Thaw Viability Research Articles

Impact of controlled ice nucleation on intracellular dehydration, ice formation and their implications on T cell freeze–thaw viability

Cryopreservation is important in manufacturing of cell therapy products, influencing their safety and effectiveness. During freezing and thawing, intracellular events such as dehydration and ice formation can impact cell viability. In this study, the impact of controlling the ice nucleation temperature on intracellular events and viability were investigated. A model T cell line, Jurkat cells, were evaluated in commercially relevant cryoformulations (2.5 and 5 % v/v DMSO in Plasma-Lyte A) using a cryomicroscopic setup to monitor the dynamic changes cells go through during freeze–thaw as well as a controlled rate freezer to study bulk freeze–thaw. The equilibrium freezing temperatures of the studied formulations and a DMSO/Plasma-Lyte A liquidus curve were determined using DSC. The cryomicroscopic studies revealed that an ice nucleation temperature of −6°C, close to the equilibrium freezing temperatures of cryoformulations, led to more intracellular dehydration and less intracellular ice formation during freezing compared to either a lower ice nucleation temperature (−10 °C) or uncontrolled ice nucleation. The cell membrane integrity and post thaw viability in bulk cryopreservation consistently demonstrated the advantage of the higher ice nucleation temperature, and the correlation between the cellular events and cell viability.

Key quality parameter comparison of mesenchymal stem cell product cryopreserved in different cryopreservation solutions for clinical applications.

Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs. MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3M/mL), 1:1 dilution (from 6M/mL), or 1:2 dilution (from 9M/mL) with PLA/5% HA, to achieve uniform concentration at 3M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis. MSCs can be cryopreserved up to 9M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.

Abstract 12 Phase I Study of Cord Tissue Derived Mesenchymal Stromal Cells in COVID-19–Related Acute Respiratory Distress Syndrome

IntroductionSARS-CoV-2 infection results in the COVID-19 disease that caused a global pandemic. In severe cases, COVID-19 leads to acute respiratory distress syndrome (ARDS), due to direct lung injury and hyperinflammatory response. COVID-related ARDS treatment now includes remdesivir, dexamethasone, and anti-inflammatory monoclonal antibodies, which have decreased the mortality rate, yet patients continue to die from sepsis or multiorgan failure and new treatments are needed. The use of mesenchymal stromal cells (MSC) offers a unique therapeutic option that may shorten time to lung injury resolution through anti-inflammatory, immune-modulatory, and regenerative mechanisms.ObjectiveThe aim of this study was to test the safety of human cord tissue-derived MSCs (hCT-MSC) in patients with COVID-related ARDS. This study was funded by The Marcus Foundation.MethodsIn this phase I multisite study, 10 adults with COVID- related ARDS were treated with 3 daily intravenous infusions of hCT-MSCs (1 million cells/kg/dose, maximum dose 100 million cells with a post thaw viability ≥70%). Patients were excluded if they had evidence of multiorgan failure, immunodeficiency, or were receiving extracorporeal membrane oxygenation or not expected to survive more than 24 hours. The primary endpoint was short-term safety of hCT-MSC infusions. The secondary endpoints included 28-day survival and changes in the Murray Lung Injury Score.ResultsFrom August to November 2020, 10 patients (7 females, 3 males; 2 Black, 6 White, 2 other; 3 Hispanic or Latino), with a median age of 61.5 years (range 39-97), were enrolled at 2 sites. There were no infusion-related or study-related adverse events. The average cell dose administered was 0.94 ± 0.29 cells/kg, and average cell viability was 85% ± 11%; 5 of 30 (17%) doses were less than the study dose, and 29 of 30 (97%) met the ≥70% viability criteria. There were 28 non-serious adverse events in 3 unique patients and 2 serious adverse events in 2 unique patients, which were expected and deemed unrelated to the study product. Five patients died: 3 by day 28 and 2 by day 90. All deaths were determined to be unrelated to the hCT-MSCs. The Murray Lung Injury Score did not appear to change over the 28-day study period.DiscussionhCT-MSCs infusions are safe in patients with COVID-related ARDS. Future studies determining their efficacy are warranted.

Effect of Ovarian Storage Temperature and Time on Post Thaw Viability and Maturation Rate of Vitrified Immature Oocytes in Sheep

BACKGROUND: Vitrification of oocytes as a method of cryopreservation is quite successful, although it is still being standardized because of structural and molecular sensitivity of oocytes to the cooling and freezing process. OBJECTIVE: To investigate the effect of ovarian storage temperature and time on post thaw viability and maturation rate of vitrified immature oocytes in sheep. MATERIALS AND METHODS: The work consisted of oocyte collection from ovaries of abattoir sheep stored at various temperature (0 °C , 4 °C and 25 °C) and time (0 h, 6 h, 12 h and 24 h) combinations and post thaw viability and in vitro maturation rate evaluation. Vitrification was done in 30% vitrification solution, using ethylene glycol and DMSO, with post vitrification evaluation after 1 week 's storage. RESULTS: Significantly higher post thaw viability was observed after storage at 0 °C for 6 h (95.3%) followed by 12 h (85%), with lowest value at 24 h (66.7%). However at 4 °C and 25 °C, values were nonsignificantly higher after 6 h (96.5 and 100% respectively) followed by 12 h (93 and 100%), with significantly lower values after 24 h (85.7 and 90.7%). At storage temperatures of 25 °C and 4 °C, a significantly higher percentage of mature oocytes was observed after 6 h (40 and 39.1%), 12 h (37.3 and 38.1%) and 24 h (34.6 and 36.4%) storage times compared to that at 0 °C (20.3% at 6 h, 14.2% at 12 h and only 13.8% at 24 h). However, at all storage temperatures, there was a tendency for the level of mature oocytes to decrease with storage time, and the levels were significantly lower than the control. CONCLUSION : Acceptable post thaw viability and in vitro maturation rates for oocytes is maintained up to 24 h in ovaries stored at 4 °C and 25 °C compared to at 0 °C, and these conditions may be used for the storage of ovaries meant for oocyte preservation.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200–300 μm) were randomly assigned into four groups. Group1 – Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2– FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2– FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (−196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

The Post-Thaw Recovery of Cryopreserved Chimeric Antigen Receptor (CAR) T-Cells during Manufacture Is Better Than That of Cryopreserved Peripheral Blood CD3+ Cells

