 described above with respect to Figure 2. For example, culture 600 is shown in Figure 6 as containing a hydrogel 608. Hydrogel 608 may contain hyaluronic acid and collagen to mimic the core components of the extracellular matrix and disease-specific cellular niches of human tissue, such as those found in breast tumors. Hydrogel 608 is shown to encapsulate a spheroid 604. Spheroid 604 may contain target cells 612, such as any of the cells described herein. Target cells 612 may be stained with a photoresponsive dye.

[0062] The arrangement of cell cultures containing spheroids 604 can facilitate tracking tumor characteristics over time. As an example, a representative circumferential figure 616 is shown in Figure 6. The representative circumferential figure 616 may correspond to size values ​​for spheroids 604, or other cells or parts, in culture 600. Culture 600 may be exposed to a treatment agent as described herein. Subsequently, the size of the circumferential figure 616, or other characteristics of culture 600, may be measured to determine changes over time. The changes may represent the effectiveness of the treatment with respect to the treatment agent compared to a control / baseline. For example, a decrease in the size of the circumferential figure 616 may provide clinicians with information regarding the effectiveness of the treatment applied to cell culture 600.

[0063] A solid cell culture 600 may be stained with one or more of the photoresponsive dyes described herein. The properties of the solid cell culture 600 and spheroids 604 may therefore be measured using the photoresponsive dyes. For illustrative purposes, referring to Figures 7A–7C, the representations of a solid cell culture 700 are shown under different conditions for the analysis of the components of the cell culture. The examples in Figures 7A–7C show spheroids of an ovarian tumor for illustrative purposes, but in other cases, other tumors may be represented using similar techniques. Referring to Figure 7A, the cell culture 700 is shown under conditions in which representation 704 is depicted or expressed by a viable cell dye. Representation 704 may suggest physical properties of viable cells, including cell location and size. Referring to Figure 7B, the cell culture 700 is shown under conditions in which representation 708 is depicted or expressed by a dead cell dye. Representation 708 may suggest physical properties of dead cells, including cell location and size. In some situations, superimposing expressions 704 and 708 may be beneficial for more accurately tracking cells as they transition from survival to death, for example. Referring to Figure 7C, a cell culture 700 is shown with expressions 704 and 708 superimposed to define expression 712. Expression 712 may be a composite visible representation of viable and dead cell pigments. Expressions 704, 708, and 712 may change over time, for example, in response to the administration of one or more treatment agents. Changes in expressions 704, 708, and 712 may be analyzed to determine the effectiveness of the treatment, as described herein.

[0064] Referring to Figures 8 and 8B, a microfluidic chip 800 is disclosed. The microfluidic chip 800 may be used in conjunction with operation 224, which involves setting a hydrogel into the chip, as described above with reference to Figure 2. The microfluidic chip 800 may be configured to mimic the environment of a human organ. For example, the microfluidic chip 800 may be configured to replicate the environment of a human organ in order to establish an environment in which cells can survive for the duration of their ex vivo period. Thus, the microfluidic chip 800 may comprise a compartment or cell culture chamber for holding cells, such as any of the cells or cell cultures described herein. The microfluidic chip 800 may include a layer that covers or shields the cell culture to block contaminants from the cell culture, while also allowing gases, such as oxygen, to reach the cell culture. The microfluidic chip 800 may also be configured to circulate growth media and treatment agents into the solid cell culture held therein. The growth media may allow cells to survive for the duration of their ex vivo period.

[0065] The microfluidic chip 800 may have a multilayer structure. Figure 8A shows an exploded view of the stacking of layers and components that may be used to form the microfluidic chip 800. Each component layer in Figure 8A is shown in plan view. Referring to Figure 8A, the base layer 802, the first body part layer 812, the second body part layer 822, the third body part layer 832, the connecting part 842, and the gas permeable layer 852 are shown. In other examples, the microfluidic chip 800 may include more or fewer layers, including one or more sterilization layers, films, adhesives, etc., which may be used to prepare a microfluidic chip 800 for drug delivery. The microfluidic chip 800 may also be associated with one or more flow restrictors 880 (Figure 8C). In the example in Figure 8A, the base layer 802 may be a substantially solid section of the chip 800, on which the other layers of the chip 800 are arranged and configured. Base layer 802 may have a size that corresponds to the size of a standard microscope slide in order to improve compatibility.

[0066] The first body layer 812, the second body layer 822, and the third body layer 832 may be layers of the body 801 defining the channel 862, the first volume 864 (including the first cell culture chamber 814), and the second volume 866 (including the second cell culture chamber 816) of the tip 800. For example, the first body layer 812 may define the first cell culture chamber 814 and the second cell culture chamber 816, which may be defined by openings or through-holes penetrating the first body layer 812. The second body layer 822 may define the first hole 824 and the second hole 826 of the second body, which may be defined by openings or through-holes penetrating the second body layer 822. Furthermore, the third body layer 832 may define the first hole 834 and the second hole 836 of the third body, which may define openings or through-holes that penetrate the third body layer 832, respectively.

[0067] As shown in the cross-sectional view of Figure 8A, the layers 812 of the first body portion, 822 of the second body portion, and 832 of the third body portion can be superimposed opposite each other so that the first cell culture chamber 814, the first pore 824 of the second body portion, and the first pore 834 of the third body portion collectively define a first volume 864. The first volume 864, including the first cell culture chamber 814, may have a substantially cylindrical shape with a closed bottom and an open top. In one example, the first cell culture chamber 814 may preferably have a diameter of about 6.75 mm. In other cases, the diameter may be greater than or less than 6.75 mm, for example less than about 5 mm, less than about 3 mm, and / or other suitable values ​​depending on the given application. Furthermore, the layers 812 of the first main body, 822 of the second main body, and 832 of the third main body can be superimposed opposite each other such that the second cell culture chamber 816, the second pore 826 of the second main body, and the second pore 836 of the third main body collectively define a second volume 866. The second volume 866, including the second cell culture chamber 816, may have a diamond or square shape with a closed bottom and an open top.

[0068] The main body 801 may also define a channel 862. For example, as shown in Figure 8A, the layer 822 of the second main body portion includes an elongated through portion 828. The elongated through portion 828 may penetrate the entire thickness of the layer 822 of the second main body portion and extend through a portion of the first opening 824 and the second opening 826 of the second main body portion. The layers 812 of the first main body portion, the layer 822 of the second main body portion, and the layer 832 of the third main body portion may be superimposed opposite each other to define a channel 862, such that the layers 812 of the first main body portion and the layer 832 of the third main body portion close the bottom and top ends of the elongated through portion 828, respectively.

[0069] The elongated through portion 828 may extend between opposing ends of the layer 822 of the second main body portion. The layer 832 of the third main body portion may cover the elongated through portion 828 and define a first lumen 833a and a second lumen 833b (each extending into the elongated through portion 828). For example, the first lumen 833a may extend into the elongated through portion 828 by penetrating the thickness of the layer 832 of the third main body portion at its first end. The second lumen 833b may extend into the elongated through portion 828 by penetrating the thickness of the layer 832 of the third main body portion at its second opposite end. The first and second lumen 833a and 833b may extend into the elongated through portion 828 to facilitate fluid connection between the channel 862 and the fluid circuit.

[0070] For example, as shown in Figure 8A, the connecting portion 842 may comprise an inlet mechanism 843a and an outlet mechanism 843b. As shown in Figure 8B, the inlet mechanism 843a and the outlet mechanism 843b may be barbs protruding from the outermost surface of the microfluidic tip 800. The inlet mechanism 843a may be alignable with and thereby receptive to the first lumen 833a and may be configured to define a fluid connection between the circulating system tube and the channel 862. The outlet mechanism 843b may be alignable with or thereby receptive to the second lumen 833b and may be configured to define a fluid connection between an additional circulating system tube and the other end of the channel 862. As described herein, this configuration may allow the tip 800 to be fluidly connected to the circulating system (in which the treatment agent circulates through the tip 800 together with the growth medium). For example, liquids or media for various purposes can be introduced into the tip 800 via the inlet mechanism 843a, flow through the tip 800 via the first lumen 833a, channel 862, and second lumen 833b, and may flow out via the outlet mechanism 843b.

[0071] Referring further to Figure 8A, the gas permeable layer 852 may include a membrane, film, or other layer configured to shield and cover the first and second volumes 864, 866 that house the first and second cell culture chambers 814, 816. The gas permeable layer 852 may be configured to shield or cover the volumes 864, 866 while allowing gases, such as oxygen, to enter and exit the cell culture chambers 814, 816 from the external environment. Thus, the cell cultures held therein may be exposed to oxygen-rich and / or oxygen-poor environments as needed, depending on the specific application. The layer 832 of the third body portion is shown as including an adhesive surface 835. As described herein, following the loading of cell cultures into the chip 800, the gas permeable layer 852 may be able to adhere to the body via the adhesive surface 835.

[0072] Referring to the cross-sectional view in Figure 8B, the tip 800 can be connected such that the base layer 802, the first body layer 812, the second body layer 822, the third body layer 832, the connecting portion 842, and the gas permeable layer 852 are superimposed on each other. In the assembled configuration, the channel 862 is shown extending through the body 801 between the first lumen 833a and the second lumen 833b. The first lumen 833a is fluidically connected to the inlet mechanism 843a. The second lumen 833b is fluidically connected to the outlet mechanism 843b. As shown in Figure 8B, the inlet mechanism 843a may be fluidically connected to the first tube 906a, and the outlet mechanism 843b may be fluidically connected to the second tube 906b. The first and second tubes 906a and 906b may be tubing of a circulating system (for example, one provided by the microfluidic device 1000 in Figures 10 and 11). The tubes 906a and 906b may be inserted into or received by the inlet mechanism 843a and the outlet mechanism 843b, respectively. The inlet and outlet mechanisms 843a and 843b may be configured to define sealed connections with the tubes 906a and 906b, respectively.

[0073] As shown in Figure 8B, the tip 800 may be configured to define a flow channel segment for the circulation system. For example, the first tube 906a is for the inlet flow F i This can be introduced into the tip 800 in the inlet mechanism 843a. Inlet flow F iThis can be a flow of a medium containing a treatment agent. The flow of the medium and / or treatment agent and / or other drugs may proceed to channel 862 defined by body 801. Channel 862 may be located adjacent to both the first cell culture chamber 814 and the second cell culture chamber 816. Thus, the flow of the medium and / or treatment agent continues through channel 862 and along and / or into portions of the first cell culture chamber 814 and / or the second cell culture chamber 816. As described herein, one or both of the first cell culture chamber 814 and / or the second cell culture chamber 816 may contain cells encapsulated in a hydrogel, for example, any of the cell and hydrogel configurations described herein. Accordingly, channel 862 may be configured to deliver growth medium and / or treatment agents to interact with the cells / hydrogel contained in each of the cell culture chambers 814 and 816. For example, a solid cell culture (including cells and hydrogel) may be placed in a first cell culture chamber 814 and / or a second cell culture chamber 816. Growth medium may be circulated through a tip 800, for example along a channel 862, so as to flow over the cells and hydrogel held therein. Thus, when a solid cell culture is introduced into the tip and growth medium circulates through it, the exposed solid cell culture can be defined within the tip. Interaction between the medium and / or treatment agent and cells may cause the cells to retain at least a portion of the medium and / or treatment agent in the cell culture chamber. The flow continues along channel 862 and, at outlet mechanism 843b, flow rate F o Data could leak from the chip.

