PANCREAS-ON-A-CHIP AND USES THEREOF
20220356428 · 2022-11-10
Inventors
- Anjaparavanda Naren (Cincinnati, OH, US)
- Kyushik Mun (Montgomery, OH, US)
- Kavisha Arora (Katy, TX, US)
Cpc classification
B01L3/502707
PERFORMING OPERATIONS; TRANSPORTING
C12M21/08
CHEMISTRY; METALLURGY
B01L2300/0816
PERFORMING OPERATIONS; TRANSPORTING
B01L3/502761
PERFORMING OPERATIONS; TRANSPORTING
International classification
C12M3/06
CHEMISTRY; METALLURGY
B01L3/00
PERFORMING OPERATIONS; TRANSPORTING
Abstract
Disclosed herein are microfluidic devices that may be used to mimic human organ systems, in particular, pancreatic function, and methods of using same. In particular, disclosed are microfluidic devices that may include a first chamber having a plurality of pancreatic ductal epithelial cells (PDECs), a second chamber having a plurality of pancreatic islets, and a permeable membrane fluidly connecting the chambers. The disclosed devices and methods may be used for the study of pancreatic cell function, for the development of therapeutics, or for the development of personalized therapeutics wherein the cells of the device are obtained from an individual in need of such treatment.
Claims
1. A microfluidic device, comprising: a first surface at least partially defining a first chamber; a plurality of pancreatic ductal epithelial cells (PDECs) received within said first chamber; a second surface at least partially defining a second chamber; a plurality of pancreatic islets received within said second chamber; and a permeable membrane fluidly connecting said first and second chambers such that said plurality of PDECs are configured to communicate with said plurality of pancreatic islets to mimic in situ pancreatic cell function.
2. The device of claim 1, wherein said PDECs and pancreatic islets are derived from an individual, wherein said individual may have a disease state selected from one or more of Acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP), and cystic fibrosis (CF).
3. The microfluidic device of claim 1, wherein said first chamber further includes a first cell culture media positioned therein, and wherein said second chamber further includes a second cell culture media positioned therein.
4. The microfluidic device of claim 3, wherein said first cell culture media and said second cell culture media comprise insulin.
5. The microfluidic device of claim 1, wherein each of said plurality of PDECs is in a monolayer.
6. The microfluidic device of claim 1, wherein said plurality of PDECs is configured to express a cystic fibrosis transmembrane conductance regulator (CFTR) protein.
7. The microfluidic device of claim 1, wherein said plurality of islets is configured to secrete insulin.
8. The microfluidic device of claim 1, wherein said permeable membrane comprises a plurality of openings extending between and fluidly connecting said first and second chambers, and wherein each of said plurality of openings has of a width of from about 5 μm to about 25 μm, or about 10 μm.
9. The microfluidic device of claim 1, wherein said first surface is in contact with said plurality of PDECs, wherein said second surface is in contact with said plurality of pancreatic islets, and wherein at least one of said first surface or said second surface at least partially includes a hydrophilic surface.
10. The microfluidic device of claim 9, wherein said hydrophilic surface is selected from poly methyl methacrylate, acrylonitrile butadiene styrene copolymer, cyclic olefin copolymer, styrene ethylene butylene styrene, collagen, or combinations thereof.
11. The microfluidic device of claim 1, wherein said first surface is in contact with said plurality of PDECs, wherein said second surface is in contact with said plurality of pancreatic islets, and wherein at least one of said first surface or said second surface has a sol-gel-modified PDMS or a collagen-coated-PDMS received thereon.
12. The microfluidic device of claim 1, wherein said first chamber includes a first branch channel and a second branch channel, wherein each of said first and second branch channels extend in a common channel plane and intersect at a first predetermined angle.
13. The microfluidic device of claim 1, wherein said first branch channel further includes a first pair of side edges extending in the common channel plane and defines a first width therebetween, wherein said second branch channel further includes a second pair of side edges extending in the common channel plane and defining a second width therebetween, and wherein the second width is narrower than the first width.
14. A method of measuring cystic fibrosis transmembrane conductance regulator (CFTR) protein function in an individual, comprising a. obtaining pancreatic ductal epithelial cells (PDECs) and pancreatic islets from said individual; b. culturing said PDECs and pancreatic islets in the device of claim 1, wherein patient-derived pancreatic ductal epithelial cells (PDECs) are co-cultured in a first chamber, and patient-derived pancreatic islet cells are cultured in second chamber; c. assaying the function of said CFTRs in a pancreatic ductal monolayer; and d. measuring insulin secretion of said pancreatic islets.
15. The method of claim 14 further comprising measuring one or more of fluid secretion from said PDECs in response to forskolin and measuring insulin secretion of said pancreatic islets in response to glucose.
16. The method of claim 14 wherein said individual has Cystic Fibrosis (CF)-related diabetes (CFRD).
17. The method of claim 14, wherein said first and/or second chamber are contacted with alcohol to determine one or both of CFTR function and endocrine function in response to said alcohol.
18. The method of claim 14, wherein said method is used to determine function of a CFTR mutation type, wherein said PDECs are known to contain said CFTR mutation type, and wherein function of one or both of said PDECs and/or pancreatic islets are correlated with said CRTR mutation type.
19. The method of claim 14, further comprising a. contacting said first or second chamber with an agent suspected of improving glucose abnormalities; and b. measuring a glucose response in said pancreatic islets in response to said contact.
20. A method of assaying a potential treatment for one or more of Acute Recurrent Pancreatitis (ARP) or Chronic Pancreatitis (CP), and Cystic Fibrosis (CF), and Cystic Fibrosis (CF)-related diabetes (CFRD), comprising a. contacting a potential therapeutic agent with one or both of said first and said second chambers of the device of claim 1; and b. detecting a desired output.
21. The method of claim 20, wherein said desired output is selected from one or both of fluid secretion from PDECs and insulin secretion from said pancreatic islets.
22. A method of making a pancreatic ductal epithelial cells (PDECs) monolayer, comprising digesting pancreatic duct tissue obtained from said individual, isolating PDECs from said digested pancreatic duct tissue, embedding said isolated PDECs in a matrix, and incubating with media until one or both of an organoid and a monolayer is formed.
23. The method of claim 22, wherein said matrix is disrupted mechanically, in the absence of trypsin, prior to said incubation with media to form said monolayer.
24. The method of claim 22, wherein said monolayer is a polarized monolayer.
25. The method of claim 22, wherein said pancreatic ductal epithelial cells express CFTR.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0006] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
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DETAILED DESCRIPTION
Definitions
[0031] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
[0032] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
[0033] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
[0034] As used herein, the term “effective amount” means the amount of one or more active components that achieves a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
[0035] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
[0036] For clarity of disclosure, the terms “upper,” “lower,” “lateral,” “transverse,” “longitudinal,” “bottom,” “top,” “right,” and “left” are relative terms to provide additional clarity to the figure descriptions provided below. The terms “upper,” “lower,” “lateral,” “transverse,” “bottom,” “top,” “right,” and “left” are thus not intended to unnecessarily limit the invention described herein.
