Raheel Samuel

Non-motile sperm cell separation using a spiral channel

Microfluidic sperm sorting has historically relied on sperm motility. However, a motility-based sperm separation technology will not work when viable, non-motile sperm need to be separated from other tissues as occurs when performing testicular sperm extraction (TESE) and microdissection testicular sperm extraction (mTESE) techniques. This work demonstrates the use of inertial microfluidics technology using spiral channels to separate sperm from blood cells. The separation method, which is label-free, does not rely on sperm motility for sorting. Basic principles of spiral channel separations were used to design a specific channel and flow parameters for separating non-motile sperm from blood. The spiral channels dimensions were: initial radius, 0.7 cm; final radius, 0.899 cm; channel width, 150 μm; channel height, 50 μm; turns of spiral, 4 turns; and space between channels, 310 μm. If sperm are modeled as a 5 μm sphere, inertial microfluidics theory suggests that the sperm could be focused and separated from red blood cells (RBCs). Channels to implement these features were validated in a series of experiments. Mixed samples of RBCs and sperm were used to test the sperm separation capability of the device with the sample injection flow rate ranging from 0.1–0.52 ml min−1. After running the sample through the spiral channel, the samples were collected from four outlets and were inspected using microscopy. The best results were obtained at a 0.52 ml min−1 flow rate and generated a concentration ratio of 81%, representing the percent of sperm collected from the two outer outlets. For the same conditions, 99% of RBCs were collected from the two inner wall outlets. Using a high speed scanner, we were able to observe the focusing of the RBCs and general focusing of the sperm. As the sperm are not a uniform shape, they did not focus in a tight band, but were collected in a general region of the channel. Nevertheless, the purification ratio for these sperm was sufficient to greatly enhance the likelihood of finding rare sperm in TESE/mTESE samples containing millions of blood cells. Sequentially processing of the samples in the system proved to further improve the ratio of sperm to blood cells.

Separation of sperm cells from samples containing high concentrations of white blood cells using a spiral channel

Microfluidic technology has potential to separate sperm cells from unwanted debris while improving the effectiveness of assisted reproductive technologies (ART). Current clinical protocol limitations regarding the separation of sperm cells from other cells/cellular debris can lead to low sperm recovery when the sample contains a low concentration of mostly low motility sperm cells and a high concentration of unwanted cells/cellular debris, such as in semen samples from patients with pyospermia [high white blood cell (WBC) semen]. This study demonstrates label-free separation of sperm cells from such semen samples using inertial microfluidics. The approach does not require any externally applied forces except the movement of the fluid sample through the instrument. Using this approach, it was possible to recover not only any motile sperm, but also viable less-motile and non-motile sperm cells with high recovery rates. Our results demonstrate the ability of inertial microfluidics to significantly reduce WBC concentration by flow focusing of target WBCs within a spiral channel flow. The estimated sample process time was more rapid (∼5 min) and autonomous than the conventional method (gradient centrifuge sperm wash; ∼1 h). A mixture of sperm/WBC was injected as the device input and 83% of sperm cells and 93% of WBCs were collected separately from two distinct outlets. The results show promise for enhancing sperm samples through inertial flow processing of WBCs and sperm cells that can provide an advantage to ART procedures such as sample preparation for intrauterine insemination.

Microfluidics: The future of microdissection TESE?

ABSTRACT Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a set of clinical protocols from which viable sperm are collected from patients (suffering from NOA), for intracytoplasmic sperm injection (ICSI). Clinical protocols associated with the processing of a microTESE sample are inefficient and significantly reduce the success of obtaining a viable sperm population. In this review we highlight the sources of these inefficiencies and how these sources can possibly be removed by microfluidic technology and single-cell Raman spectroscopy.

