Controlled-rate Freezing Research Articles

Storage Duration of Autologous Stem Cell Preparations Has No Impact on Hematopoietic Recovery after Transplantation

Peripheral blood stem cells (PBSCs) are widely used for autologous blood stem cell transplantation (ABSCT). These cells must be stored for months or even years, usually at temperatures ≤−140°C, until their use. Although several in vitro studies on CD34+ viability and clonogenic assays of PBSCs after long-term storage have been reported, only a few publications have investigated the influence of long-term storage on in vivo hematopoietic reconstitution. In this study, we retrospectively analyzed hematopoietic recovery after storage of PBSCs via controlled-rate freezing (CRF) and cryostorage in 10% DMSO at ≤−140°C in 105 patients with multiple myeloma who received high-dose melphalan before ABSCT. Three groups of PBSC transplantation (n = 247) were delineated based on the storage period: short-term (≤12 months, n = 143), medium-term (>12 and ≤60 months, n = 75), and long-term storage (>60 months, n = 29). A neutrophil increase of ≥.5 × 109/L in medium-term or long-term PBSC cryopreservation groups was observed at day 14 after ABSCT; this increase was comparable to patients who received briefly stored PBSCs (day 15). No negative effect of PBSC storage duration was observed on leucocyte or neutrophil reconstitution. Platelet reconstitutions of ≥20 × 109/L and 50 × 109/L were observed after median times of 10 to 11 and 13 to 14 days after ABSCT, respectively. No influence of PBSC storage duration on platelet recovery of ≥20 × 109/L and ≥50 × 109/L was observed in the 3 storage groups (P = .07, P = .32). The number of previous ABSCTs also had no significant impact upon hematopoietic reconstitution. In conclusion, these results indicate that long-term cryopreservation of PBSC products at vapor nitrogen temperature after CRF does not have a negative effect on hematopoietic recovery even after prolonged storage.

Detection of microRNAs in CD34+ Microparticles of Cord Blood Units

Introduction:Micropartices (MPs) are small vesicles covered by a bilayered membrane which can serve as cell-to-cell shuttles for bioactive molecules such as lipids, growth factors, microRNAs. Umbilical Cord Blood Transplant Units (UCB) has been established as a source of Hematopoietic Stem Cells (HSC) in allogeneic transplantation. Although that UCB has unique advantages of easy procurement, low risk of transmitting infections, absence of risk to donors, immediate availability, greater tolerance of HLA disparity and lower-than-expected incidence of severe graft-versus-host disease monitoring of certain parameters on UCB transplantation remains a challenging issue. MicroRNAs (miRNAs) are short non-coding RNA chains that regulate gene expression displaying important regulatory roles in many cellular processes, including differentiation, cell replication and regeneration. We have reported herein monitoring of miRNAs containing CD34+ microparticles (MPs) as a candidate parameter of the UCBs.Methods: Cord blood units (CBUs) (n=37) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controlled rate freezer (IceCube, Sy-lab). Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with a hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining were assessed. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V (AnnV+) and CD34 antibody by flow cytometry. Electron microscope photographs were obtained, after the appropriate preparation for TEM. CD34+ specific MPs as well as CD34+ cells isolation with immunomagnetic beads has been performed through MiniMACS columns (Miltenyi). Total RNA, including miRNA, was extracted from CD34+ and MPs using the miRNeasy Micro kit and miRNeasy Serum/Plasma kit (Qiagen), respectively. Samples' concentration was assessed by Qubit fluorimeter (Thermo Fisher scientific) and cDNA synthesis was achieved using miScript II RT kit (Qiagen). Quantitative Real Time PCR was performed to assess the presence of selected miRNAs in CD34+ and MPs in five independent donor samples (miScript SYBR Green PCR kit, Qiagen). Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables.Results: The AnnV+/CD34+MPs during post-processing (51,38±37,87) decreased compared to the number of pre-processing AnnV+/CD34+MPs (57,7±51,2). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p<0,000, Spearman’s rho =0,696) whereas the number of post-processing CD34 cell per unit was not statistically significant.The number of post-processing AnnV+/CD34+MPs has positive correlation to the number of BFUe pre- and post-processing (p<0,037;r=0,344 and p<0,003;r=0,474 respectively). The post-processing AnnV+/CD34+MPs has positive correlation to the number of CFU-GM pre- and post-processing (p<0,015;r=0,396 and p<0,036;r=0,347 respectively).Furthermore molecular analysis of post-processing CD34+ cells and MPs was performed for the presence of microRNA 106b, 221, 224, 517c, 518a, 519d, 520h. miRNAs 517c and 520h were detected in low levels. miRNAs 106b, 221, 224, 518a, 519d were very low or undetectable. Interestingly a different profile was observed in cell lines suggesting a line specificity of the selected miRNAs.Conclusions: Post-processing stem cell derived MPs were correlated with CFU progenitor cells (BFUe and CFU-GM). Molecular analysis confirmed the identification of line-specific miRNAs in CD34+ cells and MPs from post- processed cord blood samples. Thus miRNAs in stem cell-derived MPs could be candidate biomarkers in cord blood units. DisclosuresNo relevant conflicts of interest to declare.

