- Fundamentals for Engineering Biology Lab
BIOE 44 (Aut, Win)
- Tissue Engineering
BIOE 260 (Spr)
Independent Studies (6)
- Bioengineering Problems and Experimental Investigation
BIOE 191 (Aut, Win, Spr)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Study
BIOE 391 (Aut, Win)
- Experimental Investigation of Engineering Problems
ME 392 (Spr, Sum)
- Master's Research
MATSCI 200 (Aut, Win)
- Medical Scholars Research
MED 370 (Aut, Win)
- Bioengineering Problems and Experimental Investigation
Doctoral Dissertation Reader (AC)
Ali Kight, Elaine Lui, Zachary Sexton
Postdoctoral Faculty Sponsor
Doctoral Dissertation Advisor (AC)
Danielle Klinger, Joshua Sampson, Soham Sinha, Fredrik Samdal Solberg, Jonathan Weiss
Doctoral Dissertation Co-Advisor (AC)
Gokul Kannan, Danielle Klinger, Abena Peasah, Joshua Sampson, Jonathan Weiss, Will Yu
Biomanufacturing human tissues via organ building blocks.
Cell stem cell
2022; 29 (5): 667-677
The construction of human organs on demand remains a tantalizing vision to solve the organ donor shortage. Yet, engineering tissues that recapitulate the cellular and architectural complexity of native organs is a grand challenge. The use of organ building blocks (OBBs) composed of multicellular spheroids, organoids, and assembloids offers an important pathway for creating organ-specific tissues with the desired cellular-to-tissue-level organization. Here, we review the differentiation, maturation, and 3D assembly of OBBs into functional human tissues and, ultimately, organs for therapeutic repair and replacement. We also highlight future challenges and areas of opportunity for this nascent field.
View details for DOI 10.1016/j.stem.2022.04.012
View details for PubMedID 35523137
Programming Cellular Alignment in Engineered Cardiac Tissue via Bioprinting Anisotropic Organ Building Blocks.
Advanced materials (Deerfield Beach, Fla.)
The ability to replicate the three-dimensional myocardial architecture found in human hearts is a grand challenge. Here, we report the fabrication of aligned cardiac tissues via bioprinting anisotropic organ building blocks (aOBBs) composed of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We first generated a bioink composed of contractile cardiac aOBBs and printed aligned cardiac tissue sheets with linear, spiral, and chevron features. Next, we printed aligned cardiac macrofilaments, whose contractile force and conduction velocity increased over time and exceeded the performance of spheroid-based cardiac tissues. Finally, we highlighted the ability to spatially control the magnitude and direction of contractile force by printing cardiac sheets with different aOBB alignment. Our research opens new avenues to generating functional cardiac tissue with high cell density and complex cellularly alignment. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adma.202200217
View details for PubMedID 35451188
Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues.
Nature biomedical engineering
The generation of organoids and tissues with programmable cellular complexity, architecture and function would benefit from the simultaneous differentiation of human induced pluripotent stem cells (hiPSCs) into divergent cell types. Yet differentiation protocols for the overexpression of specific transcription factors typically produce a single cell type. Here we show that patterned organoids and bioprinted tissues with controlled composition and organization can be generated by simultaneously co-differentiating hiPSCs into distinct cell types via the forced overexpression of transcription factors, independently of culture-media composition. Specifically, we used such orthogonally induced differentiation to generate endothelial cells and neurons from hiPSCs in a one-pot system containing either neural or endothelial stem-cell-specifying media, and to produce vascularized and patterned cortical organoids within days by aggregating inducible-transcription-factor and wild-type hiPSCs into randomly pooled or multicore-shell embryoid bodies. Moreover, by leveraging multimaterial bioprinting of hiPSC inks without extracellular matrix, we generated patterned neural tissues with layered regions composed of neural stem cells, endothelium and neurons. Orthogonally induced differentiation of stem cells may facilitate the fabrication of engineered tissues for biomedical applications.
View details for DOI 10.1038/s41551-022-00856-8
View details for PubMedID 35332307
Bioprinted microvasculature: progressing from structure to function.
