JPWO2018148208A5 - - Google Patents

Description

Cross-reference of related applications

This application cites US Patent Provisional Application No. 62 / 455,881 filed February 7, 2017, claiming priority, the entire contents of which are incorporated herein by reference.

The present disclosure generally relates to a method for performing an assay for active migration and cytotoxicity of a therapeutic agent to tumor cells, such as to immune cells and / or to drug homing, migration, and tumor cytotoxicity. This method is performed in an experimental instrument that provides an opportunity for a therapeutic agent, such as an immune cell or drug, to migrate towards a tumor cell, including a tumor cell that grows into a 3D spheroid conformation. This method allows the investigation of the effects of therapeutic agents such as immune cells or drugs on tumor cells, among other uses, and homing, tumors in a single, easy-to-use, high-throughput system for more in vivo-like tests. Allows investigation of cell toxicity and tumor immune avoidance.

Traditionally, in vitro models investigating therapeutic drug homing and tumor killing activity as well as tumor immune evasion may not accurately reflect the complexity of three-dimensional (3D) tumors, two-dimensional tumor cells. It has been studied separately by utilizing the system (2D) or by studying cell toxicity using a 3D tumor cell model but without mobile components. Importantly, the barriers that immune cells and drugs need to overcome in 3D tumor cell systems are much greater than those in 2D tumor cell systems. For example, immune cells need not only need to migrate to the tumor site, but also infiltrate the 3D tumor structure to attack the target tumor cells. Beyond the physical differences between 2D and 3D systems, phenotypic differences also occur when tumor cells are cultured in 3D, and these phenotypic differences can allow for greater resistance to cytotoxicity. It is shown. Therefore, although there is growing interest in using immune cells for cancer treatment, they activate their own immune cells (eg, T cells, NK cells, B cells, etc.) to develop their own tumors. For treatments involving aggression, the effectiveness of immunotherapy is not equal for all patients or cancer types, thus requiring a better model for scientists and researchers.

Therefore, there remains a need for alternative models and methods to enable the investigation of immune cell and drug homing, tumor cytotoxicity, and tumor antigenicity in a single, easy-to-use, high-throughput system for more in vivo-like tests. It is said that.

According to various embodiments of the present disclosure, methods and experimental instruments for analyzing therapeutic agents, including immune cells and drugs, and their effects on tumor cells that grow into 3D spheroid conformations in culture are herein. It has been disclosed. This method allows the investigation of the effects of therapeutic agents such as immune cells or drugs on tumor cells, among other uses, and homing, tumors in a single, easy-to-use, high-throughput system for more in vivo-like tests. Allows investigation of cell toxicity and tumor immune avoidance.

In various embodiments, assays have been disclosed for detecting the mobility and cytotoxicity of therapeutic agents. The assay involves culturing tumor cells in a cell culture product to form spheroids , where the cell culture product comprises a chamber of structure that allows the tumor cells to grow into a 3D spheroid conformation. The assay further comprises placing an insert containing a porous membrane into a cell culture product and introducing a therapeutic agent into this insert. The assay also includes detecting the ability of the therapeutic agent to move from the insert to the cell culture product chamber and detecting the tumor cell response. In embodiments, the tumor cell response can be tumor cell lysis, penetration of a therapeutic agent into tumor cell spheroids , or measurement of changes in tumor cell physiology.

In some embodiments, the mobility of the therapeutic agent and tumor cytolysis are both detected by flow cytometry. In some embodiments, the assay further comprises detecting the penetration of a therapeutic agent, such as immune cells, into tumor cell spheroids .

In some embodiments, the therapeutic agent is a cell therapeutic agent and / or a drug. In some embodiments, the cell therapy agent comprises immune cells. In some embodiments, the immune cell is a white blood cell. In some embodiments, the immune cells are lymphocytes.

In some embodiments of the assay for detecting the mobility and cytotoxicity of a therapeutic agent, the chamber of the cell culture product comprises a side wall, an upper opening and a liquid impermeable bottom comprising at least one recessed surface. And include. In embodiments, at least a portion of the bottom surface comprises a poorly adhesive or non-adhesive material in or on at least one recess. In some embodiments, the liquid impermeable bottom containing at least one recessed surface is gas permeable. In some embodiments, the sidewalls are opaque. In some embodiments, at least a portion of the bottom is transparent.

In some embodiments of the assay for detecting the mobility and cytotoxicity of a therapeutic agent, the cell culture product comprises 1 to about 2,000 of said chambers, each chamber being physical from any other chamber. Is separated. In some embodiments, the at least one recessed surface of the chamber comprises a plurality of recessed surfaces within the same chamber.

In some embodiments of the assay for detecting the mobility and cytotoxicity of a therapeutic agent, at least one recessed surface of the chamber of the cell culture product is a hemispherical surface, 30 to about 60 degrees from the side wall to the bottom surface. Includes a conical surface with a taper of, or a combination thereof.

In some embodiments of the assay for detecting the mobility and cytotoxicity of a therapeutic agent, the sidewall surface of the chamber of the cell culture product is a vertical cylinder, a vertical cone whose diameter decreases from the top to the bottom of the chamber ( Includes a portion of a vertical conic, a vertical square shaft, or a combination thereof, having a conical transition to at least one recessed bottom surface.

In some embodiments of the assay for detecting the mobility and cytotoxicity of a therapeutic agent, the cell culture product further comprises a chamber appendage that receives a pipette tip for aspiration, which chamber appendage. Adjacent to the chamber, including a surface that communicates fluidly with the chamber, this chamber appendage has a second bottom separated above the bottom, which bottom bottoms the fluid distributed from the pipette. Distract from.

In some embodiments of the assay for detecting the mobility and cytotoxicity of a therapeutic agent, the insert comprises an insert plate.

In some embodiments of assays for detecting the mobility and cytotoxicity of therapeutic agents, at least a portion of the porous membrane is configured to simulate a biological barrier. In some embodiments, this biological barrier is the blood-brain barrier. In some embodiments, at least a portion of the porous membrane comprises an essentially confluent monolayer of microvascular endothelial cells.

Further features and benefits of the subject matter of the present disclosure are set forth in the detailed description below, which will be readily apparent to those of skill in the art, as described in detail below, the scope of claims, and the accompanying drawings. Will be recognized by implementing the subject matter of this disclosure as described herein.

Both the general description and the detailed description below may provide an embodiment of the subject matter of the present disclosure and provide an overview or concept for understanding the nature and nature of the subject matter of the present disclosure as requested. It should be understood as intended. The accompanying drawings are included to further understand the subject matter of the present disclosure, are incorporated herein and form part of this specification. The drawings show various embodiments of the subject matter of the present disclosure and serve to explain the principles and actions of the subject matter of the present disclosure in conjunction with the description. Moreover, the drawings and description are merely examples and are not intended to limit the scope of the claims.

The following detailed description of the specific embodiments of the present disclosure can be best understood by reading in conjunction with the following drawings, in which similar structures are indicated by similar reference numbers.
An embodiment of a multi-well microplate, in this case a 96-well spheroid microplate having an array of microcavities on the bottom of each well and providing multiple spheroids in each of the 96 wells, in particular a multi-well microplate. Is shown. An embodiment of a multi-well microplate, in this case a 96-well spheroid microplate having an array of microcavities on the bottom of each well and providing multiple spheroids in each of the 96 wells, particularly of a multi-well plate. Shows a single well. An embodiment of a multi-well microplate, in this case a 96-well spheroid microplate having an array of microcavities on the bottom of each well and providing multiple spheroids in each of the 96 wells, particularly in FIG. 1B. An exploded view of the bottom portion of the single well shown in Box C is shown. Diagram of an array of microcavities. An embodiment of a spheroid microplate, in this case a 96-well plate with a round bottom configured to contain a single spheroid in each of the 96 wells. An embodiment of a spheroid microplate, in this case a 96-well plate with a round bottom configured to contain a single spheroid in each of the 96 wells. Perspective view of the insert. Perspective view of the insert. Illustration of insert plates and related multi-well plates. An embodiment of the method disclosed herein is shown. An embodiment of the method disclosed herein is shown. An embodiment of the method disclosed herein is shown. Graph showing NK cell migration towards A549 / GFP tumor cells cultured in 3D according to the embodiment shown in FIG. 4B. Graph showing NK-induced cytotoxicity of A549 / GFP tumor cells cultured in 3D according to the embodiment shown in FIG. 4B. The figure of the method disclosed in this specification. The figure of the method disclosed in this specification. The figure of the method disclosed in this specification. The figure of the method disclosed in this specification. The figure of the method disclosed in this specification. The figure of the method disclosed in this specification. The figure of the method disclosed in this specification. Graph showing dose-dependent cytotoxicity of LN229 spheroids after 48 hours of direct culture with the compounds cisplatin and piperongmin. N = 12 wells / concentration from 2 independent studies. Lucifer Yellow Graph showing transparency. Graph showing Rhodamine 123 (Rh123) permeability data 5 days after culture on 96HTS Transwell in a blood-brain barrier (BBB) model. N = 120 from 3 independent studies. FIG. 11 is a graph showing LN229 cytotoxicity with and without blood-brain barrier surrogate. Percentage of survival of LN229 spheroids 48 hours after 2 hours of drug exposure with Transwell with and without BBB. Survival was assessed by standardizing drug-free controls to 100% survival. The data are shown as the average of three independent studies, N = 30 in a one-way ANOVA with Bonferroni posttest. *** = p <0.0001. Graph showing LN229 cytotoxicity without blood-brain barrier surrogate. Percentage of survival of LN229 spheroids 48 hours after 2 hours of drug exposure with Transwell without BBB. Survival was assessed by standardizing drug-free controls to 100% survival. The data are shown as the average of three independent studies, N = 30 in a one-way ANOVA with Bonferroni posttest. *** = p <0.0001. Graph showing LN229 cytotoxicity with blood-brain barrier surrogate. Percentage of survival of LN229 spheroids 48 hours after 2 hours of drug exposure with Transwell with BBB. Survival was assessed by standardizing drug-free controls to 100% survival. The data are shown as the average of three independent studies, N = 30 in a one-way ANOVA with Bonferroni posttest. *** = p <0.0001. Graphs of the screen summaries of FIGS. 12A and 12B showing the compilation of hits found with and without BBB. Results were considered "hits" if they were 3 sigma lower than the buffer response on at least two of the three independent screens. The horizontal striped hatch squares (other than buffer only) are hits that can only be seen in the absence of BBB. The shaded hatch squares are hits seen in the presence and absence of the BBB. Graphs of the screen summaries of FIGS. 12A and 12B showing the compilation of hits found with and without BBB. Results were considered "hits" if they were 3 sigma lower than the buffer response on at least two of the three independent screens. The horizontal striped hatch squares (other than buffer only) are hits that can only be seen in the absence of BBB. The shaded hatch squares are hits seen in the presence and absence of the BBB.