The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation.Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.

Variable resistance to freezing and thawing of CD34-positive stem cells and lymphocyte subpopulations in leukapheresis products

Background aimsLeukapheresis products for hematopoietic stem cell transplantation can be cryopreserved for various indications. Although it is known that CD34+ cells tolerate cryopreservation well, a significant loss of CD3+ cells has been observed, which has been ascribed to several factors, including transport, storage conditions and granulocyte-colony stimulating factor (G-CSF) administration. MethodsTo assess the tolerance of CD34+ cells and lymphocyte subpopulations for cryopreservation and thawing, the post-thaw recoveries of CD34+ cells, CD3+CD4+ cells, CD3+CD8+ cells, CD19+ cells and CD16+CD56+ cells were determined in 90 cryopreserved apheresis products, among which 65 were from G-CSF–mobilized donors, and 34 from unrelated donors that underwent transport before cryopreservation at our center. A controlled rate freezer and 5% dimethyl sulfoxide were used for cryopreservation. ResultsWe could detect statistically significant differences for CD34+ cell recovery (93.0 ± 20.7%) when compared to CD3+CD4+ cell (83.1 ± 15.4%, P = 0.014), and CD3+CD8+ cell recovery (83.3 ± 13.9%, P = 0.001). Similarly, CD19+ cell recovery (98.6 ± 15.1%) was higher than CD3+CD4+ cell (P = 2.5 × 10−7) and CD3+CD8+ cell recovery (P = 1.2 × 10−8). Post-thaw recovery rates of all cell populations were not impaired in G-CSF–mobilized products compared with non-mobilized products nor in unrelated compared with related donor products. DiscussionOur data suggest a lower tolerance of CD3+ cells for cryopreservation and demonstrate that freezing–thawing resistance thawing is cell-specific and independent from other factors that affect post-thaw recovery of cryopreserved cells. Thus, a clinical consequence may be the monitoring of post-thaw CD3+ cell doses of cryopreserved products, such as donor lymphocyte infusions.

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

A simple and efficient method for cryopreservation and recovery of viable Plasmodium vivax and P. falciparum sporozoites

Plasmodium falciparum and Plasmodium vivax sporozoites are the crucial stages of malaria parasites that initiate infection in humans. However, studies to develop new vaccines and drugs targeting these infective stages remain insufficient due to limited availability of sporozoites for research. This is a consequence of relatively few facilities that are established to produce sporozoites of human malaria parasites, sporozoites remaining viable for only a few days, and infected mosquitoes being a biohazard, making them difficult to transport. Cryopreservation of sporozoites offers the potential to alleviate these limitations and enhance sporozoite availability. These experiments were performed to evaluate methods for cryopreservation of P. vivax and P. falciparum sporozoites. Sporozoites, isolated in sterile buffer from infected mosquitoes by manual dissection of salivary glands, were cryopreserved using several types of commercially available serum-free cryoprotective solutions. The efficiency of cryopreservation was validated by a standard in vitro gliding motility assay as a measure of sporozoite activity. Viability of infective sporozoites was defined as percent gliding of sporozoites attached to the coverslip. Significant differences were observed among the cryopreservation media and protocols evaluated, with CryoStor CS2 giving the best results for both P. falciparum and P. vivax, whereas Hestar 200 worked efficiently only for P. vivax sporozoites. Further improvement in recovery of viable sporozoites would be anticipated using automated controlled-rate freezing equipment. Our results demonstrate that cryopreservation provides an alternative for experimental studies that currently rely on fresh P. falciparum and P. vivax sporozoites.