Three-dimensional (3D) bioprinting seeks to unlock the rapid generation of complex tissue constructs, but long-standing challenges with efficient in vitro microvascularization must be solved before this can become a reality. Microvasculature is particularly challenging to biofabricate due to the presence of a hollow lumen, a hierarchically branched network topology, and a complex signaling milieu. All of these characteristics are required for proper microvascular - and, thus, tissue - function. While several techniques have been developed to address distinct portions of this microvascularization challenge, no single approach is capable of simultaneously recreating all three microvascular characteristics. In this review, we present a three-part framework that proposes integration of existing techniques to generate mature microvascular constructs. First, extrusion-based 3D bioprinting creates a mesoscale foundation of hollow, endothelialized channels. Second, biochemical and biophysical cues induce endothelial sprouting to create a capillary-mimetic network. Third, the construct is conditioned to enhance network maturity. Across all three of these stages, we highlight the potential for extrusion-based bioprinting to become a central technique for engineering hierarchical microvasculature. We envision that the successful biofabrication of functionally engineered microvasculature will address a critical need in tissue engineering, and propel further advances in regenerative medicine and ex vivo human tissue modeling.
View details for DOI 10.1088/1758-5090/ac4fb5
View details for PubMedID 35086069
Reconstructing the heart using iPSCs: Engineering strategies and applications.
Journal of molecular and cellular cardiology
Induced pluripotent stem cells (iPSCs) have emerged as a key component of cardiac tissue engineering, enabling studies of cardiovascular disease mechanisms, drug responses, and developmental processes in human 3D tissue models assembled from isogenic cells. Since the very first engineered heart tissues were introduced more than two decades ago, a wide array of iPSC-derived cardiac spheroids, organoids, and heart-on-a-chip models have been developed incorporating the latest available technologies and materials. In this review, we will first outline the fundamental biological building blocks required to form a functional unit of cardiac muscle, including iPSC-derived cells differentiated by soluble factors (e.g., small molecules), extracellular matrix scaffolds, and exogenous biophysical maturation cues. We will then summarize the different fabrication approaches and strategies employed to reconstruct the heart in vitro at varying scales and geometries. Finally, we will discuss how these platforms, with continued improvements in scalability and tissue maturity, can contribute to both basic cardiovascular research and clinical applications in the future.
View details for DOI 10.1016/j.yjmcc.2021.04.006
View details for PubMedID 33895197
Flow-enhanced vascularization and maturation of kidney organoids in vitro
2019; 16 (3): 255-+
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.
View details for DOI 10.1038/s41592-019-0325-y
View details for Web of Science ID 000459804000023
View details for PubMedID 30742039
View details for PubMedCentralID PMC6488032
Voxelated soft matter via multimaterial multinozzle 3D printing.
2019; 575 (7782): 330–35
There is growing interest in voxelated matter that is designed and fabricated voxel by voxel1-4. Currently, inkjet-based three-dimensional (3D) printing is the only widely adopted method that is capable of creating 3D voxelated materials with high precision1-4, but the physics of droplet formation requires the use of low-viscosity inks to ensure successful printing5. By contrast, direct ink writing, an extrusion-based 3D printing method, is capable of patterning a much broader range of materials6-13. However, it is difficult to generate multimaterial voxelated matter by extruding monolithic cylindrical filaments in a layer-by-layer manner. Here we report the design and fabrication of voxelated soft matter using multimaterial multinozzle 3D (MM3D) printing, in which the composition, function and structure of the materials are programmed at the voxel scale. Our MM3D printheads exploit the diode-like behaviour that arises when multiple viscoelastic materials converge at a junction to enable seamless, high-frequency switching between up to eight different materials to create voxels with a volume approaching that of the nozzle diameter cubed. As exemplars, we fabricate a Miura origami pattern14 and a millipede-like soft robot that locomotes by co-printing multiple epoxy and silicone elastomer inks of stiffness varying by several orders of magnitude. Our method substantially broadens the palette of voxelated materials that can be designed and manufactured in complex motifs.
View details for DOI 10.1038/s41586-019-1736-8
View details for PubMedID 31723289
Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels.
2019; 5 (9): eaaw2459
Engineering organ-specific tissues for therapeutic applications is a grand challenge, requiring the fabrication and maintenance of densely cellular constructs composed of ~108 cells/ml. Organ building blocks (OBBs) composed of patient-specific-induced pluripotent stem cell-derived organoids offer a pathway to achieving tissues with the requisite cellular density, microarchitecture, and function. However, to date, scant attention has been devoted to their assembly into 3D tissue constructs. Here, we report a biomanufacturing method for assembling hundreds of thousands of these OBBs into living matrices with high cellular density into which perfusable vascular channels are introduced via embedded three-dimensional bioprinting. The OBB matrices exhibit the desired self-healing, viscoplastic behavior required for sacrificial writing into functional tissue (SWIFT). As an exemplar, we created a perfusable cardiac tissue that fuses and beats synchronously over a 7-day period. Our SWIFT biomanufacturing method enables the rapid assembly of perfusable patient- and organ-specific tissues at therapeutic scales.
