Manufacturable co-culture modules

Abstract

In co-culture, interactions between multiple species are analyzed. Generally, microorganisms grown in co-culture can be mixed in the same growth medium, grown in the same medium but separated by a medium exchange membrane, or grown separately and exposed to products produced by the other microorganism(s). Studies of such cultures surpass cultures grown in isolation in that they more accurately represent real-world phenomena. Described herein is a co-culture module with multiple sample volumes separated by vertical semipermeable membranes. Co-culture modules are typically manufacturable and can be disposable or reusable.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority pursuant to 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 371,763, filed August 18, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] In cocultures, interactions between multiple species are analyzed. Generally, microorganisms grown in cocultures can be either mixed in the same growth medium, grown in the same medium but separated by a medium exchange membrane, or grown separately exposed to products produced by the other microorganism(s). Studies of such cultures surpass cultures grown in isolation in that they more accurately represent real-world phenomena. Analysis of cocultures provides a better understanding of microbial dynamics in emerging research areas such as the gut microbiome. Advances in coculture laboratory techniques improve our ability to combat infections, treat disease, and proactively manage overall human health.

[0003] Currently, there is no recognized industry standard for co-culture evaluation in shape factors that allows for real-time quantitative assessment of the phenotypic behavior of individual strains in the co-culture. Most simply, co-cultures can be studied in mixed conditions, where both cultures share the same space and the same medium. However, this experiment does not allow for measurement of the growth of individual cultures.

[0004] Plate colony assays can also be used as co-culture techniques. This technique involves culturing microorganisms on a plate and observing interactions between visible colonies. However, it is not quantitative and has limited applicability to real-world microbial environments. It also does not apply to cultures grown in suspension in liquid media.

[0005] In another coculture technique, vertically stacked cultures can be grown in an arrangement where a horizontal membrane separates the cultures but allows for medium exchange. The detachable horizontal membrane and upper chamber create a method for separate optical density readings for growth measurement experiments. However, this poses a risk of contamination during manipulation and does not effectively separate the two suspension cultures. The membrane insert is designed for adherent cells, measuring how they interact with the suspension culture in the lower chamber. As such, the upper culture is generally not a microbial culture but rather some kind of mammalian or other eukaryotic sample. These inserts are also limited in their ability to adequately characterize phenotypic growth kinetics, and their vertically stacked configuration does not allow for such measurements to be made in real time. However, this is currently the most common form of coculture research, and the best-known brand is the Corning TransWell system.

[0006] A final example of a co-culture technique is the continuous medium exchange protocol, or so-called "spent medium" experiment. In this method, a microbial culture is grown, and the medium is sterilized and reused to grow a second microbial culture. In this way, metabolic products of the first microorganism can be observed to influence the behavior of the second microorganism. While this technique allows for quantification of the phenotypic behavior of each culture, it cannot capture real-time kinetics. The single point of exchange is also very different from the continuous exchange that occurs in the real world (and other co-culture techniques).

[0007] Specialized co-culture systems available to microbiologists and pathologists typically exhibit characteristics that hinder the ability to perform real-time optical readouts on plates. It is desirable for researchers to be able to dynamically monitor these cultures in a reproducible manner similar to multiwell plates with a plate reader, while simultaneously determining non-contact interactions. As the field of co-culture microbiology continues to expand in breadth and applications, it is essential to establish a standard system for performing these measurements so that researchers can compare and contrast their findings. The invention presented herein is intended to become the standard for dynamic, reproducible co-culture measurements. Summary of the Invention

[0008] In one aspect, a co-culture module is described that includes a central volume, an end cap volume, and a semi-permeable membrane, configured such that the module can interface with a multi-well plate reader.

[0009] In another aspect, a manufacturable co-culture module is described.

[0010] In another aspect, a co-culture module is described that includes a plurality of bodies forming a plurality of sample wells, at least one alignment feature between the bodies, and at least one semi-permeable membrane attached between the bodies.

