Systems and methods for co-cultivating in microplates - Patents.com

Abstract

The culture plate includes an upper surface and a plurality of well systems, the well system including a first well, a second well, a first channel in fluid communication with the first and second wells, and a third well between the first and second wells, the third well in fluid communication with the first channel, and the upper surface of the culture plate defines a well opening for each of the first, second, and third wells. A method of isolating cells in a culture plate includes filling the interior of the lower chamber with a liquid hydrogel, solidifying the hydrogel, filling the first well and / or the second well with cells, spheroids, and / or organoids, introducing the cells, spheroids, and / or organoids into the first channel to interact with the hydrogel, and adding a feeding medium to the first, second, and / or third wells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed as a PCT international patent application on March 14, 2023, and claims priority to and the benefit of U.S. Provisional Application No. 63 / 322,058, filed March 21, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Co-culture allows the generation of artificial microbial communities to demonstrate competition between microorganisms in the same environment. Co-culture also allows various cell types to be cultured together to investigate the effects of one culture system on another, which is useful when investigating the effects of one type of tissue on another, the effects of one region of the brain on another, or how specific secreted molecules lead to changes in neuronal development or physiology. For example, co-culture of different regions of spinal cord explants may reveal differential effects on the ability to attract or inhibit neurite outgrowth, or biochemical purification from explants in co-culture experiments may lead to the identification of specific molecules that, when introduced into cell lines, can express and secrete molecular guidance cues. [Brief description of the drawings]

[0003] [Figure 1A] 1A-1C are diagrams of microplate mechanisms according to various examples of the present disclosure. [Figure 1B] 1A-1C are diagrams of microplate mechanisms according to various examples of the present disclosure. [Figure 1C] 1A-1C are diagrams of microplate mechanisms according to various examples of the present disclosure.

[0004] [Figure 2A] 2A-2B are diagrams of microplate arrays according to various examples of the present disclosure. [Figure 2B]2A-2B are diagrams of microplate arrays according to various examples of the present disclosure.

[0005] [Figure 3A] 3A-3C are diagrams of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 3B] 3A-3C are diagrams of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 3C] 3A-3C are diagrams of various operations of a microplate mechanism according to various examples of the present disclosure.

[0006] [Figure 4A] 4A-4D are diagrams of microplate mechanisms according to various examples of the present disclosure. [Figure 4B] 4A-4D are diagrams of microplate mechanisms according to various examples of the present disclosure. [Figure 4C] 4A-4D are diagrams of microplate mechanisms according to various examples of the present disclosure. [Figure 4D] 4A-4D are diagrams of microplate mechanisms according to various examples of the present disclosure.

[0007] [Figure 5A] 5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 5B] 5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 5C] 5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 5D] 5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 5E] 5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 5F]5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure. [Figure 5G] 5A-5G are examples of various operations of a microplate mechanism according to various examples of the present disclosure.

[0008] [Figure 6] FIG. 6 is a flow chart illustrating a method for isolating cells according to an example of the present disclosure.

[0009] [Figure 7] FIG. 7 illustrates a block diagram of a computing device according to an example of the present disclosure. Summary of the Invention [Means for solving the problem]

[0010] Abstract In one aspect, the present technology relates to a culture plate, the culture plate includes an upper surface and a plurality of well systems, each well system includes a first well, a second well, a first channel in fluid communication with the first well and the second well, and a third well disposed between the first well and the second well, the third well in fluid communication with the first channel, and the upper surface of the culture plate defines a well opening for each of the first well, the second well, and the third well. In one example, the first channel is in fluid communication with the first well on the side of the first well and in fluid communication with the second well on the side of the second well. In another example, the third well includes an upper chamber and a lower chamber with a diameter smaller than the diameter of the upper chamber, and the first channel is in fluid communication with the lower chamber. In a further example, the first well and the second well are in fluid communication with the first channel via the first opening and the second opening, respectively, the first channel is a microchannel having an inner diameter of about 5 μm to 100 μm, the inner diameter of the first channel comprises a collagen coating, a contour of the inner diameter of the first channel is one of a circle, an ellipse, and a polygon, the first channel extends horizontally between the first well and the second well, and the first channel extends non-linearly between the first well and the second well.

[0011] In one example of the above embodiment, the culture plate further comprises a fourth well, a fifth well, and a second channel in fluid communication with the third well, the fourth well, and the fifth well. In a further example, the culture plate comprises one of a 96-well plate and a 348-well plate.

[0012] In another aspect, the present technology relates to a method for separating cells from a medium flow in a culture plate comprising a plurality of well plates, each well plate comprising at least a first well, a second well, a third well, and a first channel fluidically communicating with the first well, the second well, and the third well, the third well comprising an upper chamber and a lower chamber, the method comprising: filling the inside of the lower chamber with liquid hydrogel; solidifying the hydrogel by incubation; filling at least one of cells, spheroids, and organoids into at least one of the first well and the second well; introducing at least one of cells, spheroids, and organoids from at least one of the first well and the second well into the first channel to interact with the hydrogel; and adding a supply medium into at least one of the first well, the second well, and the third well.In one example, the interaction comprises at least one of cell attachment and organoid formation. In a further example, introducing at least one of cells, spheroids, and organoids into the first channel comprises sealing the opening of at least one of the first well and the second well with a pipette.In another example, the method further comprises accessing at least one of the first well, the second well, and the third well through its opening to manipulate the contents, the method further comprises removing air from the first channel before adding the supply medium, and filling the inside of the lower chamber with hydrogel comprises transferring the hydrogel from the upper chamber into the lower chamber.

