Patent Application
20230272321
Provided herein is an insert chip and a cell culture system including the same, adapted for culturing a plurality of cell types under various conditions.
The development of in vitro models that recapitulate in-vivo features is essential for elucidating human physiology and disease mechanisms, as well as for drug discovery. As human physiology is highly complex, such in vitro models should ideally take many parameters into account, including the following: cellular microenvironment, cell-cell communication, organ-organ interactions, and mechanical aspects such as hydrodynamic and shear stress, which are critical for the development of cellular functionality. In recent years, several in vitro modeling platforms have been developed with the capacity to capture many of these features. These platforms include Transwell (TW) cell culture inserts, which enable cells to be co-cultured over a membrane microfluidic device (Organs-on-a-Chip), which allow for both co-culturing and the application of flow and other mechanical forces organoids, which mimic 3D tissue structure; and other 3D-systems that recreate a 3D microenvironment.
Several studies have tried to combine different in vitro modeling approaches to overcome the challenges outlined above. Sip et al. (Lab Chip, 2014, 14: 302-314), for example, developed a TW with flow, which uses soft-lithography to produce PDMS microchannels which are attached to 6-well TW holders. International Patent Application Publication No. WO 2018/020274 is directed to blood brain barrier model. International Patent Application Publication No. WO 2019/219605 is directed to method for the preparation of a cell culture insert with at least one membrane.
Though these platforms somewhat constitute recapitulating in vivo environments, each has shortcomings that hinder its universal application.
There is thus a need in the art for improved insert chips that are cost effective, versatile, reusable and reliable, that can accommodate various cell populations and simulate different growth conditions.
According to some embodiments, there is provided herein an advantageous insert chip (also termed herein “Insert-Chip”) and a system including same, for culturing a plurality of different cell types.
Advantageously, the insert chip and system disclosed herein provide a modular platform which is cost efficient, easy to use, applicable to various high-throughput experiments, capable of capturing cell-cell interactions, capable of inducing flow, and compatible with high-magnification imaging procedures.
According to some embodiments, the disclosed insert-chip is a modular, inexpensive, and user-friendly chip that exposes cultured cells to a controllable flow, and that can support cell-cell interactions and co-cultures. Advantageously, the Insert-Chip can be integrated into a variety of standard well plate cell culture platforms (and/or MEA platforms.
According to some embodiments the insert chip disclosed herein provides an innovative Organ-on-a-Chip platform that can be easily fabricated (for example, with 3D printing, as detailed herein) and be integrated or used with standard cell culture systems.
According to some embodiments, as exemplified herein, the Insert-Chip has the capacity to allow the growth of different types of cells (for example, endothelial cells, epithelial cells, neuronal cells, cancer cells, and the like) under different flow patterns and/or other forces (such as, shear force), and to provide straightforward access to various types of measurements that are of importance in physiological and drug development studies, including, for example, barrier permeability.
According to some embodiments, as exemplified herein, the cell culture system disclosed herein can allow simultaneous culturing of at least two, at least three spatially distinct cell populations (i.e., the cells are physically separated).
According to some embodiments, the advantageous modularity of the Insert-Chip, coupled with its capacity to enable multiple cell-types to be co-cultured and observed under various conditions (such as, flow and shear), can simplify experimental procedures that are currently highly complex in in-vitro studies in academic and industry settings. In particular, the Insert-Chip device can facilitate the study of cell-cell interactions, such as neurovascular coupling, essential to understanding the pathogenesis of multiple diseases.
There is provided, according to some embodiments, an insert chip for cell culture, the insert chip includes:
According to some embodiments, the insert chip may further include a support structure, such as, in the form of a support ring, configured to support the porous membrane within the hollow scaffold (i.e., between inner walls thereof).
According to some embodiments, the support ring may be made of silicone, such as, for example, polydimethylsiloxane (PDMS). In some embodiments, the support ring may be associated with the membrane prior to the membrane being placed/located in the hollow scaffold. In some embodiments, the support structure maybe placed on the membrane after the membrane has been placed/positioned/located in the hollow structure.
In some embodiments, in addition to or instead of the support structure, a reducer element (insert-reducer) may be placed on the membrane, to reduce the surface area thereof, as further detailed below herein. In some embodiments, the reduced element may be made of silicone, such as, PDMS. In some embodiments, the reducer element may have a linear shape, curved shape, or the like.
According to some embodiments, the insert chip may further include a plurality of legs (pillars) extending from the bottom part/portion of the hollow scaffold and configured to position the hollow scaffold on a flat surface such that the distance between the porous membrane and the flat surface is determined by the length of the plurality of pillars. In some embodiments, the length/height of the pillars is adjustable.
According to some embodiments, the hollow scaffold may be made of a transparent material, such as, for example, a transparent polymer.
According to some embodiments, the hollow scaffold may be made of a dental resin.
There is provided, according to some embodiments, a cell culture system which includes:
According to some embodiments, the cell culture system may further include at least one inlet conduit fluidly connecting the at least one inlet to the at least one first fluid receptacle; and at least one outlet conduit fluidly connecting the at least one outlet to the at least one withdrawn fluid receptacle.