Detailed description of the invention

Here, various embodiments of the subject matter of the present disclosure are referred to in more detail, some of which are shown in the accompanying drawings. Similar numbers used in the figures refer to similar components, steps, etc. However, it is understood that the use of numbers to refer to a component in a given figure is not intended to limit the component in another figure displayed with the same number. In addition, the use of different numbers to indicate a component is not intended to indicate that the component with the different numbers cannot be the same or similar to the other numbered components.

The following description of a particular embodiment is merely exemplary in nature and is not intended to limit the scope, application, or use of the invention in any way, which may of course vary. The present invention is described in the context of the non-limiting definitions and terms contained herein. These definitions and terms are not designed to serve as a limitation to the scope or practice of the invention and are presented for purposes of illustration and illustration only. Unless otherwise specified, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms such as those defined in commonly used dictionaries should be construed to have a meaning consistent with the relevant technical arts and their meaning in connection with the present disclosure, as expressly herein. Unless it is, it is not interpreted in an idealized or overly formal sense.

Definitions As used herein, the singular forms "a", "an", and "the" include multiple referents, unless the context explicitly states otherwise. Thus, for example, reference to "bottom of structure of" includes examples of having two or more such "bottoms of structure" unless the context explicitly states otherwise.

As used herein and in the appended claims, the term "or" is generally used to include "and / or" unless the context explicitly states otherwise. The word "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

As used herein, terms such as "have, has, having", "include, include, include", "comprise, commits, combining" are open to them. Used in a comprehensive sense at the end, it generally means "including, but not limited to,".

"Arbitrary" or "arbitrarily" means that the event, situation, or component described thereafter may or may not occur, and that description causes the event, situation, or component. It means to include cases and cases that do not occur.

The terms "favorable" and "preferably" refer to embodiments of the present disclosure that may provide a particular benefit under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Moreover, the description of one or more preferred embodiments does not mean that the other embodiments are not useful and is not intended to exclude the other embodiments from the technical scope of the invention.

The range can be expressed herein as from "about" one particular value and / or "about" another particular value. When such a range is represented, the examples include from one particular value and / or to another particular value. Similarly, when the value is expressed as an approximation by the use of the antecedent "about", it is understood that a particular value forms another aspect. Further, each endpoint of the range is understood to be significant both in relation to the other endpoints and independently of the other endpoints.

Also in the present specification, the enumeration of numerical ranges by endpoints includes all numbers incorporated within that range (eg, 1 to 5 are 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Moreover, all numerical ranges described throughout the specification are all within such a wider numerical range, as if all such narrower numerical ranges were explicitly stated herein. It should be understood that it covers a narrower numerical range of. If a range of values is a particular value "greater than", "less than", etc., that value is included within that range.

Any direction referred to herein, such as "top", "bottom", "left", "right", "top", "bottom", "top", "bottom", and other directions as well. Orientations are described herein for clarity in connection with the figures and do not limit the actual device or system, or the use of that device or system. Many of the devices, products or systems described herein can be used in many orientations and orientations. The orientation descriptor used herein for a cell culture device often refers to the direction in which the device is directed for the purpose of culturing cells within the device.

It should also be noted that the description herein refers to an ingredient being "structured" or "applied" to function in a particular way. In this regard, such components are "configured" or "applied" to embody specific properties or to function in a particular way, such statements as opposed to statements of purpose of use. Structural description. More specifically, references herein to how a component is "constituent" or "applied" indicates the existing physical state of that component and thus the structural characteristics of that component. It should be interpreted as a clear statement.

As used herein, the term "cell culture" refers to keeping cells alive in vitro. The term includes continuous cell lines (eg, those with immortal phenotype), primary cell cultures, finite cell lines (eg, non-transformed cells), and in vitro including egg mother cells and embryos. Any other cell population maintained in.

As used herein, the term "in vitro" refers to an artificial environment and a process or reaction that occurs within an artificial environment. The in vitro environment can consist of, but is not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (eg, animals or cells) and processes or reactions that occur within the natural environment.

As used herein, the term "cell culture product" means any container useful for culturing cells and provides an environment for cell culture, plates, wells, flasks, mulch. Includes well plates, multi-layer flasks and perfusion systems.

In embodiments, a "well" is an individual cell culture environment provided in a multi-well plate fashion. In embodiments, the wells can be in the form of a 4-well plate, a 5-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 96-well plate, a 1536-well plate, or any other multi-well plate.

As used herein, a chamber or well or the like "structured to grow cells of interest in 3D conformation" is a cell in 3D or spheroid conformation rather than as a two-dimensional sheet of cells in culture. Means a well having a dimension or treatment, or a combination of dimensions and treatments that promotes the growth of. Treatments include, for example, treatments with low binding solutions, treatments to make the surface less hydrophobic, or sterilization treatments.

As used herein, "structured to provide" or "configured to provide" means that the product has features that provide the described results. do.

In an embodiment, a single " spheroid well" is a multi-well plate having a structure that allows cells of interest to grow as a single 3D cell mass or as a single spheroid in the single spheroid well. Can be a well. For example, 96-well plate wells (traditional 96-well plate wells are about 10.67 mm deep and have a top opening of about 6.86 mm and a well bottom diameter of about 6.35 mm.

In embodiments, " spheroid plate" means a multi-well plate having an array of single spheroid wells.

In embodiments, the wells may have an array of "microcavities". In embodiments, the "microcavity" is, for example, a microwell defining an upper opening and a bottom point, a center of the top opening, and a central axis between the bottom point and the center of the top opening. could be. In embodiments, the wells are rotationally symmetric about their axis (ie, the sidewalls are cylindrical). Alternatively, in embodiments, the wells may have a hexagonal shape as shown in FIG. 1C, or any other shape. In some embodiments, the top opening defines the diameter of the top opening from 250 μm to 1 mm, or any range within those measurements. In some embodiments, the distance from the top opening to the lowest point (depth "d") is between 200 μm and 900 μm, or between 400 and 600 μm. The array of microcavities may have different shapes, such as parabolic, hyperbolic, chevron, and cross-sectional shapes, or a combination thereof.

In embodiments, "microcavity spheroid plate" means a multi-well plate having an array of wells, each well having an array of microcavities.

In embodiments, the "round bottom" of a well or microcavity well can be part of a hemisphere, such as, for example, a hemisphere, or a horizontal cross section or slice of the hemisphere that forms the bottom of the well or microcavity.

In embodiments, the term "3D spheroid " or " spheroid " can be, for example, a ball of cells in culture rather than a flat two-dimensional sheet of cells. The terms "3D spheroid " and " spheroid " are used interchangeably herein. In embodiments, the spheroids are, for example, from about 100 to about 500 micrometers, more preferably from about 150 to about 400 micrometers, and even more preferably from about 150 to about 300 micrometers, depending on the type of cells in the spheroid . , And most preferably composed of a single cell type or multiple cell types having a diameter of about 200 to about 250 micrometers, including values and ranges in between. The spheroid diameter can be, for example, about 200 to about 400 micrometers. The maximum size of spheroids is generally constrained by diffusion considerations (eg, for an overview of spheroids and spheroid containers, see Achilli, TM, et. Al. Expert Opin. Biol. Ther. (2012) 12 (2012). See 10)).

As used herein, "tumor cells" are those that are isolated from a tumor, are derived from a tumor, cause a tumor when injected into an animal, or are injected into an animal. It means any cell derived from the cell that causes the tumor. Tumor cells can be primary tumor cells obtained from animals, including humans, or tumor cells can be cell lines or genetically engineered cells.

As used herein, "insert" means a cell culture well that fits snugly into the well of a spheroid plate or microcavity spheroid plate. This insert has a side wall and a bottom surface that define a cavity for culturing cells. The bottom surface is porous, allowing cells or chemicals to travel through the porous bottom surface and act on cells of interest growing into a 3D conformation.

As used herein, "insert plate" means an insert plate that includes an array of inserts structured to fit snugly into the array of wells in a multi-well plate.

As used herein, "therapeutic agent" means any bioactive material selected from the desired and usually beneficial or therapeutic effect. Therapeutic agents include, for example, but are not limited to: antitumor agents, immunosuppressants, immunostimulants (immune-stimulants), antiproliferative agents, antithrombins, antiplatelet substances, antilipids, anti-inflammatory agents, etc. Antibiotics, angiogenesis, anti-angiogenic, vitamins, ACE inhibitors, vasoactive substances, anti-thread splitting agents, metalloproteinase inhibitors, NO donors, estradiol, anti-sclerotic agents, hormones, It may include low molecular weight therapeutic agents commonly referred to as "drugs" which include, but are not limited to, free radical scavengers, toxins, alkylating agents alone or in combination. Therapeutic agents may also include, for example, but not limited to, biological agents including peptides, lipids, protein drugs, protein complex drugs, enzymes, oligonucleotides, ribozymes, genetics, prions, viruses, and bacteria.

