Photopatterning Proteins and Cells in Aqueous Environment Using TiO₂ Photocatalysis

The overall goal of the following experiment is to perform surface modification of a cell culture scaffold using the photo catalytic activity of titanium dioxide. This is achieved by depositing a thin layer of titanium dioxide onto a glass cover slip, and then coating it with an Octa dessaline to render the whole surface hydrophobic. As a second step, the substrate is blocked by immersing it in cell culture, medium containing serum albumin.

The albumin absorbs to the surface and makes it non permissive to cell adhesion. Next, the sample is transferred to an aqueous solution containing collagen and a focused UV light is then used to irradiate the surface. This allows collagen molecules to absorb selectively to the photo irradiated region and renders the region permissive to cell adhesion.

This process can be repeated in order to pattern multiple proteins or cell types. We show in this video how the adhesive region of PC 12 cells can be selectively modified via UV irradiation. We first had the idea for this method when we realized that Titanic dioxide photocatalysis is commercially applied as self-cleaning glass windows that remove organic contaminants in the surface upon light radiation, demonstrating the procedure will be co-head seeking a grad student from my laboratory.

Start by numbering the cover slips using a diamond scriber in a way that assists in ensuring the correct side of the sample is facing up. Then clean the cover slips first under running double distilled water, then by immersing them in piranha solution for 10 minutes. After 10 minutes, rinse the cover, slips eight times in double distilled water and dry them under nitrogen flow.

Next, set a titanium oxide target into a radio frequency sputtering system, and then attach the clean cover slips onto the sample holder using a polyamide tape, place the sample holder in the sputtering chamber and evacuate the chamber until the pressure reaches 2.0 times 10 to the minus fourth pascals. Next, introduce argon gas into the chamber and set the deposition pressure to 4.0 militar while keeping the shutter closed. Gradually increase the RF power to 70 watts.

Then open the shutter and sputter for 15 minutes To obtain a film with a thickness of 120 to 150 nanometers. Begin by drying a glass beaker and forceps in an oven at 120 degrees Celsius for 30 minutes. Next, place the cover slips into a plasma reactor and treat the titanium dioxide surface with oxygen plasma for five minutes at 200 watts with an oxygen flow of 100 standard cubic centimeters per minute to create a more hydrophilic surface.

Then remove the samples and immerse them in double distilled water to confirm that the surface is super hydrophilic and dry the surface thoroughly under nitrogen flow. Next, move the samples the dried beaker and forceps into a nitrogen filled glove bag and prepare a one millimolar solution of Octo desal trichlorethylene dissolved in toluene. Immerse the sample in the solution for one hour at room temperature to form a self-assembled monolayer on top of the titanium oxide.

Then remove any phasor molecules by fornicating the sample in toluene, acetone, ethanol. And finally, double distilled water for five minutes each. Then rinse the sample four times in fresh double distilled water and dry the surface under nitrogen flow.

Sterilize the coated cover slips by immersing the samples in 70%ethanol for five minutes. Then rinse the samples twice in sterile double distilled water. Next, place the sample in a 35 millimeter dish and add two milliliters of PC 12 growth medium.

Incubate the samples for at least three hours in a CO2 buffered incubator at 37 degrees Celsius while the serum albumin in the media absorb onto the surface of the cover slips while waiting. Set up the inverted fluorescence microscope by first turning on the arc lamp. Then insert the UV filter cube and rotate the objective lens to one with 20 x magnification.

Measure the light intensity with a UV intensity meter and calculate the irradiation time for a dose of 200 joules per square centimeter. Next, use a stage micrometer and set the diameter of the region to be irradiated to 200 microns by adjusting the field diaphragm. After the three hour incubation, supplement the medium covering the cover slips with 200 microliters of 3.0 milligrams per milliliter Type four collagen.

Then transfer the 35 millimeter dish to the microscope stage and focus the microscope onto the sample surface using the scratch marks. Irradiate an area of the cover slip with UV light at a dose of 200 joules per square centimeter. Then replace the medium with two milliliters of fresh growth, medium with NGF and without Type four collagen and place the sample back in the incubator.

Harvest cultured NGF differentiated PC 12 cells in a 15 milliliter conical tube and centrifuge the tube at 150 times G for four minutes. Then aspirate the supernatant and add one milliliter of the growth medium. Resus resuspend the cells by gently pipetting up and down using a hemo cytometer.

Count the cell density and add 3.0 times 10 to the fifth cells to a 35 millimeter dish containing the modified cover slips. Incubate the dish for one to two days in a 5%CO2 incubator at 37 degrees Celsius after one to two days of culture on the XC two modified surface. Use a microscope to confirm that the cells are attaching and growing only on the UV irradiated region of the cover slips.

Then add 67 microliters of type four collagen to the dish with the cultured cells so that the final concentration of collagen is 100 micrograms per milliliter with the microscope set up for surface patterning. Place the dish on the microscope stage and position the irradiation region adjacent to the current cell location. Irradiate the cover slip with UV light at a dose of 200 joules per square centimeter.

After irradiation, replace the medium with one devoid of collagen and place the sample back in the incubator trying to complete processing of a single sample within 30 minutes. This is a cross-sectional scanning electron microscopy image of the sputter deposited titanium dioxide film. From the observation, the thickness of the film was estimated to be approximately 150 nanometers.

Also noticeable here is the flatness of the deposited titanium dioxide film. Further analysis by atomic force microscopy revealed that the root mean square roughness of the surface was only 0.2 nanometers. The type four collagen and solution absorbs preferentially to the UV irradiated region where the albumin are displaced by the laser.

To demonstrate the versatility of this approach, other proteins such as fibrin have also been patterned using the same technique. Here cells are growing on the XC two patterned region of the cover slip and right next to them in the area defined by the dotted line. The cover slip was recently UV irradiated in situ two and coated with type four collagen.

Over the next five days, cells proliferate and migrate to cover the newly coded region. The procedure presented here can be employed to arbitrarily manipulate cells under culture and potentially to pattern multiple types of cells to construct elaborate cold co-culture systems.