Research Program
Cell Surface Interactions

Objectives
The main objective for this past year has been trying to incorporate a basement membrane and endothelial cell monolayer onto the barrier wall of our capillary flow device to simulate a more biologically relevant and realistic capillary design.  We had previously designed an in vitro capillary flow model device to assess alterations in immune cell function such as changes in cellular adhesion, morphology and chemotaxis (Fig. 1).  We have also shown that Acute Cold Restraint Stress (ACRS) causes alterations in leukocyte trafficking in vivo resulting in lymphopenia and neutrophilia in whole blood due to catecholamine effects through β1 and β2-Adrenergic Receptors (Fig. 3).  However, the mechanism of catecholamine-induced leukocyte trafficking is unknown.  We have been able to use our current device to successfully demonstrate leukocyte subset differences in rolling, adhesion, chemotaxis and diapedsis under physiological flow conditions however, it has only worked for neutrophils (Fig. 4).  Lymphocytes fail to adhere or chemotax in our device, most likely due to an inadequate adhesion surface and chemokine presentation.  Therefore, we have focused this past year on generating an endothelial cell monolayer on a basement membrane within our device to provide better control of chemokine diffusion and to provide a more realistic adhesion surface and presentation of chemokine by the endothelial cells (Fig. 2).

Methods
We have continued to evaluate diapedesis in our current capillary device but have focused mainly on incorporating a basement membrane and endothelial monolayer into the overall design.  We have been using whole blood from Control or Acute Cold Restraint Stress mice to evaluate diapedesis in an in vitro model.  Leukocytes are moved through the capillary channel under positive or negative pressure.  By controlling the fluid flow rates/pressures of the multiple inlet channels, we can focus the cells along the barrier wall where they undergo the first process of diapedesis, which is rolling.  Chemokines are introduced on the opposite side of the device, in order to stimulate up regulation of adhesion molecules and initiate the chemotaxis and/or diapedesis process, from one side of the barrier wall to the other, however our main focus this past year has been trying to incorporate a basement membrane and endothelial cell monolayer onto the barrier wall to simulate a more biologically relevant and realistic capillary design.

Bovine Aortic Endothelial Cells (BAEC) forms a monolayer with tight junctions when grown to confluence in culture.  We desired to achieve this same monolayer formation within our capillary channel.  We tried to achieve this by simply injecting and seeding endothelial cells into our device with or without a Fibronectin and allowing them to grow and proliferate over several days in culture.

Summary
We have previously shown that leukocyte subsets from whole blood have differential diapedsis requirements in an in vitro capillary physiologic flow model, which is posited to be characteristic of the in vivo process.  Blood with the red blood cells hypotonically lysed was flown down a lane with a well known leukocyte chemokine (SDF-1α) being simultaneously flown through a separate lane in the capillary device. The two lanes are separated by a barrier wall with 3-5 micron gaps (Fig. 1).  This design allowed of us to observe several different leukocyte subset phenomena within the device along with their distinct response to SDF-1α.  Leukocytes rolled through the device as a result of the fluid flow conditions either along the bottom of the device or along the barrier wall.  However, only one leukocyte subset adhered to the device or to other leukocytes in order to begin their chemotaxis and/or diapedesis process across the barrier wall towards the increased concentration of SDF-1α (Fig. 4).

Accomplishments

• Re-Design and new Fabrication procedure (Photoresist Patterning) for production of the "Capillary Flow Model"
• Successful demonstration of leukocyte rolling, adhesion, chemotaxis and diapedesis in an in vitro capillary model under physiological fluid flow conditions
• Determining the best materials (Matrigel and Hydrogels) and protocol for patterning basement membrane materials within the Capillary Device.