Microfluidic cell culture of patient-derived tumor cell spheroids

Abstract

Provided herein are methods for culturing patient-derived tumor cell spheroids in a three-dimensional microfluidic device. The method comprises mincing primary tumor sample in a medium supplemented with serum; treating the minced primary tumor sample with a composition comprising an enzyme; collecting tumor spheroids having a diameter of 10 μm to 500 μm from the enzyme treated sample; suspending the tumor spheroids in biocompatible gel; and culturing the tumor spheroids in a three dimensional microfluidic device. Methods for identifying an agent for treating cancer and microfluidic devices that allow for the simultaneous exposure of the cultured patient-derived primary tumor cell spheroids to a treatment of choice and to control treatment are also provided.

1 .- 24 . (canceled) 
     
     
         25 . A method for identifying an agent for treating cancer, the method comprising:
 culturing patient-derived tumor cell spheroids in a three-dimensional microfluidic device in the presence and absence of a first test agent, detecting a change in the tumor cell spheroid culture indicative of a condition that is likely to reduce proliferation and/or dispersion of the tumor cell spheroids in the presence of the first test agent as compared to the absence of the first test agent;   wherein if the change in the tumor cell spheroid culture is indicative of a condition that is likely to reduce the proliferation and/or dispersion of the tumor cell spheroids in the presence of the first test agent as compared to the absence of the first test agent, then the first test agent can be used to treat cancer,   wherein the culturing of patient-derived tumor cell spheroids comprises:   mincing a primary tumor sample in a medium supplemented with serum;   treating the minced primary tumor sample with a composition comprising an enzyme;   collecting tumor spheroids having a diameter of 10 μm to 500 μm from the enzyme treated sample;   suspending the tumor spheroid in biocompatible gel; and   culturing the tumor spheroids in a three dimensional microfluidic device.   
     
     
         26 . The method of  claim 25 , wherein culturing patient-derived tumor cell spheroids in the presence of the first test agent comprises introducing the first test agent into one or more fluid channels of the three-dimensional microfluidic device, wherein the three-dimensional microfluidic device comprises one or more fluid channels flanked by one or more gel cage regions, and wherein the one or more gel cage regions of the device comprises a gel in which the tumor spheroids are embedded; and culturing the tumor spheroids under suitable culture conditions. 
     
     
         27 . The method of  claim 25 , wherein the change in the tumor cell spheroid culture is detected chemically, physically, or a combination thereof. 
     
     
         28 . The method of  claim 25 , wherein the change in the tumor cell spheroid culture is detected visually. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 25 , wherein the change in the tumor cell spheroid culture is:
 i) a clustering of immune cells around one or more tumor cell spheroids in the culture; or   ii) a decrease in size and/or number of cells of one or more tumor cell spheroids in the culture.   
     
     
         31 .- 32 . (canceled) 
     
     
         33 . The method of  claim 27 , wherein the change in the tumor cell spheroid culture is determined by detection of the presence of a biological molecule secreted into the culture supernatant. 
     
     
         34 . The method of  claim 33 , wherein the biological molecule is a protein, carbohydrate, lipid, nucleic acid, metabolite, a chemokine, a cytokine, or a combination thereof. 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 25 , comprising obtaining a sample of tumor cell spheroid culture supernatant. 
     
     
         37 . The method of  claim 25 , comprising detecting a cytokine profile or chemokine profile in the tumor cell spheroid culture supernatant. 
     
     
         38 . The method of  claim 25 , wherein the first test agent inhibits epithelial-mesenchymal transition (EMT). 
     
     
         39 . The method of  claim 25 , wherein the first test agent is:
 i) a small molecule, a nucleic acid molecule, an RNAi agent, an aptamer, a protein or a peptide, an antibody or antigen-binding antibody fragment, a ligand or receptor-binding protein, a gene therapy vector, or a combination thereof;   ii) a chemotherapeutic agent, an immunomodulatory agent, or radiation;   iii) an immune modulator; and/or   iv) an immune checkpoint inhibitor.   
     
     
         40 .- 43 . (canceled) 
     
     
         44 . The method of  claim 25 , wherein the patient-derived tumor cell spheroids are cultured in the presence of a second test agent. 
     
     
         45 . The method of  claim 44 , wherein the second test agent is:
 i) an anti-cancer agent, wherein the anti-cancer agent is a chemotherapeutic agent, an immunomodulatory agent, or radiation;   ii) an immune modulator; and/or   iii) an immune checkpoint inhibitor.   
     
     
         46 .- 48 . (canceled) 
     
     
         49 . A microfluidic device comprising:
 a substrate comprised of an optically transparent material and further comprising   i) a first gel cage region and a second gel cage region   ii) a first fluid channel and a second fluid channel;   iii) one or more fluid channel inlets;   iv) one or more fluid channel outlets; and   v) a plurality of posts;   wherein   a portion of the first gel cage region is flanked by a portion of the second gel cage region, thereby creating a gel cage region-gel cage region interface region;   the first and second gel cage regions are separated by a barrier which does not allow intermixing between components present in the two gel cage regions;   a portion of the first gel cage region is flanked by all or a portion of the first fluid channel, thereby creating a first gel cage region-fluid channel interface region;   a portion of the second gel cage region is flanked by all or a portion of the second fluid channel, thereby creating a second gel cage region-fluid channel interface region; and   each gel cage region comprises one row of posts along the length of the gel cage region at the first and second gel cage region-fluid channel interfaces.   
     
     
         50 . The device of  claim 49 , wherein each gel cage region has a height of at least 500 μm. 
     
     
         51 . (canceled) 
     
     
         52 . The device of  claim 49 , wherein each gel cage region has a cuboidal shape. 
     
     
         53 . The device of  claim 49 , wherein the fluid channel outlet is configured to receive a tube and/or a needle. 
     
     
         54 . The device of  claim 54 , wherein the tube and/or needle is in fluid connection with the fluid channel such that a sample of conditioned medium can be removed from the fluid channel via the fluid channel outlet. 
     
     
         55 . A method for identifying an agent for treating cancer, the method comprising:
 a) introducing a test agent into the first fluid channel of the device of  claim 49 , wherein each gel cage region of the device comprises a gel in which the tumor spheroids are embedded; and   b) detecting a signal in the first cage region indicative of a reduction in proliferation and/or dispersion of the tumor cell spheroids in the first gel cage region as compared that in the second gel cage region;   wherein if the signal in the first cage region is indicative of a condition that is likely to reduce proliferation and/or dispersion of the tumor cell spheroids in the first gel cage region as compared that in the second gel cage region, then the agent can be used to treat cancer.   
     
     
         56 . A method for treating cancer in a subject, the method comprising:
 a) obtaining a tumor sample from the subject;   b) identifying an agent that can be used to treat cancer in the subject according to the method of  claim 25 ; and   c) administering the agent to the subject.