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

Background: Peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) for allogeneic transplantation of stem cells or donor lymphocyte infusion are frequently cryopreserved to allow cellular therapy at later time-points. Differences in the post-thaw recovery of CD34+ and CD3+ T cells in cryopreserved products have been hypothesized to depend on liquid storage time or, in the case of CD3+ T cells, on the mobilization by G-CSF.Methods: The recovery of CD3+ CD4+, CD3+ CD8+, CD19+, CD16+ CD56+, and CD34+ cells from 58 allogeneic apheresis products (among which 38 derived from G-CSF-mobilized donors) was analyzed by flow cytometry in order to evaluate the impact of the freezing/thawing process in the recovery of lymphocyte subpopulations as well as hematopoietic stem cells. In addition, cell viability was determined by measuring viable CD45+ (vCD45+) cells. All measurements were performed with aliquots that had been cryopreserved in parallel with the products using a controlled-rate freezer and 8% dimethyl sulfoxide and were stored in liquid nitrogen.Results: The post-thaw recovery was 78.6 ± 14.7% (mean±SD) for CD3+ CD4+ cells, 79.0 ± 12.9% for CD3+ CD8+ cells, 95.6 ± 15.8% for CD19+ cells, 84.6 ± 22.4% for CD16+ CD56+ cells, and 93.9 ± 24.6% for CD34+ cells. In G-CSF mobilized products, higher recovery rates were observed than in non-mobilized products, reaching statistical significance for CD3+ CD4+ T cells (82.8 ± 14.5 vs 72.1 ± 12.8%, p=0.008), CD19+ B cells (99.3 ± 15.9 vs 89.7 ± 14.1%, p=0.027), and CD16+ CD56+ NK cells (90.1% ± 19.8% vs 75.9 ± 22.4%, p=0.025). Within the lymphocyte subpopulations the post-thaw recovery was significantly lower for CD3+ CD4+ vs CD19+ (p=1.0x10-9 in G-CSF mobilized products, p=2.9x10-5 in non-mobilized products), and CD3+ CD8+ vs CD19+ (p=1.8x10-8 in G-CSF mobilized products, p=3.2x10-4 in non-mobilized products). With a ratio of vCD45+/CD45+ of 62.6 ± 18.2% in G-SCF mobilized products and 64.4 ± 18.3% in non-mobilized products no difference in the viability was observed. Spearman's analysis revealed only a weak negative correlation between liquid storage time (30 ± 14 h for G-CSF mobilized products, 22 ± 7 h for non-mobilized products) and viability (rs=-0.33, p=0.015), and between liquid storage time and CD34+ recovery (rs=-0.42, p=0.009). The post-thaw recovery of all other cell types did not decrease with longer liquid storage time.Conclusion: G-CSF mobilization and longer liquid storage time do not impair post-thaw recovery of lymphocyte subpopulations when compared to products from non-mobilized donors. However there is a slight decrease of viability with longer liquid storage time. Our results further suggest that T lymphocytes exhibit a higher sensitivity toward freezing and thawing than B lymphocytes, which may have clinical implications for cellular therapies using frozen products. DisclosuresOldenburg:SOBI: Consultancy.

