Christopher Lambert

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping

Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed “ZEG” (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising.

Drug-delivering nerve conduit improves regeneration in a critical-sized gap: LABROO et al.

Autologous nerve grafts are the current “gold standard” for repairing large nerve gaps. However, they cause morbidity at the donor nerve site and only a limited amount of nerve can be harvested. Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of GDNF can enhance axon growth and motor recovery. FK506, an FDA approved small molecule, has also been shown to enhance peripheral nerve regeneration. This paper describes the design of a novel hole‐based drug delivery apparatus integrated with a polytetrafluoroethylene (PTFE) nerve conduit for controlled local delivery of a protein such as GDNF or a small molecule such as FK506. The PTFE devices were tested in a diffusion chamber, and the bioactivity of the released media was evaluated by measuring neurite growth of dorsal root ganglions (DRGs) exposed to the released drugs. The drug delivering nerve guide was able to release bioactive concentrations of FK506 or GDNF. Following these tests, optimized drug releasing nerve conduits were implanted across 10 mm sciatic nerve gaps in a BL6 yellow fluorescent protein (YFP) mouse model, where they demonstrated significant improvement in muscle mass, compound muscle action potential, and axon myelination in vivo as compared with nerve conduits without the drug. The drug delivery nerve guide could release drug for extended periods of time and enhance axon growth in vitro and in vivo.

Experimental validation of an optofluidic platform for microbial single cell isolation and whole genome amplification for human microbiome applications

Single microbial cell genome sequencing is becoming a powerful tool for the discovery of the hidden genetic information valuable for many medical applications. One of the critical steps in single-cell genome sequencing is the physical isolation of individual cells from a highly diverse heterogeneous population. Amplifying the genome of a single microbial cell is another challenge due to the minute amount of DNA. Efforts have been directed in developing an optofluidic platform integrating advanced microscopy, optical tweezers and microfluidic technology for single cell isolation and genome amplification. Here, we investigate and evaluate the validity of this platform for single microbial cell genome amplification. The successful validation of this approach allows us to perform various single cell studies using this platform.

Abstract 134: FK506 Delivering Conduit For Peripheral Nerve Regeneration

PURPOSE: Following a peripheral nerve injury, nerve gaps require grafts or conduits to connect the two nerve ends. There is a clinical need to improve functional recovery following nerve injury and local release of neurotrophic factors is one way to improve outcomes. FK506, an FDA approved small molecule, has been shown to enhance axon growth in vitro and peripheral nerve regeneration in vivo. Systemic delivery of FK506 has numerous potentially serious side effects such as central nervous system toxicity, infection and nephrotoxicity. Local delivery of FK506 from a nerve guide could provide the neurotrophic benefits of FK506 but prevent the negative consequence of systemic delivery. Here we describe the in vitro and in vivo outcomes when using a novel drug delivery apparatus integrated with a biocompatible polytetrafluoroethylene (PTFE) based nerve conduit for controlled local delivery of FK506.