Acoustic Droplet Vitrification Method for High-Efficiency Preservation of Rare Cells

Abstract

Cryopreservation is a key step for current translational medicine including reproductive medicine, regenerative medicine, and cell therapy. However, it is challenging to preserve rare cells for practical applications due to the difficulty in handling low numbers of cells as well as the lack of highly efficient and biocompatible preservation protocols. Here, we developed an acoustic droplet vitrification method for high-efficiency handling and preservation of rare cells. By employing an acoustic droplet ejection device, we can encapsulate rare cells into water-in-air droplets with a volume from ∼pL to ∼nL and deposit these cell-containing droplets into a droplet array onto a substrate. By incorporating a cooling system into the droplet array substrate, we can vitrify hundreds to thousands of rare cells at an ultrafast speed (about ∼2 s) based on the high surface to volume ratio of the droplets. By optimizing this method with three different cell lines (a human lung cancer cell line, A549 cells, a human liver cell line, L02 cells, and a mouse embryonic fibroblast cell line, 3T3-L1 cells), we developed an effective protocol with excellent cell viability (e.g., >85% for days, >70% for months), proliferation, and adhesion. As a proof-of-concept application, we demonstrated that our method can rapidly handle and efficiently preserve rare cells, highlighting its broad applications in species diversity, basic research, and clinical medicine.

Droplet Generation, Vitrification, and Warming for Cell Cryopreservation: A Review

“…However, this drawback does not have an impact on cryopreservation because cell solutions containing cryoprotectants are not highly viscous. This method has been applied to the cryopreservation process by Xia et al…”

“…(d) Droplets are generated by acoustic printing and dropped onto the aluminum foil. Reproduced with permission from ref . Copyright 2021 American Chemical Society.…”

“…However, this drawback does not have an impact on cryopreservation because cell solutions containing cryoprotectants are not highly viscous. This method has been applied to the cryopreservation process by Xia et al 51 In the field of cryopreservation, there are other methods of droplet generation such as pipet (Figure 2d), 52 syringe pumps (Figure 2e), 53 microfluidics, 54 and nitrogen stream spray methods (Figure 2f). 55 These methods have some limitations compared to the mainstream methods.…”

In Situ Microreaction Platform Based on Acoustic Droplet Manipulation for Ultra-High-Precision Multiplex Bioassay

“…Acoustic-droplet-based microfluidic technology has been widely used in the field of life sciences due to its high biocompatibility, label-free droplet manipulation, and ease of integration. − Surface acoustic waves could be used to manipulate ultrasmall samples (∼nL) by adjusting the output frequency. In addition, the force of the acoustic waves causes minimal damage to samples (cells, proteins, and molecules), just like those widely used in fetal medical ultrasound imaging.…”

Acoustic Droplet Printing Tumor Organoids for Modeling Bladder Tumor Immune Microenvironment within a Week

“…We also proved that the organoids prepared by this platform still retain the immune microenvironment similar to that of the parent tissue within two weeks. [44][45][46] Meanwhile, research have shown that intertumoral tumorinfiltrating lymphocytes (TILs) can directly or indirectly have a large impact on multiple antitumor immune responses and the progression of various solid tumors. [47,48] Adoptive immunotherapy with autologous tumor-infiltrating lymphocytes TIL is an attractive approach to the treatment for melanoma and renal cancer, but requires the isolation and proliferation of T cells with antitumor activity.…”