[0013] In one example of the above embodiment, the culture plate further comprises a second channel that is in fluid communication with the third well, the fourth well and the fifth well, and the method further comprises: filling at least one of cells, spheroids and organoids into at least one of the fourth well and the fifth well; and adding a supply medium into at least one of the fourth well and the fifth well.In another example, the method further comprises: adding epithelial cells into the upper chamber; and filling at least one of the first well and the second well comprises filling at least one of organoids and tumor organoids into at least one of the first well and the second well.In a further example, the method further comprises: adding a differentiation medium into the upper chamber; adding progenitor cells into at least one of the first well and the second well; and imaging the healing of neural cells through the opening of the upper chamber. In yet another example, filling the liquid hydrogel includes filling the liquid hydrogel with a cell-repellent hydrogel, and the method further includes, prior to incubation, maintaining the culture plate at a desired temperature to allow the hydrogel to flow into the first channel, and filling the first channel with the hydrogel mixed with at least one of cells, spheroids, and organoids.

[0014] In another example of the above embodiment, filling the liquid hydrogel includes filling with a cell-repellent hydrogel, and the method further includes, prior to incubation, maintaining the culture plate at a desired temperature to allow the hydrogel to flow into the first channel, and filling the first channel with a medium mixed with cells capable of forming a tubular vessel. In a further example, filling the liquid hydrogel includes filling with a liquid hydrogel with a cell-repellent hydrogel, and the method further includes, prior to incubation, maintaining the culture plate at a desired temperature to allow the hydrogel to flow into the first channel, and filling the first channel with a medium mixed with cells capable of forming a pulmonary tissue or epithelium. In yet another example, the method includes maintaining the incubation until the cells settle to the bottom of the upper chamber and the pulmonary epithelium or tissue is formed.

[0015] In another example of the above embodiment, filling liquid hydrogel and filling at least one of the first well and the second well comprises filling different combinations of cells, spheroids and organoids into each of the first well, the second well and the third well, and then the first well, the second well and the third well are fluidically connected by removing the air present inside the first channel.In another example, removing air comprises one of using vacuum to suck air from the first channel and sealingly engaging pipette tip. In a further example, the filling of the liquid hydrogel, the filling of the first well and / or the second well, and the filling of the fourth well and / or the fifth well include different combinations of cells, spheroids, and organoids in each of the first well, the second well, the third well, the fourth well, and the fifth well, and the first well, the second well, and the third well are fluidically connected by removing air present inside the first channel, and the third well, the fourth well, and the fifth well are fluidically connected by removing air present inside the second channel.In a further example, the filling of the liquid hydrogel includes filling the liquid hydrogel premixed with at least one of the cells, spheroids, and organoids through the filling port into the first channel by sealingly engaging a pipette containing the premixed hydrogel with the filling port of the third well. As another example, filling the liquid hydrogel includes filling the liquid hydrogel pre-mixed with at least one of the cells, spheroids, and organoids into the first channel and the second channel through the fill port by sealingly engaging a pipette containing the pre-mixed hydrogel with the fill port of the third well.In a further example, filling the liquid hydrogel includes filling the first channel with a liquid hydrogel pre-mixed with at least one of the cells, spheroids, and organoids through the filling port by sealingly engaging a pipette containing the pre-mixed hydrogel with the filling port of the third well, and filling the second channel with a supply medium.

[0016] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will become more fully understood from the following description and drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description The current state of product development, and scientific advances in general, such as life sciences, suffer from problems with existing systems and methods that slow down product and / or scientific development cycles. For example, it is usually difficult to separate cells and cell aggregates from a media stream by a hydrogel barrier and at the same time maintain direct access to mechanically manipulate the separated cells or aggregates. It is usually difficult to simultaneously monitor and mechanically manipulate nerve cells or muscle tissue bundles or other growing structures. In addition, multi-organ systems are difficult to generate in microplate formats, for example, tubed microfluidic chips are often used.

[0018] Thus, a technical problem exists, which is the ability to separate cells and cell aggregates from the media flow with a hydrogel barrier, while simultaneously maintaining direct access to the cells and / or cell aggregates, for example to mechanically manipulate the separated cells or aggregates for experimental purposes. One solution to this technical problem may involve connecting two wells to one channel and introducing an opening in the middle of the channel to allow access to manipulate the cells in the channel. For example, the opening may have structural elements that allow the formation of a hydrogel barrier that separates the medium of the feeding well from the medium inside the channel. Thus, having an opening of the central channel accessible from the top allows the manipulation of the cells in this well.

[0019] In various examples of the present disclosure, seeding cells in the central well and monitoring the growth of cells into the channel connecting the two wells or from one side channel toward the central channel may allow, for example, cutting or manipulating cells or introducing a measurement probe into the central channel. Adding multiple channels may allow more cells to be seeded and monitored, and may also increase the complexity of the system being monitored. For example, to create a multi-organ system, various types of cells, spheroids, or organoids may be seeded into one of three different wells: a feeding well, a culture well, and a central well connected to the central channel. By creating one of multiple barriers, complex organ combinations may be replicated and studied, such as the kidney, blood-brain barrier, or liver. This may be useful, for example, for studies such as toxicology studies and brain studies. Having access to the central well may also allow the creation of, for example, a lung system, by leaving one side exposed to ambient air and the other side exposed to a medium. Thus, given the versatility of microplates, they may be used in many different applications other than those mentioned above.

[0020] Terms such as first, second, etc. may be used herein to describe various elements, but it will be understood that such elements should not be limited by these terms. Such terms are used only to distinguish one element from another. Thus, for example, a first element, first component, or first section described below may be referred to as a second element, second component, or second section without departing from the teachings of the present disclosure. Similarly, various spatial terms such as "top", "bottom", "side", etc. may be used to relatively distinguish one element from another element. However, it should be understood that components may be oriented differently without departing from the teachings of the present disclosure, for example, a lifting device may be turned on its side so that its "top" surface faces horizontally and its "side" surface faces vertically.

