High-density 3D hepatocyte spheroid platform for drug ADME studies

Abstract

The present disclosure relates to methods for evaluating the interaction of candidate compounds with 3D hepatocyte spheroids in vitro culture, including evaluating the metabolism of candidate compounds for various biochemical and molecular biological studies. The method is performed in a laboratory apparatus that combines 3D spheroid culture with micropatterning design, allowing multiple to hundreds of spheroids to be processed under the same conditions, and generating sufficient materials (e.g., parent drugs, drug metabolites, DNA, RNA and proteins of cells) and higher detection signal intensity for ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) studies. The method is particularly convenient for studying and generating accurate in vitro intrinsic clearance data, among other uses, and thus more accurately predicting in vivo clearance, particularly for low clearance compounds.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §120 to U.S. Provisional Application Serial No. 62 / 642,447, filed on March 13, 2018, the contents of which are hereby relied upon and incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to methods for evaluating the interaction of candidate compounds with 3D hepatocyte spheroids in in vitro culture, including evaluating the metabolism of candidate compounds for various biochemical and molecular biological studies. The method is performed in a laboratory apparatus, which combines 3D spheroid culture with micropatterning design, maintains the vitality and function of liver cells (e.g., hepatocytes) for a long time, but also allows the treatment of multiple to hundreds of spheroids under the same conditions, and facilitates the production of sufficient materials (e.g., parent drugs, drug metabolites, and DNA, RNA, and proteins of cells) and has a higher detection signal intensity for ADME / Tox (absorption, distribution, metabolism, excretion, and toxicity) and other related studies for more accurate detection. The method is particularly convenient for studying and generating accurate in vitro intrinsic clearance data of compounds, and thus more accurately predicting in vivo clearance, especially for compounds with low clearance. Background Art

[0004] The ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) properties of a compound are key elements in predicting the clinical success of a compound. In the drug discovery process, early ADME / Tox screening is used to select drugs that avoid problematic conditions, reduce metabolic risks, and ultimately achieve the desired dosing regimen. The increase in low-clearance compounds in drug discovery programs has challenged conventional in vitro systems used for clearance studies. The ability to predict the in vivo liver clearance of a compound using in vitro data is crucial in drug discovery and development, because it is known that the intrinsic clearance parameters of a compound are the most important parameters for determining drug half-life, oral bioavailability, dosage and dosing regimen. In addition, unexpected in vivo clearance of a candidate drug may lead to exposure and safety issues. However, due to the detection limits of existing in vitro liver models, these models cannot reliably predict the in vivo clearance or half-life of low-clearance candidate drugs.

[0005] Therefore, there is a continuous need for alternative in vitro models and methods to enable the study of the ADME / Tox properties of compounds, and more specifically, there is a continuous need for in vitro models and methods that can reliably and accurately predict the in vivo clearance or half-life of low-clearance compounds. Summary of the invention

[0006] According to various embodiments of the present disclosure, methods and laboratory apparatus for evaluating the interaction of candidate compounds with 3D hepatocyte spheroids in vitro culture are provided for various biochemical and molecular biological studies, particularly ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) studies. In various aspects, the methods and laboratory apparatus can be used to evaluate the metabolism of candidate compounds in in vitro 3D hepatocyte spheroid cultures. The methods are particularly convenient for studying and generating accurate in vitro intrinsic clearance data for compounds, and thus more accurately predicting in vivo clearance, particularly for candidate compounds with low clearance, among other uses.

[0007] In various embodiments, an assay for evaluating the interaction of one or more low clearance candidate compounds with hepatocytes is disclosed. The assay comprises: culturing hepatocytes in a cell culture article to form a sphere, wherein the cell culture article comprises a chamber. The chamber is, for example, a well of a multi-well plate or a flask. In an embodiment, the bottom of each well can be an array of microcavities. Each microcavity is structured to restrict the growth of hepatocytes in a 3D spheroid conformation. The assay also comprises: contacting the 3D spheroid hepatocytes with one or more low clearance candidate compounds. The assay also comprises: measuring the in vitro intrinsic clearance of the one or more low clearance candidate compounds. In an embodiment, the culture is a long-term culture. In some embodiments, the long-term culture is at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 7 days, at least about 14 days, at least about 21 days, or at least about 28 days.

[0008] In some embodiments, each microcavity of the chamber of the cell culture article comprises a top hole and a liquid-impermeable bottom, the bottom comprising a bottom surface. In embodiments, at least a portion of the bottom surface comprises a low-viscosity or non-viscosity material in or on the bottom surface. In some embodiments, the liquid-impermeable bottom comprising the bottom surface is gas permeable. In some embodiments, at least a portion of the bottom is transparent.

[0009] In some embodiments, the bottom surface comprises a concave bottom surface. In some embodiments, at least one concave surface of each microcavity of the chamber comprises a hemispherical surface, a tapered surface having a taper of 30 to about 60 degrees from the sidewall to the bottom surface, or a combination thereof.

[0010] In some embodiments, the chamber further comprises a side wall. In some embodiments, the side wall of the chamber comprises a vertical cylinder, a portion of a vertical cone with a decreasing diameter from the top to the bottom surface of the chamber, a vertical shaft tapering to the at least one concave bottom surface, or a combination thereof.

[0011] In some embodiments, each microchamber comprises a sidewall. In some embodiments, the sidewall of each microchamber comprises a vertical cylinder, a portion of a vertical cone that decreases in diameter from the top of the chamber to the bottom surface, a vertical square tube that tapers to the at least one concave bottom surface, or a combination thereof.

[0012] In some embodiments, the cell culture article comprises 1 to about 2000 of the chambers, wherein each chamber is physically separated from any other chamber. In embodiments, the chambers are wells of a multi-well plate. For example, the cell culture article can have 1, 6, 12, 24, 96, 384, or 1536 chambers. In some embodiments, each chamber comprises about 25 to about 1,000 of the microcavities.

[0013] In some embodiments, the in vitro intrinsic clearance of one or more low clearance candidate compounds is measured by the disappearance of the one or more candidate compounds. In some embodiments, the in vitro intrinsic clearance of one or more candidate compounds is measured by the formation of metabolites from the one or more candidate compounds.

[0014] In some embodiments, the in vitro intrinsic clearance rate of one or more candidate compounds measured is used to predict the in vivo half-life of the one or more candidate compounds. In some embodiments, the in vitro intrinsic clearance rate of one or more candidate compounds measured is used to predict the in vivo clearance rate of the one or more candidate compounds.

[0015] In some embodiments, the assay method further comprises the step of analyzing metabolites of the one or more low clearance candidate compounds, wherein the metabolites are produced during the incubation of the 3D spheroid hepatocytes and the one or more low clearance candidate compounds. In some embodiments, analyzing the metabolites of the one or more candidate compounds comprises: identifying the metabolites of the one or more candidate compounds produced during the incubation of the 3D spheroid hepatocytes and the one or more low clearance candidate compounds. In some embodiments, analyzing the metabolites of the one or more candidate compounds comprises: quantifying the metabolites of the one or more candidate compounds produced during the incubation of the 3D spheroid hepatocytes and the one or more low clearance candidate compounds.

[0016] In some embodiments, the assay method further comprises a step of analyzing the molecular, biochemical or genetic effects of the one or more low clearance candidate compounds on the 3D spheroid hepatocytes. In some embodiments, analyzing the molecular, biochemical or genetic effects of one or more low clearance candidate compounds on the 3D spheroid hepatocytes comprises: measuring gene and / or protein expression changes during incubation of the 3D spheroid hepatocytes with the one or more low clearance candidate compounds. In some embodiments, analyzing the molecular, biochemical or genetic effects of one or more low clearance candidate compounds on the 3D spheroid hepatocytes comprises: measuring DNA, RNA and / or proteins (e.g., isolated from cells, cell extracts and / or culture media) produced by the 3D spheroid hepatocytes during incubation of the 3D spheroid hepatocytes with the one or more low clearance candidate compounds.

[0017] In some embodiments, the hepatocytes comprise primary human hepatocytes. In some embodiments, the hepatocytes comprise a hepatocyte cell line.

[0018] In some embodiments, the assay method further comprises: simultaneously evaluating a plurality of candidate compounds.

[0019] In some embodiments, the 3D hepatocyte spheroids are functionally stable for at least three weeks. In some embodiments, the functional stability of the 3D hepatocyte spheroids is determined by measuring metabolic activity, cell function, gene expression, or a combination thereof. In some embodiments, the functional stability of the 3D hepatocyte spheroids is measured by CYP3A4 activity.

[0020] Other features and advantages of the subject matter of the present disclosure are set forth in the following detailed description, some of which will be readily understood by those skilled in the art based on the description or recognized by practicing the subject matter of the present disclosure described herein, including the following detailed description, claims, and drawings.

[0021] It should be understood that the foregoing general description and the following specific embodiments give embodiments of the disclosed subject matter and are intended to provide an overall review or framework for understanding the nature and characteristics of the claimed subject matter. The included drawings provide a further understanding of the disclosed subject matter, and the drawings are incorporated into and constitute a part of the specification. The drawings illustrate various embodiments of the disclosed subject matter and, together with the specification, explain the principles and operations of the disclosed subject matter. In addition, the drawings and description are merely exemplary and are not intended to limit the scope of the claims in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following detailed description of specific embodiments of the present disclosure may be best understood when read in conjunction with the following drawings, in which like structures are represented by like reference numerals, and in which:

[0023] Figure 1A , Figure 1B and Figure 1C An embodiment of a multi-well microplate is shown, in this case a 96-well spheroid microplate, having an array of microcavities on the bottom surface of each well to provide a plurality of spheroids in each of the 96 wells. Figure 1A A multiwell microplate is shown. Figure 1B A single well of a multiwell plate is shown. Figure 1C yes Figure 1B An exploded view of the area of ​​the bottom surface of a single hole shown in Box C.

[0024] Figure 2A is a diagram of an exemplary microcavity array. Figure 2B is an illustration of another exemplary microcavity array.

[0025] Figure 3A is when at 0 hours first Photograph of HepatoCells spheroids when they were manually pooled together. Figure 3B It shows that when Photograph of HepatoCells spheroids confluent after they were manually pooled and incubated for 22 hours.

[0026] Figure 4 is one embodiment of the method disclosed herein.

[0027] Figure 5 is a graph showing the CYP3A4 activity of two batches of 3D hepatocyte spheroids cultured in 96-well spheroid plates compared to the CYP3A4 activity of hepatocytes cultured in 2D.

[0028] Fig. 6A -I is a graph showing in vitro measurements of classical low clearance compounds using one embodiment of the methods disclosed herein.

[0029] Fig. 7A is a diagram showing a microcavity insert. Figure 7B is a photograph of human primary hepatocytes cultured in 24-well microchamber inserts at day 1. Figure 7C is a photograph of human primary hepatocytes cultured in 24-well microchamber inserts at day 3, demonstrating the formation of 3D spheroids.

[0030] Fig. 8A -I is a graph showing in vitro measurements of classical low clearance compounds using suspension cells. DETAILED DESCRIPTION

[0031] Various embodiments of the subject matter of the present disclosure will be described in more detail below, some of which are illustrated in the accompanying drawings. The same reference numerals used in the drawings represent the same parts, steps, etc. However, it should be understood that the use of a reference numeral to represent a component in a given figure does not limit the components marked with the same reference numeral in another figure. In addition, the use of different reference numerals to represent components is not intended to indicate that the components with different numbers cannot be the same or similar to the components with other numbers.

[0032] The following description of specific embodiments is merely exemplary in nature and is in no way intended to limit the scope of the invention, its application or use, which are of course variable. The present invention is described with respect to the non-limiting definitions and terms included herein. These definitions and terms are not intended to limit the scope or implementation of the present invention, but are provided for illustrative and descriptive purposes only. Unless otherwise defined, the meaning of all terms (including technical and scientific terms) used herein is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. It should also be understood that terms, such as those defined in common dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the present disclosure and related fields, and should not be interpreted in an idealized or overly formal sense, unless clearly defined herein.

[0033] definition

[0034] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" include plural references. Thus, for example, reference to a "structured bottom surface" includes instances having two or more such "structured bottom surfaces", unless the context clearly indicates otherwise.

[0035] As used in this specification and the appended claims, the word "or" is generally used in its sense including "and / or" unless the context clearly indicates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0036] As used herein, “have,” “having,” “having,” “containing,” “including,” “include,” “included,” “covering,” and the like are used in their open sense and generally mean “including but not limited to,” “including but not limited to,” or “including but not limited to.”

[0037] "Optional" or "optionally" means that the subsequently described event, circumstance or portion may or may not occur, and that the description includes instances where the event, circumstance or portion occurs and instances where it does not.

[0038] The terms "preferred" and "preferably" refer to embodiments of the present disclosure that can produce certain benefits under specific conditions. However, other embodiments may also be preferred under the same or other conditions. In addition, the description of one or more preferred embodiments does not mean that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0039] Herein, ranges may be expressed as starting from "about" one particular value and / or ending at "about" another particular value. When such ranges are expressed, examples include starting from a particular value and / or ending at another particular value. Similarly, when the antecedent "about" is used to indicate that a value is an approximation, it is understood that the specific value constitutes another aspect. It is also understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint.

[0040] Likewise, herein, numerical ranges described by endpoints include all numbers contained in the range (e.g., 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). It should also be understood that each numerical range given throughout this specification should include each narrower numerical range that falls within the broader numerical range, as if these narrower numerical ranges were explicitly stated herein. When a numerical range is "greater than," "less than," or a specific value, the value is included in the range.

[0041] As used herein, "structured to provide" or "configured to provide" means that the article has features that provide the stated result.

[0042] Any directions referred to herein, such as "top," "bottom," "left," "right," "up," "down," "above," "below," and other directions and orientations are described herein with reference to the accompanying drawings for clarity and are not limitations on the actual device or system or use of the device or system. Many of the devices, articles, or systems described herein can be used in a variety of directions and orientations. Directional descriptors used herein with respect to cell culture devices often refer to the direction when the device is oriented for the purpose of culturing cells in the device.

[0043] It should also be noted that descriptions herein that relate to a component being "configured to" or "adapted to" function in a particular manner. In this regard, the component is "configured to" or "adapted to" in order to specifically exhibit a particular property, or function in a particular manner, and such descriptions are structural descriptions rather than descriptions of intended use. More specifically, the manner in which a component is "configured to" or "adapted to" as described herein refers to the existing physical conditions of the component, and thus can be viewed as a limiting description of the structural features of the component.

[0044] As used herein, the term "cell culture" refers to keeping cells alive in vitro. Included in this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro, including oocytes and embryos.

[0045] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment. In vitro environments may include, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur in a natural environment.

[0046] As used herein, "long-term culture" is intended to mean that cells (e.g., but not limited to, hepatocytes) have been cultured for at least about 12 hours, optionally, at least about 24 hours, at least about 48 hours, or at least about 72 hours, at least about 96 hours, at least about 7 days, at least about 14 days, at least about 21 days, or at least about 28 days. Long-term culture facilitates the establishment of functional properties, such as metabolic pathways in the culture.

[0047] As used herein, the term "cell culture article" means any container for culturing cells, including plates, wells, flasks, multiwell plates, multilayer flasks, TRANSWELL inserts, TRANSWELL microchamber inserts, and perfusion systems that provide a cell culture environment.

[0048] As used herein, "chamber" means a cell culture container, which can be a flask or a dish or a well of a multi-well plate. In some embodiments, the cell culture article includes 1 to about 2000 of the chambers, wherein each chamber is physically separated from any other chamber. In an embodiment, the chamber is a well of a multi-well plate. For example, the cell culture article can be a flask with a single chamber having an array of microcavities on a cell culture surface or bottom surface. Alternatively, the cell culture article can be a multi-well plate with 1, 6, 12, 24, 96, 384 or 1536 chambers or wells. In some embodiments, each chamber includes about 25 to about 1,000 of the microcavities.

[0049] In embodiments, a "well" is an independent cell culture environment provided in a multi-well plate format. In embodiments, a well can be a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 96-well plate, a 538-well plate, a 1536-well plate, or a well of any other multi-well plate configuration.

[0050] In an embodiment, a single chamber can be a well of a multi-well plate that is structured to constrain the cells of interest to grow as a single 3D cell mass or a single spheroid in the single chamber. For example, a well of a 96-well plate (wells of a conventional 96-well plate) is about 10.67 mm deep, with a top hole of about 6.86 mm and a well bottom diameter of about 6.35 mm.

[0051] In embodiments, "spheroid plate" means a multiwell plate having an array of individual spheroid chambers or wells. That is, in embodiments, a multiwell plate may have a plurality of chambers or wells, wherein each chamber or well is configured to accommodate a single spheroid.

[0052] As used herein, "microwells" or "microcavities" are "structured to constrain the growth of cells of interest in a 3D conformation", and the "microwells" or "microcavities" and the like mean microwells or microcavities having a size or treatment, or a combination of size and treatment, which promotes the growth of cells in culture in a 3D or spheroid conformation rather than a two-dimensional cell sheet. Treatments include, for example, treatment with a low binding solution, treatment to reduce the hydrophobicity of the surface, or sterilization treatment.

[0053] In embodiments, a "microcavity" or "micropore" can be, for example, a micropore that defines an upper hole and a lowest point, a center of the upper hole, and a central axis between the center of the upper hole and the lowest point. In embodiments, the hole of the microcavity or micropore is rotationally symmetric about the axis (i.e., the sidewalls are cylindrical). In some embodiments, the upper hole defines a distance across the upper hole between about 250 μm and 1 mm, or any range within these measurements. In some embodiments, the distance from the upper hole to the lowest point (depth "d") is between 200 μm and 900 μm, or between 400 and 600 μm. The microcavity array can have different geometric structures, for example, parabolic, hyperbolic, V-shaped and cross-sectional geometries, or combinations thereof.

[0054] In embodiments, the holes may have an array of "microcavities". In embodiments, a "microcavity" may, for example, be a microhole defining an upper hole and a lowest point, a center of the upper hole, and a central axis between the center of the upper hole and the lowest point. In embodiments, the hole is rotationally symmetric about the axis (i.e., the sidewalls are cylindrical). In some embodiments, the upper hole defines a distance across the upper hole between 250 μm and 1 mm, or any range within these measurements. In some embodiments, the distance from the upper hole to the lowest point (depth "d") is between 200 μm and 900 μm, or between 400 and 600 μm. The microcavity array may have different geometric structures, for example, parabolic, hyperbolic, V-shaped and cross-sectional geometries, or combinations thereof.

[0055] In an embodiment, "microcavity spheroid plate" means a multi-well plate having an array of wells, and each well has an array of microcavities.

[0056] In embodiments, the "circular bottom" of a well or microcavity well may be, for example, a hemisphere, or a portion of a hemisphere, for example, a horizontal cross section or slice of the hemisphere constituting the bottom of the well or microcavity.

[0057] In an embodiment, the term "3D spheroid" or "spheroid" can be, for example, a cell ball in culture, which is not a flat two-dimensional cell sheet. The terms "3D spheroid" and "spheroid" are used interchangeably herein. In an embodiment, the spheroid comprises a single cell type or a multi-cell type, and its diameter is, for example, about 100 to about 500 microns, more preferably about 150 to about 400 microns, even more preferably about 150 to about 300 microns, most preferably about 200 to about 250 microns, including intermediate values ​​and ranges, depending on, for example, the cell type in the spheroid. The spheroid diameter can be, for example, about 200 to about 400 microns. The maximum size of the spheroid is generally limited by diffusion considerations (for a review of spheroids and spheroid containers, see Achilli, TM et al., Expert Opin. Biol. Ther. (2012) 12 (10)).

[0058] As used herein, "hepatocyte" means any cell derived from the main parenchymal tissue of the liver. The hepatocyte may be a primary hepatocyte obtained or isolated from an animal (including a human), or the hepatocyte may be a cell of a hepatocyte cell line or a primary hepatocyte origin.

[0059] As used herein, "insert" means a cell culture well that fits into the well of a spheroid plate or microchamber spheroid plate. An insert has sidewalls and a bottom surface that define a cavity for culturing cells. As used herein, "TRANSWELL microchamber insert" means an insert having an array of microcavities on the bottom surface.

[0060] As used herein, "insert plate" means an insert plate containing an array of inserts structured to fit into an array of wells of a multiwell plate. As used herein, "microcavity insert plate" means an insert plate in which each insert in an array of inserts has a bottom surface with an array of microcavities.

[0061] As used herein, "candidate compound" and the like (e.g., "compound," "compound of interest," or "drug compound") are intended to refer to any compound (exogenously administered or endogenously generated) for which it is desired to characterize the ADME / Tox properties of the compound. Exemplary candidate compounds include xenobiotic low molecular weight therapeutics and other therapeutic agents, carcinogens and environmental pollutants, and endobiotics, such as steroids, bile acids, fatty acids, and prostaglandins, which are commonly referred to as "drugs." Candidate compounds may include drugs, which include all classes of action, alone or in combination, including but not limited to: anti-tumor agents, immunosuppressants, immunostimulants, antiproliferative agents, antithrombotic agents, antiplatelet agents, antilipid agents, anti-inflammatory agents, antibiotics, angiogenic agents, anti-angiogenic agents, vitamins, ACE inhibitors, vasoactive substances, antimitotic agents, metalloproteinase inhibitors, NO donors, estradiol, anti-sclerotic agents, hormones, free radical scavengers, toxins, alkylating agents. Candidate compounds may also include, for example but in no way limited to, biologics, including but not limited to: peptides, lipids, protein drugs, protein-bound drugs, enzymes, oligonucleotides, ribozymes, genetic material, prions, viruses, and bacteria.

[0062] As used herein, "clearance" is intended to refer to the blood volume that completely clears a compound (such as, but not limited to, a drug) per unit time. Clearance is usually measured in ml / min (milliliters per minute) or ml / min / kg (milliliters per minute per kilogram).

[0063] < As used herein, "intrinsic clearance" or "Cl int (Cl 内在 )" is intended to refer to the ability of the liver to remove a compound (such as, but not limited to, a drug) when the compound is not restricted in flux and is not bound to cells or proteins in the blood. Thus, intrinsic clearance is the intrinsic ability of the liver enzymes to metabolize a drug.

[0064] As used herein, "low clearance compound" is intended to refer to a compound with a clearance of <5 ml / min / kg.

[0065] Unless otherwise stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a specific order. Therefore, when a method claim does not actually state that its steps follow a certain order or it does not specifically indicate in any other way in the claims or description that the steps are limited to a specific order, it is not intended to imply any particular order. Any single or multiple features or aspects recited in any claim may be combined with or substituted for any other features or aspects recited in any one or more other claims.

[0066] While the transitional term “comprising” may be used to disclose various features, elements, or steps of a particular embodiment, it should be understood that this implies including alternative embodiments that may be described using the transitional term “consisting of” or “consisting essentially of.

[0067] As mentioned above, the ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) properties of a compound are key elements in predicting the clinical success of a compound, and are used to select drugs that avoid problematic conditions, reduce metabolic risks and ultimately enable once-a-day dosing. Although traditional in vitro systems have achieved some success and increased the percentage of low-clearance compounds in drug discovery programs, there are still challenges. The ability to use in vitro data to predict liver metabolism and toxicity in vivo is key. In addition, even for drug candidate compounds that exhibit slow metabolism, it is also crucial to accurately distinguish drug candidate compounds based on the predicted clearance. For example, predicting the in vivo liver clearance of a compound is crucial in drug discovery and development, because it is known that the clearance parameters of a compound are important factors in determining drug half-life, oral bioavailability, dosage and dosing regimens. The ability to generate accurate in vitro intrinsic clearance data is an important element in predicting clearance in humans, because unexpected in vivo clearance of drug candidates can lead to exposure problems and safety issues.

[0068] However, existing in vitro liver models cannot reliably and accurately predict (e.g., within 2-fold or 3-fold of the actual value) the in vivo clearance or half-life of low-clearance compounds (e.g., but not limited to low-clearance drug candidates). For example, since hepatocytes contain all the oxidation / reduction, hydrolysis, and conjugated drug metabolizing enzymes present in the liver (as well as the complete hepatic clearance pathway), as well as the increased availability of fresh and cryopreserved hepatocytes, in vitro hepatocyte suspensions are a common method for predicting in vivo hepatic clearance. However, hepatocyte suspensions, as well as human liver microsomes, typically underestimate in vivo clearance, especially for low-clearance compounds. This is primarily due to the rapid loss of enzyme activity of in vitro hepatocyte suspensions and human liver microsomes over time (enzyme activity of hepatocyte suspensions is typically within 4-6 hours, while enzyme activity of human liver microsomes is limited to about 1-2 hours), and therefore, the metabolic stability of slowly metabolized, low-clearance compounds cannot be accurately evaluated. More specifically, the use of human liver microsomes typically has a lower limit of intrinsic clearance measurement of 10 ml / min / kg, while the use of human hepatocyte suspensions typically has a lower limit of intrinsic clearance measurement of 6.3 ml / min / kg. As previously mentioned, the clearance of low clearance candidate compounds is <5 ml / min / kg.