[0011] In another aspect, a co-culture module is described that includes a pair of bodies that form two sample wells, with at least one alignment feature between the bodies and at least one semi-permeable membrane attached between the bodies.

[0012] These and other aspects, which will become apparent in the detailed description that follows, are accomplished through the inventors' discovery of a novel co-culture module. [Brief explanation of the drawings]

[0013] [Figure 1]FIG. 1 is an isometric view of an assembled co-culture module 100 showing a central volume 400, two end cap volumes 200, and two semi-permeable membranes 300, and configured to interface with a multi-well plate reader. [Figure 2] FIG. 1 is an isometric exploded view of a co-culture module configured to interface with a multiwell plate reader. [Figure 3] FIG. 2 is an isometric view of the underside of the assembled co-culture module showing the internal rib network 210 and flat areas (e.g., 212) for sample interrogation. [Figure 4A] FIG. 11 is an isometric cross-sectional view of a co-culture module, with an enlarged cross-sectional view in a detailed view (right) highlighting the manufacturing features used to secure and hermetically seal the module. [Figure 4B] FIG. 1 is an isometric cross-sectional view of a co-culture module providing a pair of sample volumes, highlighting the manufacturing features used to secure and hermetically seal the module. [Figure 5] FIG. 2 is a top view of the end cap 200 showing the coordinate labels 220 of the wells. [Figure 6] FIG. 1 is an isometric view of a co-culture module providing twin sample volumes. [Figure 7] FIG. 1 is an isometric exploded view of a co-culture module providing a pair of sample volumes, highlighting the manufacturing features used to secure and hermetically seal the module. [Figure 8] FIG. 16 is an isometric view of the underside of an assembled co-culture module providing a pair of sample volumes. DETAILED DESCRIPTION OF THE INVENTION

[0014] Exemplary aspects of the present invention are described herein. Although the following detailed description contains many details for purposes of illustration, those skilled in the art will recognize that variations and modifications to the following details are within the scope of the present invention. Accordingly, the following aspects of the present invention are presented without any loss of generality and without imposing limitations on the claimed invention.

[0015] One embodiment includes a device having vertically oriented semipermeable membranes positioned between one or more pairs of wells on a single module. These vertically oriented membranes allow selective transport of molecules and / or microorganisms depending on the porosity, thickness, and material selected for the membrane.

[0016] Another aspect is a) a plurality of bodies each containing at least one sample well, the bodies being precisely aligned with one another such that the sample wells within the bodies are connected via horizontal cavities; b) at least one alignment feature disposed between the bodies that facilitates alignment of the bodies and hermetic sealing by ultrasonic welding, heat stamping, laser welding, press fit, or other industrial process for permanent bonding; c) at least one semi-permeable membrane permanently sealed to or between the bodies, aligned to completely cover at least one horizontal cavity connecting said sample wells; the membrane material is porous to selectively allow the movement of biologically relevant compounds between the wells while preventing the movement of organisms between the wells; The body and membrane comprise a multi-cavity co-culture module that are permanently attached to one another prior to use to form a single operable component.

[0017] In another embodiment, the sample wells are designed to reside in horizontal positions that correspond to the horizontal positions of one or more wells of a standard multi-well plate.

[0018] In another aspect, mechanical features are provided that facilitate alignment and temporary attachment of a horizontal cover over the top of the module. Examples of horizontal covers include rigid lids and membranes.

[0019] In another embodiment, the horizontal surface is optically transparent to facilitate measurements of optical density, absorbance, transmittance, and / or other properties involving the transmission of light through the sample.

[0020] In another embodiment, the vertical surfaces are optically opaque to facilitate measurement of fluorescence, luminescence, and / or other properties involving the emission of light from the sample.

[0021] In another aspect, the industrial process used to secure the bodies together achieves an airtight seal with the membrane material in the absence of other elements (eg, gaskets, etc.).

