Presence Of Fetal Bovine Serum Research Articles

Nanomolar concentrations of the photodynamic compound TLD-1433 effectively inactivate numerous human pathogenic viruses

The anti-viral properties of a small (≈1 kDa), novel Ru(II) photo dynamic compound (PDC), referred to as TLD-1433 (Ruvidar™), are presented. TLD-1433 had previously been demonstrated to exert strong anti-bacterial and anti-cancer properties. We evaluated the capacity of TLD-1433 to inactivate several human pathogenic viruses. TLD-1433 that was not photo-activated was capable of effectively inactivating 50 % of influenza H1N1 virus (ID50) at a concentration of 117 nM. After photo-activation, the ID50 was reduced to <10 nM. The dose of photo-activated TLD-1433 needed to reduce H1N1 infectivity >99 % (ID99) was approximately 170 nM. Similarly, the ID99 of photo-activated TLD-1433 was determined to range from about 20 to 120 nM for other tested enveloped viruses; specifically, a human coronavirus, herpes simplex virus, the poxvirus Vaccinia virus, and Zika virus. TLD-1433 also inactivated two tested non-enveloped viruses; specifically, adenovirus type 5 and mammalian orthoreovirus, but at considerably higher concentrations. Analyses of TLD-1433-treated membranes suggested that lipid peroxidation was a major contributor to enveloped virus inactivation. TLD-1433-mediated virus inactivation was temperature-dependent, with approximately 10-fold more efficient virucidal activity when viruses were treated at 37 °C than when treated at room temperature (∼22 °C). The presence of fetal bovine serum and virus solution turbidity reduced TLD-1433-mediated virucidal efficiency. Immunoblots of TLD-1433-treated human coronavirus indicated the treated spike protein remained particle-associated.

Superior migration ability of umbilical cord-derived mesenchymal stromal cells (MSCs) toward activated lymphocytes in comparison with those of bone marrow and adipose-derived MSCs.

Introduction: Mesenchymal stromal cells (MSCs) are activated upon inflammation and/or tissue damage and migrate to suppress inflammation and repair tissues. Migration is the first important step for MSCs to become functional; however, the migration potency of umbilical cord-derived MSCs (UC-MSCs) remains poorly understood. Thus, we aimed to assess the migration potency of UC-MSCs in comparison with those of bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AD-MSCs) and investigate the influence of chemotactic factors on the migration of these cells. Methods: We compared the migration potencies of UC-, BM-, and AD-MSCs toward allogeneic stimulated mononuclear cells (MNCs) in mixed lymphocyte reaction (MLR). The number of MSCs in the upper chamber that migrated toward the MLR in the lower chamber was counted using transwell migration assay. Results and discussion: UC-MSCs showed significantly faster and higher proliferation potencies and higher migration potency toward unstimulated MNCs and MLR than BM- and AD-MSCs, although the migration potencies of the three types of MSCs were comparable when cultured in the presence of fetal bovine serum. The amounts of CCL2, CCL7, and CXCL2 in the supernatants were significantly higher in UC-MSCs co-cultured with MLR than in MLR alone and in BM- and AD-MSCs co-cultured with MLR, although they did not induce the autologous migration of UC-MSCs. The amount of CCL8 was higher in BM- and AD-MSCs than in UC-MSCs, and the amount of IP-10 was higher in AD-MSCs co-cultured with MLR than in UC- and BM-MSCs. The migration of UC-MSCs toward the MLR was partially attenuated by platelet-derived growth factor, insulin-like growth factor 1, and matrix metalloproteinase inhibitors in a dose-dependent manner. Conclusion: UC-MSCs showed faster proliferation and higher migration potency toward activated or non-activated lymphocytes than BM- and AD-MSCs. The functional chemotactic factors may vary among MSCs derived from different tissue sources, although the roles of specific chemokines in the different sources of MSCs remain to be resolved.

In vitro maturation of African catfish (Clarias gariepinus, Burchell, 1822) oocytes results in viable larvae

In this study, we describe a protocol for in vitro maturation and ovulation of African catfish (Clarias gariepinus) postvitellogenic ovarian follicles, resulting in fertilizable and developmentally competent eggs. Such culture systems enable detailed studies of fish oocyte development and present a useful tool in assisted reproduction of fish. The maturation-inducing effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), human chorionic gonadotropin (hCG) and recombinant human insulin-like growth factor 1 (rhIGF-1) were evaluated based on their ability to promote ooplasm translucency and germinal vesicle breakdown (GVBD), which indicates meiosis resumption. Among the tested groups, only follicles exposed to DHP underwent GVBD in a time-dependent manner, with the highest rates (>80%) observed within 10 to 12 h of incubation. Furthermore, adding rhIGF-1 or hCG prior to or during incubations with DHP did not influence the GVBD outcome. This suggested that the oocytes acquired maturational competence prior to isolation, as high concentrations of DHP (1 μg/ml) were alone sufficient to induce complete nuclear and cytoplasmic maturation; however, only 4 ± 3% of these oocytes ovulated in culture. To promote ovulation, we incorporated prostaglandins (PGs) as an additional incubation step following DHP-induced maturation. Exposure to both prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) led to increased rupture of the follicular layer, most notably in groups with 5 μg/ml PGF2α (71 ± 8%). In addition to identifying optimal hormonal stimulation, effects of culture media pH in combination with media supplementation—in the form of bovine serum albumin (BSA) and fetal bovine serum (FBS)—were considered. Compared to results in media with pH of 7.5, significantly less follicles matured and ovulated in a more alkaline environment (pH 8.5), regardless of supplement type and concentration. Although the presence of FBS (5–20%) slightly improved ovulation rates, this benefit was not reflected in the fertilization outcomes. In vitro matured and ovulated oocytes obtained in non-supplemented media (pH 7.5) maintained their developmental competence and were successfully fertilized. The hatching rate was 39%, after which the survival rate of larvae was 8% at 72 h post fertilization (hpf).

Cellular uptake and in vitro antibacterial activity of lipid-based nanoantibiotics are influenced by protein corona.

Bacteria have evolved survival mechanisms that enable them to live within host cells, triggering persistent intracellular infections that present significant clinical challenges due to the inability for conventional antibiotics to permeate cell membranes. In recent years, antibiotic nanocarriers or 'nanoantibiotics' have presented a promising strategy for overcoming intracellular infections by facilitating cellular uptake of antibiotics, thus improving targeting to the bacteria. However, prior to reaching host cells, nanocarriers experience interactions with proteins that form a corona and alter their physiological response. The influence of this protein corona on the cellular uptake, drug release and efficacy of nanoantibiotics for intracellular infections is poorly understood and commonly overlooked in preclinical studies. In this study, protein corona influence on cellular uptake was investigated for two nanoparticles; liposomes and cubosomes in macrophage and epithelial cells that are commonly infected with pathogens. Studies were conducted in presence of fetal bovine serum (FBS) to form a biologically relevant protein corona in an in vitro setting. Protein corona impact on cellular uptake was shown to be nanoparticle-dependent, where reduced internalization was observed for liposomes, the opposite was observed for cubosomes. Subsequently, vancomycin-loaded cubosomes were explored for their drug delivery performance against intracellular small colony variants of Staphylococcus aureus. We demonstrated improved bacterial killing in macrophages, with greater reduction in bacterial viability upon internalization of cubosomes mediated by the protein corona. However, no differences in efficacy were observed in epithelial cells. Thus, this study provides insights and evidence to the role of protein corona in modulating the performance of nanoparticles in a dynamic manner; these findings will facilitate improved understanding and translation of future investigations from in vitro to in vivo.

Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

The Application of Fluorescence Anisotropy for Viscosity Measurements of Small Volume Biological Analytes.

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 μL and 100 μL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze-thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements' accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems.

Esterase-Responsive Polyglycerol-Based Nanogels for Intracellular Drug Delivery in Rare Gastrointestinal Stromal Tumors.

Rare gastrointestinal stromal tumors (GISTs) are caused by mutations in the KIT and PDGFRA genes. Avapritinib (BLU-285) is a targeted selective inhibitor for mutated KIT and PDGFRA receptors that can be used to treat these tumors. However, there are subtypes of GISTs that exhibit resistance against BLU-285 and thus require other treatment strategies. This can be addressed by employing a drug delivery system that transports a combination of drugs with distinct cell targets. In this work, we present the synthesis of esterase-responsive polyglycerol-based nanogels (NGs) to overcome drug resistance in rare GISTs. Using inverse nanoprecipitation mediated with inverse electron-demand Diels-Alder cyclizations (iEDDA) between dPG-methyl tetrazine and dPG-norbornene, multi-drug-loaded NGs were formed based on a surfactant-free encapsulation protocol. The obtained NGs displayed great stability in the presence of fetal bovine serum (FBS) and did not trigger hemolysis in red blood cells over a period of 24 h. Exposing the NGs to Candida Antarctica Lipase B (CALB) led to the degradation of the NG network, indicating the capability of targeted drug release. The bioactivity of the loaded NGs was tested in vitro on various cell lines of the GIST-T1 family, which exhibit different drug resistances. Cell internalization with comparable uptake kinetics of the NGs could be confirmed by confocal laser scanning microscopy (CLSM) and flow cytometry for all cell lines. Cell viability and live cell imaging studies revealed that the loaded NGs are capable of intracellular drug release by showing similar IC50 values to those of the free drugs. Furthermore, multi-drug-loaded NGs were capable of overcoming BLU-285 resistance in T1-α-D842V + G680R cells, demonstrating the utility of this carrier system.

Potentiality of Bone Marrow-Derived Mesenchymal Stromal Cells (BM-MSCs) to Differentiate into the Osteogenic Lineage in in Vitro Model of Tissue Regeneration

Introduction:The mesenchymal stromal cells (MSCs) have gained considerable popularity owing to the vast possibilities and lack of ethical constraints. MSCs serve as multipotent progenitors for several niche components in the bone marrow (BM) and play an essential role in the quiescence, proliferation, and differentiation of hematopoietic stem cells. Recently, MSCs from BM (BM-MSCs) represent an attractive cell source for tissue engineering, thanks to their immunomodulatory property, self-renewing and high proliferative capability. BM-MSCs have multi-lineages (adipogenesis, chondrogenesis, osteogenesis) potential and in combination with biomaterials, well support cell-attachments and stimulate extracellular matrix synthesis. For these reasons, BM-MSCs have a great potential for regenerative medicine applications. The present research aims to evaluate the ability of BM-MSCs to proliferate and differentiate toward the osteogenic lineage into innovative three-dimensional bioresorbable scaffold based on gelatin-chitosan hydrogel with a poly(lactic acid) lattice structure (PLA-CH) for regenerative medicine applications in the presence of fetal bovine serum (FBS) or human platelet lysate (HPL) with or without the osteogenic medium (OM). Methods: BM-MSCs were expanded to the passages 3 or 4 and seeded into the PLA-CH, in dry state, at a cellular density of 7x10 5 cells/scaffolds in growth medium (GM). For inducing osteogenic differentiation, cells/scaffolds constructs were cultured in 24-well plates for 4 weeks with OM consisting of a high-glucose DMEM supplemented with 10% FBS or 5% HPL, 10 −7M Dex, 25 mg/ml l-ascorbic acid, and 3mM NaH 2PO 4. For histomorphological analysis at optical microscope, scaffolds were embedded in paraffin using an automatic processor Donatello series 2 (Diapath S.p.A., Bergamo, Italy). Serial paraffin sections (5 µm thick) of each sample were cut, deparaffinized, and rehydrated, according to standard procedures and stained with hematoxylin-eosin stain for general morphology and Von Kossa (Bio-Optica, Milan, Italy) for calcium deposition. Immunohistochemistry for the osteogenic marker Osteocalcin (OSC) (Santa Cruz Biotechnologies, USA) was also performed. For Scanning Electron Microscopy (SEM) (EVO LS-10 manufactured by ZEISS) observation, samples have been progressively dehydrated through immersion in alcohol solutions. The local changes in elemental composition were investigated using the Energy Dispersive X-ray (EDX) analyzer. Results: The results showed that BM-MSCs have higher affinity to attachment, migrate and differentiate toward the osteogenic lineage inside the novel scaffold PLA-CH both with HPL and FBS as supplement in the culture media. The use of HPL for cell expansion offers the possibility to obtain a safer cellular products without xenogenic contaminants. Cells develop a large spreading area with elongated fibroblast-like morphology preferentially inside the hydrogel pores, showing great affinity with the scaffold. This is confirmed also by histomorphological analyses at optical microscope, showing homogeneous colonization of cells inside the hydrogel pores (figure 1a). The results showed the presence of calcium deposits in the mash of the scaffold with Von Kossa stain (figure 1b), moreover cells appeared well integrated with a positivity for OSC showing a clear differentiation state as osteoblasts (figure 1c). Calcium and phosphorous were detected with SEM-EDX on the samples incubated in osteogenic medium (figure 1 d,e). Conclusion: MSCs, derived from bone marrow, associated with scaffold PLA-CH proved to be biocompatible and promising for personalized medicine. In fact, BM-MSCs were able to growth, colonize and osteo-differentiate throughout the hydrogel, demonstrating their potential application for bone regenerative medicine. Figure 1. Histomorphological analysis at the optical microscope of PLA-CH with BM-hMSCs in the GM at 100 m m ( a), Von Kossa stain showing calcium deposit distribution on PLA-CH with BM-hMSCs in the OM at 30 m m ( b), immunohistochemistry for OCN with BM-hMSCs differentiated to osteoblast in the OM at 10 m m ( c). Evaluation of calcium (Ca) and phosphorous (P) with SEM-EDX of PLA-CH with BM-hMSCs in OM day 28 ( d,e).

A Method to Generate and Rescue Recombinant Adenovirus Devoid of Replication-Competent Particles in Animal-Origin-Free Culture Medium.

Adenoviruses are promising vectors for vaccine production and gene therapy. Despite all the efforts in removing animal-derived components such as fetal bovine serum (FBS) during the production of adenovirus vector (AdV), FBS is still frequently employed in the early stages of production. Conventionally, first-generation AdVs (E1 deleted) are generated in different variants of adherent HEK293 cells, and plaque purification (if needed) is performed in adherent cell lines in the presence of FBS. In this study, we generated an AdV stock in SF-BMAdR (A549 cells adapted to suspension culture in serum-free medium). We also developed a limiting dilution method using the same cell line to replace the plaque purification assay. By combining these two technologies, we were able to completely remove the need for FBS from the process of generating and producing AdVs. In addition, we demonstrated that the purified AdV stock is free of any replication-competent adenovirus (RCA). Furthermore, we demonstrated that our limiting dilution method could effectively rescue an AdV from a stock that is highly contaminated with RCA.

Serum albumin mitigated perfluorooctane sulfonate-induced cytotoxicity by affecting the cellular responses

During the wide applications of perfluorinated materials such as perfluorooctane sulfonate (PFOS) in commercial and industrial products, the potential toxicity of these engineered compounds has attracted more and more attention. As a typical environmental pollutant, PFOS could preferentially bind to albumin protein in vivo. However, the role of protein–PFOS interactions in the cytotoxicity of PFOS was not stressed enough. Herein, we investigated the interactions of PFOS with human serum albumin (HSA, the most abundant protein in human plasma) using both experimental and theoretical approaches. It was demonstrated that PFOS could mainly bind to the Sudlow site I of HSA to generate HSA-PFOS complex through hydrogen bonds and van der Waals forces. Toxicity assays with endothelial cells illustrated that the binding of HSA could significantly attenuate the intracellular uptake and subcellular distribution of PFOS, thereby inhibiting the formation of reactive oxygen species and toxicity for those HSA-bound PFOS. Similarly, the presence of fetal bovine serum in the cell culture media greatly reduced PFOS-caused cytotoxicity. Conclusively, our study reveals that the binding of albumin protein to PFOS could mitigate its toxicity by the modulation of cellular responses. The formation of protein-complexed contaminants would significantly reduce the bioavailability of these chemicals and subsequently mitigate their environmental toxicology to the human health.

