Methods and apparatus 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. 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), polystyrene, 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 500 microns. A plurality of upper and lower reservoirs may be provided to form a multi-well plate. Potential biological applications where Boyden chamber geometries are currently used include co-culture studies, tissue remodeling studies, cell polarity determinations, endocrine signaling, cell transport, cell permeability, cell invasion and chemotaxis assays.

1 . Biological measurement apparatus, comprising:
 a bottom reservoir;   a top reservoir;   a thin porous membrane separating the top and bottom reservoirs; and   
       wherein:
 the pores of the membrane are formed using a laser-based photo-machining (ablation) process; and 
 the porous membrane enables quantitative optical imaging of biological cells on the membrane without the use of fluorescent probes or optical staining. 
 
     
     
         2 . The apparatus of  claim 1 , wherein the membrane is composed of polyethylene terephthalate (PET), biaxially-oriented polyethylene terephthalate (boPET), polycarbonate, polyimide, polyether ether ketone (PEEK), or polystyrene. 
     
     
         3 . The apparatus of  claim 1 , where the porous membrane is in the range of 10 to 125 microns thick. 
     
     
         4 . The apparatus of  claim 1 , where the pores of the member are arranged in a predetermined rectangular or other geometric array. 
     
     
         5 . The apparatus of  claim 1 , where the pores of the member have a uniform and consistent spacing, density and diameter. 
     
     
         6 . The apparatus of  claim 1 , wherein the pores of the membrane have diameters in range of 1 to 15 microns. 
     
     
         7 . The apparatus of  claim 1 , wherein the pores of the membrane are spaced apart at a distance ranging from 10 to 500 microns. 
     
     
         8 . The apparatus of  claim 1 , further including a plurality of upper and lower reservoirs forming a multi-well plate. 
     
     
         9 . The apparatus of  claim 1 , wherein the membrane is coated with collagen 1, fibronectin, laminin or other extracellular matrix. 
     
     
         10 . The apparatus of  claim 1 , wherein the reservoirs are manufactured with an injection molded plastic. 
     
     
         11 . The apparatus of  claim 1 , wherein the reservoirs are manufactured with injection molded polystyrene, polycarbonate, polyethylene terephthalate (PET) or biaxially-oriented polyethylene terephthalate (boPET). 
     
     
         12 . The apparatus of  claim 1 , wherein the membrane is attached to either the top or bottom reservoir using an ultrasonic welding process or chemical bonding agent. 
     
     
         13 . The apparatus of  claim 1 , wherein the membrane is attached to either the top or bottom reservoir using laser welding, or laser mask welding. 
     
     
         14 . The apparatus of  claim 1 , wherein the size of the reservoirs, the size of the pores, the number of the pores, and the location of the pores are optimized to reduce the number of biological cells needed for a given assay precision. 
     
     
         15 . The apparatus of  claim 1 , wherein the membrane is attached to the bottom surface of the top reservoir thereby forming an insert that fits inside the bottom reservoir. 
     
     
         16 . The apparatus of  claim 1 , where a plurality of upper reservoirs are attached to a porous membrane, thereby forming a removable insert that fits inside a plurality of co-aligned bottom reservoirs forming a microplate. 
     
     
         17 . The apparatus of  claim 1 , wherein the porous membrane is substantially smooth and optically transparent to enable quantitative phase-contrast imaging of the biological cells on the membrane without the use of fluorescent probes or optical staining. 
     
     
         18 . The apparatus of  claim 1 , further including a phase contrast imaging system for performing the quantitative optical imaging of biological cells on the membrane without the use of fluorescent probes or optical staining. 
     
     
         19 . The apparatus of  claim 18 , wherein the phase contrast imaging system utilizes one of Zernike phase contrast, differential interference contrast (DIC) or Hoffman modulation contrast. 
     
     
         20 . A biological measurement method, comprising the steps of:
 providing a substantially smooth, optically transparent, thin film membrane having an upper surface and a lower surface;   forming a plurality of micropores through 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 upper surface of the membrane without the use of fluorescent probes or optical stains.   
     
     
         21 . The method of  claim 20 , including the step of using a phase-contrast technique to perform the quantitative optical imaging. 
     
     
         22 . The method of  claim 20 , including the step of using Zernike phase contrast, differential interference contrast (DIG), or Hoffman modulation contrast to perform the quantitative optical imaging. 
     
     
         23 . The method of  claim 20 , further including the use of epifluorescence microscopy. 
     
     
         24 . The method of  claim 20 , wherein the quantitative optical imaging is used for the measurement of cell migration (chemotaxis), cell invasion, cell permeability, tissue remodeling, cell polarity endocrine signaling or cell transport. 
     
     
         25 . The method of  claim 20 , 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. 
     
     
         26 . The method of  claim 20 , including the step of counting the number of cells remaining on the upper surface of the membrane over time to quantify cell chemotaxis or cell invasion. 
     
     
         27 . The method of  claim 26 , including the use of kinetic, multi-time-point quantitative optical microscopic measurements to reduce artifacts associated with transient chemical gradients in chemotaxis or chemo-invasion assays. 
     
     
         28 . The method of  claim 20 , 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 500 microns. 
     
     
         29 . The method of  claim 20 , including the step of providing a polyethylene terephthalate (PET), biaxially-oriented polyethylene terephthalate (boPET), polycarbonate, polyimide, polyether ether ketone (PEEK) or polystyrene thin film membrane. 
     
     
         30 . The method of  claim 20 , including the step of fabricating a plurality of porous membranes, each separating a respective upper and lower reservoir, thereby forming a multi-well plate. 
     
     
         31 . The method of  claim 20 , including the step of coating the membrane with collagen 1, fibronectin, laminin or other extracellular matrix. 
     
     
         32 . The method of  claim 20 , including the step of forming the upper and lower reservoirs using injection-molded polystyrene, polycarbonate, polyethylene terephthalate (PET) or biaxially-oriented polyethylene terephthalate (boPET). 
     
     
         33 . The method of  claim 20 , including the step of ultrasonically welding or chemically bonding the porous membrane to the upper or lower reservoir. 
     
     
         34 . The method of  claim 20 , including the step of laser welding or laser mask welding the porous membrane to the upper or lower reservoir. 
     
     
         35 . The method of  claim 20 , 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. 
     
     
         36 . The method of  claim 20 , 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.