[0074] Figure 8C is a cross-sectional view of a flow restrictor 880 used with a microfluidic tip 800 in a fluid circuit. The flow restrictor 880 may be introduced to promote sterility in the system, for example, by preventing contamination of the tip 800 and the cells within it. Accordingly, the flow restrictor 880 may include one or more filter elements. Accordingly, the flow restrictor 880 may further include one or more diameter reductions or changes in the flow path, among several others, to increase or alter the pressure gradient of the flow across the restrictor 880.

[0075] In the example shown in Figure 8C, the flow restrictor 880 is shown to include a first linear barb 882. The first linear barb 882 defines a channel 883 of the first barb through which it passes. The flow restrictor 880 can generally be configured to be positioned adjacent to the inlet or outlet of the microfluidic tip 800. Thus, the first linear barb 882 can be fluidically connected to one of the inlet or outlet mechanisms 843a, 843b. For example, the flow restrictor 880 can be fluidically connected to a tube received by the first linear barb 882 and one of the barbs of the inlet or outlet mechanism 843a, 843b to complete the fluid connection. The flow restrictor 880 shown in Figure 8C also includes a second linear barb 896 that defines a channel 897 of the second barb. The second linear barb 896 may be substantially similar to the first linear barb 882. The second linear barb 896 may be fluidically connected to a component of a microfluidic device (e.g., microfluidic device 1000 in Figure 10) via a tube and / or other connector. Multiple flow restrictors are available. As one example, the first flow restrictor may be used at the inlet of a given microfluidic tip, and the second flow restrictor may be used at the outlet of the microfluidic tip.

[0076] The flow restrictor 880 may include a plurality of layers, generally arranged between the first and second linear barbs 882, 896, in order to facilitate the above-mentioned functionality. For example, the flow restrictor 880 is shown to include a first layer 884 and a first hole 885, as well as an adjacent second layer 886 and two holes 887. The first linear barb 882 may be at least partially received by the first hole 885. The second hole 887 may generally be aligned with the first hole 885. The second hole 887 has a smaller diameter than the first hole 885. Thus, the channel 883 of the first barb, the first hole 885, and the second hole 887 may define a flow path together with the second hole 887, which acts to reduce or restrict the flow through the flow restrictor 880. The flow restrictor 880 is further represented as including a filter layer 888 having filter holes 889 generally adjacent to the second holes 887. The filter layer 888 may house or hold a filter 890 within the filter holes 889. Multiple constructs are possible for the filter 890. In one example, the filter 890 may include a glass microfiber filter. In some cases, multiple separation filters may be used.

[0077] Adjacent to the filter layer 888, the flow restrictor 880 is shown to include a third layer 892 and a third hole 893, and an adjacent fourth layer 894 and a fourth hole 895. The third layer 892 and the third hole 893, and the fourth layer 894 and the fourth hole 895 can generally be mirror images of the first layer 884 and the first hole 885, and the second layer 886 and the second hole 887. Thus, the diameter of the third hole 893 can generally be smaller than the diameter of the fourth hole 895. The second linear barb 896 can be at least partially received by the fourth hole 895.

[0078] Figures 9A to 9E illustrate the process of loading cell cultures into the microfluidic chip 800. Referring to Figure 9A, operation 900a is shown in which a solid cell culture (e.g., any combination of cells and hydrogels as described herein) is loaded into a first cell culture chamber 814 with a first volume 864. For example, a pipette 902 or other instrument may selectively introduce the solid cell culture 910 into the first cell culture chamber 814. In some cases, a solid cell culture may also be loaded into a second cell culture chamber 816 with a second volume 866. Referring to Figure 9B, operation 900b is shown in which the adhesive backing 879 is removed to expose the adhesive surface 835 of the body 801. The adhesive backing 879 may expose the adhesive surface 835 in preparation for attaching the body 801 to the gas permeable layer 852. Referring to Figure 9C, for example, operation 900c is shown in which the gas permeable layer 852 may be placed on the body 801. In some cases, the gas permeable layer 852 may cover the tops of both the first and second volumes 864, 866 housing the first cell culture chamber 814 and the second cell culture chamber 816 during operation 900d, as shown in Figure 9D. The gas permeable layer 852 may adhere to the main body 801 via the adhesive surface 835. Referring to Figure 9E, operation 900e is shown in which the first tube 906a is fluidically connected to the inlet mechanism or barb 843a and the second tube 906b is fluidically connected to the outlet mechanism or barb 843b.

[0079] Referring to Figures 10 and 11, the microfluidic device 1000 is shown. The microfluidic device 1000 may be configured to perform operation 232 in Figure 2, in which a solid cell culture (e.g., the desired cells and hydrogel as herein) is exposed to the treatment agent. Generally, the microfluidic device 1000 may be configured to deliver the treatment agent along with the growth medium to multiple microfluidic chips in parallel. The microfluidic device 1000 is generally fluidically isolated, and a closed circuit may be defined for each chip, separated from the circuits of other chips. The microfluidic device 1000 may be configured to deliver different treatment agents, growth media, or other solutions to the chips so that the response of the solid cell culture can be determined over time. To facilitate the above, the microfluidic device 1000 may comprise a housing 1002, a platform 1004, a staging section 1010, a drug bank 1020, and a pump 1030. The housing 1002 may provide a structural platform for various components of the microfluidic device 1000, including the pump 1030 and the drug bank 1020. The housing 1002 may also provide a structure (on which chips are placed and configured, and which are temporarily stored during the drug administration period). The housing 1002 may have one or more open sides, as shown in Figure 11. In other cases, the housing 1002 may surround one or more components of the microfluidic device 1000. In yet other cases, the housing 1002 may be omitted entirely.

[0080] The staging section 1010 may be configured to arrange and configure multiple microfluidic chips within the microfluidic device 1000. The staging section 1010 may be part of the housing 1002. In other cases, the staging section 1010 may include a raised platform, slots, or other mechanisms for receiving and securing chips at specific locations within the staging section 1010. In some cases, the staging section 1010 may include a chip tray 1012, as shown in the examples in Figures 10 and 11. The chip tray 1012 is shown as holding six microfluidic chips: a first microfluidic chip 800a, a second microfluidic chip 800b, a third microfluidic chip 800c, a fourth microfluidic chip 800d, a fifth microfluidic chip 800e, and a sixth microfluidic chip 800f. With respect to Figures 8A to 9E, each of the tips 800a to 800f may be substantially similar to the microfluidic tip 800 described above; for clarity, their redundant descriptions are omitted. Various configurations are possible, but the tip tray 1012 may include recesses or grooves configured to accommodate a single tip. For example, a given tip may be received in a groove such that movement of at least the sides of the tip is mitigated on the tray 1012. The examples in Figures 10 and 11 show the tip tray 1012 as including grooves for six tips, but other configurations are also possible, including trays that hold more or fewer tips.

[0081] The drug bank 1020 may include a plurality of reservoirs. The drug bank 1020 may include a plurality of reservoirs corresponding to a plurality of chips arranged and configured in the staging section 1010. For example, as shown in Figure 11, the drug bank may include a first reservoir 1022a, a second reservoir 1022b, a third reservoir 1022c, a fourth reservoir 1022d, a fifth reservoir 1022e, and a sixth reservoir 1022f. Reservoirs 1022a to 1022f may be configured to hold a medium, for example, any of the growth media described herein. Reservoirs 1022a to 1022f may be further configured to hold a treatment agent or substantially any other liquid for introduction into each of the chips 800a to 800f. Reservoirs 1022a to 1022f may be exposed to or capable of being exposed to additional medium and / or treatment agents so that they may be added over a long period of time. Reservoirs 1022a to 1022f may optionally be associated with a cover to shield the medium held therein from contaminants during the administration period. For example, one or more or all of reservoirs 1022a to 1022f may include a cap, such as an aluminum cap, for hermetically sealing each of the reservoirs 1022a to 1022f.

[0082] Pump 1030 may be configured to complete a fluid circuit between each of the tips 800a-800f and reservoirs 1022a-1022f. Pump 1030 may be a peristaltic pump, a pneumatic pump, and / or any other suitable pump or pumping device. Pump 1030 may be configured to define a separate fluid circuit between each of the tips 800a-800f and reservoirs 1022a-1022f for a given one of them. For example, pump 1030 may be configured to cause fluid circulation between reservoirs 1022a-1022f and tips 800a-800f without the flow in one reservoir crossing with or contaminating another reservoir. This allows for individual doping for each tip so that readings or measurements can be performed on each tip to determine the effect it receives from a particular solution held in the corresponding reservoir. Pump 1030 is shown as a single assembly, but in other cases, pump 1030 may comprise multiple pumps. Furthermore, pump 1030 is shown as having six circulation channels. In other cases, pump 1030 may define more or fewer channels.

[0083] In the example shown in Figure 11, the pump 1030, the tips 800a to 800f, and the reservoirs 1022a to 1022f are interconnected to define six circulation channels. For example, the pump 1030, the first microfluidic tip 800a, and the first reservoir 1022a are interconnected to define the first liquid circulation channel 1002a. Furthermore, the pump 1030, the second microfluidic tip 800b, and the second reservoir 1022b are interconnected to define the second liquid circulation channel 1002b. Furthermore, the pump 1030, the third microfluidic tip 800c, and the third reservoir 1022c are interconnected to define the third liquid circulation channel 1002c. Furthermore, pump 1030, the fourth microfluidic tip 800d, and the fourth reservoir 1022d are interconnected to define a fourth liquid circulation channel 1002d. Furthermore, pump 1030, the fifth microfluidic tip 800e, and the fifth reservoir 1022e are interconnected to define a fifth liquid circulation channel 1002e. Furthermore, pump 1030, the sixth microfluidic tip 800f, and the sixth reservoir 1022f are interconnected to define a sixth liquid circulation channel 1002f. The circulation channels 1002a to 1002f may be separate liquid channels that are fluidically isolated from each other. Pump 1030 can independently induce circulation along the circulation channels 1002a to 1002f. For example, the pump 1030 may cause the medium, treatment agent, etc. to circulate along the first circulation path between the first reservoir 1022a and the first tip 800a, the second reservoir 1022b and the second tip 800b, the third reservoir 1022c and the third tip 800c, the fourth reservoir 1022d and the fourth tip 800d, the fifth reservoir 1022e and the fifth tip 800e, and the sixth reservoir 1022f and the sixth tip 800f.