163 EFFECTS OF SERUM AND L-CARNITINE ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P &lt; 0.05) development to the blastocyst (19.7 ± 1.1% v. 25.3 ± 1.4%) and advanced blastocyst (9.1 ± 0.8% v. 12.4 ± 1.2%) stages. While l-carnitine did not affect blastocyst development, advanced blastocyst development was reduced (P &lt; 0.05) for l-carnitine at 3.03 mM (0 mM: 10.9 ± 1.2%, 0.75 mM: 12.2 ± 1.4%, 1.5 mM: 13.5 ± 1.5%, 3.03 mM: 7.0 ± 1.0%). Serum reduced (P &lt; 0.01) re-expansion (78.1 ± 3.4% v. 65.5 ± 3.1%, 81.0 ± 3.0% v. 68.4 ± 2.7%, 78.4 ± 3.4% v. 65.8 ± 3.1%, for 24, 48, and 72 h, respectively) and hatching (52.0 ± 4.0% v. 39.8 ± 3.6%, 61.2 ± 4.1% v. 45.4 ± 3.8%, 61.2 ± 4.1% v. 45.4 ± 3.8%, for 24, 48, and 72 h, respectively) rates at all time points. In contrast, treatment of embryos with l-carnitine during culture increased (P &lt; 0.05) post-thaw re-expansion rates at 24 and 48 h, regardless of concentration (Table 1). In conclusion, post-thaw viability of bovine embryos PIV can be improved by the addition of l-carnitine during culture. Moreover, the beneficial effects of l-carnitine on cryosurvival are not dependent on serum supplementation. Table 1.Effect of addition of l-carnitine during culture on post-thaw re-expansion and hatching rates

33 POST-THAW VIABILITY OF BOVINE EMBRYOS PRODUCED IN VITRO FOLLOWING TREATMENT WITH ASCORBIC ACID, DITHIOTHREITOL, AND CASPASE-3 INHIBITOR DURING CRYOPRESERVATION

The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P &lt; 0.05) re-expansion and hatching rates at 24, 48, and 72 h compared with embryos not treated with ascorbic acid (Table 1). The optimal concentration of ascorbic acid for post-thaw cryosurvival was 0.1 mM. In particular, embryos treated with 0.1 mM ascorbic acid during cryopreservation had increased (P &lt; 0.05) re-expansion rates at 24, 48, and 72 h, as well as hatching rates at 48 and 72 h, compared with control-treated embryos (Table 1). There was no effect of treatment with dithiothreitol or Z-DEVD-FMK on re-expansion or hatching rates at 24, 48, or 72 h after thaw. In conclusion, addition of ascorbic acid to freezing medium improves the cryosurvival of bovine embryos produced in vitro. Further research is necessary to determine whether treatment with ascorbic acid can increase pregnancy rates. Table 1.Effect of ascorbic acid during cryopreservation of bovine embryos (%, means ± standard error of the mean)

34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P &lt; 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.

Qualification of a cryogen-free controlled rate freezer for use in the delivery of cryopreserved stem cells banks for therapeutic applications

Qualification of a cryogen-free controlled rate freezer for use in the delivery of cryopreserved stem cells banks for therapeutic applications

Cryopreserved amniotic membrane as transplant allograft: viability and post-transplant outcome.

Amniotic membrane (AM) transplantation is increasingly used in ophthalmological and dermatological surgeries to promote re-epithelialization and wound healing. Biologically active cells in the epithelial and stromal layers deliver growth factors and cytokines with anti-inflammatory, anti-bacterial, anti-immunogenic and anti-fibrotic properties. In this work, confocal microscopy was used to show that our cryopreservation protocol for AM yielded viable cells in both the stromal and epithelial layers with favorable post-transplant outcome. AM was obtained from Caesarean-section placenta, processed into allograft pieces of different sizes (3 cm × 3 cm, 5 cm × 5 cm, and 10 cm × 10 cm) and cryopreserved in 10 % dimethyl sulfoxide using non-linear controlled rate freezing. Post-thaw cell viability in the entire piece of AM and in the stromal and epithelial cell layers was assessed using a dual fluorescent nuclear dye and compared to hypothermically stored AM, while surveys from surgical end-users provided information on post-transplant patient outcomes. There was no significant statistical difference in the cell viability in the entire piece, epithelial and stromal layers regardless of the size of allograft piece (p = 0.092, 0.188 and 0.581, respectively), and in the entire piece and stromal layer of hypothermically stored versus cryopreserved AM (p = 0.054 and 0.646, respectively). Surgical end-user feedback (n = 49) indicated that 16.3 % of AM allografts were excellent and 61.2 % were satisfactory. These results support the expanded clinical use of different sizes of cryopreserved AM allografts and address the issue of orientation of the AM during transplant for the treatment of dermatological defects and ocular surface disorders.

The effects of over expressing aquaporins on the cryopreservation of hepatocytes

During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt2cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments – confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt2cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt2cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability.

