We introduce a microfluidic system for simultaneously measuring single cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 hours of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1/S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis.

Direct observation of mammalian cell growth and size regulation

The investigation of human disease mechanisms is difficult due to the heterogeneity in gene expression and the physiological state of cells in a given population. In comparison to bulk cell measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. In this review, we describe the recent advances in single-cell technologies and their applications in single-cell manipulation, diagnosis, and therapeutics development.

Current Trends of Microfluidic Single-Cell Technologies.

Adipose tissue depots in distinct anatomical locations mediate key aspects of metabolism, including energy storage, nutrient release, and thermogenesis Although adipocytes make up more than 90% of adipose tissue volume, they represent less than 50% of its cellular content Here, I review recent advances in genetic lineage tracing and transcriptomics that reveal the identities of the heterogeneous cell populations constituting mouse and human adipose tissues In addition to mature adipocytes and their progenitors, these include endothelial and various immune cell types that together orchestrate adipose tissue development and functions One salient finding is the identification of progenitor subtypes that can modulate adipogenic capacity through paracrine mechanisms Another is the description of fate trajectories of monocyte/macrophages, which can respond maladaptively to nutritional and thermogenic stimuli, leading to metabolic disease These studies have generated an extraordinary source of publicly available data that can be leveraged to explore commonalities and differences among experimental models, providing new insights into adipose tissues and their role in metabolic disease

Cellular Heterogeneity in Adipose Tissues.

National Institute of Allergy and Infectious Diseases (U.S.) (Award Number 5U19AI050864-07)

On-Chip Activation and Subsequent Detection of Individual Antigen-Specific T Cells

Vision loss caused by retinal diseases affects hundreds of millions of individuals worldwide. The retina is a delicate central nervous system tissue stratified into layers of cells with distinct roles. Currently, there is a void in treatments that selectively target diseased retinal cells, and current therapeutic paradigms present complications associated with off-target effects. Herein, as a proof of concept, we introduce an in vivo method using a femtosecond laser to locally optoporate retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles (AuNPs). We provide evidence that AuNPs functionalized with an antibody toward the cell-surface voltage-gated K+ channel subunit KV1.1 can selectively deliver fluorescently tagged siRNAs or fluorescein isothiocyanate-dextran dye into retinal cells when irradiated with an 800 nm 100 fs laser. Importantly, neither AuNP administration nor irradiation resulted in RGC death. This system provides a novel, non-viral-based approach that has the potential to selectively target retinal cells in diseased regions while sparing healthy areas and may be harnessed in future cell-specific therapies for retinal degenerative diseases.

in vivo laser-mediated retinal ganglion cell optoporation using Kv1.1 conjugated gold nanoparticles

Delivery of exogenous materials or cargo such as drugs, proteins, peptides, and nucleic acids into cells is a vital segment in molecular and cellular biology for potential cellular therapy and drug-discovery applications contributing toward personalization of medicine. Over the years, drug-delivery techniques have been developed in order to gain more control over the drug dosage, targeted delivery, and to minimize side effects. The major drug-delivery techniques can be classified as viral, chemical, and physical methods. Viral vectors are prominently used for gene therapy; however, they are cell-specific and have an immune response with high toxicity. Chemical methods are often limited by the low efficiency of plasmid delivery into different cell types due to plasmid degradation and toxicity. Considering these limitations, different physical methods such as photoporation, gene gun, hydrodynamic injection, electroporation, and mechanoporation, etc., are being widely developed for highly efficient cargo delivery with low toxicity. These methods are able to create transient hydrophilic membrane pores to deliver cargos into cells using different physical energies. Currently, ex vivo cargo delivery is widely studied while few in vivo applications have been developed. Concerning several obstacles to cargo delivery into cells, this chapter mainly focuses on different physical drug-delivery techniques such as electroporation, optoporation, mechanoporation, magnetoporation, and hybrid techniques along with their working mechanisms, advantages, disadvantages, and limitations. An insight into the future prospects and real-time applications of these techniques is also discussed.

Physical approaches for drug delivery: an overview

https://link-springer-com-443.webvpn.jmu.edu.cn/content/pdf/10.1007%2F978-981-10-4857-9_3-1.pdf

Mechanoporation: Toward Single Cell Approaches

This chapter discusses in detail some of the prominent microfluidic strategies employed for cellular manipulation, analysis, and treatment and their feasibility in clinical applications. The major advantage of microfluidic techniques over other conventional methods is that they provide high-throughput devices for the manipulation and analysis of cells. Many high-throughput microfluidic devices have been used for single-cell trapping using electro-osmotic flow manipulation, microvortex manipulation, etc. The chapter discusses microfluidic-based physical techniques, such as electroporation and mechanoporation, for single-cell therapeutic and diagnostic purposes. Droplet microfluidics is frequently employed for single-cell analysis due to its high specificity and potentiality to isolate single cells. Additionally, microfluidic technologies are widely used to analyze single-cell intracellular components, such as DNA, RNA, proteins, and amino acids. Analysis of these components on a single-cell level is important due to the extreme variation in the composition of proteins within a population of cells.

Microfluidic Technologies for Cell Manipulation, Therapeutics, and Analysis

Titanium-dioxide nanotubes (TNTs) were fabricated by anodic oxidation of titanium (Ti) substrate and loaded with ciprofloxacin. A thin layer of Poly Lactic-co-Glycolic Acid (PLGA) was coated on top of the drug-loaded TNTs at various thicknesses. Field Emission Scanning Electron Microscopy (FESEM) was used to characterize the developed substrate. The FESEM images of anodized samples showed that, the native oxide layer on the surface of the Ti substrate was replaced with an array of ordered nanotubes up to ~700 nm in length. Drug release studies of ciprofloxacin were conducted in Phosphate Buffered Saline (PBS) solution for 24 h. The studies showed the ability of PLGA to slow down the drug release rate, depending on the thickness of the deposited layer. MG-63 cells (human osteosarcoma cell lines) were cultured on the TNT substrates after loading ciprofloxacin and coating with various concentrations of PLGA to show the potentiality for better osseointegration. An MTT assay was carried out to study cell viability and proliferation on these substrates. Anti-microbial studies were carried out to demonstrate the release of ciprofloxacin in treating infections against Staphylococcus epidermidis. These in-depth studies showed that local concentration of the drug released could be controlled by varying the thickness of the PLGA layer to allow for better implant osseointegration and treatment of bone infections. 

Amogh Kumar

Papers

Current Trends of Microfluidic Single-Cell Technologies.

Physical approaches for drug delivery: an overview

Mechanoporation: Toward Single Cell Approaches

Microfluidic Technologies for Cell Manipulation, Therapeutics, and Analysis

Controlled and Localized Drug Delivery Using Titania Nanotubes