The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. II: Functional activity of cryopreserved cells

Abstract

BackgroundThe cryopreservation and thawing processes are known to induce many deleterious effects in cells and might be detrimental to several cell types. There is an inherent variability in cellular responses among cell types and within individual cells of a given population with regard to their ability to endure the freezing and thawing process. The aim of this study was to evaluate the fate of cryopreserved cells within an optical cryo apparatus, the individual-cell-based cryo-chip (i3C), by monitoring several basic cellular functional activities at the resolution of individual cells.ResultsIn the present study, U937 cells underwent the freezing and thawing cycle in the i3C device. Then a panel of vital tests was performed, including the number of dead cells (PI staining), apoptotic rate (Annexin V staining), mitochondrial membrane potential (TMRM staining), cytoplasm membrane integrity and intracellular metabolism (FDA staining), as well as post-thawing cell proliferation assays. Cells that underwent the freezing - thawing cycle in i3C devices exhibited the same functional activity as control cells. Moreover, the combination of the multi-parametric analysis at a single cell resolution and the optical and biological features of the device enable an accurate determination of the functional status of individual cells and subsequent retrieval and utilization of the most valuable cells.ConclusionsThe means and methodologies described here enable the freezing and thawing of spatially identifiable cells, as well as the efficient detection of viable, specific, highly biologically active cells for future applications.

The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine

“…A good correspondence of these two parameters is demonstrated in normal cells (Afrimzon et al, 2010).…”

IVF-on-a-Chip: Recent Advances in Microfluidics Technology for In Vitro Fertilization

“…This device enabled the comparison between prefreezing and postthawing data at single-cell resolution. In a subsequent study, Afrimzon et al 83 demonstrated that the functional activity of U937 cells that were freeze-thawed on the i3C devices had no statistically significant difference from the control cells that were cryopreserved via conventional cryo-vials. Most importantly, the combination of the multiparametric analysis at a single-cell resolution and the optical and biological features of the device enabled the accurate determination of the functional status of individual cells and subsequent retrieval and utilization of the most valuable cells, which are highly desirable for on-chip cryopreservation of oocytes or embryos.…”

Microfluidics for cryopreservation

“…Later, Deutsh et al and Afrimzon et al (Afrimzon et al, 2010; Deutsch et al, 2010) introduced an individual cell-based cryo-chip (i3C) with an array of picowells (Figure 8B), enabling individual cell cryopreservation, observation, and retrieval for the first time. Furthermore, i3C is compatible with commonly used, commercially available cryostage (MDBCS 600; Linkam Instruments, Tadworth, UK), which extends its application to include programmed freezing and thawing of cells on a chip.…”

“…(C) A microfabricated chip with an incubation microchamber, microfluidic channels, and microheaters for on-chip cell cryopreservation. Reproduced from (Afrimzon et al, 2010; Deutsch et al, 2010; Li et al, 2014; Li et al, 2010) with permission.…”

“…Due to numerous intrinsic features, such as ease of mass production and specific designs and changes, ease of component integration, disposability, low cost, and requirement of smaller reagents or analyte volumes, as well as ease of rapid implementation of a high-efficiency mixture, microfluidics techniques emerged in early 1980 have been introduced for cryopreservation approaches. These fields include controlled loading/unloading of CPAs into the cells with stepwise, linear and complex CPA profiles (Heo et al, 2011; Park et al, 2011; Song et al, 2009), programmable freeze-thawing cycles (Afrimzon et al, 2010; Deutsch et al, 2010; Li et al, 2010), and low-CPA vitrification by ultra-rapid freezing (Choi et al, 2015a; He et al, 2008; Zou et al, 2013) using microfluidics. Such innovation offers a tremendous potential to revolutionize cryopreservation.…”