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The effective use of human-derived cells that are difficult to freeze, such as parenchymal cells and differentiated cells from stem cells, is crucial. A stable supply of damage-sensitive cells, such as differentiated neuronal cells, neurons, and glial cells can contribute considerably to cell therapy. We developed a serum-free freezing solution that is effective for the cryopreservation of differentiated neuronal cells. The quality of the differentiated and undifferentiated SK-N-SH cells was determined based on cell viability, live-cell recovery rate, and morphology of cultured cells, to assess the efficacy of the freezing solutions. The viability and recovery rate of the differentiated SK-N-SH neuronal cells were reduced by approximately 1.5-folds compared to that of the undifferentiated SK-N-SH cells. The viability and recovery rate of the differentiated SK-N-SH cells were remarkably different between the freezing solutions containing 10% DMSO and that containing 10% glycerol. Cryoprotectants such as fetal bovine serum (FBS), antifreeze proteins (sericin), and sugars (maltose), are essential for protecting against freeze damage in differentiated neuronal cells and parenchymal cells. Serum-free alternatives (sericin and maltose) could increase safety during cell transplantation and regenerative medicine. Considering these, we propose an effective freezing solution for the cryopreservation of neuronal cells., Graphical abstract Image 1, Highlights • The timing of freezing during cell differentiation. • More effective serum-free freezing solution for differentiated neuronal cells. • Improving the quality of damage-sensitive cells, such as differentiated neuronal cells.

Graphical abstract, Even given recent advances in nanomedicine development of breast cancer treatment in recent years and promising results in pre-clinical models, cancer nanomedicines often fail at the clinical trial stage. Limitations of conventional in vitro models include the lack of representation of the stromal population, the absence of a three-dimensional (3D) structure, and a poor representation of inter-tumor and intra-tumor heterogeneity. Herein, we review those cell culture strategies that aim to overcome these limitations, including cell co-cultures, advanced 3D cell cultures, patient-derived cells, bioprinting, and microfluidics systems. The in vivo evaluation of nanomedicines must consider critical parameters that include the enhanced permeability and retention effect, the host's immune status, and the site of tumor implantation. Here, we critically discuss the advantages and limitations of current in vivo models and report how the improved selection and application of breast cancer models can improve the clinical translation of nanomedicines.

Autologous chondrocytes in vitro expanded, are used as tools of regenerative therapies for cartilage injuries. However, inability to maintain the hyaline phenotype both in vitro and post in vivo transplantation, remains one of the major hurdles for long term efficacy under clinical settings. We have reported earlier, hyaline phenotype maintenance of both human and rabbit chondrocytes for a long duration both in vitro when cultured conditions using a Thermo-reversible Gelation Polymer (TGP) scaffold-based methodology and in vivo post-transplantation animal model of cartilage damage. Having intrigued by such encouraging outcome, we in this study, analysed the similar TGP culture environment whether would be able to allow in vitro expansion of severe osteoarthritis affected cartilage tissue from elderly patients and evaluated the cells using lectin microarray characterization for pluripotency. Cartilage tissue were obtained from patients (n = 7; age: 60–85 years) undergoing total knee arthroplasty for severe osteoarthritis. Chondrocytes were isolated and cultured in two groups: i. conventional culture without scaffold (2D) and ii. using a TGP scaffold-based culture (3D) up to 18 weeks. In addition to earlier reported findings such as maintenance of hyaline phenotype having been confirmed in this study as well, surface glycoprotein analysis by lectin microarray demonstrated that the α1-2 Fuc recognition lectin (UEA-1) (marker reported in literature for pluripotent stem cells) was found to be more highly expressed in 3D culture compared to 2D culture and even increased over time in 3D culture. We have developed an environment where osteoarthritis affected chondrocytes from the elderly could be cultured up to 18 weeks in vitro using TGP scaffold which express pluripotent cell associated surface glycoproteins compared to the conventional methodology., Highlights • Good quality chondrocytes were grown from cartilage tissue of elderly with severe osteoarthritis for 18 weeks in vitro. • Inflamed donor chondrocytes could be revived to form normal tissue in a 3D in vitro TGP scaffold environment. • Pluripotent stem cell marker UEA-1 in Lectin microarray was positive in TGP- Polymer scaffold grown cartilage.

Introduction Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). Methods Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. Results The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P, Graphical abstract Image 1, Highlights • cHep spheroids was co-cultured using canine hepatocytes, mesenchymal stem cells, and human umbilical vein endothelial cells. • The ratio of spheroid formation was improved and expression of liver related genes and protein was maintained. • The three-mix culture could promote large spheroids without impairing liver function in canine hepatocyte.

