Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition.

BACKGROUND: Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor, BioLife Solutions, Inc.), the rate of addition of two cryopreservation solutions to cord blood units (CBUs), and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS: Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e., slow addition) or as a bolus injection (n = 6; i.e., fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays, total nucleated cells, and CD34+ cell counts were compared. RESULTS: Maximum temperature excursions observed were less than 6°C, regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e., final concentration ≤ 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION: Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time, formulation consistency, and reduced DMSO in the frozen product (≤ 5%).

Direct concentration measurements of the unfrozen portion of solutions under freezing.

Phase diagrams of solutions consisting of cryoprotective agents (CPA) are very useful in cryobiology research. Those diagrams depict the points of solution concentrations at corresponding temperatures: one of essential inputs that can be utilized to compute the volume response of cell under freezing process. However, generating such plots is costly and time-consuming. A direct method is proposed in this study to determine the solution concentration of unfrozen parts at multiple sub-zero temperatures. Measurements of binary solutions, composed of water and sodium chloride, were performed and compared with published data. Ternary solutions, consisting of water, sodium chloride and dimethyl sulfoxide, were also measured. The uniqueness and advantage achieved through the usage of this method are demonstrated when phase diagrams of complex cryopreservation solutions (CryoStor solutions including CryoStor Base and CryoStor 10) are generated. The temperature range where the method is utilized is either limited by the osmometry (0-3200 mmol/kg) or by the availability of liquid samples at sub-freezing temperatures. Modified methods will be required to address the limitation of osmolality measurements and the availability of sub-freezing liquid samples at lower temperatures.

Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution.

BACKGROUND AIMS: Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS: This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. RESULTS: The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS: This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.

Phosphorylation of actopaxin regulates cell spreading and migration.

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

Cryoablation of renal cancer: variables involved in freezing-induced cell death.

The detection of renal tumors has increased significantly over recent years resulting in a greater demand for novel, minimally invasive techniques. Cryoablation has emerged as a valuable treatment modality for the management of renal cancer. In an effort to detail the effects of freezing in renal cancer, the human renal cancer (RCC) cell line, 786-O, was evaluated in vitro. 786-O cells were exposed to a range of freezing temperatures from -5 to -40 degrees C and compared to non-frozen controls. The data show that freezing to -5 degrees C did not affect 786-O cell viability, while -10 degrees C, -15 degrees C, and -20 degrees C results in a significant loss of viability (23, 70, and 91%, respectively). A complete loss of cell viability was evident at temperatures of -25 degrees C and colder. Following this analysis, variables involved in the success of cryoablation were investigated. For each of the temperatures tested, extended freeze hold times and passive thawing rates resulted in more extensive cell damage. Additionally, a double freeze-thaw cycle significantly increased cell death compared to a single cycle (62% vs. 22% at -10 degrees C; 89% vs. 63% at -15 degrees C, respectively). While these variables play an important part in the effective application of cryoablation, a molecular understanding of the cell death involved is critical to improving efficacy. Apoptotic inhibition afforded 12% (-10 degrees C), 25% (-15 degrees C), and 11% (-20 degrees C) protection following freezing. Using fluorescence microscopy analysis, the results demonstrated that apoptosis peaked at six hours post-thaw. Next, apoptotic initiating agents including 5-FU and resveratrol (RVT) applied prior to freezing exposure resulted in a significant increase in cell death compared to either application alone. Importantly, the combination of RVT and freezing was noticeably less effective when applied to normal renal cells. The results herein demonstrate the efficacy of freezing and describe a novel therapeutic model for the treatment of renal cancer that may distinguish between cancer and normal cells.

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems.

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (-5 to -40 degrees C), and cells were thawed followed by return to 37 degrees C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At -15 degrees C, PC-3 yielded approximately 55% viability versus approximately 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze-thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching -40 degrees C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze-thaw cycles, and passive thawing to provide maximum cell destruction.

Addition of anticancer agents enhances freezing-induced prostate cancer cell death: implications of mitochondrial involvement.

Recent evidence suggests that the successful treatment of prostate cancer may require adjuvant therapies. Accordingly, a better understanding of the molecular mechanisms involved in current treatments may lead to enhanced efficacy by providing a basis for adjuvant therapies. In this study, we demonstrate that the combination of sub-lethal concentrations of chemotherapeutic agents prior to freezing (-15 degrees C) in a prostate cancer cell (PC-3) model results in enhanced efficacy over either treatment alone. Morphological analysis revealed that necrosis appeared to be the prevalent mode of cell death following adjuvant (in vitro) modeling, yet molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family. Freezing results in a time-dependent increase of the antiapoptotic Bcl-2 protein, while chemotherapy results in an increase of the pro-apoptotic Bax protein. Anti-apoptotic Bcl-2 protein levels increase over 3-fold following exposure to freezing. 5-Fluorouracil (5-FU) causes pro-apoptotic Bax levels to increase 2-fold during the drug exposure. The increase in Bax was also apparent following the combination of 5-FU/freezing, while Bcl-2 levels were maintained at or below control levels. This led to a shift in the Bcl-2 to Bax ratio to a pro-death tendency. Other effective cryo/chemo combinations were also found to provide similar effects. The combination of cisplatin/freezing resulted in a 4-fold increase in the ratio of Bax to Bcl-2 when compared to controls, which represented a 2-fold increase over the 5-FU/freezing-combination model. This increase may contribute to the continued reduction in cell number observed during the 13-day recovery period. Additionally, the addition of an apoptotic caspase inhibitor was not able to protect cultures from cell death following combination treatment. In conclusion, the data suggest that both Bcl-2 and Bax may, not only, play an important role in the efficacy of the cryo/chemo combination, but also the balance between the two may determine the role and extent of system destruction.