Research Program
Cellular Microdynamics

Project #
CM5

Participating Faculty:	David Lawrence, Robert Austin, Michael Lynes
NBTC Students/Postdocs:	TJ Zieziulewicz
Other Students/Postdocs:	David Inglis

Objectives
Microfabricated devices were used to evaluate alterations of immune cell structure (i.e. size, shape, deformability) and function (adhesion, chemotaxis, cellular response). Two distinct devices were designed and fabricated in order to individually evaluate each of these cellular parameters (Fig. 1).  Alterations of immune cell structure were evaluated using a microfluidic “bump array”, which was designed to separate cells based on changes in “hydrodynamic” cell size.  This device would produce size distribution histograms comparable to that of forward light scatter in a conventional Flow Cytometer but in a much less expensive, on-chip, microfluidic device.  An in vitro capillary flow model was designed to assess alterations in immune cell function. 

Methods
Leukocyte differential size histograms were produced from a drop of human whole blood obtained from a finger prick.  The whole blood was run through the “bump array” device under constant and repeatable pressure. Cells of increasing size and fluidity get bumped further from plasma and subsequently end up in exit channels that are farther away from smaller cell types.  The channels in which the immune cells exited the device were evaluated using a fluorescence camera mounted on an inverted microscope which is connected to a recorder.  The type of immune cell and its corresponding exit channel information was extracted from the video recording and plotted in a histogram format which was compared to the histogram of the same sample produced by the flow cytometer.  The next step is to evaluate leukocyte subset distribution or separation directly within the device in real time.  The “bump array” will be used as a front end device to a GCSPRI chip, similar to the one shown in Figure 4 of the continued funding proposal.  The advantage of this setup is that leukocyte subset distribution or changes in “hydrodynamic” cell size can be determine within the device, in real time and without the use of florescent stains or markers as well as without the use of camera’s, video recorders and the laborious task of counting leukocyte subsets exiting the device.  The GCSPRI chip will have regions of interest (ROI, spotted antibodies in a set grating) with capture antibodies for each leukocyte subset in each of the “bump array” exit channels.  Therefore, as cells exit the device they will be captured by their respective capture antibody based on their subset.  Since this device will be run with the GCSPRI chip,  the cells exiting the bump array can be characterized and quantified by their capture on the ROIs. The change in the degree of the plasmonic refraction from each ROI indicates the number and type of cells evaluated in real-time.

Summary
We have shown that the “bump array” microfluidic device can effectively separate different leukocyte types in blood as well as bacterial toxin-activated from non-activated lymphocytes.  The device differentially separates leukocytes whose diameter size may only differ by a few microns or less; however, additional chemical or physical parameters, such as membrane rigidity, may influence the trafficking through the device and thus need to be assayed. The microfluidic device, which separates cells mainly by “hydrodynamic” size, produced size distribution histograms for a mixed cell sample that was comparable to the forward light scatter histogram of the same sample by Flow Cytometry (Fig. 2).  This mixed sample contained three distinct leukocyte cell types (i.e. CD4⁺, CD14⁺ and J45 T-Cells), which were known to have different cellular diameters.  This device also showed that its sensitivity is good enough to detect minor increases in cell size due to exposure to staphylococcal entertoxin B (SEB).  SEB induces some lymphocytes to become activated and become lymphoblast, which are larger in size compared to normal lymphocytes.  This device showed that it could detect this minor change in lymphocyte volume upon activation (Fig. 3)

Accomplishments

• Design and Fabrication of the “Capillary Flow Model”  and  “Bump Array” Devices
• Successful demonstration of leukocyte rolling, adhesion, chemotaxis and diapedesis in an “in vitro” capillary model under physiological fluid flow conditions
• Determined that leukocyte subsets have differential requirements to perform the adhesion, chemotaxis and diapedesis under flow conditions
• Successfully separated leukocytes by “hydrodynamic” cell sizing on a microfluidic chip that has sensitivity and resolution comparable to that of a commercial flow cytometer