[0022] In another aspect, the industrial process used to fasten objects together is ultrasonic welding.

[0023] In another embodiment, the membrane is permanently attached to at least one body via heat sealing before the bodies are permanently attached to one another.

[0024] Alternatively, the bodies are then attached to one another using ultrasonic welding.

[0025] Mounting membranes vertically in a manufacturable manner presents unique challenges to those skilled in the art. Not only do the sometimes thin and fragile membranes need to be precisely positioned, but they also require an airtight seal between themselves and multiple mating components without damaging the membrane or altering its permeability characteristics. Accordingly, another aspect includes manufacturing processes and component features that can be employed to produce co-culture systems with vertically oriented membranes.

[0026] The names of the components shown in FIGS. 1 to 8 are as follows: [Table 1]

[0027] 1-8 , another embodiment includes a module comprising a disposable co-culture module 100, one or more end cap volumes 200, one or more semi-permeable membranes 300, and up to one or more central volumes 400. In one embodiment, the co-culture module 100 is assembled to form a continuum having a perimeter 102 configured to fit, assembled with or without a lid (not shown), into instruments and devices common in laboratory and diagnostic practices for measuring microplates and similar products.

[0028] Orienting and manipulating the typically thin and fragile semipermeable membrane 300 presents unique assembly challenges, especially in vertical or near-vertical orientations designed to be secured between two or more rigid bodies. Advantages of the co-culture modules described herein arise from the types of features, materials, and component configurations used to make the co-culture module assembly 100 manufacturable on a large scale.

[0029] Another embodiment is a disposable, manufacturable co-culture module 100 designed to interface with a multi-well plate reader, comprising: a) at least one continuous feature defining the bottom perimeter of the co-culture module 102; b) at least one feature defining the upper periphery of the co-culture module 104; c) at least one feature 106 configured to bond, hermetically seal, or adhere different modular components together; d) at least one end cap volume 200, i) at least one sample well 202 for holding a microorganism sample; ii) at least one cavity 204 in the sidewall configured to interface with the semipermeable membrane 300; iii) at least one feature for alignment 206 with other components in the co-culture assembly 100; iv) at least one feature configured to plastic weld or secure 208 the end cap volume 200 to other components within the co-culture assembly 100 using a manufacturing method; v) an end cap volume comprising one or more of: at least one window or other feature 218 that allows transduction of sample parameters within the sample well 202; e) at least one semipermeable membrane 300, i) membrane morphology 302 configured to be aligned and oriented perpendicular or near perpendicular between volumes 306 within the co-culture module (see FIGS. 4A and 4B); ii) at least one feature 304 configured to align the semipermeable membrane form 302 with an alignment feature on either the end cap volume 200 and / or the central volume 400; f) at least one central volume 400, i) at least one feature configured for plastic welding or fixation 402 of the central volume(s) 400 to other components within the co-culture assembly 100; ii) at least one cavity 404 in the sidewall configured to interface with the semipermeable membrane 300; iii) at least one feature for alignment 406 with other components in the co-culture assembly 100; iv) at least one sample well 408 for holding a microorganism sample; v) at least one window or other feature 412 that allows for transduction of a sample parameter within the sample well 408; vi) an area designated for placement of company markings or writable plaques for writing or printing experiment information 414; vii) a central volume comprising one or more of: a recessed area 414 that captures spilled sample or an area that serves as a base for mechanical attachment of a lid component or sample cover;

[0030] In another aspect, the lid component or sample cover is a horizontal cover.

[0031] 3, in another embodiment, the co-culture module using the exterior rim 102 is configured with a geometry intended to fit within various laboratory plate readers or instruments. Additionally, the exterior shape may be other than rectangular in order to function and align properly within the plate reader instrument (not shown).