As used herein, "cell therapy" means any cell that may affect another cell. Cell therapy agents contact cells of interest, provide an environment that affects cells of interest, swallow cells of interest (phagocytic action), excrete chemicals that affect cells of interest, or It can act by destroying the 3D cell population provided by other methods, or it affects the cells of interest in other ways.

Unless otherwise stated, none of the methods described herein is intended to be construed as requiring the steps to be performed in a particular order. Thus, if a method claim does not actually state the order in which the steps should be followed, nor does the claim or specification specifically state in the claim or specification that it is limited to a particular order. No intention is made to infer any particular order. Any single or plural described feature or aspect in any one claim may be combined with or replaced with any other described feature or aspect in any other claim.

Various features, elements or steps of a particular embodiment can be disclosed using the transitional phrase "comprising", using the transitional phrase "consisting of" or "consisting of substantially". It should be understood to imply another embodiment that includes those that can be described.

The present disclosure describes methods and experimental instruments for analyzing therapeutic agents, including immune cells and drugs, as well as their effects on tumor cells growing in 3D spheroid conformation in culture, among others. The method allows the investigation of the effects of therapeutic agents such as immune cells or drugs on tumor cells, among other uses, and homing in a single, easy-to-use, high-throughput system for more in vivo-like testing. Allows investigation of tumor cell toxicity, and tumor immune avoidance. Unlike most current high-throughput models for immune cell migration and invasive assays that utilize tumor cells cultured in 2D, this model enables 3D tumor spheroid components for more in vivo-like testing. do.

In various embodiments, assays for detecting the mobility and cytotoxicity of therapeutic agents are disclosed. The assay method comprises culturing tumor cells in a cell culture product to form spheroids , the cell culture product comprising a chamber, eg, a well, structured to grow the tumor cells in a 3D spheroid conformation. The assay further comprises placing an insert containing a porous membrane into the cell culture product and introducing a therapeutic agent into the insert. The assay further comprises detecting, after a period of time, the ability of the therapeutic agent to move from the insert into the cell culture product chamber, and detecting tumor cell lysis. Combining such cell culture products with inserts containing porous membranes creates a model for studying 3D cancer and immune cell interactions in a single high-throughput assay. Unlike most current high-throughput models for immune cell migration and invasive assays that utilize tumor cells cultured in 2D, this model enables 3D tumor spheroid components for more in vivo-like testing. do. Another model for high-throughput 3D tumor spheroid formation and cytotoxicity assays is incompatible with the transfer and invasive component permeability supports of these assays. As a result, this model enables the investigation of immune cells and / or drug homing, tumor cytotoxicity, and tumor antigenicity in a single, easy-to-use, high-throughput system.

Cells cultured in three dimensions, such as spheroids , may exhibit more in vivo-like functionality than their counterparts cultured in two dimensions as a monolayer. In a two-dimensional cell culture system, cells can adhere to the substrate to be cultured. However, when cells are grown three-dimensionally, such as spheroids , the cells interact with each other rather than attach to the substrate. Cells cultured in three dimensions are more closely similar to in vivo tissues in terms of cell communication and development of extracellular matrix. For example, traditionally, tumor-killing activity and antigenic escape are independent by utilizing two-dimensional (2D) grown cells that may not accurately reflect the complexity of the tumor in the 3D system. Has been studied. The barrier that immune cells need to overcome in 3D and in vivo systems is the 3D system, which is much greater than the barrier in the 2D system. Not only do immune cells need to migrate to the tumor site, but they also need to infiltrate the 3D structure to attack the target cells. Moreover, beyond the physical differences between 2D and 3D systems, phenotypic differences can occur when tumor cells are cultured in 3D, which allows for greater resistance to cytotoxicity. Shown. Tumor cell spheroids therefore provide better models of cell migration, differentiation, survival, and growth, and thus provide a better system for diagnostic studies, including efficacy, pharmacology, and toxicity testing. offer.

Here, with reference to FIGS. 3A and 3B, embodiments of cell culture products comprising a chamber of structure in which tumor cells grow in a 3D spheroid conformation, eg, a spheroid plate, are shown. FIG. 3A shows an embodiment of the spheroid plate 11, in this case a 96-well plate having a rounded bottom 119 configured to contain a single spheroid in each of the 96 wells. Normally, these plates are used with the top opening 118 of the well 101 facing up, but in FIG. 3A, the plates are shown upside down to show the structure of the bottom of the well 101. FIG. 3B is a diagram of an embodiment of a spheroid microplate of a frame 130, a plurality of wells 101, each having an upper opening 118, a side wall 121, and a liquid impermeable recessed arcuate bottom surface 119. Each 3D tumor cell spheroid 25 is shown at the bottom of a separate well 101. In embodiments, the frame 130 may hold the bottom of the well above a surface such as a laboratory table or table. In some embodiments, a space may be provided between the bottom of the well 119 and the surface under the plate. In embodiments, the space may communicate with the external environment or may be closed.

In embodiments, the at least one recessed arched bottom of the chamber may have, for example, multiple adjacent recessed arched bottoms in the same well. Alternatively, as shown in FIG. 1, a multi-well plate may have a well with a flat bottom with an array of adjacent recessed arcuate bottoms or microcavities within the same well. In embodiments, the cell culture product is in a single well or multiwell plate structure, eg, a microcavity spheroid plate, with many " spheroid wells" such as multiple dimples or pits at the bottom or base of each well. could be. Multiple spheroids or spheroid wells per chamber may preferably contain, for example, a single or one spheroid per spheroid well.

Next, with reference to FIGS. 1A, 1B and 1C, one embodiment of a microcavity spheroid plate, in this case having an array of microcavities on the bottom of each well, with a plurality of spheroids in each of the 96 wells. A 96-well microcavity spheroid plate is shown. FIG. 1A shows a multi-well plate 10 with an array of wells 101. FIG. 1B shows a single well 101 of the multi-well plate 10 of FIG. 1A. The single well 101 has an upper opening 118, a bottom surface 106, and a side wall 113. FIG. 1C is an exploded view of a portion of the bottom surface 106 of the well 101 shown in box C in FIG. 1B showing an array of microcavities 112 at the bottom surface of a single well shown in FIG. 1B. Each microcavity 115 in the array of microcavities 112 has a side wall 121 and a bottom surface 116. The microcavity spheroid plates shown in FIGS. 1A, 1B and 1C, which provide an array of microcavities 112 at the bottom of each individual well 101, grow 3D spheroids in each microcavity of each individual well of the multiwell plate. Can be used to make it. By using this type of container, the user can grow a large number of spheroids in each well of the multi-well plate, thereby the same for use in assays as provided herein. A large number of spheroids that can be treated under culture and experimental conditions can be provided.

Next, with reference to FIG. 2, a diagram of an array of microcavities 112 is shown. FIG. 2 shows a well 115, each having a top opening 118, a bottom surface 119, a depth d, and a width w defined by a side wall 121. As shown in FIG. 2, the array of microcavities has a rounded bottom 119. In embodiments, the bottom surface of the microcavity can be rounded or conical, angled, flat bottom, or any shape suitable for forming 3D tumor spheroids . In embodiments, the microcavity has a rounded bottom. The rounded bottom 119 may have a transition zone 120 as a vertical side wall transitioning to the rounded bottom 119. This can be a smooth or sloping transition zone. In embodiments, the "microcavity" defines, for example, the top opening 118 and the bottom point 116, the center of the top opening, and the central axis 105 between the bottom point and the center of the top opening. It can be a microwell 115. In embodiments, the wells are rotationally symmetric about their axis (ie, the sidewalls are cylindrical). Alternatively, the wells may have other shapes, such as hexagons. In some embodiments, the top opening defines the diameter of the top opening (width w) in any range from 250 μm to 1 mm, or within their measurements. In some embodiments, the distance from the top opening to the lowest point (depth "d") is 200 μm to 900 μm, or 400 to 600 μm. The array of microcavities may have various shapes, such as parabolic, hyperbolic, chevron, and cross-sectional shapes, or a combination thereof. In embodiments, the microcavities may have a protective layer 130 beneath them that protects them from direct contact with surfaces such as laboratory tables or tables. In some embodiments, space 110 may be provided between the bottom 119 of the well and the protective layer. In embodiments, the space 110 may communicate with the external environment or may be closed.

In embodiments, the bottom of the chamber having at least one recessed arched bottom or "cup" is, for example, a hemispherical surface, a conical surface with a rounded bottom, and a similar surface shape, or a combination thereof. obtain. The chamber (eg, well) and chamber bottom (eg, well bottom or microcavity bottom) are ultimately the "gentle" roundness of spheroids such as dimples, pits, and similar recessed conical trapezoidal relief surfaces, or a combination thereof. With a tinged or curved surface, it terminates, terminates, or reaches the bottom. In embodiments, the at least one recessed surface of the cell culture product chamber comprises a hemispherical surface, a conical surface having a taper of 30 to about 60 from the side wall to the bottom surface, or a combination thereof. In some embodiments, the at least one recessed arched bottom surface depends on, for example, the selected well shape, the number of recessed arched surfaces in each well, the number of wells in the plate, and similar considerations. And, for example, a horizontal cross section or slice of a hemisphere having a diameter of about 250 to about 5,000 micrometers (ie 0.010 to 0.200 inches (254 to 5080 micrometers)) including intermediate values and ranges. Can be part of a hemisphere. Other recessed arcuate surfaces can have, for example, parabolic, hyperbolic, chevron, and similar cross-sectional shapes, or combinations thereof.

In embodiments, the cell culture product comprising a chamber, eg, a spheroid plate or a microcavity spheroid plate, is poorly adherent, ultra-low on a portion of the chamber, eg, on at least one recessed surface and / or one or more sidewalls. It may further include an adhesive or non-adhesive coating. Examples of non-adhesive materials include perfluoropolymers, olefins, or similar polymers, or mixtures thereof. Other examples include nonionic hydrogels such as agarose, polyacrylamide, or polyols such as polyether or polyvinyl alcohol such as polyethylene oxide, or similar materials, or mixtures thereof.