[0084] The tips 800a to 800f can be individually removed from the microfluidic device 1000. For example, each of the tips 800a to 800f can be removed from the microfluidic device 1000 for imaging and analysis. As described above with respect to Figure 2, the individual tips are removed and can be imaged using fluorescence microscopy or other techniques that determine, at a given point in time, the characteristics of the solid cell culture, including, but not limited to, the size, color, location, and density of the cell culture. Following imaging, the tips can be reattached to the microfluidic device 1000. The microfluidic device 1000 may continue to provide each microfluidic tip with additional treatment agents, media, etc. At a second subsequent point in time, the tips can be removed again from the microfluidic device 1000 to perform further imaging of the solid cell culture. For example, the color, size, location, and density of the cell culture, and / or various other cellular characteristics, can be detected at the second point in time. The characteristics of the solid cell culture at the second time point can be compared with those of the solid cell culture at the first time point to determine other characteristics, including cell migration distance, cell migration speed, maximum migration vector, and maximum migration speed, as described herein. To perform further imaging of the solid cell culture at the third, fourth, fifth, or subsequent time points, the chip may be repeatedly removed, for example, three, four, five times, or more times.

[0085] In one example, the width or diameter of spheroids is measured and compared at different time points. Single cells surrounding the spheroids are tracked individually or within clusters. The movement of single cells may be tracked to confirm whether cells enter or exit the spheroids. Changes in spheroid size and / or single-cell behavior can be used individually or collectively to predict the patient's response to a given treatment. Figure 12 shows an example of Report 1200, which includes images of representative cell cultures after the treatment period. Spheroids that remain stable or proliferate after exposure to the treatment may suggest a poor response, while shrinking spheroids may suggest a good response. Thus, a given image or other representation of a cell culture may be compared to an image of the culture before treatment to determine changes in spheroid size.

[0086] Referring to Figure 12, an example of report 1200 includes a column 1208a of a first cell culture, a column 1208b of a second cell culture, and a column 1208c of a third cell culture. In one example, each of columns 1208a–1208c may represent a different cell culture held by a different microfluidic chip (e.g., one of the microfluidic chips 800a–800f in Figures 10 and 11). Each cell culture in each chip may be administered with different treatment agents, different intensities or concentrations of treatment agents, and / or without treatment agents, in order to evaluate the cell culture's response to various conditions over time (e.g., as can be displayed in various three-dimensional images, as shown herein with respect to Figure 16). In the example in Figure 12, column 1208a of the first cell cultures corresponds to cell cultures for administration of the first treatment agent at a first dose (e.g., 40 nM doxorubicin), column 1208b of the second cell cultures corresponds to cell cultures for administration of the second treatment agent at a second dose (e.g., 50 nM doxorubicin), and column 1208c of the third cell cultures corresponds to cell cultures for administration of the third treatment agent at a third dose (e.g., 100 nM doxorubicin). It is recognized that specific treatment agents and doses are presented for illustrative purposes only, and substantially any other treatment agents and doses may be used as appropriate to evaluate the efficacy of the treatment.

[0087] Therefore, report 1200 includes line 1204a for the first time point and line 1204b for the subsequent time point. In the example in Figure 12, line 1204a for the first time point may correspond to the pre-medication period, for example, day 0. Line 1204b for the subsequent time point may correspond to a later time point after some time has passed since the medication period. As shown in Figure 12, line 1204b for the subsequent time point may be 1 day after medication. In other cases, line 1204b for the subsequent time point may correspond to another medication period, for example, 2, 3, 4, 5 days later. Cell cultures may be evaluated at each time point (and other time points) to assess the effectiveness of the treatment. For example, the cell culture associated with row 1208a of the first cell culture may be imaged according to any of the techniques described herein at a time associated with row 1204a of the first time point (e.g., pre-drug administration) to produce a first representation 1212a of the cell culture. The cell culture associated with row 1208a of the first cell culture may be subsequently drugged (e.g., with 40 nM doxorubicin) to produce a second representation 1212b of the cell culture, and then imaged at a time associated with row 1204b of the second time point (e.g., about one day after drug administration). Furthermore, the cell culture associated with row 1208b of the second cell culture may be imaged according to any of the techniques described herein at a time associated with row 1204a of the first time point (e.g., pre-drug administration) to produce a first representation 1216a of the cell culture. The cell culture associated with row 1208b of the second cell culture may be subsequently administered (e.g., with 50 nM doxorubicin) to produce a second representation of the cell culture 1216b, and then imaged at a time associated with row 1204b of the second time point (e.g., about one day after administration). Furthermore, the cell culture associated with row 1208c of the third cell culture may be imaged at a time associated with row 1204a of the first time point (e.g., before administration) according to any of the techniques described herein to produce a first representation of the cell culture 1220a.The cell culture associated with row 1208c of the third cell culture is subsequently administered (e.g., with 100 nM doxorubicin) to produce a second representation 1220b of the cell culture, and can be imaged at a time associated with row 1204b of the second time point (e.g., about one day after administration). Each of the second representations 1212b, 1216b, and 1220b can be evaluated using the computer vision tools described herein, including the generation of various charts, reports, graphs, etc., as described below in relation to Figures 13A to 17.

[0088] Referring to Figures 13A to 17, various charts, reports, and graphs representing the imaging and analysis operations of the computer vision system described above are shown with reference to Figure 2. The charts, reports, and graphs below are to be recognized as examples. In other cases, additional or alternative charts, reports, and graphs may be used. For example, referring to Figure 13A, chart 1300a is shown, including grid 1304a and first representation 1308a. Grid 1304a may be a three-dimensional image representing the three-dimensional space associated with a solid cell culture. First representation 1308a may represent the three-dimensional position of a specific object, such as a viable cell in three-dimensional space. In one example, chart 1300a may be generated via a two-dimensional image of a solid cell culture (where the size and location of the target cell are determined using a photoresponsive dye). The two-dimensional images may be combined to generate a three-dimensional image. Thus, the first representation 1308a may represent a composite of multiple two-dimensional images acquired at a first time point. In this example, the first representation 1308a may be acquired at a first time point (which may occur before any administration of the treatment agent). The first representation 1308a may also provide information regarding the quantity of target cells, as shown in Figure 13A.

[0089] Referring to Figure 13B, Chart 1300b is shown, including grid 1304b and a second representation 1308b. Grid 1304b may be a three-dimensional image representing the three-dimensional space associated with the solid cell culture represented by Chart 1300a after a certain period of time (e.g., a second time point). For example, the second representation 1308b may represent the three-dimensional location of a specific target, such as the viable cells mentioned above. Thus, the second representation 1308b may represent a composite of multiple two-dimensional images acquired at a second time point. In this example, the second representation 1308b may be acquired at a second time point (which may occur after a series of administrations of a treatment agent or after a period of no treatment (e.g., with respect to baseline / negative control)). The second representation 1308b may also provide information regarding the quantity of target cells, as shown in Figure 13B.

[0090] In some cases, it may be desirable to more accurately determine the three-dimensional position of target cells at different time points. For example, the three-dimensional position of target cells may be compared between a first and second time point to determine, among other features, cell migration vectors and cell migration speed. Thus, Figure 14A shows chart 1400a representing a first distribution 1420a in a three-dimensional coordinate system defined by axes 1404a, 1408a, and 1412a. The first distribution 1420a may represent a solid cell culture at a first time point (which may be before drug administration). The first distribution may be generated by integration as described herein. Chart 1400a provides information regarding the three-dimensional position of target cells within the first distribution 1420a, including distribution density. In the example in Figure 14A, the first distribution 1420a has a higher cell density with lower values ​​on axis 1412a.

[0091] Solid cell cultures associated with the first distribution 1420a may undergo one or more drug delivery procedures as described herein. The solid cell cultures may be measured at a second time point following the drug delivery procedure. Thus, Figure 14B shows chart 1400b having the second distribution 1420b in a three-dimensional coordinate system defined by axes 1404b, 1408b, and 1412b. Chart 1400b provides information regarding the three-dimensional location of target cells in the second distribution 1420b, including distribution density. In the example of Figure 14B, the second distribution 1420b has a lower cell density in the three-dimensional coordinate system compared to the first distribution shown in Figure 14A.

[0092] Figure 15 shows Chart 1500, which represents a quiver or vector diagram suggesting the three-dimensional momentum of a cell from the three-dimensional position in Figure 14A to the three-dimensional position in Figure 14B. For example, the three-dimensional position of a cell originating from a second distribution 1420b can be compared to a corresponding cell originating from a first distribution 1420a to determine the vector corresponding to the cell. The vector may correspond to the migration path and / or speed of the cell between a first time point represented by Chart 1400a and a second time point represented by Chart 1400b. Thus, in one example, a given cell originating from the first distribution 1420a may correspond to the tail of the vector, while a corresponding cell originating from the second distribution 1420b may correspond to the arrowhead of the vector. Thus, Chart 1500 may be a three-dimensional quiver plot defined by axes 1504, 1508, and 1512. The vector distribution 1520, which includes vectors relating to one or more target cells, can be plotted within chart 1500.

[0093] When plotted on chart 1500, the vector distribution 1520 may provide information about the behavior of cells during the administration of the treatment agent or in the untreated state (e.g., with respect to baseline / negative control). For example, the vector distribution 1520 may include a first region 1522 having the maximum movement vector. The maximum movement vector may correspond to the target cell that moved the greatest amount in terms of speed or position between the first and second measured time points. The vector distribution 1520 may further include a second region 1524 that may correspond to clusters of vectors that are generally larger than the other vectors in the vector distribution 1520. Thus, the second region 1524 may correspond to clusters of cells that generally moved the farthest or fastest during the administration of the treatment agent. As another example, the vector distribution 1520 may further include a third region 1526 that may correspond to clusters of vectors that are generally smaller than the other vectors in the vector distribution 1520. Thus, the third region 1526 may correspond to clusters of cells that generally moved the least distance or at the lowest speed during the administration of the treatment. These and other trends are identified within Chart 1500 and can be analyzed to determine the effectiveness of the treatment agent.

