Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

Correction: Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing.

Correction for 'Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing' by Ketaki Bachal et al., Lab Chip, 2023, 23, 261-271, https://doi.org/10.1039/D2LC00879C.

Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing.

Drug testing is a vital step in the identification of the potential efficacy of any new/existing drug and/or combinations of drugs. The conventional methods of testing the efficacy of new drugs using multiwell plates are time consuming and prone to evaporation loss and manual error. Microfluidic devices with automated generation of concentration gradients provide a promising alternative. The implementation of such microfluidic devices is still limited owing to the additional expertise and facilities required to fabricate and run these devices. Conventional microfluidic devices also need pumps, tubing, valves, and other accessories, making them bulky and non-portable. To address these problems, we have developed a method for fabricating microfluidic structures using a nonconventional technique by exploiting the Saffman-Taylor instability in lifted Hele-Shaw cells. Multi-channel structure molds with varying dimensions were fabricated by shaping ceramic polymer slurry and retaining the shape. Further using the mold thus made, polydimethyl siloxane (PDMS) devices offering static, stable, diffusion-based gradients were casted using soft lithography. We have demonstrated with COMSOL simulation, as well as using fluorescein isothiocyanate (FITC), a fluorescent dye, that the concentration gradient can be generated in this device, which remains stable for at least 5 days. Using this multichannel device, in vitro drug efficacy was validated with two drugs namely, temozolomide (TMZ) and curcumin, one FDA approved and one under research, on glioblastoma cells (U87MG). The resulting IC50 values were consistent with those reported in the literature. We have also demonstrated the possibility of conducting molecular assays post-drug testing in the device by microtubule staining after curcumin treatment on cervical cancer cells (HeLa). In summary, we have demonstrated a i) user-friendly, ii) portable, static drug testing platform that iii) does not require further accessories and can create iv) a stable gradient for a long duration. Such a device can reduce the time, manual errors, fabrication and running expenditure, and resources needed to a great extent in drug testing.

Substrate stiffness regulates the recurrent glioblastoma cell morphology and aggressiveness.

Recurrent glioblastoma is highly aggressive with currently no specific treatment regime. Therefore, to identify novel therapeutic targets for recurrent GBM, we used a cellular model developed in our lab from commercially available cell line U87MG and patient-derived cultures that allows the comparison between radiation naïve (Parent) and recurrent GBM cells generated after parent cells are exposed to lethal dose of radiation. Total RNA-seq of parent and recurrent population revealed significant upregulation of cell-ECM interactions pathway in the recurrent population. These results led us to hypothesize that the physical microenvironment contributes to the aggressiveness of recurrent GBM. To verify this, we cultured parent and recurrent GBM cells on collagen-coated polyacrylamide gels mimicking the stiffness of normal brain (Young's modulus E = 0.5kPa) or tumorigenic brain (E = 10kPa) and tissue culture plastic dishes (E ∼ 1 GPa). We found that compared to parent cells, recurrent cells showed higher proliferation, invasion, migration, and resistance to EGFR inhibitor. Using orthotopic GBM mouse model and resection model, we demonstrate that recurrent cells cultured on 0.5kPa had higher in vivo tumorigenicity and recurrent disease progression than parent cells, whereas these differences were insignificant when parent and recurrent cells were cultured on plastic substrates. Furthermore, recurrent cells on 0.5kPa showed high expression of ECM proteins like Collagen, MMP2 and MMP9. These proteins were also significantly upregulated in recurrent patient biopsies. Additionally, the brain of mice injected with recurrent cells grown on 0.5kPa showed higher Young's moduli suggesting the ability of these cells to make the surrounding ECM stiffer. Total RNA-seq of parent and recurrent cells grown on plastic and 0.5kpa identified PLEKHA7 significantly upregulated specifically in recurrent cells grown on 0.5 kPa substrate. PLEKHA7 was also found to be high in recurrent GBM patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provide an in vitro model system that captures the observed in vivo and clinical behavior of recurrent GBM by mimicking mechanical microenvironment and identifies PLEKHA7 as a novel potential target for recurrent GBM.