Simple and cost-effective fabrication of microvalve arrays in PDMS using laser cut molds with application to C. elegans manipulation in microfluidics

We present a new fabrication protocol for fabricating pneumatically controlled microvalve arrays (consisting of 100 s of microvalves) in PDMS substrates. The protocol utilizes rapid and cost-effective fabrication of molds using laser cutting of adhesive vinyl tapes and replica molding of PDMS. Hence the protocol is fast, simple and avoids cleanroom use. The results show that effective doormat-style microvalves can be easily fabricated in arrays by manipulating the stiffness of the actuating membrane through varying the valve-chamber area/shape. Three frequently used valve-chamber shapes (circle, square and capsule) were tested and all showed advantages in different situations. Circular valve chambers were best for small valves, square valves were best for medium-sized valves, and the capsule valves were best for larger valves. An application of this protocol has been demonstrated in the fabrication of a microfluidic 32-well plate for high-throughput manipulation of C. elegans for biomedical research.

Microfluidic laminate-based phantom for diffusion tensor-magnetic resonance imaging

This paper reports fabrication of a magnetic resonance imaging (MRI) phantom created by stacking of multiple thin polydimethylsiloxane (PDMS) layers. PDMS is spin coated on SU-8 molds to obtain the desired layer thickness and imprints of the microchannel patterns that define the phantom geometry. This paper also identifies the unique challenges related to the fabrication and assembly of multiple thin layers and reports for the first time assembly of a large number of thin laminates of this nature. Use of photolithography techniques allows us to create a wide range of phantom geometries. The target dimensions of the phantoms reported here are (i) a stack of 30 thin PDMS layers of 10 µm thickness (ii) curved 5 µm × 5 µm microchannels with 8.7 µm spacing, and (iii) straight 5 µm × 5 µm microchannels with 3.6 µm spacing. SEM scans of the assembled phantoms show open microchannels and a monolithic cross-section with no visible interface between PDMS layers. Based on the results of diffusion tensor magnetic resonance imaging (DT-MRI) scan, the anisotropic diffusion of water molecules due to the physical restriction of the microchannels was detected, which means that the phantom can be used to calibrate and optimize MRI instrumentation.

Advances in Microfluidics and Lab-on-a-Chip Technologies

Abstract Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high-resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities.

Instrumentation for xPCR Incorporating qPCR and HRMA

A microfluidic PCR device was developed that enables DNA amplification at speeds as fast as 2 s/cycle, with concurrent detection and amplification. Two targets were amplified from human genomic DNA. By observing the fluorescence emitted by a DNA dye while the sample is amplified, it is possible to obtain both qPCR and spatial melting information about the amplified product. The speed and integration of the device make it conducive to while-you-wait diagnostic tests that do not require post-PCR analysis.

Microfluidic—based sperm sorting & analysis for treatment of male infertility

Microfluidics technology has emerged as an enabling technology for different fields of medicine and life sciences. One such field is male infertility where microfluidic technologies are enabling optimization of sperm sample preparation and analysis. In this chapter we review how microfluidic technology has been used for sperm quantification, sperm quality analysis, and sperm manipulation and isolation with subsequent use of the purified sperm population for treatment of male infertility. As we discuss demonstrations of microfluidic sperm sorting/manipulation/analysis, we highlight systems that have demonstrated feasibility towards clinical adoption or have reached commercialization in the male infertility market. We then review microfluidic-based systems that facilitate non-invasive identification and sorting of viable sperm for in vitro fertilization. Finally, we explore commercialization challenges associated with microfluidic sperm sorting systems and provide suggestions and future directions to best overcome them.

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.

FDM 3D Printing of High-Pressure, Heat-Resistant, Transparent Microfluidic Devices

Transparent surfaces within microfluidic devices are essential for accurate quantification of chemical, biological, and mechanical interactions. Here, we report how to create low-cost, rapid 3D-printed microfluidic devices that are optically free from artifacts and have transparent surfaces suitable for visualizing a variety of fluid phenomenon. The methodology described here can be used for creating high-pressure microfluidic systems (significantly higher than PDMS-glass bonding). We develop methods for annealing Poly-Lactic Acid (PLA) microfluidic devices demonstrating heat resistance typically not achievable with other plastic materials. We show DNA melting and subsequent fluorescent imaging analysis, opening the door to other high-temperature applications. The FDM techniques demonstrated here allow for fabrication of microfluidic devices for precise visualization of interfacial dynamics, whether mixing between two laminar streams or droplet tracking. In addition to these characterizations, we include a printer troubleshooting guide and printing recipes for device fabrication to facilitate FDM printing for microfluidic device development.