Apparatus, system and method for culturing cells

Abstract

Apparatuses, systems, and methods for culturing a floating target tissue are provided. Embodiments include a first surface configured to culture a first layer of support cells and a second surface configured to culture a second layer of support cells. The first layer of support cells can be formed on a portion of the first surface and the second layer of support cells can be formed on a portion of the second surface. A floating target tissue can be added to the first layer of support cells. The second layer of support cells can be placed on the first layer of support cells such that the floating target tissue is sandwiched between the first layer of support cells and the second layer of support cells.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680058635.4 (International Application No. PCT / US2016 / 056879), filed on October 13, 2016, entitled "Apparatus, System, and Method for Culturing Cells". This application claims priority to U.S. Provisional Patent Application No. 62 / 240,612, filed on October 13, 2015, which is incorporated herein by reference. Technical Field

[0002] This application belongs to the field of cell culture and provides apparatus, methods, and systems for culturing tissues and cells. Specifically, for example, it provides apparatus, methods, and systems for culturing floating primary human tissue explants and cells under conditions that maintain primary cell type characteristics (including morphology, gene and protein expression levels, and metabolic function) even after long-term culture. Background Technology

[0003] Obesity is an increasingly common disease, affecting more than 79 million Americans. Obesity can be linked to a variety of conditions, including type 2 diabetes, heart disease, stroke, arthritis, and some cancers. Beyond its health impacts, the direct financial costs associated with treating obesity and related diseases are estimated to exceed $150 billion in the United States alone. There is currently a strong need for approval of anti-obesity treatments for human intervention.

[0004] Obesity can be described as an excessive growth of white adipose tissue (WAT) in the body. Generally, WAT can be considered an organ within the body, acting as an energy storage device to store extra calories. WAT is found throughout the body and can originate subcutaneously or from various anatomical regions, including the abdomen, chest, hips, and limbs. WAT can also be considered an endocrine organ that produces hormones to regulate various physiological systems such as hunger / satiety, glucose metabolism, and lipid metabolism. A properly functioning WAT organ is crucial. In fact, insufficient WAT can lead to illness or death.

[0005] As organs, WATs comprise mature adipocytes (wAds), which can be morphologically described as large cells with single-chambered lipid droplets comprising more than 95% of their cell volume. The presence of these large lipid droplets allows wAds to float. Human wAds may also be considered particularly fragile cells, largely due to their size. For example, human wAds range in size from about 100 to about 140 μm, which is nine (9) times the volume of rodent wAds.

[0006] Attempts to culture primary human wAds have been largely unsuccessful. Conventional in vitro culture methodologies employ techniques such as enzymatic and mechanical treatments to dissociate primary wAd tissue and isolate wAds. These treatments typically destroy or severely damage most wAds, with the majority undergoing cell lysis within 72 hours post-treatment. Consequently, there is no research model for human wAT derived from wAds.

[0007] Attempts to overcome the challenges associated with wAds culture include embedding wAds in a collagen matrix. However, the success of this technique has been limited. Figure 1 shows a micrograph of collagen-embedded human wAds stained with propidium iodide, demonstrating the induction of programmed cell death, i.e., apoptosis, after 2 days of culture. In addition to their fragility, adipocytes are also considered terminally differentiated and mitotically inert. Therefore, wAds may not proliferate in culture without altering their differentiation state (i.e., dedifferentiation).

[0008] Unlike most other model cell types (where the original cell line can be frozen for long-term storage), human WAT / wAds must be obtained fresh from the operating room or clinic and used immediately for each experiment. Therefore, researchers must rely on surgically obtained human WAT tissue as source material, which limits the accessibility of WAT / wAds to non-clinical researchers. In fact, researchers lacking relationships with clinicians may not be able to obtain human WAT. Research experiments may then be constrained by the clinician's schedule, which can be unpredictable. Furthermore, tissue acquisition can be time-consuming and often requires travel, wearing surgical gowns, and hospital approval for the investigation protocol. These barriers to obtaining source WAT have slowed the overall pace of scientific discovery and may prevent researchers from studying the biology of human WAT.

[0009] Currently, researchers rely on models, including rodent models that chemically differentiate into adipocytes or matrix / stem cell models (i.e., diffAds). However, these experimental models cannot reproduce primary human WAT biology. For example, one of the earliest identified anti-obesity pathways is controlled by the β-3-adrenergic receptor (β3-AR). Using selective β3-AR agonists, obesity and diabetes have been successfully cured in several rodent models. However, the same selective β3-AR that is successful in rodent obesity models is inactive in human β3-ARs, leading to numerous failed clinical trials.

