Fabricable co-culture module

Abstract

In co-culture, the interactions between multiple species have been analyzed. Typically, microorganisms growing in co-culture can be mixed in the same growth medium, grow in the same medium but separated by a medium exchange membrane, or grow alone when exposed to products produced by other microorganisms. The study of such cultures surpasses the study of isolated cultures as it more accurately represents the real-world phenomena. A co-culture module having a plurality of sample volumes separated by a vertical semipermeable membrane is described herein. The co-culture module is typically manufacturable, and may be disposable or reusable.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 371,763, filed on August 18, 2022. The contents of the patent application are incorporated herein by reference in their entirety. Background Art

[0003] In co-cultures, interactions between multiple species are analyzed. Typically, microorganisms grown in co-cultures are able to be mixed in the same growth medium, grown in the same medium but separated by a medium exchange membrane, or grown individually while exposed to products produced by other microorganisms. Studies of such cultures go beyond those of isolated cultures because they more accurately represent real-world phenomena. Analysis of co-cultures provides a better understanding of microbial dynamics in emerging research areas such as the gut microbiome. Advances in co-culture laboratory technology improve the ability to fight infections, treat diseases, and proactively manage overall human health.

[0004] Currently, there is no recognized industry standard for co-culture evaluation in a form factor that allows real-time quantitative assessment of the phenotypic behavior of individual strains in co-culture. At its simplest, co-culture can be studied under mixed conditions. In this case, the two cultures share the same space and the same culture medium. However, this experiment does not allow for the measurement of the growth of individual cultures.

[0005] Plate colony assays can also be used as a co-cultivation technique. This technique involves growing and observing interactions between visible microbial colonies on a petri dish. However, this is not quantitative, which limits the application of this technique to real-world microbial environments. This technique is also not suitable for cultures grown in suspension in liquid media.

[0006] In another co-culture technique, vertically stacked cultures can be grown in an arrangement in which a horizontal membrane separates the cultures but allows for medium exchange. The horizontal membrane and upper chamber can be removed, providing a way to read the optical density separately for growth measurement experiments. However, this poses a risk of contamination during operation and does not effectively separate the two suspension cultures; membrane inserts are designed for adherent cells to measure how they interact with the suspension culture in the lower chamber. Therefore, the upper culture is usually not a microbial culture, but rather some kind of mammalian or other eukaryotic sample. These inserts are also limited in their ability to fully characterize phenotypic growth dynamics, and due to the vertical stacking geometry, such measurements cannot be performed in real time. However, this is currently the most common form of co-culture research, and the best-known brand is the Corning TransWell system.

[0007] A final example of a co-cultivation technique is the serial media exchange protocol, or so-called “spent media” experiment. In this approach, a microbial culture is grown, and the media from that growth is sterilized and reused to grow a second microbial culture. In this way, it can be observed that the metabolites of the first microbe affect the behavior of the second. This technique allows for quantification of the phenotypic behavior of each culture, but does not capture real-time dynamics. The exchange at a single time point is also very different from the continuous exchange that occurs in the real world (even in other co-cultivation techniques).

[0008] Specialized co-culture systems available to microbiologists and pathologists often exhibit features that prevent optical reading of the plates in real time. Researchers would like to be able to dynamically monitor their cultures in a reproducible manner similar to multi-well plates in a plate reader, while being able to determine contact-independent interactions. As the field of co-culture microbiology continues to expand in breadth and applications, it is imperative that standard systems for performing these measurements be established so that scientists can compare and contrast their findings. The invention presented herein is intended to be the standard for dynamic and reproducible co-culture measurements. Summary of the invention

[0009] In one aspect, a co-culture module is described, comprising 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.

[0010] In another aspect, a manufacturable co-cultivation module is described.

[0011] In another aspect, a co-cultivation 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.

[0012] In another aspect, a co-culture module is described that includes a pair of bodies forming two sample wells, at least one alignment feature between the bodies, and a semi-permeable membrane attached between the bodies.

[0013] These and other aspects will become apparent in the detailed description below, which are achieved by the novel co-cultivation module discovered by the inventors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is an isometric view of an assembled co-cultivation module 100 depicting a central volume 400, two end cap volumes 200, and two semi-permeable membranes 300, such module being configured to interface with a multi-well plate reader.

[0015] Figure 2 is an exploded isometric view of a co-culture module configured to interface with a multi-well plate reader.

[0016] Figure 3 is an isometric view of the underside of an assembled co-cultivation module showing the internal ribbed network 210 and the flat area (eg, 212) for sample interrogation.

[0017] Figure 4A is an isometric cross-section of the co-culture module, with the section expanded to a detailed view (right), highlighting the fabrication features used to secure and seal the module.