[0032] Alternative embodiments include co-culture modules comprised of different combinations and permutations of the assemblies described herein. One example is a co-culture module with two end caps 200 without a central volume 400 (FIG. 6). Another example is a co-culture module configured to be constructed using three or more end caps arranged in a geometric pattern around at least one central volume 400 (not shown).

[0033] Another embodiment includes a co-culture module that is not limited to pairs of individual wells in fluid communication with a semi-permeable membrane (not shown). One example is a co-culture module with three or more wells connected in series to each other, forming a network of samples connected by semi-permeable membranes. Another example is a co-culture module in which multiple wells are connected by semi-permeable membranes to a single common well.

[0034] In embodiments where a lid (e.g., a rigid lid) is used with the module 100, the module includes a continuous external ridge 104 (see FIGS. 4A and 4B) designed to lift and restrain the lid. Another embodiment includes a ridge on the top surface of the assembled co-culture module 100 that functions to contain spillage.

[0035] Another embodiment includes a co-culture module in which the wells of each volume are labeled with an alphanumeric indicator of the well's location within the assembly (see FIG. 5, 220). Another embodiment includes these indicators being recessed or raised portions of the volume, screen printed, or alternatively left as a blank textured surface on which the user can apply their own label.

[0036] Another embodiment includes wells in either the central 400 or end cap volumes 200 having raised areas (see FIG. 5, 222) configured to align with and contact the lid, or to act as a barrier to reduce the effects of evaporation of the contained liquid sample. Another embodiment includes these raised surfaces 222 acting in conjunction with the raised perimeter 104 (see FIG. 4A, 104) to contain spillage or prevent sample contamination from one well to another.

[0037] One of the most challenging aspects of the co-culture modules described herein is the combination of manufacturing criteria that must be used to cut, seal, position, and align or attach the semi-permeable membrane 300 to the various volumes within the co-culture module assembly. Figures 2 and 4A show an embodiment of a manufacturing solution in which a membrane body 306 is attached to the central volume 400 using a heat-sealing process to attach the membrane body to the flat surface 402 surrounding the central well membrane cutout 404. The heat-sealing process forms an airtight seal between the membrane and the central volume, sealing these two components together. Once the central volume and membrane are hermetically sealed, the end cap 200 is then attached to the assembly using assembly features 208. Figure 4B shows a similar embodiment of a manufacturing solution for a module consisting of only a pair of wells.

[0038] Further embodiments include an assembly method for sealing an end cap to a central volume membrane assembly, including one or more of the following: a) A ridge feature on the end cap 208 is compressed onto another adjacent feature 402 on the central volume using a static load. In one embodiment, these features are then plastically welded together using ultrasonic welding or a similar vibration process to form an airtight seal between the end cap 200 and the central volume 400. In such an embodiment, a flat surface can be formed and supported with ribs on the exterior surface of the end cap 214 (see FIG. 3 ), and an ultrasonic horn or other vibrating tool can be used to support the assembly. b) A gasket groove formed around the central well membrane cutout 404, or in a similar location around the end cap well membrane cutout 204 (see FIG. 2). In this embodiment, an O-ring or similar gasket material is positioned in the gasket groove, and the components are ultrasonically welded together with the protrusion 206 and a mating feature on the opposing component that is intended to mate with the protrusion during the welding process 406. Similarly, instead of using an ultrasonic welding process, the components can also be mechanically attached together using similar methods, such as heat staking or other similar techniques used to form mechanical bonds between rigid plastic components. c) The two flat surfaces at location 208 and their mating elements on the central volume 400 are sealed together using a laser welding process. d) An adhesive or gasket is attached to the membrane form 302. This adhesive or gasket forms an airtight seal on one or more sides when assembled between the end cap 200 and / or central volume 400 combination.

[0039] Another embodiment includes end caps 200 and central volume 400 constructed as a single component (not shown). In this embodiment, membrane configuration 302 is rigidly attached to a frame designed to be assembled between adjacent pairs of wells or other combinations of wells.

[0040] Another embodiment includes hermetically sealing the end caps 200 to the central volume(s) 400 using plastic assembly methods such as laser welding, ultrasonic welding, or other practices described herein. In this embodiment, slots or recesses are configured to accommodate either individual membranes 302 or combinations of membrane assemblies between the aforementioned components, either as stock or attached to a rigid frame. Another embodiment includes these membranes being either permanently attached to the co-culture assembly or configured to be removable so that the module can be sterilized and reused.

[0041] Alternatively, in another aspect, the module is configured to be disposable.

[0042] Another embodiment includes the individual components that make up the modular assembly, such as end caps 200 or central volumes 400, being configured to be constructed from different combinations of materials or treatments to allow for various types of experiments to be performed in a single module. One example is an end cap with opaque walls designed for fluorescent assays combined with a central volume with transparent, tissue culture-treated walls.

[0043] Another embodiment includes the end caps 200 and / or the central volume 400 being injection molded.

[0044] Another embodiment involves molding the membrane form 302 in place on either or both of the end caps 200 or the central volume 400. When the membrane form is molded on both, the end caps and the central volume are configured to be a single component.

[0045] All references cited herein are individually incorporated by reference in their entirety. Many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

1. 1. A multi-cavity co-culture module, comprising: a) a plurality of bodies each containing at least one sample well, the bodies being precisely aligned with one another such that the sample wells within the bodies are connected via horizontal cavities; b) at least one alignment feature disposed between said bodies that facilitates alignment of said bodies and hermetic sealing by ultrasonic welding, heat stamping, laser welding, press fit, or other industrial process for permanent bonding; c) at least one semi-permeable membrane permanently sealed to or between said bodies in alignment to completely cover at least one horizontal cavity connecting said sample wells; the membrane material is porous to selectively allow the movement of biologically relevant compounds between the wells while preventing the movement of organisms between the wells; The multi-cavity co-culture module, wherein the body and the membrane are permanently attached to one another prior to use to form a single operable component.

2. The module of claim 1 , wherein the module is configured for single-use.

3. The module of claim 1 , wherein the module is configured to be sterilizable and reusable.

4. 10. The module of claim 1, wherein the sample wells are designed to reside in horizontal positions corresponding to the horizontal positions of one or more wells of a standard multi-well plate.

5. The module of claim 4 , wherein the outer dimensions of the module correspond to the outer dimensions of a standard multiwell plate.

6. The module of claim 1 , wherein the sample wells are arranged in pairs.

7. The module of claim 1 , wherein at least one of the sample wells is in fluid communication with at least two other of the sample wells to form a co-culture network of more than two of the wells.

8. The module of claim 1 , wherein mechanical features are presented to facilitate alignment and temporary attachment of a horizontal cover over the module top.

9. The module of claim 8 , wherein the horizontal cover is a rigid lid.

10. The module of claim 8 , wherein the horizontal cover is a membrane.

11. 10. The module of claim 1, wherein the horizontal surface is optically transparent to facilitate measurement of optical density, absorbance, transmittance, and / or other properties involving the transmission of light through the sample.

12. 10. The module of claim 1, wherein vertical surfaces are optically opaque to facilitate measurement of fluorescence, luminescence, and / or other properties involving emission of light from the sample.

13. The module of claim 1 , wherein a gasket is used to form the hermetic seal between the body and the membrane and is permanently held in place by the seal between the bodies.

14. The module of claim 1 , wherein the industrial process used to secure the bodies together achieves the hermetic seal with the membrane material in the absence of other elements.

15. 15. The module of claim 14, wherein the industrial process used to secure the bodies together is ultrasonic welding.

16. The module of claim 1 , wherein the membrane is permanently attached to at least one body via heat sealing before the bodies are permanently attached to one another.

17. 17. The module of claim 16, wherein the bodies are then attached to one another using ultrasonic welding.

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