Cell migration is a fundamental phenomenon associated with a vast range of basic physiological processes (such as immune cell migration), developmental processes (such as neuronal cell migration) as well as in non-communicable diseases (such as cancer) (Yamada and Sixt, 2019; SenGupta et al., 2021; Lusby et al., 2022; Merino-Casallo et al., 2022; Pawluchin and Galic, 2022). The cells respond to various chemical and biophysical cues that originate internally or externally and migrate in single cell or collective fashion (SenGupta et al., 2021; Lusby et al., 2022; Merino-Casallo et al., 2022; Pawluchin and Galic, 2022). Over the past decade many successful attempts have been made to unravel cell migration phenomenon with respect to cell behavior during migration and molecular players involved in it using experimental and simulation techniques (Grada et al., 2017; Haridas et al., 2018; Matsiaka et al., 2019; Melo et al., 2021). Especially in cancer, understanding cell migration is very important because of its principal role in metastasis. In cancer metastasis, the cell migrates away from primary tumor site and enters blood circulation (intravasation) and further migrates to secondary tissues (extravasation) to create secondary tumors, which increase complications by several orders of magnitude. Thus, metastasis is a leading cause of deaths in cancer patients (Stuelten et al., 2018; Lusby et al., 2022; Merino-Casallo et al., 2022). Therefore, study of cell migration has received prime importance in the perspective of diagnosis as well as treatment of cancer.

Apart from few exceptions such as epithelial cell migration involved in skin wound closure and cell migration on bones, all cell migration phenomena occur in 3 dimensions (3D) in normal physiological setup (Yamada and Sixt, 2019). Surrounding extracellular matrix (ECM) and neighboring cells have direct impact on the mode and rate of cell migration (Stuelten et al., 2018; Matsiaka et al., 2019; Yamada and Sixt, 2019; SenGupta et al., 2021; Lusby et al., 2022; Merino-Casallo et al., 2022; Pawluchin and Galic, 2022). In recent years, the cell migration studies have seen few technical or experimental advances such as study of cell migration in 3D environment (e.g.,: in Matrigel™ support) (Audoin et al., 2022) or in soft or hard biocompatible hydrogels (Trappmann et al., 2017), cell migration in spheroids (Zhang et al., 2022), and cell migration in microfluidic devices (Schwarz et al., 2016; Shih et al., 2019). As it is difficult to mimic 3D cell migration in laboratory setup, this phenomenon is most often studied in an in vitro 2-dimensional (2D) cell cultures (Riahi et al., 2012; Stamm et al., 2016). 2D studies provide conceptual evidence and understanding of the cell migration rate and directionality (Riahi et al., 2012; Stamm et al., 2016; Grada et al., 2017; Boyer et al., 2018; Acosta et al., 2022), but significant differences exist between 2D and 3D cell migration, so caution must be exerted when interpreting 2D migration results. 2D cell migration studies are often associated with absence and presence of additional chemical entities such as cell migration promoters (e.g.,: growth factors) or cell migration inhibitors (e.g.,: potential anticancer drug candidates). 2D cell migration assay (also known as scratch assay or wound healing assay) can be performed by various techniques as mentioned in Table 1 (Riahi et al., 2012; Stamm et al., 2016). Each technique has its own advantages and disadvantages. The common factor involved in these techniques is cell scraping (mechanical, laser- or electric current-assisted) which harms the cellular structure and associated ECM. Neighboring surviving cells may also experience mechanical or heat related stress. Such stress could induce a response from cells leading to changes in basic cellular physiology, which obscure the results obtained in these assays (Riahi et al., 2012; Stamm et al., 2016). In order to prevent the changes in cellular physiology, ibidi GmbH (Germany) has developed specialized inserts, essentially blocks of polymer, that create a cell-free area (similar to a scratch or a wound) without impacting basic cellular physiology (Reference: https://ibidi.com/content/category/21-wound-healing-and-migration (Last accessed on 26/12/2022)). These inserts work on the principle of putting up a physical barrier that prevents formation of a cell monolayer in a specific area. However, the cost of such inserts is very high. Also, these inserts are meant for single use in an experiment. Thus, in an attempt to improve this experimental technique, other types of inserts have been introduced, which work on the principle similar to the ibidi GmbH inserts. The most recent advance in such inserts were reported by Acosta et al. and Boyer et al. Even though their inserts yielded reproducible results, the technology still has some notable limitations, mentioned in Table 2 (Boyer et al., 2018; Acosta et al., 2022).