[0021] 1A-1C are diagrams of microplate mechanisms according to various examples of the present disclosure. In FIG. 1A and FIG. 1B, the microplate mechanism 100 includes a culture well 120 and a feeding well 110. For example, the feeding well 110 is a well that may hold a cell or cell aggregate, and may be referred to herein as a first well. The culture well 120 (also referred to herein as a second well) and the feeding well 110 may be connected via a channel 130, e.g., a microchannel. In one example, the channel 130 is a microchannel having an internal diameter of about 5 μm to 100 μm. In another example, the internal diameter of the channel 130 has a coating of one or more matrix proteins, e.g., structural proteins such as collagen. In yet another example, the profile of the inner diameter of the channel 130 may have various shapes, e.g., circular, elliptical, or polygonal. In a further example, the channel 130 extends horizontally between the feeding well 110 and the culture well 120. The channel 130 may extend non-linearly between the supply well 110 and the culture well 120 by having a curvature configured to aid in the formation of, for example, intestinal organoids or other types of organoids or spheroids therein. In various examples, the channel 130 may be connected to the hydrogel well 150 via the hydrogel chamber 140. For example, the hydrogel well 150 (also referred to herein as the third well) may constitute an upper chamber of the third well, and the hydrogel chamber 140 may constitute a lower chamber of the third well. In another example, the hydrogel well 150 and the hydrogel chamber 140 may not be connected to the culture well 120 or the supply well 110 except via the channel 130. In one example, the hydrogel chamber 140 includes a liquid phase guide 145. For example, the liquid phase guide 145 may be a ridge formed in the hydrogel chamber 140 to receive the channel 130.

[0022] FIG. 1C is a side view of the microplate mechanism shown in FIGS. 1A-1B according to various examples of the present disclosure. In FIG. 1C, the microplate mechanism 100 includes a feed well 110 fluidly connected to a channel 130. In various examples of the present disclosure, the connecting channel 130 is fluidly connected to the feed well 110 through an opening 160 formed in the bottom of the feed well 110. For example, the opening 160 may have a shape and size that allows a typical pipette to substantially cover the opening 160 and thereby seal the opening. In another example, the feed well 110 may be a shape and size that allows a pipette to contact its top edge to form a seal. In various examples, the hydrogel well 150 may include additional media for the hydrogel chamber 140. In other examples, the microplate mechanism 100 includes an interface 170 between the channel 130 and the hydrogel chamber 140. At the interface 170, the hydrogel present in the hydrogel chamber 140 may not be able to enter the channel 130, for example, due to the shape and / or contour of the internal cavity of the channel 130. However, the medium present in the hydrogel chamber 140 may interact with the cells and / or aggregates present in the channel 130. In another example, each of the feeding well 110, the culture well 120, and the hydrogel well 150 or the hydrogel chamber 140 may contain a different combination of cells, spheroids, and organoids.

[0023] 2A-2B are diagrams of microplate arrays 200 and 250 according to various examples of the present disclosure. In FIG. 2A, the microplate array 200 includes 48 microplate features, each including a culture well 220 and a feeding well 210 fluidly connected to a channel 230, each similar to the microplate feature 100 described above with respect to FIGS. 1A-1C. Thus, there are a total of 96 wells in the microplate feature 200 shown in FIG. 2A. In FIG. 2B, the microplate array 250 includes 192 microplate features, each including a feeding well 260 and a culture well 270 fluidly connected to a channel, each similar to the microplate feature 100 described above with respect to FIGS. 1A-1C. Thus, there are a total of 384 wells in the microplate feature 250 shown in FIG. 2B.

[0024] 3A-3C are diagrams of various operations of a microplate mechanism according to various examples of the present disclosure. In FIG. 3A, the microplate mechanism 300 is a blood-brain / gut barrier mechanism including a culture well 320 and a feeding well 310 fluidly connected to a channel 330. In operation, a hydrogel may be filled into the hydrogel well 350, and the hydrogel may be mixed with, for example, cells, spheroids, and / or organoids. In various examples, the hydrogel may be cured, for example, by heating at a desired temperature, and a medium mixed with cells, for example, epithelial cells, may be added to the feeding well 310. For example, the medium may be pressed against the bottom of the feeding well 310, for example, via a pipette, such that the medium mixed with cells may move through the channel 330 to the interface 370 of the channel 330 and the hydrogel chamber 350. Specifically, the channel 330 may be filled by first pushing out any air trapped in the channel, for example, via a pipette. Alternatively, the air may be removed by application of a vacuum. Thus, the media mixed with cells from the feeding well 310 may be pushed through the channel 330 towards the culture well 320. Thus, the cells and media inside the channel 330 may form vascular-like structures and interact with the cells, spheroids and / or organoids present in the channel 330 at the interface 370. As a result, the culture well 320, the feeding well 310 and the channel 330 are in fluidic connection, and a complex system with a barrier at the interface 370 between the channel 330 and the hydrogel well 350 may be reproduced and modeled. In various examples, the model thus created may be used to evaluate, for example, blood vessel sprouting and branching towards the organoid or tumor organoid 360, or tumor vascularization, which may be formed in the hydrogel well 350.

[0025] In FIG. 3B, in various examples of the present disclosure, the microplate mechanism 301 is a blood-brain / gut barrier mechanism including a culture well 320 and a feeding well 310 fluidly connected to each other via a channel 330. In operation, the hydrogel in the hydrogel well 350 may be mixed with, for example, cells, spheroids, and / or organoids, and media mixed with cells, such as epithelial cells 315, may be added to the feeding well 310. For example, the media and epithelial cells 315 may be pushed through the channel 330 and move between the channel 330 and the hydrogel chamber 350 at the interface 370, as described above with respect to FIG. 3A. Thus, the cells 315 and media inside the channel 330 may form a vascular-like structure, and a complex system with a barrier at the interface 370 between the channel 330 and the hydrogel well 350 may be modeled. In various examples, the model thus created may be used to evaluate blood vessel sprouting and branching towards the organoid or tumor organoid 365 that may form within the hydrogel well 350. The example shown in FIG. 3B may, for example, allow testing of compound permeability and immune cell penetration and extravasation.

[0026] In FIG. 3C, in various examples of the present disclosure, the microplate mechanism 302 mimics the mechanism for evaluating neurite outgrowth, cutting, and healing or recovery. In FIG. 3C, the culture well 320 and the feeding well 310 are fluidly connected to each other via the channel 330. In operation, differentiation medium may be added and mixed with the hydrogel in the hydrogel well 350, and progenitor cells 355 may be added to the medium in the feeding well 310. Thus, the progenitor cells 355 may be pushed or moved from the feeding well 310 to the channel 330 and near or against the interface 370 between the channel 330 and the hydrogel chamber 350. As described above with respect to FIG. 1C, in operation, the medium mixed with the progenitor cells to the feeding well 310 may be added by sealing a pipette tip containing the mixture to the bottom of the feeding well 310. Once the mixture is added to the feeding well 310, the channel 330 may be filled by removing the air present in the channel 330 and allowing the mixture of medium and cells 355 to settle into the channel 330. Thus, the feeding well 310, the culture well 320, and the channel 330 are fluidically connected, and the neuronal cells 358 may grow from the progenitor cells 355 toward the differentiation medium present in the hydrogel chamber 350. Thus, once a certain growth of the neuronal cells 358 is reached, an experiment may be performed on the neuronal cells 358. For example, injury may be induced on the neuronal cells 358, or a portion of the neuronal cells 358 may be excised, etc., to observe the healing or recovery process of the neuronal cells 358 after injury. In various examples, the observation of the healing or recovery process may be performed by the presence of an observation window above the hydrogel chamber 350.

[0027] 4A-4D are diagrams of a microplate mechanism according to various examples of the present disclosure. In FIG. 4AB, the microplate mechanism 400 includes two feeding wells 410A and 410B, two culture wells 420A and 420B, and two channels or microchannels 430A and 430B. For example, feeding well 410A, culture well 420A, and channel 430A are fluidly connected with each other, and feeding well 410B, culture well 420B, and channel 430B are fluidly connected with each other. For example, feeding well 410B, culture well 420B, and channel 430B are not fluidly connected with any of feeding well 410A, culture well 420A, or channel 430A. In various examples of the present disclosure, both microchannels 430A and 430B are in fluid communication with the same hydrogel chamber 440 and hydrogel well 450. Thus, feeding wells 410A and 410B may hold cells, progenitor cells, or aggregates, and feeding well 410A may hold cells or aggregates that are different from the cells or aggregates held in feeding well 410B. In various examples, media in feeding wells 410A and 410B may be pushed independently through microchannels 430A and 430B, respectively, to interact with any media, such as feeding media, that is mixed with the hydrogel chamber 440. Because both microchannels 430A and 430B are in contact with the same hydrogel well 440, cells in each of microchannels 430A and 430B may interact with the same media or progenitor cells present in the hydrogel well 440. Thus, two different types of interactions can be generated and observed contemporaneously or simultaneously, one between the contents of microchannel 430A and media present in hydrogel well 440, and the other between the contents of microchannel 430B and media present in hydrogel well 440. In one example, the hydrogel well 440 includes liquid phase guides 445A and 445B.For example, liquid phase guide 445A may be a raised portion formed in hydrogel well 440 to receive microchannel 430A, and liquid phase guide 445B may be a raised portion formed in hydrogel well 440 to receive microchannel 430B.

[0028] FIG. 4B is a side view of the microplate mechanism 400 shown in FIG. 4A according to various examples of the present disclosure. In FIG. 4B, the microplate mechanism 400 includes two feeding wells 410A and 410B, which are fluidly connected to microchannels 430A and 430B, respectively. In various examples of the present disclosure, the microchannel 430A is coupled to the feeding well 410A through an opening 460A formed at the bottom of the feeding well 410A. Also, the microchannel 430B is coupled to the feeding well 410B through an opening 460B formed at the bottom of the feeding well 410B. For example, the openings 460A and 460B may have a size and shape that may allow a typical pipette to substantially cover the openings 460A and 460B and thereby seal them. Alternatively, the air inside the microchannels 430A and 430B may provide the seal. In various examples, the hydrogel well 450 may contain additional media for the hydrogel chamber 440, and any media present in the hydrogel chamber 440 may interact with cells and / or aggregates present in both microchannel 430A and microchannel 430B coming from feed wells 410A and 410B, respectively. In other examples, the microplate mechanism 400 includes interfaces 470A and 470B, where interface 470A is between microchannel 430A and the hydrogel chamber 440, and interface 470B is between microchannel 430B and the hydrogel chamber 440. At interfaces 470A and 470B, media present in the hydrogel chamber 440 may not be able to enter microchannels 430A and 430B, for example, due to the shape and / or contour of the interior cavities of microchannels 430A and 430B.In one example, the interfaces 470A and 470B may also create a barrier that substantially prevents the medium from entering the microchannels 430A and 430B, respectively, due to the fact that one compartment of the microchannels 430A and 430B may be filled with the medium and another compartment of the microchannels 430A and 430B may be filled with air or another hydrogel as a result of the surface tension of the medium, for example, as further shown in FIG. 5B below. The barrier created by the interfaces 470A and 470B may be a ridge several micrometers in height. In another example, the medium present in the hydrogel chamber 440 may interact with the cells and / or aggregates present in the microchannels 430A and 430B. In another example, each of the feeding wells 410A and 410B, the culture wells 420A and 420B, and the hydrogel chamber 440 may contain a different combination of cells, spheroids, and organoids.

