CPA toxicity screening of cryoprotective solutions in rat hearts.

In clinical practice, donor hearts are transported on ice prior to transplant and discarded if cold ischemia time exceeds ∼5 h. Methods to extend these preservation times are critically needed, and ideally, this storage time would extend indefinitely, enabling improved donor-to-patient matching, organ utilization, and immune tolerance induction protocols. Previously, we demonstrated successful vitrification and rewarming of whole rat hearts without ice formation by perfusion-loading a cryoprotective agent (CPA) solution prior to vitrification. However, these hearts did not recover any beating even in controls with CPA loading/unloading alone, which points to the chemical toxicity of the cryoprotective solution (VS55 in Euro-Collins carrier solution) as the likely culprit. To address this, we compared the toxicity of another established CPA cocktail (VEG) to VS55 using ex situ rat heart perfusion. The CPA exposure time was 150 min, and the normothermic assessment time was 60 min. Using Celsior as the carrier, we observed partial recovery of function (atria-only beating) for both VS55 and VEG. Upon further analysis, we found that the VEG CPA cocktail resulted in 50 % lower LDH release than VS55 (N = 4, p = 0.017), suggesting VEG has lower toxicity than VS55. Celsior was a better carrier solution than alternatives such as UW, as CPA + Celsior-treated hearts spent less time in cardiac arrest (N = 4, p = 0.029). While we showed substantial improvement in cardiac function after exposure to vitrifiable concentrations of CPA by improving both the CPA and carrier solution formulation, further improvements will be required before we achieve healthy cryopreserved organs for transplant.

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming.

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation.

Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24-48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes.

Nanoencapsulated anti-CK2 small molecule drug or siRNA specifically targets malignant cancer but not benign cells.

CK2, a pleiotropic Ser/Thr kinase, is an important target for cancer therapy. We tested our novel tenfibgen-based nanocapsule for delivery of the inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and an siRNA directed against both CK2α and α' catalytic subunits to prostate cancer cells. We present data on the TBG nanocapsule itself and on CK2 inhibition or downregulation in treated cells, including effects on Nuclear Factor-kappa B (NF-κB) p65. By direct comparison of two CK2-directed cargos, our data provide proof that the TBG encapsulation design for delivery of drugs specifically to cancer cells has strong potential for small molecule- and nucleic acid-based cancer therapy.

Systemic administration of antisense oligonucleotides simultaneously targeting CK2α and α' subunits reduces orthotopic xenograft prostate tumors in mice.

CK2 is a highly conserved, ubiquitous, signal responsive protein serine/threonine kinase. CK2 promotes cell proliferation and suppresses apoptosis, and increased CK2 expression is observed in all cancers examined. We previously reported that direct injection of antisense (AS) CK2α phosphorothioate oligonucleotides (PTO) into xenograft prostate tumors in mice significantly reduced tumor size. Downregulation of CK2α in tumor cells in vivo appeared to result in overexpression of CK2α' protein. This suggested that in cancer cells downregulation of CK2α might be compensated by CK2α' in vivo, prompting us to design a bispecific (bs) AS PTO (bs-AS-CK2) targeting both catalytic subunits. bs-AS-CK2 reduced CK2α and α' protein expression, decreased cell proliferation, and induced apoptosis in cultured cells. Biodistribution studies of administered bs-AS-CK2 oligonucleotide demonstrated its presence in orthotopic prostate xenograft tumors. High dose injections of bs-AS-CK2 resulted in no damage to normal liver or prostate, but induced extensive cell death in tumor tissue. Intraperitoneal treatment with bs-AS-CK2 PTO decreased orthotopic tumor size and downregulated both CK2 mRNA and protein expression. Tumor reduction was accomplished using remarkably low doses and was improved by dividing the dose using a multi-day schedule. Decreased expression of the key signaling pathway proteins NF-κB p65 and AKT was also observed. We propose that the molecular downregulation of CK2 through bispecific targeting of the two catalytic subunits may be uniquely useful for therapeutic elimination of tumors.

Type I collagen structure regulates cell morphology and EGF signaling in primary rat hepatocytes through cAMP-dependent protein kinase A.

Adhesion to type 1 collagen elicits different responses dependent on whether the collagen is in fibrillar (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread and proliferate, whereas those adherent to collagen gel remain rounded and growth arrested. To explore the role of potential intracellular inhibitory signals responsible for collagen gel-mediated growth arrest, cAMP-dependent protein kinase A (PKA) was examined in hepatocytes adherent to collagen film or gel. PKA activity was higher in hepatocytes on collagen gel than on film during G1 of the hepatocyte cell cycle. Inhibition of PKA using H89 increased cell spreading on collagen gel in an EGF-dependent manner, whereas activation of PKA using 8-Br-cAMP decreased cell spreading on collagen film. PKA inhibition also restored ERK activation, cyclin D1 expression and G1-S progression on collagen gel, but had no effect on cells adherent to collagen film. Analysis of EGF receptor phosphorylation revealed that adhesion to collagen gel alters tyrosine phosphorylation of the EGF receptor, leading to reduced phosphorylation of tyrosine residue 845, which was increased by inhibition of PKA. These results demonstrate that fibrillar type 1 collagen can actively disrupt cell cycle progression by inhibiting specific signals from the EGF receptor through a PKA-dependent pathway.

The role of collagen structure in mitogen stimulation of ERK, cyclin D1 expression, and G1-S progression in rat hepatocytes.

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.