[0018] Figure 4B is an isometric cutaway view of a co-culture module providing a pair of sample volumes, highlighting the fabrication features used to secure and seal the module.

[0019] Figure 5 is a top view of the end cap 200 showing the hole coordinate labels 220 .

[0020] Figure 6 is an isometric view of a co-culture module providing a pair of sample volumes.

[0021] Figure 7 is an isometric cutaway view of a co-culture module providing a pair of sample volumes, highlighting the fabrication features used to secure and seal the module.

[0022] Figure 8 is an isometric view of the underside of an assembled co-culture module providing a pair of sample volumes. DETAILED DESCRIPTION

[0023] Exemplary aspects of the present invention are described herein. Although the following specific embodiments contain many details for illustrative purposes, it will be appreciated by those skilled in the art that variations and modifications to the following details are within the scope of the present invention. Therefore, the following aspects of the present invention are described without loss of generality and without implying the limitations of the claimed invention.

[0024] One aspect relates to a device having vertically oriented semipermeable membranes between one or more pairs of pores on a single module. These vertically oriented membranes allow for the selective transfer of molecules and / or microorganisms depending on the porosity, thickness and selected material of the membrane.

[0025] Another aspect relates to a multi-chamber co-culture module, comprising:

[0026] a) a plurality of bodies, each body comprising at least one sample well, wherein the bodies are precisely aligned with each other such that the plurality of sample wells in the bodies are connected via a horizontal cavity;

[0027] b) at least one alignment feature positioned between the bodies to facilitate alignment and hermetic sealing of the bodies by ultrasonic welding, heat stamping, laser welding, press fitting or other industrial processes to achieve permanent adhesion; and,

[0028] c) at least one semipermeable membrane permanently sealed to or between the bodies, the at least one semipermeable membrane being aligned to completely cover at least one horizontal cavity connected to the sample well;

[0029] in:

[0030] The membrane material has a porosity that selectively allows biologically relevant compounds to transfer between pores while preventing organisms from transferring between pores; and,

[0031] Prior to use, the body and membrane are permanently attached to each other to form a single operable piece.

[0032] In another aspect, the sample wells are designed to be present at horizontal positions corresponding to the horizontal positions of one or more wells of a standard multi-well plate.

[0033] In another aspect, mechanical features are provided to facilitate alignment and temporary attachment of a horizontal cover plate on top of the module.Examples of horizontal cover plates include rigid covers and membranes.

[0034] In another aspect, the horizontal surface is optically transparent to facilitate measuring optical density, absorbance, transmittance, and / or other properties involving the transmission of light through the sample.

[0035] In another aspect, the vertical surfaces are optically opaque to facilitate measuring fluorescence, luminescence, and / or other properties involving light emission from the sample.

[0036] In another aspect, the industrial process for securing the bodies together achieves the hermetic seal with the membrane material without other elements (such as, for example, gaskets).

[0037] In another aspect, the industrial process used to secure the bodies together is ultrasonic welding.

[0038] In another aspect, the film is permanently attached to at least one of the bodies via heat sealing before the bodies are permanently attached to each other.

[0039] In another aspect, the bodies are subsequently attached to each other using ultrasonic welding.

[0040] Attaching membranes vertically in a manufacturable manner presents a unique challenge to those skilled in the art. The sometimes thin and fragile membranes not only need to be precisely positioned, but also need to make an airtight seal between themselves and multiple mating components without damaging the membrane or changing its permeability characteristics. Therefore, another aspect relates to manufacturing processes and component features that can be used to produce co-culture systems with vertically oriented membranes.

[0041] Figures 1 to 8 The names of the components shown are as follows:

[0042] 100 Disposable Co-culture Module 200 End cap volume 300 Semipermeable membrane 400 Center volume 102 The lower perimeter of the module is raised 104 Module perimeter ridge 106 Module sealing interface 202 End cap hole 204 End cap hole membrane cutout 206 Volume Alignment Features 208 Welding protrusion 210 Internal rib network 212 External conduction mode window 214 Module perimeter groove 218 Internal conduction mode window on the end cap volume 220 Hole Coordinate Labels 222 Independent hole ridge 302 Membrane shape 304 Membrane alignment / fixing holes 306 Membrane mounted into assembled co-culture module 402 Membrane fixing protrusions / grooves 404 Center hole membrane incision 406 Volumetric Alignment Holes with Material Capture 408 Center hole 410 Material discharge 412 Internal conduction modal window on the central volume 414 Spill catch recess and logo placement area

[0043] like Figures 1 to 8 As depicted, another aspect relates to 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 aspect, the co-culture module 100 is assembled in such a manner as to form a continuous body having an outer perimeter 102 configured to be assembled with or without a cover into instruments and devices commonly used in laboratory and diagnostic practice for measuring microplates and similar products.

