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US-20260125627-A1 - METHOD AND SYSTEM FOR CULTIVATING CELLS IN MEDIA-EXCHANGING WELLS

US20260125627A1US 20260125627 A1US20260125627 A1US 20260125627A1US-20260125627-A1

Abstract

Methods, systems, and apparatus for cultivating cells in media-exchanging wells. In an exemplary method of cell cultivation, a device ( 50 ) may be selected that includes a row of wells ( 52 ), and a first reservoir ( 56 ) and a second reservoir ( 58 ) located at opposite ends of the row of wells ( 52 ). Each well ( 52 ) may have a lower portion and an upper portion. The lower portion of each well ( 52 ) of at least two of the wells ( 52 ) may contain a cell culture in contact with a liquid. Liquid may be transferred between the first reservoir ( 56 ) and the second reservoir ( 58 ) at least partly along a flow path ( 62 ) defined by the device and extending through the upper portion of each well ( 52 ) of the row of wells ( 52 ), such that molecules of the media are exchanged between or among the at least two wells ( 52 ).

Inventors

  • Oksana Sirenko
  • Andreas Kenda
  • Josef Atzler

Assignees

  • Oksana Sirenko
  • Andreas Kenda
  • Josef Atzler

Dates

Publication Date
20260507
Application Date
20251202

Claims (20)

  1. 1 . A method of cell cultivation, the method comprising: (a) selecting a device ( 50 ) including a row of wells ( 52 ), and a first reservoir ( 56 ) and a second reservoir ( 58 ) located at opposite ends of the row of wells ( 52 ), wherein each well ( 52 ) has a lower portion ( 70 ) and an upper portion ( 68 ), and wherein the lower portion ( 70 ) of each well ( 52 ) of at least two of the wells ( 52 ) contains a cell culture in contact with liquid ( 104 ); and (b) transferring the liquid ( 104 ) between the first reservoir ( 56 ) and the second reservoir ( 58 ) at least partly along a flow path ( 62 ) defined by the device ( 50 ) and extending through the upper portion of each well ( 52 ) of the row of wells ( 52 ), such that the liquid ( 104 ) is exchanged between or among the at least two wells ( 52 ).
  2. 2 . The method of claim 1 , wherein the flow path ( 62 ) extends through a plurality of passages ( 64 ), each passage ( 64 ) creating fluid communication between the upper portions of a pair of adjacent wells ( 52 ) of the row of wells ( 52 ) or between an upper portion of one of the reservoirs and the upper portion of an adjacent well ( 52 ) of the row of wells ( 52 ).
  3. 3 . The method of claim 1 , wherein transferring the liquid ( 104 ) includes creating turbulence that encourages intra-well mixing of liquid in the lower and upper portions of each well ( 52 ) of the row of wells ( 52 ).
  4. 4 . The method of claim 1 , wherein transferring the liquid ( 104 ) includes driving liquid flow with gravity.
  5. 5 . The method of claim 4 , wherein driving liquid flow includes tilting the device ( 50 ) to elevate the first reservoir ( 56 ) relative to the second reservoir ( 58 ), such that the liquid ( 104 ) flows along the flow path ( 62 ) to the row of wells ( 52 ) from the first reservoir ( 56 ) and from the row of wells ( 52 ) to the second reservoir ( 58 ).
  6. 6 . The method of claim 5 , wherein transferring the liquid ( 104 ) includes rocking the device ( 50 ) such that gravity drives flow of the liquid ( 104 ) alternately in opposite directions along the flow path ( 62 ) between the first reservoir ( 56 ) and the second reservoir ( 58 ).
  7. 7 . The method of claim 1 , further comprising introducing at least one three-dimensional cluster of cells into at least one of the at least two wells ( 52 ).
  8. 8 . A device ( 50 ) for cell culture, the device ( 50 ) comprising: (a) a row of wells ( 52 ) each having a lower portion and an upper portion, the lower portion being configured to contain a cell culture; and (b) a first reservoir ( 56 ) and a second reservoir ( 58 ) located at opposite ends of the row of wells ( 52 ); wherein the device ( 50 ) defines a flow path ( 62 ) for liquid that extends from the first reservoir ( 56 ) to the second reservoir ( 58 ) via the row of wells ( 52 ) and passes through the upper portion of each well ( 52 ) of the row of wells ( 52 ).
  9. 9 . The device ( 50 ) of claim 8 , wherein the lower portion of each well ( 52 ) is configured to receive liquid only via the upper portion of the well ( 52 ).
  10. 10 . The device ( 50 ) of claim 8 , wherein one or more of the wells ( 52 ) of the row of wells ( 52 ) has a flat bottom inside the one or more of the wells ( 52 ).
  11. 11 . The device ( 50 ) of claim 8 , wherein the lower portion of at least one well ( 52 ) of the row of wells ( 52 ) includes a transparent, lateral optical window ( 90 ) having an outer surface and an inner surface that are planar, and wherein each of the outer and inner surfaces is parallel to a longitudinal axis ( 60 ) of the device ( 50 ).
  12. 12 . The device ( 50 ) of claim 8 , wherein at least one well ( 52 ) of the row of wells ( 52 ) has a concave inner surface region at the bottom of the well ( 52 ).
  13. 13 . The device ( 50 ) of claim 12 , wherein the concave inner surface region is concave in a vertical plane that contains a longitudinal axis ( 60 ) defined by the row of wells ( 52 ).
  14. 14 . The device ( 50 ) of claim 12 , wherein the concave inner surface region is concave in a vertical plane that is orthogonal to a longitudinal axis ( 60 ) defined by the row of wells ( 52 ).
  15. 15 . The device ( 50 ) of claim 8 , further comprising a lid ( 100 ) configured to cover the row of wells ( 52 ), the first reservoir ( 56 ), and the second reservoir ( 58 ).
  16. 16 . The device ( 50 ) of claim 8 , wherein each well ( 52 ) of the one or more wells ( 52 ) has a bottom outer surface region that is flat or that is rounded in a pair of vertical planes that are orthogonal to one another.
  17. 17 . An apparatus comprising a frame ( 42 ) to removably hold the device ( 50 ) of claim 8 in alignment with another of the device ( 50 ).
  18. 18 . The apparatus of claim 17 , wherein a center-to-center spacing of wells ( 52 ) within each row of wells ( 52 ) is the same as a center-to-center spacing of wells ( 52 ) between adjacent rows of wells ( 52 ).
  19. 19 . The apparatus of claim 17 , further comprising a lid ( 100 ) configured to fit over at least one of the devices ( 50 ) and cover the wells ( 52 ) of the at least one of the devices ( 50 ).
  20. 20 . The apparatus of claim 19 , wherein the lid ( 100 ) is configured to cover all the wells ( 52 ) and each of the reservoirs of the device ( 50 ).

