HIGH-CONTENT IMAGING OF MICROFLUIDIC DEVICES
The present invention is related to high-content microscopy imaging of microfluidic cell culture systems. A method of high-content microfluidic device microscopy is contemplated. along with related statistical analysis and microfluidic device adaptors.
1 . A method of imaging microfluidic devices comprising:
(a) providing a microfluidic device comprising a porous membrane, said porous membrane separating a first microfluidic channel having an endothelial cell layer and a second microfluidic channel having a second cell layer;
(b) providing a microscope capable of image acquisition;
(c) taking a first set of microscopic image acquisitions;
(d) determining a focal height and locating a standard coordinate system from said first set of microscopic image acquisitions, wherein the coordinate system is located based on the location of the membrane within the microfluidic device; and
(e) taking a second set of microscopic image acquisitions based on the coordinate system located in the first set of microscopic acquisitions;
wherein said second set of microscopic image acquisitions comprise said endothelial cell layer and said second cell layer together, separated by the membrane.
2 . The method of claim 1 , wherein the microscope is a confocal microscope.
3 . The method of claim 1 , wherein the first set of microscopic acquisitions are low-resolution.
4 . The method of claim 1 , wherein the second set of microscopic acquisitions are high-resolution.
5 . (canceled)
6 . The method of claim 4 , wherein the second set of microscope acquisitions are used to evaluate the effect of an agent on the cells.
7 . The method of claim 6 , wherein the agent is a pharmaceutical.
8 . The method of claim 1 , wherein the cells are cultured for more than seven days.
9 . (canceled)
10 . The method of claim 4 , wherein the second set of microscopic acquisitions comprises a three-dimensional acquisition.
11 . (canceled)
12 . The method of claim 1 , wherein the second cell layer comprises liver cells.
13 . The method of claim 12 , wherein the liver cells are hepatocytes.
14 . The method of claim 13 , wherein the hepatocytes are human hepatocytes.
15 . The method of claim 1 , wherein the second cell layer comprises kidney cells.
16 . (canceled)
17 . (canceled)
18 . The method of claim 1 , further comprising applying flow to the channels.
19 . The method of claim 1 , wherein the second set of acquisitions, guided by the coordinate system, comprises Z stack slices through different layers of the microfluidic device.
20 - 37 . (canceled)
38 . A method of imaging microfluidic devices comprising:
(a) providing a microfluidic device comprising a membrane having pores, said membrane separating two microfluidic channels;
(b) providing a microscope capable of image acquisition;
(c) taking a set of low resolution microscopic image acquisitions;
(d) locating a standard coordinate system using said set of low resolution image acquisitions, wherein the coordinate system is located based on the location of said pores; and
(e) taking a set of high resolution microscopic acquisitions based on the coordinate system located in the first set of microscopic acquisitions.
39 - 61 . (canceled)
62 . The method of claim 38 , wherein the microfluidic device is seeded with cells.
63 . The method of claim 62 , wherein the high resolution set of microscopic image acquisitions is used to evaluate the effect of an agent on the cells.
64 - 68 . (canceled)
69 . The method of claim 62 , wherein the cells are liver cells.
70 . The method of claim 69 , wherein the liver cells are hepatocytes and sinusoidal endothelial cells.
71 . (canceled)
72 . The method of claim 62 , wherein the cells are kidney cells.
73 . The method of claim 62 , wherein the microscopic acquisitions are of individual cells.
74 . (canceled)
75 . The method of claim 62 , further comprising applying flow to the channels.
76 . The method of claim 75 , where in the flow exerts shear stress on the cells.
77 . A method of analyzing cellular phenotype changes following agent exposure comprising:
(a) providing a plurality of microfluidic devices comprising cells in microchannels, said microchannels comprising microchannel walls;
(b) providing a microscope capable of image acquisition;
(c) treating a number of said microfluidic devices with an agent and a number of said microfluidic devices with a control media;
(d) taking a first set of microscopic acquisitions;
(e) locating a standard coordinate system using the first set of microscope acquisitions, wherein the coordinate system is located based on the location of the microchannel walls within the microfluidic device;
(f) taking a second set of microscopic acquisitions based on the coordinate system located in the first set of microscopic acquisitions;
(g) making endpoint measurements of the acquisitions;
(h) fitting a regression model to the measurements;
(i) estimating a field effect based on the regression; and
(j) comparing the field effect from microfluidic devices treated with an agent verses microfluidic device treated with a control media;
wherein said high resolution microscopic acquisition comprises a three-dimensional microscopic acquisition.
78 . The method of claim 77 , wherein said regression model is a Bayesian linear regression model.
79 . The method of claim 77 , wherein said field effect is a linear field effect.
80 . The method of claim 77 , wherein the microscope is a confocal microscope.
81 . The method of claim 77 , wherein the first set of microscopic acquisitions are low-resolution.
82 . The method of claim 77 , wherein the second set of microscopic acquisitions are high-resolution.
83 . The method of claim 77 , wherein the agent is a pharmaceutical.
84 - 85 . (canceled)
86 . The method of claim 77 , wherein the three-dimensional acquisition comprises an endothelial cell layer and hepatocyte cell layer together, separated by the membrane.
87 . The method of claim 77 , wherein the cells are liver cells.
88 . The method of claim 87 , wherein the liver cells are hepatocytes and sinusoidal endothelial cells.
89 . The method of claim 88 , wherein the hepatocytes and sinusoidal endothelial cells are human hepatocytes and human sinusoidal endothelial cells.
90 . The method of claim 77 , wherein the cells are kidney cells.
91 . The method of claim 77 , wherein the microscopic acquisitions are of individual cells.
92 . The method of claim 77 , further comprising applying flow to the channels.
98 - 110 . (canceled)