Cells in tissues and organs are present in microenvironments which consist of Extra Cellular Matrix (ECM) and neighboring cells. These microenvironments affect different characteristics of cells including cell architecture, mechanics, polarity and function ^(1–6). Also, it has been shown that the arrangement and orientation of both ECMs and neighboring cells dictate cell size, cell spreading and cell morphology ⁽⁷⁾. As it is obvious, the classic cell culture cannot provide the native cell microenvironment existing naturally in different tissues. In addition, properties such as expression levels of specific genes usually come from thousands of cells. Since cells in culture vessels are usually not homogenous, these expression levels would represent cell behavior throughout the whole flask which is inaccurate.

Micropatterning of cells on surfaces have become popular since 1970s ^{(6, 8–10)}. The main advantage for culturing in a micro-patterned environment over growing cells in classical flask/dish culture is to establish a fine and sub-micron spatial pattern of certain molecules which leads to isolated single-cell culture, or forcing cells to grow in a certain geometric pattern ⁽¹¹⁾. In comparison to culturing cells in culture flasks, micropatterning is an enabling tool for analyzing cells in their microenvironment ⁽¹⁾ and in small scales (∼500 cells) ⁽¹²⁾. Although one can control the content of the culture substratum in all scales, the small-scale analysis needs much lower amount of material and enables the researchers to perform high throughput studies ^(13–15). The patterned area is small so cell niches could be controlled with different materials embedded in ECM micropatterns.

Although micropatterning methods showed that the cells could be cultivated in small populations of around 500 cells/patterns, it has been shown that cell analysis could be done even in single cells ⁽¹²⁾. There are a number of methods, which have been used to make micro-scale patterns on different surfaces ^{(1, 6, 8, 16)}. Among them, photolithography, soft lithography and stencil printing are more used by biologists to create micropatterned surfaces. Soft lithography and stencil printing are more biocompatible than photolithography, but the patterns formed with these methods are less precise than photolithography ^{(2, 8, 16)}.

Several groups have reported the uses of cellular micropatterns for different in vitro studies. Jones et al have studied the release of growth factors from micropatterns and its effect on alcohol injury. In their experiments, the cells were treated with alcohol to mimic alcohol injury in the presence of BMP7 and HGF, which were released from micropatterns containing collagen ⁽¹⁷⁾. In another experiment, Lee et al showed that the function and behavior of cultivated cells on ECM micro-patterns could be analyzed through different ways such as gene expression levels ⁽¹²⁾. In their experiment, HepG2 cells were cultivated on collagen type I micropatterns and then the cells were extracted by laser catapulting for further gene expression analysis ^{(12, 18)}. These studies reveal micropatterning as a very strong tool for both in vitro studies and tissue engineering.

In the studies mentioned earlier, one problem was attaching the ECMs to glass surface. The main issue was that cell-ECM complexes started to detach after one day of cell seeding ^{(12, 17, 18)}. In order to solve this problem, the glass surface treatment protocol was modified in order to minimize cell-ECM complex detachment rate. We have shown that cell behavior on collagen I micropattern spots are normal through the time points of this experiment. To investigate whether the cells on micropattern spots can go through transfection studies, they were also transfected with pmax-cGFP plasmid using lipofectamine reagent.

Micropatterning usefulness appears when it comes to mimicking the microenvironment present in the tissues ^{(1, 7, 8, 16)}. As mentioned earlier, preparing this microenvironment helps the in vitro studies to elucidate what happens in living organisms. Microenvironment in different tissues dictates the fate of the cells by its chemical and physical characteristics. These characteristics dictate cell division rates and cell spreading behaviors. The behavior of tissue in growth pattern and hormone diffusion depends on these characteristics as well. In a study discussed earlier ⁽¹⁷⁾ Jones et al used collagen micropatterns embedded with BMP7 and HGF growth factors to mimic hepatic microenvironments for hepatic differentiation. In other studies, micropatterning is used as a tool for high throughput studies including cell cloning ⁽¹³⁾, gene transfection ⁽¹⁵⁾, cell secretion studies ⁽¹⁴⁾ and tissue engineering ⁽¹⁹⁾.

In previous studies using ECM micropatterning, one of the main problems was that the cells could not be tracked after three days due to ECM micropattern detachment which became a trouble when it came to in vitro differentiation studies. In our method, the cells were attached to surface for 15 days after cell seeding, which made it possible to track cells for transfection, differentiation, and gene expression studies that require cell tracking of 10 days or longer. In this study, we showed that by extending the surface modification re-action (silanzation) to 1.5 hr, the detachment rate of micropatterned ECMs would be near to zero (Figure 3).

Although the process of glass surface treatment and micropatterning is time consuming and laborious, its advantage, that the cells could be tracked using optical microscopy, makes it valuable in comparison to techniques using metal coated glass surfaces (e.g. gold) ⁽²⁰⁾. The whole process of glass surface treatment and attaching the ECM proteins through lysine and cysteine groups are shown in Figure 1. Although the number of HEK and HepG2 cells seeded on collagen I spots were the same, after 12 hr, HepG2 cells were more on the spots. This shows the higher tendency of HepG2 cells in attaching to micropattern ECMs.

This differential tendency of cells for different ECMs can be used for micropatterned co-culture studies. As an example, Lee et al ⁽¹⁹⁾ used a printed pattern of collagen I and fibronectin. They cultivated mESCs (mouse Embryonic Stem Cells) and HepG2 cells sequentially on these spots (mESCs were seeded first followed by washing unattached cells, then they seeded HepG2 cells followed by another step of washing unattached cells). Since mESCs have higher tendencies for fibronectin, they would attach only to fibronectin (washed with appropriate timing) and HepG2 will only attach to collagen I micropatterns (due to higher tendency). Later, they showed that this co-culture could facilitate the differentiation of mESC cells to hepatic cells by analyzing hepatic gene expression levels.

The model in transfection studies which uses pmaxGFP vector reveals that cells could go through transfection studies while culturing on micropatterns. The transfection occurred with more than 90% of efficiency in HEK cells, which again makes the method valuable since transfection of cells located on micropatterns consumes low amount of both lipofectamine and plasmidic DNA in comparison to other techniques involved in ECM and non-viral gene ⁽²¹⁾.

In the present study, the glass surface modification method was optimized to fulfill the requirement of most in-vitro studies in the field of micropatterning. Taking all the results into account, this novel method is promising for efficient cell culture studies on micropatterened surfaces in the future.