Nanosensitive optical coherence tomography for detecting structural changes in stem cells.

Mesenchymal stromal cells (MSCs) are adult stem cells that have been widely investigated for their potential to regenerate damaged and diseased tissues. Multiple pre-clinical studies and clinical trials have demonstrated a therapeutic response following treatment with MSCs for various pathologies, including cardiovascular, neurological and orthopaedic diseases. The ability to functionally track cells following administration in vivo is pivotal to further elucidating the mechanism of action and safety profile of these cells. Effective monitoring of MSCs and MSC-derived microvesicles requires an imaging modality capable of providing both quantitative and qualitative readouts. Nanosensitive optical coherence tomography (nsOCT) is a recently developed technique that detects nanoscale structural changes within samples. In this study, we demonstrate for the first time, the capability of nsOCT to image MSC pellets following labelling with different concentrations of dual plasmonic gold nanostars. We show that the mean spatial period of MSC pellets increases following the labelling with increasing concentrations of nanostars. Additionally, with the help of extra time points and a more comprehensive analysis, we further improved the understanding of the MSC pellet chondrogenesis model. Despite the limited penetration depth (similar to conventional OCT), the nsOCT is highly sensitive in detecting structural alterations at the nanoscale, which may provide crucial functional information about cell therapies and their modes of action.

Needle to needle robot-assisted manufacture of cell therapy products.

Advanced therapeutic medicinal products (ATMPs) have emerged as novel therapies for untreatable diseases, generating the need for large volumes of high-quality, clinically-compliant GMP cells to replace costly, high-risk and limited scale manual expansion processes. We present the design of a fully automated, robot-assisted platform incorporating the use of multiliter stirred tank bioreactors for scalable production of adherent human stem cells. The design addresses a needle-to-needle closed process incorporating automated bone marrow collection, cell isolation, expansion, and collection into cryovials for patient delivery. AUTOSTEM, a modular, adaptable, fully closed system ensures no direct operator interaction with biological material; all commands are performed through a graphic interface. Seeding of source material, process monitoring, feeding, sampling, harvesting and cryopreservation are automated within the closed platform, comprising two clean room levels enabling both open and closed processes. A bioprocess based on human MSCs expanded on microcarriers was used for proof of concept. Utilizing equivalent culture parameters, the AUTOSTEM robot-assisted platform successfully performed cell expansion at the liter scale, generating results comparable to manual production, while maintaining cell quality postprocessing.

GMP-Compliant Production of Autologous Adipose-Derived Stromal Cells in the NANT 001 Closed Automated Bioreactor.

In recent years mesenchymal stromal cells (MSCs) have received a great deal of interest for the treatment of major diseases, but clinical translation and market authorization have been slow. This has been due in part to a lack of standardization in cell manufacturing protocols, as well as a lack of biologically meaningful cell characterization tools and release assays. Cell production strategies to date have involved complex manual processing in an open environment which is costly, inefficient and poses risks of contamination. The NANT 001 bioreactor has been developed for the automated production of small to medium cell batches for autologous use. This is a closed, benchtop system which automatically performs several processes including cell seeding, media change, real-time monitoring of temperature, pH, cell confluence and cell detachment. Here we describe a validation of the bioreactor in an environment compliant with current good manufacturing practice (cGMP) to confirm its utility in replacing standardized manual processing. Stromal vascular fraction (SVF) was isolated from lipoaspirate material obtained from healthy donors. SVF cells were seeded in the bioreactor. Cell processing was performed automatically and cell harvesting was triggered by computerized analysis of images captured by a travelling microscope positioned beneath the cell culture flask. For comparison, the same protocol was performed in parallel using manual methods. Critical quality attributes (CQA) assessed for cells from each process included cell yield, viability, surface immunophenotype, differentiation propensity, microbial sterility and endotoxin contamination. Cell yields from the bioreactor cultures were comparable in the manual and automated cultures and viability was >90% for both. Expression of surface markers were consistent with standards for adipose-derived stromal cell (ASC) phenotype. ASCs expanded in both automated and manual processes were capable of adipogenic and osteogenic differentiation. Supernatants from all cultures tested negative for microbial and endotoxin contamination. Analysis of labor commitment indicated considerable economic advantage in the automated system in terms of operator, quality control, product release and management personnel. These data demonstrate that the NANT 001 bioreactor represents an effective option for small to medium scale, automated, closed expansion of ASCs from SVF and produces cell products with CQA equivalent to manual processes.

Accessing depth-resolved high spatial frequency content from the optical coherence tomography signal.

Optical coherence tomography (OCT) is a rapidly evolving technology with a broad range of applications, including biomedical imaging and diagnosis. Conventional intensity-based OCT provides depth-resolved imaging with a typical resolution and sensitivity to structural alterations of about 5-10 microns. It would be desirable for functional biological imaging to detect smaller features in tissues due to the nature of pathological processes. In this article, we perform the analysis of the spatial frequency content of the OCT signal based on scattering theory. We demonstrate that the OCT signal, even at limited spectral bandwidth, contains information about high spatial frequencies present in the object which relates to the small, sub-wavelength size structures. Experimental single frame imaging of phantoms with well-known sub-micron internal structures confirms the theory. Examples of visualization of the nanoscale structural changes within mesenchymal stem cells (MSC), which are invisible using conventional OCT, are also shown. Presented results provide a theoretical and experimental basis for the extraction of high spatial frequency information to substantially improve the sensitivity of OCT to structural alterations at clinically relevant depths.

Survival/Adaptation of Bone Marrow-Derived Mesenchymal Stem Cells After Long-Term Starvation Through Selective Processes.