[0010] Similarly, certain model cell types, such as stromal and stem cells, can chemodifferentiate into adipocytes (diffAds). However, diffAds express human wAds markers only at reduced levels, including CCAAT / enhancer-binding protein α, lipoprotein lipase, fatty acid-binding protein 4, and hormone-sensitive lipase. Furthermore, diffAds-based obesity models cannot reproduce wAds function in metabolic assays measuring glycerol release, lipofuscin release, and glucose uptake.

[0011] Genetically, in vitro cell models and WATs do not share similar gene expression patterns. Figure 2 illustrates the comparison of changes in adipocyte marker gene expression between conventional culture models and primary WATs, where the genes indicated in the upper left corner are... Figure 2A , 2B Figures 2 and 2C illustrate the differences in expression patterns of the marker genes peroxisome proliferator-activated receptor γ (PPARG), fatty acid-binding protein 4 (FABP4), and hormone-sensitive lipase (HSL) in adipocytes differentiated from stem cells (diffAds) and in primary adipose tissue, respectively. Specifically, the densely filled diagonal bars (“-PPARG”) in Figure 2 represent the expression levels of stem cells differentiated into multicompartmental “adipocytes” (diffAds) using a standard protocol. The sparsely filled diagonal bars (“+PPARG”) in Figure 2 represent the expression levels of stem cells differentiated into “adipocytes” using a lentiviral construct expressing PPARG. The rightmost sparsely filled backslash bar in Figure 2, labeled with primary adipose tissue on the lower axis (“Primary Fat”), represents the expression levels in primary adipose tissue. As shown, primary adipose tissue expresses adipocyte genes at levels 10 to 100 times higher than in vitro models. Therefore, conventional models cannot reproduce the gene expression levels of primary adipose tissue. Summary of the Invention

[0012] In exemplary embodiments, apparatus, methods, and systems are provided for in vitro culture of floating tissue explants of human WAT.

[0013] Embodiments of this disclosure provide systems, methods, and apparatus for culturing floating tissues and cells when added to an aqueous culture medium. Embodiments of this disclosure provide systems, methods, and apparatus for culturing primary human tissue explants and cells obtained from an individual (e.g., a patient). Embodiments of this disclosure provide systems, methods, and apparatus for culturing human tissues and cells in a stable differentiation state over an extended period of time (e.g., several weeks).

[0014] Embodiments of this disclosure provide systems, methods, and apparatus for configuring microphysiology, such as organ-on-a-chip model systems. Embodiments of this disclosure provide methods for evaluating the effects of chemical compounds, such as drugs, on human tissue explants and cells cultured using the systems, methods, and apparatus disclosed herein.

[0015] Typically, in vitro tissue and cell culture systems use culture containers, such as petri dishes, plates, flasks, and slides, to which tissues or cells are added along with nutrient-rich culture media. In some cases, the petri dish provides a matrix for adhering tissues or cells, and the culture medium provides the necessary components to support and promote the metabolic functions of the tissues and cells added therein. Establishing a new culture of tissues or cells requires transferring the sample tissues or cells to a petri dish containing an aqueous culture medium. Non-floating tissue and cell types may remain on the surface of the culture container, where a complex process commonly referred to as cell adhesion occurs. However, some tissue and cell types are buoyant and therefore float in their culture medium rather than adhering to the surface of the petri dish. For some cell types, non-adherence can lead to cell death.

[0016] This disclosure provides systems, methods, and apparatus for in vitro culture of floating tissues and cells. In specific embodiments, the systems, methods, and apparatus described herein are applicable to the culture of all tissue and cell types, including but not limited to: white adipose tissue (WAT), brown adipose tissue (BAT), brain, nervous system tissues, thyroid gland, pancreas, spleen, cartilage, liver, kidney, and bone. Attached Figure Description

[0017] This patent or application document contains at least one drawing. A published copy of this patent or application with the drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0018] Figure 1A Microscopic images of human white adipose tissue (WAT) cultured in a collagen matrix after 2 days of routine collagen embedding are shown.

[0019] Figure 1B Microscopic images are shown, illustrating the morphological differences between differentiated "adipocytes" (diffAds) and primary adipocytes.

