Vivian K. Lee

Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.

We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.

On-Demand Three-Dimensional Freeform Fabrication of Multi-Layered Hydrogel Scaffold With Fluidic Channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186.

The integration of 3-D cell printing and mesoscopic fluorescence molecular tomography of vascular constructs within thick hydrogel scaffolds

Developing methods that provide adequate vascular perfusion is an important step toward engineering large functional tissues. Meanwhile, an imaging modality to assess the three-dimensional (3-D) structures and functions of the vascular channels is lacking for thick matrices (>2 ≈ 3 mm). Herein, we report on an original approach to construct and image 3-D dynamically perfused vascular structures in thick hydrogel scaffolds. In this work, we integrated a robotic 3-D cell printing technology with a mesoscopic fluorescence molecular tomography imaging system, and demonstrated the capability of the platform to construct perfused collagen scaffolds with endothelial lining and to image both the fluid flow and fluorescent-labeled living endothelial cells at high-frame rates, with high sensitivity and accuracy. These results establish the potential of integrating both 3-D cell printing and fluorescence mesoscopic imaging for functional and molecular studies in complex tissue-engineered tissues.

Three-dimensional bioprinting of rat embryonic neural cells

We present a direct cell printing technique to pattern neural cells in a three-dimensional (3D) multilayered collagen gel. A layer of collagen precursor was printed to provide a scaffold for the cells, and the rat embryonic neurons and astrocytes were subsequently printed on the layer. A solution of sodium bicarbonate was applied to the cell containing collagen layer as nebulized aerosols, which allowed the gelation of the collagen. This process was repeated layer-by-layer to construct the 3D cell–hydrogel composites. Upon characterizing the relationship between printing resolutions and the growth of printed neural cells, single/multiple layers of neural cell–hydrogel composites were constructed and cultured. The on-demand capability to print neural cells in a multilayered hydrogel scaffold offers flexibility in generating artificial 3D neural tissue composites.

Mesoscopic fluorescence molecular tomography of reporter genes in bioprinted thick tissue

Abstract. Three-dimensional imaging of thick tissue constructs is one of the main challenges in the field of tissue engineering and regenerative medicine. Optical methods are the most promising as they offer noninvasive, fast, and inexpensive solutions. Herein, we report the use of mesoscopic fluorescence molecular tomography (MFMT) to image function and structure of thick bioprinted tissue hosted in a 3-mm-thick bioreactor. Collagen-based tissue assembled in this study contains two vascular channels formed by green fluorescent protein- and mCherry-expressing cells. Transfected live cell imaging enables us to image function, whereas Flash Red fluorescent bead perfusion into the vascular channel allows us to image structure. The MFMT optical reconstructions are benchmarked with classical microscopy techniques. MFMT and wide-field fluorescence microscopy data match within 92% in area and 84% in location, validating the accuracy of MFMT reconstructions. Our results demonstrate that MFMT is a well-suited imaging modality for fast, longitudinal, functional imaging of thick, and turbid tissue engineering constructs.

Functional magnetic resonance imaging-mediated learning of increased activity in auditory areas.

Our earlier study indicated that functional magnetic resonance imaging (fMRI)-based detection and feedback of regional cortical activity from the auditory area enabled a group of individuals to increase the level of activation mediated by auditory attention during sound stimulation. The long-term ability to maintain an increased level of cortical activation, extending to a time period of a few weeks, however, has not been investigated. We used real-time fMRI to confirm the utility of fMRI in forming a basis for the regulation of brain function to increase the activation in the auditory areas, and demonstrated that the learned ability could be retained after a 2-week period, with additional involvement of an attention-related neural network.

