Serum-free Cell Culture Research Articles

Sericin Protein: Structure, Properties, and Applications.

Silk sericin, the glue protein binding fibroin fibers together, is present in the Bombyx mori silkworms' cocoons. In recent years, sericin has gained attention for its wide range of properties and possible opportunities for various applications, as evidenced by the meta-analysis conducted in this review. Sericin extraction methods have evolved over the years to become more efficient and environmentally friendly, preserving its structure. Due to its biocompatibility, biodegradability, anti-inflammatory, antibacterial, antioxidant, UV-protective, anti-tyrosinase, anti-aging, and anti-cancer properties, sericin is increasingly used in biomedical fields like drug delivery, tissue engineering, and serum-free cell culture media. Beyond healthcare, sericin shows promise in industries such as textiles, cosmetics, and food packaging. This review aims to highlight recent advancements in sericin extraction, research, and applications, while also summarizing key findings from earlier studies.

Expression of matrix metalloproteinases and their inhibitors in corneal stromal fibroblasts and keratocytes from healthy and keratoconus corneas.

To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas. Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant. KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively. This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC.

Life cycle assessment of Beefy-9 and Beefy-R serum-free culture media for cell-cultivated beef production

The sustainable production of cell-cultivated meat requires the use of environmentally friendly nutrients. Culture media, which provides the necessary nutrients for cell proliferation and differentiation is a major contributor to the environmental impacts of cultivated meat production. Fetal bovine serum (FBS) is problematic due to animal sourcing, ethical concerns, and high cost. Beefy-R and Beefy-9 are experimentally validated, serum-free cell culture media designed for bovine myoblast culture. While both formulations include basal media, ascorbic acid, sodium selenite, recombinant proteins, and growth factors, Beefy-9 and Beefy-R contain recombinant albumin and rapeseed protein isolates (RPI) to replace FBS, respectively. While these culture media do not include animal-sourced components, their environmental impacts have not yet been investigated. In this study, life cycle assessment (LCA) was performed to estimate the environmental burdens of Beefy-9 and Beefy-R culture media production and identify hotspots for optimization. Uncertainty was assessed through sensitivity and scenario analyses, as well as Monte Carlo simulation. Our results demonstrate that Beefy-R has significantly lower environmental impacts compared to Beefy-9 in 13 out of 18 evaluated impact categories. For instance, the global warming potential (GWP) of Beefy-R was almost five times lower when compared against that of Beefy-9 production. In Beefy-R, despite the absence of recombinant albumin, recombinant proteins and growth factors were hotspots, accounting for 41 %, 5.9 %, and 12 % of GWP, land use, and water consumption impacts, respectively. This study confirms that RPI can be a more sustainable substitute for recombinant albumin in bovine myoblast serum-free media formulations. Additional research is required to identify low-impact alternatives for other recombinant proteins and growth factors in culture media.

Abstract 6781: Maintaining the tumor microenvironment: Serum-free cell culture for an advanced 3D in vitro tissue model

Abstract Cancer research has made significant progress in recent years, especially in the field of immunotherapy that uses immune modulators. A significant portion of these novel therapeutic strategies focuses on manipulating the tumor microenvironment (TME). The TME is a complex structure that comprises various cellular elements, including cancer cells, immune cells, and structural components, such as secreted factors and extracellular matrix proteins. However, replicating the human TME in experimental murine models has proved challenging. Therefore, researchers have started to develop ex vivo cultivation protocols of tumor tissues, particularly precision-cut slices (PCS), that offer a three-dimensional representation of the TME. It is essential to select the best culture conditions to ensure the preservation of the TME that closely resembles the in vivo conditions. To further this research, a serum-free cell culture medium has been developed to propagate primary epithelial tumor cells originating from pancreatic, renal, and ovarian tissues. The optimized media formulation was successfully employed in the derivation and expansion of tumor cell lines, sourced from both primary and xenotransplanted tumors. An additional serum-free cell culture medium has been designed to cultivate primary lung cancer cells. Suitable culturing conditions have been introduced that maintain TME composition ex vivo cultured PCS prepared from fresh non-small cell lung carcinoma (NSCLC) tissue. Lung TumorMACS Medium and a serum-containing medium were compared, and it was found that the cellular composition of PCS cultivated in Lung TumorMACS was comparable to the original composition. At the same time, PCS cultivated in Lung TumorMACS showed a lower expression of stress genes, compared to the serum-containing alternative. Citation Format: Alina Siebenmorgen, Christian Wöhle, Katharina Lamfried, Lilian Martinez Carrera, Bianca Heller, Katharina Lupar, Giovanna Bergamini, Daniel Poeckel, Olaf Hardt, Benjamin Theek. Maintaining the tumor microenvironment: Serum-free cell culture for an advanced 3D in vitro tissue model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6781.