[0020] Figures 2A-2C This is a diagrammatic illustration comparing the gene expression of adipocyte identity genes between a conventional culture model of adipocytes derived from stem cells (diffAds) and primary WAT, illustrating the contrasting expression patterns of marker genes in both models. The upper left corner of the diagram indicates... Figure 2A , 2BFigures 2 and 2C illustrate the differences in expression patterns of three different marker genes. In each figure, densely backslashed bars (“Undifferentiated”) represent expression levels in undifferentiated stem cells, with each stem cell type labeled on the axis below the bar. Densely backslashed bars represent expression levels in stem cells differentiated into multicompartmental “adipocytes” (diffAds) using the standard protocol (“-PPARG”). Sparsely backslashed bars in Figure 2 represent expression levels in stem cells differentiated into “adipocytes” using a lentiviral construct expressing PPARg (“+PPARG”). The rightmost sparsely backslashed bar (“Primary Fat”) represents expression levels in primary adipose tissue.

[0021] Figure 3 This is an isometric view of a culture apparatus having a supporting cell layer disposed in a temperature-sensitive tray and an insertion device for removing the supporting cell layer from the tray, according to an exemplary embodiment of the present disclosure.

[0022] Figure 4 yes Figure 3 An isometric view of the insertion device shown, in which the supporting cell layer is attached to the base of the insertion device.

[0023] Figure 5 yes Figure 4 An isometric view of the culture apparatus shown, with the supporting cell layer attached to the base of the insertion device removed from the temperature-sensitive plate.

[0024] Figure 6 This is an isometric view of a culture apparatus, which, according to an exemplary embodiment of the present disclosure, has floating cells or tissue explants deposited on a first layer of supporting cells in a temperature-sensitive dish.

[0025] Figure 7 yes Figure 6 An isometric view of the culture apparatus shown, according to an exemplary embodiment of the present disclosure, the insertion device has a second layer of supporting cells attached to the base of the insertion device, the second layer of supporting cells being deposited on top of the tissue explant and the first layer of supporting cells.

[0026] Figure 8 This is an isometric view of white adipose tissue sandwiched between two layers of supporting cells, according to an exemplary embodiment of the present disclosure.

[0027] Figures 9A-9B Microscopic images are shown, illustrating the morphological stability of a sandwich white adipose tissue (SWAT) cell culture system according to an exemplary embodiment of the present disclosure.

[0028] in, Figure 9AMicroscopic images are shown of a SWAT culture system having a double layer of supporting cells surrounding a cluster of WAT cells, according to an exemplary embodiment of the present disclosure.

[0029] and Figure 9B Microscopic images are shown, illustrating the morphological stability of WAT cell clusters in a SWAT cell culture system according to an exemplary embodiment of the present disclosure.

[0030] Figure 10 These are a series of microscopic images showing the long-term stability of WAT cell clusters in a SWAT culture system according to an exemplary embodiment of the present disclosure.

[0031] Figure 11 This is an illustration showing that, according to an exemplary embodiment of the present disclosure, SWAT cultures have transcriptional activity and express genes associated with adipose tissue identity.

[0032] Figure 12 Microscopic images are shown, indicating that, according to an exemplary embodiment of this disclosure, SWAT cultures exhibit translational activity and express proteins associated with adipose tissue identity.

[0033] Figure 13A This is an illustration showing that, according to an exemplary embodiment of the present disclosure, SWAT cultures secrete leptin at basal levels on days 1 and 5 of culture reflecting primary WAT.

[0034] Figure 13B This is an illustration showing that, according to an exemplary embodiment of the present disclosure, SWAT cultures secrete adiponectin at basal levels on days 1 and 5 of culture, which reflect primary WAT culture.

[0035] Figure 13C This is an illustration showing that, according to an exemplary embodiment of the present disclosure, SWAT cultures undergo lipolysis in response to catecholamine stimulation after day 1 and day 5 of culture.

[0036] Figure 14 Images are shown illustrating a SWAT culture fully implanted into immunocompromised eGFP-tagged mice according to an exemplary embodiment of this disclosure. Detailed Implementation

[0037] refer to Figures 3 to 8 The image shows different views of the system used to culture floating cell types. Figure 3An exemplary embodiment of a culture apparatus 500 for culturing floating cell types is shown. The culture apparatus 500 may include a culture container 200 (e.g., a culture dish 200) and an insert 100. In some non-limiting embodiments, the culture container 200 and the insert 100 may contain materials that promote adhesion of tissue or cells to the surface of the container or insert.

