(440d) Conformal Single Cell Hydrogel Coating with Electrically Induced Tip Streaming of AC Cone

Single-cell encapsulation in a micrometre size microgel coating is a new bio-protective strategy providing precise control of stem cell niches and minimum molecular transport limitations. These encapsulated cells act as ‘microbots’ are highly instrumental in several bio-applications such as stem cell-based therapy, 3D cell culture pharmacokinetic study and tissue engineering. Furthermore, conformally microgel coated ‘microbots’ not only allow rapid diffusion of oxygen, nutrients, and cellular waste but also block immunoglobulins to mitigate innate immune response. However, despite recent advances in microfluidic technologies to allow high throughput encapsulation of single-cell, existing methods either rely on a special crosslinking agent that is pre-coated on the cell surface and subject to the variation of the cell membrane, or on expensive fluorescence-activated single-cell sorting (FACS) methods which limit their widespread use. Moreover, regular PDMS based microfluidic channels often cannot bear the high pumping pressure required to produce droplets similar to the size of the cell using the viscous hydrogel solution. Herein, we report a novel and universal method of utilizing the electrically induced tip streaming model of AC cone to enable high-throughput encapsulation of a single cell with high efficiency and without the assistance of a high shear flow.

With appropriate back pressure and a DC (direct current) electric potential applied across a liquid interface, the interface becomes charged and form a sharp conic structure with a 49-degree half-angle (Taylor cone). Despite earlier attempts to exploit the sharp conic structure to encapsulate cells, multiple cells are encapsulated in a single droplet, which is often attributed to recirculation within the Taylor cone. However, an AC field above a critical frequency of 100 kHz, related to the inverse Debye layer RC time, can produce a much sharper cone (11 degrees vs 49 degrees) due to a different polarization mechanism. Importantly, the weak charge of an AC cone cannot sustain a recirculation flow and ejects nearly electroneutral droplets that do not undergo Rayleigh fission that is detrimental to the cells. A single cell will jam the slender cone jet and induce pinch-off prior to the arrival of the next cell. This unique mechanism then allows sequential encapsulation of single cells at a throughput of 300Hz. The 300kHz operating frequency also avoids field penetration into the cell at higher (megahertz) frequencies, which has a detrimental effect on cell viability.

We demonstrate the universality of the method by using both natural (alginate and collagen) and synthetic (hyaluronic acid-functionalized with norbornene groups, NorHA) hydrogels as encapsulation material. These hydrogels have different crosslinking chemistry, for example, alginate can be crosslinked using divalent cations(e.g., Ca2+ or Mg2+), collagen at an elevated temperature to 37°C and synthetic NorHA hydrogels can be crosslinked using light-mediated thiol-norbornene chemistry. Cell viability of more than 90% was observed for breast cancer cell line MDA-MB-231 with a microgel coating of ~3-5µm using the abovementioned hydrogels. Also, an order of magnitude difference between the empty and cell-containing droplets can be effectively exploited to separate them by mild centrifugation instead of expensive FACS, yielding more than 80% single-cell encapsulation efficiency. To further demonstrate the utility of this technology across different cell types, encapsulated human multipotent mesenchymal stromal cells(hMSCs) are used were cultured in either osteogenic or adipogenic differentiation medium. Encapsulated hMSCs maintain good cell viability over an extended culture period and exhibit robust differentiation potential into osteoblasts and adipocytes. Collectively, electrically induced tip streaming enables high-throughput encapsulation (~300Hz) of single-cell with high efficiency(>80%) and universality, which are applicable for various applications in cell therapy, pharmacokinetic study, and regenerative medicine.