Description

RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 17/631,917, filed Feb. 1, 2022, which is a National Stage Application of PCT/US2020/044545, filed Jul. 31, 2020, which claims the benefit of U.S. Provisional Application No. 62/882,363, filed Aug. 2, 2019, the entire disclosures of which are incorporated herein by reference in their entireties. INTRODUCTION There is a pressing need to increase the biological relevance of cell-based assays. To meet this need, researchers have turned with growing interest to organoids. An organoid is a three-dimensional cluster of cells of different types produced in vitro and having some resemblance to an organ, such as exhibiting a realistic histology of organ-specific tissue. The cluster of cells may be generated by seeding a matrix, such as a hydrogel, with a small number of stem cells. The stem cells then proliferate, differentiate, and self-organize within the matrix, while using the matrix as a scaffold. Organoids resembling tissue from the brain, heart, intestine, kidney, liver, and stomach, among others, have been generated in “organ-on-a-chip” systems. In some cases, a plurality of different types of organoids, such as organoids representing brain, heart, and liver, are cultured in the same device under conditions of media exchange. This creates a “body-on-a-chip” system in which a set of organoids representing an organ system can interact one another at a distance via signaling molecules. Both organ-on-a-chip and body-on-a chip systems have produced promising results, thereby driving a fundamental shift from animal tests to three-dimensional (3D) cell-based models for studying biological processes, modeling disease, and testing drugs. These 3D cell-based models are attractive to researchers because they can reduce the hands-on time and cost for experiments. However, currently available technologies for organoid culture are typically very complex, not highly reproducible, often incompatible with automation, and not well characterized. New methods, systems, and apparatus are needed for cultivating separate cell cultures under conditions of media exchange to permit interaction between the cultures. SUMMARY The present disclosure provides methods, systems, and apparatus for cultivating cells in media-exchanging wells. In an exemplary method of cell cultivation, a device may be selected that includes a row of wells, and a first reservoir and a second reservoir located at opposite ends of the row of wells. Each well may have a lower portion and an upper portion. The lower portion of each well of at least two of the wells may contain a cell culture in contact with a medium. Liquid may be transferred between the first reservoir and the second reservoir at least partly along a flow path defined by the device and extending through the upper portion of each well of the row of wells, such that molecules of the media are exchanged between or among the at least two wells. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an isometric view of an exemplary cell-cultivation apparatus including multiple copies of a culture vessel that are held in alignment with one another by a frame in a standard microplate format, where each copy of the culture vessel defines a flow path for liquid through an upper region of a row of communicating wells, to permit exchange of media, and particularly molecules and/or cellular products therein, between two or more of the wells containing respective cell cultures. FIG. 2 is an isometric view of one of the copies of the culture vessel of FIG. 1 taken in isolation. FIG. 3 is a top view of the culture vessel of FIG. 2. FIG. 4 is a side view of the culture vessel of FIG. 2. FIG. 5 is a sectional view of the culture vessel of FIG. 2, taken generally along line 5-5 of FIG. 3. FIG. 6 is another sectional view of the culture vessel of FIG. 2, taken generally along line 6-6 of FIG. 3. FIG. 7 is yet another sectional view of the culture vessel of FIG. 2, taken generally along line 7-7 of FIG. 3. FIG. 8 is a view of selected aspects of an exemplary light sheet microscopy configuration for capturing an image of cells contained in one of the wells of the culture vessel of FIG. 2. FIG. 9 is a sectional view of the culture vessel of FIG. 2, taken as in FIG. 5, with the culture vessel oriented horizontally, each well and a first reservoir of the device holding a separate volume of media, and each well containing a separate 3D cell culture. FIG. 10 is another sectional view of the culture vessel of FIG. 2, taken as in FIG. 9, except with the culture vessel supported at an incline by a rocking platform after net flow of liquid through the row of wells from the first reservoir to a second reservoir of the culture vessel. FIG. 11 is still another sectional view of the culture vessel of FIG. 2, taken generally as in FIG. 10, except with the culture vessel supported at a fixed incline, to drive flow of liquid with gravity from the first