[0038] In some embodiments, the adhesion-promoting material itself may be an assembly of container 200 or insert 100. In other embodiments, adhesion-promoting material may be added to container 200 and / or insert 100. In this embodiment, the respective substrate / surface of container 200 and / or insert 100 may be coated with a protein matrix or extracellular material. Non-limiting examples of adhesion-promoting materials include, but are not limited to, poly(N-isopropylacrylamide) (pNIPAAM / pNIPAm), modified methylcellulose, and thermosensitive materials such as thermosensitive polyelectrolyte multilayer films, gelatin, collagen, hyaluronic acid, and cellulose.

[0039] In one exemplary embodiment of this disclosure, the culture container 200 may include a culture dish 200 having a base 201, sidewalls 203, and an opening 202. The base 201 of the culture dish 200 may be configured to allow the culture of at least one layer of support cells 204. The culture dish 200 may be configured to include an opening 202 for inserting an insert 100 device.

[0040] like Figure 3 As shown, the insert 100 may include a base 102 and a handle 101. The handle 101 may be configured to allow insertion through the culture dish opening 202. The base 102 may be configured to allow the culture of at least one layer of supporting cells 104.

[0041] In various embodiments, the thermosensitive layer can be added to the surface of the culture dish substrate 201 before culturing the supporting cells. The supporting cell layer 204 can be cultured on the surface of the thermosensitive layer covering the culture dish substrate 201 or the insert surface 102. See also Figure 3 Treating the surfaces of culture dish 200 and insert 100 with a temperature-sensitive layer allows for the transfer of an intact supporting cell layer from culture dish 200 or insert 100 to another culture dish or insert. See also Figure 3 , Figure 4 and Figure 5 .

[0042] In one exemplary embodiment, a first culture dish 200 having a layer of thermosensitive material on the surface of a first culture dish substrate 201 can be used to culture a supporting cell layer 204. An insert 100 can be placed through an opening 202 in the first culture dish 200 such that the surface 102 of the insert 100 can contact the supporting cell layer 204. Conditions in the culture environment can be altered to activate the thermosensitive material, such as through a temperature change, and to release the supporting cell layer 204 from the substrate surface 201 of the first culture dish 200, allowing the supporting cell layer 204 to adhere to the surface 102 of the insert 100. See also Figure 4 The supporting cells 204 are removed from the substrate surface 201 of the culture dish 200 by activating the thermosensitive material, and also attached to the surface 102 of the insert 100. Figure 5 As shown in the image.

[0043] In one exemplary embodiment, subcutaneous WAT samples can be obtained from human subjects during elective surgical procedures. In this embodiment, sample size can range from about 100 to about 5000 grams of WAT tissue. In specific embodiments, experimental samples can be separated for various experimental purposes. In one specific embodiment, a portion of the subcutaneous WAT sample (e.g., 10 grams) can be chopped, rapidly frozen, and stored as a matched primary WAT sample. In one specific embodiment, a portion of the subcutaneous WAT sample, such as 10 grams, can be stored as a matched primary WAT sample for transcriptional confirmation in a nucleic acid lysis buffer, such as RNeasy Lipid Tissue Mini Kit™ (Qiagen). In a specific embodiment, according to embodiments of this disclosure, a portion of the subcutaneous WAT sample (e.g., 25 grams) can be used to produce SWAT cultures. In one specific embodiment, when WAT is chopped, enzymatically digested, and centrifuged, a portion of the subcutaneous WAT sample (e.g., 25 grams) can be used to isolate matched supporting cells, such as adipocytes (ADSCs) for differentiation into diffAd using a standard protocol.

[0044] In an exemplary embodiment of this disclosure, primary human WAT can be isolated from a patient and mechanically fragmented into pieces 300. The WAT tissue fragments 300 are transferred to a culture dish 400 having a supporting cell layer 304 grown on a culture dish substrate 401. See also Figure 6 .

[0045] The insert 100, which has a supporting cell layer 204, can then be inserted into the culture dish 400, including the WAT tissue 300 on top of the supporting cell layer 204. See also Figure 7In this embodiment, primary human WAT tissue 300 can then be sandwiched between two layers of supporting cells 204, 304 to form a sandwiched WAT (SWAT) co-culture system 800. See also Figure 8 The upper supporting cell layer 104 can attach to the base 102 of the insert 100 and serves to maintain contact between the floating WAT tissue 300 and the lower supporting cells 204 attached to the culture dish 200 until attachment occurs. In an exemplary embodiment, attachment between the WAT tissue 300 and the supporting cell layers 104, 204 occurs within minutes. In some embodiments, the bilayer construct of the SWAT system can be entirely cellular or can contain a variety of synthetic or cell-free components.