According to some embodiments, the at least one cell culture container may be configured to contain/hold fluids at a fluid level such that the fluids contact the porous membrane, wherein the fluids include a fresh first fluid flowing therein through the at least one inlet and withdrawn therefrom through the at least one outlet at a flow rate adjusted to maintain the fluids level within the cell culture container.
According to some embodiments, the cell culture system may include a plurality of cell culture containers and a plurality of insert chips as disclosed herein, each insert chip is contained/placed/held within a cell culture container.
According to some embodiments, the plurality of cell culture containers may be selected from a multi-well plate and multi-electrode array (MEA) environment.
According to some embodiments, the first fluid is a tissue culture medium.
According to some embodiments, there is provided ab insert chip for cell culture, the insert chip includes:
According to some embodiments, the insert chip may further include a support ring, configured to support the porous membrane within the hollow scaffold.
According to some embodiments, the length of the one or more legs may be adjustable.
According to some embodiments, the insert may include a plurality of legs, wherein the length of the legs may essentially similar.
According to some embodiments, the membrane may be associated with an inner surface of the hollow scaffold.
According to some embodiments, the membrane is at least partially held between inner walls of the hollow scaffold.
According to some embodiments, the membrane may be removable.
According to some embodiments, the insert chip may further include an insert-reducer configured to reduce a surface area of the membrane on which the insert-reducer is positioned/placed on.
According to some embodiments, the insert chip may include two inlets and two outlets. According to some embodiments, each set of inlet and outlet is configured to allow passage of a separate fluid. According to some embodiments each set of inlet and outlet is configured to provide fluid flow to/over a distinct compartment of the insert chip.
According to some embodiments, the insert chip is configured to allow controlling or determining flow pattern, flow strength and/or shear forces applied on cells associated therewith.
According to some embodiments, least the hollow scaffold of the insert chip is essentially transparent.
According to some embodiments, at least the hollow scaffold is reusable.
According to some embodiments, the insert is configured to be placed or located in a culture well plate and/or multi-electrode array (MEA).
According to some embodiments, the cells may be selected from primary cells, culture cells, endothelial cells, epithelial cells, neuronal cells, cancer cells, or any combination thereof.
According to some embodiments, there is provided a cell culture system which includes:
According to some embodiments, the cell culture system may further include at least one inlet conduit fluidly connecting the at least one inlet to the at least one fluid receptacle; and at least one outlet conduit fluidly connecting the at least one outlet to the at least one withdrawn fluid receptacle.
According to some embodiments, the at least one cell culture container is configured to contain fluids at a fluid level such that the fluids contact the porous membrane, wherein the fluids include a fresh first fluid flowing therein through the at least one inlet and withdrawn therefrom through the at least one outlet at a flow rate adjusted to maintain the fluids level within the cell culture container.
According to some embodiments, the cell culture system may further include one or more pumps configured to allow fluid passage between the conduits.
According to some embodiments, the cell culture system may include a plurality of cell culture containers.
According to some embodiments, at least a portion of the plurality of cell culture containers hold/harbor an insert chip.
According to some embodiments, each of the plurality of cell culture containers comprises an insert chip.
According to some embodiments, wherein at least a portion of the insert chips in the plurality of cell culture containers are fluidly connected.
According to some embodiments, the cell culturing system is configured to allow essentially simultaneous culturing of at least two distinct cell populations.
According to some embodiments, the cell culturing system is configured to allow essentially simultaneous culturing of at least three spatially distinct cell populations.
According to some embodiments, the distinct cell populations are physically and/or spatially separated.
According to some embodiments, the cell populations may be selected from: primary cells, culture cells, endothelial cells, epithelial cells, neuronal cells, cancer cells, or any combination thereof
Other objects, features and advantages of the present invention will become clear from the following description, examples and drawings.
Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more other technical advantages may be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.
The patent or 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.
Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments may be practiced. The figures are for the purpose of illustrative description and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the disclosure. For the sake of clarity, some objects depicted in the figures are not to scale.
In the Figures:
The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art will be able to implement the teachings herein without undue effort or experimentation. In the figures, same reference numerals refer to same parts throughout. In the figures, same reference numerals refer to same parts throughout.
In the description and claims of the application, the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated.
According to some embodiments, organ-on-a-Chip platforms provide rich opportunities to observe interactions between different cell types under in vivo-like conditions, i.e., in the presence of flow. Yet, the costs and knowhow required for the fabrication and implementation of these platforms restrict their accessibility. The advantageous Insert-Chip disclosed herein is a microfluidic device that provides the functionality of an Organ-on-a-Chip platform, namely, the capacity to co-culture cells, expose the cells to flow (e.g. of culture media), and enables to observe cell-cell interactions, yet it can be easily integrated/be used with standard culture systems (e.g., well plates or multi-electrode arrays (MEA)). According to some embodiments, as detailed herein, the insert chip may be produced using stereolithograpy 3D printing and is user-friendly and reusable. Moreover, its design features overcome some of the measurement and imaging challenges characterizing standard Organ-on-a-Chip platforms. The advantageous Insert-Chip device and its capabilities are exemplified with exemplary cultures of endothelial and epithelial cells, which are co-cultured with neuronal cells, subjected to flow, and analyzed with various assays. Overall, the microfluidic device disclosed herein is a valuable platform for the investigation of biological functions, cell-cell interactions, response to therapeutics, and the like.