View details for DOI 10.1126/sciadv.aaw2459
View details for PubMedID 31523707
View details for PubMedCentralID PMC6731072
In Vitro Human Tissues via Multi-material 3-D Bioprinting
ATLA-ALTERNATIVES TO LABORATORY ANIMALS
2018; 46 (4): 209–15
This paper highlights the foundational research on multi-material 3-D bioprinting of human tissues, for which the Lewis Bioprinting team at Harvard University was awarded the 2017 Lush Science Prize. The team's bioprinting platform enables the rapid fabrication of 3-D human tissues that contain all of the essential components found in their in vivo counterparts: cells, vasculature (or other tubular features) and extracellular matrix. The printed 3-D tissues are housed within a customised perfusion system and are subjected to controlled microphysiological environments over long durations (days to months). As exemplars, the team created a thick, stem cell-laden vascularised tissue that was controllably differentiated toward an osteogenic lineage in situ, and a 3-D kidney tissue that recapitulated the proximal tubule, a subunit of the nephron responsible for solute reabsorption. This highly versatile platform for manufacturing 3-D human tissue in vitro opens new avenues for replacing animal models used to develop next-generation therapies, test toxicity and study disease pathology.
View details for DOI 10.1177/026119291804600404
View details for Web of Science ID 000451747100003
View details for PubMedID 30365335
3D printed structures for modeling the Young's modulus of bamboo parenchyma
2018; 68: 90-98
Bamboo is a sustainable, lightweight material that is widely used in structural applications. To fully develop micromechanical models for plants, such as bamboo, the mechanical properties of each individual type of tissue are needed. However, separating individual tissues and testing them mechanically is challenging. Here, we report an alternative approach in which micro X-ray computed tomography (µ-CT) is used to image moso bamboo (Phyllostachys pubescens). The acquired images, which correspond to the 3D structure of the parenchyma, are then transformed into physical, albeit larger scale, structures by 3D printing, and their mechanical properties are characterized. The normalized longitudinal Young's moduli of the fabricated structures depend on relative density raised to a power between 2 and 3, suggesting that elastic deformation of the parenchyma cellular structure involves considerable cell wall bending. The mechanical behavior of other biological tissues may also be elucidated using this approach.Bamboo is a lightweight, sustainable engineering material widely used in structural applications. By combining micro X-ray computed tomography and 3D printing, we have produced bamboo parenchyma mimics and characterized their stiffness. Using this approach, we gained insight into bamboo parenchyma tissue mechanics, specifically the cellular geometry's role in longitudinal elasticity.
View details for DOI 10.1016/j.actbio.2017.12.036
View details for Web of Science ID 000426026700008
View details for PubMedID 29294375
3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds.
Science (New York, N.Y.)
2018; 362 (6420): 1281–85
Lithographic nanofabrication is often limited to successive fabrication of two-dimensional (2D) layers. We present a strategy for the direct assembly of 3D nanomaterials consisting of metals, semiconductors, and biomolecules arranged in virtually any 3D geometry. We used hydrogels as scaffolds for volumetric deposition of materials at defined points in space. We then optically patterned these scaffolds in three dimensions, attached one or more functional materials, and then shrank and dehydrated them in a controlled way to achieve nanoscale feature sizes in a solid substrate. We demonstrate that our process, Implosion Fabrication (ImpFab), can directly write highly conductive, 3D silver nanostructures within an acrylic scaffold via volumetric silver deposition. Using ImpFab, we achieve resolutions in the tens of nanometers and complex, non-self-supporting 3D geometries of interest for optical metamaterials.
View details for DOI 10.1126/science.aau5119
View details for PubMedID 30545883
View details for PubMedCentralID PMC6423357
- Multi-photon microfabrication of three-dimensional capillary-scale vascular networks SPIE-INT SOC OPTICAL ENGINEERING. 2017
Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips
2016; 6: 34845
Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.
