Dimethyl Sulfoxide Cryopreservation Research Articles

Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation.

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.

Increased Cell Survival of Human Primary Conjunctival Stem Cells in Dimethyl Sulfoxide-Based Cryopreservation Media.

Glycerol and dimethyl sulfoxide (DMSO) are widely used cryoprotectants for freezing human cell cultures. During the manufacturing process of ocular stem cell-based autographs, ex vivo cultivated ocular cells are cryopreserved for quality control purposes in accordance with regulatory requirements. The efficiency of the cryopreservation methods is limited by their effect on cell survival and quality. We compared two cryopreservation reagents, glycerol and DMSO, for their influence on the survival and quality of human primary conjunctival cultures. We found increased cell viability after cryopreservation in DMSO compared to cryopreservation in glycerol. The clonogenic and proliferative capacity was unaffected by the cryopreservation reagents, as shown by the colony forming efficiency and cumulative cell doubling. Importantly, the percentage of p63α- and keratin 19 (K19)-positive cells following cryopreservation in DMSO or glycerol was comparable. Taken together, our results demonstrate that cryopreservation in DMSO improves cell survival compared to cryopreservation in glycerol, with no subsequent effect on cell proliferative-, clonogenic-, or differentiation capacity. Therefore, we advise the use of a 10% DMSO-based cryopreservation medium for the cryopreservation of human primary conjunctival cells, as it will improve the number of cells available for the manufacturing of conjunctival stem cell-based autografts for clinical use.

Synthetically Scalable Poly(ampholyte) Which Dramatically Enhances Cellular Cryopreservation.

The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether-alt-maleic anhydride). This poly(ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies.

Cryopreserved platelets augment the inflammatory response: role of phosphatidylserine- and P-selectin-mediated platelet phagocytosis in macrophages.

Cryopreservation in dimethyl sulfoxide and storage at -80 °C extends the shelf life of platelets to at least 2 years, allowing greater availability in rural and military areas. While cryopreserved platelets (CPPs) have been extensively characterized for coagulation and thrombin generation, reports on the mechanism of adverse reactions to CPPs transfusion are scarce. Here, we tested the hypothesis that CPPs facilitate phagocytosis by Kupffer cells and subsequently promote the inflammatory response in Kupffer cells. P-selectin expression, glycoprotein Ibα clustering and phosphatidylserine (PS) surface exposure on platelets stored at 22 °C, 4 °C and - 80 °C for 3 days were examined by flow cytometry. The phagocytosis of mepacrine-labeled platelets coincubated with THP-1 cells was examined by flow cytometry and confocal microscopy, and the release of cytokines from THP-1 cells was measured by enzyme-linked immunosorbent assay. CPPs showed a marked enhancement of exposed PS but dramatically reduced glycoprotein Ibα expression and clustering compared with platelets stored at 4 °C. Activation of THP-1 cells was stronger by CPPs than by platelets stored at 22 °C and 4 °C. CPP interference tests using annexin V and anti-P-selectin showed that CPPs induced increases in PS- and P-selectin-mediated phagocytosis, as well as secretion of the proinflammatory cytokine tumor necrosis factor-α, and interleukins IL-1β and IL-6, but a decrease in transforming growth factor-β production in THP-1 cells. Surface-exposed PS was more effective than P-selectin for the activation of THP-1 cells. CPPs triggered PS and P-selectin-mediated phagocytosis by macrophages and stimulated the inflammatory response of macrophages.