[0044] Orientation and manipulation of the typically thin and fragile semipermeable membrane 300 presents unique assembly challenges, particularly in a vertical or near vertical orientation designed to be secured between two or more rigid bodies. The benefits of the co-cultivation module described herein derive from the types of features, materials, and configurations of components used to make the co-cultivation module assembly 100 manufacturable on a large scale.

[0045] Another aspect relates to a disposable and manufacturable co-culture module 100 designed to interface with a multi-well plate reader, the module comprising: one or more of the following:

[0046] a) at least one continuous feature defining the bottom outer perimeter of the co-cultivation module 102;

[0047] b) defining at least one feature of the top outer perimeter of the co-cultivation module 104;

[0048] c) at least one feature 106 configured to bond, hermetically seal or attach different module components together;

[0049] d) at least one end cap volume 200, comprising one or more of the following:

[0050] i) at least one sample hole 202 for accommodating a microbial sample; and,

[0051] ii) at least one cavity 204 in the wall-facing side configured to interface with the semi-permeable membrane 300;

[0052] iii) at least one feature 206 for alignment with other components in the co-cultivation assembly 100;

[0053] iv) at least one feature configured for plastic welding or attaching 208 the end cap volume 200 to other components of the co-cultivation assembly 100 using a manufacturing method; and,

[0054] v) at least one window or other feature 218 that allows for switching of sample parameters within the sample well 202;

[0055] e) at least one semi-permeable membrane 300, comprising: one or more of the following:

[0056] i) a membrane type 302 configured to be aligned between volumes 306 in a co-cultivation module and oriented vertically or nearly vertically (see Figure 4A and 4B );as well as,

[0057] ii) at least one feature 304 configured to align the semi-permeable membrane 302 with an alignment feature on the end cap volume 200 and / or the central volume 400;

[0058] f) at least one central volume 400, comprising: one or more of the following:

[0059] i) at least one feature configured for plastic welding or attaching 402 the central volume 400 to other components in the co-cultivation assembly 100;

[0060] ii) at least one cavity 404 in the side facing the hole configured to interface with the semi-permeable membrane 300;

[0061] iii) at least one feature 406 for alignment with other components in the co-cultivation assembly 100;

[0062] iv) at least one sample hole 408 for receiving a microbial sample;

[0063] v) at least one window or other feature 412 that allows for switching of sample parameters within the sample well 408;

[0064] vi) a designated area for placement of a company logo or an engravable plaque for writing or printing experimental information 414;

[0065] vii) A recessed region 414 for catching spilled sample or serving as a basis for mechanical attachment of a cover member or sample cover plate.

[0066] In another aspect, the cover member or sample cover is a horizontal cover.

[0067] refer to Figure 3 In another aspect, the co-culture module using the outer edge 102 is configured to have a geometry designed to fit a variety of laboratory plate readers or instruments. In addition, the outer geometry may not be rectangular in order to properly operate and register within a plate reader instrument (not shown).

[0068] Another aspect relates to a co-cultivation module composed of different combinations and arrangements of the components described herein. An example is a co-cultivation module including two end caps 200 without a central volume 400 ( Figure 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).

[0069] Another aspect relates to a co-culture module that is not limited to a single well pair in fluid communication with a semipermeable membrane (not shown). An example is a co-culture module that includes three or more wells connected in series to form a sample network connected by a semipermeable membrane. Another example is a co-culture module in which more than one well is connected to a single common well through a semipermeable membrane.

[0070] In aspects where a cover (eg, a rigid cover) is used with the module 100, the module includes a continuous outer ridge 104 designed to elevate and restrain the cover (see Figure 4A and Figure 4B ). Another aspect relates to ridges for containing spillage on the top surface of the assembled co-cultivation module 100.

[0071] Another aspect relates to a co-culture module wherein the wells on each volume are labeled with an alphanumeric indicator of the well location within the module (see Figure 5 , 220). Another aspect relates to the indicators being recessed or raised portions of the volume, which are screen printed, or alternatively left as a blank textured surface into which the user may affix his own labels.

[0072] Another aspect relates to the hole of the central volume 400 or the end cover volume 200, which has a raised area (see Figure 5 , 222), the raised area is configured to align and contact the cover, or to act as a barrier to reduce the effects of evaporation of the contained liquid sample. Another aspect relates to the surface 222 of these raised areas and the outer periphery 104 of the raised areas (see Figure 4A , 104) work together to contain spills or prevent sample contamination from one well to another.

