Addition Of Fetal Bovine Serum Research Articles

Shape and Shear Stress Impact on the Toxicity of Mesoporous Silica Nanoparticles: In Vitro and In Vivo Evidence.

Mesoporous silica nanoparticles (MSNs) are widely used in the biomedical field because of their unique and excellent properties. However, the potential toxicity of different shaped MSNs via injection has not been fully studied. This study aims to systematically explore the impact of shape and shear stress on the toxicity of MSNs after injection. An in vitro blood flow model was developed to investigate the cytotoxicity and the underlying mechanisms of spherical MSNs (S-MSN) and rodlike MSNs (R-MSN) in human umbilical vein endothelial cells (HUVECs). The results suggested that the interactions between MSNs and HUVECs under the physiological flow conditions were significantly different from that under static conditions. Whether under static or flow conditions, R-MSN showed better cellular uptake and less oxidative damage than S-MSN. The main mechanism of cytotoxicity induced by R-MSN was due to shear stress-dependent mechanical damage of the cell membrane, while the toxicity of S-MSN was attributed to mechanical damage and oxidative damage. The addition of fetal bovine serum (FBS) alleviated the toxicity of S-MSN by reducing cellular uptake and oxidative stress under static and flow conditions. Moreover, the in vivo results showed that both S-MSN and R-MSN caused cardiovascular toxicity in zebrafish and mouse models due to the high shear stress, especially in the heart. S-MSN led to severe oxidative damage at the accumulation site, such as liver, spleen, and lung in mice, while R-MSN did not cause significant oxidative stress. The results of in vitro blood flow and in vivo models indicated that particle shape and shear stress are crucial to the biosafety of MSNs, providing new evidence for the toxicity mechanisms of the injected MSNs.

Binding of serum albumin to perfluorooctanoic acid reduced cytotoxicity

With the ubiquitous applications of perfluorinated compounds such as perfluorooctanoic acid (PFOA) in industrial and commercial products, the toxicity of these engineered materials in environmental and public health is received growing attention. As a typical organic pollutant, PFOA has been extensively found in wildlife and human bodies, and can preferentially bind to serum albumin in vivo. However, the importance of protein–PFOA interactions on the cytotoxicity of PFOA could not be stressed enough. In this study, we used both experimental and theoretical approaches, to investigate the interactions of PFOA with bovine serum albumin (BSA, the most abundant protein in blood). It was found that PFOA could mainly interact with Sudlow site I of BSA to form BSA-PFOA complex, in which van der Waals forces and hydrogen bonds played dominant roles. Moreover, the strong binding of BSA could greatly alter the cellular uptake and distribution of PFOA in human endothelial cells, and result in the decreases of reactive oxygen species formation and cytotoxicity for these BSA-coated PFOA. Consistently, the addition of fetal bovine serum into cell culture medium also significantly mitigated PFOA-induced cytotoxicity, which was attributed to the extracellular complexation between PFOA and serum proteins. Altogether, our study demonstrates that the binding of serum albumin to PFOA could reduce its toxicity by affecting the cellular responses.

Systematic comparison of culture media uncovers phenotypic shift of primary human microglia defined by reduced reliance to CSF1R signaling.

Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum-free media with brain-derived growth factors (colony stimulating factor 1 receptor [CSF1R] ligands and TGF-β1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia (hMGL). Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of hMGL in culture, with observations that diverge from those previously made in rodent microglia.

Replacing animal-derived components in in vitro test guidelines OECD 455 and 487

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns.In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis.All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium.Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

The gelatin-based liquid marbles for cell cryopreservation.