According to some embodiments, as further exemplified herein below, the advantageous insert chip can be used to perform experiments with various types of cells at various maturation stages, while allowing to simultaneously monitor various cellular functionalities even if characterized by different maturation times.
According to some embodiments, as further exemplified herein below, the advantageous insert chip can be used to form and/or study various cellular barriers. In some embodiments, utilizing the insert chip system, cells can create a barrier layer, and flow induction can enhance the barrier properties.
According to some embodiments, the insert-chip platform disclosed herein is close to “ideal” as it combines the strengths of two popular platforms, namely, Transwells (TWs) and the Organ-on-a-Chip, while overcoming some of their limitations.
TW inserts are commercially available in a range of size, easy to use, and can be used as a high-throughput tool. Yet, TWs are considered to be “static” models, as they do not have the capacity to induce flow, a crucial feature for models of vasculature and epithelial tissues. The Organ-on-a-Chip, in turn, enables flow to be induced, and can provide insight regarding organ-organ interactions; however, Organ-on-a-Chip systems are not modular, and their fabrication and implementation typically require a great deal of time and knowhow. Moreover, most chips are made of polydimethylsiloxane (PDMS), which adsorbs hydrophobic compounds, limiting the platform's applicability to drug testing. An additional shortcoming, shared by both TW and Organ-on-a-Chip systems, is the substantial difficulty in using high-resolution microscopy to investigate cell dynamics, owing to the large working distance needed for visualizing the cells.
According to some embodiments, to capture the benefits of TW inserts and Organs-on-a-Chip, while overcoming their individual and shared limitations, the advantageous insert-chip platform disclosed here utilizes new fabrication tools (3D printing) to develop an easy-to-use, customizable, microfluidic chip that, can be inserted into any standard culture platform, thereby transforming it into an advanced in vitro model platform.
Thus, according to some embodiments, there is provided an insert chip for cell culture, the insert chip includes: a hollow scaffold adapted to enclose therewithin a porous membrane, wherein the hollow scaffold comprises one or more inlets configured to deliver a fluid thereinto (or to portions/compartments thereof) and one or more outlets configured to withdraw fluids therefrom, and wherein each of said one or more inlets and one or more outlets is configured to be fluidly associated with corresponding fluid receptacle(s) and withdrawn fluid receptacle(s), respectively, through corresponding conduits; and a porous membrane having two sides, an upper side and a lower side, the porous membrane is configured to/capable of accommodating cell culture population on each side, wherein the porous membrane is positioned at a lower portion/part/region (i.e., closer to the bottom part/portion) of the hollow scaffold and is attached to an inner surface thereof. In some embodiments, the membrane is associated with a support structure (such as, a ring) that may be placed on at least a portion of the top surface of the membrane.
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According to some embodiments, the insert chip is a cylindrical Insert-Chip (as shown, for example, in
According to some embodiments, each Insert-Chip includes a cell culture chamber (hollow scaffold) having an external diameter customizable to up to 25 mm (for example, in the range of about 2-25 mm). and an inner diameter of 17 mm (for example, in the range of about 1-24 mm), with capacity of up to 2 mL (for example, in the range of about 0.1-4 ml) of fluids (such as, cellular medium or any other suitable buffer). According to some embodiments, the inlet and outlet channels on the upper portion of the chip and/or on lower portion of the chip (if present), enable the chamber to be connected to a controlled flow system (as illustrated, for example, in
According to some embodiments, as discussed herein, the bottom portion of the chip includes one or more (such as, 1-10, for example, 2, 3, 4, 5, 6, 7, 8 or more legs), modular and/or adjustable legs/pillars, which enable the device to be self-standing, while providing visual access to the membrane (e.g., for continuous microscopic visualization of cell growth) and while allowing adjusting the height or distance from the bottom portion of the culture well. According to some embodiments, the insert-chip includes a plurality of pillars/legs/short legs. According to some embodiments, the length of each pillar in the plurality of pillars is within the range of about 0.25 to 10 mm. According to some embodiments, each pillar is about 0.5-4 mm long. According to some embodiments, each pillar is about 0.75-3 mm long. According to some embodiments, each pillar is about 1-2 mm long. According to some embodiments, the plurality of pillars are of the same length. According to some embodiments, the insert chip is configured to stand on the plurality of legs, and thus can stand alone in either a well plate or multi-electrode array (MEA) environment, above a cell culture surface, thereby enabling the cells in that environment to interact with the cells in the chip. According to some embodiments, the height of the legs is adjustable. As used herein, the terms “pillar” and “leg” may interchangeably be used.
According to some embodiments, the membrane is at least partially porous. In some embodiments, the membrane is porous. In some embodiments, the porous membrane is configured to include/harbor/accommodate/hold cells cultured thereon. According to some embodiments, the porous membrane may be positioned near the base of the hollow scaffold, i.e., in close proximity to a lower portion thereof. According to some embodiments, the porous membrane is situated within the hollow scaffold with the support of supporting structure, such as, a PDMS ring. In some embodiments, the membrane can be versatile with respect of composition, size and/or shape. In some embodiments, the membrane may be interfaced to the Insert-Chip with a ring (having, for example an external diameter of about 3-25 mm (for example, about 16 mm) and inner diameter of about 2-24 mm (for example, about 13 mm). To ensure complete adherence between the membrane and the sealing ring, plasma and APTES may be used. According to some embodiments, such process facilitates long-term stability, which is important for reusing the Insert-Chip and for allowing diffusion between the two compartments.
According to some embodiments, the fluid may include any type of suitable fluid, including, for example, but not limited to: buffer, saline, growth medium, tissue culture medium, and the like, or any combination thereof. In some embodiments, different or same fluids may be used/applied for different cells/different cell populations. In some embodiments, specific characteristics of the fluids may be determined according to the performed experiment and/or type of cells. In some embodiments, such specific characteristics of the fluids may include, for example, but not limited to: composition thereof, components thereof, viscosity, ionic strength, inclusion of antibiotics, inclusion of serum, and the like, or any combination thereof.
According to some embodiments, the chip is re-usable, allows for advanced imaging and sensing, and can be used in high-throughput platforms, while providing the enabling to assess organ-organ interactions (see, for example,
According to some embodiments, the Insert-Chip has several key design aspects that overcome the current limitations of Organs-on-a-Chip, by leveraging the strengths of “static” TW inserts: The Insert-Chip is a stand-alone platform that can be integrated into almost any standard culturing platform (6, 12 or 24-well plate or MEA substrate) (
According to some embodiments, a key feature of Organs-on-a-Chip is the capacity to accommodate cell-cell interaction and diffusion between compartments. (
According to some embodiments, the insert chip facilitates spatial and/or physical separation of different/distinct/separate population of cells. According to some embodiments, the insert chip facilitates functional association/interaction between different/distinct/separate population of cells. According to some embodiments, the insert chip facilitates spatial and/or physical separation of different/distinct/separate population of cells, while optionally enabling functional association/interaction between the cells.
According to some embodiments, the Insert-Chip can enable different flow configurations (such as shown, for example, in
According to some embodiments, advantageously, the Insert-Chip can be fabricated by a standard 3D printer, using transparent materials such as a PC membrane and clear dental resin, which allow for real-time observations/visualization of cell morphology. Moreover, the advantageously, the membrane can be easily disassembled from the hollow scaffold, enabling cells to be imaged at high-resolution. Notably, this feature also enables the Insert-Chip to be reused (as demonstrated in
According to some embodiments, the insert-chip and systems including the same allows simulating or mimicking various physiological/cellular conditions, which require cell-cell interaction(s), and/or different physiological conditions, such as various follow patterns/conditions, shear forces, and the like. In some embodiments, such physiological/cellular conditions may include, for example, but not limited to: hypoxia (mimicking, for example, various stroke conditions, microbiome conditions, etc.), intra-cellular barriers, cell-cell interactions, tissue-tissue barrier (such as, blood-brain barrier), and the like, or combinations thereof. In some embodiments, as disclosed herein, the use/creation of at least two compartments within the insert chip, allows the study of cellular systems, while not being exposed to external environment (for example, under hypoxia conditions, under different gasses, fluids, and the like).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs. As used herein, the indefinite articles “a” and “an” mean “at least one” or “one or more” unless the context clearly dictates otherwise.
It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the disclosure. No feature described in the context of an embodiment is to be considered an essential feature of that embodiment, unless explicitly specified as such.
Although steps of methods according to some embodiments may be described in a specific sequence, methods of the disclosure may include some or all of the described steps carried out in a different order. The methods of the disclosure may include a few of the steps described or all of the steps described. No particular step in a disclosed method is to be considered an essential step of that method, unless explicitly specified as such.
The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting. Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the disclosure. Section headings are used herein to ease understanding of the specification and should not be construed as necessarily limiting.
As used herein, the term “about” may be used to specify a value of a quantity or parameter (e.g. the length of an element) to within a continuous range of values in the neighborhood of (and including) a given (stated) value. According to some embodiments, “about” may specify the value of a parameter to be between 99% and 101% of the given value. In such embodiments, for example, the statement “the length of the element is equal to about 1 millimeter” is equivalent to the statement “the length of the element is between 0.99 millimeters and 1.01 millimeters”.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
As exemplified herein, a 3D-printed Insert-Chips in different sizes were produced in order to demonstrate their modularity and adaptability to standard cell culture platforms commonly used in a lab. In addition, barrier tissue cells (either endothelial or epithelial cells) were cultured on top of the Insert-Chip membrane and were used to demonstrate the capacity to induce controlled flow in the Insert-Chip and to image cells with high-resolution confocal microscopy. In additional experiments, it has been demonstrated how the chip can be integrated into conventional culturing platforms, while providing the capacity to co-culture cell populations in the presence of flow. To this end, an Insert-Chip cultured with endothelial cells was inserted into an MEA containing parenchymal cells (neurons and astrocytes). Endothelial and neuronal cell functionality was demonstrated via simultaneous barrier and electrophysiological measurements. Finally, experiments with modified versions of the Insert-Chip can further improve the chip's efficiency or suitability for specific types of experiments. The results exhibit the potential and capabilities of the Insert-Chip as a straightforward yet advanced in vitro modeling platform that can benefit both academic and pharmaceutical labs.
The Insert-Chip was designed using SolidWorks CAD software (SolidWorks Corporation, MA, USA). A schematic representation of the Insert-Chip fabrication is shown in
Fabrication and assembly of additional components: SolidWorks CAD software was used to design master-molds for fabrication of the device's additional components: the support structure (PDMS support ring), and two different “reducer” components (insert-reducer) aimed at reducing the active surface area in the chip and controlling the flow. The molds were printed with a commercial polylactic acid filament using a Raise 3D Pro2 Dual Extruder 3D Printer (Raise Technologies Inc., US). Prior to printing, model surfaces were checked, and, if needed, a scaffold was added using Idea Maker software (3.6.1, Raise Technologies Inc, US). Then, the molds were filled with PDMS prepared by mixing Sylgard 184® (Dow Corning, Midland, Mich., USA) with the curing agent at a ratio of 1:10, followed by curing at 60° C. overnight. The resulting PDMS rings and reducers were cleaned in ethanol, dried at room temperature (RT), and then activated in oxygen plasma (Atto-BR-200-PCCE, Diener Electronic, Germany) for 30 s.
Polycarbonate (PC) membranes (0.4 μm pore size, it4ip S.A., Belgium), 25 μm thick, were cut to size with their protective backing on. The protective backings were then removed, and the PC membranes were rinsed with isopropanol, dried under a stream of compressed air, and activated in oxygen plasma for 2 minutes (Diener Electronic, Germany). Then, the membranes were immersed for 30 minutes in 5% aqueous solution of 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) in order to introduce amino groups at the surface of the PC membrane. Then they were washed 3 times with water and dried under a stream of compressed air.
PDMS-rings or PDMS-reducers and PC membranes were then aligned and brought into contact, gently pressed together to ensure conformational contact, and baked at 60° C. overnight. The assembled parts were then inserted into the 3D-printed microfluidic Insert-Chip.
The ready-to-use assembled chip was sterilized using 70% ethanol for 30 min, and was then washed with phosphate-buffered saline (PBS, Biological Industries) 3 times and sterilized under a UV lamp for 20 minutes.
Validation of the flow gradient inside the chip: Flow was controlled by an external peristaltic pump (IP-N 8, Ismatec, Cole-Parmer GmbH, Wertheim, Germany), and connections were in elastic tubing (inner diameter 1 mm, outer diameter 3 mm, Ismatec, Germany). The input tube was connected to the inlet of the chip, and the output tube was connected to a reservoir via the peristaltic pump.
To test the biocompatibility and the versatility of the Insert-Chip, epithelial and endothelial monolayers were cultured separately in Insert-Chips and the cells were monitored under static and flow conditions. Furthermore, in order to demonstrate the significance of the Insert-Chip, cells were also cultured on commercially available Transwells (Corning, USA). Moreover, to demonstrate how the Insert-Chip can be integrated into a more conventional cell culture environment, neuronal cells were cultured in MEAs, in which the Insert-Chip was subsequently placed.
Epithelial culture. For the epithelial model, human epithelial colorectal adenocarcinoma cells (Caco-2 cells, ATCC® HBT-37™, American Type Culture Collection, Rockville, Md., USA) were used. The passages of the Caco-2 cell line ranged from 26th to 40th. After thawing, the Caco-2 cells were cultured routinely in Dulbecco's Modified Eagle Medium (DMEM, Biological Industries), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, Biological Industries), 1% Glutamax (Gibco) and 1% Penicillin-Streptomycin-Amphotericin B (PSA, Biological Industries) solution, at 37° C. with 5% CO2 in a humidifying incubator. Cells were grown to 80-90% confluence before being transferred inside the Insert-Chip. Before seeding, the porous membrane inside the Insert-Chip was treated with Matrigel Basement Membrane Matrix (Corning) used at 1:50 ratio with the culture medium, for 30 min in the incubator. The membrane was then rinsed with culture medium and the Caco-2 cells, harvested with trypsin/EDTA solution (Biological Industries), were seeded at a density of 100.000 cells/cm2 and grown for 9-11 days, changing the medium every 4 days of cell culture.