[0037] In addition, the terms “first” and “second” are used herein to distinguish one or more portions of a device. For example, a first assembly and a second assembly may be alternatively and respectively described as a second assembly and a first assembly. The terms “first” and “second” and other numerical designations are merely exemplary of such terminology and are not intended to unnecessarily limit the invention described herein.
[0038] The instant disclosure relates to microfluidic devices and uses thereof. The microfluidic devices may be useful for a variety of different purposes, both as described herein and as would be readily understood by one of ordinary skill in the art. For example, the various embodiments of the device as described herein may be used in personalized medicines, wherein the device may be used to culture cells derived from the individual, and efficacy and/or safety of a therapeutic may be assayed using the device. The devices may be further used for tissue analysis, such as the ability to grow normal structures and cells from cells derived from an individual.
[0039] In one aspect, a microfluidic device, comprising a first surface at least partially defining a first chamber; a plurality of pancreatic ductal epithelial cells (PDECs) received within said first chamber; a second surface at least partially defining a second chamber; a plurality of pancreatic islets received within said second chamber; and a permeable membrane fluidly connecting said first and second chambers such that said plurality of PDECs are configured to communicate with said plurality of pancreatic islets to mimic in situ pancreatic cell function is disclosed. In one aspect, the PDECs and pancreatic islets may be derived from an individual. In one aspect, the PDECs and pancreatic islets may be derived from the same individual. In certain aspects, the individual may be one who has undergone a TPIAT. In certain aspects, said individual may be one having a disease selected from one or more of Acute Recurrent Pancreatitis (ARP) or chronic pancreatitis (CP), and cystic fibrosis (CF).
[0040] In one aspect, the first chamber further of the microfluidic device may include a first cell culture media positioned therein. The second chamber may further includes a second cell culture media positioned therein.
[0041] In one aspect, the first cell culture media and second cell culture media may comprise insulin.
[0042] The cell culture media may comprise the following components. For PDECs, the media may comprise Advanced DMEM/F-12 based medium containing HEPES, GlutaMAX, penicillin streptomycin, N2, B27, N-Acetylcysteine, and growth factors (Noggin, R-Spondin, and Epithermal Growth Factor). For pancreatic islets, the media may comprise Low glucose-containing based medium (DMEM; 100 mg/dL glucose) containing fatal bovine serum, penicillin streptomycin. In certain aspects, for the co-culture of PDECs and islets, the same media may be used. An exemplary media may be DMEM based, and may be used for both chambers. In one aspect, the media may be Advanced DMEM/F-12 (Advanced DMEM/F-12 contains ethanolamine, glutathione, ascorbic acid, insulin, transferrin, AlbuMAX® II lipid-rich bovine serum albumin for cell culture, and the trace elements sodium selenite, ammonium metavanadate, cupric sulfate, and manganous chloride, and is available from ThermoFisher Scientific.)
[0043] In one aspect, each of said plurality of PDECs may be in a monolayer. The monolayer may be a polarized monolayer.
[0044] In one aspect, the plurality of PDECs express a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In one aspect, the plurality of islets secrete insulin.
[0045] In one aspect, the permeable membrane may comprise a plurality of openings extending between and fluidly connecting said first and second chambers. The plurality of openings may have of a width of from about 5 μm to about 25 μm, or about 10 μm. Suitable opening sizes will be readily understood by one of ordinary skill in the art, and may vary, depending on the desired operation of the membrane and porosity. The gap of between two pores (from center to center) may be, in certain aspects, about 25 μm. The thickness of the membrane may generally be less than about 10 μm.
[0046] In one aspect, the first surface may be in contact with said plurality of PDECs, wherein said second surface is in contact with said plurality of pancreatic islets, and wherein at least one of said first surface or said second surface at least partially includes a hydrophilic surface.
[0047] In one aspect, the hydrophilic surface may be selected from poly methyl methacrylate, acrylonitrile butadiene styrene copolymer, cyclic olefin copolymer, styrene ethylene butylene styrene, collagen, or combinations thereof.
[0048] In one aspect, the first surface may be in contact with said plurality of PDECs, wherein said second surface may be in contact with said plurality of pancreatic islets, and wherein at least one of said first surface or said second surface may have a sol-gel-modified PDMS or a collagen-coated-PDMS received thereon. The device itself may comprise any suitable material as would be appreciated in the art. Materials that may be used for the disclosed microfluidic device may comprise, for example, SiO2, glass, and synthetic polymers. Synthetic polymers can, for example, comprise polystyrol (PS), polycarbonate (PC), polyamide (PA), polyimide (PI), polyetheretherketone (PEEK), polyphenylenesulfide (PPSE), epoxide resin (EP), unsaturated polyester (UP), phenol resin (PF), polysiloxane, e.g. polydimethylsiloxane (PDMS), melamine resin (MF), cyanate ester (CA), polytetrafluoroethylene (PTFE) and mixtures thereof. The synthetic polymers are optically transparent and can include, for example, polystyrol (PS), polycarbonate (PC), and polysiloxane, e.g. polydimethylsiloxane (PDMS).
[0049] In one aspect, the first chamber may include a first branch channel and a second branch channel, wherein each of said first and second branch channels may extend in a common channel plane and intersect at a first predetermined angle. Pancreatic ducts may be connected to Acinar cells and deliver digestive enzymes to the duodenum. In vivo, pancreatic ducts are spread out entire pancreas as roots with branching from a main duct, where it connects to the duodenum. The wide channel of the device, thus, is considered as a main duct and each branch channel has narrowing diameters. This arrangement may aid the study of pancreatic pressure-related disorders. Pancreatic pressure can be modelled by adjusting flow rate. In one aspect, the first branch channel may further include a first pair of side edges extending in the common channel plane and may define a first width therebetween, wherein said second branch channel may further includes a second pair of side edges extending in the common channel plane and defining a second width therebetween, and wherein the second width may be narrower than the first width.
[0050] In further aspects, a method of measuring cystic fibrosis transmembrane conductance regulator (CFTR) protein function in an individual is disclosed. In this aspect, the method may comprise obtaining pancreatic ductal epithelial cells (PDECs) and pancreatic islets from said individual; culturing said PDECs and pancreatic islets in the device disclosed herein, wherein patient-derived pancreatic ductal epithelial cells (PDECs) may be co-cultured in a first chamber, and patient-derived pancreatic islet cells may be cultured in second chamber; assaying the function of said CFTRs in a pancreatic ductal monolayer; and measuring insulin secretion of said pancreatic islets.
[0051] In one aspect, the method may further comprise measuring one or more of fluid secretion from said PDECs in response to forskolin, measuring insulin secretion of said pancreatic islets in response to glucose, and combinations thereof.
[0052] In one aspect, the individual may have Cystic Fibrosis (CF)-related diabetes (CFRD).
[0053] In one aspect, the first and/or second chamber may be contacted with alcohol to determine CFTR function and/or endocrine function in response to said alcohol.
