ELF Magnetic Field Exposure System for In Vitro Studies Based on Lee-Whiting Coils.

In order to run a series of in vitro studies on the effect of extremely low-frequency magnetic fields on cell cultures, developing and characterizing an appropriate exposure system is required. The present design is based on a two-shielded Lee-Whiting coils system. The circular design was chosen because its axial symmetry allowed for both reducing simulation unknowns and measurement points during the characterization, and additionally made the machining of the parts easier. The system can generate magnetic flux densities (B fields) up to 1 mT root-mean-square amplitude (rms) with no active cooling system in the incubator, and up to 3 mTrms with it. The double-wrapped windings with twisted pairs allow for the use of each set of coils either as exposure or control with no detectable parasitic B field in the control. The artifacts have also been analyzed; the B field in the center of the sham control chamber is about 1 µTrms for a maximum of 3 mTrms in the exposure chamber, the parasitic incident electric fields are less than 1 V/m, the temperature difference between sham and exposure chamber is less than or equal to 0.2 °C, and the typical vibration difference between sham and exposure is less than 0.1 m/s2 . © 2020 Bioelectromagnetics Society.

Construction and in vitro testing of a cellulose dura mater graft.

INTRODUCTION: Cerebrospinal fluid (CSF) leaks are a common complication after cranial and spinal surgery and are associated with increased morbidity. Despite continuous research in this field, this problem is far from solved. In this paper, we describe the construction and testing of a bacterial cellulose (BC) membrane as a new dural patch. MATERIALS AND METHODS: The synthesis of BC was performed using Gluconacetobacter hansenii (ATCC 23769) and films were sterilized by autoclaving. The membranes were seeded with human dural fibroblasts. Growth, shape, and cell viability were assessed after 4 weeks. RESULTS: Normally shaped fibroblasts were seen on the BC grafts; confocal microscopy showed cells inside the structure of the mesh. Both viable and nonviable cells were present. Cellular attachment and viability were confirmed by replating of the membranes. DISCUSSION: BC membranes are used in clinical practice to improve skin healing. In the presence of water, they form an elastic, nontoxic, and resistant biogel that can accommodate collagen and growth factors within their structure, thus BC is a good candidate for dural graft construction.

Assessing the role of selected growth factors and cytostatic agents in an in vitro model of human dura mater healing.

OBJECTIVES: Cerebrospinal fluid (CSF) leaks are a common concern in skull base surgery. Appropriate dural healing is crucial to prevent CSF leaks but the entire process has been barely understood so far. Here, we review the impact of growth factors and chemotherapeutic agents on an explant culture of human dural fibroblasts and a 3D subculture grown in a collagen mesh scaffold. METHODS: Human dural specimens were harvested during surgical procedures where they would not be further used therapeutically or diagnostically. Explant cultures were grown in Petri dishes, and subcultures were grown in collagen mesh scaffolds. Insulin, fibroblast growth factor type 2 (FGF-2), and human serum were analyzed for their effect as growth factors, whereas mitomycin C, vincristine, and colchicine were analyzed for their role as inhibitors. Cell count was used as a parameter to assess the effects of these factors. In addition, the effects of human serum were assessed using collagen mesh scaffolds. RESULTS: Insulin, FGF-2, and human serum increased culture cell count; human serum also achieved an increased number of viable fibroblasts embedded in a collagen mesh. Mitomycin C, which is a mitosis inhibitor, showed no significant effect on cell count, whereas colchicine and vincristine, which inhibit both mitosis and migration, resulted in cell growth suppression. DISCUSSION: In our model, dural defect closure is achieved through cell migration rather than through cell growth. Adding growth factors to the dural suture line or into a collagen mesh might prove useful to stimulate dural closure.

Skin fibroblasts from patients with type 1 diabetes (T1D) can be chemically transdifferentiated into insulin-expressing clusters: a transgene-free approach.

The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.

Inhibition of brain ST8SiaIII sialyltransferase leads to impairment of procedural memory in mice.

Several glycoproteins in mammalian brains contain α2,8-linked disialic acid residues. We previously showed a constant expression of disialic acid (DiSia) in the hippocampus, olfactory bulb and cortex, and a gradual decrease of expression in the cerebellum from neonatal to senile mice. Previous publications indicate that neurite extension of neuroblastoma-derived Neuro2A cells is inhibited in the presence of DiSia antibody. Based on this, we treated Neuro2A cell cultures with RNA interference for ST8SiaIII mRNA, the enzyme responsible for DiSia formation. We observed that neurite extension was inhibited by this treatment. Taking this evidence into consideration and the relationship of the cerebellum with learning and memory, we studied the role of DiSia expression in a learning task. Through delivery of pST8SiaIII into the brains of C57BL/6 neonatal mice, we inhibited the expression of ST8SiaIII. ST8SiaIII mRNA and protein expressions were analyzed by real-time PCR and western blot, respectively. In this work, we showed that pST8SiaIII-treated mice presented a significantly reduced level of ST8SiaIII mRNA in the cerebellum (p<0.01) in comparison to control mice at 8 days after treatment. It is also noted that these levels returned to baseline values in the adulthood. Then, we evaluated behavioural performance in the T-Maze, a learning task that estimates procedural memory. At all ages, pST8SiaIII-treated mice showed a lower performance in the test session, being most evident at older ages (p<0.001). Taken all together, we conclude that gene expression of ST8SiaIII is necessary for some cognitive tasks at early postnatal ages, since reduced levels impaired procedural memory in adult mice.

