Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell-cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. Recent studies have shown the potential of hydrogels to mimic native ECM. Such an engineered native-like ECM is more likely to provide cells with rational cues for diagnostic and therapeutic studies. The research for novel biomaterials has led to an extension of the scope and techniques used to fabricate biomimetic hydrogel scaffolds for tissue engineering and regenerative medicine applications. In this article, we detail the progress of the current state-of-the-art engineering methods to create cell-encapsulating hydrogel tissue constructs as well as their applications in in vitro models in biomedicine. Keywordsbiopatterning; cell-encapsulating microfluidic hydrogels; cell microenvironment; extracellular matrix; tissue engineering Mimicking the extracellular matrixCells and tissues are routinely cultured in vitro on 2D substrates [1][2][3]. However, it has been demonstrated that cells or tissues cultured on 2D substrates (e.g., tissue culture plates or flasks) do not mimic cell growth in vivo, and fail to express certain tissue-specific genes and proteins at levels comparable to those found in vivo. For instance, it has been found that cell-drug interactions in a 2D culture system do not represent the actual working mechanism in vivo. Thus, 2D culture is not appropriate to be used in in vitro drug testing models. This is due to the fact that cells and tissues in vivo are immersed within a 3D network constituting a complex extracellular environment with a highly porous nanotopography, while a 2D culture system is too simple to mimic the native environment (Table 1).From a tissue engineering (TE) standpoint, constructing a culture environment that closely mimicks the native tissue, which is composed of the extracellular matrix (ECM), soluble bioactive factors, and products of homo-and hetero-typical cell-cell interactions, is desirable to replicate tissue functions in vitro. However, this remains as one of the major challenges in TE, given the complexity of cell-ECM interactions as well as multicellular architectural features such as repeating tissue units and proper vascular structure. Cells commit to their fate by deriving a vast amount of information from this environment. As a part of the cell environment, ECM has been the most emulated component in TE studies. In native tissue, ECM is mainly a mixture of two classes of macromolecules, glycosaminoglycans and fibrous proteins (e.g., collagen, elastin, fibronectin and laminin), which self-assemble into nanofibrillar supramolecular networks that fill the extracellul...

Recently, there has been a growing interest to apply bioprinting techniques to stem cell research. Several bioprinting methods have been developed utilizing acoustics, piezoelectricity, and lasers to deposit living cells onto receiving substrates. Using these technologies, spatially defined gradients of immobilized proteins can be engineered to direct stem cell differentiation into multiple subpopulations of different lineages. Stem cells can also be patterned in a high-throughput manner onto flexible implementation patches for tissue regeneration or onto substrates with the goal of accessing encapsulated stem cell of interest for genomic analysis. Here, we review recent achievements with bioprinting technologies in stem cell research, and identify future challenges and potential applications including tissue engineering and regenerative medicine, wound healing, and genomics.

The encapsulation of mammalian cells within the bulk material of microfluidic channels may be beneficial for applications ranging from tissue engineering to cell-based diagnostic assays. In this work, we present a technique for fabricating microfluidic channels from cell-laden agarose hydrogels. Using standard soft lithographic techniques, molten agarose was molded against a SU-8 patterned silicon wafer. To generate sealed and water-tight microfluidic channels, the surface of the molded agarose was heated at 71 degrees C for 3 s and sealed to another surface-heated slab of agarose. Channels of different dimensions were generated and it was shown that agarose, though highly porous, is a suitable material for performing microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. While most cells were found to be viable upon initial device fabrication, only those cells near the microfluidic channels remained viable after 3 days, demonstrating the importance of a perfused network of microchannels for delivering nutrients and oxygen to maintain cell viability in large hydrogels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering, diagnostics, and drug screening applications.

Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.

Figure 7. Recent advances in surface functionalization methods and materials. (A) Lipid-supported surface functionalization and applications: (i) Schematic of a gold nanorod coated with a biotinylated lipid membrane and interactions with streptavidin. Adapted with permission from ref 146.

The capability to encapsulate single to few cells with micrometre precision, high viability, and controlled directionality via a nozzleless ejection technology using a gentle acoustic field would have great impact on tissue engineering, high throughput screening, and clinical diagnostics. We demonstrate encapsulation of single cells (or a few cells) ejected from an open pool in acoustic picolitre droplets. We have developed this technology for the specific purpose of printing cells in various biological fluids, including PBS and agarose hydrogels used in tissue engineering. We ejected various cell types, including mouse embryonic stem cells, fibroblasts, AML-12 hepatocytes, human Raji cells, and HL-1 cardiomyocytes encapsulated in acoustic picolitre droplets of around 37 microm in diameter at rates varying from 1 to 10,000 droplets per second. At such high throughput levels, we demonstrated cell viabilities of over 89.8% across various cell types. Moreover, this ejection method is readily adaptable to other biological applications, such as extracting data from single cells and generating large cell populations from single cells. The technique described in the current study may also be applied to investigate stem cell differentiation at the single cell level, to direct tissue printing, and to isolating pure RNA or DNA from a single cell at the picolitre level. Overall, the techniques described have the potential for widespread impact on many high-throughput testing applications in the biological and health sciences.

Complex functional materials with three-dimensional micro- or nano-scale dynamic compositional features are prevalent in nature. However, the generation of three-dimensional functional materials composed of both soft and rigid microstructures, each programmed by shape and composition, is still an unsolved challenge. Herein, we describe a method to code complex materials in three-dimensions with tunable structural, morphological, and chemical features using an untethered magnetic micro-robot remotely controlled by magnetic fields. This strategy allows the micro-robot to be introduced to arbitrary microfluidic environments for remote two- and three-dimensional manipulation. We demonstrate the coding of soft hydrogels, rigid copper bars, polystyrene beads, and silicon chiplets into three-dimensional heterogeneous structures. We also use coded microstructures for bottom-up tissue engineering by generating cell-encapsulating constructs.