Extracellular matrix protein gelatin provides higher expansion, reduces size heterogeneity, and maintains cell stiffness in a long-term culture of mesenchymal stem cells.

Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behavior of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behavior. However, the influence of ECM protein coating on long-term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 µg/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin-coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of gelatin showed less size heterogeneity among the cells when expanded from passages 3 to 9 (P3 to P9). Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins, provides a better platform for mesenchymal stem cell expansion.

Scalable large-area mesh-structured microfluidic gradient generator for drug testing applications.

Microfluidic concentration gradient generators are useful in drug testing, drug screening, and other cellular applications to avoid manual errors, save time, and labor. However, expensive fabrication techniques make such devices prohibitively costly. Here, in the present work, we developed a microfluidic concentration gradient generator (µCGG) using a recently proposed non-conventional photolithography-less method. In this method, ceramic suspension fluid was shaped into a square mesh by controlling Saffman Taylor instability in a multiport lifted Hele-Shaw cell (MLHSC). Using the shaped ceramic structure as the template, µCGG was prepared by soft lithography. The concentration gradient was characterized and effect of the flow rates was studied using COMSOL simulations. The simulation result was further validated by creating a fluorescein dye (fluorescein isothiocanate) gradient in the fabricated µCGG. To demonstrate the use of this device for drug testing, we created various concentrations of an anticancer drug-curcumin-using the device and determined its inhibitory concentration on cervical cancer cell-line HeLa. We found that the IC50 of curcumin for HeLa matched well with the conventional multi-well drug testing method. This method of µCGG fabrication has multiple advantages over conventional photolithography such as: (i) the channel layout and inlet-outlet arrangements can be changed by simply wiping the ceramic fluid before it solidifies, (ii) it is cost effective, (iii) large area patterning is easily achievable, and (iv) the method is scalable. This technique can be utilized to achieve a broad range of concentration gradient to be used for various biological and non-biological applications.

The effect of noise in an HIV infection model with cytotoxic T-lymphocyte impairment.

The human immunodeficiency virus (HIV) interacts with the immune cells within the human body, where the environment is uncertain and noisy. Stochastic models can successfully encapsulate the effect of such a noisy environment compared to their deterministic counterparts. The human immune system is complex but well-coordinated with various immune cells like C D 4T cells, dendritic cells, and cytotoxic T-lymphocyte (CTL) cells, among many others. The CTL can kill the antigenic cells after its recognition. However, the efficacy of CTL in removing the infected C D 4T cells is progressively compromised in HIV-infected individuals. This paper considers a noise-induced HIV-immune cell interaction model with immune impairment. A multiplicative white noise is introduced in the infection rate parameter to represent the fluctuations around the average value of the rate parameter as a causative effect of the noise. We analyzed the deterministic and stochastic models and prescribed sufficient conditions for infection eradication and persistence. It is determined under what parametric restrictions the asymptotic solutions of the noise-induced system will be a limiting case of the deterministic solutions. Simulation results revealed that the solutions of the deterministic system either converge to a CTL-dominated interior equilibrium or a CTL-free immunodeficient equilibrium, depending on the initial values of the system. Stochastic analysis divulged that higher noise might be helpful in the infection removal process. The extinction time of infected C D 4T cells for some fixed immune impairment gradually decreases with increasing noise intensity and follows the power law.

Compartmentalized microfluidic device for in vitro co-culture of retinal cells.