Lipolysis inhibition improves the survival of fat grafts through ameliorating lipotoxicity and inflammation.

Fat grafting is a promising technique for correcting soft tissue abnormalities, but oil cyst formation and graft fibrosis frequently impede the therapeutic benefit of fat grafting. The lipolysis of released oil droplets after grafting may make the inflammation and fibrosis in the grafts worse; therefore, by regulating adipose triglyceride lipase (ATGL) via Atglistatin (ATG) and Forskolin (FSK), we investigated the impact of lipolysis on fat grafts in this study. After being removed from the mice and chopped into small pieces, the subcutaneous fat from wild-type C57BL/6J mice was placed in three different solutions for two hours: serum-free cell culture medium, culture medium+FSK (50 μM), and culture medium+ATG (100 μM). Following centrifugation to remove water and free oil droplets, 0.3 mL of the fat particles per mouse was subcutaneously injected into the back of mice. Additionally, the subcutaneous fat grafting area was immediately injected with PBS (control group), ATG (30 mg/kg), and FSK (15 mg/kg) following fat transplantation. Detailed cellular events after grafting were investigated by histological staining, real-time polymerase chain reaction, immunohistochemistry/immunofluorescent staining, and quantification. Two weeks after grafting, grafts treated with ATG showed lower expression of ATGL and decreased mRNA levels of TNFα and IL-6. In contrast, grafts treated with ATG showed elevated expression levels of IL-4 and IL-13 compared to the control grafts. In addition, fewer apoptotic cells and oil cysts were observed in ATG grafts. Meanwhile, a higher CD206+/CD68+ ratio of macrophages and more CD31+ vascular endothelial cells existed in the 2-month ATG grafts. In comparison to the control, ATG treatment improved the volume retention of grafts, and decreased graft fibrosis and oil cyst formation. By preventing oil droplet lipolysis, pharmacological suppression of ATGL shielded adipocytes from lipotoxicity following grafting. Additionally, ATG ameliorated the apoptosis and inflammation brought on by adipocyte death and oil droplet lipolysis in grafted fat. These all indicate that lipolysis inhibition improved transplanted fat survival and decreased the development of oil cysts and graft fibrosis, offering a potential postoperative pharmacological intervention for bettering fat grafting.

Effects of Oral Cavity Stem Cell Sources and Serum-Free Cell Culture on Hydrogel Encapsulation of Mesenchymal Stem Cells for Bone Regeneration: An In Vitro Investigation.

To develop a stem cell delivery model and improve the safety of stem cell transplantation for bone regeneration, this study aimed to determine the effects of stem cell sources, serum-free cell culture, and hydrogel cell encapsulation on the growth and osteogenic differentiation of mesenchymal stem cells (MSCs) from the oral cavity. The study groups were categorized according to stem cell sources into buccal fat pad adipose (hBFP-ADSCs) (Groups 1, 4, and 7), periodontal ligament (hPDLSCs) (Groups 2, 5, and 8), and dental pulp-derived stem cells (hDPSCs) (Groups 3, 6, and 9). MSCs from each source were isolated and expanded in three types of sera: fetal bovine serum (FBS) (Groups 1-3), human serum (HS) (Groups 4-6), and synthetic serum (SS) (StemPro™ MSC SFM) (Groups 7-9) for monolayer (m) and hydrogel cell encapsulation cultures (e). Following this, the morphology, expression of MSC cell surface antigens, growth, and osteogenic differentiation potential of the MSCs, and the expression of adhesion molecules were analyzed and compared. SS decreased variations in the morphology and expression levels of cell surface antigens of MSCs from three cell sources (Groups 7m-9m). The levels of osteoblastic differentiation of the hPDLSCs and hBFP-ADSCs were increased in SS (Groups 8m and 7m) and the cell encapsulation model (Groups 1e, 4e, 7e-9e), but the promoting effects of SS were decreased in a cell encapsulation model (Groups 7e-9e). The expression levels of the alpha v beta 3 (ITG-αVβ3) and beta 1 (ITG-β1) integrins in the encapsulated cells in FBS (Group 1e) were higher than those in the SS (Group 7e). Human PDLSCs and BFP-ADSCs were the optimum stem cell source for stem cell encapsulation by using nanohydroxyapatite-calcium carbonate microcapsule-chitosan/collagen hydrogel in serum-free conditions.

Culture of Mouse Thymic Epithelial Cells in Serum-Free Medium.

Primary cell culture systems are widely used as a valuable method for analyzing the biological functions of specific cells in vitro. Recently, various serum-free primary cell culture methods have been developed that do not involve the use of animal serums. Since the thymus is comprised of many cell types, such as thymocytes, thymic epithelial cells, macrophages, and fibroblasts, thymic epithelial cells must be isolated for their functional analysis in vitro. This chapter describes the detailed protocol for the selective primary culture of thymic epithelial cells using defined serum-free medium.

The atypical antidepressant tianeptine confers neuroprotection against oxygen–glucose deprivation

Proregenerative and neuroprotective effects of antidepressants are an important topic of inquiry in neuropsychiatric research. Oxygen–glucose deprivation (OGD) mimics key aspects of ischemic injury in vitro. Here, we studied the effects of 24-h pretreatment with serotonin (5-HT), citalopram (CIT), fluoxetine (FLU), and tianeptine (TIA) on primary mouse cortical neurons subjected to transient OGD. 5-HT (50 μM) significantly enhanced neuron viability as measured by MTT assay and reduced cell death and LDH release. CIT (10 μM) and FLU (1 μM) did not increase the effects of 5-HT and neither antidepressant conferred neuroprotection in the absence of supplemental 5-HT in serum-free cell culture medium. By contrast, pre-treatment with TIA (10 μM) resulted in robust neuroprotection, even in the absence of 5-HT. Furthermore, TIA inhibited mRNA transcription of candidate genes related to cell death and hypoxia and attenuated lipid peroxidation, a hallmark of neuronal injury. Finally, deep RNA sequencing of primary neurons subjected to OGD demonstrated that OGD induces many pathways relating to cell survival, the inflammation-immune response, synaptic dysregulation and apoptosis, and that TIA pretreatment counteracted these effects of OGD. In conclusion, this study highlights the comparative strength of the 5-HT independent neuroprotective effects of TIA and identifies the molecular pathways involved.

Sequential adaptation to serum-free medium for Vero cells cultivation on ultraviolet/ozone (UVO) treated microcarrier

The adoption of serum-free media (SFM) in cell culture has gained prominence as an alternative to serum-supplemented media (SSM) owing to concerns regarding serum variability and quality assurance. Vero cells, widely utilized in viral vaccine production for their susceptibility to various viruses, were the focus of this study. Our primary objective was to adapt Vero cells from SSM to SFM and subsequently upscale the culture from T-flasks to spinner vessels using ultraviolet/ozone (UVO) treated polystyrene (PS) and polycaprolactone (PCL) microcarriers. Cell counting was conducted via haemocytometer and inverted phase microscopy. The results indicated that SFM-adapted Vero cells achieved higher maximum cell concentrations compared to those cultured in SSM, along with a shorter doubling time. However, during cultivation in spinner vessels, Vero cells exhibited lower maximum cell concentrations compared to T-flask cultures, attributed to factors such as cell inoculum size and microcarrier hydrophilicity. This study highlights the successful adaptation of Vero cells to SFM and the potential benefits in terms of cell density and growth kinetics. Furthermore, it sheds light on the challenges encountered during culture upscale, offering valuable insights for optimizing future large-scale production processes. The findings contribute to the advancement of serum-free cell culture systems and their applicability in the production of viral vaccines.