[0069] The present disclosure particularly describes methods and laboratory apparatus for evaluating the interaction of candidate compounds to 3D hepatocyte spheroids, for various biochemical and molecular biological studies, especially ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) studies. In various aspects, the method and laboratory apparatus can be used to evaluate the metabolism of candidate compounds in 3D hepatocyte spheroid culture in vitro. The method is carried out in a laboratory apparatus, which combines 3D spheroid culture with micropatterning design, so that the vitality and function of liver cells (such as hepatocytes) are maintained for a long time, but it is also allowed to process multiple to hundreds of spheroids (for example, during long-term culture) under the same conditions and culture medium, for producing enough materials (for example, parent drugs, drug metabolites and DNA, RNA and proteins of cells) and higher detection signal intensity for ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) studies, while providing a physical barrier between each 3D spheroid to prevent any spheroid fusion from occurring during culture or testing. In some embodiments of the method disclosed herein, the one or more candidate compounds are low clearance compounds. As previously mentioned, a low clearance compound is a compound with a clearance of <5 ml / min / kg. Low clearance compounds include, but are not limited to, warfarin, meloxicam, tolbutamide, diazepam, alprazolam, glimepiride, prednisolone, riluzole, and voriconazole. Unlike most existing in vitro liver models that cannot reliably predict the in vivo clearance or half-life of low clearance drug candidates, the methods disclosed herein are particularly convenient for studying and generating accurate in vitro intrinsic clearance data, among other uses, and thus more accurately predict in vivo clearance, especially for such low clearance compounds. For example, the disclosed method combining 3D spheroid culture with micropatterning design generated in vitro intrinsic clearance data for 9 low clearance compounds, with a predicted accuracy of 44% of the in vivo clearance within 2 times of the actual value, and a predicted accuracy of 67% of the in vivo clearance within 3 times of the actual value. In contrast, for the same 9 low-clearance compounds, the in vitro intrinsic clearance data generated using the 2D monolayer approach predicted in vivo clearance within 2-fold of the actual value for only 33% and within 3-fold of the actual value for 44%. In addition, compared to other co-culture systems, such as The method disclosed herein does not require animal matrix cells in 3D spheroid culture and can be completed at significantly reduced costs. In addition, compared to the in vitro rotation method, the method disclosed herein does not require repeated thawing of hepatocytes and transfer of supernatant, and therefore, the labor intensity of the method disclosed herein is significantly lower.

[0070] In various embodiments, assays for evaluating the interaction of one or more low clearance candidate compounds with hepatocytes are disclosed. In various aspects, assays for evaluating the metabolism of low clearance candidate compounds in in vitro 3D hepatocyte spheroid cultures are disclosed. The assays include culturing hepatocytes in a cell culture article to form spheroids, wherein the cell culture article includes a chamber, the chamber includes an array of microcavities, each of which is structured to constrain hepatocytes to grow in a 3D spheroid conformation. The assays also include contacting the 3D spheroid hepatocytes with one or more low clearance candidate compounds. The assay method also includes: measuring the interaction of the one or more low clearance candidate compounds with the 3D hepatocyte spheroids cultured in vitro, wherein the interaction includes but is not limited to clearance studies (uptake clearance; basolateral efflux clearance; canalicular efflux clearance; metabolite clearance), metabolite ID and metabolic stability (maternal life span), intracellular concentration of candidate compounds, protein and gene regulation (induction / inhibition) and other molecular, biochemical and genetic analysis of cultured 3D spheroid hepatocytes, toxicological effects, compound kinetics, subcellular accumulation and free or total (combination of bound and free) intracellular concentration, total bile clearance, P450 and transporter drug interactions and pharmacokinetics. In an embodiment, the measured in vitro interactions are used to predict the in vivo disposition of the one or more low clearance candidate compounds.

[0071] In some embodiments of the methods of the present disclosure, the cultured cells, including but not limited to hepatocytes forming 3D spheroids (in various aspects, formed with one or more low clearance candidate compounds) are cultured as long-term cultures. In some embodiments, the cultured cells are cultured (in various aspects, with the one or more low clearance candidate compounds) for at least about 12 hours, optionally, for at least about 24 hours, at least about 48 hours, or at least about 72 hours, at least about 96 hours, at least about 7 days, at least about 14 days, at least about 21 days, or at least about 28 days. Long-term culture promotes the establishment of functional properties, such as metabolic pathways in the culture. For example, Figure 5As shown, human primary hepatocytes from two donors (lot 299 (top) and lot 397 (bottom)) cultured in 96-well spheroid culture plates maintained higher CYP3A4 activity (activated and measured by adding testosterone) for a long time compared to hepatocytes cultured in 2D using the laboratory apparatus and methods of the present disclosure for forming 3D hepatocyte spheroids. In some embodiments of the disclosed methods, the functional stability of the cultured 3D spheroid hepatocytes lasts for at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. Functional stability can be measured as known in the art, including but not limited to measuring metabolic activity, cellular function, gene expression, or a combination thereof. In some embodiments, functional stability is measured by CYP3A4 activity.

[0072] In some embodiments, each microcavity of the chamber comprises a top hole and a liquid-impermeable bottom, the bottom comprising a bottom surface, wherein at least a portion of the bottom surface comprises a low-viscosity or non-viscosity material in or on the bottom surface. In some embodiments, the liquid-impermeable bottom comprising the bottom surface is gas-permeable. In some embodiments, at least a portion of the bottom is transparent. In some embodiments, the bottom surface comprises a concave bottom surface. In some embodiments, at least one concave bottom surface of each microcavity of the chamber 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. In some embodiments, the chamber further comprises a side wall. In some embodiments, the cell culture article comprises 1 to about 2000 of the chambers, wherein each chamber is physically separated from any other chamber. In some embodiments, each chamber comprises about 25 to about 1,000 of the microcavities. For example, but not limiting, a 6-well plate can include about 700 microcavities in one well of the 6-well plate, thus allowing 700 spheres (a total of about 1.2 million cells) in one well. Similarly, each well in a 24-well plate may include about 100-200 microchambers, while each well in a 96-well plate may contain about 50 microchambers.

[0073] Cells cultured in three-dimensional form (e.g., spheroids) can show more in vivo-like functionality than counterparts cultured in two-dimensional form as a monolayer. In a two-dimensional cell culture system, cells can be attached to a substrate on which they are cultured. However, when cells grow in three-dimensional form (e.g., spheroids), cells interact with each other rather than being attached to a substrate. With regard to the development of cell communication and extracellular matrix, cells cultured in three-dimensional form are more similar to in vivo tissues. For example, 3D spheroid culture of hepatocytes has obtained sustained hepatocyte drug metabolism activity and cell viability. Therefore, hepatocyte 3D spheroids thus provide an excellent model for ADME / Tox studies, including studying and generating accurate in vitro intrinsic clearance data, thereby more accurately predicting in vivo clearance, particularly for these low clearance compounds.

[0074] Reference now Figure 1A , Figure 1B and Figure 1C , which shows one embodiment of a cell culture article as a microchamber spheroid plate, in this case a 96-well microchamber spheroid plate, having an array of microchambers on the bottom surface of each well, and each microchamber is structured to constrain the cultured cells to grow in a 3D spheroid conformation, thereby providing multiple spheroids in each of the 96 wells. Figure 1A A multiwell plate 10 having an array of wells 110 is illustrated. Figure 1B Shows Figure 1A The single hole 101 of the multi-well plate 10 has a top hole 118, a liquid-impermeable bottom surface 106 and a side wall 113. Figure 1C yes Figure 1B FIG. 1 is an exploded view of the area of ​​the bottom surface 106 of the hole 101 shown in box C in FIG. 1 , which illustrates Figure 1B An array of microcavities 112 in the bottom surface of a single well is shown. Each microcavity 115 in the microcavity array 112 has sidewalls 121 and a bottom surface 116 that is liquid impermeable. Figure 1A , Figure 1B and Figure 1C The illustrated micro-chamber spheroid plate provides a micro-chamber array 112 in the bottom of each individual well 101, which can be used to grow individual 3D spheroids in each micro-chamber of each individual well of a multi-well plate. By using this type of container, a user can grow a large number of spheroids in each well of a multi-well plate, thereby providing a large number of liver cell spheroids that maintain long-term viability and functionality, and the large number of liver cell spheroids can be processed under the same culture and experimental conditions for use in assays as provided herein. In addition, this type of container provides a physical barrier between the individual 3D spheroids to prevent any spheroid fusion during culture or testing. Figure 3A and Figure 3B As shown, when HepatoCells-produced spheroids were manually pooled together (in Figure 3A ) and at 22 hours of incubation, the spheres were fused together (shown in Figure 3B ). Fusion of spheroids can result in a change in the ratio of exposed cell surface area to total volume. Therefore, the microwell design of the present disclosure provides a physical barrier that allows the integrity of each spheroid and test compound to be maintained during extended incubation times to allow for uniform diffusion and drug metabolism activity throughout the well.

