Single Cell Handling Research Articles

Acoustic prison for single live cell 3D multi-imaging enabled by light-sheet microscopy

Having a system capable of obtaining high resolution, quantitative, subcellular information of individual live cells through imaging, while minimizing cell damage, is of great importance in many fields of research. In recent years, optofluidic light-sheet fluorescent microscopy (LSFM) has emerged as a powerful tool providing a low photo-toxic imaging method utilising the fluidic environment offered by microfluidics. Here, the benefits of LSFM were integrated with an acoustic single cell prison for precise single cell handling and 3D multi-imaging in a semi-automated manner. The compact, monolithic, acousto-optofluidic platform employed standing surface acoustic waves (SSAWs) to sequentially trap an individual cell on either side of an imaging region, which gathered planar, cross-sectional images of the cell. A cytoplasmic stain was utilised to not only visualize the cell trajectory throughout the imaging process, but also to verify the cell viability post-acoustic exposure. A mitochondrial stain was also used to better demonstrate the resolution capabilities of the device. Through post-image processing, 3D volumetric images of the cell were reconstructed and the results between the first and second rounds of imaging were directly comparable. Furthermore, the acoustic prison advantageously positions the cells in the upper region of the channel, enabling the ability to accurately compare temporal changes in cell morphology; a capability that can lead to advancements in therapeutics and drug delivery to access responses of cells to stimuli over time.

High-precision micro-wells in polymer for single cell cloning and monitoring

Abstract STRATEC Consumables is focusing on collaborations in development of smart consumables that enable precise clinical diagnostics, single cell handling and detection of rare proteins out of complex samples. Creation of micro-and nano-features in polymers that allow high-volume replication with great accuracy to ensure reliable results in clincis and laboratories. Micro-wells are supportive to novel approaches in single cell handling, cloning, sequencing and proteomics. STRATEC’s partnership with ALS GmbH from Jena, led to the development and production of nanowell plates for automated screening and recovery of single cells, under the name “CellCelector™”. This unique system combines high-resolution fast fluorescence imaging, sensitive cell detection technology as well as patented robotics for fast recovery of pure and viable cells and is compatible with various upstream cell enrichment approaches. In order to realize the nanowell plates STRATEC Consumables made use of mastering of high aspect ratio features up to 1:10, followed by injection molding with a high pattern fidelity, assembly to a standard micro-titer plate and surface functionalization to enable the application of ALS. Together, this project shows outstanding results though tight collaboration combining capabilities to innovate in the vibrant field of single cell understanding and applications.

Abstract 133: Micro-environment on demand (MOD): A versatile technology for cognate T-cell receptor - neoantigen discovery

Abstract NCI estimates that cancer will be the leading cause of death in 2030, worldwide. Checkpoint inhibitors and adoptive cell therapies (ACTs) cost up to ~$2 million/patient and have shown durable responses in a few patients while many patients experience life-threatening side-effects. So, there is a critical need to identify cost-effective therapies to treat cancer with minimal to no side-effects. Deep mapping of cancer antigens and their cognate immune cells is one approach that has the potential for a more universal/targeted treatment. However, systematic methods do not exist to identify cancer antigens and their cognate immune cells functionally and link phenotypic readouts to antigen and immune cell DNA sequences. Here we present a single cell handling and analysis platform based on Printed Droplet Microfluidics, a new droplet microfluidic technology that enables the precise construction of cell-cell interaction experiments with single cell resolution, followed by functional and genomic analysis. At the core of the technology is a fluorescence-activated flow sorter repurposed to deterministically sort and merge droplets containing single cells, beads, reagents, and drugs, collect and assay merged droplets and resort assay-positive droplets for downstream genomic analyses. The selectivity of the sorting/merging technology enables both the construction of experiments with up to one million precise cell combinations and the performance of flexible multi-step molecular biology workflows. We will show proof-of-concept for the MOD platform that integrates three technologies after fluorescently labeled single-cells are encapsulated in droplets - droplet-based cell-sorting and merging, single-cell-based cytokine assays and selective sequencing of T-cell receptor variable regions based on cytokine signals. The technology has the potential to rapidly accelerate the rate of immunotherapy discovery by enabling a larger number of experiments to be performed with higher precision, more modes of analysis, and less starting material than competing bulk technologies. Citation Format: Maithreyan Srinivasan, Justin Madrigal, Nathan Schoepp, Russell Cole. Micro-environment on demand (MOD): A versatile technology for cognate T-cell receptor - neoantigen discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 133.

