Flow Cytometry for Adherent Cell Assays

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					                                                                  Flow Cytometry for Adherent Cell Assays
                                                                                                  Cynthia Jensen-McMullin, Stephen Ng
                                                             Faculty Advisors: G.-P. Li & Mark Bachman & Nancy Allbritton & Chris Sims
                                                Department of Electrical Engineering and Computer Science / Department of Physiology and Biophysics
                                         The Henry Samueli School of Engineering / College of Medicine / University of California, Irvine

The Problem                                                                                       Optical Detection                                                         Standard Assays
Flow cytometry and other flow-based assays are powerful                                           The surfboards are detected by using an inexpensive diode laser           Boards with cell growth are subjected to standard assays. In
technology for non-adherent cells but not for adherent cell                                       and photodiode detection scheme. As the surfboard passes, the             the images below, RBL cells grown on SU-8 plastic
lines--adherent cells need a surface to stick to. Flow                                            beam intensity to the photodiode is blocked. The output from              “surfboards” are processed through wash steps and stained
cytometry is useless for 95% of cells that matter to                                              the photodiode can be coupled to a high speed imaging system,             using Oregon Green Diacetate.
biologists and medical researchers!                                                               enabling us to
                                                                                                                                          HeNe laser
                                                                                                  view the                                                high speed
                                                                                                                                                            camera
                    sheath flow        A typical flow cell for a flow                             surfboards and
                                                                                                                            Sample
                                       cytometer. The sample flow                                 their cargo with          in

                                       contains cells in suspension                               standard optical
                                       (non-adherent).       Due      to                          measuring tools.             photodiode

                                       laminar flow, the sheath flow
                                       and sample flow do not mix.
                      sample or        Instead, pressure differences
                     core stream
                     (with cells)      are used to hydrodynamically                                Surfboards can be manipulated in                                                   Optical image of a plastic          Same image under
                                       focus the sample flow.                                      standard flow cells (e.g., flow                                                  “surfboard” with cell growth.      fluorescence excitation
                                                                                                   cytometers) were passed through a
The Solution                                                                                       micromachined acrylic flow cell. The                                     Automated Assays                                       sample stream

Go surfing of course!                                                                              blue stream represents the sample                                                                                reagent
                                                                                                                                                                                                                                     (with cells)

                                                                                                   flow (horizontal orientation).                                           We envision a system where a
                                                                                                                                                                            single cell (or cells) grown on a       laser
                                                                                                                                                                            substrate (surfboard) are
                                                                                                                                                                            transported through a                                  fluorescence
                                                                                                                                                                            programmable, automated assay                                     detector

                                                                                                                                                                            system. This system could perform
  Microfabrication techniques are          Cells are grown on the surface of the
                                                                                                                                                                            functions such as washing,              laser

used to fabricate micro-pallets with   “surfboards.” In this example, RBL cells have                                                                                        labeling, detection and sorting.
  barcodes. The “surfboards” can               been cultured on the boards.                                                                                                 Surfboards can contain bar codes                       fluorescence
         transport the cells.                                                                                                                                                                                                                 detector
                                                                                                                                       Video frames of surfboard            and would be designed such that
                                                                                                                                       transported through microfluidic     the onboard cell could be
 Surfboard Dynamics                                                                                                                    channel. Boards are readily          identified.
Laminar flow and board shape is used to guide and orient the                                                                           manipulated and imaged.
surfboards down the channels.

                                                                                                                                                                       Summary
                                                                                                  A micro-pallet system (“surfboards”) is being developed to enable high speed flow cytometry on adherent cells. Thousands of micro-
 channel
                                                                                                  surfboards will enable adherent cells to be manipulated in micro-factories that can perform automated cell analysis and sorting.
                                                                      Cell cargo on microscopic
                                                                            transport ships
                    surfboard                          laminar flow
                                                                                                    Microfabrication of surfboards                                        Optical detection of surfboards in flow channel
     Optical image of a plastic                Parabolic profile of laminar flow
                                                                                                    Tested flow of surfboards in various geometries                       Fluorescent labeling/imaging of RBL cells on surfboards
    surfboard passing through a           velocity, illustrating maximum velocity at                Growth of RBL cells on plastic surfboards confirmed
   Micromachined PMMA channel               the center and near-zero at the walls.

                                                                                                   Acknowledgements: Ruisheng Chang and the INRF staff.