As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy extend beyond early phase trials, larger scale cell manufacturing processes incorporating cryopreservation steps become a logistical necessity. Mononuclear cells (MNC) collected by leukapheresis, which serve as the pre-culture “starting” fraction (PeC) for CAR T-cell production, may need to be cryopreserved prior to cell transportation to a centralized manufacturing facility. PeC aliquots may be cryopreserved for repeat cultures in cases of primary culture failure or for patient re-dosing. Further, cryopreservation of the post-culture (PoC) CAR T-cell product is essential in order to ease post-manufacture transportation, ensure completion of regulatory testing prior to product release and avoid product wastage due to unanticipated patient-related delays. The effect of cryopreservation on CAR T-cell viability, expansion, phenotype and transduction efficiency merit evaluation prior to routine use.From 2012 to 2017, data was obtained on 160 consecutive autologous CAR T-cell cultures initiated for patient infusion on 6 clinical trials of different CAR T-cell types (1. Anti-CD19-pediatric trial (n=58), 2.Anti-GD2 (n=14), 3.Anti-BCMA (n=21), 4.Anti-CD22 (n=43), 5. Anti-CD19-adult trial (n=22), 6. Anti-CD30 (n=2)).Following autologous leukapheresis collection, PeC was either cryopreserved-thawed (PeC-C) or used fresh (PeC-F) for CAR T culture initiation, depending on patient needs. Thereafter, cell selection, transduction-expansion and harvest were performed according to trial protocol specific instructions. The harvested PoC was again cryopreserved (PoC-C) or infused fresh (PoC-F). Data on patient demographics, number, timing and duration of cryopreservation, Total Nucleated Cell (TNC) count, viability, fold expansion (FE), transduction efficiency (TE), CD3% and CD4/CD8 ratios was collected. Post thaw TNC recovery (PTR) % was calculated as TNC*viability at thaw/TNC* viability at cryopreservation.Afterdiscarding 13 products due to manufacturing failure (MF) during culture, 147CAR T infusates were prepared for pediatric or adult patients with hematologic malignancies (135, 91.8%) or solid tumors (12, 8.2%). Mean age was 27.7 years (±19.9), weight 60.7 kg (±26.3), median CAR T-cell dose of 1x106 CAR-transduced viable CD3+ cells/kg (Range: 1x105 - 1x107). Gamma-retroviral or lentiviral vectors were used for CAR delivery in 88 (59.9%) and 59(40.1%) infusates, respectively. Pre-culture lymphocyte concentration included ficoll alone (42, 28.6%), flask adherence and/or elutriation (14, 9.5%), CD3.CD28 and/or CD4/CD8 selection (68, 46.3%). Of the 147 infusates, PeC-C and PoC-C were 69 and 78, respectively. Median duration of PeC-C and PoC-C was 6 days (Range: 3-868) and 9 days (Range: 1-408), respectively. TNC and CD3 PTR were lower in PeC-C than in PoC-C (Figure). At harvest (PoC), Mean FE, TE, CD3% and CD4/CD8 ratio were 12.8 (±12.5), 54.3 (±21.9), 97.6 (±4.4) and 1.9 (±2.0) respectively. In multivariate analyses, FE, TE, CD3% and CD4/CD8 ratios at harvest were unaffected by PeC-C status or duration. TE differed by trial. Twelve out of 13 (92%) MFs were due to failure of PeC transduction and/or expansion. Of the 12 MFs, 6 products were PeC-C (50%); 6 were PeC-F (50%). Among the 6 PeC-C MFs, none of the failures could be attributed to cryopreservation. In fact, 4 of the 6 PeC-C cultures were re-initiated with an additional PeC-C and all 4 yielded a successful product.Pre-culturecryopreservation impacts viable TNC and CD3% recovery during CAR T-cell manufacture compared to post-culture cryopreservation. Within this PeC fraction, CD3+ cells appear to have better viable recovery than the whole TNC fraction. This is likely due to a higher (differential) loss of monocytes and NK cells within TNCs. Despite these findings, most products manufactured after initial cryopreservation met clinical dose requirements. FE, TE, CD3% and CD4/CD8 ratios were unaffected by initial cryopreservation. The post thaw viability of CAR T-cells cryopreserved post culture was greater than cryopreserved peripheral blood CD3+ cells. Cryopreservation at either end of CAR T-cell manufacture is a viable strategy and may ease logistical constraints associated with large scale cell production. If optional, CAR T-cells cryopreserved post manufacture may be preferable to cryopreserving the starting fraction. [Display omitted] DisclosuresKochenderfer:Bluebird bio: Research Funding; Kite Pharma: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.

Low concentrations of 3-O-methylglucose improve post thaw recovery in cryopreserved bovine spermatozoa

