Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

The overall goal of this procedure is to deposit a thin collagen assembly onto cells in a microfluidic device. This is accomplished by first modifying acidified native collagen through methylation to create net positively charged collagen molecules. The second step is to modify acidified native collagen through ation to create net negatively charged collagen molecules.

Next, the microfluidic devices are prepared and seeded with hepatocytes, which are allowed to attach and spread overnight. The final step is to deposit the collagen assembly by alternately exposing the cells to the positively and negatively charged collagen solutions to create 10 bilayers of collagen on top of the cell layer. Ultimately, phase and immunofluorescence microscopy and albumin and urea assays are used to show the development and maintenance of cell polarity and secretory function.

The main advantages of this technique are that it enables hepatocyte culture in micro devices using similar techniques to those applied in plate cultures for over 20 years. It doesn't require complex device designs and the deposited matrix is very thin on the order of 100 nanometers. We first had the idea for this method while brainstorming about how to translate class Capy cell culture techniques from plates to microfluidic devices.

The collagen sandwich methods works in open top microscopic systems, but the hydrogels used are incompatible with closed micro devices with limited channel heights. Dilute 100 milligrams of a native acidified collagen solution to a concentration of 0.5 milligrams per milliliter with ice, cold, sterile water, and place the solution on ice to prevent alation. Next, adjust the pH of the collagen solution to between nine and 10 with a few drops of one normal sodium hydroxide and gently stir the solution at room temperature for 30 minutes.

As the collagen precipitates, the solution will begin to turn cloudy, spin down the precipitated collagen solution that 3000 times G for 25 minutes. Afterwards, a clear gel-like precipitate should be visible in the bottom of the tubes, aspirate the snat, and then resuspend the precipitated collagen in 200 milliliters of methanol with 0.1 normal hydrochloric acid allow the methylation reaction to occur while stirring at room temperature for four days after the methylation centrifuge, the solution at 3000 times G for 25 minutes to pellet the methylated collagen, then aspirate and dispose of the acidified methanol supinate. Next, dissolve the methylated collagen in 25 milliliters of sterile PBS and filter the solution through a 60 micron cell strainer.

Adjust the pH of the solution to 7.3 to 7.4 using 20 microliter increments of one normal sodium hydroxide. Assess the concentration of the collagen solution using a commercial rat collagen ELIZA kit or hydroxyproline assay kit. Then dilute the solution to three milligrams per milliliter with sterile PBS.

Sterilize the methylated collagen solution by transferring it to a glass bottle with a screw cap. Carefully layering three milliliters of chloroform at the bottom of the bottle and allowing the bottle to set overnight at four degrees Celsius. The next morning.

Aseptically, remove the top methylated collagen layer. Store the methylated collagen at four degrees Celsius and use it within one month. Dilute another 100 milligrams of native acidified collagen solution to a concentration of 0.5 milligrams per milliliter with ice, cold, sterile water, and place the solution on ice to prevent dation as previously shown, adjust the pH of the collagen solution with sodium hydroxide and stir the solution at room temperature to precipitate the collagen.

Next, dissolve 40 milligrams of six cynic anhydride in 10 milliliters of acetone. Slowly add this mixture in 0.5 milliliter increments to the collagen solution while stirring and continuously monitoring the pH of the solution. Maintain the pH above 9.0 by adding one or two drops of one normal sodium hydroxide as the pH approaches 9.0 continuous stirring at room temperature for 120 minutes.

After adding all of the six cynic and hydride solution, observe the mixture become clear as the succinate collagen dissolves, continue to periodically check the pH to ensure it remains above 9.0. When all of the succinate collagen has dissolved, adjust the pH of the solution to 4.0 using 20 microliter increments of one normal hydrochloric acid. Observe the solution, become cloudy again as the succinate collagen precipitates.

Next centrifuge the solution at 3000 times G for 25 minutes. To pellet the succinate collagen asbury and dispose of the acidified snat with unreactive snic anhydride dissolve the succinate collagen with repeated pipetting in 25 milliliters of sterile PBS giving a final concentration of approximately three milligrams per milliliter. Then filter the solution through a 60 micron cell strainer, and then adjust the pH of the solution to between 7.3 and 7.4.

Using 20 microliter increments of one normal sodium hydroxide, assess the concentration of the solution using a commercial collagen rat ELIZA kit or hydroxyproline assay kit. Then dilute the solution to three milligrams per milliliter. Using sterile PBS sterilize this ated collagen solution the same way as the methylated collagen solution.

Begin by fabricating a microfluidic device using standard technique which has cell culture chambers with 100 microns tall, 0.4 to 1.5 millimeters wide and one to 10 millimeters long channels for cell growth. Use a plasma cleaner to oxidize the surfaces of the device and a glass slide. Then press the two together to form a bond.

After sterilizing the device by exposure to UV light for at least 30 minutes. Fill the chamber with 15 micrograms per milliliter, fibronectin in sterile PBS and incubate at 37 degrees Celsius for 45 minutes. Next, see the device with cells as described in the accompanying text protocol.

In a laminar flow tissue culture hood, prepare sufficient volumes of methylated and ated collagen solutions for 10 applications of each solution per device, as well as a few extrem milliliters of media. Keep the solutions on ice. Alternate flushing the devices with 20 microliters of methylated and then ated collagen solutions.

Waiting one minute between each application. Flush the device a total of 10 times per solution. Work quickly to minimize the amount of time the cells are without media while growing the layers of collagen, it is possible to observe collagen slowly accumulate at the inlet and outlet of the microfluidic device.

If the resistance to fluid flow increases, flush the device once or twice with media and then continue layering. After applying all of the layers, rinse the device twice with fresh media and return to the incubator. Methylation and ation alter the charge characteristics of the collagen molecules.

Ation removes positively charged groups and replaces them with negatively charged groups, and methylation removes negatively charged groups, creating net positively charged molecules, allowing for layer by layer deposition of the two collagen variants on top of charged surfaces such as cells. Primary hepatocytes seeded on fibronectin coated glass within a microfluidic device can be coated with this layer by layer. Collagen matrix assembly, deposition of 10 bilayers on cells creates a collagen layer thickness of approximately 140 nanometers hepatocytes without a top collagen layer by layer.

Matrix lose their differentiated phenotype contract and lift off the surface of microfluidic devices over time. In contrast, hepatocytes covered with an ultra thin collagen assembly maintain their differentiated morphology, a viability of greater than 90%and polarization over 14 days. In addition to cell viability morphology and polarization, the collagen layer by layer technique also recovers and stabilizes the function of primary hepatocytes as indicated here, After watching this video, you should have a good understanding of how to methylate and succinate collagen to create net positively and negatively charged collagen solutions and how to use them in layer by layer deposition to create thin collagen assemblies on top of cells in microfluidic devices.