[0075] Reference now Figure 2A , which shows an exemplary diagram of the microcavity array 112 . Figure 2A Microcavities 115 are illustrated, each microcavity 115 having a top hole 118, a bottom surface 119, a depth d, and a width w defined by sidewalls 121. Figure 2A and Figure 2B As shown, the microcavity array has a liquid-impermeable concave arcuate bottom surface 116. In embodiments, the bottom surface of the microcavity can be round or conical, angled, flat-bottomed, or any shape suitable for forming a 3D sphere. A round bottom is preferred. When the vertical sidewall transitions to the round bottom 119, the round bottom 119 can have a transition zone 114. It can be a smooth or angled transition zone. In embodiments, a "microcavity" can be, for example, a micropore 115 that defines an upper hole 118 and a lowest point 116, the center of the upper hole, and a central axis 105 between the center of the upper hole and the lowest point. In embodiments, the hole is rotationally symmetric around the axis (i.e., the sidewalls are cylindrical). In some embodiments, the upper hole defines a distance across the upper hole (width w), which is between 250 μm and 1 mm, or any range within these measurements. In some embodiments, the distance from the upper hole to the lowest point (depth "d") is between 200 μm and 900 μm, or between 400 and 600 μm. The microcavity arrays can have different geometries, for example, parabolic, hyperbolic, V-shaped and cross-sectional geometries, or combinations thereof. In embodiments, the microcavities can have a protective layer 130 underneath them to protect them from direct contact with surfaces such as a laboratory bench or table. In some embodiments, air spaces 110 can be provided between the bottom of the wells 119 and the protective layer. In embodiments, the air spaces 110 can be in communication with the external environment, or can be closed. 3D hepatocyte spheroids 25 are shown at the bottom of some individual microcavities 115. Now referring to Figure 2B , which shows another exemplary diagram of the microcavity array 112 . Figure 2B It is illustrated that the microcavity array 112 can have a sinusoidal or parabolic shape. This shape produces a rounded top edge or microcavity edge, which in embodiments reduces the risk of air being trapped in sharp corners or 90 degree angles at the top of the microcavity. Figure 2B As shown, in various aspects, the microcavity 115 has a top opening having a top diameter D 顶 ; The height H from the bottom 116 of the microcavity to the top of the microcavity; The diameter D of the microcavity at half the height between the top of the microcavity and the bottom 116 of the microcavity H ; and sidewalls 113. In such embodiments, the bottom of the hole is rounded (e.g., hemispherical), the diameter of the sidewall increases from the bottom to the top of the hole, and the boundaries between the holes are rounded. Thus, the top of the hole does not end at a right angle. In some embodiments, the hole has a diameter D (also referred to as D ) at a point midway between the bottom and the top. H ), the diameter D at the top of the hole 顶 and the height H from the bottom to the top of the hole. In these embodiments, D 顶 Greater than D.

[0076] In embodiments, the bottom surface of the microcavity or "cup" having the at least one concave arcuate bottom surface can be, for example, a hemispherical surface, a conical surface with a round bottom, and similar surface geometries, or a combination thereof. The bottom of the microcavity eventually terminates, ends, or bottoms out with a "spherical-friendly" rounded or curved surface (e.g., a concave truncated conical relief surface such as a pit, a depression, or a combination thereof). In embodiments, at least one concave surface of each microcavity of the chamber comprises a hemispherical surface, a conical surface having a taper of 30 to about 60 degrees from the sidewall to the bottom surface, or a combination thereof. In some embodiments, the at least one concave arcuate bottom surface can be, for example, a portion of a hemisphere, such as a horizontal portion or slice of a hemisphere, having a diameter of, for example, about 250 to about 5000 microns (i.e., 0.010 to 0.200 inches), including intermediate values ​​and ranges, depending on, for example, the selected well geometry, the number of concave arcuate surfaces in each well, the number of wells in the plate, and similar considerations. Other concave curved surfaces may have cross-sectional geometries such as parabolic, hyperbolic, V-shaped, etc., or combinations thereof.

[0077] In an embodiment, a cell culture article (e.g., a multiwell plate, a microcavity spheroid plate, a microcavity insert, a microcavity insert plate, etc.) comprising a chamber, and each chamber comprising a microcavity may also include a low-viscosity, ultra-low-viscosity, or non-viscosity coating. The coating may be, for example, on a portion of the microcavity, for example, on at least one bottom surface of each microcavity or on at least one concave bottom surface of each microcavity and / or on one or more side walls of each microcavity. Examples of non-viscosity materials include perfluorinated polymers, olefins or similar polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels (e.g., polyacrylamide), or polyethers (e.g., polyethylene oxide), or polyols (e.g., polyvinyl alcohol), or similar materials, or mixtures thereof.

[0078] In embodiments, the sidewall surface (i.e., surround) of the chamber and / or each microcavity can be, for example, a vertical cylinder or barrel, a portion of a vertical cone with a decreasing diameter from the top of the chamber to the bottom of the chamber, a vertical square cylinder or a vertical elliptical cylinder with a tapered transition, i.e., a square or elliptical shape at the top of the hole, transitioning to a cone, and ending with a bottom having at least one concave arc surface (i.e., rounded or curved), or a combination thereof. Other illustrative geometric examples include a cylinder with a hole, a cylinder with a hole and a cone, a cylinder followed by a cone, and other similar geometric structures or combinations thereof.

[0079] For example, one or more of the pore curvature in the main body and base part of the low-adhesion substrate, the microcavity, and gravity can induce tumor cells to self-assemble into spheroids. For example, individual cells can fall to the bottom of the microcavity, adhere to each other (and do not adhere to the low-binding coating surface of the micropore) and grow into spheroids. Relative to cells grown in 2D monolayers, hepatocytes maintain differentiated cell functions, which indicate a more in vivo response. In an embodiment, the spheroid can be, for example, substantially spherical, with a diameter of, for example, about 100 to about 500 microns, more preferably about 150 to about 400 microns, even more preferably about 150 to about 300 microns, most preferably about 200 to about 250 microns, including intermediate values ​​and ranges, depending on, for example, the cell type in the spheroid. The spheroid diameter can be, for example, about 200 to about 400 microns, and the upper diameter is limited by diffusion considerations.

[0080] In an embodiment, the cell culture article including the chamber and / or each microcavity in the chamber may also include an opaque sidewall and / or a gas-permeable and liquid-impermeable bottom, which includes at least one concave surface. When fluorescence imaging is used, the opaque sidewall prevents crosstalk between holes or micropores. In some embodiments, at least a portion of the bottom including at least one concave surface is transparent. Cell culture articles (e.g., microcavity spheroid plates, microcavity inserts, microcavity insert plates, etc.) with these features can provide several advantages for the method of the present disclosure, including not needing to transfer the cultured cell spheroids from one multi-well plate (wherein spheroids are formed and can be visualized) to another plate for determination, thus saving time and avoiding any unnecessary spheroid destruction. In addition, a gas-permeable bottom (e.g., a hole bottom made of a polymer having gas-permeable properties at a specific given thickness) can increase the oxygenation received by the 3D hepatocyte spheroid. An exemplary gas-permeable bottom can be formed by a certain thickness of a perfluorinated polymer or a polymer such as poly-4-methylpentane.

[0081] Representative thicknesses and ranges for gas permeable polymers can be, for example, about 0.001 inches to about 0.025 inches, 0.0015 inches to about 0.03 inches, including intermediate values ​​and ranges (where 1 inch = 25400 microns; 0.000039 inches = 1 micron). Additionally or alternatively, other materials with high gas permeability, such as polydimethylsiloxane polymers, can provide adequate gas diffusion at thicknesses of, for example, up to about 1 inch.

[0082] Reference now Figure 4 , which is a diagram of one embodiment of the disclosed methods and labware for evaluating the interaction of candidate compounds with 3D spheroid hepatocytes in in vitro culture, for use in various biochemical and molecular biological studies, particularly ADME / Tox (absorption, distribution, metabolism, excretion, and toxicity) studies. In various aspects, the disclosed methods and labware are used to evaluate the metabolism of one or more low clearance candidate compounds in in vitro 3D hepatocyte spheroid cultures. Figure 4 As shown, cells 201 of interest, such as liver cells (including but not limited to liver cells and / or non-parenchymal cells, which can be isolated from the liver) are grown 203 (i.e., cultured, which can be long-term culture) in a culture medium in a microcavity of a chamber of a cell culture article (e.g., a microcavity spheroid plate, a microcavity insert, a microcavity insert plate, etc.), the microcavity being structured to constrain the liver cells to grow in a 3D conformation. In this case and as shown, the cell culture article is a 24-well microcavity spheroid plate, and cells of interest, such as liver cells (including but not limited to liver cells and / or non-parenchymal cells) are grown 203 in a well 101 of a multiwell plate 10 having a microcavity array 112, each microcavity 115 being structured to constrain the cells of interest to grow in a 3D spheroid conformation 25, which will develop into a spheroid array, with one spheroid in each microcavity in the microcavity array on the bottom surface of the well of the multiwell plate. As the cells grow and multiply in culture, they are constrained to grow into spheroids. 25. In various aspects, a microcavity insert or microcavity insert plate can be used to culture cells of interest to grow in a 3D spheroid conformation. Fig. 7A As shown, the insert has a top hole 418, sidewalls 421 and a bottom surface 419, which form a microcavity array 420. The use of a microcavity insert or a microcavity insert plate can allow for faster formation of spheres. For example, Figure 7B (Day 1 of hepatocyte culture) and Figure 7C(Day 3 of hepatocyte culture) shows that hepatocyte spheroids can be formed within 3 days of culture using a 24-well microcavity insert. It should be understood that the insert can be obtained in many configurations, including but not limited to a 6-well microcavity insert, a 12-well microcavity insert, a 24-well microcavity insert, a 48-well microcavity insert, a 96-well microcavity insert, and an insert plate configuration in which a single plate contains multiple inserts, and the multi-well insert plate is structured to be inserted into a complementary well array in a multi-well plate.

