Evelien W. M. Kemna

High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel.

In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.

High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

In this article, we present a microfluidic device capable of successive high‐yield single‐cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single‐cell encapsulation is realized using Dean‐coupled inertial ordering of cells in a Yin‐Yang‐shaped curved microchannel using a double T‐junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro‐coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell–cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research.

Label-free, high-throughput, electrical detection of cells in droplets

Today, droplet based microfluidics has become a standard platform for high-throughput single cell experimentation and analysis. However, until now no label-free, integrated single cell detection and discrimination method in droplets is available. We present here a microfluidic chip for fast (>100 Hz) and label-free electrical impedance based detection of cells in droplets. The microfluidic glass-PDMS device consists of two main components, the droplet generator and the impedance sensor. The planar electrode pair in the main channel allows the detection of only cells and cell containing droplets passing the electrodes using electrical impedance measurements. At a measurement frequency of 100 kHz non-viable cells, in low-conducting (LC) buffer, show an increase in impedance, due to the resistive effect of the membrane. The opposite effect, an impedance decrease, was observed when a viable cell passed the electrode pair, caused by the presence of the conducting cytoplasm. Moreover, we found that the presence of a viable cell in a droplet also decreased the measured electrical impedance. This impedance change was not visible when a droplet containing a non-viable cell or an empty droplet passed the electrode pair. A non-viable cell in a droplet and an empty droplet were equally classified. Hence, droplets containing (viable) cells can be discriminated from empty droplets. In conclusion, these results provide us with a valuable method to label-free detect and select viable cells in droplets. Furthermore, the proposed method provides the first step towards additional information regarding the encapsulated cells (e.g., size, number, morphology). Moreover, this all-electric approach allows for all-integrated Lab on a Chip (LOC) devices for cell applications using droplet-based platforms.

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation.

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B‐cells and mouse myeloma (NS‐1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS‐1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.

Enzymatic Crosslinking of Polymer Conjugates is Superior over Ionic or UV Crosslinking for the On-Chip Production of Cell-Laden Microgels

Cell-laden micrometer-sized hydrogels (microgels) hold great promise for improving high throughput ex-vivo drug screening and engineering biomimetic tissues. Microfluidics is a powerful tool to produce microgels. However, only a limited amount of biomaterials have been reported to be compatible with on-chip microgel formation. Moreover, these biomaterials are often associated with mechanical instability, cytotoxicity, and cellular senescence. To resolve this challenge, dextran-tyramine has been explored as a novel biomaterial for on-chip microgel formation. In particular, dextran-tyramine is compared with two commonly used biomaterials, namely, polyethylene-glycol diacrylate (PEGDA) and alginate, which crosslink through enzymatic reaction, UV polymerization, and ionic interaction, respectively. Human mesenchymal stem cells (hMSCs) encapsulated in dextran-tyramine microgels demonstrate significantly higher (95%) survival as compared to alginate (81%) and PEGDA (69%). Long-term cell cultures demonstrate that hMSCs in PEGDA microgels become senescent after 7 d. Alginate microgels dissolve within 7 d due to Ca2+ loss. In contrast, dextran-tyramine based microgels remain stable, sustain hMSCs metabolic activity, and permit for single-cell level analysis for at least 28 d of culture. In conclusion, enzymatically crosslinking dextran-tyramine conjugates represent a novel biomaterial class for the on-chip production of cell-laden microgels, which possesses unique advantages as compared to the commonly used UV and ionic crosslinking biomaterials.

Electrofusion of single cells in picoliter droplets

We present a microfluidic chip that enables electrofusion of cells in microdroplets, with exchange of nuclear components. It is shown, to our knowledge for the first time, electrofusion of two HL60 cells, inside a microdroplet. This is the crucial intermediate step for controlled hybridoma formation where a B cell is electrofused with a myeloma cell. We use a microfluidic device consisting of a microchannel structure in PDMS bonded to a glass substrate through which droplets with two differently stained HL60 cells are transported. An array of six recessed platinum electrode pairs is used for electrofusion. When applying six voltage pulses of 2–3 V, the membrane electrical field is about 1 MV/cm for 1 ms. This results in electrofusion of these cells with a fusion yield of around 5%. The operation with individual cell pairs, the appreciable efficiency and the potential to operate in high-throughput (up to 500 cells sec−1) makes the microdroplet fusion technology a promising platform for cell electrofusion, which has the potential to compete with the conventional methods. Besides, this platform is not restricted to cell fusion but is also applicable to various other cell-based assays such as single cell analysis and differentiation assays.