The transient expression of CHIKV VLP in large stirred tank bioreactors

Abstract

Transient gene expression (TGE) bioprocesses have been difficult to scale up in large stirred tank bioreactors with volumes of more than 1.5 L. Low production levels are often observed, but the causes have not been investigated (Gutierrez-Granados et al. in Crit Rev Biotechnol 38:918–940, 2018). Chikungunya Virus-like particle (VLP), expressed by DNA–PEI transient transfection, is a representative case study for these difficulties. Clinical materials were produced in shake flasks, but the process suffered when transferred to large stirred tank bioreactors. The resulting process was not operationally friendly nor cost effective. In this study, a systematic approach was used to investigate the root causes of the poor scale up performance. The transfection conditions were first screened in ambr® 15 high throughput mini bioreactors then examined in 3 L stirred-tank systems. The studies found that production level was negatively correlated with inoculum cell growth status (P < 0.05). The pH range, DNA and PEI levels, order of the reagent addition, and gas-sparging systems were also studied and found to affect process performance. Further hydromechanical characterizations (Re, energy dissipation rates, and P/V, etc.) of shake flasks, ambr® 15, and 3-L stirred tank systems were performed. Overall, the study discovered that the shear stress (caused by a microsparger) and PEI toxicity together were the root causes of scale-up failure. Once the microsparger was replaced by a macrosparger, the scale-up was successful.

Gelam honey promotes ex vivo corneal fibroblasts wound healing

Abstract

This study evaluated the effects of Gelam honey (GH) on ex vivo corneal fibroblast ulcer model via wound healing assay, gene expression and immunocytochemistry. Corneal fibroblasts from New Zealand white rabbits were culture expanded. The corneal fibroblast wound healing capacity was observed by creating a circular wound onto confluent monolayer cells cultured in basal medium (BM), BM with GH, serum-enriched basal medium (BMS) and BMS with GH respectively. Wound healing assay and phenotypic characterization of the corneal fibroblast were performed at different stages of wound closure. Expression of aldehyde dehydrogenase (ALDH), vimentin, α-smooth muscle actin (α-SMA), lumican, collagen I and matrix metalloproteinase 12 (MMP 12) were measured at day 1, day 3 and complete wound closure day. Corneal fibroblast cultured in BMS with GH demonstrated the fastest wound closure, at day 5 post wounding. The gene expressions of ALDH and vimentin were higher than control groups while α-SMA expression was lower, in GH enriched media. The expressions of lumican, collagen I and MMP 12 were also higher in cells cultured in GH enriched media compared to the control groups. GH was shown to promote in vitro corneal fibroblast wound healing and may be a potential natural adjunct in the treatment of corneal wound.

Infant cardiosphere-derived cells exhibit non-durable heart protection in dilated cardiomyopathy rats

Abstract

Stem cells provide a new strategy for the treatment of cardiac diseases; however, their effectiveness in dilated cardiomyopathy (DCM) has not been investigated. In this study, cardiosphere-derived cells (CDCs) were isolated from infants (≤ 24 months) and identified by the cell surface markers CD105, CD90, CD117 and CD45, which is consistent with a previous report, although increased CD34 expression was observed. The molecular expression profile of CDCs from infants was determined by RNA sequencing and compared with adult CDCs, showing that infant CDCs have almost completely altered gene expression patterns compared with adult CDCs. The upregulated genes in infant CDCs are mainly related to the biological processes of cell morphogenesis and differentiation. The molecular profile of infant CDCs was characterized by lower expression of inflammatory cytokines and higher expression of stem cell markers and growth factors compared to adult CDCs. After intramyocardial administration of infant CDCs in the heart of DCM rats, we found that infant CDCs remained in the heart of DCM rats for at least 7 days, improved DCM-induced cardiac function impairment and protected the myocardium by elevating the left ventricular ejection fraction and fraction shortening. However, the effectiveness of transplanted CDCs was reversed later, as increased fibrosis formation instead of angiogenesis was observed. We concluded that infant CDCs, with higher expression of stem cell markers and growth factors, exhibit non-durable heart protection due to limited residence time in the heart of DCM animals, suggesting that multiple administrations of the CDCs or post-regulation after transplantation may be the key for cell therapy in the future.