[0083] Reference again Figure 4 Once liver cells (including but not limited to liver cells and / or non-parenchymal cells) develop into spheroids 25, liver metabolism studies, including ADME / Tox (absorption, distribution, metabolism, excretion and toxicity) studies, can be performed on one or more low clearance candidate compounds 205. For example, one or more low clearance candidate compounds can be added to each well of a multi-well plate to incubate the 3D liver cell spheroids and candidate compounds together. Then, after a suitable time, the interaction of the one or more low clearance candidate compounds with the cells is evaluated. The interaction of the one or more low clearance candidate compounds with 3D liver cell spheroids (e.g., 3D liver cell spheroids) that can be evaluated includes, but is not limited to: clearance studies (uptake clearance, basolateral efflux clearance, canalicular efflux clearance, metabolite clearance); metabolite ID and metabolic stability (maternal lifespan); intracellular concentration of candidate compounds; protein and gene regulation (induction / inhibition) and other molecular, biochemical and genetic analysis of cultured 3D spheroid liver cells; toxicological effects; compound kinetics; subcellular accumulation and free or total (bound + free) intracellular concentration; total bile clearance; P450 and transport drug interactions; and pharmacokinetics. These interactions and effects can be measured by any suitable means known in the art, including but not limited to vital staining techniques, ELISA assays, immunohistochemistry, and various conventional molecular, biochemical and genetic assays. As previously described, the methods of the present disclosure are performed in laboratory equipment that combines 3D spheroid culture with micropatterning design, so that the vitality and function of liver cells (e.g., hepatocytes) are maintained for a long time, but also allow the processing of multiple to hundreds of spheroids (e.g., during long-term culture) under the same conditions, and produce sufficient materials (e.g., parent drug, drug metabolites, and cellular DNA, RNA, and proteins) and higher detection signal intensity for the various studies listed above. The incubation period will vary depending on the one or more low clearance candidate compounds. Adjustment of the incubation time can be done in preliminary experiments and is well within the general skills of those skilled in the art.

[0084] In some embodiments, the assay method for evaluating the interaction of the one or more low clearance candidate compounds with hepatocytes comprises: culturing hepatocytes in a cell culture article to form a spheroid, wherein the cell culture article comprises a chamber, the chamber comprising a microcavity array, each microcavity array being structured to constrain the growth of hepatocytes in a 3D spheroid conformation. The assay method further comprises: contacting the 3D hepatocyte spheroid with one or more low clearance candidate compounds. After a suitable time, the assay method further comprises: measuring the in vitro intrinsic clearance of the one or more low clearance candidate compounds. The incubation time will vary depending on the candidate compound. Adjustment of the incubation time can be accomplished in preliminary experiments and is well within the general skill of those skilled in the art.

[0085] In some embodiments, the in vitro intrinsic clearance rate of one or more candidate compounds is measured by forming metabolites by the one or more candidate compounds. However, in order to calculate the intrinsic clearance rate by the formation (for example, presence and or absence) of drug metabolites, clear clearance pathways (for example, specific enzymes responsible for the removal of candidate compounds) and reliable metabolite standards are required, which are not always known or available. Therefore, in an embodiment, the in vitro intrinsic clearance rate of the one or more candidate compounds is measured by the disappearance of the one or more candidate compounds. The disappearance of the one or more candidate compounds formed by the one or more candidate compounds can be measured by any means known in the art, including but not limited to mass spectrometry, liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry or high performance liquid chromatography. In vitro clearance rate can be calculated as known in the art, such as Chan et al., (2013) Drug Metab Dispos 41: 2024–2032, which is incorporated herein by reference in its entirety.

[0086] In some embodiments, the measured in vitro intrinsic clearance of one or more candidate compounds is used to predict the in vivo half-life of the one or more candidate compounds. In some embodiments, the measured in vitro intrinsic clearance of one or more candidate compounds is used to predict the in vivo clearance of the one or more candidate compounds. For example, the in vivo hepatic clearance can be calculated from the calculated in vitro intrinsic clearance using a well-stirred model, as shown in the Examples.

[0087] In some embodiments, the assay method includes a step of analyzing metabolites of the one or more low clearance candidate compounds, wherein the metabolites are produced during the incubation of the 3D spheroid hepatocytes with the one or more low clearance candidate compounds. In some embodiments, analyzing the metabolites of the one or more candidate compounds includes: identifying the metabolites of the one or more candidate compounds produced during the incubation of the 3D spheroid hepatocytes and the one or more low clearance candidate compounds. In some embodiments, analyzing the metabolites of the one or more candidate compounds includes: quantifying the metabolites of the one or more candidate compounds produced during the incubation of the 3D spheroid hepatocytes and the one or more low clearance candidate compounds. Measuring the formation of metabolites by the one or more low clearance candidate compounds - whether identification and / or quantification of metabolites - can be measured by any means known in the art, including but not limited to mass spectrometry, liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, or high performance liquid chromatography.

[0088] In some embodiments, the assay method includes analyzing the molecular, biochemical or genetic effects of one or more low clearance candidate compounds on 3D spheroid hepatocytes. The effects of one or more low clearance candidate compounds on 3D spheroid hepatocytes during incubation can be measured by any means known in the art, including genetic, metabolic or protein analysis of cells, cell extracts or culture media, such as known visualization techniques, fluorescence measurements, ELISA assays, immunohistochemical assays and various other known conventional molecules, biochemical and genetic assays. In some embodiments, analyzing the molecular, biochemical or genetic effects of one or more low clearance candidate compounds on 3D spheroid hepatocytes includes: measuring gene and / or protein expression changes during incubation of 3D spheroid hepatocytes and the one or more low clearance candidate compounds. In some embodiments, analyzing the molecular, biochemical or genetic effects of one or more low clearance candidate compounds on 3D spheroid hepatocytes includes: measuring DNA, RNA and / or proteins (e.g., isolated from cells, cell extracts and / or culture media) produced by 3D spheroid hepatocytes during incubation of 3D spheroid hepatocytes and the one or more low clearance candidate compounds.

[0089] In some embodiments of the methods disclosed herein, candidate compounds include low molecular weight therapeutic agents of xenobiotics and other therapeutic agents, carcinogens and environmental pollutants, and endobiotics, such as steroids, bile acids, fatty acids, and prostaglandins, which are commonly referred to as "drugs". Candidate compounds may include drugs, which include all action categories, which include, but are not limited to, anti-tumor agents, immunosuppressants, immunostimulants, antiproliferative agents, antithrombotic agents, antiplatelet agents, antilipid agents, anti-inflammatory agents, antibiotics, angiogenic agents, anti-angiogenic agents, vitamins, ACE inhibitors, vasoactive substances, antimitotic agents, metalloproteinase inhibitors, NO donors, estradiol, anti-sclerotic agents, hormones, free radical scavengers, toxins, alkylating agents. Candidate compounds may also include, for example but not limited to, biological agents, which include, but are not limited to, peptides, lipids, protein drugs, protein-bound drugs, enzymes, oligonucleotides, ribozymes, genetic material, prions, viruses, and bacteria.

[0090] In some embodiments of the methods of the present disclosure, the methods include: evaluating multiple candidate compounds simultaneously. In some embodiments of the methods of the present disclosure, the methods include: exposing / contacting one or more candidate compounds to / in contact with 3D spheroid hepatocytes.

[0091] In some embodiments of the disclosed methods, hepatocytes can be any cells derived from the main parenchymal tissue of the liver. Hepatocytes can be primary hepatocytes obtained or separated from animals (including humans), or hepatocytes can be cells derived from hepatocyte lines or primary hepatocytes. In some embodiments, cell populations can be derived from one or more species, including but not limited to human cells, rat cells, mouse cells, monkey cells, pig cells, dog cells, guinea pig cells, fish cells. In addition, the cells can be fresh or frozen.

