Winter is coming: the future of cryopreservation.

The preservative effects of low temperature on biological materials have been long recognised, and cryopreservation is now widely used in biomedicine, including in organ transplantation, regenerative medicine and drug discovery. The lack of organs for transplantation constitutes a major medical challenge, stemming largely from the inability to preserve donated organs until a suitable recipient is found. Here, we review the latest cryopreservation methods and applications. We describe the main challenges-scaling up to large volumes and complex tissues, preventing ice formation and mitigating cryoprotectant toxicity-discuss advantages and disadvantages of current methods and outline prospects for the future of the field.

Postthaw characterization of umbilical cord blood: markers of storage lesion.

BACKGROUND: The continued growth in the uses of umbilical cord blood (UCB) will require the development of meaningful postthaw quality assays. This study examines both conventional and new measures for assessing UCB quality after long-term storage. STUDY DESIGN AND METHODS: The first arm of the study involved thawing UCB in storage for short (approx. 1 year) and long periods of time (>11 years). Conventional postthaw measures (colony-forming units [CFU], total nucleated cell counts, CD34+45+) were quantified in addition to apoptosis. The second arm of the study involved taking units stored in liquid nitrogen and imposing a storage lesion by storing the units in -80°C for various periods of time. After storage lesion, the units were thawed and assessed. RESULTS: In the first arm of the study, there was little difference in the postthaw measures between UCB stored for short and long periods of time. There was a slight increase in the percentage of CD34+45+ cells with time in storage and a reduction in the number of cells expressing apoptosis markers. When moved from liquid nitrogen to -80°C storage, the nucleated cell count varied little but there was a distinct decrease in frequency of CFUs and increase in percentage of cells expressing both early and late markers of apoptosis. CONCLUSION: Nucleated cell counts do not reflect damage to hematopoietic progenitors during long-term storage. Expression of caspases and other markers of apoptosis provide an early biomarker of damage during storage, which is consistent with other measures such as CFU and percentage of CD34+45+ cells.

Video analysis of osmotic cell response during cryopreservation.

Cellular response during the freeze-thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1 °C/min) to -60 °C and fast rewarming to 4 °C (100 °C/min). The ice nucleation temperature was controlled (T(n)=-8 °C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2 frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R(2)=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

Cryopreservation and quality control of mouse embryonic feeder cells.

Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed "induced pluripotent" cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are commonly used for ESC long term culture procedures and colony regeneration. These MEFs originate from different mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consistency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells. According to our findings, MEFs should be inactivated by irradiation (30Gy) and cryopreserved with optimal 10% DMSO at 1K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryopreserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture without detaching from the culture dish or disruption of the cell layer.

Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me(2)SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients. Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me(2)SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me(2)SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.

Diffusion of dimethyl sulfoxide in tissue engineered collagen scaffolds visualized by computer tomography.

Cryopreservation is a convenient method for long-term preservation of natural and engineered tissues in regenerative medicine. Homogeneous loading of tissues with CPAs, however, forms one of the major hurdles in tissue cryopreservation. In this study, computer tomography (CT) as a non-invasive imaging method was used to determine the effective diffusion of Me2SO in tissue-engineered collagen scaffolds. The dimensions of the scaffolds were 30 x 30 x 10 mm3 with a homogeneous pore size of 100 microm and a porosity of 98%. CT images were acquired after equilibrating the scaffolds in phosphate buffered saline (PBS) and transferring them directly in 10% (v/v)Me2SO. The Me2SO loading process of the scaffold could thus be measured and visualized in real time. The experimental data were fitted using a diffusion equation. The calculated effective diffusion constant for Me2SO in the PBS loaded scaffold was determined from experimental diffusion studies to be 2.4 x 10(-6) cm2/s at 20 degrees C.

Storage of human biospecimens: selection of the optimal storage temperature.

Millions of biological samples are currently kept at low tempertures in cryobanks/biorepositories for long-term storage. The quality of the biospecimen when thawed, however, is not only determined by processing of the biospecimen but the storage conditions as well. The overall objective of this article is to describe the scientific basis for selecting a storage temperature for a biospecimen based on current scientific understanding. To that end, this article reviews some physical basics of the temperature, nucleation, and ice crystal growth present in biological samples stored at low temperatures (-20°C to -196°C), and our current understanding of the role of temperature on the activity of degradative molecules present in biospecimens. The scientific literature relevant to the stability of specific biomarkers in human fluid, cell, and tissue biospecimens is also summarized for the range of temperatures between -20°C to -196°C. These studies demonstrate the importance of storage temperature on the stability of critical biomarkers for fluid, cell, and tissue biospecimens.

Effect of Me(2)SO on membrane phase behavior and protein denaturation of human pulmonary endothelial cells studied by in situ FTIR spectroscopy.

Fourier transform infrared spectroscopy (FTIR) provides a unique technique to study membranes and proteins within their native cellular environment. FTIR was used here to study the effects of dimethyl sulfoxide (Me(2)SO) on membranes and proteins in human pulmonary endothelial cells (HPMECs). Temperature-dependent changes in characteristic lipid and protein vibrational bands were identified to reveal the effects of Me(2)SO on membrane phase behavior and protein stability. At Me(2)SO concentrations equal to or below 10% (v/v), Me(2)SO was found to decrease membrane conformational disorder. At higher Me(2)SO concentrations (15% v/v), however, membrane conformational disorder was found to be similar to that of cells in the absence of Me(2)SO. This effect was observed over a wide temperature range from 90 degrees C down to -40 degrees C. Me(2)SO had no clear effects on cellular proteins during freezing. During heating, however, Me(2)SO had a destabilizing effect on cellular proteins. In the absence of Me(2)SO, protein denaturation started at an onset temperature of 46 degrees C, whereas at 15% Me(2)SO the onset temperature of protein denaturation decreased to 32 degrees C. This implies that in the presence of Me(2)SO the onset temperature of protein denaturation is lower than the normal growth temperature of the cells, which could explain the well documented toxic effect of Me(2)SO at physiological temperatures. Me(2)SO destabilizes cellular proteins during heating and decreases membrane conformational disorder over a wide temperature range.