Less Time, Less Cost: New Technique Using Cells in Just Five Hours

This study presents a simple and rapid method for forming three-dimensional cell sheets without external supports or stimuli, allowing the creation of dense, viable, and functional cellular tissues with great potential for applications in regenerative medicine and tissue engineering.

In regenerative medicine, one of the biggest challenges is to efficiently deliver living cells to injured tissue so that they can survive, remain in place, and aid in regeneration. The most common way to do this is through cell suspension injection, that is, individual cells dispersed in a liquid.

Although this technique is simple and widely used, it often has low efficacy: most cells die or are lost quickly after application, and often less than 5% of the cells remain at the site of injury long enough to exert any therapeutic effect.

To try to solve this problem, tissue engineering has begun to use support structures, called scaffolds, made of biocompatible and biodegradable materials. These supports form three-dimensional structures that help organize the cells and keep them in place. However, this approach also has important limitations.

Cell density in these grafts is usually much lower than that found in natural tissues, and the external materials used as support can provoke inflammatory or toxic responses, hindering clinical application in humans.

As an alternative, so-called "unsupported" strategies have emerged, in which the cells themselves organize to form tissue-like structures, producing their own extracellular matrix, the network of proteins that holds cells together and guides their behavior. This approach reduces the risk of immune rejection and promotes more natural cell interactions. Among these techniques are cellular spheroids, magnetic field-induced aggregation, and cell sheet engineering.

Spheroids are small, three-dimensional clusters of cells that exhibit greater survival and better cell communication after transplantation. Despite these advantages, they are difficult to produce on a large scale, have uneven size, and are complicated to administer at the site of injury.

Furthermore, just as with cells in suspension, many spheroids end up not being adequately retained in the target tissue. Some strategies attempt to join several spheroids into larger structures using supports or bioinks, but this reintroduces external materials and increases the complexity of the process.

Cellular sheets were developed to overcome many of these limitations. They consist of flat, continuous structures formed exclusively by densely packed cells, arranged in one or more layers. These sheets have a thickness similar to that of natural tissue subunits and exhibit very high cell density, comparable to that found in the human body, which is a major advantage over many traditional tissue engineering methods.

In most existing techniques, cellular sheets are produced by culturing cells until they completely cover a surface. During this process, the cells form connections with each other and produce their own extracellular matrix. Then, the entire sheet is detached from the surface as a single structure, preserving these natural interactions.

Compared to the injection of isolated cells, slides exhibit greater structural integrity, homogeneous cell distribution, and better integration with the host tissue after transplantation.

The most common method for detaching these slides uses temperature-sensitive surfaces. When the temperature is reduced, the surface changes its properties and releases the cell layer. Although it preserves cell viability, this technique is extremely sensitive to temperature variations, can be time-consuming, and, if not performed precisely, can damage the slide or compromise cell function.

Scientists who authored the study.

Other methods use pH changes, electric fields, or ultraviolet light to release the slides, but all of them involve chemical or physical stress, multiple manufacturing steps, special surfaces, and expensive equipment, and are not always compatible with all cell types.

Another problem common to these approaches is that they rely heavily on traditional two-dimensional cell culture, which does not adequately reproduce the three-dimensional environment found in body tissues. This can limit the production of the extracellular matrix and affect the normal behavior of cells.

In this context, the study presents a new, simple, and straightforward technique for the fabrication of three-dimensional cell slides in a single step. The method uses PDMS templates (a material widely used in laboratories and biologically inert) without any chemical modification.

By seeding the cells at a carefully adjusted density, close to complete surface coverage, the low adhesion of PDMS causes the cells to prefer binding to each other rather than attaching to the substrate. This process strongly stimulates cell-cell interactions, leading to the spontaneous formation of a flat, cohesive cell sheet within a few hours.

