Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars Publication date: Available online 5 October 2019 Source: Cryobiology Author(s): Vitalii Mutsenko, Ariana Barlič, Tamara Pezić, Janja Dermol-Černe, Barbara Dovgan, Bulat Sydykov, Willem F. Wolkers, Igor I. Katkov, Birgit Glasmacher, Damijan Miklavčič, Oleksandr Gryshkov Abstract Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum. In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay). Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

Exogenous carnitine application augments transport of fatty acids into mitochondria and stimulates mitochondrial respiration in maize seedlings grown under normal and cold conditions Publication date: Available online 4 October 2019 Source: Cryobiology Author(s): Hulya Turk, Serkan Erdal, Rahmi Dumlupinar Abstract This study aimed to investigate whether exogenous application of carnitine stimulates transportation of fatty acids into mitochondria, which is an important part of fatty acid trafficking in cells, and mitochondrial respiration in the leaves of maize seedlings grown under normal and cold conditions. Cold stress led to significant increases in lipase activity, which is responsible for the breakdown of triacylglycerols, and carnitine acyltransferase (carnitine acyltransferase I and II) activities, which are responsible for the transport of activated long-chain fatty acids into mitochondria. While exogenous application of carnitine has a similar promoting effect with cold stress on lipase activity, it resulted in further increases in the activity of carnitine acyltransferases compared to cold stress. The highest activity levels for these enzymes were recorded in the seedlings treated with cold plus carnitine. In addition, these increases were correlated with positive increases in the contents of free- and long-chain acylcarnitines (decanoyl-l-carnitine, lauroyl-l-carnitine, myristoyl-l-carnitine, and stearoyl-l-carnitine), and with decreases in the total lipid content. The highest values for free- and long-chain acylcarnitines and the lowest value for total lipid content were recorded in the seedlings treated with cold plus carnitine. On the other hand, carnitine with and without cold stress significantly upregulated the expression level of citrate synthase, which is responsible for catalysing the first reaction of the citric acid cycle, and cytochrome oxidase, which is the membrane-bound terminal enzyme in the electron transfer chain, as well as lipase. All these results revealed that on the one hand, carnitine enhanced transport of fatty acids into mitochondria by increasing the activities of lipase and carnitine acyltransferases, and, on the other hand, stimulated mitochondrial respiration in the leaves of maize seedlings grown under normal and cold conditions.

Foundations of modeling in cryobiology—II: Heat and mass transport in bulk and at cell membrane and ice-liquid interfaces Publication date: Available online 4 October 2019 Source: Cryobiology Author(s): Daniel M. Anderson, James Benson, Anthony J. Kearsley Abstract Modeling coupled heat and mass transport in biological systems is critical to the understanding of cryobiology. In Part I of this series we derived the transport equation and presented a general thermodynamic derivation of the critical components needed to use the transport equation in cryobiology. Here we refine to more cryobiologically relevant instances of a double free-boundary problem with multiple species. In particular, we present the derivation of appropriate mass and heat transport constitutive equations for a system consisting of a cell or tissue with a free external boundary, surrounded by liquid media with an encroaching free solidification front. This model consists of two parts–namely, transport in the “bulk phases” away from boundaries, and interfacial transport. Here we derive the bulk and interfacial mass, energy, and momentum balance equations and present a simplification of transport within membranes to jump conditions across them. We establish the governing equations for this cell/liquid/solid system whose solution in the case of a ternary mixture is explored in Part III of this series.

Measurement of grouped intracellular solute osmotic virial coefficients Publication date: Available online 3 October 2019 Source: Cryobiology Author(s): Michal W. Zielinski, Locksley E. McGann, John A. Nychka, Janet A.W. Elliott Abstract Models of cellular osmotic behaviour depend on thermodynamic solution theories to calculate chemical potentials in the solutions inside and outside the cell. These solutions are generally thermodynamically non-ideal under cryobiological conditions. The molality-based Elliott et al. form of the multi-solute osmotic virial equation is a solution theory which has been demonstrated to provide accurate predictions in cryobiological solutions, accounting for the non-ideality of these solutions using solute-specific thermodynamic parameters called osmotic virial coefficients. However, this solution theory requires as inputs the exact concentration of every solute in the solution being modeled, which poses a problem for the cytoplasm, where such detailed information is rarely available. This problem can be overcome by using a grouped solute approach for modeling the cytoplasm, where all the non-permeating intracellular solutes are treated as a single non-permeating “grouped” intracellular solute. We have recently shown (Zielinski et al., J Physical Chemistry B, 2017) that such a grouped solute approach is theoretically valid when used with the Elliott et al. model, and Ross-Rodriguez et al. (Biopreservation and Biobanking, 2012) have previously developed a method for measuring the cell type-specific osmotic virial coefficients of the grouped intracellular solute. However, the Ross-Rodriguez et al. method suffers from a lack of precision, which—as we demonstrate in this work—can severely impact the accuracy of osmotic model predictions under certain conditions. Thus, we herein develop a novel method for measuring grouped intracellular solute osmotic virial coefficients which yields more precise values than the existing method and then apply this new method to measure these coefficients for human umbilical vein endothelial cells.