A number of studies have explored the use of membrane permeable (usually metabolizable) and membrane impermeable saccharides to protect cells in general, and sperm in particular during cryopreservation. Critical concentrations for protective levels of sugars frequently range between 50 mmol/L and 500 mmol/L, where efficacy is attributed to the sugar's membrane stabilizing and glass forming attributes and colligative effects that reduce intra- and extracellular salt concentrations during freezing. Many studies on bull sperm have demonstrated that both permeating and non-permeating sugars have negligible positive effects on post-thaw viability. Recently, however, a non-metabolizable sugar, 3-O-Methylglucose (3-OMG), was shown to protect hepatocytes during liver cryopreservation at 0.1–0.3 mol/L. Because glucose is readily transported into sperm, we hypothesized that 3-OMG could be a new class of cryoprotectant to explore in bull sperm. Here we present positive results demonstrating that 3-OMG improves post thaw viability in bull sperm, and that this effect is not likely due to improved glass forming capabilities. In particular, in experiment 1, 3-OMG was added to the Tris-egg yolk-glycerol base media at levels from 0 mmol/L to 200 mmol/L. Semen from four bulls was collected and diluted with one of the cryopreservation media, cooled, and frozen following industry standard practices. Motility and mitochondrial activity were negatively impacted when concentration of 3-OMG was more than 25 mmol/L. Therefore, we explored lower concentrations in experiment 2, where semen from eight bulls was used to evaluate concentrations 5 mmol/L, 15 mmol/L and 25 mmol/L of 3-OMG compared with control. Motility and progressive motility in 5 mmol/L 3-OMG and in the control were significantly higher than 15 mmol/L and 25 mmol/L 3-OMG, whereas mitochondrial activity and acrosome integrity in 5 mmol/L 3-OMG were significantly better than the control freezing medium. In experiment 3, to evaluate whether the improved effects of 3-OMG are due to its non-metabolizing property, or due to colligative effects, we compared post-thaw viability in semen from four bulls cryopreserved with 5 mmol/L glucose, sucrose, or 3-OMG. Motility and progressive motility was significantly improved in 3-OMG compared to glucose or sucrose groups which were comparable to the EY control. In conclusion, 3-OMG at a concentration of 5 mmol/L in Tris-egg yolk-glycerol medium improves the post thaw motility, progressive motility and viability of bull sperm. The mechanism of action is not understood but because the efficacy is maximal at low concentrations, it is not likely due to improved intra- or extracellular glass forming abilities and may demonstrate a different protective mechanism than was shown in hepatocytes.

PENGARUH LAMA WAKTU PENGHITUNGAN STRAW DAN POSISI JARAK STRAW DIATAS PERMUKAAN NITROGEN CAIR TERHADAP MOTILITAS DAN VIABILITAS SEMEN BEKU SAPI MADURA POST THAWING

Aims of this research was to determine the effect of the duration of straw count and the position of straw from liquid nitrogen surface to the motility and viability of post thawing frozen Madura cattle. This study used a sample derived from Madura cattle (Pasean OO 1220) from BBIB singosari as much as 54 straw divided into 9 treatments. P1 (1cm 10 sec), P2 (1cm 20 sec), P3 (1cm 40 sec), P4 (2cm 10 sec), P5 (2cm 20 sec), P6 (2cm 40 sec), P7 (3cm 10 sec), P8 (3cm 20 sec), P9 (3cm 40 sec) and repeated as much as 6 times then observed for motility and viability of post thawing spermatozoa. The data obtained were analyzed using SPSS 16.0. The results showed that the duration of straw count and the position of straw from liquid nitrogen significant (P&lt;0,05) on post thawing motility. The position of straw from liquid nitrogen signifant (P&lt;0,05) on post thawing viability. The conclusion of this research showed that 1 cm 40 sec give the higher post thawing motility.

Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.

Effect of high nucleated cell concentration on product viability and hematopoietic recovery in autologous transplantation.