View details for DOI 10.1038/srep34845
View details for Web of Science ID 000385242900001
View details for PubMedID 27725720
View details for PubMedCentralID PMC5057112
Laser-assisted direct ink writing of planar and 3D metal architectures
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (22): 6137–42
The ability to pattern planar and freestanding 3D metallic architectures at the microscale would enable myriad applications, including flexible electronics, displays, sensors, and electrically small antennas. A 3D printing method is introduced that combines direct ink writing with a focused laser that locally anneals printed metallic features "on-the-fly." To optimize the nozzle-to-laser separation distance, the heat transfer along the printed silver wire is modeled as a function of printing speed, laser intensity, and pulse duration. Laser-assisted direct ink writing is used to pattern highly conductive, ductile metallic interconnects, springs, and freestanding spiral architectures on flexible and rigid substrates.
View details for DOI 10.1073/pnas.1525131113
View details for Web of Science ID 000376784600032
View details for PubMedID 27185932
View details for PubMedCentralID PMC4896727
Guided Homing of Cells in Multi-Photon Microfabricated Bioscaffolds
ADVANCED HEALTHCARE MATERIALS
2016; 5 (10): 1233–43
Tissues contain exquisite vascular microstructures, and patterns of chemical cues for directing cell migration, homing, and differentiation for organ development and function. 3D microfabrication by multi-photon photolithography is a flexible, high-resolution tool for generating 3D bioscaffolds. However, the combined fabrication of scaffold microstructure simultaneously with patterning of cues to create both geometrically and chemically defined microenvironments remains to be demonstrated. This study presents a high-speed method for micron-resolution fabrication of scaffold microstructure and patterning of protein cues simultaneously using native scaffold materials. By the simultaneous microfabrication of arbitrary microvasculature geometries, and patterning selected regions of the microvasculature with the homing ligand P-selectin, this study demonstrates adhesion, rolling, and selective homing of cells in defined 3D regions. This novel ability to generate high-resolution geometries replete with patterned cues at high speed enables the construction of biomimetic microenvironments for complex 3D assays of cellular behavior.
View details for DOI 10.1002/adhm.201600082
View details for Web of Science ID 000377528300015
View details for PubMedID 27059425
View details for PubMedCentralID PMC6070392
Three-dimensional bioprinting of thick vascularized tissues
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (12): 3179–84
The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.
View details for DOI 10.1073/pnas.1521342113
View details for Web of Science ID 000372488200037
View details for PubMedID 26951646
View details for PubMedCentralID PMC4812707
Synchronous Symmetry Breaking in Neurons with Different Neurite Counts
2013; 8 (2): e54905
As neurons develop, several immature processes (i.e., neurites) grow out of the cell body. Over time, each neuron breaks symmetry when only one of its neurites grows much longer than the rest, becoming an axon. This symmetry breaking is an important step in neurodevelopment, and aberrant symmetry breaking is associated with several neuropsychiatric diseases, including schizophrenia and autism. However, the effects of neurite count in neuronal symmetry breaking have never been studied. Existing models for neuronal polarization disagree: some predict that neurons with more neurites polarize up to several days later than neurons with fewer neurites, while others predict that neurons with different neurite counts polarize synchronously. We experimentally find that neurons with different neurite counts polarize synchronously. We also show that despite the significant differences among the previously proposed models, they all agree with our experimental findings when the expression levels of the proteins responsible for symmetry breaking increase with neurite count. Consistent with these results, we observe that the expression levels of two of these proteins, HRas and shootin1, significantly correlate with neurite count. This coordinated symmetry breaking we observed among neurons with different neurite counts may be important for synchronized polarization of neurons in developing organisms.
View details for DOI 10.1371/journal.pone.0054905
View details for Web of Science ID 000315100000010
View details for PubMedID 23408951
View details for PubMedCentralID PMC3569465
Electrokinetic confinement of axonal growth for dynamically configurable neural networks
LAB ON A CHIP
2013; 13 (4): 589-598
Axons in the developing nervous system are directed via guidance cues, whose expression varies both spatially and temporally, to create functional neural circuits. Existing methods to create patterns of neural connectivity in vitro use only static geometries, and are unable to dynamically alter the guidance cues imparted on the cells. We introduce the use of AC electrokinetics to dynamically control axonal growth in cultured rat hippocampal neurons. We find that the application of modest voltages at frequencies on the order of 10(5) Hz can cause developing axons to be stopped adjacent to the electrodes while axons away from the electric fields exhibit uninhibited growth. By switching electrodes on or off, we can reversibly inhibit or permit axon passage across the electrodes. Our models suggest that dielectrophoresis is the causative AC electrokinetic effect. We make use of our dynamic control over axon elongation to create an axon-diode via an axon-lock system that consists of a pair of electrode 'gates' that either permit or prevent axons from passing through. Finally, we developed a neural circuit consisting of three populations of neurons, separated by three axon-locks to demonstrate the assembly of a functional, engineered neural network. Action potential recordings demonstrate that the AC electrokinetic effect does not harm axons, and Ca(2+) imaging demonstrated the unidirectional nature of the synaptic connections. AC electrokinetic confinement of axonal growth has potential for creating configurable, directional neural networks.
