A cryopreservation method for bioengineered 3D cell culture models

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities

In vivo conditional deletion of HDAC7 reveals its requirement to establish proper B lymphocyte identity and development

Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro-B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development

Study protocol and rationale of the “Cogniaction project” a cross-sectional and randomized controlled trial about physical activity, brain health, cognition, and educational achievement in schoolchildren

Background: Education and health are crucial topics for public policies as both largely determine the future wellbeing of the society. Currently, several studies recognize that physical activity (PA) benefits brain health in children. However, most of these studies have not been carried out in developing countries or lack the transference into the education field. The Cogni-Action Project is divided into two stages, a cross-sectional study and a crossover-randomized trial. The aim of the first part is to establish the associations of PA, sedentarism, and physical fitness with brain structure and function, cognitive performance and academic achievement in Chilean schoolchildren (10–13 years-old). The aim of the second part is to determinate the acute effects of three PA protocols on neuroelectric indices during a working memory and a reading task. Methods: PA and sedentarism will be self-reported and objectively-assessed with accelerometers in a representative subsample, whilst physical fitness will be evaluated through the ALPHA fitness test battery. Brain structure and function will be assessed by magnetic resonance imaging (MRI) in a randomized subsample. Cognitive performance will be assessed through the NeuroCognitive Performance Test, and academic achievement by school grades. In the second part 32 adolescents (12–13 year-old) will be cross-over randomized to these condition (i) “Moderate-Intensity Continuous Training” (MICT), (ii) “Cooperative High-Intensity Interval Training” (C-HIIT), and (iii) Sedentary condition. Neuroelectric indices will be measures by electroencephalogram (EEG) and eye-tracking, working memory by n-back task and reading comprehension by a reading task

Polarization losses from the nonadiabatic passage of hyperpolarized solutions through metallic components

From complex-mixture analysis to in vivo molecular imaging, applications of liquid-state nuclear spin hyperpolarization have expanded widely over recent years. In most cases, hyperpolarized solutions are generated ex situ and transported from the polarization instrument to the measurement device. The sample hyperpolarization usually survives this transport, since the changes in magnetic fields that are external to the sample are typically adiabatic (slow) with respect to the internal nuclear spin dynamics. The passage of polarized samples through weakly magnetic components such as stainless steel syringe needles and ferrules is not always adiabatic, which can lead to near-complete destruction of the magnetization. To avoid this effect becoming “folklore” in the field of hyperpolarized NMR, we present a systematic investigation to highlight the problem and investigate possible solutions. Experiments were carried out on: (i) dissolution-DNP-polarized [1-13C]pyruvate with NMR detection at 1.4T, and (ii) 1.5-T-polarized H2O with NMR detection at 2.5μT. We show that the degree of adiabaticity of solutions passing through metal parts is intrinsically unpredictable, likely depending on many factors such as solution flow rate, degree of remanent ferromagnetism in the metal, and nuclear spin species. However, the magnetization destruction effects can be suppressed by application of an external field on the order of 0.1–10mT

Leveraging Magnetic Resonance Imaging to Study Biocompatible Scaffolds Diffusion and Perfusion for Lab-on-a-Chip Systems

To foster the efficient development of 3D cell culture models, we developed a strategy to assess the liquid flow perfusion through biocompatible scaffolds. Biocompatible scaffolds play a crucial role in creating 3D in vitro models for precision medicine. Therefore, it is imperative to thoroughly characterise the physical properties of these scaffolds. In this study, we leverage nuclear magnetic resonance imaging (MRI) to examine the diffusivity and perfusion properties of commonly used cryogels and hydrogels. We used deuterium oxide (i.e. heavy water) as a contrast agent to monitor the reduction in proton concentration from water within the scaffolds due to molecular motion. By analysing pixel intensity in MRI images, we extract information on the diffusion speed and perfusion efficacy of these materials. This approach allowed us to investigate passive water diffusion in a carboxymethyl cellulose cryogel and polyethylene glycol diacrylate hydrogel. The diffusion rates differed by 50% between the two scaffolds. Furthermore, we measured their perfusion properties in a PDMS microfluidic chip

Combined Homogeneous and Heterogeneous Hydrogenation with Parahydrogen to Yield Catalyst-Free Solutions of Hyperpolarized [1-13C]Succinate

