The Impact of Varying Cooling and Thawing Rates on the Quality of Cryopreserved Human Peripheral Blood T Cells.

For the clinical delivery of immunotherapies it is anticipated that cells will be cryopreserved and shipped to the patient where they will be thawed and administered. An established view in cellular cryopreservation is that following freezing, cells must be warmed rapidly (≤5 minutes) in order to maintain high viability. In this study we examine the interaction between the rate of cooling and rate of warming on the viability, and function of T cells formulated in a conventional DMSO based cryoprotectant and processed in conventional cryovials. The data obtained show that provided the cooling rate is -1 °C min-1 or slower, there is effectively no impact of warming rate on viable cell number within the range of warming rates examined (1.6 °C min-1 to 113 °C min-1). It is only following a rapid rate of cooling (-10 °C min-1) that a reduction in viable cell number is observed following slow rates of warming (1.6 °C min-1 and 6.2 °C min-1), but not rapid rates of warming (113 °C min-1 and 45 °C min-1). Cryomicroscopy studies revealed that this loss of viability is correlated with changes in the ice crystal structure during warming. At high cooling rates (-10 °C min-1) the ice structure appeared highly amorphous, and when subsequently thawed at slow rates (6.2 °C min-1 and below) ice recrystallization was observed during thaw suggesting mechanical disruption of the frozen cells. This data provides a fascinating insight into the crystal structure dependent behaviour during phase change of frozen cell therapies and its effect on live cell suspensions. Furthermore, it provides an operating envelope for the cryopreservation of T cells as an emerging industry defines formulation volumes and cryocontainers for immunotherapy products.

Spatial considerations during cryopreservation of a large volume sample.

There have been relatively few studies on the implications of the physical conditions experienced by cells during large volume (litres) cryopreservation - most studies have focused on the problem of cryopreservation of smaller volumes, typically up to 2 ml. This study explores the effects of ice growth by progressive solidification, generally seen during larger scale cryopreservation, on encapsulated liver hepatocyte spheroids, and it develops a method to reliably sample different regions across the frozen cores of samples experiencing progressive solidification. These issues are examined in the context of a Bioartificial Liver Device which requires cryopreservation of a 2 L volume in a strict cylindrical geometry for optimal clinical delivery. Progressive solidification cannot be avoided in this arrangement. In such a system optimal cryoprotectant concentrations and cooling rates are known. However, applying these parameters to a large volume is challenging due to the thermal mass and subsequent thermal lag. The specific impact of this to the cryopreservation outcome is required. Under conditions of progressive solidification, the spatial location of Encapsulated Liver Spheroids had a strong impact on post-thaw recovery. Cells in areas first and last to solidify demonstrated significantly impaired post-thaw function, whereas areas solidifying through the majority of the process exhibited higher post-thaw outcome. It was also found that samples where the ice thawed more rapidly had greater post-thaw viability 24 h post-thaw (75.7 ± 3.9% and 62.0 ± 7.2% respectively). These findings have implications for the cryopreservation of large volumes with a rigid shape and for the cryopreservation of a Bioartificial Liver Device.

A scale down process for the development of large volume cryopreservation.

The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water-ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications. This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner. It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48 h or 72 h in recovery cultures. Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation.

Advances in small molecules promoting neurotrophic function.

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons and have been implicated in the pathophysiology of numerous disease states. Although the therapeutic potential of neurotrophins has generated much excitement over the past decade, inconvenient pharmacokinetics and adverse side-effect profiles have limited the clinical usefulness of neurotrophic factors themselves. Compounds that mimic neurotrophin signaling and overcome the pharmacokinetic and side-effect barriers may have greater therapeutic potential. Here, we review the progress to date of clinical trials with direct neurotrophin modulators and describe alternative strategies to target (modulate) neurotrophin production and/or their signal transduction pathways. Particular emphasis is placed on small molecules that are able to modulate neurotrophin function in diseases of the nervous system. These alternative strategies show promise in preclinical studies, with some advancing into clinical development. Moreover, the recognition that clinically effective therapeutics, such as antidepressants and immunophilin ligands, can modulate neurotrophin function suggests that the concept of small molecule therapeutics that promote neurotrophic function may still be viable.