Irradiation at 11 kGy conserves the biomechanical properties of fascia lata better than irradiation at 25 kGy

The objective of this study was to determine the biomechanical properties of the fascia lata and the effects of three preservation methods: freezing, cryopreservation with dimethylsulfoxide solution and lyophilization; and to compare the effects of low-dose (11 kGy) and normal-dose (25 kGy) gamma-ray sterilization versus no irradiation.248 samples from 14 fasciae latae were collected. Freezing samples were frozen at −80 °C. Cryopreservation with dimethylsulfoxide solution samples were frozen with 10 cl dimethylsulfoxide solution at −80 °C. Lyophilization samples were frozen at −22 °C and lyophilized. Each preservation group were then randomly divided into 3 irradiation groups.The cryopreservation with dimethylsulfoxide solution samples had significantly worse results in all 3 irradiation conditions. Young's modulus was lower for the freezing samples (p < 0.001) and lyophilization samples groups (p < 0.001). Tear deformation was lower for the freezing samples (p = 0.001) and lyophilization samples groups (p = 0.003), as was stress at break (p < 0.001 and p < 0.001). Taking all preservation methods together, samples irradiated at 25 kGy had worse results than the 0 kGy and 11 kGy groups in terms of Young's modulus (p = 0.007 and p = 0.13) and of stress at break (p = 0.006 and p = 0.06).The biomechanical properties of fascia lata allografts were significantly worse under dimethylsulfoxide cryopreservation. The deleterious effects of irradiation were dose-dependent.

Low-molecular-weight carbohydrate Pentaisomaltose may replace dimethyl sulfoxide as a safer cryoprotectant for cryopreservation of peripheral blood stem cells.

Cryopreserved hematopoietic stem cell products are widely used for certain hematologic malignancies. Dimethyl sulfoxide (DMSO) is the most widely used cryoprotective agent (CPA) today, but due to indications of cellular toxicity, changes of the cellular epigenetic state, and patient-related side effects, there is an increasing demand for DMSO-free alternatives. We therefore investigated whether Pentaisomaltose (PIM), a low-molecular-weight carbohydrate (1 kDa), can be used for cryopreservation of peripheral blood stem cells, more specifically hematopoietic progenitor cell apheresis (HPC(A)) product. We cryopreserved patient or donor HPC(A) products using 10% DMSO or 16% PIM and quantified the recovery of CD34+ cells and CD34+ subpopulations by multicolor flow cytometry. In addition, we compared the frequency of HPCs after DMSO and PIM cryopreservation using the colony-forming cells (CFCs) assay. The mean CD34+ cell recovery was 56.3 ± 23.7% (11.4%-97.3%) and 58.2 ± 10.0% (45.7%-76.9%) for 10% DMSO and 16% PIM, respectively. The distribution of CD34+ cell subpopulations was similar when comparing DMSO or PIM as CPA. CFC assay showed mean colony numbers of 70.7 ± 25.4 (range, 37.8-115.5) and 67.7 ± 15.7 (range, 48-86) for 10% DMSO and 16% PIM, respectively. Our findings demonstrate that PIM cryopreservation of HPC(A) products provides recovery of CD34+ cells, CD34+ subpopulations, and CFCs similar to that of DMSO cryopreservation and therefore may have the potential to be used for cryopreservation of peripheral blood stem cells.

Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets.

A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. In vivo DMSO cryopreserved platelet studies published between 1972 and June of 2013 were reviewed. Assessed were the methods of cryopreservation, posttransfusion platelet responses, prevention or control of bleeding, and adverse events. Using the Department of Defense's preferred 6% DMSO cryopreservation method with centrifugation to remove the DMSO plasma before freezing at -65°C and no postthaw wash, mean radiolabeled platelet recoveries in 32 normal subjects were 33% ± 10% (52% ± 12% of the same subject's fresh platelet recoveries), and survivals were 7.5 ± 1.2 days (89% ± 15% of fresh platelet survivals). Using a variety of methods to freeze autologous platelets from 178 normal subjects, mean radiolabeled platelet recoveries were consistently 39% ± 9%, and survivals, 7.4 ± 1.4 days. More than 3000 cryopreserved platelet transfusions were given to 1334 patients. There were 19 hematology/oncology patient studies, and, in 9, mean 1-hour corrected count increments were 11 100 ± 3600 (range, 5700-15 800) after cryopreserved autologous platelet transfusions. In 5 studies, bleeding times improved after transfusion; in 3, there was either no improvement or a variable response. In 4 studies, there was immediate cessation of bleeding after transfusion; in 3 studies, patients being supported only with cryopreserved platelets had no bleeding. In 1 cardiopulmonary bypass study, cryopreserved platelets resulted in significantly less bleeding vs standard platelets. In 3 trauma studies, cryopreserved platelets were hemostatically effective. No significant adverse events were reported in any study. In summary, cryopreserved platelets have platelet recoveries that are about half of fresh platelets, but survivals are only minimally reduced. The platelets appear hemostatically effective and have no significant adverse events.