View details for DOI 10.1039/c2lc41000a
View details for Web of Science ID 000313971300012
View details for PubMedID 23314575
View details for PubMedCentralID PMC3554853
Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons
LAB ON A CHIP
2012; 12 (12): 2265-2276
Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.
View details for DOI 10.1039/c2lc21105j
View details for Web of Science ID 000304448700020
View details for PubMedID 22596091
View details for PubMedCentralID PMC3361619
Synapse microarray identification of small molecules that enhance synaptogenesis
2011; 2: 510
Synaptic function is affected in many brain diseases and disorders. Technologies for large-scale synapse assays can facilitate identification of drug leads. Here we report a 'synapse microarray' technology that enables ultra-sensitive, high-throughput and quantitative screening of synaptogenesis. Our platform enables the induction of synaptic structures in regular arrays by precise positioning of non-neuronal cells expressing synaptic proteins, while allowing neurites to grow freely around these cells. The technology increases by tenfold the sensitivity of the traditional assays, and simultaneously decreases the time required to capture synaptogenic events by an order of magnitude. It is readily incorporated into multiwell formats compatible with industrial high-throughput screening platforms. Using this technology, we screened a chemical library, and identified novel histone deacetylase (HDAC) inhibitors that improve neuroligin-1-induced synaptogenesis by modulating class-I HDACs. We also found a structure-activity relationship for designing novel potent histone deacetylase inhibitors, which can be applied towards development of new therapeutics.
View details for DOI 10.1038/ncomms1518
View details for Web of Science ID 000296787300023
View details for PubMedID 22027590
View details for PubMedCentralID PMC3544154
Large-scale analysis of neurite growth dynamics on micropatterned substrates
2011; 3 (1): 65-74
During both development and regeneration of the nervous system, neurons display complex growth dynamics, and several neurites compete to become the neuron's single axon. Numerous mathematical and biophysical models have been proposed to explain this competition, which remain experimentally unverified. Large-scale, precise, and repeatable measurements of neurite dynamics have been difficult to perform, since neurons have varying numbers of neurites, which themselves have complex morphologies. To overcome these challenges using a minimal number of primary neurons, we generated repeatable neuronal morphologies on a large scale using laser-patterned micron-wide stripes of adhesive proteins on an otherwise highly non-adherent substrate. By analyzing thousands of quantitative time-lapse measurements of highly reproducible neurite growth dynamics, we show that total neurite growth accelerates until neurons polarize, that immature neurites compete even at very short lengths, and that neuronal polarity exhibits a distinct transition as neurites grow. Proposed neurite growth models agree only partially with our experimental observations. We further show that simple yet specific modifications can significantly improve these models, but still do not fully predict the complex neurite growth behavior. Our high-content analysis puts significant and nontrivial constraints on possible mechanistic models of neurite growth and specification. The methodology presented here could also be employed in large-scale chemical and target-based screens on a variety of complex and subtle phenotypes for therapeutic discoveries using minimal numbers of primary neurons.
View details for DOI 10.1039/c0ib00058b
View details for Web of Science ID 000286109300008
View details for PubMedID 20976322
View details for PubMedCentralID PMC3173981
Construction of a femtosecond laser microsurgery system
2010; 5 (3): 395-407
Femtosecond laser microsurgery is a powerful method for studying cellular function, neural circuits, neuronal injury and neuronal regeneration because of its capability to selectively ablate sub-micron targets in vitro and in vivo with minimal damage to the surrounding tissue. Here, we present a step-by-step protocol for constructing a femtosecond laser microsurgery setup for use with a widely available compound fluorescence microscope. The protocol begins with the assembly and alignment of beam-conditioning optics at the output of a femtosecond laser. Then a dichroic mount is assembled and installed to direct the laser beam into the objective lens of a standard inverted microscope. Finally, the laser is focused on the image plane of the microscope to allow simultaneous surgery and fluorescence imaging. We illustrate the use of this setup by presenting axotomy in Caenorhabditis elegans as an example. This protocol can be completed in 2 d.
View details for DOI 10.1038/nprot.2010.4
View details for Web of Science ID 000275234900002
View details for PubMedID 20203659
View details for PubMedCentralID PMC4153993