Human Patient-Derived Brain Tumor Models to Recapitulate Ependymoma Tumor Vasculature.

Despite in vivo malignancy, ependymoma lacks cell culture models, thus limiting therapy development. Here, we used a tunable three-dimensional (3D) culture system to approximate the ependymoma microenvironment for recapitulating a patient's tumor in vitro. Our data showed that the inclusion of VEGF in serum-free, mixed neural and endothelial cell culture media supported the in vitro growth of all four ependymoma patient samples. The growth was driven by Nestin and Ki67 double-positive cells in a putative cancer stem cell niche, which was manifested as rosette-looking clusters in 2D and spheroids in 3D. The effects of extracellular matrix (ECM) such as collagen or Matrigel superseded that of the media conditions, with Matrigel resulting in the greater enrichment of Nestin-positive cells. When mixed with endothelial cells, the 3D co-culture models developed capillary networks resembling the in vivo ependymoma vasculature. The transcriptomic analysis of two patient cases demonstrated the separation of in vitro cultures by individual patients, with one patient's culture samples closely clustered with the primary tumor tissue. While VEGF was found to be necessary for preserving the transcriptomic features of in vitro cultures, the presence of endothelial cells shifted the gene's expression patterns, especially genes associated with ECM remodeling. The homeobox genes were mostly affected in the 3D in vitro models compared to the primary tumor tissue and between different 3D formats. These findings provide a basis for understanding the ependymoma microenvironment and enabling the further development of patient-derived in vitro ependymoma models for personalized medicine.

A simple and robust serum-free media for the proliferation of muscle cells

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Large-scale and cost-effective production of recombinant human serum albumin (rHSA) in transgenic Bombyx mori cocoons

HSA is considered a versatile natural cargo carrier with multiple bio-functions and applications. However, insufficient supply of HSA has limited widespread use. Although various recombinant expression systems had been applied to produce the rHSA to overcome the limited resource, cost-effective and large scale production of rHSA remains a challenge. Herein, we provide a strategy for the large-scale and cost-effective production of rHSA in cocoons of transgenic silkworms, achieving a final 13.54 ± 1.34 g/kg of rHSA yield in cocoons. rHSA was efficiently synthesized and stable over the long-term in the cocoons at room temperature. Artificial control of silk crystal structure during silk spinning significantly facilitated rHSA extraction and purification, with 99.69 ± 0.33 % purity and a productivity of 8.06 ± 0.17 g rHSA from 1 kg cocoons. The rHSA had the same secondary structure to natural HSA, along with effective drug binding capacity, biocompatibility, and bio-safe. The rHSA was successfully evaluated as a potential substitute in serum-free cell culture. These findings suggest the silkworm bioreactor is promising for large-scale and cost-effective production of high quality rHSA to meet the increased worldwide demand.

Using three-phase partitioning for the purification and recovery of antibodies from biological media

Antibodies, in particular immunoglobulin G (IgG), are one of the biopharmaceutical industry highflyers, with relevance for the treatment of several diseases. However, the recovery of antibodies from complex biological media with high quality and purity is difficult and requires multi-step and expensive approaches. Herein, we propose a cost-effective approach using three-phase partitioning (TPP) systems based on polyethylene glycol (PEG)-salt aqueous biphasic systems (ABS) with ionic liquids (ILs) as adjuvants for the purification and recovery of IgG antibodies from three biological media, i.e., human serum, and serum-containing and serum-free Chinese hamster ovary (CHO) cell culture supernatants. The economic analysis of the developed process was carried out. The results obtained using PEG-salt ABS without ILs and human serum show that IgG could be recovered either at the interphase of the TPP or in the top phase, depending on the molecular weight of the PEG. The system composed of PEG with a molecular weight of 1000 g/mol is the PEG-salt system enabling the highest purity of human polyclonal IgG at the interphase (80.7 %, with a recovery yield of 65.8 %). Still, by adding 1 wt% of the ILs tetra(n-butyl)ammonium bromide ([N4444]Br) and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), it is possible to compete or even outperform the PEG 1000-salt system with no IL regarding purity/recovery performance and production costs under given operation conditions. The best systems were then applied for the purification and recovery of monoclonal antibodies from serum-containing and serum-free culture supernatants. Improved recovery of monoclonal antibodies from serum-free Chinese hamster ovary (CHO) cell culture supernatants with a reduction of the host cell proteins (HCPs) content are obtained by introducing ILs as adjuvants in PEG-salt systems, with [N4444]Br providing the lowest production costs. Overall, TPP systems were shown to be not only cost-effective, but also robust and flexible routes to purify and recover IgG from complex biological matrices as shown here with human serum, serum-containing and serum-free cell culture supernatants.