Administering lower total product volumes with high nucleated cell (NC) concentrations may have the potential benefit of decreasing volume- and dimethyl sulfoxide (DMSO)-related patient complications, while maximizing the laboratory's freezer storage capacity. Our study is a retrospective investigation of the effect of HPC(A) products with cell concentrations greater than 3 × 108 NC/mL on clinical and product outcomes in patients undergoing autologous peripheral blood stem cell (PBSC) transplantation. A total of 113 consecutive patients with hematological malignancies who underwent autologous PBSC transplantation were included in this retrospective analysis. The primary outcomes were days to initial absolute neutrophil count (ANC) recovery and initial platelet recovery. The secondary outcomes included the storage duration, segment thaw viability, and dose of viable CD34+ cells/kg administered. Of 92 patients and 176 apheresis procedures, 81 patients received HPC(A) products with high NC concentration (4.1 × 108 NC/mL), and 11 patients received low NC concentration products (2.4 × 108 NC/mL). There were no observed differences in clinical outcomes with respect to ANC recovery (14 vs. 14 vs. 12 days) and platelet recovery (16 vs. 16 vs. 15 days) when very high NC (5.2 × 108 NC/mL) and high NC (4.1 × 108 NC/mL) groups were compared to the low NC group (2.4 × 108 NC/mL). Our retrospective investigation provides further supporting evidence that HPC(A) products with cell concentration greater than 3 × 108 NC/mL did not show detrimental effects on the clinical outcomes in patients undergoing autologous PBSC transplantation.

Correlation of the Effective Residual Vital (HPC) Dose with Engraftment Kinetics in Autologous Transplantation Amongst Myeloma and Lymphoma Patients

<h3>Introduction</h3> In clinical practice, a hematopoietic progenitor cell (HPC) dose of 2 million CD34+ cells/kg is the desired minimum needed for timely neutrophil and platelet (PLT) engraftment in autologous hematopoietic cell transplantation (HCT). Patients frequently receive higher HPC doses with the median pre-cryopreservation dose at our center being 5 million CD34+ cells/kg adjBW25%. Cryopreservation (CP) and thawing leads to cell loss through mechanisms including activation of the caspase pathway. The degree of loss in post-CP HPC doses can range from 20 to 85%; hence the cell dose that is able to effectuate engraftment is determined by the percentage of surviving cells in the initial pre-CP cell dose. This effective residual vital dose (ERVD) is the product of the pre-CP cell dose and the post thaw viability percentage. <h3>Objective</h3> Correlate viability, HPC cell dose and the ERVD to engraftment. <h3>Method</h3> We conducted a retrospective review of autologous HCT patients at our center to correlate the ERVD with engraftment. Twenty-five patients, ages 32 to 76, receiving 27 transplants had HPC infusions during 1 or 2 sessions within 3 days of each other. Diagnoses were multiple myeloma (MM) (n=20) and lymphoma (n=7). Engraftment of neutrophils and PLTs was defined using CIBMTR definitions. Viability was assessed by 7AAD on CD34+ cells. Data was analyzed with Excel Correlation Function which calculates the Pearson Product-Moment Correlation Coefficient for 2 sets of values. <h3>Results</h3> The median neutrophil and PLT engraftment times were 11 and 16.5 days, respectively. Correlation of the HPC dose of the total population with neutrophil engraftment data, revealed the r value to be -0.31 for all patients, -0.20 for lymphoma patients, and -0.32 for MM patients. Cell dose correlation with PLT engraftment revealed the r value to be -0.43 for the total population (Figure 1), -0.52 for the lymphoma subset, and -0.55 for the MM subset. We found a moderately strong correlation of ERVD with PLT engraftment with an r value of -0.56 for the entire population, i.e. as ERVD increased, time to PLT engraftment decreased (Figure 2). When separated by diagnosis (MM vs lymphoma) the ERVD correlated moderately with PLT engraftment, r value of -0.57 amongst the MM subset and -0.88 for the lymphoma subset (Figure 3). Neutrophil engraftment had a weaker correlation with the ERVD, r value of -0.33 for the entire population, -0.41 for the MM subset, and -0.68 for the lymphoma subset. <h3>Conclusion</h3> Based on our data, the ERVD has a moderately strong correlation with PLT and neutrophil engraftment in a population of MM and lymphoma patients undergoing autologous HCT. The correlation was stronger for PLT engraftment and strongest amongst patients with lymphoma. To achieve our median engraftment times for neutrophils and PLTs the optimal ERVD of approximately 3 Million CD34/Kg adjBW25% is suggested.

Trehalose uptake and dehydration effects on the cryoprotection of CHO–K1 cells expressing TRET1

Chinese hamster ovary cells (CHO–K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO–K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze–thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution. In this freezing solution, both kinds of CHO–K1 cells are independently dispersed with various amount of trehalose, and then put into the CO2 incubator for 0–6 h. After a set immersion time, the cell-suspended sample is cooled to 193 K, stored for 1 week, then quickly thawed at 310 K and its viability measured. The uptake amount of intracellular trehalose is measured before freezing. We find an upper limit for the uptake amount of trehalose when the extracellular trehalose concentration is about 400 mM, at which the freeze–thaw viability is the highest. When the extracellular trehalose concentration exceeds 400 mM, shorter immersion times are needed to obtain the maximum freeze–thaw viability. Also, longer immersion weakens the cells. Our analyses indicate that when the extracellular trehalose-concentration is less than 400 mM, the trehalose uptake occurs more slowly with less dehydration, resulting in less stress on the cell. When the extracellular trehalose concentration exceeds the saturation level, the cell is stressed by the excess dehydration due to the remaining osmotic pressure, with apoptosis occurring before freezing.

Comparison of the methods evaluating post thawing viability of peripheral blood stem cell graft

ObjectiveReliable and pratique methods are essential for rapid and accurate determination of post thawing viability of peripheral blood stem cell (PBSC) graft before hematopoietic stem cell transplantation. In this study, Trypan Blue (TP) Eosin Y (EO), and Acridine-orange-ethidium bromide (AO/EB), which are of the methods commonly used for the assessment of viability in clinic practice, were compared with the flow cytometry-7AAD (7AAD) method, which is a more sensitive method. The aim of this study is to examine which method evaluates postthawing viability in a more compatible manner with 7AAD. Materials-methodsPostthawing viability rates were examined simultaneously by means of four different methods before hematopoietic stem cell transplantation in a total of 20 PBSC graft. The results obtained from the AO/EB, TP, EO methods were evaluated with the flow cytometry-7AAD in terms of concordance. ResultsThe AO / EB was determined to be the method having the best concordance with the flow cytometry-7AAD method. Although, at a lower level compared to the AO/EB method, the EO method had a statistically significant concordance with the flow cytometry-7AAD method. No statistically significant concordance was detected between the TP method and 7AAD method in terms of viability results. ConclusionThe AO/EB method was identified to be the method having the best compatibility with the flow cytometry -7AAD method in showing the viability of the cryopreserved PBSC graft. In the viability assessment of PBCS graft using light microscopy, the EO may be preferred since is more sensitive compared to the TP method.

Seminal plasma amino acid profile in different breeds of chicken: Role of seminal plasma on sperm cryoresistance.

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’) showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

CD19-Directed CAR T-Cell (CTL019) Product Viability and Clinical Outcomes in Non-Hodgkin Lymphomas and B-Cell Acute Lymphoblastic Leukemia