Detection of the Inflammatory Cytokine IL-6 in Complex Human Serum Samples Via Rational Nanotube Surface Passivation Screening

In recent years, biosensors have emerged as a tool with strong potential in medical diagnostics. Single-walled carbon nanotube (SWCNT) based optical nanosensors have notably garnered interest due to the unique characteristics of their near-infrared fluorescence emission, including tissue transparency, photostability, and various chiralities with discrete absorption and fluorescence emission bands.The optoelectronic properties of SWCNT are sensitive to the surrounding environment, which makes them suitable for highly selective biosensing. Single-stranded (ss) DNA-wrapped SWCNTs have been reported as optical nanosensors for cancers and metabolic diseases. However, given the complexity of the human protein environment, non-specific interactions occur between SWCNT-based nanosensors and proteins. This inevitably leads to compromised selectivity of SWCNT-based nanosensors unless strategies for prevention are developed and employed.Non-covalent passivation of the ssDNA-SWCNT surface is reported as an excellent strategy to improve nanosensor selectivity in complex biological settings without causing irreversible changes to the optical properties of SWCNTs. Emerging studies have explored and successfully shown passivation using proteins and phospholipids. However, a systematic comparative study of passivating agents has not been done. In this work, we explore and compare the efficacy of select proteins, polymers, and surfactants as passivating agents.We, therefore, sought to evaluate various potential SWCNT surface passivation agents among broad classes of biomolecules and biomaterials. In the category of protein, Bovine serum albumin, dry-fat milk powder, and casein were selected due to their wide application to improve immunoassay selectivity. In the class of polymers, we selected 1 anionic and 2 cationic polymers, namely, polyethylene glycol, poly-ethylene imine, and poly-l-lysine. From surfactants, 2 phospholipids and 1 anionic surfactant: ammonium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PE2000PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE PEG phospholipids), and sodium dodecylbenzene sulfonate (SDBS) were selected.We analyzed the ability of all passivation agents to screen the non-specific interactions of ssDNA- SWCNTs in the presence of fetal bovine serum using fluorescence spectroscopy. The non-specific interactions between ssDNA-SWCNTs and proteins lead to a reorientation of the dipole moments and charge transfer in the corona phase around ssDNA-SWCNTs, leading to modulation in the center wavelength of the fluorescence as well as absorption peaks. We hence hypothesized that smaller changes in the center wavelength of the fluorescence peaks of passivated ssDNA-SWCNTs in presence of serum, lead to higher screening effect of the passivation agent towards non-specific interactions. We found that the most successful candidates were poly-L-lysine, polyethylene imine, dry-fat milk powder, and casein, in that order. Moreover, the ability to screen the interference was retained over a period of at least 3 hours. We further experimented with the four different mass ratios of passivating agents to ssDNA-SWCNTs and found that the mass ratio 50:1 for passivating agents: ss-DNA SWCNTs was most optimal.We confirmed the strength of passivation using absorption spectroscopy. We hypothesized that stronger surface saturation of the SWCNT-TAT6 is by a passivating agent in buffer conditions leads to a larger shift in the absorption peaks. We found that for the above successful passivation agents, the order of passivation strength followed the same trend as the screening abilities of the successful passivating agents, supporting this mechanistic hypothesis.Then, we evaluated an antibody-conjugated ssDNA-SWCNT nanosensor for the pro-inflammatory cytokine IL-6 with our successful passivation agents. We assessed the ability of the passivation agents to confer selective detection of IL-6 detection in clinical serum samples from patients with atherosclerosis and rheumatoid arthritis, both anticipated to have high IL-6 levels. Samples were compared to healthy human serum sample controls. We validated SWCNT fluorescence response with traditional immunoassays (ELISA).We expect this study to provide rational strategies to screen interferences from non-specific interactions and improve the selectivity of the SWCNT-based optical nanosensors for in vivo applications.