As an alternative and a straightforward cryopreservation biotechnological tool, liquid marble provides a promising cryopreservation approach. Currently, effective cell preservation mainly based on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). As state-of-the-art cryoprotectant (CPA), DMSO, has intrinsic toxicity, which is the bottleneck of its widespread application. The complex compositions of FBS have the potential risks of pathogenic microorganism contamination. However, efficient cell cryopreservation using liquid marbles, a platform independent of DMSO and FBS, has not been well investigated yet. Herein, we explore the cryoprotection role of liquid marbles based on gelatin solution. Gelatin has a superior biocompatibility, which DMSO is incomparable. During a freeze-thaw cycle, gelatin produces negligible osmotic pressure, and has high ice recrystallization inhibition (IRI) activity to induce the formation of smaller and smooth ice crystals. Moreover, the specific structure of liquid marble also provides favorable supports for cell survival. The cryopreservation efficiency of mouse fibroblasts cells L929 via the gelatin-based liquid marble was as high as 90%, and the recovered cells could maintain their normal functionalities. This work opens a new window of opportunity for non-toxic and efficient cryopreservation of liquid marbles without the need of DMSO and FBS addition.

Effect of molecular weight of chitosan on the physicochemical, morphological, and biological properties of polyplex nanoparticles intended for gene delivery

The use of viral vectors in gene therapy has many disadvantages that have been widely described. The development of new nonviral vectors using safe polymers such as chitosan provides a viable alternative. Chitosan molecular weight and nitrogen/phosphorus (N/P) ratio are key factors that determine the shape, size, and surface charge of polyplex nanoparticles (NPs) assembled by complex coacervation for gene therapy; and at the same time, their transfection efficiency is affected by these characteristics, besides the cell line used for transfection. However, there are some inconsistencies in previous reports about the effect of chitosan molecular weight on all these parameters. Enhanced Green Fluorescent Protein plasmid (pEGFP-N1) and two molecular weight (MW) chitosan (20.6 and 57.5 kDa) with a high degree of deacetylation (≥85%) were used to design and assembly nanoparticles by complex coacervation with N/P ratios of 43 and 8. NPs were characterized determining morphology by Scanning Electron Microscopy (SEM), size by Dynamic Light Scattering (DLS) and zeta-potential by Laser-Doppler velocimetry (LDV). Also, biological functionality was assessed by in vitro transfection assays. NPs assembled using the lowest MW chitosan (20.6NPs) and N/P ratio = 8 had spherical shape, smaller mean size and polydispersity index (119 ± 20 nm; 0.257, respectively), and lower zeta-potential (17.65 ± 1.21 mV). Moreover, no significant difference was observed in size, polydispersity index and zeta-potential after 30 days of storage, in contrast with NPs assembled using the 57.5 kDa chitosan (57.5NPs). In vitro transfection assays using two cervical cancer cell lines (HeLa and SiHa) showed that 20.6NPs leaded to a higher expression of Green Fluorescent Protein (GFP) in contrast to 57.5NPs for both cell lines, and expression in HeLa cells was higher than in SiHa cell line. It was confirmed that transfection efficiency depends on all these physical parameters given by MW of chitosan, cell physiology, and the addition of fetal bovine serum in the transfection formulation. These results indicate that low MW chitosan and a low N/P ratio are more suitable to design chitosan-based nonviral vectors for gene therapy, given that physicochemical and biological properties, so as the stability of these NPs are better that those formulated with chitosan of higher MW.

Functional changes in long-term incubated rat precision-cut lung slices

BackgroundRespiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days.MethodsRat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation.ResultsCultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin.ConclusionsOverall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.

In Vitro Potential Virucidal Effect Evaluation of Xibornol on Human Adenovirus Type 5, Human Rhinovirus Type 13, Human Coronavirus 229E, Human Parainfluenza Virus Type 1, and Human Respiratory Syncytial Virus.

The availability of virucidal compounds to reduce the impact of respiratory viruses is a relevant topic for public health, especially during the recent coronavirus disease (COVID-19) pandemic. Antimicrobial properties of Xibornol are known since the 1970s, but its activity on viruses is currently little explored. In this study, Xibornol activity at a fixed concentration of 0.03mg/100ml has been evaluated on five respiratory viruses (Human Adenovirus 5, Human Rhinovirus type 13, Human Coronavirus 229E, Human Parainfluenza Virus type 1, and Human Respiratory Syncytial Virus) through in vitro experiments based on adapted European standard UNI EN 14476-20019. The experiments were carried out under two different environmental conditions, one with the addition of fetal bovine serum to simulate an in vivo condition (dirty condition) and the other without the addition of any organic substances (clean condition). The viral abatement of Xibornol (expressed as Log10 reduction - LR) was statistically significant under both clean and dirty environmental conditions. Namely, in clean condition, LR ranged from 2.67 to 3.84, while in the dirty one the abatement was slightly lower (from 1.75 to 3.03). Parainfluenza Virus and Human Adenovirus were most resistant compared to the other viruses. The obtained data confirmed Xibornol activity and its use as topic substance for viral inactivation to prevent upper respiratory tract disease.

Unexpected Enhancement of Antimicrobial Polymer Activity against Staphylococcus aureus in the Presence of Fetal Bovine Serum.

Cationic and amphiphilic polymers are known to exert broad-spectrum antibacterial activity by a putative mechanism of membrane disruption. Typically, nonspecific binding to hydrophobic components of the complex biological milieu, such as globular proteins, is considered a deterrent to the successful application of such polymers. To evaluate the extent to which serum deactivates antibacterial polymethacrylates, we compared their minimum inhibitory concentrations in the presence and absence of fetal bovine serum. Surprisingly, we discovered that the addition of fetal bovine serum (FBS) to the assay media in fact enhances the antimicrobial activity of polymers against Gram-positive bacteria S. aureus, whereas the opposite is the case for Gram-negative E. coli. Here, we present these unexpected trends and develop a hypothesis to potentially explain this unusual phenomenon.

The effect of Indonesian honey Tetragonula sp. and Indonesian royal jelly Apis mellifera (Ceiba pentandra) to human preputium cell proliferation in serum-free DMEM

Fetal bovine serum (FBS) is widely used for cell culture media, especially its function as a growth supplement with high growth-promoting factors. An optimal culture medium is needed to increase protein transduction. Unfortunately, FBS reported as media contained protease and contaminated with pathogen microorganisms from an animal host. Fibroblast preputium cell is easy to culture and can be a good model for assessing the medium culture system. In this study, FBS was substituted with honey and royal jelly to find an alternative FBS. This study aimed to determine the effectivity of serum-free DMEM medium with honey from Tetragonula sp. and royal jelly from Apis mellifera (Ceiba pentandra) on the proliferation of fibroblasts preputium cells. The research design used true experimental methods. Samples were taken from healthy people. Fibroblast cells were cultured with various concentrations of honey and royal jelly (0.1%, 1%, 5%). The best result of those various concentrations continued until 9 days with continuous checking in every three days measured with Microtetrazolium (MTT) assay test. Fibroblast cells cultured in Tetragonula sp. honey and royal jelly Apis mellifera (Ceiba pentandra) 0.1% medium had a significant difference, with proliferation higher than 1% (p = 0,000) and 5% (p = 0,000), but did not exceed proliferation with FBS addition medium. Next, cells in DMEM medium with Tetragonula sp. honey and Apis mellifera royal jelly (Ceiba pentandra) 0.1% on the 3rd, 6th, and 9th (p = 0,000; p = 0,000; p = 0,000) had not similar growth to the standard medium with FBS. However, the growth on the 9th day had a significant difference with the DMEM medium without FBS. High sugar in honey can inhibit fibroblast cell proliferation. The addition of other components as needed to optimize proliferation in honey and royal jelly medium. Isolation of active ingredients in honey and royal jelly can function as an alternative development of an effective and safe substitute for FBS.

Use of MTT Assay for Proliferation of U937 Cell Line and its Inter-rater Reliability - Best Taken with a Grain of Salt

Background Biomedical research frequently employs immortal human cell lines to study various physiological and pathological topics of interest. An important problem that is often ignored is the question of reliability of observations made while using a particular assay on cell lines, especially MTT Assay. Recent studies have questioned its reliability by highlighting the diverse intracellular environmental factors that affect accuracy of MTT assay results. Objective To assess the inter-rater reliability of MTT assay while observing the differential effect of addition of fetal bovine serum to the culture medium on proliferation of U937 cell line using Intraclass correlation coefficient. Method U937 cell suspension prepared by the tutor to maintain uniformity of immortalized cell line was used. Four trainee investigators who participated in the study underwent rigorous training in pipetting and plating methods for four consecutive days prior to start of the experiment. Each trainee investigator followed the same protocol and all procedures were conducted simultaneously. At the end of the experiment, inter observer reliability was calculated. Result Interrater reliability calculated by using intra class correlation coefficient with two way mixed effect model was found to be poor (p = 0.173). For growth with 10% fetal bovine serum, inter-rater reliability was 0.258 for each item and 0.58 for the average of the triplicates, whereas for 0% fetal bovine serum it was 0.374 for each item and 0.70 for average of the values (p=0.104). Conclusion Though MTT assay is considered the gold standard test for assessment of cell proliferation and viability, the inter-rater reliability of this assay might be poor and needs further investigation.

Growing cattle embryos beyond Day 8 – An investigation of media components

The growth of viable cattle embryos in culture to stages beyond the hatching blastocyst is of interest to developmental biologists wishing to understand developmental events beyond the first lineage decision, as well as for commercial applications, because a lengthening of the culturing time allows more time for diagnostic tests on biopsies, whereas extended survival can be used as a better assay system for monitoring developmental potential. We here report on a novel extended culture medium for embryo growth until embryonic day (Day) 12. We used a non-invasive morphological characterisation system that scored viability, inner cell mass (ICM) grade, hatching and embryo and ICM diameter. The basal medium was based on published uterine fluid concentrations of amino acids, carbohydrates and electrolytes. Addition of fetal bovine serum was necessary and the additive ITSX greatly improved culture success. We tested the inclusion of a seven-growth factor cocktail consisting of Activin A, Artemin, BMP4, EGF, FGF4, GM-CSF/CSF2 and LIF, as well as omission of individual components of the cocktail. In the context of the growth factor cocktail, Artemin and BMP4 provided the greatest benefit, while FGF omission had more positive than negative effects on embryo characteristics. Lastly, replacement of ITSX by B27-additive led to the most successful culture of embryos, in all media permutations.

Efficient, Non-Viral and Reproducible Protocol for Stable Knockdown of Genes in Mantle Cell Lymphoma Cell Lines

