Cryopreservation of Adherent Cells: Strategies to Improve Cell Viability and Function After Thawing

Abstract

The commonly applied cryopreservation protocols routinely used in laboratories worldwide were developed for simple cell suspensions, and their application to complex systems, such as cell monolayers, tissues, or biosynthetic constructs, is not straightforward. In particular for monolayer cultures, cell detachment and membrane damage are often observed after cryopreservation. In this work, combined strategies for the cryopreservation of cells attached to Matrigel-coated well plate's surfaces were investigated based on cell entrapment in clinicalgrade, ultra-high viscosity alginate using two cell lines, neuroblastoma N2a and colon adenocarcinoma Caco-2, with distinct structural and functional characteristics. As the cryopreservation medium, serum-free CryoStor solution was compared with serum-supplemented culture medium, both containing 10% DMSO. Using culture medium, entrapment beneath an alginate layer was needed to improve cell recovery by minimizing membrane damage and cell detachment after thawing; nevertheless, up to 50% cell death still occurred within 24 h after thawing. The use of CryoStor solution represented a considerable improvement of the cryopreservation process for both cell lines, allowing the maintenance of high postthaw membrane integrity as well as full recovery of metabolic activity and differentiation capacity within 24 h postthawing; in this case, entrapment beneath an alginate layer did not confer further protection to cryopreserved Caco-2 cells, but was crucial for maintenance of attachment and integrity of N2a neuronal networks.

Cryopreservation of adhered mammalian cells on a microfluidic device: Toward ready‐to‐use cell‐based experimental platforms

“…First, we evaluated survival of HeLa, NIH3T3, and MCF-7 cells after cryopreservation. In contrast, the low survival rates on the control culture dishes are consistent with previous papers (Malpique et al, 2009;McKay et al, 2002;Miyamoto et al, and the ready-to-use (lower) cell-based experimental platforms. In the same sampling area of the cell culture zone, live (PI negative and Hoechst positive) cells and dead (PI positive) cells were counted.…”

“…One conclusion of these studies is that the adhered cells are more susceptible to cryoinjury than suspended cells. To cut this lengthy incubation process, considerable effort has been made to cryopreserve cells in the adhered state (Malpique et al, 2009;McKay et al, 2002;Miyamoto et al, 2009;Shoji et al, 2007). In a conventional protocol, the cryopreserved cells are thawed, washed, and seeded onto a dish surface.…”

“…Therefore, the cells are usually cryopreserved in the suspended state, although most of mammalian cells exhibit their intrinsic phenotypes only in the adhered state. These methods generally relied on coating of the cell-adhering surfaces (McKay et al, 2002;Miyamoto et al, 2009;Shoji et al, 2007) or entrapment of the cells under a hydrogel layer (Malpique et al, 2009). It takes at least overnight incubation for recovery of stable adhesion (Fig.…”

Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

“…In addition, we recently demonstrated that cell encapsulation in alginate is a valuable strategy for improving cell viability and the integrity of cell monolayers and neurospheres after freeze/thawing, since cells are protected against mechanical damages during ice crystallization and the risk of disrupting cell-cell and cell-matrix contacts are reduced through immobilization within the hydrogel [29], [30]. Despite such success in many (stem) cell types, studies describing the microencapsulation of hESCs are still limited [12], [25], [31].…”

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

“…Different transportation strategies have been investigated [30] and a significant amount of research has been carried out to understand the effects of cold storage and cryopreservation on the viability of cells [31][32][33]. The study carried out within the research reported here has investigated the effects of the combination of cold storage and mechanical stress during cold transport of an hMSC suspension without the presence of cryoprotectant.…”