In embodiments, the sidewall surface (ie, perimeter) has, for example, a vertical cylinder or shaft, a portion of a vertical cone whose diameter decreases from the top of the chamber to the bottom of the chamber, a conical transition, ie square or square at the top of the well. It can be a vertical square shaft or a vertical oval shaft, or a combination thereof, that is oval, transitions to a cone, and has at least one recessed arched surface, i.e., terminating at a rounded or curved bottom. Other exemplary shapes include perforated cylinders, perforated conical cylinders, first cylinders and later cones, and other similar shapes, or combinations thereof.

For example, a low-attainment substrate, well curvature at the body and base of the cell culture product chamber, and one or more of gravity can self-assemble tumor cells into spheroids . Tumor cells maintain differentiated cell function, which indicates a more in vivo-like response compared to cells grown in a single layer. In embodiments, the spheroids are, for example, from about 100 to about 500 micrometers, more preferably from about 150 to about 400 micrometers, even more preferably from about 150 to about 300 micrometers, and depending on the cell type in the spheroid , for example. Most preferably it can be, for example, substantially a sphere, which is about 200 to about 250 micrometers and has a diameter including values and ranges in between. The spheroid diameter can be, for example, from about 200 to about 400 micrometers, and the upper diameter is constrained by diffusion considerations.

In embodiments, the cell culture product may further comprise a gas permeable and liquid permeable bottom containing an opaque sidewall and / or at least one recessed surface. In some embodiments, at least a portion of the bottom, including at least one recessed surface, is transparent. Wellplates with such characteristics perform an assay (eg, measurement of therapeutic drug lysis and migration) from a single multiwell plate (in which spheroids can be formed and visualized) the tumor cell spheroids . Provides some benefit for the methods of the present disclosure, including eliminating the need to transfer to another plate to, thus saving time and avoiding any unnecessary destruction of spheroids . be able to. In addition, a gas permeable bottom (eg, a well bottom made of a polymer with gas permeable properties at a particular given thickness) allows tumor spheroids to receive an increased oxygen supply. Can be done. An exemplary gas permeable bottom can be formed from a polymer such as perfluoropolymer or poly4-methylpentane at a particular thickness. Typical thicknesses and ranges of gas permeable polymers range from about 0.001 inches (25.4 micrometers) to about 0.025 inches (635 micrometers), 0.0015 inches (38.1 micrometers), for example. ) To about 0.03 inch (762 micrometers), including intermediate values and ranges (where 1 inch = 25,400 micrometers; 0.000039 inches = 1 micrometer). Additional or alternative, other materials with high gas permeability, such as polydimethylsiloxane polymers, may provide sufficient gas diffusion at thicknesses up to, for example, about 1 inch (25,400 micrometers). can.

In embodiments, the cell culture product can further include a chamber appendage, a chamber expansion region, or an auxiliary side chamber for receiving the pipette tip for suction, which chamber appendage or chamber extension (eg, eg). , Side pockets) can be, for example, an integral surface adjacent to the chamber and in fluid communication with the chamber. The chamber appendage may have a second bottom that is separated from the liquid impermeable bottom of the chamber. The chamber appendage and the second bottom of the chamber appendage can be separated from the liquid impermeable bottom of the chamber, for example at higher or relative heights. The second bottom of the chamber appendage diverts the fluid distributed from the pipette from the liquid impermeable bottom of the chamber to avoid disruption or disruption of the spheroids .

Assays for detecting the mobility and cytotoxicity of a therapeutic agent further include placing an insert containing a porous membrane in the cell culture product and introducing the therapeutic agent into the insert. In embodiments, the insert comprising the porous membrane can be placed in a portion of the chamber, in a portion of the chamber appendage, or in both the chamber and the chamber appendage portion. Inserts containing a porous membrane can be placed in the top of the chamber, in the top of the chamber formed by the porous membrane, or in one or both chambers of therapeutic agents such as immune cells or drugs placed in both chambers, near the clear bottom. Provides isolation or isolation (at least first) from tumor cell spheroids in the lower part.

4A and 4B are perspective views of the insert 400. The insert 400 shown in FIGS. 4A and 4B is a Corning Transwell® insert. As shown in FIGS. 4A and 4B, the insert has an upper opening 418, side walls 421 and bottom surface 419 forming the cavity 420. As shown in FIG. 4C, these inserts 400 can be provided in the form of insert plate 401, in which case a single plate 401 comprises multiple inserts 400 and a multi-well insert plate is a multi-well plate 11. The structure is such that it is inserted into a complementary array of wells inside. Inserts are available in many forms. In embodiments, these inserts have a porous bottom surface that is sufficiently porous to allow small molecules such as drugs, proteins, vectors, or other materials to pass through the bottom surface 119 but not cells. In a further embodiment, the insert has a porous bottom surface 419 with pores of sufficient diameter for cells such as cell therapies, including immune cells, to migrate through the bottom surface.

The porous membrane of the insert can be made of a variety of materials, including but not limited to, truck-etched membranes or woven or non-woven porous materials. The material of the porous membrane can be treated or coated to be more adhesive or non-adhesive to the cells, or treated or for any other desirable coating to support cell culture. Can be coated. The treatment can be performed by a number of methods known in the art, including plasma discharge, corona discharge, gas plasma discharge, ion shock, ionization irradiation, and high intensity UV light. The coating can be introduced by any suitable method known in the art, including printing, spraying, condensation, radiant energy, ionization techniques or immersion. The coating may then provide either covalent or non-covalent sites. Such sites can be used to bind moieties such as cell culture components (eg, proteins that promote growth or adhesion). In addition, the coating can also be used to enhance cell attachment (eg, polylysine). Alternatively, cell non-adhesive coatings such as those described above can be used to prevent or inhibit cell junctions. The porous membrane may be gamma-ray sterilized. Such inserts are generally available from Corning (“Transwell”) or Millipore® (Millicell® or Ultracell®).

In some embodiments, the porous membrane may be treated or coated such that at least a portion of the porous membrane has a structure that simulates the blood-brain barrier (BBB), as is known in the art. .. In some embodiments, at least a portion of the porous membrane comprises, but is not limited to, an essentially confluent monolayer of endothelial cells such as microvascular endothelial cells. In some embodiments, the microvascular endothelial cells are brain endothelial cells. In some embodiments, the endothelial cells contained in the microvascular endothelial cells are used in combination with astrocytes and / or pericytes. Such a model is useful for studying brain cancer, which is one of the most difficult cancers to treat due to the BBB acting as the defense system of the brain itself. BBB, which should protect the brain from potential toxins, often prevents conventional therapies, such as chemotherapy, from reaching brain tumors. Traditionally, in vitro compound permeability high throughput testing with BBB is an assay for radiolabeled or fluorofore-labeled compounds, as the radiolabeled or fluorofore-labeled compound passes through a cell monolayer growing on a permeable support system. Has been limited to. Unfortunately, the labeling itself can affect the assay and the ability to determine consequent tumor cytotoxicity must be investigated independently. The methods and data disclosed herein provide a three-dimensional (3D) model for studying the BBB transport of therapeutic agents (eg, cytotherapeutic agents, drugs, or biological agents) and the resulting brain tumor cell toxicity of the therapeutic agents. Shown, enabling investigation of homing, tumor cell toxicity, and tumor immunoavoidance in a single, easy-to-use, high-throughput system for more in vivo-like testing. Unlike most current high-throughput models for immune cell migration and invasive assays that utilize tumor cells cultured in 2D, this model enables 3D tumor spheroid components for more in vivo-like testing.

In certain aspects of the assay of the present disclosure for detecting the mobility and cytotoxicity of a therapeutic agent, the present disclosure is combined with a Corning 96HTS Transwell permeability support system to provide an environment for performing the assay. The use of Corning spheroid microplates is provided. Corning spheroid microplates are multi-well cell culture microplates with a round bottom well shape and are coated on the Corning Ultra-Low Attainment surface so that they are highly reproducible singles placed in the center of each well. Can form multicellular tumor spheroids . Corning 96 HTS Transwell is a permeable support used for high-throughput drug transport, as well as cell migration and invasive studies.

5A-5C show embodiments of the disclosed methods for detecting the mobility and cytotoxicity of therapeutic agents. As shown in FIG. 5A, cells of interest 525 are grown in medium 500 in well 101 having a structure in which cells of interest such as tumor cells are grown in 3D conformation. In this case, well 101 is a single single spheroid well. In an embodiment, the well 101 is one well of a 96-well plate, for example, one well of a Corning 96-well spheroid plate. In embodiments, cells of interest 525, such as tumor cells, can grow in wells of a multi-well plate (see FIG. 1C), where each well 101 has an array of microcavities 112 and each microcavity. Is a structure that grows cells of interest in 3D conformation, resulting in an array of spheroids , one for each microcavity in the array of microcavities on the bottom of the wells of the multiwell plate. As cells grow and propagate in culture, they grow as spheroids 25. Over time, spheroid 25 is generated. FIG. 5A shows the formation of spheroid 25. Once the tumor cells have developed into spheroid 25, the cell culture insert 400 is placed in well 101 as shown in FIG. 5B. In embodiments, the insert may be an insert plate as shown in FIG. 4B. A therapeutic agent (eg, a cell therapeutic agent, drug, or biological agent), in this case the cell therapeutic agent 30, is added to the cavity 420 of the insert 400. In this form, together with an insert containing a therapeutic agent (eg, a cell therapeutic agent, drug, or biological agent) such as therapeutic agent cells 30, and a well 101 containing cells 525 of interest for 3D conformation such as 3D tumor spheroids . Incubate to. Then, after a suitable time, the effect of the therapeutic agent (eg, cell therapeutic agent, drug, or biological agent), in this case the cell therapeutic agent 30, on cells of interest such as 3D tumor spheroids , as well as cells from the insert. The transfer of the therapeutic agent to the assay chamber of the culture product is measured by the assay. This incubation period depends on the type of assay performed and the therapeutic agent used in this assay, such as a cell therapeutic agent, drug, or biological agent. This incubation period also depends on the pore size used for the membrane in the insert 400. The larger the pore size, the faster the therapeutic agent is distributed and therefore the shorter the incubation period, but with the potential for non-specific results. Adjustment of incubation time can be performed in advance experiments and is within the general skill of the art. As shown in FIG. 5C, the effect of the therapeutic agent 30 on the tumor spheroid 25 can then be measured. For example, in FIG. 5C, in an embodiment, the assay can measure the lysis of spheroid 25.

8A, 8B, 8C, 8D, 8E, 8F and 8G detect the mobility and cytotoxicity of therapeutic agent 30 in a model comprising the use of a layer of cells 300 on a porous membrane 302 that is part of an insert 400. It is a figure of the embodiment of the method of this disclosure for this. In these embodiments, the layer of cells 300 on the porous membrane 302 of the insert 400 is configured to simulate the blood-brain barrier (BBB).

First, as shown in FIG. 8A, cells 300 used to simulate BBB are seeded on the porous membrane 302 of the cell culture insert 400 in the culture medium 500. These cells are, for example, endothelial cells. After a period of time, seeded cells 300, such as endothelial cells, form a confluent monolayer (2D layer) of cells 306 that simulates BBB. Once a confluent monolayer of cells 306 has been formed, the insert 400 can be placed in the spheroid well 101 as shown in FIG. 8C.

Similar to FIG. 5, cells of interest, such as tumor cells, also have a structure in well 101 such that the cells of interest grow in 3D conformation to form spheroid 25, as shown in FIG. 8B. Grow in 500. As shown in FIG. 8B, the well 101 is a single, single spheroid well. In embodiments, cells of interest, such as tumor cells, are wells of a multi-well plate having an array of microcavities, each of which has a structure in which each microcavity grows the cells of interest in a 3D conformation (see Figure 1C). It can grow in (as shown in FIGS. 1A, 1B and 1C), resulting in a large number of spheroids , one for each of the microcavities in the array of microcavities on the bottom of the wells of the multiwell plate. .. As cells grow and proliferate in culture, they are grown as spheroids , resulting in spheroid 25, as shown in FIG. 8B. Once the tumor cells have become spheroids 25 and a confluent monolayer of endothelial cells 306 that simulates BBB has been formed, cell culture inserts 400 are placed in wells 101 as shown in FIG. 8C.

Also with reference to FIG. 8C, a therapeutic agent (eg, a cell therapeutic agent, drug, or biological agent), in this case drug 310, is added to the cavity 312 of the insert 400, as in FIG. In this form, a well containing a therapeutic agent 310 (eg, a cell therapeutic agent, drug, or biological agent), in this case an insert 400 containing the drug 310, and cells of interest for 3D conformation such as 3D tumor spheroid 25. Incubate together. This is shown in FIGS. 8D and 8E. The incubation period depends on the therapeutic agent used in the assay, such as a cell therapeutic agent, drug, or biological agent. This also depends on the pore size used for the membrane 302 in the insert 400. The larger the pore size, the faster the distribution of the therapeutic agent 310 and therefore the shorter the incubation period, but with the potential for non-specific results. Adjustment of incubation time can be done in advance experiments and is within the normal skill of the art. A layer of cells 306 on a porous membrane 302 configured to simulate the blood-brain barrier (BBB) can be used to assess the ability of a therapeutic agent to cross the blood-brain barrier.

Then, after a suitable time, the effect of the therapeutic agent 310 on cells of interest, such as 3D tumor spheroid 25, can be measured. For example, as shown in FIG. 8F, destruction or lysis of spheroid 25 is shown. This disruption can be measured by measuring changes in the integrity of the spheroid 25 in the assay chamber 315 of the cell culture product. Alternatively, as shown in FIG. 8G, spheroid 25 is shown to be intact after the incubation period, while cell function can be measured to measure changes in the physiological function of the cells constituting spheroid 25. .. For example, the cytotoxic effect of the drug 310 or the penetration of the drug 310 into the 3D tumor spheroid 25 can be measured by measuring changes in tumor cell physiology.

The transfer of therapeutic agent 310 from the insert 400 to the assay chamber 315 of the cell culture product can be measured. For example, the therapeutic agent 310 is introduced into the device's insert chamber 312 and then evaluated for the concentration of the drug 310 as measured from the assay chamber 315 to determine how many drug 310s cross the blood-brain barrier.

Effects on 3D tumor spheroids and transfer of therapeutic agents from inserts to chambers of cell culture products are known in the art including visualization of cells, cell extracts, or media, fluorescence measurements, genes, metabolism or protein analysis. It can be measured by any means of. For example, migration of immune cells (if the therapeutic cells are immune cells) and cytotoxicity (or changes in the physiological function of tumor cells), eg, but not limited to, appropriate as known in the art. It can be evaluated by flow cytometry by using different stains and can be used in the examples herein. As a further example, the transfer of a drug or biological agent can be measured by using a labeled drug or biological agent, including a radiolabeled drug or biological agent or a fluorescently labeled drug or biological agent. Penetration or tumor cell lysis of a therapeutic agent, eg, a cell therapeutic agent, drug, or biological agent, into 3D tumor spheroids can be detected, for example, by visualization and / or fluorescence measurement, without limitation. For example, 3D tumor spheroids and infiltrating cells can be fixed and divided, and the cells can be stained by conventional histological techniques as is known in the art.

Various tumor cell types can be cultured to form 3D tumor spheroids 25. Cancer cells used in tumor cell types that can be cultured to form 3D tumor spheroids 25 include tumors, neoplasms, cancers, precancerous conditions, any cells derived from cell lines, or an unlimited number. Included are any other sources of cells that have the potential to expand and grow. Cancer cells are of natural origin or are artificially generated. Cancer cells can infiltrate and metastasize to other tissues when placed in an animal host. Cancer cells further include any malignant cells that have invaded and / or metastasized to other tissues. One or more cancer cells in the context of an organism are also any other used in the art to describe cells in a cancer, tumor, neoplasm, growth, malignant tumor, or cancerous state. It can also be called by term.

Cancers that act as tumor cell type sources that can be cultured to form 3D tumor spheroids 25 are, but are not limited to, solid tumors such as fibrosarcoma, mucinosarcoma, liposarcoma, chondrosarcoma. , Bone sarcoma, spinal cord tumor, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, lymphoma, mesopharyngeal tumor, Ewing tumor, smooth myoma, rhizome myoma, colon Cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, brain cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, Flat epithelial cancer, basal cell cancer, adenocarcinoma, sweat adenocarcinoma, sebaceous adenocarcinoma, papillary cancer, papillary adenocarcinoma, cystic adenocarcinoma, medullary cancer, bronchial cancer, renal cell carcinoma, Liver cancer, bile duct cancer, chorionic villi, semi-norma, embryonic cancer, Wilms tumor, cervical cancer, uterine cancer, testis cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial cancer, gliosis Tumors, polyplastic glioblastomas, stellate cell tumors, medullary blastomas, cranopharyngeal tumors, lining tumors, pine fruit tumors, hemangioblastomas, acoustic neuromas, oligodendroglioma, meningeal tumors, skin cancers, Examples include melanoma, neuroblastoma, and retinal blastoma.

Further cancers that act as cancer cell sources include, but are not limited to, blood-infectious cancers such as acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, and acute lymphoblastic leukemia. Sexual T-cell leukemia, acute myeloblastic leukemia, acute premyeloblastic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute macronuclear blastomelic leukemia, acute myelogenous leukemia, acute Nonlymphatic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hair cell leukemia, multiple myeloma, lymphoblastic leukemia, myeloid leukemia, lymphocytic leukemia, bone marrow Includes sexual leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, Waldenstrem macroglobulinemia, heavy chain disease, and true erythrocytosis. In some embodiments of the methods for detecting the mobility and cytotoxicity of therapeutic agents disclosed herein, a 3D tumor spheroid comprises one or more cancer cells. In some embodiments, the cancer cells are cryopreserved. In some embodiments, one or more cells are actively dividing.

In some embodiments, the method comprises a culture medium (eg, nutrients (eg, proteins, peptides, amino acids) known in the art, energy (eg, carbohydrates), essential metals and minerals (eg, calcium, magnesium, etc.). Iron, phosphates, sulphates), buffers (eg phosphates, acetates), pH change indicators (eg phenol red,
Includes bromocresol purple), selective agents (eg, chemicals, antibacterial agents, etc.).

In some embodiments of the methods of the present disclosure for detecting the mobility and cytotoxicity of a therapeutic agent, the therapeutic agent is a cell therapeutic agent, drug, and / or biological agent.

In some embodiments, the cell therapy agent comprises immune cells. In some embodiments, the immune cell is a leukocyte (eg, neutrophil, macrophage, dendritic cell, or monocyte). In some embodiments, the immune cell is a lymphocyte (eg, a natural killer cell, or a lymphocyte, such as a T cell, a B cell, and more particularly a cytotoxic T cell).

In the methods of the present disclosure for detecting the mobility and cytotoxicity of a therapeutic agent containing a known anticancer drug or a substance that appears to have potential anticancer activity, such as a novel molecular entity or a novel chemical entity. Various drugs 310 can be tested. Anticancer drugs include, but are not limited to: ashibicin; acralubicin; accordazole hydrochloride; acronin; adzelesin; aldesroykin; altretamine; ambomycin; amethanetron acetate; aminoglutetimid; amsacrine; anastro. Zol; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicartamide; bisanthren hydrochloride; bisnafide dimesylate (bisnafido dimesylate); bizerecin; Bropirimine; Busulfan; Cactinomycin; Calsterone; Carasemido; Calvetimer; Carboplatin; Carmustin; Carbisin Hydrochloride; Carselcin; Sedefingol; Chlorambusyl; Cysplatin; Cladribine; Citarabin; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanin; dezaguanine mesylate; diazikun; docetaxel; doxorubicin; doxorubicin hydrochloride; (Duazomycin); edatorexate; eflomitine hydrochloride; elsamitrucin; enloplatin; empromart; epipropidine; epirubicin hydrochloride; erbrozole; esorbicin hydrochloride; estramustin; estramic acid phosphate Etoprin; Fadrazole Hydrochloride (Fadrosol); Fazarabin; fenretinide; floxuridine; Fludurabin Phosphate; Fluorouracil; Flurositabin; Hoskidone; Hostriecin (fostriecin) Sodium; Gemusitabin; Gemusitabin; Iphosphamide; ilmohocin; interleukin II (recombinant interleukin II, or rIL2) Includes), Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-Ia; Interferon γ-Ib; Iproplatin; Irinotecan hydrochloride; Lanleotide acetate; Retrosol; Riarosol; Lometrexol Sodium; Romustin; Losoxanthron Hydrochloride; Masoprocol; Mytancin; Mechloretamine Hydrochloride; Megastrol Acetate; Melengestrol Acetate; Melfalan; Menogaryl; Mercaptopurine; Metotrexate; ); Mitochromin; mitodilin; mitomalcin; mitomycin; mitosper; mittan; mitoxanthron hydrochloride; mycophenolic acid; nocodazole; nogaramycin; olmaplatin; oxythran; paclitaxel; Pentamustine; Pepromycin sulfate; Perphosphamide; Pipobroman; Piposulfan; pyroxantrone; Plicamycin; Promethane; Porfimer sodium; Porphyromycin; Prednimustin; Procarbazine hydrochloride; Puromycin; Pyrazofluin; Ribopurin; Logletimide; Saffingol; Saffingol Hydrochloride; Semstine; Simtrazen; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Talysomycin; Tecogalan sodium; Tegafur; Teloxanthron hydrochloride; Temoporfin; Teniposide; Teloxylone; Testlactone; Thiamipurine; Thioguanin; Thiotepa; Thiazofluin; Thirapazamine; Tremiphen citrate; Trestron acetate; Trisiribin phosphate; Trimethrexate; Trexate; tryptreline; tubrosole hydrochloride; uracil mustard; uredepa; vaporotide; verteporfin; bin blastin sulfate; bin sulfate Christine; vindesine; vindesine sulphate; vinepidin sulphate; binglisine sulphate; vinleurosine sulphate; vinorelbine tartrate; vinrosidine sulphate; vinrosidine sulphate; Other anti-cancer agents include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; avirateron; acralbisin; acylfluben; adecpenol; adzelesin; aldesroykin; ALL-TK antagonists. Altretamine; ambamustin; amidox; amifostine; aminolevulinic acid; amurubicin; amsacrine; anagrelide; anastrozole; androglaholide; angiogenesis inhibitor; antagonist D; antagonist G; antarellix; anti-dorsalyzing morphology Forming protein-1; anti-androgen, prostate cancer; anti-estrogen; anti-neoplastic drug; antisense oligonucleotide; affidicholine glycine acid; apoptosis gene modulator; apoptosis regulator; aprinoic acid; ara-CDP-DL-PTBA; arginine deaminase Athraculin; Atamestan; Atrimstin; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azacetron; Azatoxin; Azatyrosine; Baccatin III derivative; Baranol; Batimastat; BCR / ABL antagonist; Benzochlorin; Porin; β-lactam derivative; β-alethin; betaclamycin B; bethulic acid; bFGF inhibitor; bicartamide; bisantren; bisaziridinyl spermin; bisnaphthide; Carcipotriol; Carhostin C; Camptothecin derivative; Canalipox IL-2; Capecitabin; Carboxamide-amino-triazole; Carboxamide triazole; CaRest M3; CARN 700; Cartilage-derived inhibitor; Carzelsin; Casein kinase inhibitor (ICOS); Castanospermin; secropin B; cetrorelix; chlorin; chloroquinoxalin sulfonamide; cicaprost; cis-porphyrin; cladribine; chromifen analog; clotrimazole; collismycin A; colismycin B; combretastatin A4; combretastatin Analog; Conagenin; Cranvecidin 816; Cristanol; Cryptophycin 8; Cryptophycin A derivative; Krasin A; Cyclopenta Anthraquinone; cycloplatum; cypemycin (chorionic villi); citalabine ocphosphate; cell lytic factor; cytostatin; dacriximab; decitabin; dehydrodidemnin B; deslorerin; dexamethasone; dexphosphamide; dexolazoxane; B; diddox; diethylnorspermin; dihydro-5-azacitidine; 9-dihydrotaxol; dioxamycin; diphenylspiromstin; docetaxel; docosanol; drasetron; doxifludarabine; droroxyphen; dronabinol; duocarmycin SA; evselen Ecomstin; Edelhosin; Edrecolomab; Eflornitin; Elemen; Emiteflu; Epirubicin; Epristeride; Estrogen analog; Estrogen agonist; Estrogen antagonist; Etanidazole; Ethoposide phosphate; Exemestane; Dole; Fresellastin; Fluasterone; Fludarabine; Fluorodaunornicin hydrochloride; Holphenimex; Formestan; Hostriecin; Fotemstin; Gadrinium texaphyllin; Gallium nitrate; Galocitabine; Ganirelix; Zeratinase inhibitor; Helegrin; Hexamethylene bisacetamide; Hypericin; Ivandronic acid; Idalbisin; Idoxyphen; Idramantone; Ilmohosin; Iromastat; Imidazoacridones; Imikimod; Immunostimulatory peptide; Insulin-like growth factor 1 receptor inhibitor Interferon agonist; Interferon; Interleukin; Jobenguan; Iododoxorbicin; 4-Ipomeanaal; Iropractin; Irsogladine; Isobengazole; Isohomohalicondorin B; Itasetron; Jaspraquinolide (jasplacinolide) ) F; Lameralin-N Triacetate; Lanleothide; Reinamycin; Lenograstim; Lentinan Sulfate; Lept Rustatin; retrozole; leukemia inhibitor; leukocyte α-interferon; leuprorelin + estrogen + progesterone; leuprorelin; levamisol; rialozole; linear polyamine analog; lipophilic disaccharide peptide; lipophilic platinum compound; lysoclinamide 7 Lovaplatin; Lombricin; Lometrexol; Lonidamine; Losoxantrone; Lovastatin; Loxorubine; Lartotecan; Lutethium texaphyllin; Lysophylline; Soluble peptide; Maitansin; (Maspin); Matrysin inhibitor; Matrix metalloproteinase inhibitor; Menogaryl; Melvalon; Meterin; Methioninase; Metoclopramid; MIF inhibitor; Mifepriston; Miltehosin; Millimostim; Mismatched double-stranded RNA; Mitoguazone; Mitractor; Analog; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody human chorionic villi gonadtropin; monophosphoryl lipid A + myobibacterium; Multidrug resistance gene inhibitor; Multitumor inhibitor 1 system therapy; Mustard anticancer drug; Mikaperoxide B; Mycobacteria cell wall extract; Miliapolon; N-acetyldinalin; N-substituted benzamide; Nafalerin; Nagres chip; Naroxone + Pentazocin; napavin; naphterpin; naltograstim; nedaplatin; nemorphicin; neridronic acid; neutral endopeptidase; nitamide; nisamicin; nitrogen monoxide modulator; nitrooxide antioxidant; nitrullyn; O6 -Benzylguanin; octreotide;


Oxenone; oligonucleotides; onapriston; ondancetron; ondancetron; oracin; oral cytokine inducer; olmaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analog; paclitaxel derivative; parauamine; palmitoyl lysoxin Pamidronic acid; Panaxitriol; Panomiphen; Parabactin; Pazelliptine; Pegasparkase; Perdecin; Pentsanpolysulfate sodium; Pentostatin; Pentrozole; Perflubron; Perphosphamide; Perylyl alcohol; Phenazineomycin; Phosphatase Inhibitors; Pisibanil; Pyrocarpine Hydrochloride; Pyralbisin; Pyrytrexim; Placetin A; Placetin B; Plasminogen Activator Inhibitors; Platinum Complexes; Platinum Compounds; Platinum-Triamine Complex; Porphymer Sodium; Porphyromycin; Prednison; -Acridone; Prostaglandin J2; Proteasome inhibitor; Protein A system immunomodulator; Protein kinase C inhibitor; Protein kinase C inhibitor, Microalgae; Protein tyrosine phosphatase inhibitor; Purinucleoside phosphorylase inhibitor; Purpurin; Pyrazoloacrindin Pyridoxilized hemoglobin polyoxyethylene conjugate; raf antagonist; lartitrexed; ramosetron; ras farnesyl protein transferase inhibitor; ras inhibitor; ras-GAP inhibitor; demethylated reteriptin; renium Re186 etidronate; lysoxin; ribozyme; RII retinamide; Logretimid; rohitukine; romlutide; rokinimex; rubiginone B1; ruboxil; saffingol; sinetopin; SarCNU; sarcophytol A; sargromostim; Sdi1 mimic; semstin; Substances; Signal conversion inhibitors; Signal conversion modulators; Single-chain antigen-binding proteins; Sizophyllan; Sobuzoxane; Sodium borocaptate; Sodium phenylacetate; Solverol; Somatomedin-binding proteins Sonermin; Sparfosic acid; Spicamycin D; Spiromustin; Sprenopentin; Spongestatin 1; Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipimamide; Stromelysin inhibitor; Sulfinocin; Superactive vasoactive intestinal peptide antagonist; Sladista; Slamine Swinesonin; Synthetic glycosamminglican; Talimstin; Tamoxiphenmethiodide; Tauromustin; Tazarotene; Tecogalan sodium; Tegafur; Tellapyrylium; Telomerase inhibitor; Temoporfin; Temozolomide; Taribrastine; thiocholalin; thrombopoetin; thrombopoetin mimic; timalfacin; timopoetin receptor agonist; timotrinan; thyroid stimulating hormone; ethylethiopurpurintin; tirapazamine; titanosendichloride; topsetin; tremiphen; Trethinoin; Triacetyluridine; Trisiribin; Trimethrexate; Tryptrelin; Tropicetron; Turosteride; Tyrosine kinase inhibitor; tyrphostins; UBC inhibitor; Ubenimex; Growth inhibitor derived from urogenital sinus; Urokinase receptor antagonist; Vapreotide; variolin B; vector system, erythrocyte gene therapy; veraresol; veramine; verdins; verteporfin; binorelbin; binxartin; vitaxin; borozole; zanoteron; zenoterone; ; And dinostatin stimalamers.