[0094] The systems and techniques of this disclosure can be used to monitor responses to multiple different treatment agents. For example, solid cell cultures can be prepared using any of the techniques described herein. A portion of the solid cell cultures can be deposited in different microfluidic chips, for example, one or more of the six microfluidic chips shown in Figures 10 and 11. The microfluidic device 1000 can be operated to circulate a flow of different solutions (e.g., different treatment agents) to each cell culture in the microfluidic chip. Thus, solid cell cultures can be defined chip by chip, using different solutions or treatment agents on the cells / hydrogels initially deposited in the chip. For example, the microfluidic device 1000 can circulate a flow of different treatment agents to each chip. In some cases, one of the chips may not receive any treatment agent and may only receive growth medium as a control case. The responses of cell cultures exposed to various different treatment agents can be compared to determine the effectiveness of the treatment. For example, once the characteristics of each cell culture are clearly defined, these characteristics can be compared and analyzed to determine which treatment agent was most effective in treating the target cells.

[0095] A visual representation of this comparison is shown in Figure 16. For example, Figure 16 represents an example of a report 1600 that includes visualizations of different drug administration procedures performed on a given patient sample. Report 1600 may include information on the effects of a particular treatment agent on a patient sample over a period of time. In Figure 16, chart 1600 may include a first tip column 1608a, a second tip column 1608b, a third tip column 1608c, a fourth tip column 1608d, a fifth tip column 1608e, and a sixth tip column 1608f. In one example, each of columns 1608a-1608f may represent a different microfluidic tip (e.g., one of the microfluidic tips 800a-800f in Figures 10 and 11). The microfluidic tip may initially contain a solid cell culture, e.g., any of the cell cultures specified herein (which may include target tumor cells). Microfluidic chips can be administered or treated with treatment agents to define the cell cultures exposed within each chip. In one example, each microfluidic chip may be administered with a different treatment agent to determine the effectiveness of a particular treatment. The response of the cell cultures exposed in the microfluidic chips can be measured over a period of time. Thus, Chart 1600 may include row 1604a for a first time point, row 1604b for a second time point, and row 1604c for the final time point. In the example in Figure 16, row 1604a for the first time point may correspond to the first day, row 1604b for the second time point may correspond to the second day, and row 1604c for the final time point may correspond to subsequent days, such as the third, fourth, or fifth day.

[0096] Each microfluidic chip, represented by a row of chips, can be imaged and analyzed for each time point, represented by a row of time points. For example, a microfluidic chip can be imaged to determine its color, size, location, density, and / or other characteristics at a given first time point, corresponding to row 1604a of the first time point. The imaging can be a two-dimensional image or a three-dimensional image (which may be generated by integrating two-dimensional images together). The imaging can be used to generate a representative chart 1612a containing information about the characteristics of the solid cell culture at the first time point, as shown in Figure 16. Chart 1612a may be substantially similar to charts 1300a and 1300b described above for Figures 13A and 13B. Furthermore, the microfluidic chip can be subsequently imaged at a given second time point (which may correspond to row 1604b of the second time point), for example, following a drug administration round via a treatment agent. Therefore, imaging can be used to generate a representative chart 1612b containing information about the characteristics of the solid cell culture at a second time point. Furthermore, the microfluidic chip can be subsequently imaged at a third given time point (which may correspond to the final time point row 1604c), for example, following another drug delivery round via a treatment agent. Therefore, imaging can be used to generate a representative chart 1612c containing information about the characteristics of the solid cell culture at the final time point.

[0097] The response of solid cell cultures to the first microfluidic chip can be analyzed over time to determine the efficacy of the treatment administered to the first microfluidic chip, as described herein. Chart 1600 and the corresponding analysis may allow for comparison of the efficacy of treatments across multiple different treatments (including combinations of drugs administered simultaneously and / or sequentially to cell cultures). For example, substantially similar to column 1608a of the first chip, column 1608b of the second chip may include representative chart 1618a at row 1604a of the first time point, representative chart 1618b at row 1604b of the second time point, and representative chart 1618c at row 1604c of the final time point. Furthermore, substantially similar to the first column of chips 1608a, the third column of chips 1608c may include a representative chart 1622a at the first time point in row 1604a, a representative chart 1622b at the second time point in row 1604b, and a representative chart 1622c at the final time point in row 1604c. Furthermore, substantially similar to the first column of chips 1608a, the fourth column of chips 1608d may include a representative chart 1626a at the first time point in row 1604a, a representative chart 1626b at the second time point in row 1604b, and a representative chart 1626c at the final time point in row 1604c. Furthermore, substantially similar to the first column of chips 1608a, the fifth column of chips 1608e may include a representative chart 1630a at the first time point in row 1604a, a representative chart 1630b at the second time point in row 1604b, and a representative chart 1630c at the final time point in row 1604c. Furthermore, substantially similar to the first column of chips 1608a, the sixth column of chips 1608f may include a representative chart 1634a at the first time point in row 1604a, a representative chart 1634b at the second time point in row 1604b, and a representative chart 1634c at the final time point in row 1604c.

[0098] Regarding the final time point row 1604c, report 1600 includes representative charts 1612c, 1618c, 1622c, 1626c, 1630c, and 1634c for each of the microfluidic chips at the final time point. The final time point may represent the conclusions for the administration of various treatment agents. Thus, the information in each of the representative charts 1612c, 1618c, 1622c, 1626c, 1630c, and 1634c can be compared to determine which cell culture shows the best or most effective response to a given treatment agent in relation to the control / baseline. For example, representative chart 1612c may show information corresponding to the cell culture's response to the first treatment agent at the final time point; representative chart 1618c may show information corresponding to the cell culture's response to the second treatment agent at the final time point; representative chart 1622c may show information corresponding to the cell culture's response to the third treatment agent at the final time point; representative chart 1626c may show information corresponding to the cell culture's response to the fourth treatment agent at the final time point; representative chart 1630c may show information corresponding to the cell culture's response to the fifth treatment agent at the final time point; and representative chart 1634c may show information corresponding to the cell culture's response to the sixth treatment agent (or an untreated control, which may be applied to any single or multiple microfluidic chips) at the final time point. Thus, characteristics such as the color, size, density, and count of target cells can be compared across the representations shown in row 1604c at the final time point to determine the effectiveness of the treatment. For example, if the expression shows a decrease in tumor cell density or viable cell count at the final point in time, row 1604c, the treatment agent used in the treatment of the microfluidic chip that resulted in that expression can be determined to have high treatment efficacy among the treatment agents.

[0099] For example, the response of cell cultures to a given treatment agent can be used to determine cell viability. For instance, the viability of both responsive and non-responsive cells can be determined and plotted to determine the effectiveness of the treatment. Referring to Figure 17, Chart 1700 is shown, representing cell viability for responsive cells, e.g., BRCA mutants, and non-responsive cells, e.g., BRCA wild-type. Referring to Figure 17, Chart 1700 includes a dataset axis 1704 and a viability axis 1708. A first distribution 1710 with a first spread 1724 is shown along with a second distribution 1720 with a second spread 1722. To provide information and aid in determining the effectiveness of the treatment, Chart 1700 can be used to compare the viability of responsive cells in the first distribution 1710 with the viability of non-responsive cells in the second distribution 1720.

[0100] To help the reader understand the various functionalities of the embodiments discussed herein, flow diagrams in Figures 18–21 illustrate each of processes 1800, 1900, 2000, and 2100, respectively. The specific steps (and order of steps) of the methods presented herein are illustrated and discussed, but other methods consistent with the teachings presented herein (including more, fewer, or different steps than those illustrated) are also envisioned and incorporated into this disclosure. Referring to Figure 18, process 1800 is disclosed. Process 1800 relates to a method for forming a solid cell culture, such as the method described with respect to Figure 2. In operation 1804, target cells are isolated from a patient sample. Referring to Figures 2 and 3, for example, a patient sample 304 may be obtained. Sample 304 is delivered to the laboratory and may contain samples containing various cancer types. Cell isolation kits based on digestive enzymes, hemolytic solutions, and other selection or filtration steps may be used to isolate viable cells while removing blood and contaminants. The output may be a viable mixed cell population containing various cells derived from the primary tumor, including cancer cells, normal / non-transformed cells, stromal cells, and immune cells.

[0101] In operation 1808, stained cells are formed from cells isolated by staining target cells with a photoresponsive dye. For example, referring to Figures 2, 4, and 7, cells may be stained using either or both a viable cell dye and a dead cell staining dye. The dyes may be photoresponsive dyes. For example, cells may be stained with a first photoresponsive dye to facilitate the tracking of viable cells. Cells may be further stained with a second photoresponsive dye to facilitate the tracking of dead cells. In some cases, the photoresponsive dye is configured to cause a change in color in the stained cells when the stained cells transition from viable to dead cells. In operation 1810, spheroids may be formed from stained cells. Spheroids or organoids may also include cancer cells, normal / non-transformed cells, stromal cells, and / or immune cells formed from patient-derived tissue or tumor samples. Tissue slices, cores, surgical excisions, xenografts, and / or biopsies may also be used. In operation 1812, the stained cells are encapsulated. Referring, for example, to Figures 2 and 6, the stained cells, such as isolated cells (or spheroids formed from operation 1810), may be contained in hydrogel 608. Hydrogel 608, which can promote cell proliferation, may be configured to replicate components of human tissue. For example, as described herein, hyaluronic acid and collagen may be used to mimic the core components of the extracellular matrix of human tissue and disease-specific cell niches, such as those found in breast tumors.

[0102] Various types of cell cultures are recognized as being usable and / or formed in conjunction with process 1800 in Figure 18. For example, the culture step of process 1800 may include culturing dissociated cells in a hydrogel. Dissociated cells may be formed via two-dimensional or three-dimensional cell cultures. In some cases, the dissociated cells may be a single cell population, while in other cases multiple cell types may be used. For example, the dissociated cells may be, as one example, cancer cells derived from a tissue or tumor sample, normal / untransformed cells, stromal cells, and / or immune cells.

[0103] Referring to Figure 19, process 1900 is disclosed. Process 1900 relates to a method of loading onto a microfluidic chip, such as the method presented above with respect to Figures 9A to 9F. In operation 1904, a solid cell culture is configured to be placed within the cell culture chamber of the microfluidic chip. The microfluidic chip comprises a body defining the cell culture chamber and a channel extending across the cell culture chamber and between the inlet and outlet of the microfluidic chip. For example, referring to Figures 9B and 9C, a solid cell culture 910 is configured to be placed within the first cell culture chamber 814 of the microfluidic chip 800. The microfluidic chip 800 comprises a body 801 defining the first cell culture chamber 814 and a channel 862 positioned along or adjacent to the first cell culture chamber 814 and extending between the inlet mechanism 843a and the outlet mechanism 843b.