[0054] In one aspect, the method may be used to determine function of a CFTR mutation type, wherein said PDECs are known to contain said CFTR mutation type, and wherein function of one or both of said PDECs and/or pancreatic islets are correlated with said CRTR mutation type.
[0055] In one aspect, the method may further comprise contacting said first or second chamber with an agent suspected of improving glucose abnormalities; and measuring a glucose response in said pancreatic islets in response to said contact.
[0056] In one aspect, a method of assaying a potential treatment for one or more of Acute Recurrent Pancreatitis (ARP) or Chronic Pancreatitis (CP), and Cystic Fibrosis (CF), and Cystic Fibrosis (CF)-related diabetes (CFRD), is disclosed. In this aspect, the method may comprise
[0057] contacting a potential therapeutic agent with one or both of said first and said second chambers of the device disclosed herein; and
[0058] detecting a desired output. In one aspect, the desired output may be selected from one or both of fluid secretion from PDECs and insulin secretion from said pancreatic islets.
[0059] In one aspect, a method of making a pancreatic ductal epithelial cells (PDECs) monolayer is disclosed. In this aspect, the method may comprise digesting pancreatic duct tissue obtained from said individual, isolating PDECs from said digested pancreatic duct tissue, embedding said isolated PDECs in a matrix, preferably a basement membrane matrix, and incubating with media until one or both of an organoid and a monolayer is formed, preferably wherein said organoid or monolayer forms a duct-like structure. “Matrix,” as used herein, includes substances or mixtures of substances, which enhance proliferation, differentiation, function or organoid or organ formation of cells. Matrix material may be coated on surfaces or may be provided in voluminous applications to optimize cell attachment or allow three-dimensional cultures. Matrix usable in the context of the present invention can take a variety of shapes comprising, e.g. hydrogels, foams, fabrics or non-woven fabrics. The matrix material may comprise naturally occurring matrix substances like extracellular matrix proteins, for example, collagens, laminins, elastin, vitronectin, fibronectin, small matricellular proteins, small integrin-binding glycoproteins, growth factors or proteoglycans or may include artificial matrix substances like non degradable polymers such as polyamid fibres, methylcellulose, agarose or alginate geles or degradable polymers, e.g. polylactid. The term “matrix” may include basement membrane matrix more commonly known in the trade as “Matrigel.®”
[0060] In one aspect, the matrix may be disrupted mechanically, in the absence of trypsin, prior to said incubation with media to form said monolayer. In general, in order to obtain monolayer of epithelial cells from organoids in 3-dimensional matrix (e.g., Matrigel), Matrigel is broken down by pipetting up and down using 1 mL trypsin EDTA following wash out cell culture media with cold PBS. After 10 min incubation with the EDTA, organoids may be transferred with EDTA to 15 mL tube, followed by pipetting up and down to separate organoids into single cells. Cell culture media containing FBS may be added, followed by spinning down to obtain a cell pellet. The cells may then be resuspended with fresh cell culture media and plated on a flat surface or trans-well membrane. In one aspect, the disclosed methods may be carried out in the absence of trypsin. The matrix (Matrigel) may be fragmented by pipetting (mechanical disruption) using growth media in the culture maintaining the organoids. The organoids, matrix (Matrigel), and media may then be transferred into a 1.5 mL tube, followed by pipetting to separate the organoids from matrix. Following centrifugation at 14000 rpm for about 3 min three layers can be observed: organoids, matrix, and media (from bottom to top of the tube). The media and matrix may then be discarded, and organoids resuspended with fresh media and plated on a flat surface or trans-well membrane. A monolayer of PDECs can be obtained, keeping tight-junctions. In one aspect, the monolayer may be a polarized monolayer.
EXAMPLES
[0061] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example
[0062] Cystic fibrosis (CF) is a genetic disorder caused by defective CF Transmembrane Conductance Regulator (CFTR) function. Insulin producing pancreatic islets are located in close proximity to the pancreatic duct and impaired cell-cell signaling between pancreatic ductal epithelial cells (PDECs) and islet cells may be causative in CF. Disclosed herein, in one aspect, is an in vitro co-culturing system, termed a “pancreas-on-a-chip.” Further disclosed are methods for the microdissection of patient-derived human pancreatic ducts from pancreatic remnant cell pellets, followed by the isolation of PDECs. Applicant has found that defective CFTR function in PDECs directly reduced insulin secretion in islet cells significantly. The disclosed pancreatic function monitoring tool may be useful for, inter alia, the study of CF-related disorders in vitro, as a system to monitor cell-cell functional interaction of PDECs and pancreatic islets, characterize appropriate therapeutic measures and further the understanding of pancreatic function.
[0063] To investigate the role of CFTR in CFRD and functional correlation between PDECs and islet cells, Applicant isolated PDECs and pancreatic islets from pancreatitis patients, who underwent total pancreatectomy with islet autotransplantation (TPIAT).sup.15. Applicant cultured these cell types in a microfluidic device, such as a microfluid device (10) (see
[0064] Prior to development of the pancreas-on-a-chip described herein in more detail, early versions of the microfluidic device (not shown) more generally were developed in 1979 as a miniature gas chromatograph.sup.16 and it has been exponentially innovated in functionality and design. In recent decades, microfluidics have been used as an in vitro model system for cell culture.sup.17-19, because of its high reproducibility, ability to mimic function and structure of organs, and some unique applications such as real-time PCR.sup.20, single-cell western blot.sup.21, wearable sensor.sup.22, and organ-on-a-chip.sup.23,24. Pancreas-on-a-chip, such as represented in one example by the microfluidic device (10) (see
[0065] Results
[0066] Isolation of patient-derived PDECs and pancreatic islets. Pancreatitis patients who have a debilitating course of acute recurrent or chronic pancreatitis may undergo TPIAT to relieve their pain and incapacitation, as shown in
[0067] Hence, disclosed herein are methods for isolating, culturing, and expanding patient-derived ductal epithelial cells and pancreatic islets, which may be used, for example, to provide a platform for the development of personalized medicine in pancreas-related disorders such as CFRD.
[0068] Characterization of PDECs.
[0069] PDECs are one of the most abundant cell type present in the pancreas. Applicant intended to confirm whether the isolated cells from the pancreatic remnant cell pellet were indeed PDECs. Isolated pancreatic ductal organoids (
[0070] Functional measurements in PDECs and pancreatic islets.
[0071] PDECs are reported to have the highest expression of CFTR in the body.sup.1,11-13. CFTR function in the pancreas has a critical role in maintaining fluid and pH within the pancreatic duct to deliver digestive enzymes secreted by acinar cells into the duodenum that are important for digestive function in the intestine. CFTR function was monitored in pancreatic ductal organoids in response to the cAMP-activating agonist forskolin (FSK; 10 μM). In this assay, CFTR function was reported as a measure of fluid secretion calculated by the ratio of luminal volume to that of the entire organoid.sup.26. During treatment with FSK, CFTR channels open, and chloride ions are pumped into the lumen creating an osmotic driving force for water to follow. Thus, fluid secretion is increased resulting in expansion of luminal volume. Fluid secretion was compared before and after treatment with FSK for 2 h, as shown in
[0072] Endocrine Function In Vitro.
