System and method for microfluidic cell culture

Abstract

Microfluidic devices and methods for perfusing a cell with perfusion fluid are provided herein, wherein the gravitational forces acting on the cell to keep the cell at or near a retainer or a retaining position exceed the hydrodynamic forces acting on the cell to move it toward an outlet. Also provided, are methods for assaying cell products within the microfluidic device.

1 . A method, the method comprising:
 (a) retaining a cell at a retaining position within a microfluidic chamber having a chamber volume;   (b) flowing a perfusing fluid through the microfluidic chamber, wherein the perfusing fluid enters the chamber through an inlet at an inlet position and exits the chamber through an outlet at an outlet position; and   (c) measuring a cell product produced by the cell within the microfluidic chamber;   wherein the perfusing fluid has a greater velocity laminar flow adjacent the inlet and outlet positions than at the retaining position, and wherein a first region of the chamber is spaced apart from the retaining position, wherein the first region is interposed directly between the inlet and outlet positions.   
     
     
         2 . The method of  claim 1 , wherein measuring the cell product may be selected from one or more of: lineage staining; cell-surface protein staining; antibody staining; enzymatic assay; RT-PCR analysis; PCR analysis; sequencing; functional assay; and bead capture assay to characterize the cells. 
     
     
         3 . A method, the method comprising:
 (a) retaining a cell at a retaining position within a microfluidic chamber;   (b) flowing a perfusion fluid into the microfluidic chamber through an inlet;   (c) flowing the perfusion fluid out of the microfluidic chamber through an outlet wherein the outlet is positioned such that gravitational forces acting on the cell to keep it at or near the retaining position exceed hydrodynamic forces acting on the cell to move it toward the outlet; and   (d) measuring a cell product produced by the cell within the microfluidic chamber.   
     
     
         4 . The method of  claim 3 , wherein measuring the cell product may be selected from one or more of: lineage staining; cell-surface protein staining; antibody staining; enzymatic assay; RT-PCR analysis; PCR analysis; sequencing; functional assay; and bead capture assay to characterize the cells. 
     
     
         5 . A method, the method comprising:
 a. retaining a cell in a volume of perfusion fluid, wherein the volume is less than about 10 nL;   b. placing the volume of perfusion fluid in fluid communication with a reservoir fluid, wherein the reservoir fluid has a volume greater than the volume of perfusion fluid; and   c. measuring a cell product produced by the cell within the perfusion fluid.   
     
     
         6 . The method of  claim 5 , wherein measuring the cell product may be selected from one or more of: lineage staining; cell-surface protein staining; antibody staining; enzymatic assay; RT-PCR analysis; PCR analysis; sequencing; functional assay; and bead capture assay to characterize the cells. 
     
     
         7 . The method of  claim 2 , further comprising selecting cell clones based on cell characteristics. 
     
     
         8 . The method of  claim 7 , wherein cell characteristics are selected from one or more of: quantity of secreted product, quality of secreted product, proliferation, morphology, gene expression, fluorescent reporter, surface proteins, genealogical pedigree, viability, apoptosis, autophagy, metabolism, clone homogeneity, and clone heterogeneity. 
     
     
         9 . A microfluidic device for perfusing a cell with perfusion fluid, the device comprising:
 a chamber, having:   (i) at least one inlet;   (ii) at least one outlet; and   (iii) a cell retainer;   wherein the inlet and the outlet are in fluid communication with the cell retainer, and wherein the outlet is positioned such that, when the device is being perfused, gravitational forces acting on the cell to keep it at or near the retainer exceed hydrodynamic forces acting on the cell to move it toward the outlet.   
     
     
         10 . A microfluidic device for perfusing a cell with perfusion fluid, the device comprising:
 a chamber, having:   (i) at least one inlet;   (ii) at least one outlet; and   (iii) a cell retainer;   wherein the inlet and the outlet are in fluid communication with the cell retainer; and   wherein a first region of the chamber is interposed directly between the inlet and outlet positions and is spaced apart from the cell retainer.   
     
     
         11 . The microfluidic device of  claim 9 , further comprising a perfusion fluid comprising one or more of the following: an immunostaining agent; an enzymatic reagent; a dye; and a functionalized bead. 
     
     
         12 . The microfluidic device of  claim 11 , wherein
 (a) the immunostaining agent is selected from one or more of: monoclonal antibodies; polyclonal antibodies; fluorophores; and blocking solution;   (b) the enzymatic reagent is selected from one or more of: horseradish peroxidase; and luminol;   (c) the dye is selected from one or more of: rhodamine; fluorescein; calcein; Hoescht; Trypan blue; propidium iodide; and Giemsa solution;   (d) the functionalized bead is selected from one or more of: magnetic beads; polymer beads; protein A-coated beads; and protein G-coated beads.   
     
     
         13 . A method of culturing a cell and measuring cell products within a microfluidic chamber, the method comprising:
 (a) retaining the cell within the microfluidic chamber at a concentration of ≧10,000 cells per mL;   (b) flowing a perfusing fluid through the microfluidic chamber; and   (c) measuring a cell product produced by the cell within the microfluidic chamber.   
     
     
         14 . The method of  claim 13 , wherein the cell product is secreted by the cell. 
     
     
         15 . The method of  claim 13 , wherein the cell product is quantified directly or indirectly by an intracellular fluorescent reporter. 
     
     
         16 . The method of  claim 13 , wherein the measuring of the cell product is selected from one or more of the following: antibody staining; enzymatic assaying; functional assaying, surface capturing, or bead capturing. 
     
     
         17 . The method of  claim 13 , wherein the cell or clone is maintained under culture conditions that allow subsequent clonal expansion. 
     
     
         18 . The method of  claim 13 , wherein the cell within the microfluidic chamber is at a concentration selected from one of the following: ≧20,000 cells per mL; ≧30,000 cells per mL; ≧40,000 cells per mL; ≧50,000 cells per mL; ≧60,000 cells per mL; ≧70,000 cells per mL; ≧80,000 cells per mL; ≧90,000 cells per mL; ≧100,000 cells per mL; ≧125,000 cells per mL; and ≧250,000 cells per mL. 
     
     
         19 .- 28 . (canceled) 
     
     
         29 . The method of  claim 13 , wherein measuring the secretion product occurs between 5 minutes and 6 hours from the retaining of the cell within the microfluidic chamber. 
     
     
         30 . The method of  claim 4 , further comprising selecting cell clones based on cell characteristics. 
     
     
         31 . The method of  claim 6 , further comprising selecting cell clones based on cell characteristics. 
     
     
         32 . The microfluidic device of  claim 10 , further comprising a perfusion fluid comprising one or more of the following: an immunostaining agent; an enzymatic reagent; a dye; and a functionalized bead.