Physical mechanisms of cell rearrangements: from tissue liquidity to artificial organ structures

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on March 25, 2009)Vita.Includes bibliographical references.Thesis (Ph.D.) University of Missouri-Columbia 2006.Dissertations, Academic -- University of Missouri--Columbia -- Physics.This research presents a study of biological self-assembly in which we create 3D living functional tissue structures by exploiting the self organizing capacity of cells and tissues. Tissues composed of adhesive and motile cells mimic the behavior of viscoelastic liquids on both global and local scales. We exploited the concept of tissue liquidity to engineer tissue structures of relevant geometries encountered in the living organism. Embedding model tissue fragments in the form of spherical cell aggregates into biocompatible hydrogels, we demonstrated that by optimizing the cell-cell and cell-gel interactions, upon fusion long lived tissue structures emerge. We developed a rapid prototyping technique, "bioprinting", and automated devices capable to produce standardized "bioink" particles in the form of cell aggregates. The tissues created with our bioprinter fused into biologically relevant geometries and showed functional characteristics. Our efforts represent an important step toward building complex organ modules via biological self-assembly

Cell-matrix interaction in tissue patterning

Abstract only availableIn vivo pattern formation during morphogenesis is dependent upon the migration of cells. Cell movements are directed by the local structure of the surrounding extracellular matrix. It has been shown experimentally that ligament-like straps form in collagen gel due to the local tension created in the matrix by tissue explants [1]. For a comprehensive understanding of this phenomenon, the sprouting behavior of these aggregates was qualitatively studied in six different configurations embedded in a collagen gel. The biological motivation for this study was to observe how the interplay between the collagen matrix, simulating the extracellular matrix, and the cells affect pattern formation in tissues. The understanding of tissue patterning as a result of cell-matrix interaction has important implications for tissue engineering. Spherical aggregates were prepared from Chinese Hamster Ovary Cells as described previously [2]. The six configurations (triangular, square, hexagonal, bulls eye, dodecagon, and two adjacent aggregates) were built by manually placing spherical aggregates into collagen gels. Photographs of the evolving patterns were taken at regular time intervals for one hundred and eighty hours under a phase contrast microscope. Sprouting was delayed until a critical tension was reached in the collagen matrix. Once sprouting began, a clear bias was shown for migration of cells toward other aggregates creating a cellular bridge between aggregates in close proximity. Sprouting occurred toward each aggregate in a specific pattern exhibiting anisotropy due to the depletion of local collagen fibers in areas adjacent to the cellular bridges. In most aggregates, a void in cell sprouting was apparent on either side of the cellular bridge. The large-scale patterns exhibited in this experiment were found to be linked to local cell-matrix interactions. [1] Sawhney, R.K, Howard, J, Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels. Journal of Cell Biology. Vol 157, 6, 2002, pp. 1083-1091. [2] K. Jakab, A. Neagu, V. Mironov, R.R. Markwald and G. Forgacs. Engineering biological structures of prescribed shape using self-assembling multicellular systems. PNAS, vol. 101, 9, pp. 2864-2869, 2004.NSF-REU Biosystems Modelin

Role of Physical Mechanisms in Biological Self-Organization

URL:http://link.aps.org/doi/10.1103/PhysRevLett.95.178104 DOI:10.1103/PhysRevLett.95.178104Organs form during morphogenesis, the process that gives rise to specialized biological structures of specific shape and function in early embryonic development. Morphogenesis is under strict genetic control, but shape evolution itself is a physical process. Here we report the results of experimental and modeling biophysical studies on in vitro biological structure formation. Experimentally, by controlling the interaction between cells and their embedding matrices, we were able to build living structures of definite geometry. The experimentally observed shape evolution was reproduced by Monte Carlo simulations, which also shed light on the biophysical basis of the process. Our work suggests a novel way to engineer biological structures of controlled shape.This work was supported by NSF (IBN-0083653; FIBR-0526854) and NASA (NAG2-1611)

Magnetic tweezers for intracellular applications

doi:10.1063/1.1599066 http://rsi.aip.org/rsinak/v74/i9/p4158_s1We have designed and constructed a versatile magnetic tweezer primarily for intracellular investigations. The micromanipulator uses only two coils to simultaneously magnetize to saturation micron-size superparamagnetic particles and generate high magnitude constant field gradients over cellular dimensions. The apparatus resembles a miniaturized Faraday balance, an industrial device used to measure magnetic susceptibility. The device operates in both continuous and pulse modes. Due to its compact size, the tweezers can conveniently be mounted on the stage of an inverted microscope and used for intracellular manipulations. A built-in temperature control unit maintains the sample at physiological temperatures. The operation of the tweezers was tested by moving 1.28 μm diameter magnetic beads inside macrophages with forces near 500 pN.This work was partially supported by the NSF ~Grant No. DBI-9730999!

