Methods for lymphatic vessel culture and gene transfection

Abstract

Objective: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself.

Methods: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells.

Results: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels.

Conclusions: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

Figure 1

The parameters of lymphatic contractile activity in isolated segments of the rat thoracic duct after culture under non-pressurized conditions. Day 0 – parameters of lymphatic contractile activity in control group of non-cultured lymphatic segments (n=6); Days 3-6 - parameters of lymphatic contractile activity in group of lymphatic segments cultured for 3-6 days (n=7); Days 7-9 – similar parameters for segments cultured for 7-9 days (n=8). *Indicates significant differences (P ≤ 0.05) within each group between parameters at an initial level of transmural pressure of 1 cm H₂O versus all other levels of transmural pressure during the acute functional tests.

Figure 2

The parameters of lymphatic contractile activity in isolated segments of the rat mesenteric lymphatic vessels after culture under non-pressurized conditions. Day 0 – parameters of lymphatic contractile activity in control group of non-cultured lymphatic segments (n=5); Days 7-9 - parameters of lymphatic contractile activity in group of lymphatic segments cultured for 7-9 days (n=10). *Indicates significant differences (P ≤ 0.05) within each group between parameters at initial level of transmural pressure of 1 cm H₂O and at all other levels of transmural pressure during the acute functional tests; ◇ Indicates significant differences (P ≤ 0.05) between lymphatic tone index values in Day 0 and Days 7-9 groups.

Figure 3

Lymphatic tone in isolated segments of the rat mesenteric lymphatic vessels cultured at normal (A - 3 cm H₂O; n=5) or high (B - 10 cm H₂O; n=4) intraluminal pressures. *Indicates significant differences (P ≤ 0.05) within each group (A and B) between lymphatic tone index at day 0 of culture (control group) and at all other days of culture; shown separate for each level of transmural pressure during the acute functional tests; ◇ Indicates significant differences (P ≤ 0.05) between lymphatic tone index values in groups A and B for each given pressure/day pair.

Figure 4

Contraction amplitude in isolated segments of the rat mesenteric lymphatic vessels cultured at normal (A - 3 cm H₂O; n=5) or high (B - 10 cm H₂O; n=4) intraluminal pressures. *Indicates significant differences (P ≤ 0.05) within each group (A and B) between contraction amplitude at day 0 of culture (control group) and at all other days of culture; shown separate for each level of transmural pressure during the acute functional tests; ◇ Indicates significant differences (P ≤ 0.05) between contraction amplitude values in groups A and B for each given pressure/day pair.

Figure 5

Contraction frequency in isolated segments of the rat mesenteric lymphatic vessels cultured at normal (A - 3 cm H₂O; n=5) or high (B - 10 cm H₂O; n=4) intraluminal pressures. *Indicates significant differences (P ≤ 0.05) within each group (A and B) between contraction frequency at day 0 of culture (control group) and at all other days of culture; shown separate for each level of transmural pressure during the acute functional tests; ◇ Indicates significant differences (P ≤ 0.05) between contraction frequency values in groups A and B for each given pressure/day pair.

Figure 6

Fractional pump flow in isolated segments of the rat mesenteric lymphatic vessels cultured at normal (A - 3 cm H₂O; n=5) or high (B - 10 cm H₂O; n=4) intraluminal pressures. *Indicates significant differences (P ≤ 0.05) within each group (A and B) between fractional pump flow at day 0 of culture (control group) and at all other days of culture; shown separate for each level of transmural pressure during the acute functional tests; ◇ Indicates significant differences (P ≤ 0.05) between fractional pump flow values in groups A and B for each given pressure/day pair.

Figure 7

Examples of effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels. Vessel diameters are ~ 120-140 μm. Average 2-D projections of the stacks of confocal images, taken at steps of 0.5 μm through the vessels pressurized at 5 cm H₂O in the standard isolated vessel chamber. a. Transfection of mostly lymphatic endothelial cells was performed using solution containing 1.1*10¹⁰ viral particles/ml which was injected only inside of non-denuded vessel; b. Transfection of only lymphatic muscle cells in endothelium-denuded vessels using a concentration of 1.5*10¹⁰ viral particles/ml only intraluminally; c. Transfection of both lymphatic endothelial and muscle cells required a solution outside the vessel containing 1.1*10¹⁰ viral particles/ml together with an intraluminal solution containing 1.5*10¹⁰ viral particles/ml.

Figure 8

An example of the normal phasic contraction patterns at three different pressures was observed prior to demonstration of GFP expression by lymphatic muscle cells in the same isolated rat mesenteric lymphatic vessel. Average 2-D projections of the stacks of confocal images, taken at steps of 0.5 μm through the vessel while pressurized at 5 cm H₂O.