[0029] 4C-4D are diagrams of microplate arrays 405 and 415 according to various examples of the present disclosure. In FIG. 4C, the microplate array 405 includes 24 microplate features, each of which includes two culture wells 420A and 420B and two feeding wells 410A and 410B fluidly connected to two microchannels 430A and 430B via microchannels 430A and 430B, respectively, each of which is similar to the microplate feature 400 described above with respect to FIG. 4A. Thus, there are a total of 96 wells in the microplate feature 405 shown in FIG. 4C. In FIG. 4D, the microplate array 415 includes 96 microplate features, each of which is similar to the microplate feature 400 described above with respect to FIG. 4A. Thus, there are a total of 384 wells in the microplate feature 415 shown in FIG. 4D.

[0030] 5A-5G are examples of the operation of a microplate mechanism according to various examples of the present disclosure. In FIG. 5A, a microplate mechanism 500, such as the microplate mechanism 400 shown in FIG. 4A above, may be part of a microplate array, such as the microplate array 405 shown in FIG. 4C. In this example, the microplate mechanism 500 is configured to generate a lung model and includes feed wells 510A and 510B fluidly connected to a hydrogel well 550 via microchannels 530A and 530B, respectively. In one example, a cell repellent hydrogel 545 may be held in the bottom of the hydrogel well 550, such as in the hydrogel chamber 540 of the hydrogel well 550. During operation, the microplate mechanism 500 may be kept at a desired temperature to solidify the hydrogel 545 at the interface between the hydrogel chamber 540 and the microchannels 530A and 530B.

[0031] In FIG. 5B, the microplate mechanism 500 further holds a second hydrogel 555 within the portion of the microchannels 530A and 530B that fluidly connect to the supply wells 510A and 510B. For example, the second hydrogel 555 can be matrigel or a substitute thereof, for example, mixed with cells, spheroids, and / or organoids. In another example, one of the microchannels 530A and 530B is filled with a medium mixed with cells capable of creating vessel-like tubes. The other of the microchannels 530A and 530B can also be filled with the same medium mixed with cells capable of creating vessel-like tubes, but can also be filled with other mediums and / or other cells, for example, as a drug delivery medium.

[0032] In FIG. 5C, an additional medium or media 535 may be added to the hydrogel well 550. For example, the medium 535 may be capable of creating lung epithelium or tissue within the hydrogel well 550. In FIG. 5D, the microplate mechanism 500 is incubated or maintained at a desired temperature or temperature range until the cells in the medium 535 shown in FIG. 5C settle to the bottom of the hydrogel chamber 540 and form a membrane 538 as shown in FIG. 5D. FIG. 5D also shows that the medium 535 may remain on top of the newly formed membrane 538. In FIG. 5E, the medium 535 that remained on top of the membrane 538 may be removed. As a result, the membrane 538, also referred to herein as a cell layer, may be exposed to the ambient air within the hydrogel well 550. In various examples, in addition to the epithelial cells, other different types of lung cells may be added to create a multi-component lung system with epithelial cells on top and lung tissue cells below the epithelial cells. In another example, a thin gel layer may be added to separate both the epithelial cells and the tissue cells. In another example, in FIG. 5F, more hydrogel 545 may be added through hydrogel well 550 to, for example, substantially completely fill hydrogel chamber 540, such that membrane 538 is located at the bottom of hydrogel well 550. Thus, membrane 538 becomes formed at the bottom of hydrogel well 550. In the configuration shown in FIG. 5F, pipetting of media and cells in microchannels 530A and 530B or removal of liquid from hydrogel well 550 becomes easier. In another example, in FIG. 5G, when both microchannels 530A and 530B hold hydrogel 555 therein, such as matrigel or matrigel substitute, the need for a cell repellent hydrogel, such as hydrogel 545 described above in FIG. 5F, may be eliminated. As a result, certain cell types, such as cells in membrane 538, may settle directly to the bottom of hydrogel chamber 540, and hydrogel well 550 may only contain air to directly expose membrane 538 to air. For example, the membrane 538 that sinks to the bottom of the hydrogel chamber 540 may be coated with, for example, a 3D matrix.In one example, the cells of membrane 538 may be in contact with matrigel or matrigel substitute 555 present in both microchannels 530A and 530B at interfaces 570A and 570B between microchannels 530A and 530B and hydrogel chamber 540.

[0033] In various examples of the present disclosure, multiple other assays, also referred to as plate reader assays, may be configured for imaging. For example, in angiogenesis applications, tumor grafts or growth factors may be mixed, for example, in the feeding well 510, and endothelial cell layers may be mixed in the culture well 520. In various examples, cells may migrate to the feeding well 510. In another example, in neurogenesis or neurodegeneration applications, growth factors or neurotoxic agents may be mixed to study neurite outgrowth as described above with reference to FIG. 3C. When studying tumor cell escape / invasion, migration and wound healing, migration of cells between wells, such as feeding well 510 or culture well 520, may be studied. In yet another example, stem cell transplantation of fibroblasts into organoids, as well as T cell infiltration, monocyte migration or other migration assays may be studied. In further examples, epithelial and / or mesodermal cell migration processes, as well as any other known or new assays including co-culture may be studied. In other examples, beating heart assays, neural spheroids or mini-brain assays with washing may be observed. In yet other examples of multi-tissue toxicity assessment, interactions in systems such as liver-brain, liver-heart, kidney-liver, gut-liver, etc. may be observed and studied via the co-culture microplate setup of the present disclosure. In further examples, stem cell differentiation and self-renewal in response to compounds, toxins, growth factors, inflammation, metabolic switches may be observed. Thus, various examples of the present disclosure allow for the evaluation of secreted factors and the observation of the migration of cells, which may be separated by using either horizontal slits that allow the cells to migrate or vertical wide slits for feeding and exchange of media and growth factors.

