# Bottom‐Up Ice Growth Geometry Attenuates Shear Stress and Improves the Cryopreservation of Hematopoietic Stem/Progenitor Cells Under Low DMSO Concentrations

## TL;DR

A new freezing method called bottom-up freezing improves cell survival during cryopreservation by reducing stress and allowing lower use of a chemical called DMSO.

## Contribution

Bottom-up freezing geometry enables high cell viability with low DMSO concentrations by controlling ice growth and shear stress.

## Key findings

- Bottom-up freezing achieved high cell viability with 2.5% DMSO, matching conventional methods with 10% DMSO.
- CFD simulations showed conventional freezing creates higher shear stress at low DMSO concentrations.
- Bottom-up freezing preserved the clonogenic potential of hematopoietic stem/progenitor cells as effectively as conventional methods.

## Abstract

Studies on cell cryopreservation have been limited by the complexity of the freezing process and challenges on controlling ice formation, managing cooling rates, and optimizing cryoprotectant concentrations. The objective of this study is to evaluate the impact of bottom‐up and conventional radial freezing on the viability of mammalian cells, using mouse hybridoma cells and human umbilical cord blood (hUCB) derived mononuclear cells (MNCs) as models. UCB‐derived MNCs were selected for this study because these contain hematopoietic stem and progenitor cells (HSPCs), which hold significant clinical relevance. The study combines experimental assays, including cell viability assays and flow cytometry characterization, with Computational Fluid Dynamic (CFD) simulations. A bottom‐up freezing geometry sustained high cell viability, even at dimethyl sulfoxide (DMSO) concentrations below 5% (v/v), whereas conventional radial freezing led to lower cell viability when DMSO is used below such concentrations threshold. This observation is particularly relevant for cell‐based therapies. CFD simulations for conventional radial freezing elucidated that for such method the ice formed at the top of the vial is of high porosity for media with 10% (v/v) DMSO, but of low porosity for lower DMSO concentrations. The simulations show that the latter conditions can result in an increase in shear stress on cells, by up to an order of magnitude. Overall, this study provides a rational for 10% (v/v) DMSO being the optimal reported concentration for conventional freezing methods, as a result of poor control of ice growth direction and higher mechanical stresses at lower DMSO concentrations. Experimental results show that bottom‐up freezing method, using only 2.5% (v/v) DMSO, allow to reach cell viabilities as high as the ones obtained with conventional radial freezing protocols at 10% (v/v) DMSO. In addition, bottom‐up freezing method with 2.5% DMSO preserves the clonogenic potential of HSPCs within hUCB‐derived MNCs comparably to conventional radial freezing protocol with 10% DMSO. Importantly, the results support the recommendation to use cell cryopreservation strategies, such as bottom‐up freezing, that enable the use of lower DMSO concentrations by controlling the direction of heat transfer.