[0092] In some embodiments of the disclosed method, non-parenchymal cells may be included with the cultured hepatocytes or co-cultured with the cultured hepatocytes. Non-parenchymal cells may include, but are not limited to, Kupffer cells, hepatocytes (including but not limited to ITO cells, sinusoidal endothelial cells, bile duct cells), stromal cells (including but not limited to fibroblasts and pericytes), immune cells (including but not limited to T cells, neutrophils, macrophages, dendritic cells, eosinophils, mast cells) and stem cells (including but not limited to liver progenitor cells, embryonic stem cells and hematopoietic stem cells). These cell populations may be derived from one or more species, including but not limited to human cells, rat cells, mouse cells, monkey cells, pig cells, dog cells, guinea pig cells, fish cells. In addition, the cells may be fresh or frozen. In some embodiments of the disclosed method, animal stromal cells are not required in 3D spheroid culture.

[0093] In some embodiments, a matrix or scaffold may be used during the culture of hepatocytes. Thus, during the culture period, the hepatocytes (and possibly other co-cultured cells, including non-parenchymal cells) may be embedded, mixed or covered with a matrix or scaffold. Representative matrices or scaffolds include any suitable natural or synthetic matrices or scaffolds, such as, but not limited to, collagen, laminin and

[0094] In some embodiments, the method includes a culture medium [e.g., including nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulfates), buffers (e.g., phosphates, acetates), pH change indicators (e.g., phenol red, bromocresol purple), selection agents (e.g., chemicals, antimicrobial agents), etc.], which are known in the art.

[0095] aspect

[0096] Various aspects of methods, systems, compositions and kits are described herein. An overview of some selected examples of these methods, systems, compositions and kits is provided below.

[0097] In the first aspect is an assay method for evaluating the interaction of one or more low clearance candidate compounds with hepatocytes, the method comprising:

[0098] Culturing hepatocytes in a cell culture article to form spheroids, wherein the cell culture article comprises a chamber, the chamber comprising an array of microcavities, each microcavity being structured to constrain hepatocytes to grow in a 3D spheroid conformation to form hepatocyte spheroids; wherein each microcavity of the chamber comprises:

[0099] a top aperture and a liquid-impermeable bottom, the bottom comprising a bottom surface, wherein at least a portion of the bottom comprises a low-adhesion or non-adhesion material in or on the bottom surface;

[0100] contacting the 3D hepatocyte spheroids with one or more low clearance candidate compounds; and

[0101] The in vitro intrinsic clearance of the one or more low clearance candidate compounds is measured.

[0102] The second aspect is the method according to the first aspect, wherein the culture is long-term culture.

[0103] A third aspect is a method as described in aspect 1 or 2, wherein the long-term culture is at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 7 days, at least about 14 days, at least about 21 days, or at least about 28 days.

[0104] Aspect 4 is a method as described in any one of aspects 1-3, wherein the liquid-impermeable bottom including the bottom surface is breathable.

[0105] Aspect 5 is the method of any one of aspects 1-4, wherein the bottom surface comprises a concave bottom surface.

[0106] A sixth aspect is a method as described in any one of aspects 1 to 5, wherein at least a portion of the bottom is transparent.

[0107] A seventh aspect is the method of aspect 5, wherein the concave surface comprises a hemispherical surface, a tapered surface having a taper of 30 to about 60 degrees from the side wall to the bottom surface, or a combination thereof.

[0108] Aspect 8 is a method as described in any one of aspects 1-7, wherein each microcavity of the chamber further includes a side wall.

[0109] Aspect 9 is a method as described in aspect 8, wherein the side wall surface includes a vertical cylinder, a portion of a vertical cone whose diameter decreases from the top to the bottom surface of the chamber, a vertical square tube that tapers to a concave bottom surface, or a combination thereof.

[0110] Aspect 10 is a method as described in any of aspects 1-9, wherein the cell culture article comprises 1 to about 2000 of the chambers, wherein each chamber is physically separated from any other chamber.

[0111] Aspect 11 is a method as described in any one of aspects 1-10, wherein the in vitro intrinsic clearance of the one or more low clearance candidate compounds is measured by the disappearance of the one or more low clearance candidate compounds.

[0112] Aspect 12 is a method as described in any one of aspects 1-11, wherein the in vitro intrinsic clearance of the one or more low clearance candidate compounds is measured by the formation of metabolites by the one or more low clearance candidate compounds.

[0113] Aspect 13 is a method as described in any one of aspects 1-12, wherein the measured in vitro intrinsic clearance of the one or more low clearance candidate compounds is used to predict the in vivo half-life of the one or more low clearance candidate compounds.

[0114] A fourteenth aspect is a method as described in any one of aspects 1-13, wherein the in vivo clearance rate of the one or more low clearance candidate compounds is predicted using the in vitro intrinsic clearance rate of the one or more low clearance candidate compounds.

[0115] Aspect 15 is a method as described in any one of aspects 1-14, which also includes a step of analyzing metabolites of the one or more low clearance candidate compounds, wherein the metabolites are produced during incubation of 3D spheroid hepatocytes and the one or more low clearance candidate compounds.

[0116] Aspect 16 is a method as described in aspect 15, wherein analyzing the metabolites of the one or more candidate compounds includes: identifying the metabolites of the one or more candidate compounds produced during incubation of 3D spheroid hepatocytes with the one or more low clearance candidate compounds.

[0117] Aspect 17 is a method as described in aspect 15, wherein analyzing the metabolites of the one or more candidate compounds includes: quantifying the metabolites of the one or more candidate compounds produced during incubation of 3D spheroid hepatocytes with the one or more low clearance candidate compounds.

[0118] The 18th aspect is a method as described in any one of aspects 1-17, which further includes the step of analyzing the molecular, biochemical or genetic effects of the one or more low clearance candidate compounds on the 3D spheroid hepatocytes.

[0119] Aspect 19 is a method as described in aspect 18, wherein analyzing the molecular, biochemical or genetic effects of the one or more low clearance candidate compounds on the 3D spheroid hepatocytes includes: measuring the DNA, RNA and / or protein produced by the 3D spheroid hepatocytes during incubation of the 3D spheroid hepatocytes and the one or more low clearance candidate compounds.

[0120] Aspect 20 is a method as described in any one of aspects 1-19, wherein the hepatocytes include primary human hepatocytes.

[0121] Aspect 21 is a method as described in any one of aspects 1-20, wherein the hepatocytes include a hepatocyte cell line.

[0122] Aspect 22 is a method as described in any one of aspects 1-21, further comprising evaluating multiple candidate compounds simultaneously.

[0123] A twenty-third aspect is a method according to any one of aspects 1 to 22, wherein the functionality of the 3D spheroid hepatocytes is stable for at least 3 weeks.

[0124] Aspect 24 is the method of aspect 23, wherein the functional stability of the 3D spheroid hepatocytes is determined by measuring metabolic activity, cell function, gene expression, or a combination thereof.

[0125] Aspect 25 is the method of aspect 24, wherein the functional stability is measured by CYP3A4 activity.

[0126] Example

[0127] The following examples are intended to illustrate specific features and aspects of the disclosure and should not be construed as limiting the scope thereof.

[0128] Example 1

[0129] Primary human hepatocyte (PHH) high density (HD) spheroid culture

[0130] Pre-wet the spheroid insert: Place the pipetting aid on low dispersion speed. Using a 5 mL pipette and positioning the tip against the side wall at the bottom, add 2 mL of medium to a 6-well spheroid insert, each with approximately 700 microwells, if using William E Medium + (WEM+) (no FBS). Use a 200 ul pipette tip to blow away any air bubbles trapped in the microwells. Inspect the plate under a microscope to ensure all air bubbles are removed.

[0131] PHHs were plated onto 6-well high-density sphere inserts (~700 microwells / insert). Thaw PHHs according to standard procedures and resuspend in WEM plate culture medium. PHH cells were counted and the cell density was adjusted to 0.35e6 / ml with WEM plate culture medium. The pipetting aid was set at a low dispersion speed for paving PHHs. Most of the culture medium was removed from the pre-wetted insert with a 5mL pipette and then immediately plated. A uniform cell suspension was prepared by gently pipetting up and down. Using a 5mL pipette and positioning the tip against the side wall at the bottom, 2mL of cell suspension (700K cells, ~1000 cells / microwell) was added to the 6-well sphere insert. The plate was placed in a fume hood for 15-20 minutes. The plate was examined under a microscope with a 4x objective to ensure that a similar number of cells were deposited in each microwell.

