Micro-Sampling for Cell, Tissue, and Micro-Organism Monitoring

Abstract

Cell and organ (or tissue) cultures provide a micro-environment with respect to nutrients, gas exchange, and scaffolding in order to encourage specific cell function, and in some cases to mimic in-vivo cellular expression under in-vitro conditions. We describe apparatus and methods to chemically, spatially, and temporally measure diffusible molecules produced, or used by cells or tissues in culture. In this manner, mechanisms of cell-cell interaction and other chemical signaling, detailed biochemical pathways, and the action of potential pharmaco-therapy agents can be better understood at a molecular level. In addition to basic science, the technical advantages of process monitoring and control can be applied to optimize culture products in bioreactors. Embodiments of this device are intended to simulate and monitor [input and output] the behavior of vascular capillary beds in higher species vascular systems.

1 - 12 . (canceled) 
     
     
         13 . A method for analyzing, culturing, and monitoring of cellular samples comprising:
 a. holding the cellular sample introduced into a sample holding substrate,   b. delivering an input media via an input means to nurture or stimulate said sample,   c. controlling said delivery of said input media with spatial, temporal, and material precision,   d. segmenting fluidic output via an output from said cellular sample for analysis,   e. controlling said collection of said fluidic output with spatial, temporal, and material precision, and   f. analyzing said fluidic output to monitor the steady-state or dynamic cellular activity;   wherein biochemical and/or biophysical behavior of the cellular sample under variable and controlled conditions is monitored.   
     
     
         14 . The method as described in  claim 13 , further comprising maintaining cellular sample viability by an external support system and/or spatially, temporally, and/or compositionally controlling mass transfer into and out of regions of the cellular sample. 
     
     
         15 . The method as described in  claim 13 , wherein cellular sample regions are individually analyzed for binding affinities for chemical species delivered from the input media. 
     
     
         16 . The method as described in  claim 13 , wherein the cellular sample is explanted tissue from clinical samples, wherein the sample is analyzed for diagnostic biomarkers under enhancements from reagents delivered to the sample. 
     
     
         17 . The method as described in  claim 13 , wherein environment impact studies and toxicology studies are performed by introducing target species to selected cell types for monitoring cell behavior as a function of target species. 
     
     
         18 . The method as described in  claim 13 , wherein geometry of the input means and output conforms to a permeable tubular geometry functioning as a simulated vascular system or capillary bed. 
     
     
         19 . The method as described in  claim 13 , wherein a material composition of the input media degrades, kills, and/or lyses a region of the cellular sample in a controlled manner to monitor biochemical components of degradation including cytoplasm, nucleus, mitochondria, and membranes. 
     
     
         20 . (canceled) 
     
     
         21 . The method as described in  claim 13 , wherein the cellular sample is composed of one or more regions of cell populations of histotypic and/or organotypic cell types, nurtured to re-aggregate and assume a three dimensional culture. 
     
     
         22 . The method as described in  claim 21 , wherein the three dimensional culture is inoculated with a different cell line or virus using spatially selective input means such that a region of the three dimensional culture has contact or close population with the different cell line or virus. 
     
     
         23 . The method as described in  claim 21 , wherein the three dimensional cell culture is dosed in a spatially and/or temporally selective manner using chemical agents to analyze the output products from specific cellular regions. 
     
     
         24 . The method as described in  claim 13 , wherein the cellular sample is composed of one or more integrated populations of histotypic and/or organotypic cells where extracellular chemical signaling between populations can be measured using spatially selective output analysis. 
     
     
         25 . The method as described in  claim 13 , wherein cellular sample regions are individually analyzed for the metabolites produced in response to a chemical species delivered from the input media. 
     
     
         26 . The method as described in  claim 13 , wherein the culture volume used to sustain a living cell aggregate composed of different cell line regions is interfaced to a means of selectively delivering extracellular fluids to an observation barrier to measure compositional differences in the spatially resolved culture output in order to characterize cell:cell chemical communication. 
     
     
         27 . The method as described in  claim 13 , wherein the output comprises an output barrier comprising a plurality of apertures and a permeable material positioned upstream relative to the plurality of apertures, the permeable material configured for selective passage of the fluidic product. 
     
     
         28 . The method as described in  claim 13 , wherein the sample holding substrate comprises a scaffold configured to promote cellular adherence and three-dimensional cellular growth of the cellular sample. 
     
     
         29 . The method as described in  claim 13 , wherein the input means comprises an aperture array addressed and controlled using mechanical or electro-fluidic means to produce multiple input pathways for variable input media to be delivered to the cellular sample on a spatially and temporally resolved basis. 
     
     
         30 . The method as described in  claim 13 , wherein the input means comprises an array of tubes addressed and controlled using mechanical or electro-fluidic means to produce multiple input pathways for variable input media to be delivered to the cellular sample on a spatially and temporally resolved basis. 
     
     
         31 . The method as described in  claim 13 , wherein the output comprises an output barrier comprising an aperture array addressed or controlled to produce segmented flow to multiple output pathways in fluid communication with an analyzer on a spatially and temporally resolved basis. 
     
     
         32 . The method as described in  claim 13 , wherein the output barrier comprises an array of tubes addressed and controlled using mechanical or electro-fluidic means to produce multiple output pathways in fluid communication with an analyzer on a spatially and temporally resolved basis. 
     
     
         33 . The method as described in  claim 13 , wherein analyzing the fluidic output comprises generating a chemical map of the cellular sample based on individual analysis results performed on the fluidic output from each of a plurality of apertures of an output barrier of the output. 
     
     
         34 . A method for mapping the composition of one or more differential extra-cellular components of a living cell aggregate with respect to position across said aggregate and time relative to introduction of selective input media, comprising:
 holding a living cell aggregate in a culture volume, the living cell aggregate comprising one or more aggregate regions,   delivering input media to the living cell aggregate via an addressable and controllable input barrier comprising a plurality of apertures in fluid communication with the culture volume and configured to provide spatial and temporal fluidic control of the input media into the culture volume, such that selective and differential input media is delivered to at least one of the one or more aggregate regions   segmenting extracellular fluidic output comprising a product of biological activity resulting from the living cell aggregate responding to the input media via an addressable and controllable output barrier comprising a plurality of apertures, each of the plurality of apertures of the output barrier corresponding to at least one of the plurality of apertures of the input barrier proximate a same region of the living cell aggregate, wherein each of the plurality of apertures of the output barrier is configured to receive the extracellular fluidic output resulting from the living cell aggregate responding to the input media provided to the corresponding at least one of the plurality of apertures of the input barrier, and   analyzing, via an analyzer in fluid communication with the output barrier, the extracellular fluidic output from each of the plurality of apertures of the output barrier individually.