The investigation is focused on the development of a compartmentalized microfluidic device for coculturing the cells of crucial retinal cellular layers and assessing cell-to-cell interactions. A perfusion-based microfluidic co-culture device was employed and computationally validated for determining the pressure drop and fluid flow rate within the device microchannels. Fabrication was performed using PDMS polymer and coating of fibronectin and collagen facilitated adherence of the cells over the glass surface. Microfluidic device successfully supported cell proliferation, under continuous perfusion of 1 µl min-1 flow rate. The barrier integrity of this coculture was confirmed by evaluating the permeability of fluorescently labeled molecules. The coculture expressed characteristic phenotypic protein markers like recoverin, PAX6, for retinal precursor cells, and RPE65 for retinal epithelial cells. The coculture also exhibited basal expression of TNF-α under normal conditions. Differentiated photoreceptor cells positively expressed rhod inherently possess sensitivity toward violet/blue light, which was validated in R28 cells by exposure to light having a wavelength of 405 nm, which significantly decreased cell viability via increased TNF-α production and reduced rhodopsin expression. This proof-of-concept investigation proved the functionality of the retinal coculture, which may be used as an appropriate perfusion-based, preclinical tool for the evaluation of novel retinal drugs and delivery systems.

Biomimicked large-area anisotropic grooves fromDracaena sanderianaleaf enhances cellular alignment and subsequent differentiation.

Cellular alignment is important for the proper functioning of different tissues such as muscles or blood vessel walls. Hence, in tissue engineering, sufficient effort has been made to control cellular orientation and alignment. It has been shown that micro-and nanoscale anisotropic topological features on cell culture substrates can control cellular orientation. Such substrates are fabricated using various lithography techniques such as photolithography and soft lithography. Although such techniques are suitable for creating patterns in small areas to establish a proof-of-concept, patterning large areas with intricate features is an unsolved problem. In this work, we report that a replica of the groove-like anisotropic patterns of the abaxial side of aDracaena sanderiana(bamboo) leaf can be used for large-area patterning of cells. We imprinted the leaf on polydimethylsiloxane (PDMS) and characterised its surface topography using scanning electron microscopy. We further cultured bone marrow human mesenchymal cells (BM-hMSCs), skeletal muscle cells (C2C12), and neuroblastoma cells (SHSY5Y) on the patterned PDMS on which the cells orient along the direction of the grooved pattern. Further, we observed enhanced neuronal differentiation of SHSY5Y cells on biomimicked pattern compared to flat PDMS as measured by percentage of cells with neurites, neurite length and the expression of neuronal differentiation marker beta-III tubulin (TUJ1). This process is simple, frugal, and can be adopted by laboratories with resource constraints. This one-step technique to fabricate large-area anisotropic surface patterns from bamboo leaves can be used as a platform to study cellular alignment and its effect on various cellular functions, including differentiation.

Mitigating the transmission of infection and death due to SARS-CoV-2 through non-pharmaceutical interventions and repurposing drugs.

The Covid-19 pandemic has put the world under immeasurable stress. There is no specific drug or vaccine till now that can cure the infection or protect people from the infection of coronavirus. It is therefore prudent to use the existing resources and control strategies in an optimal way to contain the virus spread and provide the best possible treatments to the infected individuals. Use of the repurposing drugs along with the non-pharmaceutical intervention strategies may be the right way for fighting against the ongoing pandemic. It is the objective of this work to demonstrate through mathematical modelling and analysis how and to what extent such control strategies can improve the overall Covid-19 epidemic burden. The criteria for disease elimination & persistence were established through the basic reproduction number. A case study with the Indian Covid-19 epidemic data is presented to visualize and illustrate the effects of lockdown, maintaining personal hygiene & safe distancing, and repurposing drugs. It is shown that India can significantly improve the overall Covid-19 epidemic burden through the combined use of NPIs and repurposing drugs though containment of spreading is difficult without serious community participation.

"Viscotaxis"- directed migration of mesenchymal stem cells in response to loss modulus gradient.

