Microfluidic transfection of mRNA into human primary lymphocytes and hematopoietic stem and progenitor cells using ultra-fast physical deformations.

Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.

A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss.

Spheroid cultures have been often used to simulate and understand in situ biological occurrences with potential to be further applied to therapeutic approaches, such as cell transplantation. However, traditional lab-scale techniques hardly reached the needed large scale production of cell spheroids, thus limiting their versatility in many biomedical fields. Microscale technologies have rapidly improved in the last decade, and contributed to the large scale production of cell spheroids with high controllability and reproducibility. Nonetheless, the existing microwell culture platforms are problematic due to unwanted cellular adhesion to the substrate as well as due to substantial amounts of cell loss. In this study, we have developed a novel configuration of cylindrical type polyethylene glycol (PEG) hydrogel microwells featuring inverted-pyramidal openings (iPO). Highly refined microstructures of our novel microwell could be fabricated by our optimized micro-electro mechanical system protocols consisting of a silicon (Si) wet/dry etching, Si-to-polydimethylsiloxane substrate bonding, and the established soft-lithography techniques. The iPO, the PEG hydrogel, and the cylindrical geometry of our microwell successfully (1) avoided inefficient washing steps after cell seeding, (2) achieved the complete resistance to cellular adhesion on the microwell substrate, and (3) made all seeded cells readily gathered and jam-packed to form cell spheroids with uniform size, respectively. The maximal sizes of cell spheroids were confined to below 200 µm according to the size of microwells used in this study. The efficiency testing for cell spheroid formation was conducted in comparison with other types of microwells that have been often used in the fields. The results showed that our novel microwell platform effectively reached almost zero percent of cell loss while mass-producing human mesenchymal stem cell spheroids with highly precise control over spheroid's size and cell number. We believe that this study could deliver an effective method to extend the practical usability of cell spheroids in a variety of biomedical applications.