Introduction The biological role of specific genes can be investigated using gene silencing by cell transfection with small interfering RNA (siRNA), which leads to degradation of the corresponding mRNA and reduced target protein expression. The study of biological functions in modified cell cultures often requires a long-lasting knockdown (KD) of gene expression, which can be obtained by viral delivery of short hairpin RNA (shRNA) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 systems. However, these methods are laborious and require high level of laboratory safety. Hematopoietic cells, typically grown in suspension, are known to be difficult to transfect. Previous efforts to transfect lymphocytes using liposomes (H Guven et al, ExpHem2005) showed limited transfection efficiencies of only 10-30%. Transfection by electroporation leads to decreased viability, and therefore poses a risk of unintentional selection of cell subsets. Transfection of leukemia cells with siRNAs using Accell siRNA Delivery Media showed KD efficiencies of 56-65% and a viability of 57-95% (H Larsen et al,ExpHem2011). However, this was only investigated 24-72h post-transfection. By whole transcriptome sequencing, we previously identified a number of candidate genes, includingLILRA4, PTPRJ and SYNE2, which were significantly upregulated in mantle cell lymphoma (MCL) cells compared to B cells from healthy donors (MH Hansen et al, ExpHem 2020). To investigate the biological function of these genes, an efficient and stable KD was required. Aim The aim of this study was to identify the most effective non-viral method for siRNA-mediated knockdown in MCL cells. Methods The MCL cell lines Mino, Jeko-1 and Granta 519, were transfected using three different methodologies; Lipofectamine 2000 (Invitrogen), Electroporation with Amaxa Nucleofector (Lonza), and Accell siRNA Delivery Media (Dharmacon). For Accell delivery a pool of 4 siRNAs targeting each gene was used (SMARTpool siRNA, Dharmacon). For the others, 2 pre-designed siRNAs (Ambion) targeting each gene were used. For all, a positive control siRNA targetingGAPDHwas included and different conditions tested e.g. siRNA concentration, incubation time and addition of serum. KD efficiency was evaluated by qPCR usingABLandGUSreference genes. Viability of cells was measured either by flow cytometry staining with a live/dead marker or by trypan blue. Optimization of the Accell protocol was performed in Granta 519 cells transfected with SMARTpool siRNAs targeting eitherSYNE2,LILRA4,orGAPDH. KD efficiency was evaluated with qPCR and viability was counted with trypan blue. Results Using Lipofectamine, at 24h a non-reproducible and non-efficient KD (<40%) was achieved forLILRA4in the Jeko-1 cell line, while only 5-22% gene silencing was achieved for Mino. Using electroporation, a KD efficiency of 45-70% was achieved in Mino at 24h forGAPDH,LILRA4andPTPRJ, while inferior efficiency (<15%) and viability were observed in Jeko-1 cells. However, in the Mino cell line,PTPRJwas almost fully re-expressed (80% expression) after 48h. Using transfection with Accell delivery medium,GAPDH,LILRA4,andPTPRJwere successfully knocked down in all tested cell lines with a median efficiency of 75% (range 50%-98%) after 72h. When performing KD in Accell delivery medium, the viability of the cells decreased after 72h and dropped to 26-39% at day 5 and 6, and a decrease was found in the total number of cells, suggesting that proliferation may also be affected. By adding 10% fetal bovine serum (FBS) to this medium after 48h, the viability was kept above 66.7% for up to 6 days, and the total number of cells was comparable to that observed in normal culture medium, while the KD efficiency was still preserved. In Ganta 519 cells with addition of FBS, an average KD efficiency of 67% was achieved forLILRA4(range 56-72%) at 72h-5 days, while the average KD efficiency ofSYNE2was 77% (range: 67-95%) at 72h-5 days. Conclusion This study propose that transfection with SMARTpool siRNAs in Accell Delivery Medium with addition of 10% FBS 48h post transfection is an applicable procedure for siRNA-mediated KD in MCL suspension cell lines. This protocol permits efficient, non-viral, and stable KD across different MCL cell lines without affecting the cells' ability to proliferate and survive, and is therefore suitable for investigating biological processes. Disclosures No relevant conflicts of interest to declare.

Protein Binding and Stability of Drug Candidates: The Achilles' Heel in In Vitro Potency Assays.

In the present scenario of drug discovery, several screening filters ensure a rigorous nomination of clinical candidates. One of these screens is the determination of IC50, the concentration of drug at half-maximal inhibitory concentration, also known as a potency assay. However, various nuances pertaining to the design, execution, and interpretation of in vitro potency results suggest a sizeable opportunity for the generation of erroneous data. The focus areas of this article include: (1) examining the requirement for the addition of serum albumin in in vitro potency assays, (2) problems encountered with cell lysates, and (3) drug candidate stability concerns during in vitro potency assays/high-throughput screening. Based on this assessment, the interpretation of the data generated using cell-based systems (i.e., lysates with or without the addition of fetal bovine serum) should be carried out with caution for in vitro potency testing, and the inclusion of a correction factor for non-specific protein binding should be considered. The addition of serum albumin to a cell-free system should be restricted to drugs having high protein binding (≥ 90%). Additionally, stability assessment of analytes should be considered to avoid dubious in vitro potency outcomes due to degraded material or active metabolite(s).