59. High concentration ice nucleation for controlled rate freezing of complex biological systems

Cryopreservation of human organs would facilitate long term organ banking and potentially go a long way to remedying the global shortage of organs for transplantation. To date, almost all attempts at cryopreservation of whole organs have used vitrification methods. Controlled rate freezing, which is commonly used for freezing of various types of cell suspensions, is unlikely to work well for whole organs as ice crystals will inevitably nucleate and grow to damaging sizes. Here, we propose an alternative to vitrification for the cryopreservation of whole organs. By introducing very large numbers of small ice nucleating particles to an organ it might be possible to induce formation of very large numbers of small, relatively harmless crystals, rather than small numbers of large, and therefore damaging ice crystals. The ice nucleating particles used would have to be less than 50 nm in diameter, and would have to nucleate ice at a high and very uniform temperature. The minimum known size of effective ice nucleating particles has been falling rapidly of late, meaning that particles of the requisite size and effectiveness are now known, although it is likely that further work will have to be conducted to generate sufficiently uniform ice nucleation temperatures. Uniformity of crystal size is of vital importance as larger ice crystals are more thermodynamically stable than smaller ones and as such will tend to grow at the expense of smaller crystals, rapidly producing ice of a damaging size. To achieve this, an organ would need to be supercooled very uniformly so that nucleation occurred at a similar time throughout. Once extracellular nucleation had been triggered the careful control of temperature would be needed to ensure optimal dehydration of cells for successful cryopreservation. It is anticipated that the size of crystals can be tuned by varying the concentration of ice nucleating particles. On rewarming, a uniform ice crystal size will prevent excessive recrystallization of large ice crystals and the ensuing tissue damage that is often observed during vitrification procedures. Overall, it might be possible to use a controlled rate freezing type method with larger, more complex systems than is currently possible. Although such an approach provides numerous potential benefits there are many uncertainties that will need to be explored through experiment. It is proposed that such experiments could start with light microscopy of dyed cryoprotectant solutions to determine if ice crystals of suitably small size can indeed be formed before moving onto cell suspensions, tissues and whole organs.

24. Diffusion limited ice crystallisation + a comment on liquidus tracking

Diffusion limited ice crystallisation. During conventional cryopreservation many of the changes which occur in a CPA solution following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of ionic species, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature. In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a nonlinear manner with temperature. We have measured the viscosity of the residual unfrozen solution that cells are exposed to during conventional freezing, and the effect of high viscosities encountered on the diffusion of water at a constant temperature during freezing and during cooling at different linear rates has been estimated. At rates of cooling faster than 100 °C min−1 the diffusion distance during freezing was calculated to be less than 15 μ m. Validation of the diffusion calculations were confirmed by examination of the ultrastructure of the freeze concentrated matrix in samples prepared at a range of cooling rates. At a critical rate of cooling, water diffusion becomes limited by the high viscosity and the composition of the freeze concentrated matrix deviates from that of the equilibrium phase diagram, the effects on cell viability of this non equilibrium state are demonstrated. Liquidus tracking. In 1965 Farrant proposed the “liquidus tracking” method of preservation in which cryoprotectant is added progressively during cooling so that samples remain on or above the liquidus line of the equilibrium phase diagram. Low temperatures protect against cryoprotectant toxicity at high concentrations. This method has also been termed the equilibrium approach to vitrification. However due to practical issues associated with high viscosities and mixing at low temperatures the method was largely abandoned. Recently there has been revived interest in the technique as applied to the preservation of cartilage and encapsulated hepatocytes and also in ultrastructural studies where the method is known as progressive lowering of temperature (PLT). Planer products have modified their controlled rate freezer to deliver the process for cartilage. Asymptote are developing equipment for this process which will allow preservation of clinical biopsies with native immunology which can be immunolabelled and from which viable cells may be isolated for adoptive cell therapies and personalised medicine.

Preserved in vivo reconstitution ability of PBSCs cryopreserved for a decade at -80 °C.