[0046] In one exemplary embodiment, 0.5–1 mm fragments of major human WAT tissue are sandwiched between two layers of supporting cells (e.g., adipose-derived stromal cells (ADSCs)) to form the SWAT co-culture system described herein. The supporting cells, such as ADSCs, can be cultured on standard tissue culture plates coated with a thermosensitive matrix. The SWAT culture system disclosed herein can also be a standard culture medium. Examples of standard culture media include at least low-glucose DMEM, approximately 10% newborn calf serum, and approximately 1% penicillin / streptomycin antibiotic solution.

[0047] In exemplary embodiments, the SWAT system described herein can be used as a test model for any external factor or system intended to modify the biology or physiology of adipose tissue or adipocytes. In various embodiments, the test factor can be introduced into cell culture media, and its effects can be evaluated in isolated human adipocytes or fragments of primary human WAT. Non-limiting embodiments of the test factor may include, but are not limited to, pharmaceutical compounds, recombinant or natural viruses, recombinant or isolated nucleic acid constructs, expression vectors, siRNA constructs, microRNA constructs, genetic tools, bacteria, and environmental modulation, including regulation of temperature, pressure, and gas.

[0048] like Figure 9A The micrograph shows a SWAT culture established between two layers of supporting cells. SWAT was added to the bottom layer of unlabeled supporting cells, and a top layer of supporting cells expressing enhanced green fluorescent protein (eGFP) was added. Figure 9A As shown, WAT cluster cells are sandwiched between a double layer of supporting cells (e.g., eGFP-negative (bottom layer) and eGFP-positive (top layer)) to form an exemplary SWAT culture system. Furthermore, Figure 9B Microscopic images of SWAT cultures over time are shown. WAT cell clusters within SWAT cultures maintained their morphological stability for at least 47 days, or approximately 6.7 weeks. See also... Figure 9B .

[0049] In various embodiments, the SWAT co-culture system described herein exhibits long-term viability and stability, which are important characteristics of microphysiological models of terminally differentiated cells, including WAT. In one exemplary embodiment of this disclosure, in Figure 10 The staining of the SWAT clusters in the sample demonstrated long-term morphological stability. Figure 10 In A, the structural stability of WAT cell clusters was elucidated by confining neutral lipids to WAT cells, even after 51 days in SWAT cultures. Similarly, propidium iodide staining of WAT cells in SWAT cultures was demonstrated as follows: Figure 10 B indicates a negative result. Propidium iodide-negative WAT cells demonstrate that, after at least 18 days of SWAT culture, WAT cells did not undergo programmed cell death, i.e., apoptosis. Finally, lipophilic staining of adipocytes in SWAT co-cultures further demonstrates the long-term viability of the systems and methods described herein. See also Figure 10 C. Contrary to conventional methods, exemplary embodiments of this disclosure demonstrate that WAT cell clusters within SWAT cultures retain their intracellular structures, such as Figure 10 A is feasible and does not lead to programmed cell death, for example... Figure 10 B, and remain for different groups of people.

[0050] Figure 11 This study demonstrates that the SWAT system described herein maintains the gene expression profile of at least six (6) key adipocyte identity genes, including: activating receptor γ (PPARγ), a major regulator of adipocyte differentiation / identity; fatty acid-binding protein 4 (FABP4), a transcription factor essential for terminal adipocyte differentiation; CCAAT / enhancer-binding protein α (CEBPa), which delivers long-chain fatty acids and retinoic acid to nuclear receptors; lipoprotein lipase (LPL), an enzyme that hydrolyzes triglycerides; hormone-sensitive lipase (HSL), which hydrolyzes stored triglycerides into free fatty acids; and adiponectin (ADIPOQ), a central adipokine controlling lipid metabolism and insulin sensitivity. Experimentally, total RNA was collected from SWAT cultures, and expression levels were compared with those of subject-matched primary SWAT using reverse transcription polymerase chain reaction (RT-PCR). At the transcriptional level, the SWAT culture system of this disclosure maintains adipose tissue identity. See also Figure 11 .

[0051] At the translational level, the SWAT culture system disclosed herein also maintains adipocyte proteins. For example... Figure 12As shown, immunocytochemical staining of SWAT cultures demonstrated the production of proteins that are adipocyte markers, including PPARγ, FABP4, β-3 adrenergic receptors (B3-AR) associated with adipocyte lipolysis, and perillipin, which are known to be lipid droplet-associated proteins and are coated with lipid droplets in adipocytes.