Heparin interferes with the uptake of liposomes in glioma

In glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors influencing cellular uptake of liposomes in glioma cells are poorly understood. Heparin and heparin analogues are commonly used in glioma patients to decrease the risk of thrombo-embolic events. Our results show that heparin inhibits pegylated liposome uptake by U87 glioma and GL261 cells in a dose dependent manner in vitro, and that heparin-mediated inhibition of uptake required presence of fetal bovine serum in the media. In a subcutaneous model of glioma, Cy5.5 labeled liposomes could be detected with in vivo imaging after direct intra-tumoral injection. Ex-vivo analysis with flow cytometry showed a decreased uptake of liposomes into tumor cells in mice treated systemically with heparin compared to those treated with vehicle only.

Significant improvement of bone marrow-derived MSC expansion from MDS patients by defined xeno-free medium

BackgroundRobust and reliable in vitro and in vivo models of primary cells are necessary to study the pathomechanisms of Myelodysplastic Neoplasms (MDS) and identify novel therapeutic strategies. MDS-derived hematopoietic stem and progenitor cells (HSPCs) are reliant on the support of bone marrow (BM) derived mesenchymal stroma cells (MSCs). Therefore, isolation and expansion of MCSs are essential for successfully modeling this disease. For the clinical use of healthy MSCs isolated from human BM, umbilical cord blood or adipose tissue, several studies showed that xeno-free (XF) culture conditions resulted in superior growth kinetics compared to MSCs cultured in the presence of fetal bovine serum (FBS). In this present study, we investigate, whether the replacement of a commercially available MSC expansion medium containing FBS with a XF medium is beneficial for the expansion of MSCs derived from BM of MDS patients which are often difficult to cultivate.MethodsMSCs isolated from BM of MDS patients were cultured and expanded in MSC expansion medium with FBS or XF supplement. Subsequently, the impact of culture media on growth kinetics, morphology, immunophenotype, clonogenic potential, differentiation capacity, gene expression profiles and ability to engraft in immunodeficient mouse models was evaluated.ResultsSignificant higher cell numbers with an increase in clonogenic potential were observed during culture of MDS MSCs with XF medium compared to medium containing FBS. Differential gene expression showed an increase in transcripts associated with MSC stemness after expansion with XF. Furthermore, immunophenotypes of the MSCs and their ability to differentiate into osteoblasts, adipocytes or chondroblasts remained stable. MSCs expanded with XF media were similarly supportive for creating MDS xenografts in vivo as MSCs expanded with FBS.ConclusionOur data indicate that with XF media, higher cell numbers of MDS MSCs can be obtained with overall improved characteristics in in vitro and in vivo experimental models.

Cannabinoids as Potential Cancer Therapeutics: The Concentration Conundrum.

Background: Studies have reported that cannabinoids, in particular Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), significantly reduce cancer cell viability in vitro. Unfortunately, treatment conditions vary significantly across reports. In particular, a majority of reports utilize conditions with reduced serum concentrations (0-3%) that may compromise the growth of the cells themselves, as well as the observed results. Objectives: This study was designed to test the hypothesis that, based on their known protein binding characteristics, cannabinoids would be less effective in the presence of fetal bovine serum (FBS). Moreover, we wished to determine if the treatments served to be cytotoxic or cytostatic under these conditions. Methods: Six cancer cell lines, representing two independent lines of three different types of cancer (glioblastoma, melanoma, and colorectal cancer [CRC]), were treated with 10 μM pure Δ9-THC, CBD, KM-233, and HU-331 for 48 h (in the presence or absence of FBS). Cell viability was measured with the MTT assay. Dose-response curves were then generated comparing the potencies of the four cannabinoids under the same conditions. Results: We found that serum-free medium alone produces cell cycle arrest for CRC cells and slows cell growth for the other cancer types. The antineoplastic effects of three of the four cannabinoids (Δ9-THC, CBD, and KM-233) increase when serum is omitted from the media. In addition, dose-response curves for these drugs demonstrated lower IC50 values for serum-free media compared with the media with 10% serum in all cell lines. The fourth compound, HU-331, was equally effective under both conditions. A further confound we observed is that omission of serum produces dramatic binding of Δ9-THC and CBD to plastic. Conclusions: Treatment of cancer cells in the absence of FBS appears to enhance the potency of cannabinoids. However, omission of FBS itself compromises cell growth and represents a less physiological condition. Given the knowledge that cannabinoids are 90-95% protein bound and have well-known affinities for plastic, it may be ill-advised to treat cells under conditions where the cells are not growing optimally and where known concentrations cannot be assumed (i.e., FBS-free conditions).

