Methods and Apparatuses for Patient-Derived Micro-Organospheres

Abstract

Micro-Organospheres, including Patient-Derived Micro-Organospheres (PMOSs), apparatuses and methods of making them, and apparatuses and methods of using them. Also described herein are methods and systems for screening a patient using these Patient-Derived Micro-Organospheres, including personalized therapies.

1 . A method, the method comprising:
 forming an unpolymerized mixture comprising a dissociated tissue sample and a fluid matrix material, wherein the dissociated tissue sample comprises a single biopsy or resected patient sample, and wherein the dissociated tissue sample comprises tumor cells and one or more of mesenchymal cells, endothelial cells, fibroblasts, and immune cells;   forming a plurality of spherical droplets of the unpolymerized mixture by moving a continuous stream of the unpolymerized mixture at a first flow rate across one or more streams of an immiscible fluid moving at a second flow rate that is different than the first flow rate;   polymerizing the droplets to form a plurality of Patient-Derived Micro-Organospheres each having a diameter of between 50 and 500 μm with between 1 and 500 dissociated cells distributed therein; and   culturing the plurality of Patient-Derived Micro-Organospheres for between 1-14 days of forming the droplets to form structured clusters of cells replicating structures of tissue from which they were biopsied or resected.   
     
     
         2 . The method of  claim 1 , comprising modifying the dissociated cells within the dissociated tissue sample prior to forming the droplets. 
     
     
         3 . The method of  claim 1 , wherein forming the plurality of droplets comprises forming a plurality of droplets of the unpolymerized mixture of uniform size with less than 25% variation in size. 
     
     
         4 . The method of  claim 1 , wherein the dissociated tissue sample comprises dissociated cells that are not stem cells. 
     
     
         5 . The method of  claim 1 , wherein the dissociated tissue sample comprises a biopsy sample from a metastatic tumor. 
     
     
         6 . The method of  claim 1 , wherein the dissociated tissue sample comprises a clinical tumor sample, further wherein the clinical tumor sample comprises both cancer cells and stroma cells. 
     
     
         7 . The method of  claim 1 , wherein the dissociated tissue sample are distributed within the fluid matrix material to a density of less than 5×10 6  cells/ml. 
     
     
         8 . The method of  claim 1 , comprising immediately removing the immiscible fluid from the Patient-Derived Micro-Organospheres after forming the plurality of Patient-Derived Micro-Organospheres. 
     
     
         9 . The method of  claim 1 , wherein the dissociated tissue sample is combined with the fluid matrix material within six hours of removing a tissue sample from a patient. 
     
     
         10 . The method of  claim 1 , wherein the immiscible fluid is maintained at a temperature of greater than 20 degrees C. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , comprising screening the plurality of Patient-Derived Micro-Organospheres with a plurality of drug compositions within 14 days of acquiring the biopsy or resected patient sample. 
     
     
         13 . The method of  claim 1 , comprising screening the plurality of Patient-Derived Micro-Organospheres with hundreds of drug compositions within 14 days of acquiring the biopsy or resected patient sample. 
     
     
         14 . The method of  claim 1 , wherein culturing comprises culturing the plurality of Patient-Derived Micro-Organospheres for between 2-14 days of forming the droplets to form budding clusters of cells replicating the structures of the tissue from which they were biopsied or resected. 
     
     
         15 . The method of  claim 1 , wherein the dissociated tissue sample comprises an epithelial adenocarcinoma tissue, further wherein culturing comprises culturing the plurality of Patient-Derived Micro-Organospheres for between 2-14 days of forming the droplets to form a hollow structures of cells replicating the structures of the tissue from which they were biopsied or resected. 
     
     
         16 . The method of  claim 1 , wherein the dissociated tissue sample comprises an epithelial adenocarcinoma tissue, further wherein culturing comprises culturing the plurality of Patient-Derived Micro-Organospheres for between 2-14 days of forming the droplets to form a hollow structures of cells replicating the structures of the epithelial adenocarcinoma tissue from which they were biopsied or resected. 
     