Plasma Amine Oxidase-Induced Nanoparticle-to-Nanofiber Geometric Transformation of an Amphiphilic Peptide for Drug Encapsulation and Enhanced Bactericidal Activity.

Patients with cancer have reduced immune function and are susceptible to bacterial infection after surgery, chemotherapy, or radiotherapy. Spherical nanoparticles formed by the self-assembled peptide V6K3 can be used as carriers for poorly soluble antitumor drugs to effectively deliver drugs into tumor cells. V6K3 was designed to achieve nanoparticle-to-nanofiber geometric transformation under induction by plasma amine oxidase (PAO). PAO is commercially available and functionally similar to lysyl oxidase (LO), which is widely present in serum. After the addition of fetal bovine serum (FBS) or PAO, the secondary structure of the peptide changed, while the spherical nanoparticles stretched and transformed into nanofibers. The conversion of the self-assembled morphology reveals the susceptibility of this amphiphilic peptide to subtle chemical modifications and may lead to promising strategies to control self-assembled architecture via enzyme induction. Enzymatically self-assembled V6K3 had bactericidal properties after PAO addition that were surprisingly superior to those before PAO addition, enabling this peptide to be used to prevent infection. The amphiphilic peptide V6K3 displayed antitumor properties and low toxicity in mammalian cells, demonstrating good biocompatibility, as well as bactericidal properties, to prevent bacterial contamination. These advantages indicate that enzymatically self-assembled V6K3 has great biomedical application potential in cancer therapy.

Bioactive Fibrin Scaffolds for Use in Musculoskeletal Regenerative Medicine

Abstract Autologous fibrin matrices derived from the Leukocyte and Platelet Rich Plasma (L-PRP) and Leukocyte and Platelet Rich Fibrin (L-PRF) techniques present great potential to act as a bioactive scaffold in regenerative medicine, contributing to the maintenance of cell viability, proliferation stimulus and differentiation. In contrast, there are few studies that characterize the bioactive potential of these fibrin scaffolds by considering the process of production. The objective of this work was to characterize the intrinsic potential of maintaining cell viability of different fibrin scaffolds containing platelets and leukocytes. In order to achieve that, blood samples from a volunteer were collected and processed to obtain fibrin clots using the suggested techniques. To characterize the potential for in vitro viability, mesenchymal stem cells from human infrapatellar fat were used. The scaffolds were cellularized (1x105 cells/scaffolds) and maintained for 5 and 10 days under culture conditions with Dulbecco's Modified Eagle Medium, without addition of fetal bovine serum, and subsequently subjected to analyses by Fourrier transform infra-red spectroscopy, circular dichroism and fluorescence microscopy. The results demonstrated distinct intrinsic potential viability between the scaffolds, and L-PRP was responsible for promoting higher levels of viability in both periods of analysis. No viable cells were identified in the fibrin matrix used as controls. These results allow us to conclude that both fibrin substrates have presented intrinsic potential for maintaining cell viability, with superior potential exhibited by L-PRP scaffold, and represent promising alternatives for use as bioactive supports in musculoskeletal regenerative medicine.

Optimization of the Protocol for Isolation of Exosome and Secretome of CACO-2 Colorectal Adenocarcinoma Cells from Conditioned Media for Subsequent Mass Spectrometric Analysis