For the flow condition, the tubing was sterilized by perfusing 70% ethanol throughout the entire system at a flow rate of 5 μL/min for 2 hours. Following that, PBS was flushed into the entire system for an additional 2 hours at the same flow rate. Next, the solution containing Matrigel was flowed inside the Insert-Chip to coat the porous membrane, and the device was then incubated for 30 min. After incubation, the device was perfused with cell culture medium, and then the Caco-2 cells were seeded into the Insert-Chip. Next, the entire system was placed in the incubator, and the peristaltic pump was activated to perfuse culture medium at a constant flow rate of 5 μL/min, for 2 days, to ensure the establishment of an intact monolayer of Caco-2 cells.
Endothelial culture. For the endothelial model, Human Umbilical Vein Endothelial cells (HUVEC, PromoCell GmbH, Heidelberg, Germany) were used. After thawing, the HUVEC were expanded in low-serum endothelial cell growth medium (PromoCell), at 37° C. with 5% CO2 in a humidifying incubator, and used at passage p3-p5. Cells were grown to 80-90% confluence before being transferred inside the device. Before seeding, the PC membrane was treated with Entactin-Collagen IV-Laminin (ECL) Cell Attachment Matrix (Merck) diluted in DMEM (10 μg/cm2), for 1h in the incubator. Then, the HUVEC, harvested using a DetachKit (Promocell), were seeded inside the Insert-Chip at a density of 250.000 cells/cm2 and grown for 3-5 days. In the flow condition, the tubing was cleaned and sterilized as described above. Next, the solution containing ECL Matrix was flowed inside the chip and incubated for 1 h, and then cells were seeded. Then, the entire system was placed in the incubator, and the peristaltic pump was activated to perfuse culture medium at a constant flow rate of 5 μL/min, overnight, to ensure the establishment of an intact monolayer of HUVEC.
Cancer cells line. To develop a tri-culture system, cancer cell lines (U87 glioblastoma and SH-SY5Y neuroblastoma cell lines, ATCC®) were used. After thawing, the U87 cells were cultured similarly to the epithelial cells and after reaching 80% confluency, they were seeded on the membrane. The SH-SY5Y cells were cultured in RPMI-F12 Medium (Biological Industries), supplemented with 10% FBS, 7.5% Sodium bicarbonate (Sigma-Aldrich), 1% Glutamax and 1% Gentamycin (Gibco) solution, at 37° C. with 5% CO2 in a humidifying incubator. Cells were grown to 80-90% confluence before being transferred inside the multi-well plate (Corning, USA), after being harvested with trypsin/EDTA solution (Biological Industries).
Neuronal culture. Primary dissociated cultures were obtained from postnatal rats (p2-p3) as previously described. All experiments were approved by the local authority and performed in accordance with Israeli law. All efforts were made to minimize animal suffering and to reduce the number of animals used. Neuronal hippocampal cells were plated on MEAs (Multi Channel Systems, Reutlingen, Germany) for network investigation. Prior to cell seeding, the MEA substrates were treated with polyethyleneimine (PEI, Sigma-Aldrich) in Borate buffer (Sigma-Aldrich) overnight at 4° C. Then, the substrates were rinsed 4 times with distilled water, sterilized with UV for 1 h and treated with laminin (20 μg/mL, Sigma-Aldrich) diluted in plating medium containing Neurobasal Medium (Gibco), supplemented with FBS (5%, Biological Industries), B27 (2%, Gibco), Glutamax (1%, Gibco) and PSA (1%, Biological Industries), for 4 h, at 37° C.
Neuronal hippocampal cells were then plated on coated MEA substrates in a plating medium and incubated at 37° C. in a humidified atmosphere enriched with 5% CO2. After 24 h had passed since seeding, the medium was replaced (80%) with serum-free neurobasal medium, supplemented with B27 (2%), Glutamax (1%), PSA (1%) and Gentamycin (1%, Gibco). Culture medium was renewed (50%) every 3 days from seeding. Plating was carried out at a nominal density of 70,000 cells/cm2. Cultures were then used for experiments after 9-12 days in vitro (DIV).
Computational Fluid Dynamics (CFD) Model. CFD simulations were conducted to characterize the flow in the chip and to determine the influence of the chip LH (leg height) on the diffusion of mass. The fluid volume was derived from the chip geometries corresponding to the reduced and non-reduced configurations for the flow simulations, while a container was added in which the chip is submerged for the diffusion simulations. The geometries were meshed in ANSYS GAMBIT 19 R3 with the final elements number shown in Table 1. All the simulations were conducted in ANSYS fluent 19 R3 using the constant laminar flow assumption for the flow simulations at two flow rates: 5 μL/min and 50 μL/min. The diffusion was modeled through the convection diffusion equation assuming constant diffusivity and mass production rate (see solved equations below). Since there are many configurations possible in the chip, a simple configuration was elected where the cells are located at the bottom of the reduced container producing CO2 at an arbitrary constant rate (0.0054 mmol/m2/sec) while there are no cells anywhere else and there is no membrane. The CO2 diffusivity was taken to be 2.3e−9 m2/sec and only one flow rate of 5 μL/min was used. Finally, both steady state simulations to derive the final concentration gradients in the chips as well as transient simulations for 360-time steps of 1 second (6 minutes total) were performed to estimate the time scales involved and to produce movies of the diffusion process.