Molecular mechanisms of neurite growth with AMPA receptor potentiation.

Positive allosteric modulation of AMPA receptor function has therapeutic potential in a number of psychiatric disorders and neurodegenerative diseases. AMPA receptor potentiators can induce neurite sprouting in vivo. Using a strategy of combined morphological and biochemical analyses, we investigated the effect of the AMPA receptor potentiator LY404187 on neurite growth in the SH-SY5Y human neuroblastoma cell line. LY404187 (0.1-10 microM) increased average neurite length and neurofilament expression when co-administered with s-AMPA. Co-incubation with s-AMPA and LY404187 also increased Trk receptor expression. All actions of LY404187 were sensitive to AMPA receptor blockade by the selective antagonist CNQX (10 microM). Antibody sequestration of BDNF attenuated neurite growth following AMPA receptor potentiator administration, suggesting that LY404187 increases neurite length in vitro by a BDNF mediated mechanism. AMPA receptor potentiation activates multiple intracellular neurochemical cascades and the present report identifies BDNF as one key mediator of the neurotrophic effects of AMPA receptor potentiation.

Increased expression of macrophage receptor with collagenous structure (MARCO) in mouse cortex following middle cerebral artery occlusion.

Ischaemia induces activation of resident microglia and infiltration of peripheral monocyte/macrophage cells into the central nervous system. The role of scavenger receptors, receptors critical to the recognition and clearance of cell debris, has not been investigated during cerebral ischaemia. MARCO is an inducible member of the scavenger receptor family unique to cells of monocytic lineage and is a cell surface marker that plays a critical role in the differentiation of monocytes to dendritic cells. To understand the role of MARCO in cerebral ischaemia, we investigated its expression in mice following middle cerebral artery (MCA) occlusion. No MARCO mRNA expression was observed in naive mouse brain. There was no significant increase in expression of MARCO mRNA following transient occlusion (60min) of the MCA at any time point up to 24 h. However, a significant, marked increase in MARCO mRNA expression was observed at 24 h in the cortex of mouse brains after a permanent occlusion of the MCA. The increased expression of MARCO mRNA at 24 h after prolonged ischaemia is consistent with its putative role in the clearance of debris and/or degenerating cells after severe ischaemia and supports previous publications showing the presence of dendritic cells around permanently occluded lesions.

Leukocyte populations and steroid receptor expression in human first-trimester decidua; regulation by antiprogestin and prostaglandin E analog.

CONTEXT: Progesterone acting via its cognate receptor is critical to maintaining a viable endometrial environment for implantation and pregnancy. During medical termination of pregnancy, the biological effect of progesterone is pharmacologically withdrawn and prostaglandins administered exogenously. Leukocytes within the uterus are the effector cells of an inflammatory response and play important roles in both tissue breakdown and remodeling. OBJECTIVE: The aim of this study was to identify the separate and combined effects of the antiprogestin Mifepristone (single dose, 200 mg) and the prostaglandin E (PGE) analog (gemeprost) on leukocyte populations and steroid receptor expression in human first-trimester decidua. PATIENTS: Eighty women were recruited from the termination of pregnancy service with a gestational age of between 35 and 65 d at the time of surgical termination of pregnancy. MAIN OUTCOME MEASURES: Immunohistochemistry was used to measure macrophage (CD68 +ve), neutrophil (neutrophil elastase +ve), and uterine natural killer cell (CD56 +ve) populations and progesterone (PR(A) and PR(B)), estrogen (ERalpha and ERbeta), and androgen receptor (AR) expression. RESULTS: After administration of both antiprogestin and the PGE analog, macrophage and neutrophil numbers were significantly increased, whereas natural killer cell numbers were unchanged. Antiprogestin and PGE analog coadministration also significantly decreased PR and ERalpha immunoreactivity but had no effect on androgen receptor or ERbeta receptor expression. PGE analog alone was also capable of reducing PR expression. CONCLUSIONS: In this study, we demonstrate that the inflammatory response induced by antiprogestin in combination with PGE analog is accompanied by both increases in macrophages and neutrophils numbers and decreases in PR and ERalpha expression in human first-trimester decidua.

