Devices and methods for generation and culture of 3d cell aggregates

Abstract

The present disclosure relates to apparatuses, systems and methods for culturing cells. In particular, devices and methods are provided for generation and culture of 3d cell aggregates.

What is claimed is: 
     
         1 . A cell culture substrate comprising an array of microwells, each microwell comprising an opening into the microwell defined by a rounded top well edge, at least one microwell side-wall, and a rounded well-bottom. 
     
     
         2 . The cell culture substrate of  claim 1  wherein each microwell, in cross-section, has a sinusoidal well shape. 
     
     
         3 . The cell culture substrate of  claim 1  further comprising one or more ridges, wherein the ridge is round, angular, needle-shaped or hexagonal. 
     
     
         4 . The cell culture substrate of  claim 1  further comprising one or more fissures, wherein the fissure is round, angular, needle-shaped or hexagonal. 
     
     
         5 . The cell culture substrate of  claim 1  wherein the rounded well bottom has a thickness of between 10 and 100 μm. 
     
     
         6 . The cell culture substrate of  claim 1  wherein walls of the microwells are relatively thicker proximate to the opening into the microwell and relatively thinner at the bottom of the microwell. 
     
     
         7 . The cell culture substrate of  claim 1  wherein the substrate comprises 2-10000 of said microwells per square centimeter of surface of said cell culture substrate. 
     
     
         8 . The cell culture substrate of  claim 1  wherein at least the rounded well-bottom comprises a non-adherent surface. 
     
     
         9 . The cell culture substrate of  claim 1  wherein at least the rounded well-bottom comprises gas permeable material. 
     
     
         10 . The cell culture substrate of  claim 1  wherein the microwell sidewalls are discontinuous. 
     
     
         11 . The cell culture substrate of  claim 10  wherein the microwell sidewalls are corrugated. 
     
     
         12 . The cell culture substrate of  claim 1  wherein the cell culture substrate comprises at least a portion of a cell culture container. 
     
     
         13 . The cell culture substrate of  claim 12  wherein the cell culture container is selected from the group consisting of a multiwell plate, a dish, a flask, a tube, a multi-layer flask, a soft-sided flask and a bag. 
     
     
         14 . The cell culture substrate of  claim 1 , wherein microwells are configured to allow fluid communication between at least one of said microwells and a single liquid reservoir. 
     
     
         15 . The cell culture substrate of  claim 12  wherein the cell culture container further comprises a mesh placed on top of the array of microwells. 
     
     
         16 . The cell culture substrate of  claim 13  wherein the cell culture container further comprises a mesh placed on top of the array of microwells. 
     
     
         17 . The cell culture substrate of  claim 1  wherein the opening into the microwell has an effective diameter D top , the rounded well-bottom has a nadir, and each microwell has one or more sidewalls; the one or more sidewalls have a height H extending from the nadir of the rounded well-bottom to the opening into the microwell, wherein each microwell has an effective diameter halfway between the opening into the microwell and the nadir of the rounded well-bottom of D half-way , and wherein the ratio of D top :D half-way  is 1.5:2.5. 
     
     
         18 . The cell culture substrate of  claim 17  wherein D top  is from 200 μm to 500 μm. 
     
     
         19 . The cell culture substrate of  claim 17  wherein height H is from 100 μm to 500 μm. 
     
     
         20 . The cell culture substrate of  claim 17  wherein H=0.7 to 1.3 D half-way . 
     
     
         21 . The cell culture substrate of  claim 17 , wherein D half-way  is 200 to 1000 μm. 
     
     
         22 . A method of culturing spheroids comprising: charging the cell culture container of  claim 10  with culture media and spheroid forming cells.