[0034] FIG. 6 is a flow chart illustrating a method of isolating cells according to an example of the present disclosure. In FIG. 6, method 600 includes a number of operations 610-650, which are described further below. For the sake of convenience only, method 600 is described through the use of at least an exemplary system 700, described below, in combination with the microplate mechanism described above. However, it is recognized that method 600 may be performed by any suitable system. In FIG. 6, operation 610 includes filling a liquid hydrogel inside a lower chamber of a hydrogel well. For example, the hydrogel well, also referred to herein as a third well, is part of a culture plate that includes a plurality of well plates, each well plate including one or two first wells, one or two second wells, and a channel in fluid communication with one or two first wells, one or two second wells, and the third well. As an example, one or more of the first wells are feeding wells, and one or more of the second wells are culture wells. For example, the third well includes an upper chamber and a lower chamber, and during operation 610, liquid hydrogel is filled inside the upper chamber. The term "liquid hydrogel" refers to a state of hydrogel close to liquid that maintains its gel properties. During operation 610, filling the inside of the lower chamber of the hydrogel well with liquid hydrogel includes filling the upper chamber of the hydrogel well with liquid hydrogel and transferring the liquid hydrogel from the upper chamber to the lower chamber of the hydrogel well. As another example, during operation 610, the hydrogel well may be filled with a cell repellent hydrogel, for example, to prevent cells, spheroids, or organoids from contaminating the hydrogel. In another example, the hydrogel well may be filled with differentiation media or other cells, spheroids, and / or organoids. As a further example, filling the hydrogel well may include filling the hydrogel well with epithelial cells.In another example, filling the hydrogel well during operation 610 includes filling a liquid hydrogel pre-mixed with a combination of cells, spheroids, and organoids through a fill port of the third well, for example, by sealingly engaging a pipette containing the pre-mixed hydrogel with the fill port.

[0035] During operation 620, according to various examples of the present disclosure, the method 600 includes solidifying the liquid hydrogel, for example, by incubation. For example, the culture plate may be maintained at a desired temperature or range of temperatures for a sufficient time to harden the hydrogel. Thus, during operation 620, the liquid hydrogel undergoes a phase transition from a substantially liquid state to a substantially solid state. In this case, the substantially solid state includes a state in which the hydrogel is stiffer than when in the liquid phase, but maintains its gel-like properties.

[0036] During operation 630, according to various examples of the present disclosure, the first or second well is filled with cells, spheroids, and / or organoids. For example, the feeding well and / or culture well may be filled with cells, spheroids, organoids, and / or tumor organoids. In one example, to fill the feeding well and / or culture well with cells, spheroids, or organoids, the opening of the feeding well and / or culture well may be sealed, for example, with a pipette, prior to filling the cells, spheroids, organoids, and / or tumor organoids, thereby pushing air out of the feeding well and / or culture well. In various examples, filling the liquid hydrogel during operation 610 and filling the first and second wells during operation 630 may include filling each of the first well, the second well, and the hydrogel well with a different combination of cells, spheroids, and organoids. As another example, although operation 610 is described before operation 630, operation 630 may occur before, after, or contemporaneously with operation 610, according to various examples.

[0037] In another example of the present disclosure, the co-culture plate mechanism includes a second channel in fluid communication with the hydrogel well and also in fluid communication with the fourth well and the fifth well. For example, the fourth well can be another feeding well, and the fifth well can be another culture well. In this case, operation 630 also includes filling the fourth well and / or the fifth well with at least one of cells, spheroids, or organoids. Operation 630 can also include adding a feeding medium to any one or more of the first well, the second well, the fourth well, and the fifth well. In another example, any one or more of the first well, the second well, the fourth well, and the fifth well can be filled with cell precursors. As an example, if epithelial cells are filled into the hydrogel well during operation 610, the feeding well can be filled with organoids and / or tumor organoids. As a further example, if the hydrogel wells are filled with differentiation medium, the feed wells may be filled with progenitor cells. As yet another example, if the hydrogel wells are filled with a water-repellent hydrogel, the culture plate is maintained at a desired temperature to allow the hydrogel to flow through the microchannels. In various examples, filling the liquid hydrogel during operation 610 and filling the first, second, fourth, and fifth wells during operation 630 may include filling each of the first well, the second well, the hydrogel well, or the third well, the fourth well, and the fifth well with a different combination of cells, spheroids, and / or organoids.

[0038] During operation 640, cells, spheroids and / or organoids are introduced into the channels, also referred to as microchannels, which are in communication with the feeding well, the hydrogel well and the culture well. In an example where the microplate includes two feeding wells, two culture wells and two microchannels, cells, spheroids and / or organoids are introduced into both microchannels during operation 640. For example, the cells, spheroids and / or organoids introduced into both microchannels can be different from each other, one microchannel can have one type of cells, spheroids and / or organoids, and the other microchannel can have a different type of cells, spheroids and / or organoids. In one example, introducing cells, spheroids and / or organoids into one microchannel or two microchannels includes sealing the opening of the feeding well that is fluidly connected to the microchannel, for example with a pipette, and pushing air out of the microchannel before filling the microchannel with cells, spheroids and / or organoids. As a further example, during operation 640, a hydrogel may be loaded into a first channel and a feed medium may be loaded into a second channel.

[0039] During operation 650, in various examples of the present disclosure, a feeding medium may be added to the feeding well and / or the culture well. In various examples, when the feeding medium is added, an interaction may occur between the cells, spheroids, and / or organoids present in the channel and the cells introduced into the hydrogel well. For example, due to the interface between the hydrogel in the hydrogel well and the medium in the channel, the medium in the channel does not enter the hydrogel well, and the hydrogel and / or medium in the hydrogel well does not enter the channel. Thus, the interaction may occur at the interface between the microchannel and the hydrogel well. In various examples, this interaction may be observed through the opening of the hydrogel well. In other examples, the cells, spheroids, and / or organoids interacting at the interface between the microchannel and the hydrogel well may be controlled, excised, damaged, and otherwise manipulated, for example, to observe their response to various changes or trauma, among other experiments. For example, if a neuronal cell is being observed, the neuronal cell may be intentionally damaged and the healing or recovery of the neuronal cell may be observed through the opening of the hydrogel well.

