Developing a 3D bio-printed human skin model

Unravelling the pathophysiological mechanisms of skin disease relies on representative skin models. However, current laboratory skin models have acknowledged limitations which impede translation to the clinic. The need for a stratified 3D cellular co-culture with control over spatial organization to represent the complexities of human skin more realistically is therefore highly desirable. 3D bio printing has recently generated physiologically relevant human skin models (Baltazar et al. 2020). However, current bio printing technologies are typically expensive, difficult to operate, and have low customisation ability, thus hindering widespread accessibility (Ioannidis et al. 2020). Custom-built, low-cost 3D bio-printing platforms have been recently reported for the production of 3D cell culture and tissue models (Cubo et al. 2016a; Reid et al. 2016; Kahl et al. 2019; Ioannidis et al. 2020). It is therefore hypothesised that recreating the structure of human skin through developing a cost-effective flexible 3D bio-printing technology is feasible. The aim of this study is to develop a 3D-bio-printed human skin model using a low-cost flexible cell-printing platform. Preliminary 2D cell culture studies were conducted using an immortalized keratinocyte cell line to establish the optimum culture conditions. Cells were maintained in a proliferative or differentiated state by varying the calcium concentration to mimic the physiological epidermal calcium gradient (Wilson et al. 2007; Bikle et al. 2012). Morphology and specific biochemical markers of differentiation were studied in each condition. A bespoke LEGO® 3D bio-printer, capable of encapsulating high cell densities and creating 2D and 3D arrangements of cells, was built in parallel to the cell culture experiments. Cells maintained in low calcium exhibited proliferative characteristics whereas cells in higher concentrations of calcium were induced to become more differentiated, recapitulating the effect of the calcium gradient in the epidermis. The programmed custom-built LEGO® 3D bio-printer was optimized to generate high-resolution 2D and 3D complex patterns of bio-ink. Using the custom-built 3D bio-printer, the cells were successfully encapsulated in bio-material droplets and printed. Microscopy images and a cell viability assay indicated homogenous cell dispersion and high cell viability (87.5%) within the bio-printed material. Keratinocytes were successfully 3D bio-printed in an 18-layered squared lattice and imaged showing high cell viability. These initial results provide a platform for manufacture of single and mixed cell ii culture populations with a defined 3D organization, akin to the human skin. The adaptability and flexibility of the custom-built LEGO® 3D bio-printer has the potential to enhance the complexity of the skin tissue model. Therefore, a first prototype of the LEGO® 3D bio-printing platform has been developed demonstrating a printing resolution at the sub-millimeter scale, providing a cost-effective novel 3D bio-printing technology for the production of human skin models

Development and evaluation of a low‐cost lego 3d bioprinter: from building‐blocks to building blocks of life

The development of low‐cost accessible technologies for rapid prototyping of mechanical components has democratised engineering tools for hobbyists and researchers alike. The development of analogous approaches to fabrication of soft‐matter, and biologically compatible materials containing living cells, is anticipated to be similarly enabling across multiple fields of biological research. LEGO toy construction bricks represent low‐cost, precision engineered, and versatile construction materials for rapid prototyping. This study demonstrates construction of a benchtop LEGO 3D bioprinter for additive layer manufacture of a 3D structure containing viable human skin cells within a hydrogel scaffold. 3D bioprinted structures are formed from the deposition of microfluidically generated bio‐ink droplets containing live keratinocyte skin cells, representing components toward an artificial skin model. Fluid flow rates and printer speed, together with bio‐ink gelation rate, determine droplet packing arrangement in the bioprinted structures. The printing of 3D structures containing multiple bio‐inks is demonstrated and live cells are imaged in the resulting bioprints. Fluid delivery can be achieved using LEGO pumps and readily available, or home‐3D‐printed, microfluidic components, therefore avoiding the need for any specialist microfluidic hardware. Build instructions are described to enable easy uptake, modification and improvement by other laboratories, as well provide an accessible platform for learning and education. Affordable, accessible, and easy to use tools for 3D bioprinting are anticipated to open opportunities for a greater number of research labs to work with 3D cell culture and bio‐printed materials, with bioprinting expected to assist in better understanding of disease, contribute to tissue engineering and repair, and enable personalised medicine through the printing of cultured patient cells. The presented approach is not only an easily accessible laboratory tool for bioprinting, but also provides a learning system for mechanical construction, robotics, coding, microfluidics and cell biology, making it a versatile platform for research, education, and science engagement

Distinct expression profiles and functions of Kindlins in breast cancer

Abstract Background Kindlin-1, − 2, and − 3 are the three members of the Kindlin family. They are best known as regulators of integrin functions, contributing to fundamental biological processes such as cell survival, adhesion and migration. Their deregulation leads to diverse pathologies including a broad range of cancers in which both, tumor-promoting and tumor-inhibiting functions have been described. Methods To better characterize Kindlins implication in breast cancer, in vitro experiments were performed in a series of cancer cell lines. We first assessed their expression profiles and subcellular distributions. Then, their involvement in breast cancer cell morphology, migration and invasion was verified by examining phenotypic changes induced by the depletion of either isoforms using RNA interference. An expression study was performed in a series of breast cancer patient derived xenografts (n = 58) to define the epithelial and stromal contribution of each Kindlin. Finally, we analyzed the expression levels of the three Kindlins in a large series of human breast tumors, at the RNA (n = 438) and protein (n = 129) levels and we evaluated their correlation with the clinical outcome. Results We determined that Kindlin-1 and Kindlin-2, but not Kindlin-3, were expressed in breast tumor cells. We uncovered the compensatory roles of Kindlin-1 and -2 in focal adhesion dynamics and cell motility. Remarkably, Kindlin-2 had a predominant effect on cell spreading and Kindlin-1 on cell invasion. In line with these experimental observations, Kindlin-1 overexpression was associated with a worse patients’ outcome. Notably, Kindlin-3, expressed by tumor infiltrating leukocytes, also correlated with a poor prognosis of breast cancer patients. Conclusion This study demonstrates that each one of the Kindlin family members has a different expression profile emphasizing their redundant and complementary roles in breast tumor cells. We highlight the specific link between Kindlin-1 and breast cancer progression. In addition, Kindlin-3 overexpression in the tumor microenvironment is associated with more aggressive breast tumors. These results suggest that Kindlins play distinctive roles in breast cancer. Kindlins may be useful in identifying breast cancer patients with a worst prognosis and may offer new avenues for therapeutic intervention against cancer progression

Resolvin D1 regulates epithelial ion transport and inflammation in cystic fibrosis airways

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Instructions for the construction of a Low cost Lego 3D bioprinter

Parts list and an instructional video for the recreation of a Lego 3D bioprinter. When referencing please use: Moukachar, A. et al. Development and Evaluation of a Low-Cost LEGO 3D Bioprinter: From Building-Blocks to Building Blocks of Life. Advanced Materials Technologies 8, 2100868 (2023).The work was supported by funding from: British Skin Foundation (BSF) PhD studentship grant (grant number: 059/s/16) H2020- EU.1.2.2. – FET Proactive Grant agreement ID: 824060 – "ACDC" Cardiff Institute for Tissue Engineering and Repair (CITER) The Cardiff Undergraduate Research Opportunities Programme (CUROP) Cardiff University Tissue Engineering and Regenerative Medicine M.Sc and M.Pharm student project scheme

Resolvin D1 regulates epithelial ion transport and inflammation in cystic fibrosis airways

International audienc