Polyvinylpyrrolidone (PVP) Mitigates the Damaging Effects of Intracellular Ice Formation in Adult Stem Cells

Abstract

The objective of this work was to assess the effect of 10% (w/v) polyvinylpyrrolidone (PVP) on the pattern of intracellular ice formation (IIF) in human adipose tissue derived adult stem cells (ASCs) in the absence of serum and other cryoprotective agents (CPAs). The freezing experiments were carried out using a fluorescence microscope equipped with a Linkam cooling stage using two cooling protocols. Both the cooling protocols had a common cooling ramp: cells were cooled from 20 degrees C to -8 degrees C at 20 degrees C/min and then further cooled to -13 degrees C at 1 degrees C/min. At this point we employed either cooling protocol 1: the cells were cooled from -13 degrees C to -40 degrees C at a pre-determined cooling rate of 1, 5, 10, 20, or 40 degrees C/min and then thawed back to 20 degrees C at 20 degrees C/min; or cooling protocol 2: the cells were re-warmed from -13 degrees C to -5 degrees C at 20 degrees C/min and then re-cooled at a pre-determined rate of 1, 5, 10, 20, or 40 degrees C/min to -40 degrees C. Almost all (>95%) of the ASCs frozen in 1x PBS and protocol 1 exhibited IIF. However, almost none (<5%) of the ASCs frozen in 1x PBS and protocol 2 exhibited IIF. Similarly, almost all (>95%) of the ASCs frozen in 10% PVP in PBS and protocol 1 exhibited IIF. However, ~0, ~40, ~47, ~67, and ~100% of the ASCs exhibited IIF when frozen in 10% PVP in PBS and utilizing protocol 2 at a cooling rate of 1, 5, 10, 20, or 40 degrees C/min, respectively.

Adult multipotent stromal cell cryopreservation: Pluses and pitfalls

“…Cryopreservation of adult MSCs is central to their development, availability, and use. The practice is relatively new in veterinary medicine, and its use will continue to grow . It is clear that fresh and frozen MSCs are not identical, although differences are not fully established.…”

“…Human ASC post‐thaw cell viability with freezing medium containing MC in Dulbecco's modified Eagle's medium (DMEM) is greater than DMEM alone, but lower than that containing 80% serum, 10% DMSO, and 10% DMEM . Another popular CP is polyvinylpyrrolidone (PVP), a nontoxic, high molecular weight polymer . The concentration of dissolved PVP increases in extracellular fluid as ice forms at −10°C to −20°C to create an osmotic gradient that draws fluid out of cells .…”

“…The concentration of dissolved PVP increases in extracellular fluid as ice forms at −10°C to −20°C to create an osmotic gradient that draws fluid out of cells . Despite lower intracellular fluid, intracellular ice formation may occur at low PVP levels, and high concentrations of 20%‐40% can cause excessive cellular dehydration, cell necrosis, and membrane damage . Human ASC viability and plasticity appears to be maintained with 10% PVP .…”

A numerical study on distributions during cryoprotectant loading caused by laminar flow in a microchannel

“…3,7 Typical cryopreservation protocols aim to remove intracellular water to avoid damaging intracellular ice formation (IIF). 8 This is usually accomplished by exposing the sample to a cryoprotective agent (CPA) to create an osmotic pressure gradient. These chemicals can be either permeable CPAs, which penetrate the cellular membrane and replace intracellular water, or impermeable CPAs, which dehydrate the cell by drawing out intracellular water.…”

“…3,9 Complicating the process further, the CPAs themselves can be toxic to cells. 8,10,11 Two potential methods of cryopreservation are generally employed for cryopreservation: freezing and vitrification. The former uses lower CPA concentrations and slower cooling rates, which minimize osmotic shock and cytotoxicity effects, while being more susceptible to IIF.…”