A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The overall goal of this procedure is to study the interactions between blood and endothelial cells using a novel blood endothelial chamber assay. This is accomplished by first collecting blood from a healthy volunteer by open system venipuncture. In the second step, the blood is carefully layered onto heparin surface coated blood chambers coupled to endothelial cell culture plated slides.

Next, these blood endothelial cell chambers are gently rotated to facilitate the interaction between the blood and endothelial cells. Ultimately, the cell to cell interactions between the blood and endothelial cells and the resulting thrombin antithrombin complex production can be assessed by immunofluorescence microscopy and Eliza respectively. The main advantage with this technique over existing methods like those using animal models is that here a human model system can be used to investigate vascular disease Before beginning the experiment.

Couple an 18 gauge hypodermic needle to a two millimeter inner diameter piece of heparin surface coated silicone tubing. The plastic surfaces are coated with heparin in order to prevent unspecific activation of coagulation. For example, in this image, a clot is clearly visible in this uncoated chamber.

After 30 minutes of blood contact, treating the chamber with heparin surface protects the blood from uncontrolled activation, ensuring that the results are due to the endothelial cell blood interactions. Only After connecting the tubing, carefully collect a blood sample from a healthy donor by open system venipuncture into a 50 milliliter heparin surface coated tube. Supplement the collected blood with unfractionated heparin to reach a final concentration of 0.75 IUs per milliliter and invert the tube two to three times for gentle mixing.

For analysis of the initial blood protein concentration, carefully mix one milliliter of blood per tube in micro centrifuge tubes containing 30 microliters of 10 millimolar K three EDTA, and place the tubes on ice. Then use a heparin surface coated pipette tip to transfer 1.5 milliliters of blood into each heparin surface coated blood endothelial cell chamber, taking care to let the blood flow slowly and without creating bubbles to avoid activation. Next, remove the plastic walls from the endothelial cell culture slides according to the manufacturer's instructions, and gently wash the endothelial cells.

With PBS, we move as much of the wash from the cells as possible without letting the cultures dry out and place the slides on top of the blood chambers with the cells facing the blood. Then using plastic slides for additional support. To avoid breakage of the cell culture slides, place a clamp around each blood endothelial cell chamber to secure the slides.

Now, rotate the chambers on a rotating wheel in a 37 degree Celsius water bath at 0.5 Gs for 30 minutes. After the rotation, dismantle the blood endothelial cell chambers, wash the cell culture slides in PBS and fix the cells in ice cold 1%paraldehyde for 10 minutes. Finally, for evaluation of the activated blood protein concentration, mix one milliliter of blood from each chamber in micro centrifuge tubes containing 30 microliters of 0.34 molar K three EDTA, and place the tubes on ice for a maximum of one hour.

Then after spinning down the cells, store the plasma in new micro centrifuge tubes at minus 70 degrees Celsius until further analysis. Activation of the coagulation factors is more likely to occur in stagnant blood as the probability of an interaction between the activated factors is increased. Indeed, the volume of blood without any movement by air bubbles is two and a half times larger.

When 1.75 milliliters of blood is added to the chamber compared to when 1.5 milliliters of blood is used, the measured thrombin antithrombin values suggest an increased thrombin antithrombin formation with an increased blood volume as well. Interestingly, when ve are incubated with either volume of whole blood, no differences are noted between the two groups suggesting that the endothelial cells may modulate the activation of the coagulation to study the effect of TNF alpha on leukocyte recruitment. In this experiment, qve were treated with 20 nanograms per milliliter of TT NF alpha four hours prior to blood contact, after which the number of CD 16 positive cells was quantified.

Recruitment of the CD 16 positive cells increased significantly with TNF alpha treatment from 100 to 256 CD 16 positive cells per square millimeter and 1.5 liters of blood, and from 172 to 378 and 1.75 milliliters of blood. The formation of the thrombin antithrombin complexes was then measured in the corresponding plasma samples from the blood incubated with either TNF alpha treated or untreated cells. As expected, the TNF alpha stimulated cells induced an approximate doubling of the thrombin antithrombin complexes as compared to the untreated controls After its development.

This technique will pull the way for researchers in the field of immunology to explore, complement and coagulation in an in vitro model using human primary cells and human whole blood.