Introduction: CTL019 is an anti-CD19 genetically modified autologous T-cell immunotherapy developed at the University of Pennsylvania (Penn) that was recently approved for treatment of relapsed/refractory pediatric and young adult B-cell acute lymphoblastic leukemia (ALL) and adult relapsed/refractory diffuse large B-cell lymphoma (DLBCL) as tisagenlecleucel (Novartis). For ALL, the FDA-approved dose is 0.2 to 5.0 x 106 CAR-positive viable T cells per kg of body weight for patients ≤ 50 kg or 0.1 to 2.5 x 108 CAR-positive viable T cells for pts > 50 kg; for DLBCL, the FDA-approved dose is 0.6 to 6.0 x 108 CAR-positive viable T cells. For CTL019 manufactured at Penn, the dose is determined by flow cytometric staining of CAR-positive T cells, which are cryopreserved in product bags along with replicate aliquots of the final formulation in vials, simultaneously cryopreserved for release testing. The CTL019 product release criteria include a post thaw viability assessment using a vial of replicate aliquot of the final formulation for Trypan blue exclusion or dual fluorescence automated cell counting (Luna-FL, Logos Biosystems). There are no published data examining the relationship between CTL019 viability release testing and clinical outcomes.Methods: We analyzed CTL019 post thaw viability release testing in patients treated on one prospective single institution clinical trial of CD19-expressing non-Hodgkin lymphomas (NHL) (NCT02030834) and two single-institution prospective pediatric ALL clinical trials (NCT01626495 and NCT02906371). Patients were assessed for response to therapy and CAR T-cell expansion. Receiver operating characteristic (ROC) curves were constructed for prediction of complete responses based on sensitivity and specificity of CAR T-cell product post thaw viability release test results.Results: 39 pts with relapsed/refractory NHL (24 diffuse large B-cell lymphoma and 15 follicular lymphoma) were enrolled and received the protocol-specified dose of CTL019. Best response rate was 56% (22/39) complete responses (CR). 123 pts with relapsed/refractory pediatric ALL were enrolled and received the protocol-specified dose of CTL019. Best response rate was 96% (118/123) CR/complete remission with incomplete blood count recovery (CRi). For patients with NHL infused with CTL019, product % viability had a median of 89.8% viability (range: 73.7%-97.7%); product % viability quintiles were as follows: 20%-tile=81.7%, 40%-tile=88.3%, 60%-tile=91.1%, 80%-tile=94.8%). ROC area for NHL patients was 0.47 (95%CI: 0.28-0.65). For patients with ALL infused with CTL019, product % viability had a median of 89.3% viability (range: 56.0%-98.4%); product % viability quintiles were as follows: 20%-tile=82.3%, 40%-tile=87.5%, 60%-tile=90.9%, 80%-tile=94.4%). ROC area for ALL patients was 0.52 (95%CI: 0.32-0.71). For patients with NHL, progression-free survival (PFS) was not significantly influenced by product viability release test results by Cox proportional hazards (HR: 1.0, 95%CI: 0.94-1.09, p=0.7). For patients with NHL, peak CAR T-cell expansion was not significantly correlated with product viability release test results (r2=0.12, p=0.5). Data collection for Cox analysis to investigate the effect of release test viability on PFS and correlation of release test viability with peak CTL019 expansion in ALL is ongoing and will be presented.Conclusions: Our data suggest that, within the ranges obtained in these trials, there is no clear dose-response relationship between CTL019 product viability release test results and clinical response rates in pediatric and young adult ALL or DLBCL. [Display omitted] DisclosuresChong:Novartis: Consultancy. Levine:Cure Genetics: Consultancy; Brammer Bio: Consultancy; CRC Oncology: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Siegel:Novartis: Research Funding. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. June:Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Schuster:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees.

Comparative efficiency of goat mesenchymal stem cell isolation from bone marrow and bone chip

Bone marrow mesenchymal stem cell (BM-MSC) is a type of adult stem cell that offers tremendous potential in clinical use and gene-based therapies. The use of mesenchymal stem cells (MSCs) in animal models has shown promising evidences for the cure of certain human diseases. However, in animals certain obstacles like lack of standardised isolation methods and tissues used to derive MSCs often leading to lower yield of MSCs along with cumbersome characterisation steps can hamper their use, particularly for autologous stem cell therapy. In the present experiment, MSCs were isolated from bone marrow and cancellous bone chip collected from iliac crest of goats. The yield of MSCs derived from bone marrow and bone chip was compared and MSCs derived from both the sources were characterized based on cell morphology, plastic adherence, colony-forming property and expression of cell surface markers. Furthermore, these cells were cryopreserved and evaluated for post thaw viability. Interestingly, the yield of bone chip-derived MSCs was ∼3000 times higher when compared with bone-marrow-derived MSCs. MSCs derived from both the sources were plastic adherent, displayed fibroblastoid morphology, exhibited MSC-specific marker expression profile (CD105+CD90+CD45−CD34−), and possessed colony forming capacity when cultured at lower densities. Thus, it was concluded that bone chip could be used as an efficient tissue source for MSC isolation in goats.

