The overall goal of this procedure is to set up a silk film in vitro culture system. This is accomplished by first producing the silk solution for making the desired film. The second step is to cast the silk solution onto the silicone rubber molding surface and allow the film to dry down.
The silk film is subsequently processed for cell culture. Next, the culture system is assembled using sterile technique. The final step is to seed the silk films with the desired cell type.
Ultimately, the silk film culture surfaces can be either directly imaged or assayed within the culture. Plates or samples may be removed for fixation and further processing as needed. The main advantage of this technique over existing methods such as culture on standard tissue culture plastic, is that silk film biomaterials are highly biocompatible and can be readily transferred by researchers to downstream in vivo and in vitro applications.
This method can be used to answer key questions in the fields of cell biology and biomaterials, such as how can surface topography be used to affect cell response? Though this method can provide insight into corneal epithelial response, it can also be applied to other systems where initial in vitro experiments can provide preliminary answers to more complex in vivo systems. To begin fabrication of silicone rubber molds obtain the desired topographic surface for casting For this demonstration.
A standard 100 millimeter etched silicon wafer is used way out. PDMS potting and catalyst solution in a nine to one ratio, as provided from a purchased kit. Mix the solutions thoroughly to initiate the curing process.
Following placement of the silicon wafer surface within a casting dish, weigh out 4.5 grams of PDMS solution onto the silicon wafer. After the PDMS solution has spread over the wafer evenly, cover the wafer with a 100 millimeter diameter Petri dish lid and place it at 10 millimeters of mercury for two hours. To degas air bubbles from the PDMS solution, place the casting setup onto a flat surface in a 60 degree Celsius oven overnight the following day, place the cured PDMS silicon wafer into a 100%ethanol bath.
Before removal of the PDMS, begin removing the PDMS from the wafer by using a razor blade to lift the circumference edge using forceps Gently pull off the PDMS within the 100%ethanol bath. Taking care not to tear the silicone rubber casting punch out individual PDMS molds using a 14 millimeter hole punch. This diameter is designed to fit the wells of a 24 well plate.
To prepare the silk solution, bring two liters of distilled water to a boil. In a glass speaker, cut five grams of bombex Maori silkworm cocoons into thirds, dispose of extensively contaminated cocoons as indicated by excessive insect particulates coating the inner cocoon surface. Add 4.24 grams of sodium carbonate slowly to boiling water volume to prevent boiling over.
Once the sodium carbonate has dissolved, add the cocoon pieces to the boiling water and stir with a Teflon coated stir bar for 40 minutes. After boiling, carefully drain the distilled water into the sink and wr out the silk extract by hand to remove excess water. Then wash the silk extract by placing it in a plastic beaker with one liter of distilled water and stirring for 20 minutes.
Repeat this washing process with fresh water twice. Ring out the washed silk extract by hand and place the silk fiber extract inside a chemical hood to allow drying for a 12 hour period. The next day, weigh the dried silk fibers, which are typically approximately 3.5 grams.
Then prepare a 9.3 molar lithium bromide solution for a 20%weight to volume solution of silk according to the written protocol. Following placement of the silk extract into the beaker. Pour the lithium bromide solution over the silk fibers.
Make sure that the silk fibers are immersed within the solution. Using a lab spatula, then place the dissolved silk into a 60 degree Celsius oven for four hours. After four hours, use an appropriate size syringe to draw up 12 milliliters of the silk solution.
Following placement of an 18 gauge needle. On the end of the syringe, inject the solution into a dialysis cassette. Subsequent to cassette filling, draw the remaining air out of the cassette with the emptied syringe.
Place the filled dialysis cassette into one liter of distilled water. Change the water after one hour, four hours, eight hours, and then three times every 12 hours for a total of six changes. After dialysis.
Slowly collect the silk solution from the cassettes with the syringe and transfer to centrifuge tubes. Centrifuge the solution at 10, 000 g and four degrees Celsius for 20 minutes following centrifugation. Place the super natin into a new tube.
After repeating this process a second time, pipette two 0.5 milliliter silk solution samples into separate small whey dishes. Place the whey dishes inside a 60 degree Celsius dry oven for 12 hours or until the silk solution drives. The bulk silk solution can be stored at four degrees Celsius.
Weigh the remaining solid silk film from both samples to measure silk protein concentration density of the solution. Prepare PDMS casting surfaces by using clear tape to remove any dust. Next, clean the PDMS substrates by soaking them in 100%ethanol for one minute.
Then remove them and allow to dry. To produce a 50 micrometer thick film spread 70 microliters of 8%silk solution onto PDMS molds. Using a liquid spreading tool such as a one milliliter syringe tip, allow the silk films to dry uncovered on a laminar flow clean bench by running an airflow pressure of 150 pascals for a period of 90 minutes or until films are dry.
Once films are dry, place the entire set of films including PDMS molds into a water and kneeling chamber for more than four hours to produce a water insoluble film. This is typically an empty desiccated chamber with water in the bottom of the basin, pulled at a 25 kilopascals vacuum following removal of the silk films from the water and kneeling chamber. Work at a laminar flow clean bench to prepare a 70%ethanol bath in a 35 millimeter Petri dish.
Then place any control substrates and stainless steel rings into the dish for at least 10 minutes in order to sterilize them. Next, remove the silk films from the PDMS molds using forceps. Dip them into 70%ethanol and place each film into a single well of a 24 well plate prefilled with one milliliter of 70%ethanol.
Ensure that the pattern side is facing up to allow for cell adhesion To ensure success in this step, it's important that the silk films are placed correctly and sterilely within the wells Place the stainless steel rings on top of the SILT films and let them incubate for 10 minutes Following sample Film wash as instructed in the text, prepare the cell line for seating as directed there. In this demonstration, a human corneal limbal epithelial cell line is used as a final step Sample 500 microliters of HCLE suspension per well, typically using a density of 10, 000 cells per square centimeter and proceed to run the desired experimental protocol. Shown here are scanning electron micrographs of HCLE cell line, adhering to patterned and flat silk films at day two.
In culture HCLE, cultures continue to proliferate to confluence on patterned and flat surfaces by day eight. In culture here, patterned and flat silk films culture, HCLE cells are compared to glass control substrates on day one, day four, and day eight. In culture quantification with SQU nucleic acid content and MTT metabolic activity assay data demonstrate HCLE viability on both patterned and flat silk film substrates when compared to glass control surfaces over time.
Shown here is time lapse phase contrast imaging of HCLE cells migrating over a patterned silk film surface. During an 18 hour time period, cells were seated at a density of 10, 000 cells per square centimeter and cultured for two hours before imaging for comparison time lapse phase contrast imaging of HCLE cells migrating over a flat control surface is shown during an 18 hour time period under the same culture conditions. Once the silk solution and silicone molding is prepared, this technique can be done in eight hours if it is performed properly Following this procedure.
Other methods such as immunofluorescence scanning, electron microscopy and molecular biology techniques such as western blots and immunoassays along with chemical assays such as cell viability, cell proliferation, and metabolic rate can be assessed to better understand cell response upon these various custom design services. After watching this video, you should have a good understanding of how to make silk solution, create customized pattern, silk films and culture. Any cell type on these substrates.