Expression, localization, and signaling of prostaglandin F2 alpha receptor in human endometrial adenocarcinoma: regulation of proliferation by activation of the epidermal growth factor receptor and mitogen-activated protein kinase signaling pathways.

Prostaglandin F(2 alpha)(PGF(2 alpha)) is a bioactive lipid biosynthesized by cyclooxygenase (COX) enzymes and mediates its biological activity via the heptahelical G(q)-coupled PGF(2 alpha)receptor (FP receptor). This study investigated the expression and molecular signaling of the FP receptor in human endometrial adenocarcinomas. Real-time RT-PCR and Western blot analysis confirmed FP receptor expression in endometrial adenocarcinoma of all grades and differentiation. The expression of FP receptor was up-regulated in all endometrial adenocarcinomas compared with normal endometrium. The site of FP receptor expression was localized by in situ hybridization and immunohistochemistry to the neoplastic epithelial cells in all adenocarcinomas. Treatment of endometrial adenocarcinoma explants with PGF(2 alpha) resulted in mobilization of inositol phosphate signaling, indicating functional FP receptor expression. We investigated whether PGF(2 alpha) could trans-activate the epidermal growth factor receptor (EGFR) and trigger the MAPK signaling pathway. Treatment of adenocarcinoma explants and endometrial adenocarcinoma cells (Ishikawa) with PGF(2 alpha)-phosphorylated EGFR, triggered MAPK signaling and enhanced the proliferation of Ishikawa cells. Inactivation of phospholipase C, EGFR kinase, and MAPK kinase with specific inhibitors abolished PGF(2 alpha)-induced trans-activation of EGFR, MAPK signaling, and Ishikawa cell proliferation. These data suggest that PGF(2 alpha)-FP receptor promote endometrial tumorigenesis via a phospholipase C-mediated phosphorylation of the EGFR and MAPK signaling pathways.

Prostaglandin (PG) F(2alpha) receptor expression and signaling in human endometrium: role of PGF(2alpha) in epithelial cell proliferation.

Prostaglandin (PG) F(2alpha), a member of the prostanoid bioactive lipid family, is secreted by human endometrium throughout the menstrual cycle and is present in both menstrual fluid and medium of endometrial explants in culture. PGF(2alpha) mediates its effects through a seven-transmembrane G-protein-coupled receptor (FP). The aim of this study was to examine the temporal expression, signaling, and role of FP receptor in the human endometrium. Quantitative RT-PCR analysis demonstrated highest expression of FP receptor in the mid- to late-proliferative phase, compared with early-proliferative and secretory phase endometrium. In situ hybridization studies localized FP receptor mRNA expression to the epithelial cell compartment during the mid- to late-proliferative phase. Moreover, treatment of endometrial tissue with 1-100 nM PGF(2alpha) induced a concentration-dependent increase in inositol phosphate mobilization, indicating functional FP receptor expression. The Ishikawa human endometrial epithelial cell line was used to investigate further the signaling and role of PGF(2alpha) in endometrial epithelial cells. Ishikawa cells endogenously express the FP receptor, and treatment with 1-100 nM PGF(2alpha) elicits a concentration-dependent increase in inositol phosphate release. Moreover, treatment of Ishikawa cells with 100 nM PGF(2alpha) induced phosphorylation of ERK1/2 that was abolished when cells were cotreated with 50 micro M PD98059 (MAPK kinase inhibitor) or 10 micro M U73122 [phospholipase C (PLC) inhibitor]. Treatment of Ishikawa cells with PGF(2alpha) for 24 h induced a significant concentration-dependent increase in Ishikawa cell proliferation. Coincubation of the cells with 50 micro M PD98059 or 2 micro M U73122 demonstrated that PLC inhibition significantly reduced PGF(2alpha)-induced proliferation, whereas MAPK kinase inhibition had no effect. In summary, these studies demonstrate increased FP receptor expression in endometrial epithelial cells during the proliferative phase of the menstrual cycle and identify a role for PGF(2alpha) in epithelial cell proliferation via a PLC-dependent pathway.