Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells

Abstract

Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4–7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2–3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.

Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells

“…They grew exponentially (in exponential phase) while P2D11 cells had entered a deceleration phase, and P2D28 had almost no proliferation. Previous studies have shown that induced pluripotent stem cells (iPSC) were also found to be best cryopreserved at the exponential growth phase [37]. We therefore hypothesized that exponential phase was the optimal growth phase for RPE cryopreservation and then addressed this hypothesis by comparison of the recovery rate, viability rate and attachment rate for cells frozen at different phases of the growth.…”

Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics

“…They are of potential use as therapeutics in tissue regeneration (e.g., neural or ocular regeneration) and in disease modeling to study the pathology of diseases at a cellular level [ 139 ]. Procedures for cryopreservation of iPSCs usually fall into two categories: slow cooling or vitrification, and further it is of importance to distinguish between freezing of single cells or cell aggregates and the exact mode of freezing and thawing [ 140 , 141 ].…”

Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality Controls for Commercial Applications

“…In cryopreservation facilities, it has been reported that the microbial load should be controlled before freezing sperm in order to prevent cross-contamination in liquid nitrogen [172]. Freezing sperm from brood stocks that are not infected or have the least pathogen in their sperm will be the best solution.…”