An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes Publication date: Available online 1 October 2019 Source: Cryobiology Author(s): Xiaojie Guo, Zhongrong Chen, Kashan Memon, Xiaoyu Chen, Gang Zhao Abstract Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.

Effect of short-term cold temperature stress on development, survival and reproduction of Dysdercus koenigii (Hemiptera: Pyrrhocoridae) Publication date: Available online 30 September 2019 Source: Cryobiology Author(s): Muhammad Sarmad, Aiman Ishfaq, Hina Arif, Syed Muhammad Zaka Abstract Red cotton bug Dysdercus koenigii F. (Hemiptera: Pyrrhocoridae), is found destructive pest in various cotton growing areas. Under natural conditions insects are highly subjected to thermal stresses. In present work the developmental duration and survival rate of all immature stages, adult longevity and reproduction of D. koenigii by exposed to rapid changes in very low temperatures were studied. When 3 h short-stress of low temperatures (12–0 °C) was given to different stages of D. koenigii, the results revealed that survival rate of all stages were significantly reduced. Survival rate of female was significantly higher than male after exposed to cold temperature stress. Mating percentage, fecundity and hatching percentage were decreased significantly with the decrease of short-term cold temperature stress. Based on these results, we concluded that the developmental duration, survival rate and reproduction of D. koenigii significantly affected when they exposed to short term cold temperature stress.

PRE-GRAFTING histological studies OF SKIN grafts cryopreserved in α helix antarctic yeast oriented anti-freeze peptide (Afp1m) Publication date: Available online 30 September 2019 Source: Cryobiology Author(s): M.S. Khan, S.M. Ibrahim, A.A. Adamu, M.B.A. Rahman, M.Z.Abu Bakar, M.M. Noordin, L.M. Yusof Abstract A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing anti-freeze proteins (AFP). Antifreeze -Peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of −10 °C and −20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (−10 and −20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p < 0.01) among the different concentrations at −10 and −20 °C. In conclusion, the integrity of cryopreserved skin grafts with lower concentrations of Afp1m (0.5, 1 and 2 mg/mL) or at −20 °C was not maintained. The present study attested that Afp1m is a good cryoprotective agent for the cryopreservation of skin graft. Higher Afp1m concentrations (5 and 10 mg/mL) at −10 °C found to be suitable for the future in vivo study using (SD) rat skin grafts.

Leukapheresis cell concentration adjustment required for a successful recovery of HSC after cryopreservation Publication date: Available online 26 September 2019 Source: Cryobiology Author(s): Rebar N. Mohammed, Dereen Ahmad, Najmaddin Khoshnaw, Dastan O. Hassan, Hanar A. Abdulrahman, Michele Vacca, Marco Possenti, Diana Faruq, Harem Omer, Salah Muhamad, Ignazio Majolino, Dosti N. Othman Abstract The recovering of an adequate number of hematopoietic stem cells after cryopreservation is considered pivotal for successful transplantation. Various factors could influence the recovery of HSC following processing and cryopreservation. Therefore, leukapheresis product from thirty patients was cryopreserved in 10% DMSO in cryopreservation bags for their autologous bone marrow transplantation, and 2 ml were cryopreserved in cryovials for post-thaw viability assessment by flow cytometry. The percentage of viable HSCs recovered post-cryopreservation in leukapheresis product was significantly influenced by the concentration of the total nucleated cells cryopreserved per volume. Patients receiving a higher rate of viable HSCs resulted in earlier engraftment of both neutrophils and platelets, so they have been discharged earlier from the hospital. Furthermore, Storage temperature and duration played a role in the recovery of these cells and for the support of the findings, age of the patient at the time of collection and sex did not show any impact on the recovery of this HSC post-cryopreservation. In conclusion, various influencing factors must be taken into consideration during the cryopreservation of HSCs, especially for poor mobilizing patients with a low number of collected hematopoietic stem cells.

Myelinosome-like vesicles in human seminal plasma: A cryo-electron microscopy study Publication date: Available online 21 September 2019 Source: Cryobiology Author(s): M. Yefimova, E. Bere, A.S. Neyroud, B. Jegou, N. Bourmeyster, C. Ravel Abstract Seminal plasma is particularly rich in extracellular vesicles. Myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. Our aim was to determine the presence of myelinosomes in human seminal plasma. Transmission electron microscopy and cryo electron microscopy analysis of standard myelinosome preparation from TM4 Sertoli cells and human seminal plasma samples. We have specified by cryo-EM the morphological aspect of “standard” myelinosomes isolated from the culture media of TM4 Sertoli cells. Vesicles with the same morphological appearance were revealed in human seminal plasma samples. Human seminal plasma contains a population of large EV (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. Defining the specific biomarkers and functionalities of myelinosome in human seminal plasma are the concerns to be addressed in our further research.

Nylon mesh cryodevice for bovine mature oocytes, easily removable excess vitrification solution Publication date: Available online 21 September 2019 Source: Cryobiology Author(s): Shoichiro Chinen, Takahiro Yamanaka, Kenyu Nakayama, Hiroki Watanabe, Yoshitake Akiyama, Masumi Hirabayashi, Shinichi Hochi Abstract The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-μm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-μm group (39%) was higher than that in the 77-μm group (28%), and the yield in the 57-μm group (31%) was the intermediate. The 37-μm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-μm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes.