Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The overall goal of this procedure is to develop an in vitro model of smooth muscle contractile function that can be used to study the mechanisms of vascular dysfunctions, such as cerebral vasospasms over disease relevant timescales. This is accomplished with long-term vascular muscular thin films by first preparing elastomer substrates by serially spin coating cover slips with a strip of piam followed by PDMS. The second step is to use a microfluidic deposition device to serially deposit genin and fibronectin onto the surface.

In order to provide guidance cues for tissue self-organization on vascular, muscular thin films, the final step is to perform a contractility assay by cussing vascular, muscular thin films inducing contraction and relaxing contraction while imaging the sample. Ultimately, this approach yields tissues that maintain structural fidelity and functional contractility for up to two weeks in culture. The main advantage of this technique over existing methods like micro contact printed muscular thin films, is that tissue integrity and functional contractility are maintained for weeks where tissues constructed using previous methods begin to degrade after three to four days.

In culture, This method provides a platform for studying key questions in vascular disease, such as how the functional mechanics of arteries are altered during chronic disease progression. Here we are studying vascular mechanics, but this method can be applied to other model organ systems such as organ chip technologies for recapitulate, cardiac or skeletal muscle function. We first had the idea for this method when we aimed to study slow developing vascular dysfunctions and found that current methods either could not be used to quantify vascular smooth muscle cell function, or did not maintain integrity for a sufficient period of time.

Jin Microfluidic deposition provides a template for cell self-organization in a way similar to that of micro contact printing, but also allows pattern tissues to maintain their structure and integrity for timescales appropriate for studying vascular remodeling. To begin clean a batch of 25 millimeter diameter glass cover slips as described in the accompanying text protocol cover the edges of the cleaned cover slips by placing adhesive tape on their sides, leaving an exposed strip of glass in the center. Next, cut the cover slips free from the dish and place them one at a time onto a spin coated chuck.

Using forceps, add 150 microliters of a 10%solution of poly N isop profile. Acrylamide also called poly pam or Piam dissolved in one butanol. Be sure to completely cover the exposed area of the cover slip.

Then turn on the spin coter and ramp the speed for 10 seconds to 3000 RPMs. Wait for five seconds and then ramp for 10 seconds to 6, 000 RPMs. Wait at 6, 000 RPMs for one minute and then ramp back down to 3000 RPMs over 10 seconds.

Holding it there for five seconds before finishing the spin coating cycle. Once dry, carefully remove the adhesive tape from all of the cover slips, leaving the full cover slip exposed with a thin layer of piam coating in the center of the glass cover slip. Next, spin coats a layer of Degas PDMS onto each cover.

Slip and cure them in an oven at 90 degrees Celsius for at least 1.5 hours. Design a tissue microfluidic photo mask and then prepare a pattern silicon wafer as described in the accompanying text protocol. Once patterned, ate the wafer using standard techniques and place it feature side up in a Petri dish.

Next mix and DGAs 100 grams of PDMS With a 10 to one based crosslinker ratio, pull the PDMS into the dish completely and evenly covering the patterned wafer. Place the dish in a vacuum desiccate for approximately 30 minutes until all air bubbles are removed. Then cure the PDMS in the dish at 90 degrees Celsius for at least 1.5 hours.

Once cured, cut away excess PDMS and slowly peel the remaining patterned PDMS away from the pattern side of the wafer. Cut away the excess PDMS from around the patterns using a razor blade and shape the devices in rectangles to aid removal of the device in later steps. Next, use a one millimeter surgical biopsy punch to form both the inlet and outlet holes.

Sonicate microfluidic devices in 70%ethanol for at least 30 minutes prior to each use. Place up to 10 vascular muscular thin film substrate cover slips in an ultraviolet ozone cleaner for eight minutes. Next, prepare a five milligram per milliliter genin solution by adding one milliliter of sterile double distilled water to a five milligram container of lyophilized genin.

Mix the solution using a vortex mixer and set the vial aside at room temperature for at least 30 minutes. Place the cleaned microfluidic devices feature side down onto each cover. Slip one at a time.

Press down firmly on the devices to ensure a tight seal to the PDMS coated cover slips. Quickly place a drop of 70%ethanol at the inlet of each device for device priming. After five to 10 minutes, carefully aspirate the excess ethanol at the inlet and immediately replace it with approximately 100 microliters of PBS.