It should be understood that the methods and concepts of the present disclosure can be extended to further co-culture models. For example, although we have described a simulated blood-brain barrier, cells grown on a porous membrane can be configured to simulate any biological barrier. A "biological barrier" is a biological membrane associated with a physiological protective barrier, including, but not limited to, a blood-brain barrier, lung barrier, placenta barrier, epidermal barrier, eye barrier, olfactory barrier, gastroesophageal tract. It may include barriers, mucosa, blood-urinary barriers, air-tissue barriers, blood-bile barriers, oral barriers, anal-rectal barriers, vaginal barriers, and urinary tract barriers. Further biological barriers include blood-milk barriers, blood-testicle barriers, and mucosal barriers such as vaginal mucosa, urethral mucosa, anal mucosa, buccal mucosa, sublingual mucosa, and rectal mucosa.

Furthermore, cells that are cultured in cell culture products to form spheroid 25 are not limited to tumor or cancer cells. Various cell types can be cultured. In some embodiments, the spheroid comprises a single cell type. In some embodiments, the spheroid comprises multiple cell types. In some embodiments that grow multiple spheroids , each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown. The cells grown on spheroids may be native cells or modified cells (eg, cells containing one or more unnatural genetic modifications). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem cell or precursor of any desired state of differentiation (eg, pluripotent, multi-potent, fate determined, immortalization, etc.). Cells (eg, embryonic stem cells, iPS cells). In some embodiments, the cells are disease cells or disease model cells. Cells are, but are not limited to, adrenal gland, bladder, blood vessels, bones, bone marrow, brain, cartilage, cervix, keratin, endometrial, esophagus, gastrointestinal, immune system (eg, T lymphocytes, B lymph). Spheres, leukocytes, macrophages, and dendritic cells), liver, lungs, lymph vessels, muscles (eg, myocardium), nerves, ovaries, pancreas (eg, island cells), pituitary gland, prostate, kidneys, salivary glands, skin, tendons. It can be or can be derived from any desired tissue or organ type, including, testis, and thyroid. In some embodiments, the cells are mammalian cells (eg, humans, mice, mice, rabbits, dogs, cats, cows, pigs, chickens, goats, horses, etc.).

Moreover, the effects of therapeutic agents tested on spheroids in cell culture chambers are not limited to cytotoxicity (eg, cell lysis). The methods and concepts can be applied to drug discovery, characterization, efficacy testing, and toxicity testing. Such tests include, but are not limited to, pharmacological efficacy assessments, carcinogenicity assessments, medical contrast agent characterization, half-life assessments, radiation safety assessments, genotoxicity studies, immunotoxicity studies, reproduction and Growth tests, drug interaction evaluations, dose evaluations, adsorption evaluations, predisposition evaluations, metabolism evaluations, release studies, etc. can be mentioned. As is known to those of skill in the art, for specific tests (eg, hepatic cells for liver toxicity, proximal renal tubule epithelial cells for kidney toxicity, vascular endothelial cells for vascular toxicity, neuronal and glial cells for neurotoxicity, cardiac toxicity, Specific cell types can be used with respect to myocardial cells, skeletal muscle cells for rhabdomyolysis, etc.). The effect of therapeutic agents on spheroid cells is not limited, but can be any number, including membrane integrity, intracellular metabolic content, mitochondrial function, lysosomal function, apoptosis, genetic alterations, gene expression differences, etc. Can be evaluated for the desired parameters of.

Thus, using the methods and concepts of the present disclosure, for example, but not limited to, any drug or virus (if the therapeutic agent is a drug or virus) is the blood-brain barrier or placenta barrier (porous). To study the ability of the membrane to cross the blood-brain barrier or placenta barrier), as well as its subsequent effects (eg, cytotoxicity) and penetration into tumor spheroids in the cell culture product chamber. Can be done. Further, as an example, using the methods and concepts of the present disclosure, a therapeutic agent for viral infection of spheroid cells (if the therapeutic agent is a virus) or embryonic development (eg, if the spheroid is composed of embryonic stem cells). You can study the effect of.

The following examples serve to illustrate certain preferred embodiments and embodiments of the present disclosure and should not be construed as limiting their scope.

Example 1
Cell Culture A549 / GFP cells (Cell Biolabs, Inc. Catalog No. AKR-209) are modified DMEM (IMDM) (Corning) of Iskove supplemented with 10% bovine fetal serum (FBS) (Corning Catalog No. 35-010-CV). 2,000 cells per well were seeded in 96-well spheroid microplates (Corning Catalog No. 4515) in Catalog No. 10-016-CM). The next day, the medium was replaced with 30 ng / mL human SDF-1α (SDF-1α) / CXCL12 (Shenandoah Biotechnology Inc ™ Catalog No. 100-20) or 200 μL IMDM 10% FBS containing a vehicle control. NK92-MI cells were stained with 80 μM cell Tracker ™ Blue CMHC Dye (Molecular Probes ™ Catalog No. C2110) for 1 hour and at the same time 2 μg / mL prostaglandin E2 (PGE2) (Tocris) in serum-free IMDM. Treated with catalog number 2296) or vehicle control for 1 hour. HTS Transwell-96Well Permeable Support was placed in 96-well spheroid plates (schematic diagrams are shown in FIGS. 1A and 4C). NK-92 MI cells were then resuspended in serum-free IMDM and seeded in the apical chamber of the insert at 50,000 cells / insert. After 24 hours, the inserts were removed and spheroid microplates were treated for flow cytometry. Briefly, the medium is removed and replaced with 150 μL of TrypLE ™ Select Enzyme (10X) (Gibco ™ Catalog No. A1217701) so that the spheroids can be degraded into single cells with minimal pipetting. It was incubated at 37 ° C until it became. Cells were then analyzed by flow cytometry using Miltenyi Biotec MacsQuant®.

Evaluation of NK-92MI (NK) Cell Migration NK-92MI migration and tumor-killing activity of A549 / GFP cells utilizes commercially available Corning HTS 96 Transwell inserts with Corning 96-well spheroid microplates, as shown in FIGS. 4 and 5. And evaluated. The presence of certain immune cells in the malignant tumor structure has been shown to correlate with increased patient survival. Unlike the 2D in vitro model, which is more commonly used to study immune cell toxicity, a 3D model can be used to observe immune cell penetration into tumor spheroids . FIG. 6 is a graph showing the migration of NK cells to A549 / GFP tumor cells cultured in 3D according to the embodiment shown in FIG. Data are shown by averaging two independent studies, N = 24 in a one-way ANOVA with Bonferroni posttest. *** = p <0.0001 and ** = p <0.001. FIG. 6 shows how immune cell migration can be enhanced by the addition of chemokines such as SDF-1α and by the addition of inhibitors such as PGE2. NK transfer was significantly increased when the chemoattractant SDF-1α was present in the spheroid microplate with tumor spheroids . FIG. 7 is a graph showing NK-induced cytotoxicity of A549 / GFP tumor cells cultured in 3D according to the embodiments shown in FIGS. 4A, 4B, and 5. Conversely, when NK cells were exposed to PGE2, a known inhibitor of immune cell function often secreted by cancer cells as a form of antigenic escape, migration was significantly reduced. Tumor killing activity correlates well with migration data, with the lowest viability of A549 / GFP tumor cells observed in NK cells exposed to PGE2-free SDF-1α (FIG. 7).

Example 2
Blood-brain barrier model MDCKII / MDR1 cells were obtained from Dr. Piet Borst (Netherlands Cancer Institute, Amsterdam, Netherlands), and 100 μL of Dulvecco'added with 10% fetal bovine serum (FBS) (Corning catalog number 35-010-CV). 100,000 cells per cm2 in s Modification of Eagle's Medium (DMEM) (Corning Catalog No. 10-013-CM) were seeded in HTS96-Well Transwell (Corning Catalog No. 3391 or 3977). The media were changed 24 hours prior to the assay and they were cultured for 5 days. Single layer integrity was assessed by Lucifer Yellow permeability (FIG. 10A) (Sigma Catalog No. L0144) and Rhodamine 123 Transport (FIG. 10B) (Sigma Catalog No. R8004). Immunostaining of MDCKII / MDR1 monolayers was performed to confirm the presence of tight junction proteins ZO1 (Thermo Fisher Catalog No. 339188) and Occludin (Thermo Fisher Catalog No. 331588) according to the manufacturer's protocol (data not shown). did.

Glioma globulans LN229 cells (ATCC® Catalog No. CRL-2611 ™) were routinely cultured in DMEM containing 10% FBS. Cells were collected in Accutase® cell isolation solution (Corning Catalog No. 25-058-CI) and seeded in 96-well spheroid microplates with 1,000 cells per well for 24 hours prior to assay (FIG. 8B).

