Automated collection of a specified number of cells

Abstract

Embodiments of the disclosed subject matter provide an automated method and system to isolate and collect cells using computerized analysis of images of cells and their surroundings obtained from a digital imaging device or system. Embodiments of the disclosed subject matter make use of a “microwell array,” which can comprise a formed, elastomeric grid of indentations or “wells.” Many, most, or all of the wells in a microwell array can contain a releasable, microfabricated element, which can be referred to as a “raft.” Embodiments of the disclosed subject matter provide a system and method for cell collection that includes computerized identification and collection of rafts with isolated single cells or a specific group or groups of cells, eliminating the need for continuous human identification and selection.

1 . An automated, computer-controlled method for monitoring and collecting selected cells, the method comprising:
 i) using a system, imaging a microwell array comprising a plurality of cell rafts at a first time subsequent to a plurality of cells having been seeded into the microwell array, and at one or more times later than the first time, to identify at least one selected cell raft that contains a cell undergoing a cellular process, the system comprising:
 an imaging device configured for obtaining the images of the microwell array; 
 an actuator configured for releasing the cell rafts from the microwell array; 
 a mechanical cell raft collector; and 
 a computer system comprising at least one processor and memory, the computer system configured for:
 obtaining the images of the microwell array using the imaging device; 
 identifying, by analyzing the images of the microwell array, the at least one selected cell raft that contains a cell undergoing a cellular process, storing a location of the selected cell raft on the microwell array, and assigning the selected cell raft to a mapped location of a collection plate; 
 controlling the actuator to release the selected cell raft from the microwell array; and 
 controlling the mechanical cell raft collector to collect the selected cell raft after release from the microwell array and controlling the mechanical cell raft collector to deposit the selected cell raft at the mapped location of the collection plate; 
 
   ii) using the system, releasing the at least one selected cell raft that contains the cell undergoing the cellular process; and   iii) using the system, collecting the at least one selected cell at the mapped location of the collection plate.   
     
     
         2 . The automated, computer-controlled method of  claim 1 , wherein the at least one selected cell raft that contains the cell undergoing the cellular process is an individual cell or a clonal colony of cells. 
     
     
         3 . The automated, computer-controlled method of  claim 2 , wherein the individual cell or the clonal colony of cells harbors or has been exposed to a specific transgene, a siRNA, or a small molecule. 
     
     
         4 . The automated, computer-controlled method of  claim 2 , wherein the individual cell or the clonal colony of cells is displaying one or more properties comprising extreme gene expression, cell proliferation, or extended survival. 
     
     
         5 . The automated, computer-controlled method of  claim 1 , wherein the plurality of cells are from a diverse cell population and the at least one selected cell raft that contains the cell undergoing the cellular process is an individual cell or a clonal colony of cells with unique temporal characteristics. 
     
     
         6 . The automated, computer-controlled method of  claim 5 , wherein the diverse cell population is from a tumor. 
     
     
         7 . The automated, computer-controlled method of  claim 1 , wherein the plurality of cells are target dendritic cells, and further comprising seeding T cells onto the array, wherein the at least one selected cell raft that contains the cell undergoing the cellular process is an individual T cell showing a high rate of cytotoxicity against the target dendritic cells. 
     
     
         8 . The automated, computer-controlled method of  claim 1 , wherein identifying the at least one selected cell raft comprises detecting, for each cell raft, a marker depicted in a sub-image of the cell raft and gating the cell rafts based on detecting the marker. 
     
     
         9 . The automated, computer-controlled method of  claim 8 , wherein the detection of the marker is based on intensity of a color channel, a size, or both. 
     
     
         10 . The automated, computer-controlled method of  claim 8 , comprising monitoring a localization pattern or an abundance of the marker, or both, within the at least one selected cell by the imaging of the microwell array. 
     
     
         11 . The automated, computer-controlled method of  claim 10 , wherein the localization pattern of the marker within the at least one selected cell is monitored in response to a stimulus to the plurality of cells. 
     
     
         12 . The automated, computer-controlled method of  claim 8 , wherein the marker is detected with an optically detectable probe. 
     
     
         13 . The automated, computer-controlled method of  claim 12 , wherein the optically detectable probe comprises: i) a dye, alone or in combination with one or more chemical or affinity moieties, or ii) a gene expressing the marker engineered to include a fluorescent motif, a green fluorescent protein, an antigen, a His×6, an enzymatic activity, a luciferase, or an alkaline phosphatase. 
     
     
         14 . The automated, computer-controlled method of  claim 13 , wherein the one or more chemical or affinity moieties comprises a nucleic acid with complementary sequence to the marker sequence or an antibody against the marker protein. 
     
     
         15 . The automated, computer-controlled method of  claim 1 , wherein the analyzing of the one or more images of the microwell array comprises:
 identifying and storing a size of the microwell array;   identifying and storing an optimal focus position and an optimal exposure for the microwell array; and   sectioning the microwell array using the processor and storing a translation required to match a set of microwells to a field of view.   
     
     
         16 . The automated, computer-controlled method of  claim 1 , wherein the identifying of the at least one selected cell raft comprises:
 segmenting, using the processor, the plurality of rafts of the microwell array; and   counting, using the processor, cell nuclei per raft for the plurality of rafts.   
     
     
         17 . The automated, computer-controlled method of  claim 16 , wherein the counting of the cell nuclei further comprises performing, using the processor, a watershed transform to define the cell nuclei relative to a fluorescence threshold. 
     
     
         18 . The automated, computer-controlled method of  claim 1 , wherein analyzing the images comprises storing the images and associating each of the images with a respective capture time of the image. 
     
     
         19 . The automated, computer-controlled method of  claim 1 , wherein obtaining the images of the microwell array comprises illuminating the at least one cell raft with light of a specified wavelength and detecting a fluorescence signature of the cellular process. 
     
     
         20 . The automated, computer-controlled method of  claim 19 , wherein the fluorescence signature is a result of the cell undergoing the cellular process expressing an enhanced green fluorescent protein (EGFP) reporter gene. 
     
     
         21 . The automated, computer-controlled method of  claim 19 , wherein identifying, by analyzing the images of the microwell array, the at least one selected cell raft that contains the cell undergoing the cellular process comprises presenting, in a graphical user interface, a graph of the fluorescence signature in the images over time to provide a temporal evolution of the cellular process.