[0052] In addition to expressing gene and protein markers associated with adipocytes, the SWAT cultures of this disclosure also perform basic endocrine functions. In some embodiments of this disclosure, it may be necessary to maintain the function of tissues and cells in the culture model as closely as possible to native tissues. In various embodiments, the SWAT clusters retain their native endocrine functions. Primary human SWAT is an endocrine tissue that secretes at least two hormones, including leptin and adiponectin. Based on standardized quantitative ELISA assays, such as… Figure 13A and Figure 13B As shown, after one (1) and five (5) days of culture, SWAT cultures secreted leptin and adiponectin at levels similar to those of WAT cultured in subjects. See also Figure 13A and Figure 13B .

[0053] Furthermore, the SWAT culture system described in this paper responds to exogenous signals and undergoes lipolysis at a level similar to that of primary WAT. See also Figure 13C Lipolysis is the core function of WAT, a process that converts stored fat into metabolic fuel. In vivo, lipolysis occurs at a basal rate and is upregulated by catecholamines. In vitro, lipolysis can be quantified by measuring the amount of free glycerol released using the standard Bradford total protein assay and normalizing the total protein level. In one specific embodiment of this disclosure, SWAT cultures were exposed to 100 μM trichosin + 1 μM epinephrine for three (3) hours. After 1 and 5 days of culture, SWAT cultures responded to the stimulus with lipolysis at levels similar to those of primary WAT.

[0054] In another exemplary embodiment, the SWAT culture system described herein can maintain native functionality after at least ten days of SWAT culture. Figure 14 As shown, SWAT cultures were harvested ten days after incubation and subcutaneously injected into immunocompromised eGFP-tagged mice (NOD-scid IL2Rγnull). It is known that implanted tissue must either recruit a new blood supply, i.e., induce angiogenesis, or the tissue will die within 48 hours, i.e., necrosis and subsequent liquefaction by the host. In one exemplary embodiment, SWAT grafts were reharvested from their mouse hosts 10 days after subcutaneous injection. The injected SWAT tissue is readily visible to the naked eye upon visual inspection. See also... Figure 14(Reharvested). Furthermore, histological analysis showed that the SWAT graft retained the structural characteristics of WAT and did not express endogenous eGFP from the mouse host. See also Figure 14 (SWAT and Neg). These data indicate that SWAT cultures retain sufficient native function even after ten days in SWAT culture to enable SWAT tissue transplantation. Because recruiting a new host blood supply, i.e., angiogenesis, is a highly complex process, this data further suggests that the SWAT system described in this paper can be a robust microphysiological model of human SWAT.

[0055] Exemplary embodiments of this disclosure provide a system that allows for the study of effective anti-obesity strategies. It is previously understood that only brown adipose tissue (BAT) is capable of burning energy in a process known as thermogenesis. However, in rodent and diffAds models, it is known that white adipocytes (wAds) can be induced to become thermogenic light brown / “brite” adipocytes (brAds), which can be biochemically identified based on the upregulation of uncoupling protein 1 (UCP1) in response to elevated intracellular cyclic AMP (cAMP) levels. Specifically, UCP1 induction converts WAT into thermogenic cells and leads to changes in cell morphology.

[0056] Morphologically, brAds have transitioned from a large, single-compartment phenotype associated with WAT cells to a multi-compartment phenotype. The WAT-specific origin of brAds has been confirmed through rodent lineage tracing studies: brAds are myogenic factor 5 (Myf5) negative, while brown adipocytes (bAds) share a Myf5+ lineage with skeletal muscle cells. Browning has been observed in most subcutaneous and visceral WAT repositories in rodents. In rodent models, the weight loss resulting from browned WAT can be profound. Therefore, the SWAT culture system disclosed herein provides a microphysiological model system for evaluating controlled browning of cultured primary human WAT as a feasible and effective anti-obesity strategy.

[0057] The embodiments disclosed herein provide systems and methods for studying the biochemistry of browning pathways identified in rodent and diffAds models, which can be controlled by β-3-adrenergic receptors (B3-AR), cryoretin receptors, cardiac natriuretic peptide receptors, Janus inhibitor kinase 3 (JAK3), and Notch 1. Each of these endogenous biochemical pathways provides numerous candidate targets for drug intervention. Several compounds brown WAT in rodents and diffAds. In rodents, chemically induced browning of WAT has successfully improved obesity and cured type 2 diabetes. Therefore, the SWAT culture system disclosed herein provides a microphysiological model system for evaluating candidate drugs in primary human WAT tissue.