Cryopreservation of tissue-engineered epithelial sheets in trehalose

Tissue-engineered epidermal membranes are useful for clinical wound healing. To facilitate these products in the clinic, optimized storage methods need to be developed. We studied the efficiency of extracellular trehalose at various concentrations for cryopreserving human tissue-engineered epidermal membranes compared with that of dimethyl-sulfoxide (DMSO) used by most organ banks for cryopreserving skin grafts and artificial skin substitutes. Keratinocyte (KC) viability, proliferation and marker expression following cryopreservation in trehalose were examined with similar results to those using DMSO. Trehalose concentration (0.4 m) was optimized according to the described cellular activities following cryopreservation. Artificial epidermal substitutes were then cryopreserved in trehalose at the optimized concentration. Cell viability, growth factor secretion and wound healing properties of cryopreserved artificial epidermal substitutes using nude mice were examined and compared with those of DMSO cryopreservation. Cryopreservation with trehalose enhanced human KC viability in suspension and artificial skin substitutes. In addition, trehalose cryopreservation provided fast recovery of EGF and TGF-β1 secretion by KCs after thawing. When transplanted into nude mice, trehalose-cryopreserved artificial skin repaired skin defects in a similar manner to that of a non-cryopreserved control. Moreover, trehalose-cryopreserved artificial skin resulted in engraftment and wound closure that was significantly enhanced compared with that of DMSO-cryopreserved epidermal membranes. The results indicate that the use of trehalose improves cryopreservation of tissue-engineered epithelial sheets.

Pooled peripheral blood mononuclear cells provide an optimized cellular substrate for human immunodeficiency virus Type 1 isolation during acute infection

Cellular substrate (CS) composed of phytohemagglutinin (PHA)-stimulated PBMNCs is required for isolating primary human immunodeficiency virus Type 1 from plasma (PHIV) during early acute infection. The transmitted founder PHIV is neutralization-sensitive and uses chemokine-receptor 5 to infect CD4-positive PBMNCs. Therefore, PHIV cocultured with optimized CS would enable efficient virologic diagnosis, vaccine development, and tests for broadly neutralizing antibodies (bnAb). Fifteen leukapheresis donations were used to isolate CD4-enriched PBMNCs. Individual or three or four donors' pooled cells were stimulated with PHA, and both CSs were evaluated in parallel cocultures using five different PHIV isolates from nucleic acid testing-positive anti-HIV-negative donations. Feasibility of coculturing PHIV with pooled PBMNC-CS after dimethyl sulfoxide cryopreservation was investigated. The cell-free supernatants from individual CSs contained mean HIV-p24 antigen varying between 20.35 and 85.54 ng/mL, while that from pooled CSs ranged between 57.54 and 88.10 ng/mL. Thus, PBMNCs pooled from multiple donors provide an optimal CS for coculturing PHIV. Cryopreserved PBMNC-CS for PHIV cocultures is promising for systematic biosynthesis of PHIV. Pooled CSs after cryopreservation were functional for PHIV replication and enable on-demand diagnostic cocultures, preparing panels of PHIV stocks for bnAb testing and developing an envelope-subunit vaccine candidate analogous to hepatitis B surface antigen, proven successful in preventing hepatitis B virus infection.

Viability of small preantral ovarian follicles from domestic cats after cryoprotectant exposure and cryopreservation.

