Microfluidic platform for the rapid production of organoids/spheroids for compound screening

Abstract

The present disclosure provides a method of producing uniformly sized organoids/multicellular spheroids using a microfluidic device having an array of microwells. The method involves several successive steps. First, a microfluidic device containing parallel rows of microwells that are connected with a supplying channel is filled with a wetting agent. The wetting agent is a liquid that is immiscible in water. For example, the wetting agent may be an organic liquid such as oil. In the next step, the agent in the supplying channel and the microwells is replaced with a suspension of cells in an aqueous solution that contains a precursor for a hydrogel. Next, the aqueous phase in the supplying channel is replaced with the agent, which leads to the formation of an array of droplets of cell suspension in the hydrogel precursor solution, which were compartmentalized in the wells. The droplets are then transformed into cell-laden hydrogels. Subsequently, the agent in the supplying channel is replaced with the cell culture medium continuously flowing through the microfluidic device and the cells within the hydrogels are transformed into multicellular spheroids.

1 .- 35 . (canceled) 
     
     
         36 . A microfluidic device for producing multicellular aggregates in hydrogel scaffolds comprising:
 at least one row having at least one microwell, each of the at least one microwell having a diameter and a height;   for each row, a supplying channel having a height, the supplying channel spanning along a length of the row and having an entry opening at one end of the supplying channel and an exit opening at the opposite end of the supplying channel,   wherein each microwell is in flow connection with the corresponding supplying channel.   
     
     
         37 . The microfluidic device of  claim 36  wherein the at least one microwell is a plurality of microwells. 
     
     
         38 . The microfluidic device of  claim 36  wherein the height of each microwell is about larger than the diameter of each microwell. 
     
     
         39 . The microfluidic device of  claim 38  wherein the height of each microwell is about at least 20% larger than the diameter of the each microwell. 
     
     
         40 . The microfluidic device of  claim 39  wherein the height of each microwell is greater than the height of the supplying channel. 
     
     
         41 . The microfluidic device of  claim 40  wherein a ratio of the height of each microwell to the height of the supplying channel is at least about 4.5. 
     
     
         42 . The device of  claim 41  wherein the ratio of the height of the microwells to the height of the supplying channel is between about 4.5 to about 5.5. 
     
     
         43 .- 51 . (canceled) 
     
     
         52 . The microfluidic device of  claim 36  wherein each microwell is connected to the supply channel downstream of the microwell. 
     
     
         53 . The microfluidic device of  claim 36  wherein each microwell is cylindrical in shape. 
     
     
         54 . The microfluidic device of  claim 39  wherein the microwell has a diameter between about 100-1000 um and a height between about 120-1200 um. 
     
     
         55 . The microfluidic device of  claim 36  wherein the microfluidic device is for use with a wetting agent. 
     
     
         56 . The microfluidic device of  claim 41  wherein the microfluidic device is for use with a fluorinated oil as a wetting agent. 
     
     
         57 . The microfluidic device of  claim 56  wherein the microfluidic device is made with polydimethylsiloxane (PDMS). 
     
     
         58 . The microfluidic device of  claim 37  wherein the diameter of the plurality of microwells is varied, thereby producing the multicellular aggregates having a diversity of diameters. 
     
     
         59 . The microfluidic device of  claim 37  wherein the height of each microwell is about larger than the diameter of each microwell. 
     
     
         60 . The microfluidic device of  claim 59  wherein the height of each microwell is about at least 20% larger than the diameter of the each microwell. 
     
     
         61 . The microfluidic device of  claim 60  wherein the microwell has a diameter between about 100-1000 um and a height between about 120-1200 um. 
     
     
         62 . The microfluidic device of  claim 37  wherein the height of each microwell is greater than the height of the supplying channel. 
     
     
         63 . The microfluidic device of  claim 62  wherein a ratio of the height of each microwell to the height of the supplying channel is at least about 4.5. 
     
     
         64 . The microfluidic device of  claim 62  wherein the ratio of the height of the microwells to the height of the supplying channel is between about 4.5 to about 5.5.