Printing of Three-Dimensional Tissue Analogs for Regenerative Medicine

Three-dimensional (3-D) cell printing, which can accurately deposit cells, biomaterial scaffolds and growth factors in precisely defined spatial patterns to form biomimetic tissue structures, has emerged as a powerful enabling technology to create live tissue and organ structures for drug discovery and tissue engineering applications. Unlike traditional 3-D printing that uses metals, plastics and polymers as the printing materials, cell printing has to be compatible with living cells and biological matrix. It is also required that the printing process preserves the biological functions of the cells and extracellular matrix, and to mimic the cell–matrix architectures and mechanical properties of the native tissues. Therefore, there are significant challenges in order to translate the technologies of traditional 3-D printing to cell printing, and ultimately achieve functional outcomes in the printed tissues. So it is essential to develop new technologies specially designed for cell printing and in-depth basic research in the bioprinted tissues, such as developing novel biomaterials specifically for cell printing applications, understanding the complex cell–matrix remodeling for the desired mechanical properties and functional outcomes, establishing proper vascular perfusion in bioprinted tissues, etc. In recent years, many exciting research progresses have been made in the 3-D cell printing technology and its application in engineering live tissue constructs. This review paper summarized the current development in 3-D cell printing technologies; focus on the outcomes of the live printed tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions of 3-D cell printing technology are also discussed.

Three-dimensional cell-hydrogel printer using electromechanical microvalve for tissue engineering

In this study, we report a newly developed three-dimensional (3D) biological printer using non-contact, electromechanical microvalves with a nozzle diameter of 150 µm. To control and utilize this printer for life science applications, we developed an easy-to-use control software with a graphic user interface (GUI). First, using the printer, we tested the viability of dispensed mammalian cells after printing, and there was no significant difference in viability between dispensed cells and conventionally plated cells. Next, we constructed a 3D hydrogel scaffold by printing collagen hydrogel precursor layer-by-layer with linear patterns of gelatin inside. Using the same scheme, neurons were printed and patterned in multi-layered collagen scaffold. The on-demand capability to print cells and hydrogels in multi-layered hydrogel scaffold offers flexibility in generating artificial 3D tissue composites.

Venous Endothelial Marker COUP-TFII Regulates the Distinct Pathologic Potentials of Adult Arteries and Veins

Arteries and veins have very different susceptibility to certain vascular diseases such as atherosclerosis and vascular calcification. The molecular mechanisms of these differences are not fully understood. In this study, we discovered that COUP-TFII, a transcription factor critical for establishing the venous identity during embryonic vascular development, also regulates the pathophysiological functions of adult blood vessels, especially those directly related to vascular diseases. Specifically, we found that suppression of COUP-TFII in venous ECs switched its phenotype toward pro-atherogenic by up-regulating the expression of inflammatory genes and down-regulating anti-thrombotic genes. ECs with COUP-TFII knockdown also readily undergo endothelial-to-mesenchymal transition (EndoMT) and subsequent osteogenic differentiation with dramatically increased osteogenic transcriptional program and calcium deposition. Consistently, over-expression of COUP-TFII led to the completely opposite effects. In vivo validation of these pro-atherogenic and osteogenic genes also demonstrates a broad consistent differential expression pattern in mouse aorta vs. vena cava ECs, which cannot be explained by the difference in hemodynamic flow. These data reveal phenotypic modulation by different levels of COUP-TFII in arterial and venous ECs, and suggest COUP-TFII may play an important role in the different susceptibilities of arteries and veins to vascular diseases such as atherosclerosis and vascular calcification.

3D Bioprinting and 3D Imaging for Stem Cell Engineering

Three-dimensional (3D) bio-printing, a technology to create 3D tissue through layer-by-layer approach, offers great capacity to engineer tissue with desired cells, growth factors and biomaterial scaffolds in spatial patterns to mimic the native tissue architecture. With its flexibility and power, the 3D bio-printing technology can also be used to control stem cell fate and creating 3D stem cell niches. Meanwhile, 3D bio-printed tissues often incorporate thick opaque scaffold, dense population of cells, and are often large in size (1–100 mm). Thus, there are significant difficulties in visualizing the biological events within thick tissue constructs using current microscopic techniques. To elucidate the interaction of stem cells with the microenvironment in tissue engineering applications, it is necessary to develop novel molecular imaging techniques to non-invasively observe stem cell fate, cell-cell interactions, and structural features of an engineered tissue in real time. In this chapter, we review the usage of bio-printing technologies in stem cell and tissue engineering application, and the most recent development in the optical molecular imaging techniques for thick tissue imaging.