1M
Solved Equations:
Momentum
∂/∂t(ρv{right arrow over ( )})+∇(ρv{right arrow over ( )}v{right arrow over ( )})=−∇p+∇·(t=)+ρg{right arrow over ( )}+F{right arrow over ( )} (eq. 1)
τ==μ[(∇v{right arrow over ( )}+v{right arrow over ( )}{circumflex over ( )}T)−⅔∇·v{right arrow over ( )}I] (eq. 2)
dρ/dt+∇·(ρv{right arrow over ( )})=0 (eq. 3)
τ_w=μ∂u/∂n (eq. 4)
∂c/∂t=∇·(D∇c)−∇·(uc)+R (eq. 5)
Fixation, immunocytochemistry, and confocal imaging—HUVEC, Caco-2 and the cancer cells lines were rinsed in PBS and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) for 20 minutes at RT. Immunocytochemistry was carried out after permeabilization with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min at RT and blocking for 30 min in FBS (5%) in PBS. Primary antibodies were applied overnight in PBS at 4° C. The following primary antibodies were used for immunocytochemistry experiments: rabbit anti-ZO-1 (Abcam) and rabbit anti-CD-31 (Abcam), to stain the zona occludens-1 (a key component of tight junctions) in Caco-2 cells and the endothelial cell adhesion molecule 1 in HUVEC, respectively; mouse anti-GFAP (Abcam), to stain the Glial Fibrillary Protein in U87 cells; Phalloidin-iFluor 488 (Abcam), to stain actin in SY-SY5Y cells. Cells were then washed three times in PBS and stained with the secondary antibody for 1 h at RT. The secondary antibodies were anti-rabbit Alexa Fluor-488 (Invitrogen) and anti-mouse Alexa Fluor-594 (Invitrogen). After being washed four times with PBS, cells were mounted on a 0.17-mm-thick glass coverslip using DAPI-Fluoromount-G® (SouthernBiotech), to stain the nuclei. Imaging was carried out using an inverted confocal microscope (Olympus FV3000-IX83), with appropriate filter cubes and equipped with 2×/0.08 NA, 10×/0.3 NA, 20×/0.8 and 60×/1.42 NA objectives. For imaging the entire channel within the PDMS-reducer, images were acquired by sequential tile scanning. Image reconstruction and processing were done using open-source ImageJ software.
Trans-epithelial endothelial electrical resistance (TEER). The barrier properties of the epithelial/endothelial monolayer were evaluated with TEER measurements along the cellular growth period. TEER was measured with the Millicell ERS-2 Voltohmmeter (Merck Millipore). TEER values (MΩ cm2) were calculated and compared to those obtained in an Insert-Chip not containing cells, considered as blank, and were obtained from 4 different individual experiments, with 2 Insert-Chips used in each experiment.
Permeability Assay. HUVECs and Caco-2 were cultured on the Insert-Chip in static and under-flow condition. Permeability of the monolayer was assessed by measuring leakage of Fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich) administered to the upper compartment of the Insert-Chip at different time points. One hour after adding dextran, the fluorescence intensity of the medium in the lower compartment was measured by a fluorescent plate reader (Multiskan Go, Thermo Scientific), at an excitation of 492 nm and emission of 518 nm (2 Insert-Chip for each condition)
MEA recording. Neuronal network extracellular recordings were carried out using the MEA60 system (Multi Channel Systems). Primary hippocampal cultures were plated on Titanium Nitride (TiN) MEAs with 60 electrodes (30 μm dimeter, 200 μm inter-electrode spacing). Raw data were monitored and recorded by using the commercial software MCRack (Multi Channel Systems), at 37° C., in the presence of cell culture medium. The recorded events were analyzed offline with NeuroExplorer 5.127 software (Nex Technologies, Colorado, USA).
Statistical analysis. The results are presented as the mean±SD. Statistically significant differences among multiple groups were evaluated by two-way ANOVA, followed by the Holm-Sidak test for multiple comparison (GraphPad Prism 8.4.3). A statistically significant difference between two data sets was assessed and P<0.05 was considered statistically significant.