Antitumor effects of seleno-short-chain chitosan (SSCC) against human gastric cancer BGC-823 cells

Abstract

Seleno-short-chain chitosan (SSCC) is a derivative of chitosan. In the present study, we sought to investigate the underlying antitumor mechanism of SSCC on human gastric cancer BGC-823 cells in vitro. MTT assay suggested that SSCC exhibited a dose-dependent inhibitory effect on the proliferation of BGC-823 cells. We found the SSCC-treated cells showed typical morphological characteristics of apoptosis in a dose dependent manner by observing on microscope. Annexin V-FITC/PI double staining and cell cycle assay identified that SSCC could induce BGC-823 cells apoptosis by triggering G2/M phase arrest. Our research provided the first evidence that SSCC could effectively induce the apoptosis of BGC-823 cells via an intrinsic mitochondrial pathway, as indicated by inducing the disruption of mitochondrial membrane potential (MMP), the excessive accumulation of reactive oxidative species (ROS), the increase of Bax/Bcl-2 ratio and the activation of caspase 3, caspase 9 and cytochrome C (Cyt-C) in BGC-823 cells. These combined results clearly indicated that SSCC could induce BGC-823 cells apoptosis by the involvement of mitochondrial signaling pathway, which provided precise experimental evidence for SSCC as a potential agent in the prevention and treatment of human gastric cancer.

Process optimization for the rapid production of Enterovirus 71

Abstract

Enterovirus 71 (EV71) infection can cause hand–foot-and-mouth disease (HFMD). Inactivated EV71 vaccine was effective to prevent EV71 derived HFMD. A highly efficient and economical process for producing EV71 is needed. In our study, the epidemic strain of EV71 (EV71-2013ZJHFMD) was obtained and purified. The Vero cells were cultured for production of EV71. The mini-bioreactor vessel (Amprotein Inc., China) packed with a 0.6 g polymer fiber carrier was used to determine the best seeding cell density, multiplicity of infection (MOI) and temperature. Then the optimized procedure was further applied in a 10 L disposable perfusion bioreactor ACPB (AmProtein Current Perfusion Bioreactor). The Vero cell culture and viral titer were monitored. The seeding density of 1.5 × 10⁷ cells per 0.6 g disk was considered to be the most appropriate for the culture. The best MOI was 0.1 and the temperature was 32 °C. The total cell number increased from 1.5 × 10⁹ to 3.0 × 10¹⁰. The maximum viral titers reached 1.0 × 10⁸/mL 3 days post-infection in our optimized special culture procedure (serum-free during the harvest period, supplemented with 0.25% Lactalbumin Hydrolysate). The total volume of the harvested supernatant was 25 L and the total virus yield was 1.93 × 10¹². The procedure using Vero cells grown on polymer fiber paper carriers was effective for the large-scale production of EV71.

In vitro cytogenetic assessment and comparison of vildagliptin and sitagliptin

Abstract

Vildagliptin and sitagliptin are commonly used antidiabetic drugs. Chromosomal aberration (CA), sister chromatid exchange (SCE) and cytokinesis-block micronucleus (CBMN) assays were employed to assess and compare cytotoxic and genotoxic effects of these drugs. Peripheral lymphocytes were exposed to 125 μg/ml, 250 μg/ml and 500 μg/ml of vildagliptin and 250 μg/ml, 500 μg/ml and 1000 μg/ml of sitagliptin for 24 h and 48 h with and without exogenous metabolic activation. At the end of the study, it was determined that these drugs and their metabolites had no genotoxic effects on CA, SCE and CBMN. On the other hand, parallel to the increase in dose, vildagliptin showed weak cytotoxicity on the mitotic index, and depending on its increase in dose; sitagliptin caused potential cytotoxicity and cytostatic effect on the mitotic index, nuclear division index and proliferation index. Due to their cytotoxic and cytostatic potential, these drugs inhibit cell proliferation.