Directed cell migration plays a crucial role in physiological and pathological conditions. One important mechanical cue, known to influence cell migration, is the gradient of substrate elastic modulus (E). However, the cellular microenvironment is viscoelastic and hence the elastic property alone is not sufficient to define its material characteristics. To bridge this gap, in this study, we investigated the influence of the gradient of viscous property of the substrate, as defined by loss modulus (G″) on cell migration. We cultured human mesenchymal stem cells (hMSCs) on a collagen-coated polyacrylamide gel with constant storage modulus (G') but with a gradient in the loss modulus (G″). We found hMSCs to migrate from high to low loss modulus. We have termed this form of directional cellular migration as "Viscotaxis". We hypothesize that the high loss modulus regime deforms more due to creep in the long timescale when subjected to cellular traction. Such differential deformation drives the observed Viscotaxis. To verify our hypothesis, we disrupted the actomyosin contractility with myosin inhibitor blebbistatin and ROCK inhibitor Y27632, and found the directional migration to disappear. Further, such time-dependent creep of the high loss material should lead to lower traction, shorter lifetime of the focal adhesions, and dynamic cell morphology, which was indeed found to be the case. Together, findings in this paper highlight the importance of considering the viscous modulus while preparing stiffness-based substrates for the field of tissue engineering. STATEMENT OF SIGNIFICANCE: While the effect of substrate elastic modulus has been investigated extensively in the context of cell biology, the role of substrate viscoelasticity is poorly understood. This omission is surprising as our body is not elastic, but viscoelastic. Hence, the role of viscoelasticity needs to be investigated at depth in various cellular contexts. One such important context is cell migration. Cell migration is important in morphogenesis, immune response, wound healing, and cancer, to name a few. While it is known that cells migrate when presented with a substrate with a rigidity gradient, cellular behavior in response to viscoelastic gradient has never been investigated. The findings of this paper not only reveal a completely novel cellular taxis or directed migration, it also improves our understanding of cell mechanics significantly.

Biomimicked hierarchical 2D and 3D structures from natural templates: applications in cell biology.

Intricate structures of natural surfaces and materials have amazed people over the ages. The unique properties of various surfaces also created interest and curiosity in researchers. In the recent past, with the advent of superior microscopy techniques, we have started to understand how these complex structures provide superior properties. With that knowledge, scientists have developed various biomimicked and bio-inspired surfaces for different non-biological applications. In the last two decades, we have also started to learn how structures of the tissue microenvironment influence cell function and behaviour, both in physiological and pathological conditions. Hence, it became essential to decipher the role and importance of structural hierarchy in the cellular context. With advances in microfabricated techniques, such complex structures were made by superimposing features of different dimensions. However, the fabricated topographies are far from matching the complexities presentin vivo. Hence, the need of biomimicking the natural surfaces for cellular applications was felt. In this review, we discuss a few examples of hierarchical surfaces found in plants, insects, and vertebrates. Such structures have been widely biomimicked for various applications but rarely studied for cell-substrate interaction and cellular response. Here, we discuss the research work wherein 2D hierarchical substrates were prepared using biomimicking to understand cellular functions such as adhesion, orientation, differentiation, and formation of spheroids. Further, we also present the status of ongoing research in mimicking 3D tissue architecture using de-cellularized plant-based and tissue/organ-based scaffolds. We will also discuss 3D printing for fabricating 2D and 3D hierarchical structures. The review will end by highlighting the various advantages and research challenges in this approach. The biomimickedin-vivolike substrate can be used to better understand cellular physiology, and for tissue engineering.

Persistence and extinction of species in a disease-induced ecological system under environmental stochasticity.

Population extinction is a serious issue both from the theoretical and practical points of view. We explore here how environmental noise influences persistence and extinction of interacting species in presence of a pathogen even when the populations remain stable in its deterministic counterpart. Multiplicative white noise is introduced in a deterministic predator-prey-parasite system by randomly perturbing three biologically important parameters. It is revealed that the extinction criterion of species may be satisfied in multiple ways, indicating various routes to extinction, and disease eradication may be possible with the right environmental noise. Predator population cannot survive, even when its focal prey strongly persists if its growth rate is lower than some critical value, measured by half of the corresponding noise intensity. It is shown that the average extinction time of population decreases with increasing noise intensity and the probability distribution of the extinction time follows the log-normal density curve. A case study on red grouse (prey) and fox (predator) interaction in presence of the parasites trichostrongylus tenuis of grouse is presented to demonstrate that the model well fits the field data.

