Methods for improving in vitro measurements using boyden chambers

Abstract

Apparatus and methods to improve the Boyden chamber used in cellular biological measurements, allowing quantitative optical microscopy of biological cells in situ without using fluorescent probes or optical staining. In the preferred embodiment, a thin porous membrane separating top and bottom reservoirs includes an array of precisely positioned micropores pores manufactured using a laser-based photo-machining (ablation) process. The membrane may be composed of polyethylene terephthalate (PET), polycarbonate, polyimide, polyether ether ketone (PEEK) or other appropriate material. The pores formed in the membrane may have diameters in the range of 1 to 15 microns and spaced apart at a distance ranging from 10 to 200 microns. A plurality of upper and lower reservoirs may be provided to form a multi-well plate. The invention finds application in a wide range of potential biological applications where Boyden chamber geometries are currently used including co-culture studies, tissue remodeling studies, cell polarity determinations, endocrine signaling, cell transport, cell permeability, cell invasion and chemotaxis assays.

1 . A biological measurement method, comprising the steps of:
 providing a thin film membrane;   forming a plurality of micropores in the membrane using a laser-based photo-machining (ablation) process;   using the membrane to separate upper and lower fluid-containing reservoirs; and   performing quantitative optical imaging of biological cells on the membrane without using fluorescent probes or optical stains.   
     
     
         2 . The method of  claim 1 , wherein the step of imaging is performed with one of Zernike phase contrast, differential interference contrast (DIC) or Hoffman modulation contrast. 
     
     
         3 . The method of  claim 1 , further including the use of epifluorescence microscopy. 
     
     
         4 . The method of  claim 1 , wherein the method is used for the measurement of cell migration (chemotaxis), cell invasion, cell permeability, tissue remodeling, cell polarity endocrine signaling or cell transport. 
     
     
         5 . The method of  claim 1 , wherein the step of quantitative imaging involves a morphological assessment of shape, and/or the counting of the cells on the surface of the membrane and/or the identification of a particular cell type within a mixed cell population. 
     
     
         6 . The method of  claim 1 , wherein the step of counting the cells remaining on the top side of the membrane at multiple time points is used to quantify the amount of cell chemotaxis or cell invasion. 
     
     
         7 . The method of  claim 1 , wherein kinetic, multi-time point quantitative optical microscopic measurements are to reduce artifacts associated with transient chemical gradients in chemotaxis or chemo-invasion assays. 
     
     
         8 . The method of  claim 1 , wherein the step of forming a plurality of micropores in the membrane includes forming pores with diameters in range of 1 to 15 microns and spaced apart at a distance ranging from 10 to 200 microns. 
     
     
         9 . The method of  claim 1 , wherein the step of providing a thin film membrane includes providing a polyethylene terephthalate (PET), polycarbonate, polyimide or polyether ether ketone (PEEK) thin film. 
     
     
         10 . The method of  claim 1 , including the step of fabricating a plurality of porous membranes, each separating a respective upper and lower reservoir, thereby forming a multi-well plate. 
     
     
         11 . The method of  claim 1 , including the step of:
 coating the membrane with collagen  1 , fibronectin, laminin or other extracellular matrix.   
     
     
         12 . The method of  claim 1 , including the step of:
 forming upper and lower reservoirs using injection-molded polystyrene, polycarbonate or polyethylene.   
     
     
         13 . The method of  claim 1 , including the step of:
 ultrasonically welding or chemically bonding the porous membrane to the upper or lower reservoir.   
     
     
         14 . The method of  claim 1 , including the step of:
 attaching the membrane to the bottom surface of the top reservoir, thereby forming a removable insert that fits inside the bottom reservoir.   
     
     
         15 . The method of  claim 1 , including the step of:
 optimizing the pore diameter, pore locations and timing of data acquisition in order to reduce artifacts associated with a transient diffusion gradient in chemotaxis or chemo-invasion assays.