[0073] Pancreatic islets were isolated from the pancreatic remnant cell pellet of the same patient source as for PDECs and cultured in vitro using the methodology as described above (
[0074] Microfluidic Device.
[0075] Using human tissue has its limitations, including limited availability and a very low viable cellular yield. The short-circuit current (Isc) assay is the gold-standard method to monitor CFTR function in real time; however, it requires approximately 1.3×10.sup.5 cells, and takes approximately 2 weeks to achieve a fully covered-polarized monolayer of epithelial cells on the trans-well membrane (33 mm.sup.2).
[0076] Here, Applicant developed a highly sensitive microfluidic device to monitor CFTR function from PDECs and insulin secretion from pancreatic islets cultured on the chip as shown in
[0077] Pancreas-On-a-Chip to Study CF-Related Disorders.
[0078] Applicant could detect that there is an interface between the ductal cells and islets based on H&E staining performed in a small piece (1 cm.sup.2) of non-treated tissue isolated from the head of the pancreas of a TPIAT patient (
[0079] Next, Applicant tested how CFTR function may affect insulin secretion from the islet cells in this double-channel chip system. Applicant measured secreted insulin in 1 h increments from pancreatic islets in the bottom chamber following stimulation or inhibition of CFTR channel function (
TABLE-US-00001 TABLE 1 TPIAT Patient Summary Mutations Age FEV1 Patients Allele 1 Allele 2 Gender (years) Sweat (%) BMI Patient 1 SPINK1 None F 14 ND ND 34 (pN34S) Patient 2 CFTR None M 8 2 ND 17 (Δ508) Patient 3 None None M 13 ND ND 23 Patient 4 PRSS1 (R122H) None F 9 16 ND 18 Patient 5 CFTR None F 4 21 ND 15 (1454G > C Het) Patient 6 CFTR (R170H) SPINK1 M 18 19 ND 21 (pN34S) Patient 7 None None F 13 46 108 25 Patient 8 CPA1 None F 13 ND ND 28 Patient 9 CFTR (Δ508), CFTR F 15 51 114 20 SPINK1 (R117H)
[0080] Discussion
[0081] Applicant has successfully isolated patient-derived pancreatic ductal organoids following TPIAT and has generated a freezing and reviving protocol for pancreatic ductal epithelial cells. Pancreatic ductal organoids demonstrated growth into large spheres over time. The organoids cultured in 3D matrix allows for the efficient harvest of pure pancreatic ductal epithelial cells among multiple cell types that are present in the pancreatic remnant cell pellet. The organoids can be grown effectively from a limited number of cells to form a functional unit. The 3D organoid formation with luminal area internally has been observed in other organs, including lung.sup.27, liver.sup.28, and intestine.sup.29. This is a repeated observation of duct-like formation from the pancreatic ductal organoids. This ductal formation may further be used to elucidate mechanisms involved in the development of the pancreatic duct in vivo.
[0082] Pancreas-on-a-chip mimics in situ pancreatic cell function and interface compared to conventional human cell culture model. The chip allows mimicking of fluid flow in vivo by setting a perfusion system in a cell culture incubator or on a microscope, relevant mechanical cues in cellular signaling, and allows tissue-issue interface (i.e., duct-islet) to study cell-cell signaling.sup.30. Pancreas-on-a-chip helps answer the fundamental question in CFRD: is loss of CFTR function in PDECs primary to CI-RD development. Based on the data, it is indeed the case. Surprisingly, the absolute amount of insulin was around 50% decreased during inhibition of CFTR channel function. In the human pancreas, the organ system is extremely complex in physiological and pathological perspectives. However, Applicant has found that CFTR channel function plays an important role in maintaining endocrine function and may provide insight into the etiology of CFRD. To investigate the crosstalk between PDECs and pancreatic islets, metabolism studies of these two cell types may be performed. CFRD is a serious complication in CF patients who in general have disordered glucose metabolism with increasing risk with advancing age.
[0083] Using this in vitro chip model, CFRD and glucose imbalance can be studied in CF individuals, assay variability in the glucose measures in these individuals, determine correlation of glucose levels with the CFTR mutation type, and test small-molecule interventions (i.e., approved CFTR modulators) that may improve glucose abnormalities in the patient samples. Applicant's data based on the effect of CFTR-specific inhibitor and lack of function mutation in CFTR strongly suggests that CFTR function modulates insulin secretion that underlies the pathology of CFRD. This patient-derived in vitro model system also allows the development of personalized medicine with highly sensitive measurements of epithelial and/or endocrine functions from the pancreatic cells. Because the cells cultured in the chip are all patient derived, Applicant can easily and quickly obtain other clinically relevant measures using this model in safe manner Alcohol abuse has been reported to lead to dysfunction and degradation of CFTR protein on the apical membrane of the epithelium.sup.31. Using this chip model, Applicant can monitor CFTR function and/or endocrine function in response to alcohol in a dose-dependent manner that is not possible in patients. The microfluidic device can be set up for multiple analyses, including functional assays and microscopic measurements in real time. This in vitro model system will facilitate drug discoveries. However, the polydimethylsiloxane (PDMS) used for the cell culture chambers has a challenging property, which is that the hydrophobic PDMS absorbs hydrophobic small molecules.sup.32,33. After oxygen plasma treatment, it changes to highly hydrophilic.sup.34. However, it recovers to hydrophobic over time.sup.35. The hydrophobic surface interferes with cell adhesion on the substrate.sup.36. Alternatively, other materials for fabricating the micro-fluidic device have been adopted, such as poly methyl methacrylate.sup.37, acrylonitrile butadiene styrene copolymer.sup.33, cyclic olefin copolymer.sup.38, and styrene ethylene butylene styrene.sup.39. However, those materials also have limitations when mimicking human organ systems due to their rigidity and brittleness, leading to a difficult fabrication process. PDMS-based microfluidic devices can be maintained as a hydrophilic surface for weeks after treatment with oxygen plasma.sup.34. It has also been shown that sol-gel-modified PDMS.sup.40 and bovine serum albumin-coated-PDMS.sup.41 can minimize absorbance of hydrophobic drugs by PDMS. Alternatively, collagen coating of the chamber can be utilized to increase cell adhesion, as is used in the model system.
[0084] In summary, Applicant has isolated and cultured patient-derived pancreatic cells, PDECs, and pancreatic islets from the same patient. This efficient and highly reproducible method allows the study of pancreatic disorders. Moreover, the in vitro model system, pancreas-on-a-chip, allows for the investigation of the crosstalk between PDECs and islet cells in the development of disease pathologically and physiologically. This pancreas-on-a-chip model system, with its highly sensitive profile, can allow for early diagnosis and individual diagnosis that may help prevent or reduce the progression of disorders such as CFRD, and additionally, can afford the opportunity for drug discovery and personalized medicine in such disorders.
[0085] Methods
[0086] Human Studies.
[0087] Human tissue, pancreatic remnant cell pellets were collected according to standard research protocols approved by the Institutional Review Board and Department of Pathology at Cincinnati Children's Hospital (IRB: 2014-6279; renewed 27 Nov. 2017).
[0088] Cell Culture Media.
[0089] For PDECs, advanced Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12) (Invitrogen; Ser. No. 12/634,010) with 10 mM HEPES (Invitrogen; #15630-080), GlutaMAX (lx; Invitrogen; #35050-061), and penicillin streptomycin (PS) (lx; Invitrogen; #15140-122) was used as base organoid media(I). Organoid media (II) contains N2 (lx; Invitrogen; #17502-048), B27 (lx; Invitrogen; #17504-044), and 1 mMN-acetylcysteine (Sigma; #A7250-100G) in organoid media (I). Organoid media (III) contains growth factors, supplements 100 ng/mL epidermal growth factor (R&D System; #236-EG-200), 50 ng/mL R-spondin (R&D System; #4645-RS-025/CF), and 100 ng/mL Noggin (R&D System; #6057-NG-025) in organoid media (II). ROCK inhibitor, 10 μM Y-27632 (BDBiosciences; #562822), was added for the first 4 days followed by isolation of pancreatic ductal epithelial cells. Islet cells were cultured in RPMI-1640 (Invitrogen; No. 61/870,036) containing PS(lx), 10% fetal bovine serum (FBS) (Atlanta Biologicals; #S11150 premium), and 10 μM Y-27632 for the first day of isolation. The RPMI-1640 media were switched to low glucose-containing DMEM (Invitrogen; #11885-084; 100 mg/dL glucose) with PS (lx) and 10% FBS from the second day of isolation. High-glucose-containing DMEM (Invitrogen; #11960-044; 450 mg/dL glucose) was used to stimulate pancreatic islets.
[0090] Isolation of pancreatic ductal organoids and islet.
[0091] Pediatric patients with severe acute recurrent or chronic pancreatitis undergo TPIAT. During the TPIAT, the excised pancreas was surgically dissected and digested to isolate pancreatic islets for infusion into the liver through the portal vein. Applicant obtained discarded pancreatic remnant cell pellets following isolation of pancreatic islets. The pancreatic remnant cell pellet still contains pancreatic islets, ductal epithelial cells, and acinar cells (
[0092] Obtaining monolayer of pancreatic ductal epithelial cells.
[0093] Pancreatic ductal organoids were grown over time in Matrigel (
[0094] Freezing and reviving pancreatic ductal epithelial cells.
[0095] To cryopreserve PDECs, PDEC monolayers were trypsinized with 0.5% trypsin EDTA (1×; Invitrogen; #15400-054) at 37° C. for 10 min to detach cells after washing cells with PBS and transferred to 15 mL tube containing 5 mL organoid media (I) with 10% FBS and 10 μM Y-27632. Cell pellets were obtained after centrifuging at 233×g for 5 min. The supernatant was discarded and cells were re-suspended with freezing media (Invitrogen; Ser. No. 12/648,010) containing 10 μM Y-27632. Epithelial cells were transferred to a cryopreservation tube and placed on dry ice immediately and stored at −80° C. For long-term storage, the cells were stored in liquid nitrogen. To revive PDECs, the cells were thawed quickly at 37° C. and all supernatants were transferred to 15 mL tube containing 5 mL organoid media (I) with 10 μM Y-27632. After centrifugation at 233×g for 5 min, the supernatant was discarded. Appropriate organoid media (II) with Matrigel were added and 50 μL Matrigel was plated with cells on plates to form organoid structure as before. The Matrigel was covered with organoid media (III) containing 10 μM Y-27632 after incubation at 37° C. for 15 min.
[0096] Fabrication of Pancreas-On-a-Chip.
[0097] Applicant's customized microfluidic device was designed to mimic ductal structure having branches with narrowing diameters (
[0098] After cooling down to room temperature, a negative photoresist SU-8 (Microchem; #Y131269) is applied to the wafer using a spin coater (Specialty Coating Systems; #6800) by the following process: (1) Place the wafer on the vacuum chuck of the spin coater and drop appropriate SU-8 on the wafer. (2) Ramp up to 500 rpm for 10 s and hold for 10 s. (3) Increase the speed to 1000 rpm for 10 s and hold it for 15 s for 140 μm thickness of cell culturing chamber in the chip. (4) Speed down to 0 rpm for 10 seconds. The wafer is placed on the hot plate and baked at 65° C. for 10 min and at 95° C. for 30 min serially. The wafer is exposed to ultraviolet (UV) light (wavelength: 365 nm; exposure energy:240mJ/cm2) through a patterned photomask for 20 s after cooling down to room temperature. The wafer is baked on the hot plate at 65° C. for 1 min and at 95° C. for 20 min and is cooled down to room temperature. The wafer is immersed into SU-8 developer (Fisher Scientific; #NC9901158) for development process of unexposed area to UV light. After completion of development, the wafer is washed with IPA and dried with filtered air. The patterned silicon wafer is then baked on the hot plate at 150° C. for 30 min After cooling down to room temperature, the patterned wafer can be used as a mold. These standard photo-lithography procedures can be carried out in in a 100-class clean room. For this microfluidic device, Applicant used flexible, transparent, and low-cost materials, PDMS (Ells Worth Adhesive; #4019862). Viscous PDMS is mixed with a curing kit at the ratio of 10:1 (wt %) and degassed in a desiccator to remove bubbles. In the meantime, the patterned silicon wafer is treated with trichloro silane (Sigma-Aldrich; #448931) for 30 min in another desiccator to assist peeling off the patterned PDMS layer from the wafer. The uncured PDMS is cast onto the wafer and cured at 60° C. for at least 4 h. The solidified patterned PDMS layer is peeled off from the wafer and holes are created at both ends of the cell culture area for seeding and feeding cells. The patterned PDMS layer and a cover glass are treated with oxygen plasma for 30 s using Tergeo Plasma Cleaner (PIE Scientific) and immediately assembled together. It is placed on the hot plate at 120° C. for 30 min to seal completely the single-channel chip. The activated surface of the patterned PDMS layer and cover glass by plasma treatment becomes highly hydrophilic with polar characteristics.sup.34. This enhances the bonding process of the two surfaces. Pancreas-on-a-chip is comprised of top and bottom layers for cell culture chambers and a thin layer of porous membrane to separate the two chambers as double-channel chip. Patterned PDMS layers of top and bottom chambers are prepared as described previously for single-channel chip. Holes were created through the PDMS layer of the top chamber for seeding and feeding cells before assembly with the porous membrane. For the thin layer of porous membrane, a mold was fabricated of uniformly arranged cylinders, with 10 μm diameters, 25-μm gaps, and 40-μm thickness, on a silicon wafer through the photolithography. The wafer is coated with trichloro silane in the desiccator for 30 min. In the meantime, RTV615 (Momentive; #9480), which shows large linear behavior of stain and promotes fabrication of a thin layer uniformly comparing to PDMS.sup.44,45, is mixed with a curing kit at the ratio of 5:1 (wt %) and degassed in the desiccator for 30 min. The patterned wafer was placed on the spin coater and spun after covering the pattern with degassed RTV615 as the standard for 10 μm thickness of porous membrane; thus, (1) ramp up to 500 rpm for 10 s and hold for 10 s; (2) increase the speed to 3000 rpm for 10 s and hold it for 5 min; (3) speed down to 0 rpm for 10 s. Leave the wafer at room temperature for 10 min for uniform surface and incubate at 60° C. for 10 min for partial solidification of the surface. After incubation, the top PDMS layer was placed, patterned face down, directly onto the cylinders and slightly pressed onto the PDMS layer for contacting the surface of top layer to the partially cured RTV615. The top chamber is incubated with the porous membrane on the wafer overnight and cooled down to room temperature. The top chamber with the porous membrane is peeled from the wafer and holes created through the porous membrane to connect to the bottom chamber only. The top chamber and bottom chamber are aligned after oxygen plasma treatment and placed on the hotplate at 120° C. for 30 min to seal the double-channel chip. Before seeding cells, the cell culture chambers were sterilized with 70% EtOH for 10 min and washed with autoclaved water using a needle (BD Biosciences; #305175; 20 G) and syringe (BD Biosciences; #309657; 3 mL). The chambers were coated with 50 μg/mL collagen (Sigma-Aldrich; #C3867-1VL) for 1 h at 37° C. and washed with PBS to increase cell adhesion. The microfluidic device was connected to a peristaltic pump (Cole-Parmer; #ISMATEC Reglo ICC) with tubing (Cole-Parmer; #97619-09) and supplied organoid growth media (III) at the flow rate of 1 μL/min to feed cells continuously. To feed cells manually, a syringe and needle was used.
[0099] Culture Cells in the Microfluidic Device.
[0100] Monolayers of PDECs were treated with 0.5% Trypsin EDTA (lx) at 37° C. for 10 min and floating cells were transferred to a 15 mL tube containing 5 mL organoid media (I) with 10% FBS and 10μMY-27632. The supernatant was discarded after spinning down at 233×g for 5 min (4° C.) and cells were re-suspended with 120 μL organoid media (III) containing 10 μM Y-27632. Cells were transferred (2×10.sup.5 cells/mL; 10,000 cells/chip) in the cell culture chamber coated with collagen (50 μg/mL) using a syringe and needle through a tubing (5 cm length) inserted through the PDMS layer. After overnight incubation at 37° C., 5% CO2 media were refreshed. Pancreatic islets in 24-well plate were washed with PBS and incubated with 200 μL 0.5% Trypsin EDTA (lx) at 37° C. for 3 min for trypsinization. Pancreatic islets were transferred to a 1.5 mL tube filled with culture media. The supernatant was discarded after centrifugation at 8600×g (microcentrifuge) for 3 min and cells were re-suspended with 120 μL media. Pancreatic islets were transferred (300 islets/mL; 15 islets/chip) into the cell culture chamber using a syringe and needle. Media were refreshed after the pancreatic islets attached onto the surface of the chip.
[0101] Immunofluorescence Microscopy.
[0102] Pancreatic ductal organoids in Matrigel were fixed with 3.7% formaldehyde for 15 min at room temperature and the Matrigel was broken down by pipetting with 1 mL EtOH. The organoids were embedded into HistoGel (Invitrogen; #HG-4000-012) and were first examined by gold-standard morphological section and H&E stain. Paraffin-sectioned organoids were deparaffinized for immunofluorescence microscopy. For a monolayer of PDECs on a trans-well membrane or a pancreas-on-a-chip, cells were fixed with 3.7% for maldehyde for 15 min at room temperature. Cells were then permeabilized using lx permeabilization solution (eBioscience; #00-8333-56) for 8 min at room temperature and washed three times with PBS for 5 min each. Cells were then blocked using 1% goat serum (Sigma-Aldrich; #A8806-5G) for 1 h at room temperature and incubated with primary antibodies (diluted in antibody diluent (Invitrogen; #TA-125-ADQ) 1:100), anti-CFTR R1104 (Eric Sorscher lab, CF Center, University of Alabama, Birmingham, Ala., USA [presently, Emory University, Atlanta, Ga., USA]), anti-ZO-1 (BD Biosciences; #610967), anti-ENaC (Invitrogen; #PA1-920A), anti-KRT 19 (Invitrogen; #MA5-12663), anti-E cadherin (Cell Signaling Technology; #3195), anti-insulin (Cell Signaling; #C27C9), and anti-glucagon (Sigma; #G2654) overnight at 4° C. Cells were washed three times with PBS for 5 min each and incubated with secondary antibodies (Invitrogen; Alexa Fluor 488 or 568; 1:500) for 1 h at room temperature. Alexa Fluor 488 Phalloidin (Invitrogen; #A12379; 1:50) was employed to the secondary antibody for F-actin staining fol-lowing washing three times with PBS for 5 min each. Cells were incubated with DAPI solution (Invitrogen; #D1306; 1:500) for 20 min for nucleus staining and washed with PBS. For the trans-well membrane, cut edge of the membrane and transferred the membrane with cells onto a glass slide oriented cell-side up. Cells were then mounted in Vecta-shield mounting medium (Vector Labs; #H-1000). A cover slip was placed onto the cells and fixed with nail polish. For the pancreas-on-a-chip, cell culture chambers were separated manually by hands followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) solution for 20 min. The porous membrane with cells remained on the upper layer. One drop of mounting solution was applied onto the cells and a coverslip was placed for imaging. Fluorescence images were obtained using a confocal microscope (Olym-pus FV1200). Combined images were created using an Image J software provided by NIH.
[0103] Extract RNA from Pancreatic Ductal Organoids.
[0104] Organoid growth media were discarded and the Matrigel was broken down by pipetting with 1 mL PBS. Pancreatic ductal organoids were picked and transferred to 1.5 mL RNA-free tube manually using a 200 μL pipette. The supernatant and Matrigel were discarded after microcentrifuge at 16,800×g for 5 min and RNA was extracted using an Ambion miRNA Isolation Kit (Invitrogen; #AM1561) using the protocol provided by Ambion.
[0105] Monitoring CFTR Function.
[0106] CFTR function of pancreatic ductal organoids was monitored using the fluid secretion assay.sup.26 in response to an intracellular cAMP-activating agonist (FSK; 10 μM) for 2 h at 37° C. Fluid secretion was calculated by measuring the volume ratio of luminal area over the entire organoid pre-treatment and post treatment with FSK. Fluid secretions were monitored at day 4 after isolation of organoids with at least 20 organoids. The area of lumen and outer sphere was measured using the Image J software. Pancreatic ductal organoids were transferred, when their diameter reached 500 μm, onto trans-well membranes (Corning; #3470), 10 organoids each as previously described. The ductal epithelial cells transformed into a polarized monolayer from spheroids on the trans-well membrane within 2 weeks. Transepithelial electrical resistance was measured using epithelial volt-ohm meter (World Precision Instruments, #EVOM and #STX2) and the trans-well membrane was mounted in an Ussing chamber when the resistance was over 1000 Ω/cm2. Cells were bathed in Ringer's solution (mM) for apical side (pH 7.2): 0.12 NaCl, 25 NaHCO.sub.3,3.3 KH.sub.2PO.sub.4, 0.83 KH.sub.2PO.sub.4, 1.2 CaCl.sub.2, 1.2 MgCl.sub.2, 141 Na-gluconate, and 10 mannitol, and for basolateral side (pH 7.2): 120 NaCl, 25 NaHCO.sub.3, 3.3 KH.sub.2PO.sub.4, 0.83 KH.sub.2PO.sub.4, 1.2 CaCl.sub.2, 1.2 MgCl.sub.2, and 10D-glucose maintained the temperature of the bath using circulate system as 37° C..sup.26,46. CFTR function was monitored in real time in response to current changing by FSK. When the current showed a stable baseline, 10 μM FSK was added to the apical side for CFTR channel opening. For CFTR channel closing, CFTR channel inhibitor, CFTR.sub.inh-172 (20 μM), was applied to the apical side. CFTR function of PDECs was monitored using iodide efflux assay.sup.47. Cell culture media were washed out with 136 mM NaNO.sub.3 and incubated with 136 mMNaI for 1 h at 37° C. After 1-h incubation, cells were washed with 300 μL of NaNO.sub.3 (136 mM) and the supernatant was collected with 136 mM NaNO.sub.3 using a syringe and needle. A 1.5 mL tube was placed on a digital weighing scale and the supernatant was dropped into the tube with recording the weight for approximately 20 μL of each sample. The first 10 samples were collected with 136 mM NaNO.sub.3 and the other 10 samples with 136 mM NaNO.sub.3 containing 10 μMFSK. Iodide concentration was calculated using an electrolyte detector (Thermo Orion; #420) with electrode probe filled with specific iodide-sensitive electrolyte (Invitrogen; #900063). The electrode was immersed in 5 mL of 100 mM NaNO.sub.3 (stirred) to detect iodide. Voltage change was measured by adding each sample serially. A standard curve was obtained using 10 μM, 100 μM, and 1 mM NaI.
[0107] Monitoring Insulin Secretion.
[0108] Cell culture media were discarded just before collection for the measurement. Media (60 μL) were collected and incubated with refreshed media for 1 h at 37° C., 5% CO2. After 1-h incubation, an additional 60 μL media were collected. Collected media were placed on ice until ready to assay. For stimulation of pancreatic islets, 450 mg/dL glucose-containing media (instead of 100 mg/dL) were used. Insulin secretion was monitored by measuring concentration of insulin in the culture media using ELISA (Invitrogen; #KAQ1251) following a protocol provided by the company. To monitor insulin secretion from pancreas-on-a-chip, Applicant co-cultured PDECs in the top chamber and pancreatic islets in the bottom chamber. Base media for PDECs, advanced DMEM/F12, contains insulin, which can affect the concentration of insulin secreted by pancreatic islets in the bottom chamber. It was switched to DMEM (same as pancreatic islets media). Two chips were prepared, Chip A and Chip B, to employ agonist (10 μM FSK) or inhibitor (20 μM CFTR.sub.inh-172) of CFTR channel on the PDECs. The chips were incubated at 37° C. for 1 h and 60 μL media were collected from the bottom chamber. FSK (Chip A) and CFTR.sub.inh-172 (Chip B) were employed on the top chambers and the chips were incubated at 37° C. for 1 h. Sixty microliters of media was collected from the pancreatic islets on the bottom chamber. For Chip A, the media in the bottom chamber were switched to high-glucose-containing media (450 mg/dL) and the chip was incubated at 37° C. for 1 h. For Chip B, a combination of FSK and CFTR.sub.inh-172 were added to the top chamber and the chip was incubated at 37° C. for 1 h. The chip was incubated with high-glucose-containing media at 37° C. for 1 h. Sixty microliters of media were collected from the pancreatic islets in the bottom chamber.
[0109] Statistical Analysis.
[0110] Data were derived from at least three independent replicates. The level of marginal significance, p-value, was calculated using two-tailed Student's t test for pairwise comparison and one-way analysis of variance with Bonferroni adjustment for multiple variations. A p value <0.05 was considered significant. Reporting summary
[0111] Exemplary Pancreatic Microfluidic Device
[0112]
[0113] As shown in
[0114] In order to more effectively mimic pancreatic duct-like structures as discussed above, the top chamber (22) successively narrows from the first top end channel (30) toward the second top end channel (44) at each of the first, second, third, fourth, and fifth top branch channels (32, 34, 36, 38, 40). More particularly, the first top branch channel (32) includes a first pair of top edges (54) extending in the common plane of the upper plate (12) and defining a first top width therebetween. Similarly, the second, third, fourth, and fifth, and sixth top branch channels (34, 36, 38, 40, 42) respectively include second, third, fourth, fifth, and sixth pairs of top edges (58, 60, 62, 64, 66) and respectively define second, third fourth, fifth, and sixth top widths. The first, second, third, fourth and fifth top widths successively narrow such that the the fifth top width is smaller than the fourth top width, the fourth top width is smaller than the third top width, the third top width is smaller than the second top width, and the second top width is smaller than the first top width. As used herein, the term “edges” generally refers to opposing sides of a channel of top chamber (22) that define a width therebetween, such as any one or more of top branch channels (32, 34, 36, 38, 40, 42), and is not intended to unnecessarily limit the invention described herein.
[0115] In addition, the first, second, third, fourth, and fifth top branch channels (32, 34, 36, 38, 40, 42) of the present example also respectively include first, second, third, fourth, fifth, and sixth depths that are respectively equal to the first, second, third, fourth, fifth, and sixth top widths. In instances where the depths are widths of respective top branch channels are equal, the first, second, third, fourth, fifth, and sixth top widths may also be referred to as first, second, third, fourth, fifth, and sixth top diameters. The first, second, third, fourth, fifth, and sixth depths respectively extend from the first, second, third, fourth, fifth, and sixth pairs of top edges (54, 58, 60, 62, 64, 66) to a top chamber floor, portions of which may be generally flat between the first, second, third, fourth, fifth, and sixth pairs of top edges (54, 58, 60, 62, 64, 66) and/or curved between the first, second, third, fourth, fifth, and sixth pairs of top edges (54, 58, 60, 62, 64, 66).
