A modular microfluidic array
A modular microfluidic array system efficiently disaggregates cell clusters using laminar flow and autoclavable components, addressing the challenges of enzymatic treatments and open air exposure in current methods, ensuring sterility and scalability.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- UNIVERSITY OF ROCHESTER
- Filing Date
- 2026-01-07
- Publication Date
- 2026-07-16
Smart Images
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Abstract
Description
Attorney Docket Number 161118.08701 / UR 6-25030A MODULAR MICROFLUIDIC ARRAYCROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional Patent Application No.63 / 743,119, filed Jan. 8, 2025, which is incorporated by reference herein in its entirety.BACKGROUND
[0002] Many methods of scaled cell culture require cells to be grown in suspension. These suspensions include organoids, embryo bodies, neurospheres, gliospheres, and other forms in which cells grow as expanding clusters. In such preparations, clusters may need to be disaggregated or otherwise reduced in size, so as to allow nutrient access to the cluster interior and prevent the internal concentration of growth factors, cytokines and other agents that might influence the differentiated fate of the cells therein. Current methodologies require enzymatic treatments (e.g., using trypsin or collagenase that digest pieces of tissue to release target cells), or physical chopping with moving blades, that necessitate open air exposure during processing, resulting in systems not readily compliant with good manufacturing practice. This document describes methods and systems that address issues such as those discussed above, and / or other issues.SUMMARY
[0003] The present disclosure describes embodiments related to disaggregating cell clusters. A disaggregation system includes a first reservoir configured to contain a solution including multiple cell clusters. The system further includes a cluster processing system. The cluster processing system includes at least one separation device having a first set of separationAttorney Docket Number 161118.08701 / UR 6-25030elements spaced apart from each other, each separation element including an edge portion configured to apply a separation force to cell clusters that contact the edge portion. The cluster processing system also includes one or more channels in fluid communication with the first reservoir, wherein the one or more channels are configured to receive the solution from the first reservoir and direct the solution to pass through a corresponding separation device, causing at least one cell cluster of the plurality of cell clusters to contact the edge portion of one or more separation elements, causing the at least one cell cluster to disaggregate into at least two separate cell clusters.
[0004] Implementations of the disclosure may include one or more of the following optional features. In some examples, the one or more channels are configured to receive the solution from the first reservoir and direct the solution to pass through the corresponding separation device without exposing the solution to ambient air. The one or more channels may include multiple channels, each having a corresponding separation device. Each separation device may include an array of first separation elements that are evenly spaced from each other by a distance. The at least one separation device may include at least one vibrating separation element. In some examples, each of the separation elements includes a cutting blade and the edge portion of each of the separation elements includes a sharpened edge of the cutting blade. The at least one separation device may include components that are autoclavable.
[0005] In some examples, the at least one separation device includes a second set of separation elements spaced apart from each other, the second set of separation elements disposed downstream of the first set of separation elements, such that the solution passes through the first set of separation elements and the second set of separation elements. The first set of separation elements and the second set of separation elements may have a different spacing and / or may be disposed at a different angular orientation. In some examples, the disaggregation system furtherAttorney Docket Number 161118.08701 / UR 6-25030includes a second reservoir and a fluid pump configured to pump the solution from the first reservoir to the one or more channels, causing the solution to pass through the corresponding separation device to the second reservoir. The fluid pump may be configured to cause laminar flow of the solution through the one or more channels. The fluid pump may cause the solution to pass through the corresponding separation device using electroosmosis or surface acoustic waves.
[0006] A method of disaggregating cell cultures includes receiving a disaggregation system. The method further includes adding the solution including the cell clusters to the first reservoir. The method further includes applying pressure to the solution in the first reservoir, causing the solution to flow into the one or more channels and through the corresponding separation device. The method further includes receiving the at least two separate cell clusters in a second reservoir.
[0007] Implementations of the disclosure may include one or more of the following optional features. In some examples, causing the solution to flow into the one or more channels may include causing laminar flow of the solution in the one or more channels.
[0008] The details of one or more aspects of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the techniques described in this disclosure will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Fig. 1 shows an example disaggregation system.
[0010] Fig. 2 shows an example fluid channel and separation device.
[0011] Fig. 3 shows an array of fluid channels.Attorney Docket Number 161118.08701 / UR 6-25030
[0012] Fig. 4a and 4b show views of an example separation device.
[0013] Fig. 5 shows a flowchart for an example method according to an embodiment.
[0014] In the drawings, like reference numbers generally indicate identical or similar elements. Additionally, generally, the left-most digit(s) of a reference number identifies the drawing in which the reference number first appears.DETAILED DESCRIPTION
[0015] As used in this document, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning(s) as commonly understood by one of ordinary skill in the art. As used in this document, the term “comprising” (or “comprises”) means “including (or includes), but not limited to.” When used in this document, the term “exemplary” is intended to mean “by way of example” and is not intended to indicate that a particular exemplary item is preferred or required.
[0016] In this document, when terms such as “first” and “second” are used to modify a noun or phrase, such use is simply intended to distinguish one item from another and is not intended to require a sequential order unless specifically stated. The term “about” when used in connection with a numeric value, is intended to include values that are close to, but not exactly, the number. For example, in some embodiments, the term “about” may include values that are within + / - 10 percent of the value.
[0017] The present disclosure relates generally to methods and systems of disaggregating cell cultures. In particular, the disclosed disaggregation system provides for directing cell cultures (e g., contained in a solution) to a separation device configured to disaggregate the cultures. In some embodiments, the separation device is configured to be readily sterilized between uses, e.g.,Attorney Docket Number 161118.08701 / UR 6-25030in an autoclave, and may not contain any moving parts. After separation, the disaggregated cell cultures may then be collected, such that the entire process occurs without exposing the solution to ambient air. Furthermore, the system may include an array of separation devices operating in parallel, allowing the process to scale so as to achieve arbitrarily large rates of disaggregation.
[0018] Fig. 1 shows an example disaggregation system 100. A first reservoir 102 contains a solution 104 which includes cell cultures. In some examples, the reservoir 102 is a bioreactor, i.e., a vessel or device that supports a biological environment for the purpose of carrying out biochemical reactions or other biological processes. For example, the reservoir 102 may contain 150-200 milliliters of solution 104, including about 1,000 to 1,500 cell clusters 220. However, the reservoir 102 can also be a culture dish or plate or other container suitable for nurturing and / or storing cell cultures. Regardless of the form of the reservoir, at least some of the cell cultures may be larger than an ideal size. That is, a cluster 220 may be too large to allow for effective nutrient access to the interior of the cluster 220. For example, the clusters 220 may be up to about 250 microns in diameter and may have approximately spherical symmetry, whereas a desired size may be approximately 100 microns. To address this issue, the system also includes a processing system 200 (shown in more detail in Fig. 2). The processing system 200 is configured to receive the solution 104 from the first reservoir 102 and cause at least some of the cell clusters 220 to be separated into smaller cell clusters 220. After the clusters 220 are disaggregated by the processing system 200, the solution 104 (and the disaggregated cell clusters 220) may be collected into a second reservoir 108, such as a sterile receptacle compatible with further cell culturing.
[0019] As described above, the entire process may be carried out without exposing the solution 104 to ambient air. That is, the solution 104 may be forced (e.g., pumped) from the first reservoir 102, through the processing system 200, and into the second reservoir 108. In someAttorney Docket Number 161118.08701 / UR 6-25030implementations, after a first phase of disaggregation, the solution 104 (now containing disaggregated cell cultures 220) is directed back to the processing system 200 for a subsequent phase of disaggregation before being collected in the second reservoir 108. Furthermore, the disaggregation system 100 may include any number of such subsequent passes through the processing system 200. In some examples, the solution 104 containing disaggregated call clusters 220 may be collected in the first reservoir 102, e.g., after a predetermined number of passes through the processing system 200 and / or after a predetermined period of time.
[0020] Fig. 2 shows an example processing system 200. As shown, the processing system 200 includes a fluid channel 210 through which the solution 104, and associated cell clusters, e.g., 220a, 220b pass. That is, the fluid channel 210 is in fluid communication with the first reservoir 102. As shown, the fluid channel 210 leads to a separation element 230 which causes cell clusters 220 to split (e.g., disaggregate) into separate, smaller cell clusters (e.g., 220c, 220d). For simplicity, the 220 clusters are shown as disaggregated by a single separation element 230. However, a larger separation device (400, Fig. 4) may be formed from multiple separation elements 230. For example, a separation device 400 may include multiple separation elements 230 spaced apart from each other. The fluid channel 210 directs the solution 104 (and associated clusters 220) to pass through the separation device 400 and the multiple separation elements 230. In this case, each of the multiple separation elements 230 may cause cell clusters 220 to split (e.g., disaggregate) into separate, smaller cell clusters (e.g., 220c, 220d), as described above with respect to a single separation element 230.