[0058] Exemplary embodiments of this disclosure provide in vitro systems that can allow the study and evaluation of the effects of chemical compounds (e.g., drugs) on human WAT and other floating cell types. Non-limiting exemplary drug candidates may include, but are not limited to: β-3 adrenergic receptor agonists and antagonists, such as 1&3-AR; migrabegron, an FDA-approved fourth-generation β-3 agonist for overactive bladder syndrome, but known to activate BAT in humans; CL-316243, a specific β-3 agonist (e.g., 1&1, 1&2, 1&3 = 0:1:100,000), which improved obesity in obese diabetic yellow KK mice; L-796568, a benzylsulfonamide family specific β-3 agonist (e.g., 1&1, 1&2, 1&3 = 1:230:660), which improved energy expenditure but did not produce significant anti-obesity effects in obese human men; and BRL 26830A, a β-3 agonist that showed significant improvement in weight loss in a double-blind trial.

[0059] In other exemplary embodiments, the culture system described herein can maintain the natural function of other floating cells in the culture. Endogenous biochemical pathways can be evaluated by applying and assessing the effects of exogenous stimuli (e.g., chemical compounds) to assess drug interventions. Regardless of tissue type or species of origin, any type of floating cell can be a candidate for use in the embodiments of this disclosure. Exemplary embodiments of floating tissue and cell types that can be used for evaluation using the devices, systems, and methods disclosed herein include, but are not limited to: hepatocytes, kidney tissues and cells, brain tissues and cells, thyroid tissues and cells, spleen tissues and cells, liver tissues and cells, central and peripheral nerve tissues and cells, and immune tissues and cells. Moreover, floating cells can be obtained from any source organism. Exemplary source organisms can include, but are not limited to: plants, animals, protists, fungi, archaea, and eubacteria. Other exemplary sources of tissues or cells for evaluation using the devices, systems, and methods disclosed herein include, but are not limited to: humans, mice, rats, monkeys, dogs, cats, pigs, non-human primates, and fish.

[0060] Exemplary embodiments of this disclosure provide systems and methods for studying the biological responses of exemplary, non-limiting cell types. For example, the type of floating tissue established may include neuronal tissue. Neuronal tissue may not readily adhere to the surface of the culture dish if, for example, excessive air bubbles are introduced into the aqueous culture medium. Therefore, the floating tissue culture apparatus, systems, and methods disclosed herein can be directly applied to the study of neural tissue.

[0061] Embodiments of this disclosure provide apparatus, systems, and methods for culturing neuronal tissue, which may include embryonic or adult neuronal tissue. In one exemplary embodiment, this disclosure provides a model system that can be used to assess neurogenesis. In other embodiments, this disclosure provides a system for assessing the progression of neuronal diseases.

[0062] In exemplary embodiments, the devices, systems, and methods of this disclosure can be used to evaluate the biochemical pathways leading to neuronal disease commonly known as Alzheimer's disease (AD) and the effects of various pharmacological interventions. For example, central to AD disease is the differential processing of the integrated membrane protein amyloid precursor protein (APP) between normal and disease states. In the normal state, APP is first cleaved by α-secretase to produce sAPP and a C83 carboxyl-terminal fragment. The presence of sAPP is associated with normal synaptic signaling and contributes to synaptic plasticity, learning and memory, emotional behavior, and neuronal survival. In the disease state, APP is sequentially cleaved by α-secretase and γ-secretase to release an extracellular fragment called A40 / 42. This neurotoxic fragment frequently aggregates and leads to A40 / 42 oligomerization and plaque formation. Aggregation of A40 / 42 results in blocked ion channels, disruption of calcium homeostasis, mitochondrial oxidative stress, impaired energy metabolism and aberrant glucose regulation, and ultimately neuronal cell death. The microphysiological system of this disclosure provides a model for rapidly and efficiently evaluating in vitro floating neuronal tissue while maintaining the neuronal tissue in a native state.

[0063] This disclosure provides devices, systems, and methods for evaluating biochemical pathways involved in cardiovascular disease (CVD). Cardiovascular disease (CVD) remains the leading cause of death in the United States, causing more than 600,000 deaths annually and incurring direct costs approaching $300 billion annually. Hypertension (HTN) and obesity are the two most common and modifiable risk factors for CVD. HTN affects 29.1% of U.S. adults, and successful treatment of blood pressure can reduce the risk of CVD by 20–50%. Obesity is more prevalent than HTN, affecting 36% of adult Americans and is considered a global epidemic. However, despite the availability of several classes of antihypertensive drugs, no widely effective anti-obesity drugs have been approved for patient use.

[0064] In exemplary embodiments, the apparatus, systems, and methods of this disclosure can be used to evaluate overlapping biochemical pathways involved in cardiovascular disease and obesity. For example, the pathogenesis of HTN typically involves the overactivation of the renin-angiotensin system (RAS). RAS overactivation is also associated with obesity, a disease involving excessive growth of glutathione (WAT). Furthermore, the RAS shares biochemical signaling pathways that overlap with the obesity biochemical pathway, as demonstrated by the fact that: (i) molecular components of the RAS are present in adipose tissue, (ii) WAT secretes angiotensinogen (AGT), (iii) angiotensin II (Ang II) can induce adipogenesis in isolated adipocytes and differentiated adipocytes (diffAds), and (iv) Ang II stimulation inhibits lipolysis in isolated human adipocytes, thus favoring adipogenesis.

[0065] Furthermore, the embodiments disclosed herein demonstrate that SWAT cultures can maintain the expression of RAS pathway components. For example, using RT-PCR, it was determined that SWAT cultures retained the expression of key RAS components (n = 5): (i) SWAT AGT expression: 62% (range 47-79%) of primary WAT; SWAT ACE expression: 58% (range 45-71%) of primary WAT; SWAT AT1R expression: 14% (range 6-19%) of primary WAT; SWAT AT2R expression: 231% (range 72-617%) of primary WAT; SWAT renin: no detectable expression. In addition, regarding endocrine function, as determined by enzyme-linked immunosorbent assay (ELISA), SWAT secretes AGT, leptin, and adiponectin. Finally, SWAT secretes 77 to 204 ng AGT / mg total protein, and Ang II was not identified in the culture medium derived from the cultured SWAT by ELISA. In summary, these data suggest that RAS overactivation may drive lipogenesis in a systemic and autocrine manner. In the embodiments, RAS inhibition by existing approved drug therapies can improve hypertension and obesity. Embodiments of this disclosure provide apparatus, systems, and methods for studying this system.

[0066] Although embodiments are described with reference to various implementations and uses, it will be understood that these embodiments are illustrative and the scope of the invention is not limited thereto. Many variations, modifications, additions, and improvements are possible. Furthermore, any steps described herein can be performed in any desired order, and any desired steps can be added to or removed.

Claims

1. A microphysiological system, comprising: A culture comprising an aqueous culture medium and a floating tissue sandwiched between two layers of supporting cells, wherein the floating tissue floats relative to the aqueous culture medium and comprises primary human white adipose tissue (WAT); The exogenous reagents were prepared to be added to the WAT culture; and At least one detector is configured to assess physiological changes in WAT resulting from the addition of the exogenous reagent to the culture. The supporting cells are adipose-derived stromal cells (ADSCs), and the supporting cells are attached to the substrate / surface of the culture container, wherein the culture container contains a material that promotes the adhesion of tissues or cells to the substrate / surface.

2. The system of claim 1, wherein the culture is operable to maintain the sandwiched WAT in a live differentiated state for at least three days.

3. The system of claim 1, wherein the exogenous reagent comprises at least one of chemicals, pharmaceuticals, nucleic acids, proteins, viruses, lipids, and carriers.

4. The system of claim 1, wherein the detector comprises at least one of an optical detector, a biochemical detector, and an energy detector.

5. The system of claim 1, wherein the physiological changes include intracellular changes, extracellular changes, transcriptional changes, translational changes, post-transcriptional changes, post-translational changes, secretory changes, and functional changes.

6. The system of claim 1, wherein the microphysiological system comprises an organ-on-a-chip system.

7. The system of claim 1, wherein the WAT and two layers of supporting cells are immersed in an aqueous culture medium.

8. The system of claim 1, wherein the floating tissue comprises an organ.

9. The system of claim 1, wherein the floating tissue comprises a tissue explant.

10. The system of claim 1, wherein the floating tissue comprises 0.5-1 mm fragments of tissue sandwiched between two layers of supporting cells.

11. The system of claim 1, wherein the exogenous agent comprises a candidate drug, and wherein the system is effective for evaluating the candidate drug in WAT.

12. The system of claim 1, wherein the physiological changes include browning of WAT.

13. The system of claim 1, wherein the culture is operable to maintain the sandwiched WAT in a live terminally differentiated state for at least one day.

14. The system of claim 1, wherein the culture is operable to maintain the sandwiched WAT in a live terminally differentiated state for at least five days.

15. The system of claim 1, wherein the culture is operable to maintain the sandwiched WAT in a live terminally differentiated state for at least ten days.

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