(T1330-03-15) Viable Eukaryotic Cell Dry Powders Prepared by Thin Film Freeze-Drying

Purpose: Eukaryotic cells are commonly used in biomedical research. Cell-based therapies are also used in clinics (e.g., CAR-T cells for tumor therapy). Cells are stored and shipped as a frozen liquid suspension, formulated in dimethyl sulfoxide (DMSO) as an intracellular cryoprotectant, in ultra-low temperatures (~ -190°C). However, DMSO may cause severe adverse effects and even fatalities when cells are infused to cancer patients. Moreover, cells require ultra-cold chain for storage and distribution using a liquid nitrogen container, which is bulky, and exposure to its vapor is hazardous and requires special precautions. This research aimed to test the feasibility of using thin-film freeze-drying (TFFD) to produce DMSO-free dry powders of eukaryotic cells that can be stored at refrigerated temperatures while maintaining cell viability.

Methods: Various cell lines (J774A.1 mouse macrophages, THP-1 human monocytes, B16-F10 mouse melanoma cells, M-Wnt mouse mammary tumor cells) were used to test the feasibility. Trehalose (4-30%), in combination with various antioxidants and polymers, was used as lyoprotectants. Cells in suspension were dropped onto a cryogenically cooled surface to form frozen thin films that were then lyophilized to remove water. The viability of the cells was determined using trypan blue and MTT assays.

Results: It was found that trehalose alone at concentrations ranging from 7.5 to 15% incubated with cells for 4-6 h prior to TFFD was able to maintain 40-50% cell viability. Combining trehalose with epigallocatechin gallate and polyvinyl pyrrolidone (PVP-40) in proper concentrations improved the cell viability to above 90%. The viability of the cells was preserved after the cell powders were stored in refrigerated temperatures for 5 weeks. Finally, using MTT assay, it was shown that B16-F10 cells reconstituted from a TFFD powder can proliferate after being incubated in cell culture medium.

Conclusion: It is concluded that TFFD has the potential to be applied to convert eukaryotic cells to dry powders, in the absence of DMSO, for storage without the need for ultra-cold temperatures.

References: 1. Zhang M, Oldenhof H, Sydykov B, Bigalk J, Sieme H, Wolkers WF. Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity. Sci Rep. 2017; 7 (1): 6198.
2. Wolkers WF, Tablin F, Crowe JH. From anhydrobiosis to freeze-drying of eukaryotic cells. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 2002 Mar 1;131(3):535-43.
3. Rockinger U, Funk M, Winter G. Current approaches of preservation of cells during (freeze-) drying. Journal of Pharmaceutical Sciences. 2021 Aug 1;110(8):2873-93.

Acknowledgements: ZC and ROW report financial support by TFF Pharmaceuticals, Inc. ZC reports a relationship with TFF Pharmaceuticals, Inc. that includes equity or stocks and funding. ROW reports a relationship with TFF Pharmaceuticals, Inc. that includes consulting or advisory, equity or stocks, and funding.