Viscoelastic substrate decouples cellular traction force from other related phenotypes.

Survival and maintenance of normal physiological functions depends on continuous interaction of cells with its microenvironment. Cells sense the mechanical properties of underlying substrate by applying force and modulate their behaviour in response to the resistance offered by the substrate. Most of the studies addressing cell-substrate mechanical interactions have been carried out using elastic substrates. Since tissues within our body are viscoelastic in nature, here we explore the effect of substrate's viscoelasticity on various properties of mesenchymal stem cells. Here, we used two sets of polyacrylamide substrates having similar storage modulus (G' = 1.1-1.6 kPa) but different loss modulus (G" = 45 Pa and 300 Pa). We report that human mesenchymal stem cells spread more but apply less force on the viscoelastic substrate (substrate with higher loss modulus). We further investigated the effect of substrate viscoelasticity on the expression of other contractility-associated proteins such as focal adhesion (FA) proteins (Vinculin, Paxillin, Talin), cytoskeletal proteins (actin, mysion, intermediate filaments, and microtubules) and mechano-sensor protein Yes-Associated Protein (YAP). Our results show that substrate viscoelasticity decouples cellular traction from other known traction related phenotypes.

Hyperthermia induced disruption of mechanical balance leads to G1 arrest and senescence in cells.

Human body temperature limits below 40°C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40°C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40°C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40°C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared with cells incubated at 37°C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40°C than at 37°C. The mitotic cells at 40°C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia down-regulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40°C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40°C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses.

Mathematical perspective of Covid-19 pandemic: Disease extinction criteria in deterministic and stochastic models.

The world has been facing the biggest virological invasion in the form of Covid-19 pandemic since the beginning of the year 2020. In this paper, we consider a deterministic epidemic model of four compartments based on the health status of the populations of a given country to capture the disease progression. A stochastic extension of the deterministic model is further considered to capture the uncertainty or variation observed in the disease transmissibility. In the case of a deterministic system, the disease-free equilibrium will be globally asymptotically stable if the basic reproduction number is less than unity, otherwise, the disease persists. Using Lyapunov functional methods, we prove that the infected population of the stochastic system tends to zero exponentially almost surely if the basic reproduction number is less than unity. The stochastic system has no interior equilibrium, however, its asymptotic solution is shown to fluctuate around the endemic equilibrium of the deterministic system under some parametric restrictions, implying that the infection persists. A case study with the Covid-19 epidemic data of Spain is presented and various analytical results have been demonstrated. The epidemic curve in Spain clearly shows two waves of infection. The first wave was observed during March-April and the second wave started in the middle of July and not completed yet. A real-time reproduction number has been given to illustrate the epidemiological status of Spain throughout the study period. Estimated cumulative numbers of confirmed and death cases are 1,613,626 and 42,899, respectively, with case fatality rate 2.66% till the deadly virus is eliminated from Spain.

Hydrogel scaffold with substrate elasticity mimicking physiological-niche promotes proliferation of functional keratinocytes.

High numbers of autologous human primary keratinocytes (HPKs) are required for patients with burns, wounds and for gene therapy of skin disorders. Although freshly isolated HPKs exhibit a robust regenerative capacity, traditional methodology fails to provide a sufficient number of cells. Here we demonstrated a well characterized, non-cytotoxic and inert hydrogel as a substrate that mimics skin elasticity, which can accelerate proliferation and generate higher numbers of HPKs compared to existing tissue culture plastic (TCP) dishes. More importantly, this novel method was independent of feeder layer or any exogenous pharmaceutical drug. The HPKs from the hydrogel-substrate were functional as demonstrated by wound-healing assay, and the expression of IFN-γ-responsive genes (CXCL10, HLADR). Importantly, gene delivery efficiency by a lentiviral based delivery system was significantly higher in HPKs cultured on hydrogels compared with TCP. In conclusion, our study provides the first evidence that cell-material mechanical interaction is enough to provide a rapid expansion of functional keratinocytes that might be used as autologous grafts for skin disorders.

Methyl-ß-cyclodextrin, an actin depolymerizer augments the antiproliferative potential of microtubule-targeting agents.

Methyl-ß-cyclodextrin (MCD), an established pharmacological excipient, depolymerizes the actin cytoskeleton. In this work, we investigated the effect of MCD-mediated actin depolymerization on various cellular phenotypes including traction force, cell stiffness, focal adhesions, and intracellular drug accumulation. In addition to a reduction in the contractile cellular traction, MCD acutely inhibits the maturation of focal adhesions. Alteration of contractile forces and focal adhesions affects the trypsin-mediated detachment kinetics of cells. Moreover, MCD-mediated actin depolymerization increases the intracellular accumulation of microtubule-targeting agents (MTAs) by ~50% with respect to the untreated cells. As MCD treatment enhances the intracellular concentration of drugs, we hypothesized that the MCD-sensitized cancer cells could be effectively killed by low doses of MTAs. Our results in cervical, breast, hepatocellular, prostate cancer and multidrug-resistant breast cancer cells confirmed the above hypothesis. Further, the combined use of MCD and MTAs synergistically inhibits the proliferation of tumor cells. These results indicate the potential use of MCD in combination with MTAs for cancer chemotherapy and suggest that targeting both actin and microtubules simultaneously may be useful for cancer therapy. Importantly, the results provide significant insight into the crosstalk between actin and microtubules in regulating the traction force and dynamics of cell deadhesion.

Soft substrate maintains proliferative and adipogenic differentiation potential of human mesenchymal stem cells on long-term expansion by delaying senescence.

Human mesenchymal stem cells (hMSCs), during in vitro expansion, gradually lose their distinct spindle morphology, self-renewal ability, multi-lineage differentiation potential and enter replicative senescence. This loss of cellular function is a major roadblock for clinical applications which demand cells in large numbers. Here, we demonstrate a novel role of substrate stiffness in the maintenance of hMSCs over long-term expansion. When serially passaged for 45 days from passage 3 to passage 18 on polyacrylamide gel of Young's modulus E=5 kPa, hMSCs maintained their proliferation rate and showed nine times higher population doubling in comparison to their counterparts cultured on plastic Petri-plates. They did not express markers of senescence, maintained their morphology and other mechanical properties such as cell stiffness and cellular traction, and were significantly superior in adipogenic differentiation potential. These results were demonstrated in hMSCs from two different sources, umbilical cord and bone marrow. In summary, our result shows that a soft gel is a suitable substrate to maintain the stemness of mesenchymal stem cells. As preparation of polyacrylamide gel is a well-established, and well-standardized protocol, we propose that this novel system of cell expansion will be useful in therapeutic and research applications of hMSCs.

Low Cost and Lithography-free Stamp fabrication for Microcontact Printing.

Microcontact printing (µCP) is a commonly used technique for patterning proteins of interest on substrates. The cells take the shape of these printed patterns. This technique is used to explore the effect of cellular morphology on their various functions such as survival, differentiation, migration, etc. An essential step for µCP is to fabricate a stamp from a silicon mould, prepared using lithography. Lithography is cost intensive and needs a high level of expertise to handle the instrumentation. Also, one stamp can be used to print patterns of one size and shape. Here, to overcome these limitations, we devised a low-cost fabrication technique using readily available objects such as injection needles and polystyrene beads. We patterned the C2C12, myoblasts cells on the shapes printed using lithography-free fabricated stamps. We further exploited the surface curvature of the stamp to vary the size of the print either by changing the applied load and/or the substrate stiffness. We showed that the print dimension could be predicted well by using JKR theory of contact mechanics. Moreover, some innovative improvisations enabled us to print complex shapes, which would be otherwise difficult with conventional lithography technique. We envisage that this low cost and easy to fabricate method will allow many research laboratories with limited resources to perform exciting research which is at present out of their reach.