98 - Post Thaw Viability and Engraftment of Cryopreserved HPCs with Increased Nucleated Cell Concentration

98 - Post Thaw Viability and Engraftment of Cryopreserved HPCs with Increased Nucleated Cell Concentration

Measuring post thaw viability to validate expiry of HPCs in extended cryopreservation storage

Measuring post thaw viability to validate expiry of HPCs in extended cryopreservation storage

A09 Session 2: Biopreservation of biologics: Translation from stem cells to cancer : Cryobiological biophysics and freeze–thaw viability of the HL-1 cardiac muscle cell line: Implications for cryoablation studies

Atrial fibrillation (AF) is the irregular beating of the heart affecting an estimated 2.7 million Americans and responsible for 26 billion dollars of the nation’s annual healthcare cost. Cryoablation is a clinically approved technique for the treatment of paroxysmal AF in which an ablation catheter rapidly cools the cardiac tissue. Through freezing, discrete lesions are created in the region where the pulmonary veins join with the atria in order to terminate the conduction of abnormal electrical signals responsible for AF. While the efficacy of AF cryoablation is well known, the thermal thresholds and mechanisms of cryoinjury that determine efficacy are not known thereby hindering development of the next generation of AF cryoablation systems. This study begins to address this by improving understanding of the mechanisms and thermal thresholds of cryodestruction in a cardiac muscle cell line (HL-1). Specifically, we determine biophysical parameters governing known mechanisms of injury (membrane permeability and likelihood of intracellular ice formation, IIF). Additionally we correlate these biophysics and thermal history (cooling rate and end = temperature) to post-thaw viability in vitro in HL-1s guide optimal lesion formation and thus cryoablation device design. HL-1 cells were cultured using previously published methods. Cells were harvested using trypsinization and kept as a suspension on ice until needed. A temperature stage was used in conjunction with a light microscope to observe freezing of the samples. The controlled thermal history included 3 variables including: cooling rate (0.5 to 130 °C/min), end temperature (−5 to −60 °C), and hold time (0–5 min). These were varied in an experimental matrix to determine the importance of each. Before cooling, samples were pre-nucleated by applying a chilled needle on the outer edge of the sample. Change in water content within cells (cellular dehydration) was measured based on observed changes to projected cell area during freezing. IIF events were measured based on observing a sudden change in opacity (darkening) of the cell interior. Measured data were applied to water transport and nucleation models to determine membrane permeability and IIF nucleation parameters. Additionally, post-thaw viability of samples were determined after a rapid thaw and 15 min incubation period using a fluorescence membrane dye exclusion assay. For cases when the end temperature was set as −20 °C the highest viability of around 15% was observed at a cooling rate of 5 °C/min, while slower or faster cooling rates resulted in reduced viability. Subsequent measurements with cooling rate set as 5 °C/min and varied end temperatures resulted in viability values around 94%, 72%, 15%, 6% and less than 5% at −5 °C, −10 °C, −20 °C, −40 °C, and −60 °C, respectively. Our results suggest a large difference in post-thaw cell viability dependent on cooling rate and end temperature, while difference in hold times (0, 1, and 5 min) appeared less important. The application of this work to pulmonary vein freezing numerical models, and to future work in more translational systems such as attached (monolayer, artificial constructs) cell systems and tissues will be discussed.

Post thaw viability of cryopreserved HPC with increased nucleated cell concentration

Post thaw viability of cryopreserved HPC with increased nucleated cell concentration