Overview of the biofabrication technique for unsupported cell sheets and its capabilities. (A) Schematic representation of the fabrication process: (i) High-density cells are seeded onto untreated, non-adherent PDMS molds. (ii) Cell sheet formation progresses through self-assembly, beginning with cell-cell junctions, followed by ECM deposition and cell-ECM interactions. (B) Versatility of the method: (i) Generation of cell sheets in customizable shapes; (ii) Stacking to form thicker, tissue-like structures; and (iii) Modular assembly of building blocks into larger, more complex structures.

Molecular analyses showed that this process is associated with high expression of E-cadherin, a protein fundamental for cell adhesion, confirming that sheet formation is primarily driven by intercellular junctions. Viability tests demonstrated that cells remain alive and functional, and histological analyses confirmed the progressive production of extracellular matrix, including collagen.

In addition to being simple and quick, the technique has proven to be highly versatile. It allows the fabrication of blades of different sizes and shapes, adapted to the geometry of the mold, enabling the creation of customized grafts.

It is also possible to stack several blades to form thicker tissues or combine different cell types in organized co-cultures. The method worked consistently with a wide variety of cells, including established cell lines, primary cells, and stem cells of murine, bovine, and human origin.

Cell sheet created in the Selvaganapathy laboratory. Modular assembly of individual cell sheets into complex structures for advanced tissue modeling.

In short, this approach offers a practical, fast, and low-cost alternative to traditional cell sheet fabrication methods. By avoiding the need to modify surfaces, alter culture conditions, or use specialized equipment, it facilitates the production of functional cell tissues in common laboratory environments.

This significantly expands the potential application of cell sheets in tissue engineering, regenerative medicine, disease modeling, and even emerging areas such as lab-grown meat production.

READ MORE:

Rapid scaffold-free cell sheet formation and their patterning as building blocks of complex 3D tissue constructs 

Maedeh Khodamoradi, Seyedaydin Jalali, Maria Fernanda Hutter, Yufei Chen, Faraz Chogan, Alisa Douglas, Graham Rix, Bhavishya Challagundla, Margarita Elloso, Marc G. Jeschkeabcd, and P. Ravi Selvaganapathy

Royal Society of Chemistry. Lab Chip. 03 Dec 2025

DOI: 10.1039/d5lc00678c

Abstract:

Three-dimensional (3D) cell cultures offer superior potential in replicating native tissue microenvironments by better supporting cell–cell and cell–extracellular matrix (ECM) interactions that are critical for guiding cellular behavior and functionality in engineered tissues. Among 3D approaches, scaffold-free techniques have gained attention for their ability to produce high-cellular density, and well-organized tissue-like constructs. In particular, cell sheets are uniquely suited for regenerative applications due to their contiguous architecture, large-area coverage, and integration potential with host tissues. However, current biofabrication methods for cell sheet production often require altering culture conditions (e.g., temperature, pH) or applying external stimuli (e.g., magnetic or electrical fields), which can damage cells, compromise sheet integrity, or demand costly, non-adaptable equipment. Here, we present a rapid, self-assembly-based technique using unmodified polydimethylsiloxane (PDMS) molds as culture vessels. When seeded at a critical cell density, adherent cells spontaneously self-assemble into planar 3D cell sheets within 6 hours, without substrate modification or specialized equipment. Our qRT-PCR analysis revealed significant upregulation of E-cadherin in cell sheets, confirming that cell-cell adhesion, rather than cell-substrate anchorage, drives sheet formation. We showed that our technique is versatile, supporting the creation of large-area and patterned sheets, stacked multi-layer constructs, and co-culture configurations. Notably, fibroblast cell sheets, demonstrated progressive ECM production, with histological analysis confirming collagen deposition over time. Overall, our approach preserves cell viability and function while offering a simple, rapid, and cost-effective alternative to conventional methods for fabricating cell sheets. This platform holds broad potential for applications in tissue engineering, regenerative medicine, disease modeling, and cultivated meat production.