A new model for dura mater healing: human dural fibroblast culture.

OBJECTIVE: Dura mater healing is crucial to prevent cerebrospinal fluid (CSF) leaks after neurosurgical procedures. Biological mechanisms leading to dural closure are only partially understood and have been studied in animals exclusively. We studied an in vitro model of dural closure which uses human cells. MATERIALS AND METHODS: We used human dura intended for disposal after surgery. Explant primary cultures were performed. Cells were characterized through common staining and immunohistochemistry. A cell growth curve was elaborated and the effect of dexamethasone on cell count was assessed. Spongostan®, oxidized regenerated cellulose and autologous plastic materials were also evaluated for their effect on cellular growth. RESULTS: All specimens showed growth in fusiform cells, which project pseudopods and fuse into spindles. Cells showed desmin and vimentin positivity, and were negative for all the other stains, behaving phenotypically like fibroblasts. No collagen base was necessary for cell growth. Dexamethasone decreased cell count in the primary culture as well as in the explant, and reduced the cell proliferation marker Ki-67. Spongostan® was successfully used as a graft, and fibroblast cultures were additionally developed with muscle, pericranium, galea, and fascia. Oxidized cellulose induced cell death by lowering the pH of the solution. DISCUSSION: According to the findings, unlike mini-pigs and rabbits, in humans, dural fibroblast sensitivity to collagen seems to be lower. Dexamethasone inhibits fibroblast invasion, which is the biological base of wound dehiscence in cranial surgery. Although Spongostan is useful, Surgicel® can lower the media pH, thereby inhibiting cellular growth.

Differential expression of Shh and BMP signaling in the potential conversion of human adipose tissue stem cells into neuron-like cells in vitro.

The nervous system (NS) has a limited self-repair capability and adult neurogenesis is limited to certain regions of the brain. This generates a great interest in using stem cells to repair the NS. Previous reports have shown the differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) in neuron-like cells when cultures are enriched with growth factors participating in embryonic and adult neurogenesis. Therefore, it could be thought that there exists a functional parallelism between neurogenesis and neuronal differentiation of ASCs. For this reason, the goal of this work was to study the differential gene expression of Shh and BMP genetic pathways involved in cell fate determination and proliferation. In this study we demonstrated that hASCs are endowed with active Hedgehog and BMP signaling pathways through the expression of genes of both cascades and that their expressions are downregulated after neuronal induction. This idea is in accordance with the facts that Shh and BMP signaling is involved in the maintenance of cells with stem cells properties and that proliferation decreases during the process of differentiation. Furthermore, Noggin expression was detected in induced hASCs whereas there was no expression in noninduced cells, which indicates that these cells are probably adopting a neuronal fate because noggin diverts neural stem cells from glial to neuronal fate. We also detected FM1-43 and synaptophisin staining, which is evidence of the presence of putative functional presynaptic terminals, a neuron-specific property. These results could partially contribute to the elucidation of the molecular mechanisms involved in neuronal differentiation of adult human nonneural tissues.

Culture and characterization of human hepatocytes obtained after graft reduction for liver transplantation: a reliable source of cells for a bioartificial liver.

This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 x 10(6) and 38 x 10(6) cells/g of tissue (median value 14.73 x 10(6) cells/g), and the viability was 61.17 +/- 27.43%. The total number of cells ranged between 57.6 x 10(6) and 12 150 x 10(6) cells (median value: 740 x 10(6) cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 +/- 1.77 micro g/mL). Urea production was detected during the first week (peak value at day 6: 17.12 +/- 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver.

Ammonium detoxification performed by porcine hepatocyte spheroids in a bioartificial liver for pediatric use: preliminary report.

Bioartificial liver devices are alternative therapies for patients suffering from acute hepatic failure or metabolic defects. Here, we show a bioartificial device, developed with a cartridge used for pediatric hemofiltration and spheroids of porcine hepatocytes housed in the extracapillary space of the cartridge. The cartridge was attached to a robotic arm that supplied a continuous, oscillatory movement. It was connected through the capillary circulation to a neonatal membrane oxygenator contain-ing human blood supplemented with ammonium and diazepam. A decrease in ammonium concentration was observed, reaching an almost 70% decrease upon 9 h of operation. In addition, urea was detected and diazepam metabolism proved from the fourth hour of operation. It is worth mentioning that the system described was assembled with commercially available components for current clinical use. The setup may be done in a short period, thus eliminating long-term culture times and the need for cell anchoring to matrices.