UHF-Dielectrophoresis Crossover Frequency as a New Marker for Discrimination of Glioblastoma Undifferentiated Cells

This paper introduces the first results of dielectric spectroscopy characterization of glioblastoma cells, measuring their crossover frequencies in the ultra-high-frequency range (above 50 MHz) by dielectrophoresis (DEP) techniques. Experiments were performed on two glioblastoma lines U87-MG and LN18 that were cultured following different conditions, in order to achieve different phenotypic profiles. We demonstrate here that the presented DEP electrokinetic method can be used to discriminate the undifferentiated from the differentiated cells. In this study, microfluidic lab-on-chip systems implemented on bipolar-complementary oxide semiconductor technology are used allowing single cell handling and analysis. Based on the characterizations of their own intracellular features, both the selected glioblastoma (GBM) cell lines cultured in distinct culture conditions have shown clear differences of DEP crossover frequency signatures compared to the differentiated cells cultured in a normal medium. These results support the concept and validate the efficiency for cell characterization in glioblastoma pathology.

All-optical trapping, relocation, and manipulation of nanoparticles using SOI ring resonators

Optical tweezers have opened new doors in microscale research such as single-cell handling and analysis. To develop this technique to nanoscale applications, a number of near-field techniques have been introduced, but they suffer from issues and limitations. Here, we propose simple schemes built by SOI-based optical ring resonators that have efficient trapping and precise transporting functionalities, which are applicable for optical manipulation of nanoparticles. The distribution of the optical intensity excited in a ring resonator and its resultant optical forces on the nanoparticles are calculated numerically. According to the calculations, a subwavelength polystyrene particle as small as 12 nm in radius can be trapped with a depth of 10  kBT by an input power of 10 mW and conveyed to a specified location along the ring with nanometer precision. In addition, we show that the introduced schemes are capable of analyzing physical properties of biostructures such as the binding force of a DNA molecule.

Combining dielectrophoresis and computer vision for precise and fully automated single-cell handling and analysis.

With the advent of single-cell technologies comes the necessity for efficient protocols to process single cells. We combine dielectrophoresis with open source computer vision programming to automatically control the trajectories of single cells inside a microfluidic device. Using real-time image analysis, individual cells are automatically selected, isolated and spatially arranged.

Modular microfluidics for double emulsion formation.

For many engineering applications such as manipulating two phase flows, generating single and double emulsions, and passively propelling liquids through channels, control over the surface energy of microfluidic channels is essential. In particular, double emulsion formation, which benefits from alternating hydrophobic and hydrophilic sections of channel, represents a challenge in fabricating controlled microfluidic channel surface properties. As double emulsions find further applications in single-cell handling and analysis, straightforward methods for generating them increase in value. Here, we present a method for generating double emulsions in microfluidic channels fabricated from modular fluidic blocks. By using a vapor-phase polymer coating technology-initiated chemical vapor deposition-we are able to fabricate blocks with varying surface properties. Assembling these blocks together then creates step-like changes in surface energy within a microchannel.

Microfluidic actuators based on temperature-responsive hydrogels

The concept of using stimuli-responsive hydrogels to actuate fluids in microfluidic devices is particularly attractive, but limitations, in terms of spatial resolution, speed, reliability and integration, have hindered its development during the past two decades. By patterning and grafting poly(N-isopropylacrylamide) PNIPAM hydrogel films on plane substrates with a 2 μm horizontal resolution and closing the system afterward, we have succeeded in unblocking bottlenecks that thermo-sensitive hydrogel technology has been challenged with until now. In this paper, we demonstrate, for the first time with this technology, devices with up to 7800 actuated micro-cages that sequester and release solutes, along with valves actuated individually with closing and opening switching times of 0.6±0.1 and 0.25±0.15 s, respectively. Two applications of this technology are illustrated in the domain of single cell handling and the nuclear acid amplification test (NAAT) for the Human Synaptojanin 1 gene, which is suspected to be involved in several neurodegenerative diseases such as Parkinson’s disease. The performance of the temperature-responsive hydrogels we demonstrate here suggests that in association with their moderate costs, hydrogels may represent an alternative to the actuation or handling techniques currently used in microfluidics, that are, pressure actuated polydimethylsiloxane (PDMS) valves and droplets.

Automated single cell isolation from suspension with computer vision

Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.

Technologies for Single-Cell Isolation.

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Microfluidic construction of minimalistic neuronal co-cultures

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency Epatt was >75% during lengthy in chip culture, with ∼85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

1A2-O07 画像処理による1滴1細胞プリンティングの自動化に関する研究(ナノ・マイクロ流体システム)

Automation of piezoelectric inkjet-based single cell printing is the target of this study. Piezoelectric inkjet technology capability of high speed printing is the main advantage of this technology over other technologies. Therefore, this technology has gained attention in recent field of tissue engineering. Additionally, this technology was successfully developed for single cell handling. However, automation system for the single cell handling was not yet developed. In this study, utilizing open source image processing library, OpenCV, to process image of the inkjet head, automatic cell detection system was successfully developed. Finally, combining cell detection system and cell printing system, automation of single cell printing system with 98% successful ratio was successfully developed.