[0116] Also in order to more effectively mimic pancreatic duct-like structures as discussed above, each of the first, second, third, fourth, and fifth top branch channels (32, 34, 36, 38, 40, 42) intersects adjacent top branch channels (32, 34, 36, 38, 40, 42) as applicable at predetermined angles. In this respect, the first and second top branch channels (32, 34) intersect at a first top predetermined angle, the second and third top branch channels (34, 36) intersect at a second top predetermined angle, the third and fourth top branch channels (36, 38) intersect at a third top predetermined angle, the fourth and fifth top branch channels (38, 40) intersect at a fourth top predetermined angle, and the fifth and sixth top branch channels (40, 42) intersect at a fifth top predetermined angle. As used herein, “predetermined angle” refers to an angle that is neither 0 degrees nor 180 degrees such that adjacent first, second, third, fourth, and fifth top branch channels (32, 34, 36, 38, 40, 42) are non-parallel relative to each other, although non-adjacent top branch channels (32, 34, 36, 38, 40, 42) may be parallel in some examples. In the present example, the first, second, third, fourth, and fifth top predetermined angles are oriented such that the first, second, third, fourth, and fifth top branch channels (32, 34, 36, 38, 40, 42) laterally zigzag back and forth while also longitudinally projecting from the first end top channel (30) to the second end top channel (44) such that the first and sixth top branch channels (32, 42) are parallel to each other.
[0117] Similar to the upper plate (12),
[0118] In order to more effectively mimic pancreatic duct-like structures as discussed above, the bottom chamber (26) successively narrows from the first bottom end channel (130) toward the second bottom end channel (144) at each of the first, second, third, fourth, and fifth bottom branch channels (132, 134, 136, 138, 140). More particularly, the first bottom branch channel (132) includes a first pair of bottom edges (154) extending in the common plane of the lower plate (14) and defining a first bottom width therebetween. Similarly, the second, third, fourth, fifth, and sixth bottom branch channels (134, 136, 138, 140, 142) respectively include second, third, fourth, fifth, and sixth pairs of bottom edges (158, 160, 162, 164, 166) and respectively define second, third fourth, fifth, and sixth bottom widths. The first, second, third, fourth, and fifth bottom widths successively narrow such that the the fifth bottom width is smaller than the fourth bottom width, the fourth bottom width is smaller than the third bottom width, the third bottom width is smaller than the second bottom width, and the second bottom width is smaller than the first bottom width. Again, as used herein, the term “edges” generally refers to opposing sides of a channel of bottom chamber (26) that define a width therebetween, such as any one or more of bottom branch channels (132, 134, 136, 138, 140, 142), and is not intended to unnecessarily limit the invention described herein.
[0119] In addition, the first, second, third, fourth, and fifth bottom branch channels (132, 134, 136, 138, 140, 142) of the present example also respectively include first, second, third, fourth, fifth, and sixth depths that are respectively equal to the first, second, third, fourth, fifth, and sixth bottom widths. In instances where the depths are widths of respective bottom branch channels are equal, the first, second, third, fourth, fifth, and sixth bottom widths may also be referred to as first, second, third, fourth, fifth, and sixth bottom diameters. The first, second, third, fourth, fifth, and sixth depths respectively extend from the first, second, third, fourth, fifth, and sixth pairs of bottom edges (154, 158, 160, 162, 164, 166) to a bottom chamber floor, portions of which may be generally flat between the first, second, third, fourth, fifth, and sixth pairs of bottom edges (154, 158, 160, 162, 164, 166) and/or curved between the first, second, third, fourth, fifth, and sixth pairs of bottom edges (154, 158, 160, 162, 164, 166).
[0120] Also in order to more effectively mimic pancreatic duct-like structures as discussed above, each of the first, second, third, fourth, and fifth bottom branch channels (132, 134, 136, 138, 140, 142) intersects adjacent bottom branch channels (132, 134, 136, 138, 140, 142) as applicable at predetermined angles. In this respect, the first and second bottom branch channels (132, 134) intersect at a first bottom predetermined angle, the second and third bottom branch channels (134, 136) intersect at a second bottom predetermined angle, the third and fourth bottom branch channels (136, 138) intersect at a third bottom predetermined angle, the fourth and fifth bottom branch channels (138, 140) intersect at a fourth bottom predetermined angle, and the fifth and sixth bottom branch channels (140, 142) intersect at a fifth bottom predetermined angle. Again, as used herein, “predetermined angle” refers to an angle that is neither 0 degrees nor 180 degrees such that adjacent first, second, third, fourth, and fifth bottom branch channels (132, 134, 136, 138, 140, 142) are non-parallel relative to each other, although non-adjacent bottom branch channels (132, 134, 136, 138, 140, 142) may be parallel in some examples. In the present example, the first, second, third, fourth, and fifth bottom predetermined angles are oriented such that the first, second, third, fourth, and fifth bottom branch channels (132, 134, 136, 138, 140, 142) laterally zigzag back and forth while also longitudinally projecting from the first end bottom channel (130) to the second end bottom channel (144) such that the first and sixth bottom branch channels (132, 142) are parallel to each other.
[0121] With continued reference to
[0122] Each of the portions of the upper and lower plates (12, 14) respectively defining the top and bottom chambers (22, 26) is formed of polydimethylsiloxane (PDMS) although alternative materials may be used to at least some extent as discussed herein. Upon oxygen plasma treatment, the PDMS becomes hydrophobic such that the upper and lower inner surfaces (18, 20) are hydrophobic surfaces. Absorption of hydrophobic drugs may be further reduced by modifying these upper and lower inner surfaces (18, 20) with sol-gel, bovine serum albumin, and/or collagen as further discussed above.
[0123]
[0124] With respect to
[0125]
[0126] While the present examples of top and bottom chambers (22, 26) have a variety of channels (30, 32, 34, 36, 38, 40, 42, 44, 130, 132, 134, 136, 138, 140, 142, 144), such as 16 distinct channels, that successively narrow and zigzag with predetermined widths and angles, it will be appreciated that alternative numbers of such channels may be similarly used and arranged in alternative examples. The invention is thus not intended to be unnecessarily limited to the particular top and bottom chambers (22, 26) shown and described herein.
[0127] To this end, more particular details of the present top and bottom chambers (22, 26) are shown in
[0128]
TABLE-US-00002 First Top Width approximately 1 mm Second Top Width approximately 0.886 mm Third Top Width approximately 0.798 mm Fourth Top Width approximately 0.718 mm Fifth Top Width approximately 0.646 mm Sixth Top Width approximately 0.656 mm First Top Predetermined Angle approximately 170 degrees Second Top Predetermined Angle approximately 160 degrees Third Top Predetermined Angle approximately 160 degrees Fourth Top Predetermined Angle approximately 160 degrees Fifth Top Predetermined Angle approximately 170 degrees
[0129]
TABLE-US-00003 First Bottom Width approximately 1 mm Second Bottom Width approximately 0.886 mm Third Bottom Width approximately 0.798 mm Fourth Bottom Width approximately 0.718 mm Fifth Bottom Width approximately 0.646 mm Sixth Bottom Width approximately 0.656 mm First Bottom Predetermined Angle approximately 170 degrees Second Bottom Predetermined Angle approximately 160 degrees Third Bottom Predetermined Angle approximately 160 degrees Fourth Bottom Predetermined Angle approximately 160 degrees Fifth Bottom Predetermined Angle approximately 170 degrees
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[0174] All percentages and ratios are calculated by weight unless otherwise indicated.
[0175] All percentages and ratios are calculated based on the total composition unless otherwise indicated.
[0176] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[0177] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”
[0178] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
[0179] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.