[0040] FIG. 7 illustrates a block diagram of a computing device according to various embodiments. In the illustrated example, the computing device 700 may include a bus 702 or other communication mechanism of similar functionality for communicating information, and at least one processing element 704 (collectively referred to as processing element 704) coupled to the bus 702 for processing information. As will be appreciated by those skilled in the art, the processing element 704 may include multiple processing elements or cores, which may be packaged as a single processor or in a distributed configuration. Additionally, multiple virtual processing elements 704 may be included in the computing device 700 to provide control or management operations for the microplate mechanisms 100-500 or method 600 described above.

[0041] The computing device 700 may also include one or more volatile memories 706 coupled to the one or more buses 702 for use by the at least one processing element 704, which may include, for example, random access memory (RAM) or other dynamic memory components. The computing device 700 may further include static non-volatile memory 708, such as read only memory (ROM) or other static memory components, coupled to the bus 702 for storing information and instructions used by the at least one processing element 704. A storage component 710, such as a storage disk or storage memory, may be provided for storing information and instructions used by the at least one processing element 704. As will be appreciated, the computing device 700 may include a distributed storage component 712, such as a networked disk or other storage resource available to the computing device 700.

[0042] The computing device 700 may be coupled to one or more displays 714 for displaying information to a user. An optional user input device 716, such as a keyboard and / or touch screen, may be coupled to the bus 702 for communicating information and command selections to the at least one processing element 704. An optional cursor control or graphic input device 718, such as a mouse, trackball, or cursor direction keys, for communicating graphical user interface information and command selections to the at least one processing element. The computing device 700 may further include input / output (I / O) components, such as serial, digital, network connections, or other input / output components to enable intercommunication with other computing components and various components of the microplate mechanisms 100-500 or methods 600 described above.

[0043] In various embodiments, the computing device 700 can be connected to one or more other computer systems via a network to form a networked system. Such a network can include, for example, one or more private networks or a public network such as the Internet. In a networked system, one or more computer systems can store data and provide data to other computer systems. The one or more computer systems that store and provide data may be referred to as a server, or in a cloud computing scenario, a cloud. The one or more computer systems may include, for example, one or more web servers. Other computer systems that send and receive data to and from the server or cloud may be referred to as, for example, a client or cloud device. Various operations of the microplate mechanisms 100-500 or method 600 described above may be assisted by the operation of a distributed computing system.

[0044] The computing device 700 may be operable to control the operation of the components of the microplate mechanism 100-500 or method 600 described above, for example through a communications device such as communications device 720, and to process data provided from a data source as described above with respect to the microplate mechanism 100-500 or method 600. In some examples, analytical results are provided by the computing device 700 in response to the at least one processing element 704 executing instructions contained in memory 706 or 708 and performing operations on received data items. Execution of instructions contained in memory 706 and / or 708 by the at least one processing element 704 can enable the microplate mechanism 100-500 or method 600 to perform the methods described herein.

[0045] The term "computer-readable medium" as used herein refers to any medium that participates in providing instructions to processing elements 704 for execution. Such media may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as disk storage device 710. Volatile media include dynamic memory, such as memory 706. Transmission media include coaxial cables, copper wire and fiber optics, including the wires that comprise bus 702.

[0046] Common forms of computer readable media or computer program products include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape or any other magnetic medium, a CD-ROM, a digital video disk (DVD), a Blu-ray disk, any other optical medium, a thumb drive, a memory card, RAM, PROM, and EPROM, Flash EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0047] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to the processing element 704 for execution. For example, the instructions may initially be carried on a magnetic disk of a remote computer. The remote computer may load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to the computing device 700 may receive the data on the telephone line and convert the data to an infrared signal using an infrared transmitter. An infrared detector coupled to the bus 702 may receive the data carried in the infrared signal and place the data on the bus 702. The bus 702 carries the data to memory 706, from which the processing element 704 retrieves and executes the instructions. The instructions received by memory 706 and / or memory 708 may optionally be stored on storage device 710 either before or after execution by the processing element 704.

[0048] According to various embodiments, instructions operable to be executed by a processing element to perform the method are stored on a computer-readable medium. The computer-readable medium may be a device that stores digital information. For example, the computer-readable medium includes a compact disc read-only memory (CD-ROM) as known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing the instructions that are configured to be executed.

[0049] This disclosure has described several examples of the technology with reference to the accompanying drawings, in which only some of the possible examples are shown. However, other aspects may be embodied in many different forms and should not be construed as being limited to the examples set forth herein. Rather, these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the possible examples to those skilled in the art.

[0050] Although specific examples have been described herein, the scope of the technology is not limited to those specific examples. Those skilled in the art will recognize other examples or improvements that are within the scope of the technology. Thus, specific structures, operations, or media are disclosed as examples only for illustration purposes. Examples according to the technology can also combine elements or components that are generally disclosed but not explicitly illustrated in combination, unless otherwise specified herein. The scope of the technology is defined by the following claims and any equivalents thereof.

[0051] The following is claimed:

Claims

1. A culture plate comprising: The top surface and A plurality of well systems, each well system comprising: A first well, A second well, a first channel in fluid communication with the first well and the second well; and a third well disposed between the first well and the second well, the third well being in fluid communication with the first channel, and the top surface of the culture plate defining a well opening for at least one of the first well, the second well, and the third well; a culture plate comprising: a plurality of wells;

2. The culture plate of claim 1 , wherein the first channel is in fluid communication with the first well on a side of the first well and in fluid communication with the second well on a side of the second well.