Abstract 56: Unlocking primary cell culture for more relevant in vitro models

Abstract Tumor cell lines are commonly used to investigate biological and translational questions. However, many cell lines have been cultured in serum-containing medium for decades, continuously accumulating genetic and epigenetic aberrations. Without a doubt, these cell lines have very little in common with the tumor they originate from, resulting in low biological and clinical relevance. Despite these drawbacks, cell lines are still the most frequent research tool when it comes to the use of ex vivo models. Yet, in light of higher consistency, reproducibility, and biological relevance, more and more researchers are paying attention to which cells and culture medium to use. Although primary cell cultures certainly reflect cellular and genetic heterogeneity of the original tumor, finding a suitable medium that supports their growth and maintains genetic heterogeneity remains challenging. To this end, we developed serum-free cell culture media to cultivate primary epithelial tumor cells of pancreatic, renal, and ovarian origin. Notably, generating in vitro models with high biological relevance was successful when using our media during the derivation and expansion of tumor cell lines from primary and xenotransplanted tumors. Here, we present another serum-free cell culture media supporting the in vitro growth of primary colon adenocarcinoma. Using this optimized medium, we had a 42% success rate in establishing a primary cell culture for a given tumor sample, higher than for any other media tested in our hands. Next to 2D cell culture, our formulation supports 3D tumoroid formation when cells are embedded in a basement membrane matrix. Furthermore, targeted sequencing of 486 tumor-associated genes revealed that more than 99% of the detected genetic variants in an early passage (p11) were also present after extended time in culture (p53). In summary, our new serum-free medium enables the efficient derivation and expansion of primary tumor and xenograft-derived colon adenocarcinoma cell cultures in 2D and 3D, yet retaining their genetic identity. Citation Format: Benjamin Theek, Anne Franke, David Agorku, Katharina Lamfried, Nojan Jelveh, Michael Linnebacher, Lilian Martinez Carrera, Olaf Hardt. Unlocking primary cell culture for more relevant in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 56.

The effect of serum-free media on the metabolic yields and growth rates of C2C12 cells in the context of cultivated meat production

Successful commercialization of cultivated meat products as alternative sources of dietary protein for a growing global population will require development of efficient and inexpensive serum-free cell culture media. It is known that serum-free media elicit dissimilar cell growth behavior compared to serum-based media, but data comprehensively exploring the effects of serum-free culture on the nutrient requirements of cultivated meat-relevant cell types have yet to be reported. We performed spent media analysis of C2C12 cells growing in Essential 8 serum-free media and in conventional serum-containing media. Data indicated that although the cell growth rates were similar in the two media over seven days, there were significant differences in the utilization rates of some key nutrients such as glucose, glutamine, glycine, and cystine. We extrapolated from the data that roughly 250–275 g of amino acids and 1100–1500 g of glucose would be required to produce 1 kg of C2C12 cells, though these requirements may change when using more optimized cells, media, and feed strategies. Our results highlight the influence of serum and serum replacements on cell metabolism, and indicate that previous data from metabolic studies performed using serum-containing media may not directly translate to serum-free systems.

Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

The Mycotoxin De-Epoxy-Deoxynivalenol (DOM-1) Increases Endoplasmic Reticulum Stress in Ovarian Theca Cells.