About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection. This is a potentially rich source of genetic material if ova could be preserved and grown in vitro, especially from rare or endangered species that die abruptly or are ovariectomized for medical reasons. The aims of this study were to examine cryoprotectant toxicity and then the potential of successfully cryopreserving preantral cat follicles. In the initial toxicity trial, isolated cat follicles (40-90 micron) were exposed to dimethylsulfoxide, glycerol, 1,2-propandiol or ethylene glycol at 0 degrees C for 15 min. Follicle viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258 at O h, and after 18 h and 1 week of culture. Percentages of follicles with intact oocytes and granulosa cells were similar (P > 0.05) among control (no cryoprotectant), dimethylsulfoxide, 1,2-propandiol and ethylene glycol treatments at all time points, but were reduced (P < 0.05) after glycerol exposure. On the basis of this finding, dimethylsulfoxide and 1,2-propandiol were used to cryopreserve intact follicles, and post-thaw viability was assessed by supravital staining and 5-bromo-2'-deoxyuridine uptake into oocytes and granulosa cells during culture. Of control (noncryopreserved) follicles, 31.4% +/- 2.9%, 18.8% +/- 1.9% and 16.2% +/- 1.6% were intact after O h, 18 h and 1 week of culture, respectively. Uptake of 5-bromo-2'-deoxyuridine occurred in approximately 20% of follicles at all time points. On the basis of the presence of both a healthy oocyte and granulosa cells, cryopreservation in dimethylsulfoxide or 1,2-propandiol allowed approximately 19% of follicles to survive. Approximately 10% demonstrated clear evidence of cell activity that was sustainable for 1 week. In conclusion, the cat ovary contains a population of preantral follicles that are not adversely affected by short-term exposure to most conventional cryoprotectants. Furthermore, there is a subpopulation of these follicles capable of surviving cryopreservation, remaining structurally intact and physiologically active after thawing.

Acquired secretion defect in platelets after cryopreservation in dimethyl sulfoxide.

Despite the use of preservatives, platelets are severely damaged during cryopreservation and, following freezing, function poorly in a number of in vitro tests. We report here that cryopreserved platelets show diminished aggregation in response to collagen. This may be a consequence of a secretion defect as evidenced by a 20 to 30 percent loss of dense- and alpha-granule content (p less than 0.05) and an impaired secretion mechanism. Analysis of adenine nucleotides confirmed the defect in dense granule adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content (storage pool), but in addition revealed a 50 percent fall in cytosolic ATP (metabolic pool). In contrast, the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + adenosine monophosphate), was normal. We concluded that platelet cryopreservation leads to a secretion defect, probably as a result of activation during freezing and thawing procedures.

Long-term Patency of Vein Grafts Preserved in Liquid Nitrogen in Dimethyl Sulfoxide

Autogenous canine jugular veins were stored in 15% dimethyl sulfoxide (DMSO) in liquid nitrogen vapor for one to 28 days and then implanted in the carotid artery as autografts. The patency rate at one year was 62.5-87.5%. The patency rate of fresh jugular vein autografts placed in the carotid artery for one year was 75%. Similar autografts stored in liquid nitrogen vapor for one to 28 days without the cryopreservative DMSO exhibited a zero to 12.5% patency rate at one year. Scanning electron microscope studies revealed preservation of theendothelium in DMSO protected veins and a damaged or sloughed endothelium in veins frozen without DMSO cryopreservation.

Viable vein graft preservation

Homologous vein grafts are becoming increasingly useful in patients without suitable autologous saphenous veins for peripheral arterial bypass procedures [4, 81. However, fresh veins are the only tissue that has been used successfully in homograft vascular reconstruction in terms of long-term patency [4]. The advantages of a reliable method of long-term storage of viable vein segments and the ultimate creation of a vein “bank” are therefore apparent. Dimethylsulfoxide (DMSO) is a low molecular weight compound that diffuses readily across cell membranes [2] and has been shown to be an effective cryoprotectant avoiding cellular dehydration, a major cause of cell death during freezing [2]. The DMSO has been used for successful cryopreservation of other tissues [3, 61, but has not been utilized for vein graft preservation. This study assesses a method for the longterm preservation of viable vein segments by freezing in liquid nitrogen with DMSO cryopreservation.