[0104] In operation 1908, the gas permeable membrane is positioned over the volume housing the cell culture chambers, while the inlets and outlets remain exposed for connection to the circulating system. Referring, for example, to Figures 9D and 9E, the gas permeable layer 852 is positioned on the body 801. The gas permeable layer 852 may adhere to the body 801 via the adhesive surface 835. As shown in Figure 9E, the gas permeable layer 852 may cover both the first volume 864 and the second volume 866 housing the first cell culture chamber 814 and the second cell culture chamber 816, while allowing the first cell culture chamber 814 and the second cell culture chamber 816 to be exposed to the gas in the ambient air surrounding the microfluidic tip 800.

[0105] Referring to Figure 20, process 2000 is disclosed. Process 2000 relates to a method for operating a microfluidic chip. In operation 2004, the microfluidic chip is fluidically connected to a microfluidic device to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump. The microfluidic chip contains a cell culture, and the reservoir contains a medium. Referring, for example, to Figures 10 and 11, microfluidic chips 800a to 800f may be fluidically connected to a microfluidic device 1000. The microfluidic device 1000 may be operated to define a fluid circuit 1002a to 1002f between each of the microfluidic chips 800a to 800f and one of the corresponding reservoirs 1022a to 1022f.

[0106] In operation 2008, a flow of the medium is induced through the circuit so that the medium interacts with the solid cell culture in the microfluidic chip and defines the cell culture exposed within the microfluidic chip. For example, referring to Figures 10 and 11, the microfluidic device 1000 can activate the pump 1030 to induce a flow of the medium through each of the fluid circuits 1002a to 1002f. The pump 1030 may be configured to individually control the circulating flow through each of the fluid circuits 1002a to 100f. This may allow the microfluidic device 1000 to administer different treatment agents to each of the microfluidic chips 800a to 800f so that the response to each of the different treatment agents can be measured. For example, in operation 2012, the response of a solid cell culture to a medium is analyzed. Referring, for example, to Figures 11, 13A, 13B, and 16, a given microfluidic chip may be removed and fluidically detached from the microfluidic device 1000. The microfluidic chip may be imaged or otherwise analyzed at a given point in time when the microfluidic chip is fluidically detached. As one example, the microfluidic chip may undergo a fluorescence microscopy process. Fluorescence microscopy may capture images of the cell culture on a given microfluidic chip under conditions that allow a photoresponsive dye to detect target cells, e.g., dead cells and / or viable cells. Confocal microscopy, bright-field microscopy, and lattice-sheet microscopy may be used in addition to other imaging techniques. In one example, the image may be a two-dimensional image. As described herein, the two-dimensional image may be captured in a manner that represents layers of the cell culture. The layers may be stacked on top of each other or combined together to form a three-dimensional image.

[0107] In another example, a given microfluidic chip may be re-fluidically connected to a microfluidic device to administer a further medium containing an additional treatment agent. Thus, Method 2000 may further include the steps of inducing another flow of the medium through the circuit at a second time point so that the medium interacts with the cell culture in the microfluidic chip, and analyzing the subsequent response of the cell culture to the medium. The step of analyzing the subsequent response of the cell culture may include imaging used to generate two-dimensional or three-dimensional images of the cell culture, as described herein. Images of the cell culture at the first and second time points may be analyzed to determine the effectiveness of the treatment. They may also be analyzed at additional time points, as described herein. As one example, Method 2000 may include the step of analyzing images at a first time point to determine the quantity of a first viable / dead cell population. Method 2000 may further include the step of analyzing images at a second time point to determine the quantity of a second viable / dead cell population. To determine a change in the quantity of cell populations that suggests the effectiveness of the treatment, the quantity of a first cell population and the quantity of a second cell population may be compared. As another example, method 2000 may include the steps of analyzing an image at a first time point to determine the location of a first cell population, and analyzing an image at a second time point to determine the location of a second cell population. To determine a change in the location of cell populations that suggests the effectiveness of the treatment, the location of the first cell population and the location of the second cell population may be compared.

[0108] Referring to Figure 21, process 2100 is disclosed. Process 2100 relates to the analysis of solid cell cultures over time. Process 2100 may be performed in whole or in part using a computing device 2200, which is described below with reference to Figure 22. Various operations may be performed after computing device 2200 (or multiple computing devices 2200) to receive data obtained from a system (e.g., a microfluidic system and a chip) and to use this data to assess the effectiveness of the treatment and the patient response. In operation 2104, a first response of the solid cell culture to a growth medium containing the treatment agent is determined. The solid cell culture is held in a cell culture chamber of a microfluidic chip. Referring to Figures 9B and 13A, for example, the first response of the cell culture 910 may be determined at a first time point. The first response may be determined by executing instructions on a non-transient computer-readable medium using one or more processing elements of computing device 2200. For example, a cell culture 910 may be analyzed using a computing device 2200 to determine the color, number, size, density, and / or other characteristics of the cells, which may be represented by the first representation 1308a in Figure 13A, at a first time point. The first response may be the response of the cell culture 910 before administration with the treatment agent. More specifically, in one implementation, the computing device 2200 may receive data corresponding to an image taken by one or more of the components described herein, in which case the image may be analyzed by utilizing image analysis or computer vision algorithms that can identify, for example, the pixel color and distribution corresponding to the cells, in order to determine the color, number, size, and density of the cells.

[0109] In operation 2108, an additional response of the cell culture to the medium is determined. For example, referring to Figures 9B and 13B, an additional response of the cell culture 910 may be determined at a second, third, fourth, fifth time point, etc. The additional response may be determined by executing instructions on a non-temporary computer-readable medium using one or more processing elements of the computing device 2200. For example, the cell culture 910 may be analyzed using the computing device 2200 to determine the number, color, size, density, and / or other characteristics of cells that can be represented by one or more of the distributions in Figure 16 at substantially any subsequent time point. The additional response may be the response of the cell culture 910 following administration with a treatment agent. In some implementations, the computing device 2200 may utilize computer vision and image analysis algorithms that can identify cells in one or more captured images and extract information that can be used to determine response information, such as color, intensity, etc.

[0110] In operation 2112, the first and additional responses (e.g., a second response, a third response, a fourth response, a fifth response, etc.) are compared to determine the effectiveness of the treatment. The first and additional responses may be compared by executing instructions on a non-temporal computer-readable medium using one or more processing elements of the computing device 2200, such as one or more image analysis or computer vision algorithms, as described above. For example, referring to Figure 16, as one example, the color, size, quantity, density, and / or other characteristics of a cell culture may be compared between a first time point and a subsequently measured time point using the computing device 2200. A relative increase or decrease in one or more of these characteristics may indicate the effectiveness of the treatment brought about by the corresponding treatment agent. Other characteristics, including but not limited to the color of the cell culture, the pixel intensity of the image of the cell culture, the shape of the cell culture, the size of the cell culture, the position of cells in the cell culture, or the quantity of cells in the cell culture, may also be measured and compared with respect to the first and second responses.

[0111] Therefore, the first and / or second response may include taking images, such as two-dimensional or three-dimensional images of cells. Method 2100 may further include the step of determining one or more of the following from the images: cell viability, cell proliferation, and cell location. With respect to spheroids / organoids, Method 2100 may further include the step of determining cell viability using color and / or color ratios to predict the treatment response. For example, image analysis can extract color and / or intensity information and associate them with cell viability based on color mapping, machine learning, lookup tables, etc. However, the method for determining cell viability may vary depending on the type of image analysis or computer vision algorithm used.

[0112] A first color (e.g., red) representing viable cells and a second color (e.g., green) representing dead cells are comparable at one, two, or more time points to determine, for example, cell death within a spheroid. In other words, the system can analyze color information over time at various pixel locations that can be associated with cell death within a spheroid. Such information can be used as a predictive metric (e.g., viable-to-dead cell ratio). In other cases, other techniques may be used to determine cell viability. Method 2100 may further include the step of predicting a patient response to a treatment agent based on one or more of cell viability, cell proliferation, or cell location, for example, as described herein with respect to operation 252 in Figure 2. In some cases, as described herein, an image representing a first response may be compared with an image representing a second response using a computing device 2200 to facilitate the prediction of the patient response. For example, data obtained from two images may be analyzed by a computer device 2200 to determine, through image analysis, differences in multiple responses (suggesting that one response may be better or better than others in terms of cancer cell death). Thus, method 2100 may further include a step of comparing images to determine, for one or more cells, one or more of the cell migration speed or cell migration distance over time. For example, a pixel analysis method may be used to generate a distance vector, which can then be compared for length or dimensions to determine the cell migration distance. The cell migration speed and / or cell migration distance over time may represent the patient's response to the treatment agent. The maximum or minimum values ​​of the migration vector or speed may also be computer-calculated from this perspective. In some cases, cell migration speed and / or distance may be determined with respect to a subset of the most invasive cells, including, but not limited to, the most invasive cells accounting for 5% of the cell culture, the most invasive cells accounting for 2% of the cell culture, and the most invasive cells accounting for 1% of the cell culture.Additionally or alternatively, cell migration speed and / or distance can be computer-calculated with respect to a subset of cells expressing specific biomarkers.

[0113] The analysis of operation 2112 may be performed with respect to a single cell. For example, the change in the position of a single cell may be tracked between a first time point and a second time point. In this example, the cell may be tagged or identified in one or more images, and then tracked over time when determining its movement between different image frames. In other cases, operation 2112 may be performed with respect to weighted cell measurements. For example, a weighting coefficient may be used based on how much influence each feature of a cell culture has on predicting a treatment response. As an example, the likelihood of a patient responding to treatment A may be based on the patient with the lowest score of X. Score X is calculated by having three-quarters of the score derived from the cell viability of the culture and one-quarter of the score derived from the cell migration speed of the culture. A weighted cell measurement may be a composite score of several individual measurements (e.g., cell viability, migration distance, etc.).

[0114] In some cases, the images of the first and second responses described above may be images of spheroids or organoids. Thus, the radius, diameter, or circumference of the spheroid or organoid can be measured or determined using the images and compared across multiple time points. For example, a computing device can analyze the images to determine an approximate perimeter of the spheroid or organoid, and then calculate the circumference, diameter, or other measurements with respect to the area and / or volume of the spheroid or organoid being evaluated. Additionally or alternatively, analysis of dissociated / single cells can be achieved using similar techniques. For example, dissociated / single cells can be tracked and measured using substantially any relevant metrics as described herein. Illustratively, the count and / or location, migration distance, migration speed, etc., of single cells detached from a spheroid can be determined and analyzed according to the techniques described herein to determine the effectiveness of the treatment. Therefore, Method 2100 may include a step of predicting the patient's response to the treatment based on a comparison of a first radius or first diameter in each of the first and second images with a second radius or second diameter. Additionally or alternatively, surgical excisions, tissue slices, xenografts, and / or core needle biopsies, which may have length, width, and height, may be used. Therefore, the length, width, or height of the tissue slices, surgical excisions, xenografts, and / or core needle biopsies may be measured using images and compared across multiple time points. Therefore, Method 2100 may include a step of predicting the patient's response to the treatment based on a comparison of a first length, first width, or first height in each of the first and second images with a second length, second width, or second height.

[0115] Figure 22 shows an example of computing device 2200. The schematic diagram of computing device 2200 shown in Figure 22 may comprise a system, component, module, assembly, and subassembly configured to perform or carry out any of the technologies and processes described herein. Thus, computing device 2200 may include any suitable hardware (e.g., computing devices, data centers, switches), software (e.g., applications, system programs, engines), network components (e.g., communication paths, interfaces, routers), etc. (not necessarily shown for clarity) used to facilitate any suitable operation disclosed herein.

[0116] As shown in Figure 22, the computing device 2200 may include a processing unit or element 2208a operationally connected to the computer memory 2212 and the computer-readable medium 2216. The processing unit 2208a may be operationally connected to the memory 2212 and the computer-readable medium 2216 components via an electronic bus or bridge (e.g., a system bus 2210). The processing unit 2208a may include one or more computer processors or microcontrollers configured to perform operations in response to computer-readable instructions. The processing element 2208a may be the central processing unit of the computing device 2200. Additionally or alternatively, the processing unit 2208a may be other processors within the device, including application-specific integrated chips (ASICs) and other microcontroller devices.

[0117] Memory 2212 may include various types of non-temporary computer-readable storage media, such as random-access memory (RAM), read-only memory (ROM), erasable programmable memory (e.g., EPROM and EEPROM), or flash memory. Memory 2212 is configured to store computer-readable instructions, sensor values, and other persistent software elements. Computer-readable media 2216 may also include various types of non-temporary computer-readable storage media, such as hard drive storage devices, solid-state storage devices, portable magnetic storage devices, or other similar devices. Computer-readable media 2216 may also be configured to store computer-readable instructions, sensor values, and other persistent software elements.

[0118] In this example, the processing unit 2208a is operable to read computer-readable instructions stored in memory 2212 and / or on the computer-readable medium 2216. The processing unit 2208a can be modified to perform the operations or functions described above with respect to Figures 1 to 21. The computer-readable instructions may be provided as a computer program product, a software application, etc. As shown in Figure 22, the computing device 2200 may also include a display 2218. The display 2218 may include a liquid crystal display (LCD), an organic light-emitting diode (OLED) display, an LED display, etc. The computing device 2200 may also include a battery 2224 configured to provide power to the components of the computing device 2200. The battery 2224 may include one or more energy storage cells linked together to provide an internal power source. Thus, the battery 2224 may be a component of the power supply 2228 (for example, including a charging system or other circuitry that supplies power to the components of the computing device 2200).

[0119] The computing device 2200 may also include one or more sensors 2240 that can be used to detect touch and / or force input, environmental conditions, orientation, position, or several other aspects of the computing device 2200. The computing device 2200 may also include a camera 2232 configured to capture digital images or other optical data. The computing device 2200 may also include a communication port 2244 configured to transmit and / or receive signals or electrical communications to and from an external or separate device. The communication port 2244 may be configured to connect to an external device via a cable, adapter, or other type of electrical connector. In some embodiments, the communication port 2244 may be used to connect the computing device 2200 to a computing device and / or other suitable accessories configured to send and / or receive electrical signals.

[0120] Other examples and forms of implementation are included within the scope and spirit of this disclosure and the appended claims. For example, a mechanism for performing a function may be physically located in various locations, including arrangements in which several parts of the function are performed in different physical locations. Also, where used herein, including within the claims, "or" in an enumeration of items preceding "at least one of" represents a non-associative enumeration, for example, the enumeration "at least one of A, B, or C" means A or B or C or AB or AC or BC or ABC (i.e., A and B and C). Furthermore, the term "exemplary" does not imply that the examples described are preferable or better than other examples.

[0121] The above description uses a special nomenclature for illustrative purposes and to ensure a thorough understanding of the described embodiments. However, it will be obvious to those skilled in the art that specific details are unnecessary for practicing the described embodiments. Therefore, the above description of the specific embodiments described herein is presented for illustrative and illustrative purposes only. The above description is not intended to be exhaustive or to limit the embodiments to the exact form disclosed. Based on the above teachings, it will be obvious to those skilled in the art that many modifications and variations are possible. Another aspect of the present invention may be as follows: [1] A method for analyzing solid cell cultures over time, The first step is to determine the first response of a solid cell culture to a growth medium containing a treatment agent, wherein the solid cell culture is held in a cell culture chamber of a microfluidic chip. A step to determine the second response of the solid cell culture to the growth medium, The steps include comparing the first and second responses to determine the effectiveness of the treatment, and A method that includes this. [2] The method according to [1], wherein one or both of the first response or the second response include at least one of the following: one or more colors of a solid cell culture, pixel intensity of an image of a solid cell culture, shape of a solid cell culture, size of a solid cell culture, position of cells in a solid cell culture, or number of cells in a solid cell culture. [3] The method according to [1], wherein the effectiveness of the treatment is expressed as the viability of cells in a solid cell culture responding to the treatment agent. [4] The method according to [1], wherein the step of determining a first response includes taking an image of a solid cell culture. [5] A step to determine cell viability from images, A step of predicting the patient's response to a treatment agent based on cell viability, and The method according to [4], further comprising: [6] Steps to determine cell proliferation from images, A step of predicting the patient's response to a treatment agent based on cell proliferation. The method according to [4], further comprising: [7] A step of determining the cell position from the image, A step of predicting the patient's response to a treatment agent based on cell location, The method according to [4], further comprising: [8] The image is the first image, The method according to [4], wherein the step of determining a second response includes taking a second image of a solid cell culture. [9] A step of comparing the first image with the second image to determine the cell migration distance of cells in a solid cell culture over time, A step of predicting the patient's response to a treatment agent using cell migration distance. The method according to [8], further comprising:

[10] A step of comparing the first image with the second image to determine the cell migration speed of cells in a solid cell culture over time, A step of predicting the patient's response to a treatment agent using the cell migration speed. The method according to [8], further comprising:

[11] A step of comparing the first image with the second image to determine the migration distance of multiple cells over time, defining all cells or a subset of cells in a solid cell culture, A step of predicting the patient's response to the treatment agent using the distance traveled, and The method according to [8], further comprising:

[12] A step of determining the migration speed of multiple cells over time, defining all cells or a subset of cells in a solid cell culture by comparing the first image with the second image, A step of predicting the patient's response to the treatment agent using the speed of movement. The method according to [8], further comprising:

[13] The method according to

[11] or

[12] , wherein the plurality of cells comprises a subset of cells that are the most invasive cells, accounting for 5% of the plurality of cells.

[14] The method according to

[11] or

[12] , wherein the plurality of cells comprises a subset of cells that are the most invasive cells, accounting for 2% of the plurality of cells.

[15] The method according to

[11] or

[12] , wherein the plurality of cells comprises a subset of cells that are the most invasive cells, accounting for 1% of the plurality of cells.

[16] The method according to

[11] or

[12] , wherein the plurality of cells include a subset of cells that express a specific biomarker.

[17] A step of comparing the first image with the second image to determine the cell having the maximum migration vector in the solid cell culture over time, A step of predicting the patient's response to the treatment agent using the maximum movement vector. The method according to [8], further comprising:

[18] A step of comparing the first image with the second image to determine the cell having the maximum migration speed in the solid cell culture over time, A step of predicting the patient's response to the treatment agent using the maximum travel speed. The method according to [8], further comprising:

[19] The method according to [8], wherein one or both of the steps of determining a first response or determining a second response include determining the characteristics of a single cell.

[20] The step of determining the first response includes determining the characteristics of a single cell in the first period, The step of determining the second response includes determining the characteristics of a single cell in the second period following the first period, The method according to

[19] , wherein the step of predicting a patient response to a treatment agent is based on a comparison of measured characteristics of single cells in a first period and a second period.

[21] Either the first image or the second image, or both, include a composite and / or weighted cell measurement. The method according to [8], further comprising the step of predicting a patient response to a treatment agent based on a composite and / or weighted cell measurement.

[22] The first image includes the first composite and / or weighted cell measurement, The second image includes a second composite and / or weighted cell measurement. The method according to

[21] , wherein the step of predicting a patient response to a treatment agent is based on a comparison of a first composite and / or weighted cell measurement with a second composite and / or weighted cell measurement.

[23] The first image or either or both of the second images include information relating to the radius, diameter, or circumference of the spheroid or organoid, The method according to [8], further comprising the step of predicting a patient response to a treatment agent based on the radius, diameter, or circumference of a spheroid or organoid.

[24] The first image includes the first radius, first diameter, or first circumference of the spheroid or organoid, The second image includes the second radius, second diameter, or second circumference of the spheroid or organoid. The method according to

[23] , wherein the step of predicting a patient response to a treatment agent is based on a comparison between a first radius, a first diameter, or a first circumference and a second radius, a second diameter, or a second circumference.

[25] The first image or either or both of the second images include information relating to one or more of the length, width, or height of the surgical excision, tissue slice, xenograft, or core needle biopsy, The method according to [8], further comprising the step of predicting a patient response to a treatment agent based on the length, width, or height of a surgical excision, tissue slice, xenograft, or core needle biopsy.

[26] The first image includes a first length, first width, or first height of a surgical resection, tissue slice, xenograft, or core needle biopsy. The second image includes the second length, second width, or second height of the surgical excision, tissue slice, xenograft, or core needle biopsy. The method according to

[25] , wherein the step of predicting a patient response to a treatment agent is based on a comparison of a first length, first width, or first height with a second length, second width, or second height.

[27] The method according to [1], wherein the comparison step includes executing instructions on a non-temporary computer-readable medium using one or more processing elements of a computer to determine the effectiveness of the treatment, the effectiveness of the treatment corresponds to a treatment option applied to a patient of solid cell culture origin.

[28] A step to determine a third response of a solid cell culture to a growth medium, A step of comparing the first, second, and / or third responses to determine the effectiveness of the treatment. The method according to [1], further comprising:

[29] A step to determine the fourth response of a solid cell culture to the growth medium, A step of comparing the first, second, third, and / or fourth responses to determine the effectiveness of the treatment. The method according to

[28] , further comprising:

[30] A step to determine a fifth response of a solid cell culture to a growth medium, A step of comparing the first, second, third, fourth, and / or fifth responses to determine the effectiveness of the treatment. The method according to

[29] , further comprising:

[31] Isolating target cells from patient samples, By staining isolated cells with a photoresponsive dye, stained cells are formed from isolated cells, and By encapsulating stained cells within a hydrogel, The method according to [1], further comprising the step of forming a solid cell culture.

[32] The method according to

[31] , wherein the photoresponsive dye is configured to cause discoloration in the stained cells when the stained cells transition from living cells to dead cells.

[33] A step of isolating target cells from a patient sample, The process involves staining isolated cells with a photoresponsive dye to form stained cells from the isolated cells, The steps include encapsulating stained cells within a hydrogel and A method for forming a solid culture containing [a specific substance].

[34] The method according to

[33] , wherein the hydrogel comprises hyaluronic acid and collagen configured to mimic the core components and disease-specific cell niches of the extracellular matrix of human tissue.

[35] The method according to

[33] , further comprising the step of culturing dissociated cells obtained from a patient sample in a hydrogel.

[36] The method according to

[35] , wherein the culturing step further comprises forming a two-dimensional cell culture of dissociated cells.

[37] The method according to

[35] , wherein the culturing step further comprises forming a three-dimensional cell culture of dissociated cells.

[38] The method according to

[35] , wherein the culturing step further comprises forming a cell culture of a single cell population.

[39] The method according to

[35] , wherein the culturing step further comprises forming a cell culture from a plurality of cell types.

[40] The method according to

[35] , wherein the dissociated cells include cancer cells, stromal cells, immune cells, or normal / non-transformed cells.

[41] The method according to

[35] , further comprising the step of culturing cancer cells, normal / non-transformed cells, stromal cells, and immune cells.

[42] The method according to

[35] , wherein dissociated cells are isolated from a patient-derived tissue sample.

[43] The method according to

[35] , wherein dissociated cells are isolated from a tumor sample derived from a patient.

[44] The method according to

[33] , further comprising the step of forming a spheroid or organoid.

[45] The method according to

[44] , further comprising the step of culturing a spheroid or organoid in a hydrogel.

[46] The method according to

[44] , wherein the spheroid or organoid comprises cancer cells, stromal cells, immune cells, normal / non-transformed cells, or stem cells.

[47] The method according to

[44] , wherein the culturing step further comprises forming a co-culture of cancer cells, normal / non-transformed cells, stem cells, stromal cells, and immune cells.

[48] ​​The method according to

[44] , wherein isolated cells are isolated from a patient-derived tissue sample.

[49] The method according to

[44] , wherein isolated cells are isolated from a tumor sample derived from a patient.

[50] The method according to

[33] , further comprising the step of processing a patient sample using a digestive enzyme-based operation, a hemolytic solution, or a selective operation to isolate target cells from the patient sample.

[51] The method according to

[33] , wherein the photoresponsive dye is configured to enable tracking of target cells via fluorescence microscopy.

[52] The method according to

[33] , wherein the photoresponsive dye is configured to stain the mitochondria of a target cell in order to track viable cells.

[53] The method according to

[33] , wherein the photoresponsive dye is configured to stain the nucleus of a target cell in order to track dead cells.

[54] The step of forming stained cells is To track viable cells, isolated cells are stained with a first photoresponsive dye configured to stain the mitochondria of target cells, To track dead cells, isolated cells are stained with a second photoresponsive dye configured to stain the nucleus of target cells. The method according to

[33] , further comprising:

[55] The method according to

[33] , wherein the photoresponsive dye is configured to cause discoloration in the stained cells when the stained cells transition from living cells to dead cells.

[56] The method according to

[33] , wherein the target cells are tumor cells, and the tumor includes breast cancer, colorectal cancer, lung cancer, kidney cancer, pancreatic cancer, ovarian cancer, brain cancer, or gastric cancer.

[57] The method according to

[33] , wherein the patient sample comprises tissue slices, surgical excisions, xenografts, and / or core needle biopsies.

[58] The method according to

[57] , further comprising the step of culturing tissue slices, surgical excisions, xenografts, and / or core needle biopsies in a hydrogel.

[59] A method for loading onto a microfluidic chip, The step involves arranging a solid cell culture within a cell culture chamber of a microfluidic chip, wherein the microfluidic chip comprises a body defining the cell culture chamber and a channel extending across the cell culture chamber and between the inlet and outlet of the microfluidic chip. The steps include: positioning a gas permeable membrane over the cell culture chamber while the inlets and outlets remain exposed to connect to the circulatory system; A method that includes this.

[60] The method according to

[59] , wherein the step of arranging and composing the cell culture further comprises dropping a certain amount of solid cell culture into a cell culture chamber using a pipette, the solid cell culture being in a hydrogel.

[61] The solid cell culture is the first solid cell culture, and the cell culture chamber is the first cell culture chamber. The method according to

[59] , further comprising the step of arranging a second solid cell culture in a second cell culture chamber of a microfluidic chip, wherein the second cell culture is in a hydrogel and the second cell culture chamber is defined by a body and fluidly connected to a channel between an inlet and an outlet.

[62] The method according to

[61] , wherein the first cell culture chamber has a first volume and the second cell culture chamber has a second volume different from the first volume.

[63] The method according to

[61] , wherein the first cell culture chamber has a first shape and the second cell culture chamber has a second shape different from the first shape.

[64] The method according to

[59] , further comprising the step of removing backing from a microfluidic tip, following the step of positioning.

[65] The method according to

[59] , wherein the placement step further includes the step of adhering the gas permeable membrane to the main body and the step of covering the cell culture chamber.

[66] The method according to

[59] , further comprising the step of fluidically connecting a microfluidic chip to a microfluidic device.

[67] A main body that defines the channel and the cell culture chamber fluidly connected to the channel, A connecting portion that is attached to the main body and defines an inlet and an outlet, wherein a channel extends between the inlet and the outlet, and a cell culture chamber is positioned between them, defining a flow path, A gas permeable membrane covering the cell culture chamber and A microfluidic chip equipped with this feature.

[68] The chip according to

[67] , wherein the channel is configured to deliver growth medium to a cell culture chamber.

[69] The chip according to

[67] , wherein the cell culture chamber is configured to accept a solid cell culture, and optionally, the cell culture chamber is configured to accept a liquid cell culture.

[70] The tip according to

[67] , wherein the cell culture chamber has a substantially cylindrical shape.

[71] The tip according to

[70] , wherein the substantially cylindrical shape has a diameter of about 6.75 mm.

[72] The chip according to

[67] , wherein the main body defines a cell culture chamber having a closed bottom end and an open top end, and a gas permeable membrane covers the open top end of the cell culture chamber.

[73] The chip according to

[67] , wherein the gas permeable membrane is attached to the main body portion with an adhesive.

[74] The cell culture chamber is the first cell culture chamber, The main body further defines a second cell culture chamber that is fluidly connected to a channel along a flow path between the first cell culture chamber and the inlet or outlet of the connecting portion. The chip described in

[67] above.

[75] The chip according to

[74] , wherein the first and second cell culture chambers are configured to accept solid cell cultures, and optionally, the cell culture chambers are configured to accept liquid cell cultures.

[76] The chip according to

[74] , wherein the first cell culture chamber has a first volume and the second cell culture chamber has a second volume different from the first volume.

[77] The chip according to

[74] , wherein the first cell culture chamber has a first shape and the second cell culture chamber has a second shape different from the first shape.

[78] The chip according to

[67] , wherein the main body has a multilayer structure.

[79] The multilayer structure A first main body layer defining the cell culture chamber, A second main body layer is connected to the first main body layer, defines a channel, and is fluidly connected to the cell culture chamber. A third layer of the main body connects to the second layer of the main body on the opposite side of the first layer of the main body and defines an opening on the cell culture chamber. The chip described in

[78] above, comprising:

[80] The connecting portion is joined to the third main body layer, The third layer of the main body is A first lumen is fluidly connected to the inlet of the connecting portion and extends to the flow channel of the layer of the second main body portion, A second lumen is fluidly connected to the outlet of the connecting section and extends to the flow channel of the second main body layer. The chip described in

[79] further defines the chip.

[81] The tip according to

[67] , wherein the inlet and outlet define the barb of the tube.

[82] The chip according to

[67] , wherein the main body comprises an acrylic material or a silicone-based material.

[83] A drug bank comprising a plurality of reservoirs, wherein each of the plurality of reservoirs is configured to hold a growth medium, A staging section configured to arrange and configure multiple microfluidic chips corresponding to multiple reservoirs, A pump that can be fluidly connected to multiple reservoirs and multiple microfluidic tips, A fluid circuit is defined between each corresponding pair of multiple reservoirs and multiple microfluidic chips. For each corresponding pair, a pump is used to cause the circulation of the growth medium through a fluid circuit. A microfluidic device equipped with the following features.

[84] The device according to

[83] , wherein each of the reservoirs of the multiple reservoirs is fluidly isolated from one another.

[85] The device according to

[83] , wherein the fluid circuits are fluidically isolated from each other.

[86] The device according to

[83] , wherein the pump is further configured to selectively induce the circulation of growth medium through individual fluid circuits of a plurality of fluid circuits.

[87] The device according to

[83] , further comprising one or more flow restrictors that are fluidically connectable to a microfluidic chip and configured to filter contaminants.

[88] The device according to

[83] , wherein multiple reservoirs are exposed to the atmosphere for the purpose of loading treatments into the reservoirs and can be airtightly sealed with caps for the purpose of sterilization.

[89] The device according to

[88] , wherein multiple reservoirs are configured to contain one or more treatment agents during the operating period of the pump.

[90] The device according to

[83] , wherein the drug bank comprises a tray configured to hold a plurality of reservoirs in a substantially upright position.

[91] The device according to

[83] , further comprising a tube for fluidically connecting the pump to each reservoir and each microfluidic tip housed in a staging section.

[92] A method for operating a microfluidic chip, The steps include: fluidically connecting a microfluidic chip to a microfluidic device in order to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump, wherein the microfluidic chip contains a solid cell culture and the reservoir contains a growth medium; The steps include: causing a flow of growth medium through the circuit so that the growth medium interacts with the solid cell culture in the microfluidic chip; A step to analyze the response of solid cell cultures to growth medium and A method that includes this.

[93] The method according to

[92] , wherein the growth medium contains a treatment agent.

[94] The method according to

[92] , wherein the step of fluidically connecting further includes fluidically connecting a first tube portion to an inlet of a microfluidic tip, the first tube portion being connected to a second tube portion which is fluidly connected to a reservoir, and the first and second tube portions define a common tube.

[95] The method according to

[94] , wherein the step of fluidically connecting further includes fluidically connecting a third tubular portion to an outlet and reservoir of a microfluidic tip in order to define a fluid circuit, thereby defining a flow path through which a microfluidic device passes.

[96] The microfluidic chip comprises a cell culture chamber that holds a hydrogel containing target cells, The channel traverses the cell culture chamber, The step of inducing flow further includes inducing a flow of growth medium along the channel while the hydrogel restrains the target cells to prevent them from escaping from the cell culture chamber. The method described in

[94] above.

[97] The method according to

[92] , wherein the circuit is a closed circuit.