Blood-brain barrier / glioma sphere model test As shown in FIGS. 8A-8G, the blood-brain barrier / glioma sphere model test involves the use of a porous membrane configured to simulate the blood-brain barrier. It was carried out in one embodiment of the method of the present disclosure for detecting the mobility and cytotoxicity of a therapeutic agent in a model. First, as shown in FIG. 8A, MDCKII / MDR1 cells used to simulate BBB were seeded on the porous membrane 302 of the cell culture insert 304. LN229 tumor cells were grown in Corning 96 well spheroid plates (as shown in FIG. 8B). Once a confluent monolayer of MDCKII / MDR1 endothelial cells simulating BBB on spheroid -generated LN229 tumor cells (as shown in FIG. 8B) and BBB on the porous membrane of cell culture inserts (as shown in FIG. 8A) is formed. , Cell culture inserts were placed in wells. With reference to FIG. 8C, a drug, such as cisplatin or piperongmin, was added to the cavity of the insert for 2 hours. After drug incubation, cell culture inserts were removed and tested for monolayer integrity (data not shown). Spheroids were cultured for an additional 2 days (as shown in FIGS. 8D and 8E) and then analyzed for tumor cell lysis by staining, for example CellTiter-Glo® 3D (as shown in FIG. 8F; after treatment). Spheroid physiology (as shown in FIG. 8G) was also tested. FIG. 9 is a graph showing dose-dependent cytotoxicity of LN229 spheroids after 48 hours of direct culture with the compounds cisplatin and piperongmin. N = 12 wells per concentration. Two independent studies were performed in. FIG. 11 is a graph showing LN229 cell toxicity of cisplatin or piperongmin in the presence or absence of blood-brain barrier surrogate. 48 hours after 2 hours of drug exposure by Transwell with and without BBB. Survival percentage of LN229 spheroids is shown. Survival was assessed by standardizing drug-free controls to 100% viability. Data are shown as the average of three independent studies and centralized dispersion analysis with Bonferroni posttest. N = 30. *** = p <0.0001. FIGS. 12A and 12B show representative screens from the Tocris library showing hits seen with BBB with (FIG. 12B) and without BBB (FIG. 12A). The upper dotted line is the mean buffer control and the lower dotted line represents 3 sigmas lower than the buffer response. FIGS. 13A and 13B show the hit compilation seen with and without BBB. FIG. 12B is a schematic graph of the screen. Hits considered whether they were 3 sigmas lower than the buffer response in at least 2 of the 3 independent screens. Horizontal striped hatching. Boxes (other than buffer alone) were hits found only in the absence of BBB. Boxes with diagonal hatching were hits seen in the presence and absence of BBB. The data presented therein may include a combination of Corning spheroid microplates with an HTS Transwell 96-well permeable support, resulting in glioma globules (or other 3D tumor spheroids ) cytotoxicity by the methods of the present disclosure. While also investigating, we show that a novel 3D model that can distinguish between compounds that can pass through the BBB and those that cannot pass through the BBB will be possible.

All publications and patents referred to herein are incorporated herein by reference. It will be apparent to those skilled in the art that various changes and variations can be made to the techniques of the invention without departing from the gist and scope of the disclosure. Although disclosures have been described in the context of certain preferred embodiments, it should be understood that the requested disclosure should not be unnecessarily limited to such particular embodiments. The invention is within the scope of the appended claims and their equivalents, as those skilled in the art can come up with changes, combinations, sub-bindings and variations of the disclosed embodiments incorporating the gist and gist of the invention. It should be interpreted as including all of the above.

Hereinafter, preferred embodiments of the present invention will be described in terms of terms.

Embodiment 1
An assay for detecting the mobility and cytotoxicity of therapeutic agents:
a) Tumor cells are cultured in a cell culture product containing a chamber structured to grow the tumor cells into a 3D spheroid conformation to form spheroids .
b) An insert containing a porous membrane is placed in the cell culture product and a therapeutic agent is introduced into the insert.
An assay method comprising c) detecting the mobility of the therapeutic agent from the insert into the cell culture product chamber and d) detecting the tumor cell response.

Embodiment 2
The chamber is:
Side wall,
A liquid impervious bottom containing a top opening and at least one recessed surface, wherein at least a portion of the bottom contains a low adhesive or non-adhesive material in or on the at least one recessed surface. The assay method according to embodiment 1, comprising an impermeable bottom.

Embodiment 3
The assay method according to embodiment 1 or 2, wherein the liquid impermeable bottom comprising at least one recessed surface is gas permeable.

Embodiment 4
The assay method according to embodiment 2, wherein the sidewall is opaque.

Embodiment 5
The assay method according to any one of embodiments 2 to 4, wherein at least a portion of the bottom is transparent.

Embodiment 6
The assay method according to any one of embodiments 2 to 5, wherein the cell culture product comprises 1 to about 2000 of the chambers, each chamber being physically separated from each other.

Embodiment 7
The assay method according to any one of embodiments 2 to 6, wherein the at least one recess comprises a plurality of recesses in the same chamber.

8th embodiment
The invention according to any one of embodiments 2 to 7, wherein the at least one recessed surface comprises a hemispherical surface, a conical surface having a taper of 30 to about 60 degrees from the side wall to the bottom surface, or a combination thereof. Assay method.

Embodiment 9
The sidewall surface is a vertical cylinder, a portion of a vertical cone whose diameter decreases from the top of the chamber to the bottom, a square shaft perpendicular to the at least one recessed bottom with a conical transition, or a combination thereof. 2. The assay method according to embodiment 2.

Embodiment 10
The product further comprises a chamber appendage for receiving the pipette tip for suction, the chamber appendage is adjacent to the chamber and includes a fluid communication surface with the chamber, the chamber appendage is from the bottom surface. The assay method according to embodiment 2, wherein the second bottom is separated upward, and the second bottom diverts the fluid distributed from the pipette from the bottom.

Embodiment 11
The assay method according to any one of embodiments 1 to 10, wherein the insert comprises an insert plate.

Embodiment 12
The assay method according to any one of embodiments 1 to 11, wherein at least a portion of the porous membrane is configured to simulate the blood-brain barrier.

Embodiment 13
The assay method according to embodiment 12, wherein at least a portion of the porous membrane comprises an essentially confluent monolayer of microvascular endothelial cells.

Embodiment 14
The assay method according to any one of embodiments 1 to 13, wherein both the mobility of the therapeutic agent and the tumor cell response are detected by flow cytometry.

Embodiment 15
The assay method according to any one of embodiments 1 to 14, wherein detection of the tumor cell response comprises detecting penetration of the therapeutic agent into the tumor cell spheroid .

Embodiment 16
The assay according to any one of embodiments 1 to 15, wherein detection of a tumor cell response comprises measuring tumor cytolysis.

Embodiment 17
The assay method according to any one of embodiments 1 to 16, wherein the therapeutic agent comprises a drug or a cell therapeutic agent.

Embodiment 18
The assay method according to embodiment 17, wherein the therapeutic agent comprises white blood cells or lymphocytes.

Embodiment 19
The assay method according to any one of embodiments 10 to 18, wherein the therapeutic agent is a drug.

10 Multiwell Plate 11 Spheroid Plate 25 3D Tumor Cell Spheroid
30, 310 Therapeutic agent 101 Well 105 Central axis 106 Bottom 110 Space 112 Microcavity 113 Side wall 115 Microwell 116 Bottom, bottom point 118, 418 Top opening 119, 419 Bottom 120 Transition zone 121, 421 Side wall 130 Frame, protective layer 300, 306, 525 Cell 302 Porous Membrane 304, 400 Insert 312, Cavity, Insert Chamber 315 Assay Chamber 401 Insert Plate 420 Cavity 500 Medium w Width d Depth

Claims (18)

An assay for detecting the mobility and cytotoxicity of therapeutic agents:
a) Tumor cells are cultured in a cell culture product containing a chamber structured to grow the tumor cells into a 3D spheroid conformation to form spheroids .
b) An insert containing a porous membrane, at least in part containing an essentially confluent monolayer of microvascular endothelial cells and configured to simulate the blood-brain barrier, is placed in the cell culture product to provide a therapeutic agent. Introduced into the insert
An assay method comprising c) detecting the mobility of the therapeutic agent from the insert into the cell culture product chamber and d) detecting the tumor cell response. The chamber
Side wall,
A liquid impervious bottom containing a top opening and at least one recessed surface, wherein at least a portion of the bottom contains a low adhesive or non-adhesive material in or on the at least one recessed surface. The assay method of claim 1, comprising an impermeable bottom. The assay method of claim 1 or 2, wherein the liquid impermeable bottom comprising at least one recessed surface is gas permeable. The assay method of claim 2, wherein the sidewalls are opaque. The assay method according to any one of claims 2 to 4, wherein at least a part of the bottom is transparent. The assay method according to any one of claims 2 to 5, wherein the cell culture product comprises 1 to 2000 of the chambers, each chamber being physically separated from each other. The assay method according to any one of claims 2 to 6, wherein the at least one recessed surface comprises a plurality of recessed surfaces in the same chamber. 17. Assay method. A vertical cylinder with a vertical wall surface, a portion of a vertical cone whose diameter decreases from the top to the bottom of the chamber, and a vertical square shaft having a conical transition to the at least one recessed bottom, or a combination thereof. The assay method of claim 2, comprising: The product further comprises a chamber appendage for receiving the pipette tip for suction, the chamber appendage comprising a surface adjacent to the chamber and in fluid communication with the chamber, the chamber appendage from the bottom surface. The assay method according to claim 2, wherein the assay method has a second bottom that is separated upward, and the second bottom diverts the fluid distributed from the pipette from the bottom. The assay method according to any one of claims 1 to 10, wherein the insert comprises an insert plate. The assay method according to any one of claims 1 to 11, further comprising removing the insert from the cell culture product and detecting monolayer integrity after incubation for a period of time after b). The assay method according to any one of claims 1 to 12 , wherein both the mobility of the therapeutic agent and the tumor cell response are detected by flow cytometry. The assay method according to any one of claims 1 to 13 , wherein detection of the tumor cell response comprises detecting penetration of the therapeutic agent into the tumor cell spheroid . The assay method according to any one of claims 1 to 14 , wherein detection of the tumor cell response comprises measuring tumor cytolysis. The assay method according to any one of claims 1 to 15 , wherein the therapeutic agent comprises a drug or a cell therapeutic agent. 16. The assay method of claim 16 , wherein the therapeutic agent comprises white blood cells or lymphocytes. The assay method according to any one of claims 1 to 16 , wherein the therapeutic agent is a drug.

Images