In order to overcome these limitations of inserts for 2D cell migration assays, we designed and 3D printed an alternative novel cell insert with a characteristic shape (Figure 1) that would allow reproducible creation of cell-free area, easy and hassle-free usage with possibility of imaging while insert is placed in well. To validate our inserts, we carried out an in vitro cell migration assay in cell culture medium supplemented with cell migration promoter (epidermal growth factor (EGF)) (Kim et al., 2015), a cell migration inhibitor (colchicine) (Wang et al., 2019), and an anticancer drug (doxorubicin hydrochloride) (Thorn et al., 2011). For bulk image analysis we also developed a new image analysis pipeline. Our overall results depict a modified, novel, and improved design for cell inserts and its applicability in high throughput study of cell migration in 2D cell culture setup with high reproducibility.

As stated previously, there are several methods available for performing cell migration studies; however, they suffer from serious drawbacks such as limited reproducibility, destruction of cell monolayer and/or ECM secreted by cells, mechanical or thermal stress, and stress related genetic and/or biochemical changes in cells. Commercial cell inserts that were meant to overcome these drawbacks are very costly and applicable for one time use only. Therefore, we have designed and 3D printed cell inserts with arguably superior characteristics. For 3D printing, we selected high temperature resin to yield the sturdy and autoclave compatible cell inserts. Among the two latest state of the art cell inserts meant for studying cell migration, one provides simple experimental setup owing to its small size and simple design. However, it does not allow for visualization of cells under a microscope in the presence of cell inserts (Acosta et al., 2022). On the other hand, the second bulky cell inserts offer the possibility of cell visualization, but this requires the use of extra devices such as well plate locking and stabilizing clamps (Boyer et al., 2018). Without these clamps, the cell inserts may not fit inside plate and may move during handling. This could lead to compromised reproducibility in the cell migration assays. The use of additional clamps with the inserts makes the procedure cumbersome for high throughput screening. Also, such extra devices increase overall 3D printing cost. By contrast, our cell insert; due to the “plus sign” design at the top that creates a four-quadrant space, allows for microscopy cell visualization in real time, while the insert is kept in the well plate. The “plus sign” design on the top and the “cylindrical” design below it in our cell inserts allow the user to replace the cell culture medium over the course of an experiment. Our inserts need no additional “holding” equipment owing to their simpler design and smaller size. Hence, we would like to argue that our cellinserts are easier to use and more suitable for reproductible and high throughput cell migration assays with real time monitoring. They also allow for exploring additional experimental parameters via easy change of the growth medium. No significant differences in the viability of cells grown in presence of these inserts as compared to control group proves that our inserts are perfectly compatible with A549 cells. Unlike the methods used to create cell-free area such as laser treatment, mechanical stamping, scratching with pipette tip, our cell inserts did not seem to alter the basic physiology of human cells. This guarantees that the findings of studies with our inserts would be solely the result of the cellular response to the medium supplemented with various chemicals. After confirming zero leakage under cell inserts and uniformity of cell-free areas in all wells, we validated them with cell migration study under various treatments. But, before actual cell migration analysis, it is imperative that the image analysis should be hassle-free, easy, and automated for several images. To enable that, we created a macro script in ImageJ platform. Our macro script yielded comparable results (in terms of determination of percent cell-free area) with another available ImageJ program called “Wound healing size tool” (Suarez-Arnedo et al., 2020). Our macro script proved to be superior than “Wound healing size tool” because unlike this tool, it allowed uniform analysis of hundreds of images in one go.