The conditioned media in which the tumor cells are cultured represents a model object for studying of secretome and proteomic composition of exosomes. This pool of proteins is of particular interest in terms of the search for potential markers of malignant diseases. The efficiency of the isolation of exosomes and secretome from the cell culture media largely determines the success of their analysis by the mass spectrometric method. In this paper, we have applied various approaches to isolate secretome and exosomes originated from Caco-2 colorectal adenocarcinoma cells. The combination of ultrafiltration and centrifugation provided a deep proteomic analysis of the secretome and exosomes obtained from the one initial volume of conditioned medium without the addition of fetal bovine serum. Applying tandem mass spectrometric analysis we have identified 436 secreted proteins. In exosomes, the characteristic proteins ALIX, CD63, syntenin, lactadherin (MFGE8) were identified. Using targeted mass spectrometry, the exosome markers CD82, CD9 and HSPA8 were determined at the levels of 0.08 ± 0.03 fmol/μg, 0.13 ± 0.03 fmol/μg and 2.6 ± 0.02 fmol/μg of total protein, respectively. Among the secreted proteins, there were many markers associated with tumor progression and metastasis, such as DAG1, PODXL, LRRN4, TGFBI, IL6ST, DSC1, DSG1, and NPC2.

Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles

ABSTRACTRecent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background.

Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts.

The postwarming recovery culture, as one of the steps in cryopreservation process, is directly correlated with the survival and quality of embryos. Generally, recovery medium includes undefined serum or serum components that may cause the instability of results and other problems. The objective of this study was to evaluate the effect of knockout serum replacement (KSR) as a substitute for serum during recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Fetal bovine serum (FBS) was used as a positive control. The expanded blastocysts on day 5 were vitrified by the Cryotop method, and recovered with 10% (v/v) KSR or 10% (v/v) FBS for 48 hours after warming. Survival and hatching rates of vitrified blastocysts were significantly increased by KSR or FBS supplementation. The vitrified blastocysts recovered in KSR or FBS exhibited significantly decreased percentages of membrane damage and apoptosis, and increased total cells. Addition of KSR or FBS during recovery culture significantly reduced reactive oxygen species levels, and improved mitochondrial activity and adenosine triphosphates content in the vitrified blastocysts. Vitrification did not affect the gene expression of PCNA, CDX2, and CPT1, but significantly increased mRNA levels of POU5F1 and uPA. KSR added to the recovery medium significantly upregulated mRNA levels of PCNA and CPT1, and downregulated POU5F1 mRNA levels. The expression levels of PCNA, CDX2, CPT1, and uPA in vitrified blastocysts were significantly upregulated by addition of FBS to recovery medium. Moreover, the BAX: BCL2L1 ratio was similar between fresh and vitrified blastocysts, and KSR or FBS supplementation had no effect on the value. In conclusion, our data showed that KSR supplementation during recovery culture can improve the development and quality of vitrified parthenogenetic porcine blastocysts. These findings provide a useful reference that KSR could be used to replace FBS as a defined serum supplement for recovery culture of vitrified blastocysts.

Structural and Functional Stability of DNA Nanopores in Biological Media.

DNA nanopores offer a unique nano-scale foothold at the membrane interface that can help advance the life sciences as biophysical research tools or gate-keepers for drug delivery. Biological applications require sufficient physiological stability and membrane activity for viable biological action. In this report, we determine essential parameters for efficient nanopore folding and membrane binding in biocompatible cell media. The parameters are identified for an archetypal DNA nanopore composed of six interwoven strands carrying cholesterol lipid anchors. Using gel electrophoresis and fluorescence spectroscopy, the nanostructures are found to assemble efficiently in cell media, such as LB and DMEM, and remain structurally stable at physiological temperatures. Furthermore, the pores’ oligomerization state is monitored using fluorescence spectroscopy and confocal microscopy. The pores remain predominately water-soluble over 24 h in all buffer systems, and were able to bind to lipid vesicles after 24 h to confirm membrane activity. However, the addition of fetal bovine serum to DMEM causes a significant reduction in nanopore activity. Serum proteins complex rapidly to the pore, most likely via ionic interactions, to reduce the effective nanopore concentration in solution. Our findings outline crucial conditions for maintaining lipidated DNA nanodevices, structurally and functionally intact in cell media, and pave the way for biological studies in the future.