MiR-214 - 3p regulates the viability, invasion, migration and EMT of TNBC cells by targeting ST6GAL1

Abstract

MiR-214-3p is concerned with the outcomes of various tumors, such as liver cancer, bladder cancer, etc. However, the role and target of miR-214-3p in triple negative breast cancer (TNBC) is not fully understood. This study took this as the entry point, with a view to find a potential target for TNBC. The expressions of miR-214-3p in TNBC tissues and cell lines were detected, and the effects of miR-214-3p inhibitor on the viability, migration, invasion and epithelial mesenchymal transition (EMT) of TNBC cells were further analyzed. The potential target of miR-214-3p were predicted and verified, as well as the effects of target silencing on the TNBC cells were also measured. MiR-214-3p was abnormally elevated in both TNBC tissues and cell lines, especially in MDA-MB-468 cells. Low-expression of miR-214-3p restrained the survival, migration, invasion and EMT of TNBC cells. ST6GAL1 was the target gene of miR-214-3p, and its expression level increased with the low-expression of miR-214-3p. ST6GAL1 expression was abnormally reduced in both TNBC tissues and cell lines. The silence of ST6GAL1 promoted the viability, migration, invasion and EMT of TNBC cells, which could be reversed by miR-214-3p inhibitor. The down-regulation of miR-214-3p could suppress the viability, migration, invasion and EMT of TNBC cells though targeting ST6GAL1, which might be a potential target for future treatment of TNBC. Up-regulation of miR-214-3p could promote the EMT of non-TNBC cells.

Early integration of Design of Experiment (DOE) and multivariate statistics identifies feeding regimens suitable for CHO cell line development and screening

Abstract

In Chinese Hamster Ovary (CHO) cell lines, the establishment of the ideal fed-batch regimen promotes metabolic conditions advantageous for the bioproduction of therapeutic molecules. A tailored, cell line-specific feeding scheme is typically defined during process development (PD) activities, through the incorporation of Design of Experiment (DOE) and late stage cell culture approaches. The feeding during early stage cell line development (CLD) was a simplified “one-fits-all” design, inherited from PD lab, that didn’t account for CLD needs of throughput and streamlined workflow. The “one-fits-all” efficiency was not routinely verified when novel technologies were incorporated in CLD and sub-optimal feeding carried the risk of not selecting the most desirable cell lines amenable to late stage PD. In our work we developed the DOE-feed method; a streamlined, three-stages framework for identifying efficient feeding schemes as the CLD technologies evolved. We combined early stage cell culture input data with late-stage techniques, such as statistical modelling, principal component analysis (PCA), DOE and Prediction Profiler. Novel in our DOE-feed work, we deliberately anticipated the application of statistics and approached the method development as an early-stage, continuously updated process, by building iterative datasets and statistically interpreting their responses. We capitalized on the statistical models defined by the DOE-feed methodology to study the influence of feeds on daily productivity and growth and to extrapolate feeding-schemes that improved the cell line screening. The DOE-feed became a methodology suited for CLD needs at AbbVie, and optimized the early stage screening, reduced the operational hands-on time and improved the overall workstream efficiency.

Bioactive molecule Icariin inhibited proliferation while enhanced apoptosis and autophagy of rat airway smooth muscle cells in vitro

Abstract

Icariin is the main active compound extracted from epimedium Flavonoids (EFs) and involved in regulation of cell behaviors (proliferation, apoptosis, and autophagy etc.) for many cell types, but the effect of Icariin on airway smooth muscle cells (ASMCs) is still unknown. The aim of the present study is to examine the role of Icariin on rat ASMCs proliferation, apoptosis and autophagy. CKK8 assay showed that Icariin inhibited rat ASMCs growth in dose-time-dependent manner, and the flow cytometry assay showed that the Icariin interfered with ASMCs cell cycle, when treated with Icariin, S phase shortened while G2 phase extended, cyclin E1 and cyclinA1 gene and protein expression decreased. Next apoptosis was detected, Flow cytometry and TdTmediated dUTP Nick-End Labeling (TUNEL) assay showed that Icariin promoted ASMCs apoptosis, and enhanced apoptosis protein cleaved-caspase-3 expression. Finally, it was found Icariin induced rat ASMCs autophagy, with enhancement expression of autophagy marker LC3 II. In conclusion, Icariin inhibited ASMCs proliferation while promoted apoptosis and autophagy, revealing its potential role in treatment of airway remodeling in asthma.