In actual cell migration assay, for validation of these inserts, we used various treatments. Among these treatments, DMEM, FBS, and EGF had positive impacts on cell migration; whereas colchicine and doxorubicin had negative impact. The DMEM supplies basic nutrients (amino acids, mineral salts, and vitamins) for growth and proliferation of cells whereas medium supplemented serum provides additional nutrients such as growth factors, proteins, immunoglobulins, and several small molecules (Kwon et al., 2016). FBS is the most widely accepted serum supplement for cell culture experiments. Owing to availability of growth factors from serum, cells proliferate and migrate with faster rate filling the cell-free area, as it is evident from Figure 5. EGF is a well-known growth factor that promotes and regulates growth and proliferation of primary cells and immortalized cell lines (Kim et al., 2015; Ohnishi et al., 2017). Its positive effect on cell migration; and especially in case of A549 cell line, has been well documented (Lauand et al., 2013; Castro-Aceituno et al., 2016). EGF binds to Epidermal Growth Factor Receptor (EGFR) and show several growth promoting actions such as improved cell proliferation, cell growth and cell differentiation, improved ECM synthesis and ECM remodeling, increased protein synthesis and protein phosphorylation, and increased cell migration (Lauand et al., 2013; Kim et al., 2015; Ohnishi et al., 2017). Accordingly, EGF-supplemented cells migrated faster than those given only DMEM and FBS. Colchicine, originally used as an anti-inflammatory agent, is a microtubule binding alkaloid with potent anticancer activity (Wang et al., 2019). Its exact anticancer mechanism is not yet fully known. It is believed to bind with microtubules and destabilize the microtubule network in the cell (Wang et al., 2019; Oh et al., 2022). Disturbed microtubule network impairs cell migration. Colchicine also induces caspase-3-mediated apoptosis via suppressing the PI3K/Akt/mTOR signaling pathway in some cancer cell lines (Huang et al., 2015; Balkrishna et al., 2019; Zhang et al., 2019; Oh et al., 2022). Specifically in A549 cell line, it binds strongly with microtubule assembly (reported binding constant 0.5 µM), depolarizes the assembly, induce ROS and p53 signaling pathway, and increases proapoptotic to antiapoptotic protein ratio that ultimately leads to cell death (Bhattacharya et al., 2016). Doxorubicin is a known FDA approved anticancer drug that binds with DNA and hinders its repair via topoisomerase-II-enzyme (Thorn et al., 2011; Bojko et al., 2019; Kciuk et al., 2023). It also induces oxidative stress that leads to cell membrane damage, inactivation of proteins, and DNA damage. Even though doxorubicin does not have a direct impact on microtubule assembly, it reduces cell migration probably via inhibition of protein synthesis and prolyl 4-hydroxylase enzyme activity (Thorn et al., 2011; Bojko et al., 2019; Kciuk et al., 2023).

Thus, all these results (Figure 5; Supplementary Figures S1–S3), altogether further strengthen our findings of cell viability assay, where we claimed that presence of our inserts does not affect cellular physiology and cell viability. Rather, cells only respond to given treatment and this observation makes our cell inserts perfectly suitable for high throughput cell migration experiments. Overall, our novel cell inserts yielded highly reproducible results for cell migration study of A549 cell line in presence of known standards such as EGF, colchicine, and doxorubicin. In presence of these agents, the obtained results are similar to what reported in literature for other cancer cell lines, which validate these novel inserts.

Our overall results suggest that these novel cell inserts provide a simple, fast, and highly reproducible method for cell migration analysis. These inserts are compatible with autoclaving, and with performing high throughput experiments. Due to the special design, these inserts address the main drawbacks of previous devices, namely, they allow real time monitoring of the progressing cell monolayer with a brightfield microscope, easy exchange of medium without interrupting the experiment, and very simple operation, suitable for inexperienced users. Moreover, the newly developed very simple ImageJ macro script allows rapid analysis of thousands of images in reproducible and error-free way. Based on the presented data, we propose that our cell insert design coupled with the new image analysis method provides a clear technological advancement in the field of 2D cell migration studies in terms of simplicity, reproducibility, and robustness.