[0073] One of the most challenging aspects of the co-cultivation modules described herein is the combination of manufacturing practices that must be used to cut, seal, position, and align or attach the semipermeable membrane 300 to the various volumes in the co-cultivation module assembly. Figure 2 and Figure 4A Aspects of a manufacturing solution are shown where a membrane body 306 is attached to a central volume 400 using a heat sealing process to attach it to a flat surface 402 surrounding a central hole membrane cutout 404. The heat sealing process forms an airtight seal between the membrane and the central volume, sealing the two components together. Once the central volume and membrane are sealed, the end cap 200 is then attached to the assembly using assembly components 208. Figure 4B Similar aspects of the manufacturing solution for a module consisting of only a pair of holes are shown.

[0074] Additional aspects relate to a method of assembling an end cap to a center volume membrane assembly comprising one or more of the following.

[0075] a) A ridged feature on the end cap 208 is compressed onto another adjacent feature 402 on the central volume using a static load. In one aspect, 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 this aspect, the end cap 214 (see Figure 3 ) is formed with a flat surface on its outer surface and supported with ribs to support the component using an ultrasonic horn or other vibrating tool.

[0076] b) A gasket groove is formed around the center hole film cutout 404 or at a similar location around the end cap hole film cutout 204 (see Figure 2 ). In this regard, an O-ring or similar gasket material is positioned into 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 engage the protrusion during the welding process 406. Likewise, instead of using an ultrasonic welding process, similar methods may be used to mechanically attach the components together, such as heat staking or other similar techniques for forming mechanical bonds between rigid plastic components.

[0077] c) The two flat surfaces at location 208 and their mating components on the central volume 400 are sealed together using a laser welding process.

[0078] d) Attaching an adhesive or gasket to the membrane type 302. When this adhesive or gasket is assembled between the combination of the end caps 200 and / or the central volume 400, it forms an airtight seal on one or more faces.

[0079] Another aspect involves configuring the end caps 200 and the central volume 400 as a single component (not shown). In this regard, the membrane type 302 is rigidly attached to a frame designed to be assembled between adjacent pairs of holes or other combinations of holes.

[0080] Another aspect involves hermetically sealing the end cap 200 to the central volume 400 using a plastic assembly method such as laser welding, ultrasonic welding, or other practices described herein. In this regard, slots or grooves are configured between the above components to accommodate individual membranes 302 or combinations of membrane assemblies, either as raw materials or attached to a rigid frame. Another aspect involves permanently attaching these membranes to the co-culture assembly or configuring them in a removable manner so that the module can be sterilized and reused.

[0081] Alternatively, in another aspect, the module is configured for single use.

[0082] Another aspect relates to the individual components that make up the assembly of the module, such as the end caps 200 or the central volume 400, being configured such that they are constructed of different combinations of materials or treatments to allow various types of experiments to be performed using a single module. One example is an end cap with opaque walls—designed for fluorescence analysis—combined with a central volume with transparent, tissue culture treated wells.

[0083] Another aspect involves the end cap 200 and / or the central volume 400 being injection molded.

[0084] Another aspect involves molding the membrane type 302 in place onto the end caps 200 or the central volume 400 or both. In the case where the membrane type is molded into both, the end caps and the central volume will be configured as a single component.

[0085] All references listed herein are incorporated herein by reference alone. In view of the above teachings, many modifications and variations of the present invention are possible. Therefore, it should be understood that within the scope of the appended claims, the present invention may be implemented differently than as specifically described herein.

Claims

1. A multi-chamber co-culture module, comprising: a) a plurality of bodies, each body comprising at least one sample well, wherein the bodies are precisely aligned with each other such that the plurality of sample wells in the bodies are connected via a horizontal cavity; b) at least one alignment feature positioned between the bodies to facilitate alignment and hermetic sealing of the bodies by ultrasonic welding, heat stamping, laser welding, press fitting or other industrial processes to achieve permanent adhesion; and, c) at least one semipermeable membrane permanently sealed to or between the bodies, the at least one semipermeable membrane being aligned to completely cover at least one horizontal cavity connected to the sample well; in: The membrane material has a porosity that selectively allows biologically relevant compounds to transfer between pores while preventing organisms from transferring between pores; and, Prior to use, the body and membrane are permanently attached to each other to form a single operable piece.

2. A module according to 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. The module according to claim 1, wherein the sample wells are designed to be present at 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 match the outer dimensions of a standard multiwell plate. The module according to claim 1 , wherein the sample wells are arranged in pairs.

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

8. The module of claim 1 wherein mechanical features are provided to facilitate alignment and temporary attachment of a horizontal cover plate on top of the module.

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

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

11. 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 transmission of light through a sample.

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

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

14. The module of claim 1 wherein said industrial process for securing said body together achieves said hermetic seal with said membrane material without additional elements.

15. A module according to 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 each other.

17. A module according to claim 16, wherein the bodies are subsequently attached to each other using ultrasonic welding.

Images