Most Organs-on-a-Chip or microfluidic devices are fabricated from PDMS, which is biocompatible, transparent, and has good gas permeability. However, a major limitation of PDMS is its hydrophobicity, which causes substantial absorption of hydrophilic materials. Moreover, in some cases, chip fabrication requires specific knowhow and facilities. To overcome these challenges, stereolithograpy 3D printing was used for fabricating the Insert-Chip. The use of 3D-printing enables the design of the desired platform to be quickly modified, and it reduces the need for multi-step fabrication needed in “standard” Organs-on-a-Chip. Furthermore, the use of 3D-printing reduces the fabrication time of the Organ-on-a-Chip from several days to a few hours, as well as the possibility to use not-absorbing materials. The Insert-Chip is made only from 3 parts (
An important feature of the Insert-Chip is the fact that “one-design fits all”, i.e., the chip is modular and can be integrated with existing platforms. One of the strengths of the “standard” dual-channel Organ-on-a-Chip platform is that it provides the capacity to observe cell-cell interactions. With the Insert-Chip, cell-cell interactions can take place between the cells plated on the insert chip membrane and the cells cultured in the well into which the device is inserted. The characteristics of these interactions are mainly determined by the flow rate, pore size of the membrane, and the distance between the two cell populations (the distance between the membrane and the bottom of the plate). As the Insert-Chip is fabricated via 3D printing, all these parameters can be adjusted in accordance with experimental requirements. For example,
Moreover, when multiple Insert-Chips are placed next to each other (
This feature can contribute substantially to the study of human physiology and pharmacokinetics and pharmacodynamics, for which organ-organ interactions are crucial, yet highly challenging to mimic in vitro.
The aim of the study was to demonstrate the use of the Insert-Chip as a modular “Epithelium-on-a-Chip” (Caco-2 cells) or “Endothelium-on-a-Chip” (HUVEC) (
Cell growth and barrier development was monitored over 4 and 9 days (from 1 to 4 or from 1 to 9 DIV), until the Caco-2 cells and HUVEC formed complete confluent monolayers (
Both methods provide complementary information on the barrier properties, as TEER provides a quick, non-invasive and real-time indication of barrier properties; while fluorescent assays can provide information on how the permeability changes with the molecular weight; it is important to note that the design of the Insert-Chip allows for the use of commercial TEER systems. TEER measurements were used to compare our Insert-Chip system to the ones measured on commercially available Transwells.
No significant differences were found in Caco-2 cells cultured under flow (from 151.2±6.2 Ωcm2 to 600.0±70.7 Ωcm2) compared to the ones grown without flow (from 171.2±6.2 Ωcm2 to 590.0±11.5 Ωcm2) or on Transwells (from 202.5±5.01 cm2 to 600.5±40.0 Ωcm2,
To validate the culturing system/platform, permeability measurements were done without cells and cells that were cultured with and without flow. This was performed by quantifying the rate at which water soluble fluorescein isothiocyanate (FITC)-dextran was transported across the endothelium and epithelium to the bottom compartment of the Insert-Chip upon addition at the upper one (
High-resolution imaging is an indispensable tool for studying the structure and the dynamics of cells. Unfortunately, it is highly challenging to do high-magnification imaging with “standard” dual-channel Organs-on-a-Chip, as the typical working distance of 40×, 60× objectives is 170-200 μm, and the distance of the membrane where cells are cultured from the bottom of the Chip is usually above 300 μm. To overcome this challenge, the Insert-Chip was designed such that the membrane can be easily removed from the chip (
Though the basic design of the Insert-Chip allows for the application of flow, and the use of relatively small quantities of cells, it was sought to enable the number of cells used to be further reduced, as well as to provide more precise control over the shear forces applied to the cells. To do so, a “reducer” (insert-reducer) made of PDMS that easily can be placed in the chip (
Nevertheless, the shear increases with the flow and can be brought to higher levels in both the reduced (
In these experiments, two reducers were used, one with a linear shape (
To demonstrate the use of the reducer, Caco-2 cells were cultured in the Insert-Chip with the linear shape reducer (
As most of the parenchyma is surrounded by barrier layer, there is a need for creating such co-culture systems which allow to culture barrier layer and parenchymal, while assessing their functionality. an Organ-on-a-Chip with multiple sensors, in which it is possible to simultaneously measure both barrier function via TEER and the electrical activity of excitable cells, using MEAs was demonstrated by Maoz et al., Lab Chip, 17: 2944, 2017. However, this platform requires custom fabrication and is therefore less accessible than commercial platforms. The Insert-Chip was designed to overcome this challenge; that is, it can be integrated into a commercial MEA platform (
It is to note that such experiments are challenging to carry out with “standard” Organs-on-a-Chip, not only because of the technological aspect but also because of the biological aspect, which requires that both cell populations be at the same stages of maturation and functionality, which might be hard to coordinate. For example, it takes 1-3 days for the HUVEC to create a fully functional barrier; however, it takes at least 10 days to achieve robust neuronal activity. Use of the Insert-Chip enables the experimenter to culture each of the cell populations separately, and to combine them-by inserting the Insert-Chip into the MEA plate-only when both populations are mature.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs. As used herein, the indefinite articles “a” and “an” mean “at least one” or “one or more” unless the context clearly dictates otherwise.
It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 278594 | Nov 2020 | IL | national |
This application is a Continuation of PCT Patent Application No. PCT/IL2021/051317 having International filing date of Nov. 8, 2021, which claims the benefit of priority of Israeli Patent Application No. 278594 filed on Nov. 9, 2020. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL2021/051317 | Nov 2021 | US |
| Child | 18144937 | US |