[0132] Example 2

[0133] 3D PHH High-Density Spheroid Drug Clearance Assay

[0134] Replace half of the medium with WME + medium: Place the pipetting aid on low dispersion speed. Using a 5 mL pipette, slowly draw up 1 mL of old medium from each insert. Position the tip of a 1000 μL pipette against the top of the insert side wall and slowly add 1 mL of pre-warmed WME + medium and allow the medium to flow down the side wall. Wait 5-10 minutes between each half medium change and repeat this half medium change 3 more times. Examine the PHHs under a microscope for sphere formation.

[0135] Preparation of 2X clearance assay solutions, wherein the final DMSO concentration is less than 0.2%, is shown in Table 1.

[0136] Table 1: 2X Clearance Assay Solution

[0137]

[0138] Incubate the candidate compound to be tested for clearance with the cultured spheroids: Set the pipetting aid to a low dispersion speed. Using a 5 mL pipette, slowly remove 1 mL of old medium from each insert. Position the tip of a 1000 μL pipette against the top of the insert side wall and slowly add 1 mL of the 2X substrate solution containing the candidate compound to be tested for clearance and allow the medium to flow down the side wall. After 5-10 minutes, return the culture to the 37°C incubator. Start timing the incubation at this time.

[0139] Sampling: 40 μL / well of stop solution Labetalol (0.2uM in 0.05% FA / 50% ACN) was aliquoted in the assay plate and placed on ice. At the desired assay incubation time (0 hour, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, or any value or range therebetween), 40uL sample was drawn from each incubation well, mixed thoroughly with the stop solution, and stored at -80°C.

[0140] LC / MS analysis: Liquid chromatography / mass spectrometry is used to measure the amount of candidate compounds as known in the art.

[0141] Example 3

[0142] Data analysis and clearance calculation

[0143] Calculate the elimination rate constant (k el ): As known in the art, the percentage of candidate compounds remaining in the culture is calculated by dividing the mass spectrometry peak area at a certain incubation time by the mass spectrometry peak area at zero. The natural logarithm of the percentage of candidate compounds remaining is plotted against time, and k el Calculated as the absolute value of the slope.

[0144] Calculate the consumption half-life of the candidate compound. Once the k of the candidate compound is determined el , the consumption half-life of the candidate compound is determined by Equation 1, which is known in the art.

[0145] T 1 / 2 =0.692 / k el (1)

[0146] Calculate the in vitro clearance rate (Cl int ): As known in the art, the in vitro clearance of the candidate compound is calculated using the in vitro half-life of Equation 1 using the scaling factor of Equation 2:

[0147]

[0148] Calculate the predicted in vivo hepatic clearance (CL h ): The in vivo hepatic clearance was calculated from the in vitro clearance determined by Equation 2 using the well-stirred model Equation 3 as known in the art.

[0149]

[0150] Example 4

[0151] result

[0152] like Figures 6A-6I As shown, the methods and laboratory apparatus of the present disclosure are capable of measuring classical low clearance compounds, such as warfarin ( Fig. 6A )、Meloxicam( Figure 6B )、Tolbutamide( Figure 6C )、Diazepam( Fig.6D )、Glimepiride( Fig. 6E ), alprazolam ( Fig. 6F ), prednisolone ( Figure 6G ), Riluzole ( Figure 6H ) and voriconazole ( Fig.6I ). In addition, as shown in Table 2, the disclosed method combining 3D spheroid culture with micropatterned design generated in vitro intrinsic clearance data for 9 low clearance compounds, with a prediction accuracy of 44% of the in vivo liver clearance within 2 times of the actual value, and a prediction accuracy of 67% of the in vivo liver clearance within 3 times of the actual value. In contrast, for the same 9 low clearance compounds, the in vitro intrinsic clearance data generated by the 2D monolayer method only predicted the in vivo clearance of 33% within 2 times of the actual value, and the prediction accuracy of 44% of the in vivo clearance was within 3 times of the actual value. Therefore, unlike most existing in vitro liver models that cannot reliably predict the in vivo clearance or half-life of low clearance drug candidates, the disclosed data demonstrate that the disclosed method, in addition to other uses, is particularly convenient for studying and generating accurate in vitro intrinsic clearance data for low clearance compounds, and thus more accurately predicts in vivo clearance, especially for such low clearance compounds. In contrast, PHH grown in suspension are not able to measure classical low-clearance compounds such as warfarin ( Fig. 8A )、Meloxicam( Figure 8B)、Tolbutamide( Figure 8C )、Diazepam( Fig.8D )、Glimepiride( Fig. 8E ), alprazolam ( Figure 8F ), prednisolone ( Figure 8G ), Riluzole ( Figure 8H ) and voriconazole ( Figure 8I ).

[0153] Table 2: Predicted CL of low clearance compounds in 2D hepatocyte culture and 3D hepatocyte spheroid culture h contrast

[0154]

[0155]

[0156] All publications and patents mentioned in the above specification are incorporated herein by reference. It is obvious to those skilled in the art that various modifications and changes can be made to the technology of the present invention without departing from the spirit and scope of the present disclosure. Although the present disclosure has been described in conjunction with specific preferred embodiments, it should be understood that the present disclosure should not be unduly limited to these specific embodiments as described in the claims. Because those skilled in the art can combine the spirit and essence of the technology of the present invention to make various improvements, combinations, sub-item combinations and changes to the disclosed embodiments, the technology of the present invention should be considered to include all the contents within the scope of the attached claims and their equivalents.

Claims

1. An assay for evaluating the interaction of one or more candidate compounds with hepatocytes, the method comprising: Culturing hepatocytes in a culture medium in a cell culture article to form a plurality of spheroids in a chamber of the cell culture article, wherein the cell culture article comprises a plurality of chambers, each chamber comprising an array of microcavities, each microcavity being structured to constrain the hepatocytes to grow in a 3D spheroid conformation so that they do not fuse with spheroids in other microcavities during culture or testing; wherein each microcavity of the chamber comprises: a side wall; a top aperture and a liquid-impermeable bottom, the bottom comprising a concave surface, wherein at least a portion of the bottom comprises a low-adhesion or non-adhesion material in or on the concave surface; a) contacting the 3D spheroid hepatocytes in the microchamber with one or more candidate compounds by adding the one or more candidate compounds to the culture medium in the chamber; b) sampling the culture medium in the chamber at one or more assay incubation times and c) measuring the in vitro intrinsic clearance of the one or more candidate compounds in the one or more samples of the assay incubation time, wherein the concave surface comprises a hemispherical surface, a tapered surface having a taper of 30 to about 60 degrees from the side wall to the bottom surface, or a combination thereof, wherein the side wall surface comprises a vertical cylinder, a portion of a vertical cone whose diameter decreases from the top to the bottom surface of the chamber, a vertical square tube that tapers to a concave bottom surface, or a combination thereof, wherein each chamber is physically separated from any other chamber, wherein the one or more candidate compounds are low clearance candidate compounds, and Among them, the clearance rate of low clearance compounds is less than 5ml / min / kg.

2. The assay method according to claim 1, wherein The liquid-impermeable bottom including the concave surface is breathable.

3. The assay method according to claim 2, wherein The side walls are opaque.

4. The assay method according to claim 2, wherein At least a portion of the bottom is transparent.

5. The assay method according to claim 1, wherein The cell culture article comprises from 1 to about 2000 of the chambers.

6. The assay method according to claim 1, wherein The in vitro intrinsic clearance of one or more low clearance candidate compounds is measured by the disappearance of the one or more candidate compounds.

7. The assay method according to claim 1, wherein The in vitro intrinsic clearance of one or more candidate compounds is measured by the formation of metabolites from the one or more candidate compounds.

8. The assay method according to claim 1, wherein The measured in vitro intrinsic clearance rate of the one or more candidate compounds is used to predict the in vivo half-life of the one or more candidate compounds.

9. The assay method according to claim 1, wherein The measured in vitro intrinsic clearance rate of one or more candidate compounds is used to predict the in vivo clearance rate of one or more candidate compounds.

10. The assay method according to claim 1, wherein Hepatocytes are primary human hepatocytes.

11. The assay method according to claim 1, wherein Hepatocytes are cells of the liver lineage.

12. The assay method according to claim 1, further comprising: Evaluate multiple candidate compounds simultaneously.

13. The assay method according to claim 1, wherein The functional stability of 3D spheroid hepatocytes persisted for at least 3 weeks.

14. The assay method according to claim 1, wherein The functional stability of 3D spheroid hepatocytes is determined by measuring metabolic activity, cell function, gene expression, or a combination thereof.

15. The assay method according to claim 1, wherein Functional stability was measured by CYP3A4 activity.

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