Bioprinting: Development of a novel approach for engineering three-dimensional tissue structures [abstract]

Abstract only availableBioprinting is a tissue engineering technique in which spherical cell aggregates, the "bio-ink", are deposited into biocompatible hydrogels, the "bio-paper", by a 3-axis "bio-printer". The aggregates can be deposited into essentially any 3D configuration, and when comprised of adhesive and motile cells aggregate fusion occurs. This self-organizing, liquid-like nature of these tissues is described on a molecular basis by The Differential Adhesion Hypothesis (DAH). The techniques we have developed are quite unique because of the high degree of automation that has been incorporated into our processes and the variety of engineered tissues that we are capable of creating. Despite automation, the creation of aggregates remains a nontrivial and time intensive process. The entire process of aggregate formation from initial cell culture to mature aggregate ready to be loaded into the printer takes approximately five days. This time is a limiting factor in the potential use of bio-printing as a source of on-demand tissues for clinical applications. A solution to this potential problem lies in the cryopreservation of aggregates. Freezing mediums and freezing protocols were tested and the effect of the freezing process on aggregate fusion was determined. An alternate solution to expedite the bioprinting process could lie in the printing of cell 'sausages', tightly packed cylinders of cells. In this method aggregate preparation is forgone. Elimination of this step could allow for increased time in tissue creation. Cell sausage printing provides another technique that could be incorporated into the fabrication of complex tissues. Our experiments in this novel and developing technology of bioprinting represent steps towards building complex tissues via self-assembly.McNair Scholars Progra

The evolving technology of bio-printing

Abstract only availableBio-printing is a novel method of tissue engineering that uses living cell spheroids as the 'bio-ink' and biocompatible gels as the 'bio-paper' with a three dimensional printer that deposits these aggregates into the gel with great precision. The deposited aggregates fuse into three dimensional tissue structures of the desired conformation due to the liquid like nature of cells and tissues, serving as the driving force of biological self assembly. Successful results from previous experiments and theoretical modeling of the fusion process prompted the development of a standardized and automated method that increases the speed, accuracy and reproducibility of printing. To fulfill these requirements, a cell packer, an aggregate cutter and bio-printer was developed, calibrated and tested. The tools produced more uniform and spherical aggregates as compared to the manual protocols, allowing the standard size and shape necessary for rapid and precise printing. The printed structures (ring and grid-like arrangements of aggregates) fused into toroids and compact sheets, fundamental building blocks of a living organism. The precision of the printing, combined with the cell packer and aggregate cutter makes bio-printing a feasible technology. The automated process using organ specific cells could allow histologically analogous tissues to be produced and used for tissue repair and regeneration.Life Sciences Undergraduate Research Opportunity Progra

Multiscale computational analysis of Xenopus laevis morphogenesis reveals key insights of systems-level behavior

<p>Abstract</p> <p>Background</p> <p>Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment.</p> <p>Results</p> <p>We have developed a computational simulation of mesendoderm migration in the <it>Xenopus laevis </it>explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/β-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors.</p> <p>Conclusion</p> <p>Our computational framework couples two different spatial scales in biology: intracellular with multicellular. In our simulation, events at one scale have quantitative and dynamic impact on events at the other scale. This integration enables the testing and identification of key systems-level hypotheses regarding how signaling proteins affect overall tissue-level behavior during morphogenesis in an experimentally verifiable system. Applications of this approach extend to the study of tissue patterning processes that occur during adulthood and disease, such as tumorgenesis and atherogenesis.</p

Organ printing as an information technology

Funding Information: This work has been sponsored by the São Paulo Research Foundation (FAPESP), The Brazilian Institute of Biofabrication (INCT-BIOFABRIS) and National Council for Scientific and Technological Development (CNPq). Publisher Copyright: © 2015 Published by Elsevier Ltd.Organ printing is defined as a layer by layer additive robotic computer-aided biofabrication of functional 3D organ constructs with using self-assembling tissue spheroids according to digital model. Information technology and computer-aided design softwares are instrumental in the transformation of virtual 3D bioimaging information about human tissue and organs into living biological reality during 3D bioprinting. Information technology enables design blueprints for bioprinting of human organs as well as predictive computer simulation both printing and post-printing processes. 3D bioprinting is now considered as an emerging information technology and the effective application of existing information technology tools and development of new technological platforms such as human tissue and organ informatics, design automation, virtual human organs, virtual organ biofabrication line, mathematical modeling and predictive computer simulations of bioprinted tissue fusion and maturation is an important technological imperative for advancing organ bioprinting.publishersversionPeer reviewe

Tissue Engineering by Self-Assembly and Bioprinting of Living Cells

Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion