The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells.

Arterial Smooth Muscle Function after Prolonged Exposure to a Medium Containing Dimethyl Sulfoxide (Me2SO) and Storage at -196°C

Rational engineering of an antifreeze-based biomaterial with dual-enhanced ice-binding and delivery for high-efficiency cryopreservation.

Influence of G-CSF Mobilization and Liquid Storage Time on Post-Thaw Recovery of Lymphocyte Subpopulations and CD34+ Cells in Cryopreserved Apheresis Products

This study examined the post-thaw recovery of Jurkat cells cryopreserved in three combinations of five osmolytes including trehalose, sucrose, glycerol, mannitol, and creatine. Cellular response was characterized using low-temperature Raman spectroscopy, and variation of post-thaw recovery was analyzed using statistical modeling. Combinations of osmolytes displayed distinct trends of post-thaw recovery, and a nonlinear relationship between compositions and post-thaw recovery was observed, suggesting interactions not only between different solutes but also between solutes and cells. The post-thaw recovery for optimized cryoprotectants in different combinations of osmolytes at a cooling rate of 1°C/min was comparable to that measured with 10% dimethyl sulfoxide. Statistical modeling was used to understand the importance of individual osmolytes as well as interactions between osmolytes on post-thaw recovery. Both higher concentrations of glycerol and certain interactions between sugars and glycerol were found to typically increase the post-thaw recovery. Raman images showed the influence of osmolytes and combinations of osmolytes on ice crystal shape, which reflected the interactions between osmolytes and water. Differences in the composition also influenced the presence or absence of intracellular ice formation, which could also be detected by Raman. These studies help us understand the modes of action for cryoprotective agents in these osmolyte solutions.

This study examined the post-thaw recovery of Jurkat cells cryopreserved in single osmolyte solutions containing sucrose, glycerol or isoleucine, as well as in a combination of the three osmolytes. Cell response was determined using low temperature Raman Spectroscopy and variation in post-thaw recovery with composition was analyzed using statistical modeling. Post-thaw recovery of Jurkat cells in single osmolyte was low. A combination of the osmolytes displayed a non-linear relationship between composition and post-thaw recovery, suggesting that interactions exist between the different solutes. The post-thaw recovery for an optimized multicomponent solution was comparable to that observed using 10% dimethyl sulfoxide and a cooling rate of 1 °C/min. Statistical modeling was used to characterize the importance of each osmolyte in the combination and test for interactions between osmolytes. Higher concentrations of glycerol increase post-thaw recovery and interactions between sucrose and glycerol, as well as sucrose and isoleucine improve post-thaw recovery. Raman images clearly demonstrated that damaging intracellular ice formation was observed more often in the presence of single osmolytes as well as non-optimized multi-component solution compositions.

BackgroundHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored.MethodshiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium l-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function.ResultshiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5 °C/min and a low nucleation temperature of -8 °C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw.ConclusionsIt was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.

BackgroundHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored.MethodshiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium I-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function.ResultshiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5°C/min and a low nucleation temperature of −8°C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw.ConclusionsIt was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.

Variable recovery of cryopreserved hematopoietic stem cells and leukocyte subpopulations in leukapheresis products

Impact of pre- and post-thaw processing on recovery and fitness of cord blood mononuclear cells as starting material for cell therapy.

Context-aware synthetic biology by controller design: Engineering the mammalian cell.

Cell therapies such as allogenic CAR T-cell therapy, natural killer cell therapy and stem cell transplants must be cryopreserved for transport and storage. This is typically achieved by addition of dimethyl sulfoxide (DMSO) but the cryoprotectant does not result in 100% cell recovery. New additives or technologies to improve their cryopreservation could have major impact for these emerging therapies. l-Proline is an amino acid osmolyte produced as a cryoprotectant by several organisms such as the codling moth Cydia pomonella and the larvae of the fly Chymomyza costata, and has been found to modulate post-thaw outcomes for several cell lines but has not been studied with Jurkat cells, a T lymphocyte cell line. Here we investigate the effectiveness of l-proline compared to d-proline and l-alanine for the cryopreservation of Jurkat cells. It is shown that 24-hour pre-freezing incubation of Jurkat cells with 200 mM l-proline resulted in a modest increase in cell recovery post-thaw at high cell density, but a larger increase in recovery was observed at the lower cell densities. l-Alanine was as effective as l-proline at lower cell densities, and addition of l-proline to the cryopreservation media (without incubation) had no benefit. The pre-freeze incubation with l-proline led to significant reductions in cell proliferation supporting an intracellular, biochemical, mechanism of action which was shown to be cell-density dependent. Controls with d-proline were found to reduce post-thaw recovery attributed to osmotic stress as d-proline cannot enter the cells. Preliminary analysis of apoptosis/necrosis profiles by flow cytometry indicated that inhibition of apoptosis is not the primary mode of action. Overall, this supports the use of l-proline pre-conditioning to improve T-cell post-thaw recovery without needing any changes to cryopreservation solutions nor methods and hence is simple to implement.

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

HypothesisIn concentrated suspensions, the dynamics of colloids are strongly influenced by the shape and topographical surface characteristics of the particles. As the particles get into close proximity, surface roughness alters the translational and rotational Brownian motions in different ways. Eventually, the rotations will get frustrated due to geometric hindrance from interacting asperities. ExperimentsWe use model raspberry-like colloids to study the effect of roughness on the translational and rotational dynamics. Using Confocal Scanning Laser Microscopy and particle tracking, we simultaneously resolve the two types of Brownian motion and obtain the corresponding Mean Squared Displacements for varying concentrations up to the maximum packing fraction. FindingsRoughness not only lowers the concentration of the translational colloidal glass transition, but also generates a broad concentration range in which the rotational Brownian motion changes signature from high-amplitude diffusive to low-amplitude rattling. This hitherto not reported second glass transition for rough spherical colloids emerges when the particle intersurface distance becomes comparable to the roughness length scale. Our work provides a unifying understanding of the surface characteristics’ effect on the rotational dynamics during glass formation and provides a microscopic foundation for many roughness-related macroscale phenomena in nature and technology.

Fish embryonic stem (ES) cells derived from of blastulae (64 cell stage embryo) of Labeo rohita were propagated in culture and retained their ES cell-like properties after cryogenic storage (-196 degrees C, i.e., liquid nitrogen). Toxic effect of DMSO (dimethyl sulphoxide) on stem cells during preservation process has been reported to restrict therapeutic applications. In this study we reduced the concentration of DMSO and added the non-toxic cryoprotective agent (CPA) trehalose. Cryopreservation of ES cell colonies was done at 5, 25 and 52 passages with 0.2 M trehalose and 0.8 M (DMSO). A combination of both the cryoprotective agents (non-toxic and toxic) demonstrated better survival and recovery of ES cells than the DMSO used alone. Use of this CPA combination in the freezing media gave an optimum viability of more than 83 % in a slow freezing protocol. Trehalose showed a definite advantage over DMSO in terms of viability and intactness of ES cell colonies with evenly distributed morphology. There was no significant difference observed in the expression levels of cell surface markers like stage specific embryonic antigen-1 (SSEA-1) and alkaline phosphatase (ALP) between early and late passages after 60 days of post-thawing. More than 90 % of the ES cell colonies showed extensive expression of ALP and positive expression of SSEA-1 from an early stage of ES cells culture up to passage 52 (in our study) in the presence of leukemia inhibitory factor (LIF) and without feeder cells. Further, thawed ES cells showed a normal karyotype and maintained an undifferentiated state through out the study. This study on ES cell cryopreservation and subsequent retention of stem cell properties without feeder cells using a non-toxic cryoprotectant trehalose would be highly useful for future in vitro differentiation, manipulation of fish ES cells and as a model for mammalian ES cell culture.

piggyBac Transposon-mediated Long-term Gene Expression in Mice