Dynamics of cell attachment: adhesion time and force.

Due to its biological significance, cell adhesion to biomaterial surfaces or scaffolds is the key step in biomedical applications. Here, we describe two sensitive and facile methods that quantify the kinetic and mechanic properties of the entire cell attachment process characterized by two parameters: Adhesion Time T(Ad) and Adhesion Force F(Ad). We demonstrate that both methods can be applied to any adherent cell type (e.g., stem or cancer cells), tissue-engineered substrate, and culture condition in a fast, effective, and reproducible manner. Additional investigations about the role of the extracellular matrix and the formation of focal contacts help in acquiring further interpretations of these parameters from biological and mechanical points of view.

Skin tissue generation by laser cell printing.

For the aim of ex vivo engineering of functional tissue substitutes, Laser-assisted BioPrinting (LaBP) is under investigation for the arrangement of living cells in predefined patterns. So far three-dimensional (3D) arrangements of single or two-dimensional (2D) patterning of different cell types have been presented. It has been shown that cells are not harmed by the printing procedure. We now demonstrate for the first time the 3D arrangement of vital cells by LaBP as multicellular grafts analogous to native archetype and the formation of tissue by these cells. For this purpose, fibroblasts and keratinocytes embedded in collagen were printed in 3D as a simple example for skin tissue. To study cell functions and tissue formation process in 3D, different characteristics, such as cell localisation and proliferation were investigated. We further analysed the formation of adhering and gap junctions, which are fundamental for tissue morphogenesis and cohesion. In this study, it was demonstrated that LaBP is an outstanding tool for the generation of multicellular 3D constructs mimicking tissue functions. These findings are promising for the realisation of 3D in vitro models and tissue substitutes for many applications in tissue engineering.

Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration.

Recent study showed that mesenchymal stem cells (MSC) could inhibit apoptosis of endothelial cells in hypoxic condition, increase their survival, and stimulate the angiogenesis process. In this project we applied Laser-Induced-Forward-Transfer (LIFT) cell printing technique and prepared a cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSC (hMSC) in a defined pattern for cardiac regeneration. We seeded HUVEC and hMSC in a defined pattern on a Polyester urethane urea (PEUU) cardiac patch. On control patches an equal amount of cells was randomly seeded without LIFT. Patches were cultivated in vitro or transplanted in vivo to the infarcted zone of rat hearts after LAD-ligation. Cardiac performance was measured by left ventricular catheterization 8 weeks post infarction. Thereafter hearts were perfused with fluorescein tomato lectin for the assessment of functional blood vessels and stored for histology analyses. We demonstrated that LIFT-derived cell seeding pattern definitely modified growth characteristics of co-cultured HUVEC and hMSC leading to increased vessel formation and found significant functional improvement of infarcted hearts following transplantation of a LIFT-tissue engineered cardiac patch. Further, we could show enhanced capillary density and integration of human cells into the functionally connected vessels of murine vascular system. LIFT-based Tissue Engineering of cardiac patches for the treatment of myocardial infarction might improve wound healing and functional preservation.

Laser printing of three-dimensional multicellular arrays for studies of cell-cell and cell-environment interactions.

Utilization of living cells for therapies in regenerative medicine requires a fundamental understanding of the interactions between different cells and their environment. Moreover, common models based on adherent two-dimensional cultures are not appropriate to simulate the complex interactions that occur in a three-dimensional (3D) cell-microenvironment in vivo. In this study, we present a computer-aided method for the printing of multiple cell types in a 3D array using laser-assisted bioprinting. By printing spots of human adipose-derived stem cells (ASCs) and endothelial colony-forming cells (ECFCs), we demonstrate that (i) these cell spots can be arranged layer-by-layer in a 3D array; (ii) any cell-cell ratio, cell quantity, cell-type combination, and spot spacing can be realized within this array; and (iii) the height of the 3D array is freely scalable. As a proof of concept, we printed separate spots of ASCs and ECFCs within a 3D array and observed cell-cell interactions in vascular endothelial growth factor-free medium. It has been demonstrated that direct cell-cell contacts trigger the development of stable vascular-like networks. This method can be applied to study complex and dynamic relationships between cells and their local environment.

Dispensing pico to nanolitre of a natural hydrogel by laser-assisted bioprinting.

BACKGROUND: Laser-assisted bioprinting of multi-cellular replicates in accordance with CAD blueprint may substantially improve our understandings of fundamental aspects of 3 D cell-cell and cell-matrix interactions in vitro. For predictable printing results, a profound knowledge about effects of different processing parameters is essential for realisation of 3 D cell models with well-defined cell densities. METHODS: Time-resolved imaging of the hydrogel jet dynamics and quantitative assessment of the dependence of printed droplet diameter on the process characteristics were conducted. RESULTS: The existence of a counterjet was visualised, proving the bubble collapsing theory for the jet formation. Furthermore, by adjusting the viscosity and height of the applied hydrogel layer in combination with different laser pulse energies, the printing of volumes in the range of 10 to 7000 picolitres was demonstrated. Additionally, the relationship between the viscosity and the layer thickness at different laser pulse energies on the printed droplet volume was identified. CONCLUSIONS: These findings are essential for the advancement of laser-assisted bioprinting by enabling predictable printing results and the integration of computational methods in the generation of 3 D multi-cellular constructs.

Laser printing of stem cells for biofabrication of scaffold-free autologous grafts.

Stem cells are of widespread interest in regenerative medicine due to their capability of self-renewal and differentiation, which is regulated by their three-dimensional microenvironment. In this study, a computer-aided biofabrication technique based on laser-induced forward transfer (LIFT) is used to generate grafts consisting of mesenchymal stem cells (MSCs). We demonstrate that (i) laser printing does not cause any cell damage; (ii) laser-printed MSC grafts can be differentiated toward bone and cartilage; (iii) LIFT allows printing of cell densities high enough for the promotion of chondrogenesis; (iv) with LIFT three-dimensional scaffold-free autologous tissue grafts can be fabricated keeping their predefined structure, and (v) predifferentiated MSCs survived the complete printing procedure and kept their functionality. We believe that our results will find important applications in stem cell biology and tissue engineering.

Laser printing of skin cells and human stem cells.

Laser printing based on laser-induced forward transfer (LIFT) is a new biofabrication technique for the arrangement of biological materials or living cells in well-defined patterns. In the current study, skin cell lines (fibroblasts/keratinocytes) and human mesenchymal stem cells (hMSC) were chosen for laser printing experiments due to their high potential in regeneration of human skin and new application possibilities of stem cell therapy. To evaluate the influence of LIFT on the cells, their survival rate, their proliferation and apoptotic activity, and the DNA damages and modifications of their cell surface markers were assessed and statistically evaluated over several days. The cells survived the transfer procedure with a rate of 98% +/- 1% standard error of the mean (skin cells) and 90% +/- 10% (hMSC), respectively. All used cell types maintain their ability to proliferate after LIFT. Further, skin cells and hMSC did not show an increase of apoptosis or DNA fragmentation. In addition, the hMSC keep their phenotype as proven by fluorescence activated cell sorting (FACS) analysis. This study demonstrates LIFT as a suitable technique for unharmed computer-controlled positioning of different cell types and a promising tool for future applications in the ex vivo generation of tissue replacements.