PBSC products for auto- and allografting can be cryopreserved in liquid nitrogen with controlled-rate freezing until their use. Alternatively, they can be stored at -80 °C in a mechanical chest freezer, but it remains to be clarified whether PBSCs can be stored for the long term. We evaluated viability and functions of PBSCs cryopreserved for more than 10 years with this simplified method. Although recovery rate and viability of CD34(+) cells were significantly decreased, myeloid differentiation potential and in vivo reconstitution and self-renewal potential of CD34(+) cells in a xenogeneic engraftment assay were maintained for more than 10 years. These results indicate that PBSCs can be stored at -80 °C for years. Although accumulation of clinical engraftment data is required to confirm our results, this simplified cryopreservation will thus meet the increasing worldwide demand for PBSC transplantation in a region with limited resources.

Method validation for automated isolation of viable peripheral blood mononuclear cells.

This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood, and compare the two methods. The manual method was optimized for whole blood centrifugation speed, gradient type (Ficoll, Leucosep, CPT), and freezing method (Mr Frosty, Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield, viability, recovery, white blood cell (WBC) subpopulation distribution, gene expression, and lymphoblastoid cell line (LCL) transformation. An initial centrifugation of whole blood at 2000 g was considered optimal for further processing, allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield, viability, and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality, quantity, and WBC subpopulation distribution were seen between the two freezing methods, and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity, recovery, viability, WBC subpopulation distribution, gene expression, and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential. We validated the first fully automated method for isolating viable PBMCs, including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection.

Cryopreservation of boar sperm comparing different cryoprotectants associated in media based on powdered coconut water, lactose and trehalose

In swine spermatozoa, the damage caused by cryopreservation is more severe than other species, provoking reduced potential for fertilization. Adjustments in the freezing extender composition may be an important alternative to increase its efficiency. The objective of this study was to test the efficiency of different cryoprotectant solutions during cryopreservation of swine semen with a controlled cooling curve. Three cryoprotectant solutions (5% dimethylformamide, 3% glycerol and the combination of these two cryoprotectants) were used in association with three base media (powdered coconut water, lactose and trehalose), constituting nine different treatments. The semen was frozen using a controlled-rate freezer (TK-3000). After thawing, semen was evaluated for total sperm motility, vigor, morphology, plasma membrane integrity and acrosome integrity. Cryopreservation with the controlled curve using an automated system showed satisfactory results, guaranteeing practicality and repeatability for the process of freezing swine sperm. With this curve, the solutions of lactose, trehalose and powdered coconut water associated with glycerol, as well as the solution of coconut water containing dimethylformamide, presented higher quality of sperm compared to the other solutions. Powdered coconut water associated with dimethylformamide appears as a new solution for swine sperm cryopreservation. The freezing controlled curve used in this study allowed standardization of the cryopreservation technique.

Comparison of two different methods in the cryopreservation of Eurasian perch (Perca fluviatilis) sperm

Two different cryopreservation methods were compared and an optimal dilution ratio for the use of controlled-rate freezer (CRF) was established for Eurasian perch (Perca fluviatilis) sperm. Progressive motility (72±15%) and curvilinear velocity (VCL, 146±11μm/s) of sperm cryopreserved with CRF did not reduce significantly compared to fresh sperm [progressive motility (90±4%), VCL (173±24μm/s)]. On the other hand, progressive motility (62±15%) and VCL (120±21μm/s) of sperm cryopreserved with the conventional floating frame technique were significantly lower when compared to the fresh control. Sperm in both cryopreserved groups showed significantly higher straightness [STR, CRF (84±4%), frame (84±2%)] than in the fresh control group (68±4%). Perch sperm cryopreserved with CRF at a dilution ratio of 1:20 showed significantly higher progressive motility (49±6%) than at a ratio of 1:5 (39±6%) and showed significantly higher VCL (129±11μm/s) than at dilution ratios of 1:10 (112±17μm/s) and 1:5 (115±9μm/s).

Conservation of Mouse Models Through Embryo Freezing

The ability to interrogate the entire coding sequence of the mouse combined with the tools to manipulate the genome has firmly established the mouse as the model organism of choice for studying the causes of human disease. Consequently, a huge number of novel mouse models are generated each year to support active research programs. However, it is neither ethically justifiable, nor economically viable to maintain mouse colonies on the shelf that are not part of active research programs. This means that novel mouse lines have to be preserved in some way. If this is not done and the line is simply killed off, the genetics will be lost to future generations of scientists. This article describes the current practices used in cryopreservation laboratories to archive and recover mouse embryos frozen using controlled-rate freezing and vitrification techniques.