The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

Adolescent polycystic ovary syndrome (PCOS) is characterized by androgen excess and oligomenorrhea, and commonly driven by hepato-visceral fat excess (“central obesity”) ensuing from a mismatch between prenatal and postnatal nutrition, on a background of genetic susceptibility. There is no approved treatment for adolescent PCOS. We report the pooled results of 2 pilot studies in nonobese girls with PCOS (N = 62, age 15.8 years) that compared the effects of randomized treatment for 1 year, either with an oral estro-progestogen contraceptive (OC), or with a low-dose combination of spironolactone-pioglitazone-metformin (SPIOMET, targeting the excess of ectopic fat). Auxological and endocrine-metabolic variables (including fasting insulin, androgens, high-molecular-weight adiponectin [HMW-adiponectin], and microRNA [miR]-451a), body composition (dual x-ray absorptiometry) and hepato-visceral fat (magnetic resonance imaging) were assessed on- and posttreatment. Data from menstrual diaries were combined with weekly salivary progesterone measurements to infer ovulation rates during the second and fourth quarter of the posttreatment year. OC and SPIOMET treatment reduced the androgen excess comparably, and had no differential effects on total-body lean or fat mass. However, SPIOMET was accompanied by more broadly normalizing effects, including on hepato-visceral fat and on circulating insulin, HMW-adiponectin, and miR-451a. On average, there were 3-fold more ovulations post-SPIOMET than post-OC; normovulation was only observed after SPIOMET; anovulation was >10-fold more prevalent post-OC. Pooled results of randomized studies in nonobese adolescent girls with PCOS indicate that SPIOMET treatment leads to an overall healthier, more insulin-sensitive condition—with less ectopic fat—than OC treatment, and to a more normal posttreatment ovulation rate.

Primary cultures of brain cell neurospheres are valuable in vitro models for neurotoxicology and brain cell research. Such applications would greatly benefit from the development of efficient cryopreservation protocols that assure the availability of viable and genetically stable stocks of functional neurospheres. In this work we aimed at developing an integrated strategy allowing for long-term culture and cryopreservation of brain cell neurospheres with high viability and reduced recovery time postthawing. Microencapsulation in clinical-grade, ultrahigh viscous, highly purified alginate uniformly cross-linked with Ba(2+) was evaluated as the main strategy to avoid the commonly observed loss of cell-cell and cell-matrix interactions with consequent aggregate's fragmentation and decrease in cell viability that occurs postthawing. Brain cells isolated from 16-day-old fetal rats were cultured in spinner vessels as neurospheres, encapsulated at the 5th day of culture, and cryopreserved at day 19. Culture characterization and assessment of postthawing recovery, concerning cell metabolism, aggregate's cell type composition, and neuron-astrocyte interactions were performed through analysis of membrane integrity, metabolic activity assays, and immunohistochemistry. Our results show that the encapsulation process does not affect cell viability's central metabolism; neither cell differentiation nor cell extensions into cell networks are usually observed between neurons and astrocytes within the neurosphere structure. Neurosphere encapsulation resulted in reduced recovery time postthawing and significantly less fragmentation. Further, the use of serum-free CryoStor™ solution provided further protection for both nonencapsulated and encapsulated aggregates compared with serum-supplemented culture medium as the cryopreservation medium.

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The commonly applied cryopreservation protocols routinely used in laboratories worldwide were developed for simple cell suspensions, and their application to complex systems, such as cell monolayers, tissues, or biosynthetic constructs, is not straightforward. In particular for monolayer cultures, cell detachment and membrane damage are often observed after cryopreservation. In this work, combined strategies for the cryopreservation of cells attached to Matrigel-coated well plate's surfaces were investigated based on cell entrapment in clinicalgrade, ultra-high viscosity alginate using two cell lines, neuroblastoma N2a and colon adenocarcinoma Caco-2, with distinct structural and functional characteristics. As the cryopreservation medium, serum-free CryoStor solution was compared with serum-supplemented culture medium, both containing 10% DMSO. Using culture medium, entrapment beneath an alginate layer was needed to improve cell recovery by minimizing membrane damage and cell detachment after thawing; nevertheless, up to 50% cell death still occurred within 24 h after thawing. The use of CryoStor solution represented a considerable improvement of the cryopreservation process for both cell lines, allowing the maintenance of high postthaw membrane integrity as well as full recovery of metabolic activity and differentiation capacity within 24 h postthawing; in this case, entrapment beneath an alginate layer did not confer further protection to cryopreserved Caco-2 cells, but was crucial for maintenance of attachment and integrity of N2a neuronal networks.

Prepubertal AGA girls had significantly greater BAT activation index as compared to AGA boys; this difference was not observed in SGA subjects. Higher BAT activation associated with a lower amount of visceral fat and with a favorable metabolic profile. Long-term follow-up is needed to determine whether those differences relate to pubertal timing, and to the development of obesity and metabolic disorders.

PCOS in adolescent girls was preceded by marked z score increments between weight at birth and BMI at PCOS diagnosis, thus corroborating the notion that PCOS development is driven by a mismatch between prenatal weight gain and postnatal weight gain.

Deficient vascularization is a major driver of early islet graft loss and one of the primary reasons for the failure of islet transplantation as a viable treatment for type 1 diabetes. This study identifies the protein tyrosine phosphatase 1B (PTP1B) as a potential modulator of islet graft revascularization. We demonstrate that grafts of pancreatic islets lacking PTP1B exhibit increased revascularization, which is accompanied by improved graft survival and function, and recovery of normoglycemia and glucose tolerance in diabetic mice transplanted with PTP1B-deficient islets. Mechanistically, we show that the absence of PTP1B leads to activation of hypoxia-inducible factor 1α–independent peroxisome proliferator–activated receptor γ coactivator 1α/estrogen-related receptor α signaling and enhanced expression and production of vascular endothelial growth factor A (VEGF-A) by β cells. These observations were reproduced in human islets. Together, these findings reveal that PTP1B regulates islet VEGF-A production and suggest that this phosphatase could be targeted to improve islet transplantation outcomes.