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

Identification and gene expression profiling of human gonadotrophic pituitary adenoma stem cells

BackgroundGonadotrophic pituitary adenoma is a major subtype of pituitary adenoma in the sellar region, but it is rarely involved in the hypersecretion of hormones into blood; thus, it is commonly regarded as “non-functioning.” Its tumorigenic mechanisms remain unknown. The aim of this study was to identify human gonadotrophic pituitary adenoma stem cells (hPASCs) and explore the underlying gene expression profiles. In addition, the potential candidate genes involved in the invasive properties of pituitary adenoma were examined.MethodsThe hPASCs from 14 human gonadotrophic pituitary adenoma clinical samples were cultured and verified via immunohistochemistry. Genetic profiling of hPASCs and the matched tumor cells was performed through RNA-sequencing and subjected to enrichment analysis. By aligning the results with public databases, the candidate genes were screened and examined in invasive and non-invasive gonadotrophic pituitary adenomas using Real-time polymerase chain reaction.ResultsThe hPASCs were successfully isolated and cultured from gonadotrophic pituitary adenoma in vitro, which were identified as positive for generic stem cell markers (Sox2, Oct4, Nestin and CD133) via immunohistochemical staining. The hPASCs could differentiate into the tumor cells expressing follicle-stimulating hormone in the presence of fetal bovine serum in the culture medium. Through RNA-sequencing, 1352 differentially expressed genes were screened and identified significantly enriched in various gene ontologies and important pathways. The expression levels of ANXA2, PMAIP1, SPRY2, C2CD4A, APOD, FGF14 and FKBP10 were significantly upregulated while FNDC5 and MAP3K4 were downregulated in the invasive gonadotrophic pituitary adenomas compared to the non-invasive ones.ConclusionGenetic profiling of hPASCs may explain the tumorigenesis and invasiveness of gonadotrophic pituitary adenoma. ANXA2 may serve as a potential therapeutic target for the treatment of gonadotrophic pituitary adenoma.

Cultivation, cryopreservation and resuscitation of Theileria annulata transformed cells in serum-free media.

Tropical theileriosis is a protozoan disease caused by Theileria annulata that affects cattle in Northern Africa, the Middle East and Asia where vector ticks of the genus Hyalomma occur. Various measures are applied to control the disease, including vaccination with attenuated T. annulata schizonts. Cultivation of T. annulata schizonts is mainly conducted in media containing Fetal Bovine Serum (FBS), which has some disadvantages such as costs, batch- to-batch variation and ethical concerns. In this study, we conducted three experiments to evaluate the ability of (1) T. annulata strains grown in RPMI with 10% FBS (RPMI-FBS) to adapt and grow in serum-free media (i.e., HL-1, RPMI without FBS supplementation, ISF-1, and M199), (2) a T. annulata strain grown in ISF-1 and subsequently frozen in this medium to grow in ISF-1 again after long-term storage in liquid nitrogen, and (3) a T. annulata strain freshly isolated from infected bovine lymphocytes to growin ISF-1, also after cryopreservation. Cell numbers, schizont index, the viability and generation doubling time were calculated in all experiments. In the first experiment, the Hessiene and Beja cell lines from Tunisia previously cultivated in RPMI-FBS and adapted to serum-free media continued to grow significantly better in RPMI-FBS compared to the serum-freemedia. In the second experiment, a Tunisian cell line (Hessiene) cryopreserved in ISF-1 with 5%[v/v] dimethylsulfoxide (DMSO) grewbetter after thawing in RPMI-FBS compared to ISF-1 with a highly significant difference in cell growth (p < 0.001), whereas the third experiment showed that the Ankara cell line had similar growth characteristics in both RPMI-FBS and ISF-1 before and after thawing, with a shorter generation doubling time in ISF-1 than in RPMI-FBS (p = 0.23). Our findings suggest that freshly isolated cells can be propagated, frozen and thawed in serum-free media such as ISF-1, but once cells are adapted to cultivation in the presence of FBS or resuscitated from frozen storage, propagation in serum-free media may not perform as well as cultivation in RPMI-FBS.