We show that catalyst-free aqueous solutions of hyperpolarized [1-13C]succinate can be produced using parahydrogen-induced polarization (PHIP) and a combination of homogeneous and heterogeneous catalytic hydrogenation reactions. We generate hyperpolarized [1-13C]fumarate at 23% 13C polarization via PHIP with a homogeneous ruthenium catalyst, and subsequently remove the toxic catalyst and reaction side products via a purification procedure. Following this, we perform a second hydrogenation reaction to convert the fumarate into succinate using a solid Pd/Al2O3 catalyst. The catalyst is filtered off to yield a clean aqueous solution containing [1-13C]succinate at 11.9% 13C polarization for the hyperpolarized molecules. In this proof-of-principle demonstration we simplified the purification procedure by adding unpolarized fumarate to the mixtures so the observed succinate polarization was lower, but this step is not necessary for applications. This inexpensive polarization protocol has a turnover time of a few minutes, and represents a major advance for in vivo applications of [1-13C]succinate as a hyperpolarized contrast agent

Real-Time Polarimetry of Hyperpolarized ¹³C Nuclear Spins Using an Atomic Magnetometer

We introduce a method for nondestructive quantification of nuclear spin polarization, of relevance to hyperpolarized spin tracers widely used in magnetic resonance from spectroscopy to in vivo imaging. In a bias field of around 30 nT we use a high-sensitivity miniaturized 87Rb-vapor magnetometer to measure the field generated by the sample, as it is driven by a windowed dynamical decoupling pulse sequence that both maximizes the nuclear spin lifetime and modulates the polarization for easy detection. We demonstrate the procedure applied to a 0.08 M hyperpolarized [1–13C]-pyruvate solution produced by dissolution dynamic nuclear polarization, measuring polarization repeatedly during natural decay at Earth’s field. Application to real-time and continuous quality monitoring of hyperpolarized substances is discussed

Ammonium quantification (AQua) in human plasma by 1 H‐NMR for staging of liver fibrosis in alcohol‐related liver disease and non‐alcoholic fatty liver disease

Liver fibrosis staging is a key element driving the prognosis of patients with chronic liver disease. Currently, biopsy is the only technique capable of diagnosing liver fibrosis in patients with alcohol‐related liver disease (ArLD) and nonalcoholic fatty liver disease (NAFLD) unequivocally. Noninvasive (e.g. plasma‐based) biomarker assays are attractive tools to diagnose and stage disease, yet must prove that they are reliable and sensitive to be used clinically. Here, we demonstrate proton nuclear magnetic resonance as a method to rapidly quantify the endogenous concentration of ammonium ions from human plasma extracts and show their ability to report upon early and advanced stages of ArLD and NAFLD. We show that, irrespective of the disease etiology, ammonium concentration is a more robust and informative marker of fibrosis stage than current clinically assessed blood hepatic biomarkers. Subject to validation in larger cohorts, the study indicates that the method can provide accurate and rapid staging of ArLD and NAFLD without the need for an invasive biopsy

The transcriptional repressor HDAC7 promotes apoptosis and c-Myc downregulation in particular types of leukemia and lymphoma

The generation of B cells is a complex process requiring several cellular transitions, including cell commitment and differentiation. Proper transcriptional control to establish the genetic programs characteristic of each cellular stage is essential for the correct development of B lymphocytes. Deregulation of these particular transcriptional programs may result in a block in B-cell maturation, contributing to the development of hematological malignancies such as leukemia and lymphoma. However, very little is currently known about the role of transcriptional repressors in normal and aberrant B lymphopoiesis. Here we report that histone deacetylase 7 (HDAC7) is underexpressed in pro-B acute lymphoblastic leukemia (pro-B-ALL) and Burkitt lymphoma. Ectopic expression of HDAC7 induces apoptosis, leads to the downregulation of c-Myc and inhibits the oncogenic potential of cells in vivo, in a xenograft model. Most significantly, we have observed low levels of HDAC7 expression in B-ALL patient samples, which is correlated with the increased levels of c-Myc. From a mechanistic angle, we show that ectopically expressed HDAC7 localizes to the nucleus and interacts with the transcription factor myocyte enhancer factor C (MEF2C) and the corepressors HDAC3 and SMRT. Accordingly, both the HDAC7-MEF2C interaction domain as well as its catalytic domain are involved in the reduced cell viability induced by HDAC7. We conclude that HDAC7 has a potent anti-oncogenic effect on specific B-cell malignancies, indicating that its deregulation may contribute to the pathogenesis of the disease

In vivo conditional deletion of HDAC7 reveals its requirement to establish proper B lymphocyte identity and development

Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro-B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development