Micropatterns are a tool that researchers use to control the spatial arrangement of cells on a substrate (surface) by adhering the cell onto a substrate. One of the most common methods to micropattern adhesive proteins which cells attach to is microcontact printing (µCP). Originally done using gold as the substrate, we are now able to perform µCP on cheaper surfaces, such as glass, polystyrene, and polydimethylsiloxane (PDMS, or rubber).
We will go over the procedure for a PDMS substrate.
Making the Master and Stamp
First, we make make a "master" which is generally made of silicon. The master is a piece of material which has been carved by shooting a beam of electrons through a chromium photomask (which resembles the slides from old projectors). Principally, a beam of electrons or UV light is shot over the entire photomask film, but then in the areas where there is the chromium ink, the beam does not travel through. This apparatus is over the silicon wafer, which has been covered by a photoresist, a material which goes away or is solidifies when hit by the beam. Thus, we are able to make a pattern. This technique can achieve patterns that have a resolution of several nanometers (see figure one).
Now that we have this master with engravings, we can begin the patterning process. First, we have to make a replica of the silicon wafer using PDMS (rubber). To do this, we put liquid PDMS (PDMS prepolymer) on top of the master silicon wafer with a curing agent and bake the entire thing. Eventually, we can peel the PDMS off the master and it is ready for use. This PDMS replica is called a stamp, and is used exactly like a regular stamp.
The stamp is turned so that the carved side is facing upwards. We then add an adhesive protein (which the cell will stick to) onto the top of the stamp. Then, we evaporate the protein and end up with a layer of the protein that is a single layer thick, called a monolayer. We can then stamp the PDMS stamp onto our substrate (which is ALSO made of PDMS). In the places that were carved, the protein prepolymer was not printed onto the substrate surface, whilst in the other areas they were (Figure 2)
Preparation of the Substrate
As mentioned before, the surface where we hope to have cell adherence is called the substrate, and in this case it is made of PDMS. Except not entirely. Instead, what we have is is a piece of glass. Then we spin coat the PDMS prepolymer so that there is a thin, flat layer of PDMS. Spin coating is when you have a wheel spinning so quickly that once the prepolymer of PDMS is put in the middle, it spreads into a thin disk on the glass. We also add fetal cow serum (FCS), which provides food for the cells to survive on the substrate.
Printing Patterns
As mentioned before, the PDMS stamps have been prepared so that they can print a pattern of protein which cells can stick to. However, most researchers also add another chemical who's job it is to prevent cells from sticking to an area. This is done by putting the post-printed substrate in a solution containing the anti-adhesive. The anti-adhesive is most commonly a pluronic, which has a structure containing a polyethylene glycol (PEG) piece. This piece is hydrophobic, and the cells do not stick to it. Thus we have a pattern with both stick and ati-stick regions, ensuring we have patterned cell adhesion.
Adhesion Complexities of Wettability
This is all well, except for problems that arise in wettability, or how well water sticks to it. It is the same as being hydrophilic. The PDMS substrate is hydrophobic. Pluronics - the anti-adhesive molecules - adhere to the PDMS because it is hydrophobic. However, proteins do not adhere to such hydrophobic surfaces, so it does not adhere to the PDMS. Both are needed in this method.
Thus, a compromise has to be made. This compromise is done by tuning the wettability of the PDMS substrate so that it is less hydrophobic so that the proteins can now adhere to it, but not so much that the pluronics stop adhering to it. This is shown in figure 3.
Cell Adhesion
Now that we have a micropatterned surface with both an adhesive protein pattern and a non adhesive pluronic backfill, when the cells are added, cells will only stick to the adherent areas. After adding the cells, we wash off the non-adhered cells using a phosphate buffer saline (PBS). We now have a pattern with adhered cells that will last from 1-3 days.
Sources
We will go over the procedure for a PDMS substrate.
Making the Master and Stamp
First, we make make a "master" which is generally made of silicon. The master is a piece of material which has been carved by shooting a beam of electrons through a chromium photomask (which resembles the slides from old projectors). Principally, a beam of electrons or UV light is shot over the entire photomask film, but then in the areas where there is the chromium ink, the beam does not travel through. This apparatus is over the silicon wafer, which has been covered by a photoresist, a material which goes away or is solidifies when hit by the beam. Thus, we are able to make a pattern. This technique can achieve patterns that have a resolution of several nanometers (see figure one).
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| Figure 1: How the creation of a photomask works. This is how it is used in electronics. In biology we use silicon instead of the "Fused Silica: and we do not use the drawn chromium layer. Instead, the photomask has parts with the chromium, and others without, dictating where the UV can penetrate. |
The stamp is turned so that the carved side is facing upwards. We then add an adhesive protein (which the cell will stick to) onto the top of the stamp. Then, we evaporate the protein and end up with a layer of the protein that is a single layer thick, called a monolayer. We can then stamp the PDMS stamp onto our substrate (which is ALSO made of PDMS). In the places that were carved, the protein prepolymer was not printed onto the substrate surface, whilst in the other areas they were (Figure 2)
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| Figure 2: The process of making the stamp and using it. In this picture, the Elastomeric Stamp is the same thing as the PDMS |
As mentioned before, the surface where we hope to have cell adherence is called the substrate, and in this case it is made of PDMS. Except not entirely. Instead, what we have is is a piece of glass. Then we spin coat the PDMS prepolymer so that there is a thin, flat layer of PDMS. Spin coating is when you have a wheel spinning so quickly that once the prepolymer of PDMS is put in the middle, it spreads into a thin disk on the glass. We also add fetal cow serum (FCS), which provides food for the cells to survive on the substrate.
Printing Patterns
As mentioned before, the PDMS stamps have been prepared so that they can print a pattern of protein which cells can stick to. However, most researchers also add another chemical who's job it is to prevent cells from sticking to an area. This is done by putting the post-printed substrate in a solution containing the anti-adhesive. The anti-adhesive is most commonly a pluronic, which has a structure containing a polyethylene glycol (PEG) piece. This piece is hydrophobic, and the cells do not stick to it. Thus we have a pattern with both stick and ati-stick regions, ensuring we have patterned cell adhesion.
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| An Example to What a pattern looks like |
This is all well, except for problems that arise in wettability, or how well water sticks to it. It is the same as being hydrophilic. The PDMS substrate is hydrophobic. Pluronics - the anti-adhesive molecules - adhere to the PDMS because it is hydrophobic. However, proteins do not adhere to such hydrophobic surfaces, so it does not adhere to the PDMS. Both are needed in this method.
Thus, a compromise has to be made. This compromise is done by tuning the wettability of the PDMS substrate so that it is less hydrophobic so that the proteins can now adhere to it, but not so much that the pluronics stop adhering to it. This is shown in figure 3.
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| Figure 3: This picture shows how the different materials adhere to the micropatterns as the wettability changes. In it, 0% OH is completely hydrophobic, whereas 100% OH is completely hydrophilic. 40% OH is where both the pluronic and the protein stick to the micropattern. Note that cells stick to the proteins, so if the protein adhered, so did the cells. |
Now that we have a micropatterned surface with both an adhesive protein pattern and a non adhesive pluronic backfill, when the cells are added, cells will only stick to the adherent areas. After adding the cells, we wash off the non-adhered cells using a phosphate buffer saline (PBS). We now have a pattern with adhered cells that will last from 1-3 days.
Sources
Wettability: here
Procedure: here
Procedure: here
Comparative Study: here