Deoxynivalenol (DON) is a major mycotoxin present in animal feed and negatively affects growth and reproduction in farm species, including pigs and cattle. The mechanism of DON action involves the ribotoxic stress response (RSR), and it acts directly on ovarian granulosa cells to increase cell death. In ruminants, DON is metabolized to de-epoxy-DON (DOM-1), which cannot activate the RSR but has been shown to increase cell death in ovarian theca cells. In the present study, we determined if DOM-1 acts on bovine theca cells through endoplasmic stress using an established serum-free cell culture model and to assess whether also DON activates endoplasmic stress in granulosa cells. The results show that DOM-1 increased the cleavage of ATF6 protein, increased the phosphorylation of EIF2AK3, and increased the abundance of cleaved XBP1 mRNA. Activation of these pathways led to an increased abundance of mRNA of the ER stress target genes GRP78, GRP94, and CHOP. Although CHOP is widely associated with autophagy, inhibition of autophagy did not alter the response of theca cells to DOM-1. The addition of DON to granulosa cells partially increased ER stress pathways but failed to increase the abundance of mRNA of ER stress target genes. We conclude that the mechanism of action of DOM-1, at least in bovine theca cells, is through the activation of ER stress.

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30µm). To reduce the supernatant volume and process time, the clarified sample was concentrated by ultrafiltration either using tangential flow filtration or centrifugal-based filtration units, depending on the process scale. The final purification step at laboratory-scale, was carried out by density gradient ultracentrifugation, the recovery of which was compared with chromatographic purification at large-scale. The virus preparations were analyzed using dynamic light scattering to verify the virus size and transmission electron microscopy for purity and virus morphology. Density gradient ultracentrifugation allowed the recovery of≥80% infectious particles and reduced the contaminant DNA and host cell proteins relatively to standard ultracentrifugation pelleting using a sucrose cushion. At large-scale, weak and strong anion-exchangers were tested and compared. The best columns allowed infectious virus recoveries as high as 77% and eliminated 92% of host cell proteins.

Comparison of blocking reagents for antibody microarray-based immunoassays on glass and paper membrane substrates.

Background noise due to nonspecific binding of biomolecules on the assay substrates is one of the most common challenges that limits the sensitivity of microarray-based immunoassays. Background signal intensity usually increases when complex biological fluids are used because they have a combination of molecules and vesicles that can adsorb onto substrate surfaces. Blocking strategies coupled with surface chemistries can reduce such nonspecific binding and improve assay sensitivity. In this paper, we conducted a systematic optimization of blocking strategies on a variety of commonly used substrates for protein measurement in complex biofluids. Four blocking strategies (BSA, non-fat milk, PEG, and a protein-free solution) coupled with four surface chemistries (3-glycidoxypropyltrimethoxysilane (GPS), poly-L-lysine (PLL), aminoalkylsilane (AAS), and nitrocellulose (NC)) were studied for their effect on background, microspot, and net signal intensities. We have also explored the effect that these blocking strategies have when proteins in complex samples (plasma, serum, cell culture media, and EV lysate) are measured. Irregular spot morphology could affect signal extraction using automated software. We found that the microspots with the best morphology were the ones printed on GPS glass surfaces for all immunoassays. On NC membrane, the protein-based blocking strategies yielded the highest net fluorescent intensity with the antigen contained in PBS, plasma, serum, and serum-free cell culture media. Differently, with EV lysate samples, Pierce™ protein-free blocker yielded the best net signal intensity on both GPS and NC surfaces. The choice of blocking strategies highly depends on the substrate. Moreover, the findings discovered in this study are not limited to microarray-based immunoassays but can provide insights for other assay formats.

O-GlcNAc-Mediated Regulation of Galectin Expression and Secretion in Human Promyelocytic HL-60 Cells Undergoing Neutrophilic Differentiation.

In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all-trans retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular O-GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of O-GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for LGALS9 and LGALS12. The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by O-GlcNAc-reducing ATRA and DON while O-GlcNAc-elevating thiamet G (O-GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that O-GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.