[0021] In an embodiment having multiple separation elements 230 that are spaced apart from each other by a distance, the spacing between separation elements 230 may define the largest expected disaggregated cell cluster 220. Furthermore, each fluid channel 210 may have multipleAttorney Docket Number 161118.08701 / UR 6-25030corresponding separation devices 400. In some examples, each fluid channel 210 has two or more cascading separation devices 400. For example, the cascading separation devices 400 may have progressively smaller spacing between separation elements 230. In such a configuration, each separation device 400 may disaggregate the clusters 220 into smaller and smaller clusters 220. For example, a first separation device 400 may have separation elements 230 spaced 250 microns apart, and a subsequent separation device 400 may have separation elements 230 spaced 125 microns apart. In other examples, a subsequent separation device 400 may include separation elements 230 that are rotated with respect to the separation elements 230 of the first separation device 400 (e.g., by 90 degrees or other advantageous angular orientation).
[0022] The separation element 230 may have a sharpened or pointed leading edge 232 configured to separate the cell clusters 220 into smaller clusters 220 while avoiding damage to individual cells in the cluster 220. That is, the clusters 220 may be forced through the fluid channel 210 until the clusters 220 contact (e.g., impinge on) the leading edge 232 of the separation element 230. As the clusters 220 are forced into the leading edge 232, the separation element 230 may cause the clusters 220 to separate into smaller units, e.g., by applying a slicing force. The force may be sufficient to disaggregate the cluster 220 at a cell boundary, while avoiding damage to individual cells. In other examples, the separation element 230 may be a thin wire. That is, the leading edge 232 may not be sharpened or pointed, but the diameter of the wire may be small enough to effectively separate the clusters 220 into smaller units. In some examples, one or more separation elements 230 are configured to vibrate, e.g., to facilitate separation. In some examples, the vibration is longitudinal with respect to the flow of the solution 104. In other examples, the vibrating separation element 230 may reciprocate along the axis of flow of the solution 104.Attorney Docket Number 161118.08701 / UR 6-25030
[0023] The fluid channel 210 may be sized and / or shaped based on expected sizes of cell clusters 220 and / or desired sizes of disaggregated cell clusters 220. As shown the fluid channel 210 narrows (in a direction of flow) to be approximately the same size as a cell cluster 220. In other examples, the channel 210 may be sized to be an average expected size of a cell cluster 220 plus one or more standard deviations of expected cluster size. Other relative sizes of the channel 210 with respect to typical cluster sizes are also within the scope of this disclosure. As shown, each cluster 220 has spherical symmetry. Thus, the channel 210 is wide enough to accommodate the cluster 220 in any orientation. In other examples, the cluster 220 may be wider in one dimension than another dimension. In this example, the cluster 220 may need to rotate or otherwise reorient in order to fit within the channel 210. In this configuration, the cell clusters 220 necessarily impinge on the leading edge 232 of the separation element 230 disposed at a downstream portion of the fluid channel 210. Similarly, in configurations having multiple separation elements 230, the cell clusters 220 may necessarily impinge on the leading edge 232 of multiple separation elements 230. Thus, the maximum size of disaggregated clusters 220 may be defined by the spacing between the separation elements 230.
[0024] The disaggregation system 100 may include a pump to force the solution 104 through the fluid channel 210. That is, the pump may apply suction and / or pressure to force the solution 104 into the fluid channel 210 (or fluid channels 210 in configurations having multiple fluid channels 210). That is, the rate of flow in the channels 210 may be configured by the pressure (and / or suction) applied to the solution 104 by the pump. In other examples, each fluid channel 210 is configured to pump the solution 104 internally. For example, a disaggregation system 100 may use electroosmosis to induce the solution 104 to move through the channel 210. In other examples, the fluid channel 210 may be configured to create surface acoustic waves (e.g., using aAttorney Docket Number 161118.08701 / UR 6-25030piezo material) to induce the solution 104 to move through the channel 210. Other microfluidic pumping techniques are also within the scope of this disclosure.