Phenotyping of M1 and M2a Macrophages and Differential Expression of ACE-2 on Monocytes by Flow Cytometry: Impact of Cell Culture Conditions and Sample Processing.

Macrophages are ubiquitously distributed throughout the various tissues of the body and perform many functions including the orchestration of inflammatory responses against pathogens by classically activated M1 macrophages and the regulation of wound healing and tissue remodeling by anti-inflammatory, alternatively activated M2 macrophages. The responsibility for these pleiotropic functions lies in the expression of a myriad of surface receptors unique to given subsets of macrophages. Much of what we know about the function of human macrophage subsets has been gleaned by studying in vitro generated macrophages matured in the presence of GM-CSF or M-CSF and polarized with different cytokines. Oftentimes, culture conditions, such as the type of serum used, the duration of the culture, and the use of polarizing cytokines, vary between studies making direct comparisons difficult. Sample preparation and processing (e.g., Ficoll® enrichment of leukocytes from whole blood) can also influence gene expression on human monocytes. Furthermore, overlap in surface marker expression can make it difficult to distinguish between different macrophage subsets.We directly compared the expression of over 20 different surface markers on M1 and M2a macrophages cultured in either serum-free media or in the presence of fetal bovine serum or human AB serum and found that the presence or type of serum used affected the expression of several markers such as CD200R1 and CD32. Moreover, we compared the expression of these surface markers on polarized and unpolarized macrophages and determined that polarization was critical to the expression of several of these markers including CD38 and SLAM F7. Differences in sample processing can alter the expression of surface markers, such as ACE-2, on monocytes. We observe that ACE-2 expression is higher on human whole blood CD14+ monocytes versus Ficoll®-enriched CD14+ monocytes derived from PBMCs (peripheral blood mononuclear cells), where expression can be reduced by up to 50%. These results indicate that differences in serum, culture media, and sample processing can alter gene expression in both human macrophages and monocytes. Importantly, the results of these studies significantly expand our knowledge of the phenotypic differences between human M1 and M2a macrophages and demonstrate the importance of culture conditions in generating these phenotypes.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.

Nano-Sized Polyelectrolyte Complexes Formed between Poly(vinyl benzyl trimethyl ammonium chloride) and Insulin

Novel biohybrid homo-polyelectrolyte-based nanocarriers were formed by the complexation of insulin (INS) with a biocompatible and cationic polyelectrolyte, namely, poly(vinyl benzyl trimethylammonium chloride) (PVBTMAC). According to light-scattering techniques, the hydrophilic PVBTMAC homo-polyelectrolyte forms single chains in aqueous media. The resulting biohybrid PVBTMAC/INS nanocarriers were formed via electrostatic co-assembly. The effects of polyelectrolyte structure and content on the characteristics of the formed PVBTMAC/INS complexes were studied. A significant aggregation tendency of the PVBTMAC/INS complexes was observed, based on the physicochemical results, especially at high protein concentration, corroborating the effective electrostatic interaction of INS with the cationic polyelectrolyte. The physicochemical properties of the formed PVBTMAC/INS nanocarriers depended on the concentration of the stock polymer and INS solutions. A neat PVBTMAC homo-polymer and PVBTMAC/INS nanocarriers demonstrated good serum stability in the presence of fetal bovine serum (FBS) proteins. Fluorescence spectroscopy (FS) studies revealed no INS conformational changes after its complexation with the cationic PVBTMAC polyelectrolyte. The obtained PVBTMAC/INS complexes demonstrated considerable and promising characteristics for potential use as insulin delivery systems.