     
         17 . The method of  claim 1 , comprising monitoring the corresponding flow rates of the unpolymerized mixture and the immiscible fluid to maintain a continuous and uninterrupted stream of unpolymerized mixture during the method. 
     
     
         18 . The method of  claim 1 , comprising pressurizing the unpolymerized mixture. 
     
     
         19 . The method of  claim 1 , wherein the fluid matrix material comprises a substrate basement membrane matrix. 
     
     
         20 . The method of  claim 1 , comprising culturing the plurality of Patient-Derived Micro-Organospheres for between 2-10 days of forming the droplets to form structured clusters of cells replicating the structures of the tissue from which they were biopsied or resected. 
     
     
         21 . The method of  claim 1 , wherein the polymerizing comprises crosslinking the fluid matrix material. 
     
     
         22 . The method of  claim 1 , wherein the fluid matrix material is chemically crosslinkable or photo-crosslinkable. 
     
     
         23 . The method of  claim 1 , wherein the dissociated tissue sample consists of freshly biopsied dissociated cells. 
     
     
         24 . The method of  claim 23 , wherein the dissociated tissue sample has been taken from a patient within 12 hours of forming the Patient-Derived Micro-Organospheres. 
     
     
         25 . The method of  claim 1 , wherein dissociated tissue sample comprises the immune cells, and wherein the immune cells comprise one or more of T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages dendritic cells, and combinations thereof. 
     
     
         26 . The method of  claim 1 , wherein the Patient-Derived Micro-Organospheres contain more than one cell type. 
     
     
         27 . The method of  claim 26 , wherein the unpolymerized mixture includes only the dissociated tissue sample and the fluid matrix material. 
     
     
         28 . The method of  claim 1 , wherein at least some of the Patient-Derived Micro-Organospheres are cryopreserved before or after the culturing, and wherein the unpolymerized mixture comprises a freezing medium. 
     
     
         29 . A composition of matter comprising a plurality of cryopreserved Patient Derived Micro-Organospheres, wherein each Patient-Derived Micro-Organosphere has a spherical shape having a diameter of between 50 pm and 500 pm, wherein the Patient-Derived Micro-Organospheres have less than a 25% variation in size, and wherein each Patient-Derived Micro-Organosphere comprises a polymerized base material, and between about 1 and 500 dissociated primary cells distributed within the polymerized base material that have been passaged less than six times, whereby heterogeneity of the cells within the Patient-Derived Micro-Organospheres is maintained. 
     
     
         30 . A microfluidic device for generating Patient-Derived Micro-Organosphere, the microfluidic device comprising:
 a chip structure having inlets and outlets for receiving a first fluid and a second fluid, the chip structure defining a plurality of channels having:
 a first channel configured to receive the first fluid, the first fluid comprising an unpolymerized mixture; 
 a second channel configured to receive the second fluid, the second fluid comprising an immiscible fluid that is immiscible with the unpolymerized mixture; and 
 a third channel fluidly coupled to the first and second channels at a junction region, the junction region configured to combine the first and second fluids into a single combined fluid; 
   a flow sensor operably coupled to a portion of the plurality of channels;   a pump operably coupled to a portion of the plurality of channels; and   a controller operably coupled to the flow sensor and the pump, the controller configured to control the flow sensor and the pump to move the first, second, and combined fluids in the plurality of channels such that the unpolymerized mixture moves at a first flow rate and the immiscible fluid moves at a second flow rate that is different from the first flow rate.   
     
     
         31 . The method of  claim 1 , comprising:
 forming hundreds of the Patient-Derived Micro-Organospheres from a single biopsy;   marking or labeling cells in the Patient-Derived Micro-Organospheres; and   imaging the Patient-Derived Micro-Organospheres.