After in vivo transplantation, mesenchymal stem cells (MSC) face an ischemic microenvironment, characterized by nutrient deprivation and reduced oxygen tension, which reduces their viability and thus their therapeutic potential. Therefore, MSC response to models of in vitro ischemia is of relevance for improving their survival and therapeutic efficacy. The aim of this study was to understand the survival/adaptive response mechanism that MSC use to respond to extreme culture conditions. Specifically, the effect of a long-term starvation on human bone marrow (hBM)-derived MSC cultured in a chemically defined medium (fetal bovine serum-free [SF] and human SF), either in hypoxic or normoxic conditions. We observed that hBM-MSC that were isolated and cultured in SF medium and subjected to a complete starvation for up to 75 days transiently changed their behavior and phenotype. However, at the end of that period, hBM-MSC retained their characteristics as determined by their morphology, DNA damage resistance, proliferation kinetic, and differentiation potential. This survival mode involved a quiescent state, confirmed by increased expression of cell cycle regulators p16, p27, and p57 and decreased expression of proliferating cell nuclear antigen (PCNA), Ki-67, mTOR, and Nanog. In addition, Jak/STAT (STAT6) antiapoptotic activity selected which cells conserved stemness and that supported metabolic, bioenergetic, and scavenging requirements. We also demonstrated that hBM-MSC exploited an autophagic process which induced lipid ß-oxidation as an alternative energy source. Priming MSC by concomitant starvation and culture in hypoxic conditions to induce their quiescence would be of benefit to increase MSC survival when transplanted in vivo. Stem Cells 2019;37:813-827.

Sleep to Lower Elevated Blood Pressure: A Randomized Controlled Trial (SLEPT).

BACKGROUND: Impaired sleep quality is common and associated with an increased risk of cardiovascular disease (CVD), thought to be mediated through adverse effects on established vascular risk factors, particularly hypertension. We determined if a web-delivered sleep intervention (sleep-hygiene education, stimulus control, and cognitive behavioral therapy) reduces blood pressure compared to vascular risk factor education (standard care) alone. METHODS: Phase II randomized, blinded, controlled trial of 134 participants without CVD with mild sleep impairment and blood pressure 130-160/<110 mm Hg. The primary outcome was the difference in the mean change in 24-hour ambulatory systolic blood pressure (SBP) over 8 weeks between intervention and control groups. Secondary outcomes included measures of sleep quality and psychosocial health, namely Insomnia Severity Index (ISI), Pittsburgh Sleep Quality Index (PSQI), Beck Depression Inventory (BDI), and Beck Anxiety Inventory (BAI). RESULTS: Participants in the sleep intervention group showed significantly greater improvements in sleep quality, including ISI [difference in mean improvement 2.8; 95% confidence interval (CI), 1.3-4.4], PSQI (1.1; 95% CI, 0.1-2.2), sleep condition indicator (0.8; 95% CI, 0.2-1.4), and psychosocial health, including BDI (2.0; 95% CI, 0.3-3.7) and BAI (1.4; 95% CI, 0.02-2.8). The mean improvement in 24-hour ambulatory SBP did not differ between the sleep intervention (0.9 mm Hg) and control (0.8 mm Hg) arms, (difference in mean improvement 0.1; 95% CI, -3.4 to 3.2). CONCLUSION: A simple, low-cost, web-delivered sleep intervention is feasible and significantly improves sleep quality and measures of psychosocial health in individuals with mild sleep impairment but does not result in short-term improvements in blood pressure.

A Role for Adrenergic Receptors in the Uterotonic Effects of Ergometrine in Isolated Human Term Nonlaboring Myometrium.

BACKGROUND: Ergometrine is a uterotonic agent that is recommended in the prevention and management of postpartum hemorrhage. Despite its long-standing use, the mechanism by which it acts in humans has never been elucidated fully. The objective of this study was to investigate the role of adrenoreceptors in ergometrine's mechanism of action in human myometrium. The study examined the hypothesis that α-adrenoreceptor antagonism would result in the reversal of the uterotonic effects of ergometrine. METHODS: Myometrial samples were obtained from women undergoing elective cesarean delivery. The samples were then dissected into strips and mounted in organ bath chambers. After the generation of an ergometrine concentration-response curve (10 to 10 M), strips were treated with increasing concentrations of ergometrine (10 to 10 M) alone and ergometrine (10 to 10 M) in the presence of phentolamine (10 M), prazosin (10 M), propranolol (10 M), or yohimbine (10 M). The effects of adding ergometrine and the effect of drug combinations were analyzed using linear mixed effects models with measures of amplitude (g), frequency (contractions/10 min), and motility index (g×contractions/10 min). RESULTS: A total of 157 experiments were completed on samples obtained from 33 women. There was a significant increase in the motility index (adding 0.342 g × counts/10 min/µM; 95% confidence interval [CI], 0.253-0.431, P < .001), amplitude (0.078 g/µM; 95% CI, 0.0344-0.121, P = 5e-04), and frequency (0.051 counts/10 min/µM; 95% CI, 0.038-0.063, P < .001) in the presence of ergometrine. The α-adrenergic antagonist phentolamine and the more selective α1-adrenergic antagonist prazosin inhibited the ergometrine mediated increase in motility index, amplitude, and frequency (-1.63 g × counts/10 min/µM and -16.70 g × counts/10 min/µM for motility index, respectively). CONCLUSIONS: These results provide novel evidence for a role for α-adrenergic signaling mechanisms in the action of ergometrine on human myometrial smooth muscle in the in vitro setting. Information that sheds light on the mechanism of action of ergometrine may have implications for the development of further uterotonic agents.