[0025] In some examples, the pumping pressure and / or the size of the fluid channel 210 may be configured to avoid turbulent flow of the solution 104 through the channel 210. That is, the disaggregation system 100 as a whole may be configured such that the flow of the solution 104 is dominated by laminar flow, at least as the solution 104 passes though the fluid channel(s) 210. Furthermore, the disaggregation system 100 may be configured to achieve a high throughput, i.e., a high flow rate of solution 104 through the processing system 200 that is consistent with avoiding disruption (e.g., harm) to the clusters 220 (and or cells with the clusters 220) caused by turbulent flow.
[0026] Referring now to Fig. 3, a processing system 200 is shown that includes four fluid channels 210, each of which has a corresponding separation device (400, Fig. 4). As shown, each separation device 400 includes one separation element 230. However, as discussed above, each separation device 400 may include multiple separation elements 230. Furthermore, although the embodiment of Fig. 3 includes four fluid channels 210a-d (and four separation elements 230a-d), configurations having greater or fewer fluid channels 210 and / or greater or fewer separation elements 230 are also within the scope of this disclosure. In some examples, the processing system 200 includes many parallel fluid channels 210 to allow for high throughput. That is, the solution 104 in the first reservoir 102 may be simultaneously directed to all the parallel fluid channels 210, allowing for parallel disaggregation.
[0027] Fig. 4A shows an example separation device 400 having multiple separation elements 230. As perhaps can be best seen in the enlarged view of Fig. 4B, the example separation device of Fig. 4A includes six separation elements 230a-f. As shown, each separation element 230Attorney Docket Number 161118.08701 / UR 6-25030includes a cutting blade having a sharpened or pointed leading edge 232a-f. The separation elements 230 are arranged in an array and are clamped in a holder 234. The separation elements 230 may be spaced apart from each other by spacers and / or gaskets that define the spacing between the separation elements 230. The holder 234 may include guides 236 such as rods or similar elements configured to help keep the separation elements 230 aligned. Although the example separation device 400 includes six separation elements 230, separation devices 400 having more or fewer separation elements 230 are also within the scope of this disclosure. Furthermore, the spacing between separation elements 230 may be uniform or may be non-uniform. That is, the separation elements 230 may be evenly spaced apart or may be spaced apart by varying distances. Although the example separation device 400 of Figs. 4A and 4B show six separation elements 230, other configurations of separation devices 400 are also within the scope of this disclosure, including separation device 400 having a greater number or a smaller number of separation elements 230. For example, a separation device 400 may include hundreds of separation elements 230 or even more.
[0028] The holder 234, guides 236, and / or blades 230 may be made from autoclavable materials (e.g., stainless steel and / or polymers) to allow for sterilization. That is, the separation device 400 and / or components of the separation device 400 may be configured to withstand temperatures typically used to sterilize medical equipment. Alternatively (or additionally) the separation device 400 and / or components of the separation device 400 may be configured to withstand ultraviolet radiation and / or chemical sterilization agents. In an embodiment, the blades 230 are made from stainless steel and are spaced apart from each other by polymer separators or gaskets. The holder 234 and guides 236 may also be stainless steel, so that the entire unit can be autoclaved with or without disassembly.Attorney Docket Number 161118.08701 / UR 6-25030
[0029] Fig. 5 shows a flowchart 500 for an example method of disaggregation cell cultures. At step 502, the method includes receiving a disaggregation system 100. In some examples, the disaggregation system 100 is similar to, or the same as, the example disaggregation system 100 illustrated in Fig. 1. At step 504, the method includes adding a solution 104 (including cell clusters 220) to the first reservoir 102 of the disaggregation system 100. As described above, the reservoir 102 may be a bioreactor, a cell culture disk / plate, a flask, and so forth. In the case of a flask, adding the solution 104 to the flask may include simply decanting the solution 104 into the flask. At step 506, the method includes applying pressure to the solution 104 in the first reservoir 102, causing the solution 104 to flow into the one or more channels 210 and through the corresponding separation device 400. As described above, applying pressure to the solution 104 in the reservoir 102 may cause the solution 104 to pass through the one or more channels 210 at a rate that depends on the amount of pressure. The rate of flow may also depend on other factors, such as the number of channels 210, the aggregate width of all channels 210, the viscosity of the solution 104, and so forth. In some examples, causing the solution 104 to flow into the one or more channels 210 includes causing laminar flow of the solution 104 in the one or more channels 210. At step 508, the method includes receiving the at least two separate cell clusters 220 in a second reservoir 108.
