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

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.

A Structure Shaped by Fire, but Also Water: Ecological Consequences of the Variability in Bark Properties Across 31 Species From the Brazilian Cerrado.

Bark is a structure involved in multiple physiological functions, but which has been traditionally associated with protection against fire. Thus, little is known about how the morpho-anatomical variations of this structure are related to different ecological pressures, especially in tropical savanna species, which are commonly subjected to frequent fire and drought events. Here we evaluated how the structural and functional variations of bark are related to the processes of resilience and resistance to fire, as well as transport and storage of water in 31 native species from the Brazilian Cerrado. Because of their thick bark, none of the trees analyzed were top-killed after a severe fire event. The structural and functional variations of the bark were also associated with water storage and transport, functions related to properties of the inner bark. In fact, species with a thicker and less dense inner bark were the ones that had the highest water contents in the wood, bark, and leaves. Lower bark density was also related to higher stem hydraulic conductivity, carbon assimilation, and growth. Overall, we provide strong evidence that in addition to protection from fire, the relative investment in bark also reflects different strategies of water use and conservation among many Cerrado tree species.