[98] A step of fluidically connecting a second microfluidic chip to a microfluidic device in order to define a second fluid circuit between a second microfluidic chip, a second reservoir, and a pump, wherein the second microfluidic chip contains a second solid cell culture and the second reservoir contains a second growth medium, The steps include: causing a second flow of the second growth medium through the second circuit so that the second growth medium interacts with the cell culture in the second microfluidic chip; A step of analyzing the response of a second solid cell culture to a second growth medium, The method according to

[92] , further comprising:

[99] The method according to

[98] , wherein the growth medium and the second growth medium each contain different treatment agents.

[100] The method according to

[98] , further comprising the step of comparing the response of a solid cell culture with the response of a second solid cell culture to determine the effectiveness of the treatment. The method according to

[98] , wherein the

[101] circuit and the second circuit are fluidly isolated from each other.

[102] The method according to

[98] , wherein the pump is configured to independently control the flow and the second flow.

[103] The method according to

[92] , further comprising the step of fluidically disconnecting the microfluidic chip from the microfluidic device before the step of analysis.

[104] Following the step of analysis, The steps include fluidically connecting the microfluidic chip to the microfluidic device in order to define a fluid circuit between the microfluidic chip, reservoir, and pump, The steps include: causing another flow of growth medium through the circuit so that the growth medium interacts with the solid cell culture in the microfluidic chip; The method according to

[103] , further comprising:

[105] The method according to

[104] , further comprising the step of analyzing the subsequent response of a solid cell culture to a growth medium, following the step of inducing another flow.

[106] The method according to

[105] , further comprising the step of determining the effectiveness of the treatment by comparing the response of the solid cell culture to the growth medium with the subsequent response of the solid cell culture to the growth medium.

[107] The step of analyzing the response of a solid cell culture to a growth medium includes determining the quantity of a first cell population, The method according to

[106] , wherein the step of analyzing the subsequent response of a solid cell culture to a growth medium includes determining the quantity of a second cell population.

[108] The method according to

[107] , further comprising the step of comparing the quantity of a first cell population with the quantity of a second cell population to determine a change in the quantity of cell populations that suggests the effectiveness of the treatment.

[109] The step of analyzing the response of a solid cell culture to a growth medium includes determining the location of a first cell population, The method according to

[106] , wherein the step of analyzing the subsequent response of a solid cell culture to a growth medium includes determining the location of a second cell population.

[110] The method according to

[109] , further comprising the step of comparing the location of a first cell population with the location of a second population to determine a change in the location of the cell population that suggests the effectiveness of the treatment.

[111] The method according to

[92] , wherein the step of analysis includes performing a fluorescence microscopy operation on a solid cell culture on a microfluidic chip.

[112] The method according to

[92] , wherein the step of analysis includes collecting a three-dimensional image of a solid cell culture on a microfluidic chip.

[113] The method according to

[92] , wherein the step of analysis includes collecting a two-dimensional image in the z-stack of solid cell cultures on a microfluidic chip.

[114] The method according to

[92] , wherein the analytical step includes analyzing multiple responses of a solid cell culture to a growth medium over time.

[115] The method according to

[114] , wherein the analysis step is performed daily.

[116] The method according to

[92] , wherein the step of analysis includes performing a confocal microscopy operation on a solid cell culture on a microfluidic chip.

[117] The method according to

[92] , wherein the step of analysis includes performing bright-field microscopy operations on a solid cell culture on a microfluidic chip.

[118] The method according to

[92] , wherein the step of analysis includes performing a lattice light sheet microscopy operation on a solid cell culture on a microfluidic chip.

[119] The method according to

[92] , wherein the step of analysis includes executing instructions on a non-transient computer-readable medium using one or more processing elements of a computer to determine the effectiveness of the treatment of growth medium treatment agents on solid cell cultures.

Claims

1. A method for analyzing ex vivo solid cell cultures over time, The steps include determining a first response of a solid cell culture to a growth medium containing a treatment agent, and determining the first position coordinates of at least one single cell of the solid cell culture, including a first x-position coordinate, a first y-position coordinate, and a first z-position coordinate, thereby ensuring that the solid cell culture is retained within the cell culture chamber of a microfluidic chip. The steps include determining the second response of a solid cell culture to a growth medium by determining the second position coordinates of at least one first single cell of the solid cell culture, including a second x-position coordinate, a second y-position coordinate, and a second z-position coordinate. The step of determining the effectiveness of the treatment by comparing a first response and a second response, wherein comparing the first response and the second response means comparing a first position coordinate and a second position coordinate to determine the difference between the first position coordinate and the second position coordinate, and the difference between the first position coordinate and the second position coordinate is at least one between the first x position coordinate and the second x position coordinate, between the first y position coordinate and the second y position coordinate, or between the first z position coordinate and the second z position coordinate, and the difference indicates the cell migration distance over time of at least a first single cell. A method that includes this.

2. The method according to claim 1, wherein one or both of the first response or the second response include at least one of the following: one or more colors of a solid cell culture, pixel intensity of an image of a solid cell culture, shape of a solid cell culture, size of a solid cell culture, position of cells in a solid cell culture, or number of cells in a solid cell culture.

3. The method according to claim 1, wherein the effectiveness of the treatment is expressed as the viability of cells in a solid cell culture responding to the treatment agent, and the method of analysis does not use light scattering.

4. The method according to claim 1, wherein the step of determining a first response includes taking an image of a solid cell culture using a microscope.

5. The steps to determine cell viability from images, A step of predicting the patient's response to a treatment agent based on cell viability, and The method according to claim 4, further comprising:

6. Steps to determine cell proliferation from images, A step of predicting the patient's response to a treatment agent based on cell proliferation. The method according to claim 4, further comprising:

7. The steps include determining the cell location from the image, A step of predicting the patient's response to a treatment agent based on cell location, The method according to claim 4, further comprising:

8. The image is the first image, The method according to claim 4, wherein the step of determining a second response includes taking a second image of a solid cell culture using a microscope.

9. The method according to claim 8, further comprising determining the time-dependent cell migration distance of at least one first single cell by comparing a first image with a second image, and using the cell migration distance to predict a patient response to a treatment agent.

10. The steps include comparing the first image with the second image to determine the cell migration speed of at least the first single cell in the solid cell culture over time, A step of predicting the patient's response to a treatment agent using the cell migration speed. The method according to claim 8, further comprising:

11. The first image and the second image are compared to determine the migration distance of multiple cells over time, defining all cells or a subset of cells in a solid cell culture. A step of predicting the patient's response to the treatment agent using the distance traveled, and The method according to claim 8, further comprising:

12. The first image and the second image are compared to determine the migration speed of multiple cells over time, defining all cells or a subset of cells in a solid cell culture. A step of predicting the patient's response to the treatment agent using the speed of movement. The method according to claim 8, further comprising:

13. The method according to claim 11, wherein the plurality of cells include a subset of cells that are the most invasive cells, accounting for 5% of the plurality of cells.

14. The method according to claim 11, wherein the plurality of cells include a subset of cells that are the most invasive cells, accounting for 2% of the plurality of cells.

15. The method according to claim 11, wherein the plurality of cells include a subset of cells that express a specific biomarker.

16. The first image and the second image are compared to determine the cell with the maximum migration vector in the solid cell culture over time. A step of predicting the patient's response to the treatment agent using the maximum movement vector. The method according to claim 8, further comprising:

17. The first image and the second image are compared to determine, over time, the cell with the maximum migration speed in the solid cell culture. A step of predicting the patient's response to the treatment agent using the maximum travel speed. The method according to claim 8, further comprising:

18. The step of determining the first response includes determining the characteristics of at least the first single cell at the first time period, The step of determining the second response includes determining the characteristics of at least the first single cell in the second period following the first period, The method according to claim 1, wherein the step of predicting a patient response to a treatment agent is based on a comparison of measured characteristics of at least a first single cell in a first time period and a second time period.

19. Either or both of the first or second image include a composite and / or weighted cell measurement. The method according to claim 8, further comprising the step of predicting a patient response to a treatment agent based on a composite and / or weighted cell measurement.

20. The first image includes the first composite and / or weighted cell measurement, The second image includes a second composite and / or weighted cell measurement. The method according to claim 19, wherein the step of predicting a patient response to a treatment agent is based on a comparison of a first composite and / or weighted cell measurement with a second composite and / or weighted cell measurement.

21. The first image or either or both of the second images include information related to the radius, diameter, or circumference of the spheroid or organoid. The method according to claim 8, further comprising the step of predicting a patient response to a treatment agent based on the radius, diameter, or circumference of a spheroid or organoid.

22. The first image includes the first radius, first diameter, or first circumference of the spheroid or organoid. The second image includes the second radius, second diameter, or second circumference of the spheroid or organoid. The method according to claim 21, wherein the step of predicting a patient response to a treatment agent is based on a comparison between a first radius, a first diameter, or a first circumference and a second radius, a second diameter, or a second circumference.

23. The first image or either or both of the second images include information relating to one or more of the length, width, or height of a surgical excision, tissue slice, xenograft, or core needle biopsy. The method according to claim 8, further comprising the step of predicting a patient response to a treatment agent based on the length, width, or height of a surgical excision, tissue slice, xenograft, or core needle biopsy.

24. The first image includes a first length, first width, or first height of a surgical resection, tissue slice, xenograft, or core needle biopsy. The second image includes the second length, second width, or second height of the surgical excision, tissue slice, xenograft, or core needle biopsy. The method according to claim 23, wherein the step of predicting a patient response to a treatment agent is based on a comparison of a first length, a first width, or a first height with a second length, a second width, or a second height.

25. The method according to claim 1, wherein the comparison step includes executing instructions on a non-temporary computer-readable medium using one or more processing elements of a computer to determine the effectiveness of the treatment, the effectiveness of the treatment corresponds to a treatment option applied to a patient whose origin is a solid cell culture.

26. A step to determine the third response of a solid cell culture to the growth medium, A step of comparing the first, second, and / or third responses to determine the effectiveness of the treatment. The method according to claim 1, further comprising:

27. A step to determine the fourth response of a solid cell culture to the growth medium, A step of comparing the first, second, third, and / or fourth responses to determine the effectiveness of the treatment. The method according to claim 26, further comprising:

28. A step to determine the fifth response of a solid cell culture to the growth medium, A step of comparing the first, second, third, fourth, and / or fifth responses to determine the effectiveness of the treatment. The method according to claim 27, further comprising:

29. Isolating target cells from patient samples, By staining isolated cells with a photoresponsive dye that causes a change in color in stained cells when the stained cells transition from living cells to dead cells, stained cells are formed from isolated cells, and By encapsulating stained cells within a hydrogel, The method according to claim 1, further comprising the step of forming a solid cell culture.

Images