3. the third well comprises: an upper chamber and a lower chamber having a size smaller than that of the upper chamber; an internal first ridge configured to receive the first channel; The culture plate of claim 1 or claim 2, wherein the first channel is in fluid communication with the lower chamber.

4. The culture plate according to any one of claims 1 to 2, wherein the first well and the second well are in fluid communication with the first channel via a first opening and a second opening, respectively.

5. The culture plate according to any one of claims 1 to 2, wherein the first channel is a microchannel having an inner diameter of about 5 µm to 100 µm.

6. The culture plate of any one of claims 1 to 2, wherein the inner diameter of the first channel is provided with a protein coating.

7. The culture plate of any one of claims 1 to 2, wherein the inner diameter profile of the first channel is one of a circle, an oval, and a polygon.

8. The culture plate according to any one of claims 1 to 2, wherein the first channel extends horizontally between the first well and the second well.

9. The culture plate according to any one of claims 1 to 2, wherein the first channel extends non-linearly between the first well and the second well.

10. a fourth well; and a fifth well; and a second channel in fluid communication with the third well, the fourth well, and the fifth well; the third well includes a second ridge therein configured to receive the second channel; The culture plate according to any one of claims 1 to 2.

11. The culture plate according to any one of claims 1 to 2, wherein the culture plate comprises one of a 96-well plate and a 348-well plate.

12. 1. A method for separating cells from a medium stream in a culture plate comprising a plurality of well plates, each well plate comprising at least a first well, a second well, a third well, and a first channel in fluid communication with the first well, the second well, and the third well, the third well including an upper chamber and a lower chamber, the method comprising: Filling the interior of the lower chamber with a liquid hydrogel; allowing the hydrogel to solidify by incubation; Filling at least one of the first well and the second well with at least one of cells, spheroids, and organoids; Introducing the at least one of cells, spheroids, and organoids from the at least one of the first well and the second well into the first channel to interact with the hydrogel; adding a feeding medium to said at least one of said first well, said second well, and said third well.

13. 13. The method of claim 12, wherein the interaction comprises at least one of cell attachment and organoid formation.

14. 14. The method of claim 12 or claim 13, wherein introducing at least one of cells, spheroids, and organoids into the first channel comprises sealing an opening of at least one of the first well and the second well with a pipette.

15. 14. The method of any one of claims 12-13, further comprising accessing at least one of the first well, the second well, and the third well through an opening therein to manipulate its contents.

16. The method of any one of claims 12 to 13, further comprising removing air from the first channel before introducing the at least one of cells, spheroids, and organoids into the first channel.

17. The method of any one of claims 12 to 13, wherein filling the interior of the lower chamber with the hydrogel comprises transferring the hydrogel from the upper chamber into the lower chamber.

18. the culture plate further comprising a second channel in fluid communication with the third well, the fourth well, and the fifth well, and the method further comprising: Filling at least one of the fourth well and the fifth well with at least one of cells, spheroids, and organoids; The method of any one of claims 12 to 13, further comprising adding a feeding medium to at least one of the fourth well and the fifth well.

19. adding epithelial cells to the lower chamber; 14. The method of any one of claims 12 to 13, wherein filling at least one of the first well and the second well comprises filling at least one of the first well and the second well with organoids and tumor organoids.

20. adding differentiation medium to the lower chamber; adding progenitor cells to said at least one of said first well and said second well; The method of any one of claims 12 to 13, further comprising imaging the healing of the nerve cells through the opening in the upper chamber.

21. and wherein filling the liquid hydrogel comprises filling a cell-repellent hydrogel, the method comprising:

14. The method of any one of claims 12-13, further comprising, prior to said incubation, maintaining said culture plate at a desired temperature to allow said hydrogel to flow through said first channel.

22. 14. The method of any one of claims 12 to 13, further comprising filling the first channel with a medium mixed with cells capable of forming tab vessels within the first channel.

23. The method of any one of claims 12 to 13, further comprising filling the first channel with a medium mixed with cells capable of forming a pulmonary membrane.

24. The method of any one of claims 12 to 13, further comprising maintaining the incubation until the cells settle to the bottom of the upper chamber and the lung membrane is formed.

25. Filling the liquid hydrogel and filling at least one of the first well and the second well comprises filling each of the first well, the second well, and the third well with a different combination of cells, spheroids, and organoids; The method of any one of claims 12 to 13, wherein the first well, the second well, and the third well are then fluidly connected by removing air present inside the first channel.

26. 14. The method of claim 12, wherein removing the air comprises one of using a vacuum to remove the air from the first channel and sealingly engaging a pipette tip.

27. wherein filling the liquid hydrogel, filling at least one of the first well and the second well, and filling at least one of the fourth well and the fifth well comprises filling each of the first well, the second well, the third well, the fourth well, and the fifth well with a different combination of cells, spheroids, and organoids; the first well, the second well, and the third well are fluidly connected by removing air present inside the first channel; 14. The method of claim 12, wherein the third well, the fourth well, and the fifth well are fluidly connected by removing air present inside the second channel.

28. 14. The method of claim 12, wherein filling the liquid hydrogel comprises filling the first channel with a liquid hydrogel pre-mixed with at least one of cells, spheroids, and organoids through a fill port of the third well by sealingly engaging a pipette containing the pre-mixed hydrogel with the fill port.

29. 14. The method of any one of claims 12 to 13, wherein filling the liquid hydrogel comprises filling the first channel and the second channel with a liquid hydrogel pre-mixed with at least one of cells, spheroids, and organoids through a fill port of the third well by sealingly engaging a pipette containing the pre-mixed hydrogel with the fill port.

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