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US-12624327-B2 - Cell culture insert

US12624327B2US 12624327 B2US12624327 B2US 12624327B2US-12624327-B2

Abstract

A cell culture insert for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts. The cell culture insert includes a porous membrane and one or more sidewalls that are non-adherent to cells and cause the cells in the insert to associate with each other and form spheroids.

Inventors

  • Gregory Roger Martin
  • Allison Jean Tanner

Assignees

  • CORNING INCORPORATED

Dates

Publication Date
20260512
Application Date
20240415

Claims (9)

  1. 1 . An apparatus for culturing spheroids, comprising: a multiwell cell culture plate comprising wells, each well being a reservoir; and an insert for at least one well of the multiwell cell culture plate, wherein the insert comprises: a body having a first open end; a second end wherein the second end defines an opening; and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls comprise a lower sidewall portion proximate the second end; wherein the lower sidewall portion of the one or more sidewalls is sloped; wherein the opening of the second end comprises a porous membrane, the porous membrane comprising an array of microwells; wherein each microwell of the insert comprises a diametric dimension in a range from 100 μm to 1000 μm, a sidewall having a depth in a range from 100 μm to 500 μm, and the sidewall and a porous membrane bottom of each microwell are non-adherent to cells to promote cells cultured in the microwell to form spheroids.
  2. 2 . The apparatus of claim 1 , wherein the sidewall of each microwell is sloped.
  3. 3 . The apparatus of claim 1 , wherein the porous membrane bottom is arcuate.
  4. 4 . The apparatus of claim 1 , wherein the insert further comprises a ledge extending around the perimeter of the first open edge.
  5. 5 . The apparatus of claim 1 , wherein at least a portion of the reservoir is gas permeable.
  6. 6 . The apparatus of claim 1 , wherein the diametric dimension is in a range from 100 μm to 500 μm.
  7. 7 . The apparatus of claim 1 , wherein the diametric dimension is in a range from 200 μm to 750 μm.
  8. 8 . The apparatus of claim 1 , wherein the multiwell cell culture plate comprises polystyrene, polypropylene, polyethylene, or polyvinyl chloride.
  9. 9 . The apparatus of claim 1 , wherein the sidewall of each microwell is non-adherent to cells.

Description

This application is a continuation of U.S. patent application Ser. No. 17/691,511 filed on Mar. 10, 2022 which is a continuation of U.S. patent application Ser. No. 15/492,730 filed on Apr. 20, 2017, which is a continuation application of International Patent Application Serial No. PCT/US15/58053 filed on Oct. 29, 2015, which claims the benefit of priority to U.S. Provisional Application Ser. No. 62/069,996, filed on Oct. 29, 2014, the contents of which are relied upon and incorporated herein by reference in their entirety, and the benefit of priority under 35 U.S.C. § 120 is hereby claimed. FIELD The present disclosure relates to apparatuses, systems and methods for culturing cells. TECHNICAL BACKGROUND The present disclosure relates generally to cell culture inserts for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts. Spheroids are three-dimensional (3D) cell clusters that can provide more in vivo-like functions to the cells than cells cultured as monolayers in 2D cell culture systems. For certain cell types, such as hepatocytes, spheroids can attain and retain better in vivo-like functionality than their 2D cultured counterparts. BRIEF SUMMARY In accordance with various embodiments of the present disclosure, cell culture inserts for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts are described. In some embodiments, a cell culture insert as described herein can be nested in another cell culture insert or another cell culture insert can be nested in a cell culture insert as described herein. In various embodiments, the disclosure describes a cell culture insert having a body and a porous membrane. The body has a first open end, a second end wherein the second end defines an opening having a diametric dimension in a range from 100 μm to 1000 μm (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and all values and ranges therein between; e.g., 200 to 500, 200 to 700, 400 to 600, etc.), and one or more sidewalls extending from the first open end to the second end. The one or more sidewalls, or a portion thereof, are sloped (e.g., greater than 5 degrees from perpendicular relative to first or second ends; e.g., >10°, >20°, >30°, >40°, >50°, etc.). The sidewalls, if sloped, are preferably sloped such that the diameter at the second is less than the diameter at the first open end. A porous membrane is disposed over the opening of the second end. In various embodiments, the disclosure describes a cell culture insert having a body and a porous membrane. The body has a first open end, a second end wherein the second end defines an opening, and one or more sidewalls extending from the first open end to the second end. The one or more sidewalls, or a portion thereof, are sloped. The porous membrane is disposed over the opening of the second end and is non-adherent to cells. In various embodiments, the disclosure describes a permeable support device configured to be at least partially inserted into a reservoir of a cell culture device. The permeable support device comprises a first well having a tapered shape and bottom at least partially defined by a first permeable support. In various embodiments, the disclosure described a cell culture insert. The insert includes a body having a first open end, a second end, and one or more sidewalls extending from the first open end to the second end. The second end comprises a substrate having an array of microwells defining wells with a porous membrane at the bottom, wherein at least a portion of a substrate having an array of microwells defining a well is sloped. In various embodiments, the disclosure describes a nested permeable support device comprising a first well having a tapered shape and a bottom portion at least partially defined by a first permeable support, and a reservoir having a bottom located below the first well. In embodiments the reservoir is made from gas permeable material or has an array of microwells or both. In various embodiments, the disclosure describes a nested permeable support device comprising a first well, a second well and a third well. The first well has a bottom, wherein at least a portion of the bottom is formed by a first porous membrane. The second well has a bottom, wherein at least a portion of the bottom is formed by a second porous membrane. A portion of either the first or the second permeable support may comprise an arcuate shape. The second well and the second permeable support are located below the first well and the first permeable support. The third well has a non-liquid permeable, gas permeable bottom which is located below the second well and the second permeable support. In another embodiment, the disclosure describes a nested permeable support device comprising a first well, a second well, and a third well. The first well has a substrate forming a microwell array, t