Graphical abstract, Asthma is a complicated lung disease, which has increased morbidity and mortality rates in worldwide. There is an overlap between asthma pathophysiology and mitochondrial dysfunction and MSCs may have regulatory effect on mitochondrial dysfunction and treats asthma. Therefore, immune-modulatory effect of MSCs and mitochondrial signaling pathways in asthma was studied. After culturing of MSCs and producing asthma animal model, the mice were treated with MSCs via IV via IT. BALf's eosinophil Counting, The levels of IL-4, −5, −13, −25, –33, INF-γ, Cys-LT, LTB4, LTC4, mitochondria genes expression of COX-1, COX-2, ND1, Nrf2, Cytb were measured and lung histopathological study were done. BALf's eosinophils, the levels of IL-4, −5, −13, −25, –33, LTB4, LTC4, Cys-LT, the mitochondria genes expression (COX-1, COX-2, Cytb and ND-1), perivascular and peribronchial inflammation, mucus hyper-production and hyperplasia of the goblet cell in pathological study were significantly decreased in MSCs-treated asthma mice and reverse trend was found about Nrf-2 gene expression, IFN-γ level and ratio of the INF-γ/IL-4. MSC therapy can control inflammation, immune-inflammatory factors in asthma and mitochondrial related genes, and prevent asthma immune-pathology.

Objective The miR-200–Zeb1 axis regulates the epithelial-to-mesenchymal transition (EMT), differentiation, and resistance to apoptosis. A better understanding of these processes in diabetes is highly relevant, as β-cell dedifferentiation and apoptosis contribute to the loss of functional β-cell mass and diabetes progression. Furthermore, EMT promotes the loss of β-cell identity in the in vitro expansion of human islets. Though the miR-200 family has previously been identified as a regulator of β-cell apoptosis in vivo, studies focusing on Zeb1 are lacking. The aim of this study was thus to investigate the role of Zeb1 in β-cell function and survival in vivo. Methods miR-200 and Zeb1 are involved in a double-negative feedback loop. We characterized a mouse model in which miR-200 binding sites in the Zeb1 3′UTR are mutated (Zeb1200), leading to a physiologically relevant upregulation of Zeb1 mRNA expression. The role of Zeb1 was investigated in this model via metabolic tests and analysis of isolated islets. Further insights into the distinct contributions of the miR-200 and Zeb1 branches of the feedback loop were obtained by crossing the Zeb1200 allele into a background of miR-141–200c overexpression. Results Mild Zeb1 derepression in vivo led to broad transcriptional changes in islets affecting β-cell identity, EMT, insulin secretion, cell–cell junctions, the unfolded protein response (UPR), and the response to ER stress. The aggregation and insulin secretion of dissociated islets of mice homozygous for the Zeb1200 mutation (Zeb1200M) were impaired, and Zeb1200M islets were resistant to thapsigargin-induced ER stress ex vivo. Zeb1200M mice had increased circulating proinsulin levels but no overt metabolic phenotype, reflecting the strong compensatory ability of islets to maintain glucose homeostasis. Conclusions This study signifies the importance of the miR-200–Zeb1 axis in regulating key aspects of β-cell function and survival. A better understanding of this axis is highly relevant in developing therapeutic strategies for inducing β-cell redifferentiation and maintaining β-cell identity in in vitro islet expansion., Highlights • The miR-200-Zeb1 double negative feedback loop is essential for pancreatic β-cell identity and function. • Disruption of the miR-200-Zeb1 axis leads to broad transcriptional changes affecting many aspects of β-cell biology. • Disruption of miR-200-mediated inhibition of Zeb1 protects against ER stress. • Derepression of Zeb1 leads to impaired reaggregation of dissociated islets and perturbed insulin secretion.

Obtaining T cells by reprogramming is one of the major goals in regenerative medicine. Here, we describe a protocol for generating functional T cells from Hoxb5-expressing pro/pre-B cells in vivo. This protocol includes the construction of Hoxb5 recombinant plasmids, retroviral packaging, isolation and viral transduction of pro/pre-B cells, cell transplantation, and phenotypic analysis of induced T cells. The procedure is reproducible and straightforward, providing an approach for generating induced T cells for translational research. Keywords: Hoxb5, Retrovirus, Pro/pre-B cells, T cells

Summary Precision medicine strives to delineate disease using multiple data sources—from genomics to digital health metrics—in order to be more precise and accurate in our diagnoses, definitions, and treatments of disease subtypes. By defining disease at a deeper level, we can treat patients based on an understanding of the molecular underpinnings of their presentations, rather than grouping patients into broad categories with one-size-fits-all treatments. In this review, the authors examine how precision medicine, specifically that surrounding genetic testing and genetic therapeutics, has begun to make strides in both common and rare cardiovascular diseases in the clinic and the laboratory, and how these advances are beginning to enable us to more effectively define risk, diagnose disease, and deliver therapeutics for each individual patient., Central Illustration