[0030] While the invention has been described with specific embodiments, other alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it will be intended to include all such alternatives, modifications, and variations within the spirit and scope of the appended claims.
Claims
Attorney Docket Number 161118.08701 / UR 6-25030CLAIMS:
1. A disaggregation system (100) comprising:a first reservoir (102) configured to contain a solution (140) comprising a plurality of cell clusters (220); anda cluster processing system (200) comprising:at least one separation device (400) comprising a first plurality of separation elements (230) spaced apart from each other, each first separation element (230) comprising an edge portion (232) configured to apply a separation force to cell clusters (220) that contact the edge portion (232); andone or more channels (210) in fluid communication with the first reservoir (102), wherein the one or more channels (210) are configured to receive the solution (140) from the first reservoir (102) and direct the solution (140) to pass through a corresponding separation device (400), causing at least one cell cluster (220) of the plurality of cell clusters (220) to contact the edge portion (232) of one or more of the first plurality of separation elements (230), causing the at least one cell cluster (220) to disaggregate into at least two separate cell clusters (220).
2. The disaggregation system (100) of claim 1, wherein the one or more channels (210) are configured to receive the solution (140) from the first reservoir (102) and direct the solution (140) to pass through the corresponding separation device (400) without exposing the solution (140) to ambient air.
3. The disaggregation system (100) of any of claims 1 through 2, wherein the one or more channels (210) comprise a plurality of channels (210), each of the plurality of channels (210) having a corresponding separation device (400).
4. The disaggregation system (100) of any of claims 1 through 3, wherein each separation device (400) comprises an array of first separation elements (230) that are evenly spaced from each other by a distance.Attorney Docket Number 161118.08701 / UR 6-250305. The disaggregation system (100) of any of claims 1 through 4, wherein the at least one separation device (400) comprises at least one vibrating separation element (230).
6. The disaggregation system (100) of any of claims 1 through 5, wherein:each of the first plurality of separation elements (230) comprises a cutting blade; and the edge portion (232) of each of the first plurality of separation elements (230) comprises a sharpened edge of the cutting blade.
7. The disaggregation system (100) of any of claims 1 through 6, wherein the at least one separation device (400) comprises components that are autoclavable.
8. The disaggregation system (100) of any of claims 1 through 7, wherein the at least one separation device (400) comprises a second plurality of separation elements (230) spaced apart from each other, the second plurality of separation elements (230) disposed downstream of the first plurality of separation elements (230), such that the solution (140) passes through the first plurality of separation elements (230) and the second plurality of separation elements (230).
9. The disaggregation system (100) of claim 8, wherein the first plurality of separation elements (230) and the second plurality of separation elements (230) have a different spacing and / or are disposed at a different angular orientation.
10. The disaggregation system (100) of any of claims 1 through 9, further comprising: a second reservoir (102); anda fluid pump configured to pump the solution (140) from the first reservoir (102) to the one or more channels (210), causing the solution (140) to pass through the corresponding separation device (400) to the second reservoir (102).
11. The disaggregation system (100) of claim 10, wherein the fluid pump is configured to cause laminar flow of the solution (140) through the one or more channels (210).Attorney Docket Number 161118.08701 / UR 6-2503012. The disaggregation system (100) of any of claims 10 through 11, wherein the fluid pump causes the solution (140) to pass through the corresponding separation device (400) using electroosmosis or surface acoustic waves.
13. A method of disaggregation cell cultures, the method comprising:receiving the disaggregation system (100) of any of claims 1 through 12;adding the solution (140) comprising the plurality of cell clusters (220) to the first reservoir (102);applying pressure to the solution (140) in the first reservoir (102), causing the solution (140) to flow into the one or more channels (210) and through the corresponding separation device (400); andreceiving the at least two separate cell clusters (220) in a second reservoir (102).
14. The method of claim 13, wherein causing the solution (140) to flow into the one or more channels (210) comprises causing laminar flow of the solution (140) in the one or more channels (210).