Towards Single-Cell Analysis For Pharmacokinetics

Traditional 'macroscopic' pharmacokinetics (PK) investigates the fate of drugs or toxicants administered externally to living organisms, described by the extent and rate of absorption, distribution, metabolism and excretion. However, how a single cell affects a specific pharmaceutical after administration still remains a largely untouched area, primarily due to the technical restrictions imposed by minute amounts of chemicals involved. With the fast development of high-temporal and spatial-resolution detection techniques and single-cell handling techniques, it becomes possible to pursue single-cell PK. This review summarizes useful methodological and experimental techniques to investigate PK at the level of the single cell, including the microfluidics-based single-cell manipulation and the MS and electrochemical methods for single-cell analysis.

High-throughput single-cell manipulation system for a large number of target cells

A sequential and high-throughput single-cell manipulation system for a large volume of cells was developed and the successive manipulation for single cell involving single-cell isolation, individual labeling, and individual rupture was realized in a microhydrodynamic flow channel fabricated by using two-dimensional simple flow channels. This microfluidic system consisted of the successive single-cell handlings of single-cell isolation from a large number of cells in cell suspension, labeling each isolated single cell and the lysate extraction from each labeled single cell. This microfluidic system was composed of main channels, cell-trapping pockets, drain channels, and single-cell content collection channels which were fabricated by polydimethylsiloxane. We demonstrated two kinds of prototypes for sequential single-cell manipulations, one was equipped with 16 single-cell isolation pockets in microchannel and the other was constructed of 512 single-cell isolation pockets. In this study, we demonstrated high-throughput and high-volume single-cell isolation with 512 pocket type device. The total number of isolated single cells in each isolation pocket from the cell suspension at a time was 426 for the cell line of African green monkey kidney, COS-1, and 360 for the rat primary brown preadipocytes, BAT. All isolated cells were stained with fluorescence dye injected into the same microchannel successfully. In addition, the extraction and collection of the cell contents was demonstrated using isolated stained COS-1 cells. The cell contents extracted from each captured cell were individually collected within each collection channel by local hydrodynamic flow. The sequential trapping, labeling, and content extraction with 512 pocket type devices realized high-throughput single-cell manipulations for innovative single-cell handling, feasible staining, and accurate cell rupture.

Single Cell Deposition and Patterning with a Robotic System

Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.

Nanomanipulation Using Silicon Photonic Crystal Resonators

Optical tweezers have enabled a number of microscale processes such as single cell handling, flow-cytometry, directed assembly, and optical chromatography. To extend this functionality to the nanoscale, a number of near-field approaches have been developed that yield much higher optical forces by confining light to subwavelength volumes. At present, these techniques are limited in both the complexity and precision with which handling can be performed. Here, we present a new class of nanoscale optical trap exploiting optical resonance in one-dimensional silicon photonic crystals. The trapping of 48 nm and 62 nm dielectric nanoparticles is demonstrated along with the ability to transport, trap, and manipulate larger nanoparticles by simultaneously exploiting the propagating nature of the light in a coupling waveguide and its stationary nature within the resonator. Field amplification within the resonator is shown to produce a trap several orders of magnitude stronger than conventional tweezers and an order of magnitude stiffer than other near-field techniques. Our approach lays the groundwork for a new class of optical trapping platforms that could eventually enable complex all-optical single molecule manipulation and directed assembly of nanoscale material.

Lab‐on‐substrate technology platform

AbstractA technology platform based on standard PCB technology has been developed for cost‐effective fabrication of biological and medical test devices. Aluminum, polyimide and Pyralux have been selected as materials with focus on bio‐ and process compatibility. A process flow consisting of lamination, Al structuring by wet etching, microwell and via formation by laser drilling and via metallization was developed. Surface treatments have been evaluated for hydrophobic and hydrophilic modification of the materials. The technology was demonstrated with a dielectrophoresis enhanced microwell device for single cell handling and detection of cell‐to‐cell interaction as needed for the improvement of tumor therapy. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Microtechnologies and nanotechnologies for single-cell analysis

Many efforts are currently underway to try and mimic the properties of single cells with the aim of designing chips that are as efficient as cells. However, cells are nature's nanotechnology engineering at the scale of atoms and molecules, and it might be better to envision a microchip that utilizes a single cell as an experimentation platform. A novel, so-called laboratory-in-a-cell concept has been described, where advantage is taken of micro- and nanotechnological tools to enable precise control of the biochemical cellular environment; these tools also offer the possibility to analyse the composition of single cells. Methods for single-cell handling and analysis are being developed and will be required for this concept to progress further.