Use a vacuum to draw PBS through to the outlet of the devices to rinse away the ethanol. Stop when only a small amount of PBS remains at the inlet. Then place 60 microliters of the five milligram per milliliter genin solution at each inlet, draw the genin solution through the devices replacing a vacuum aspirator tip at the outlet.

Stop the suction when only a small amount of solution remains at the inlet. Next place drops of PBS at both the inlet and outlet to prevent the solution from drying. During incubation, move the dish containing the devices to a humidified oven or incubator set to 37 degrees Celsius and incubate for four hours.

During incubation, re suspend fibronectin to a concentration of 50 micrograms per milliliter in sterile double distilled water and place it on ice for at least 30 minutes after the incubation with Jin. Draw through or, but a minimal amount of PBS at the inlet. Next place, 100 microliters of a 50 microgram per milliliter fibronectin solution.

At each inlet, draw the solution through the devices using a vacuum aspirator tip. At the outlet, move the uncovered dish containing the devices to an oven or incubator set to 37 degrees Celsius and incubate for 24 hours. Following incubation carefully remove the devices from the cover slips by slowly peeling the device at a corner while lightly grasping the cover slip in the opposite hand place the cover slips into sterile six well dishes.

Add at least five milliliters of penicillin streptomycin solution to each well, and then place the dishes in a sterile incubator at 37 degrees Celsius for at least 30 minutes. After sterilization, aspirate the penicillin streptomycin solution and seed the cover slips with cultured human umbilical artery vascular smooth muscle cells at a concentration of 80, 000 cells per square centimeter. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for at least 24 hours prior to testing.

To begin testing, place a tissue sample in a 100 millimeter dish. Add sterile prewarm tyro solution at pH 7.4 to cover the sample. Using a razor blade makes several parallel cuts through the cell sheet perpendicular to the pipe arm edge.

Make cuts that yield wider tissue sections about two millimeters thick. That will be the vascular muscular thin films alternating with thin strips to be removed later. Then rotate the dish 90 degrees and make two straight parallel cuts in the middle of the tissue parallel to the strip of piam.

Remove the thin strips in between vascular, muscular, thin films to prevent adjacent films from making contact. Next place the dish in a temperature controlled platform on the stereo microscope stage and begin to capture time-lapse, transmitted, and fluorescent light images are desired intervals throughout the treatment assay. Serially treat the vascular muscular thin films with 50 nanomolar of endothelium, one for 20 minutes to induce contractions, followed by 100 micromolar of HA 10 77 for 30 minutes to induce relaxation upon cooling to below 32 degrees Celsius.

Piam dissolves releasing the vascular muscular thin films. Measurement of projection length can be converted to a radius of curvature, is used to calculate the average stress in the cell layer on the film shown here at the sequential transmitted light images of a representative contractility assay. These images can be converted to stress values from which the basal tone and induced contractility are calculated.

In this video, a representative time lapse of a full contractility assay is shown. Muscular thin films reach contractile equilibrium, then contract further upon addition of 50 nyla molar endothelium. One films relax after addition of 100 micromolar ha 10 77.

The slight drop in basal tone and tissue contractility at the end of the assay is likely the direct result of the reduced number of cells composing the tissue. Since serum starved vascular smooth muscle cells do not proliferate shown here are phase contrast images of smooth muscle cells grown on genin modified surfaces at various sacrifice time points ranging from one day to two weeks. Immunohistochemistry was used here to highlight the confluence and cellular alignment of the tissues by staining the F actin filaments colored green following one, four and seven days of serum starvation.

Quantitative assessment of tissue confluence over this time range showed minimal deterioration on these surfaces. The alignment of the f actin filaments was quantified and it was confirmed that alignment was maintained over the two weeks. In culture, Once mastered this in vitro tissue fabrication technique can be done in two days.

While attempting this procedure, it's important to remember to start microfluidic deposition a day prior to anticipated cell seeding and to maintain a constant physiological temperature during fin film experiments. Following this procedure, traditional biochemistry methods like Western blotting and PCR can be performed to study changes in protein and gene expression to correlate cell phenotype with functional behavior. This technique paves the way for researchers to explore progression of slow developing vascular dysfunctions like cerebral vasospasm, hypertension and atherosclerosis, and in vitro models of the human arterial wall.