Suspension Culture In Serum-free Medium Research Articles

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.

A new nodavirus-negative Trichoplusia ni cell line for baculovirus-mediated protein production.

Cell lines derived from Trichoplusia ni (Tn) are widely used as hosts in the baculovirus-insect cell system (BICS). One advantage of Tn cell lines is they can produce recombinant proteins at higher levels than cell lines derived from other insects. However, Tn cell lines are persistently infected with an alphanodavirus, Tn5 cell-line virus (TnCLV), which reduces their utility as a host for the BICS. Several groups have isolated TnCLV-negative Tn cell lines, but none were thoroughly characterized and shown to be free of other adventitious viruses. Thus, we isolated and extensively characterized a new TnCLV-negative line, Tn-nodavirus-negative (Tn-NVN). Tn-NVN cells have no detectable TnCLV, no other previously identified viral contaminants of lepidopteran insect cell lines, and no sequences associated with any replicating virus or other viral adventitious agents. Tn-NVN cells tested negative for >60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma. Finally, Tn-NVN cells grow well as a single-cell suspension culture in serum-free medium, produce recombinant proteins at levels similar to High Five™ cells, and do not produce recombinant glycoproteins with immunogenic core α1,3-fucosylation. Thus, Tn-NVN is a new, well-characterized TnCLV-negative cell line with several other features enhancing its utility as a host for the BICS.

Role of ovarian tumor stem-like cells sorted from human epithelial ovarian cancer SKOV3 cells in vasculogenic mimicry formation

To isolate tumor stem-like cells from human epithelial ovarian cancer SKOV3 cells and explore their role in the formation of vascularization mimicry (VM). SKOV3 cells were passaged to the 7th generation by suspension culture in serum-free medium, and the percentages of CD133- and CD117-positive cells in the 1st, 3rd, 5th and 7th generations were analyzed using flow cytometry. The proliferative activity of the cells sorted from the 7th generation SKOV3 cells was assessed with colony formation assay. A three-dimensional cell culture model was established to compare the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2 (MMP-2) and MMP-9 in the two groups were detected using real-time PCR and Western blotting. Some SKOV3 cells formed typical cell spheres with suspension growth in serum-free medium and were passaged to the 7th generation. Flow cytometry revealed that the percentage of CD133-positive cells increased with cell passaging. The cloning efficiency of the sorted cells was significantly higher than that of the parental SKOV3 cells (50.33% vs 5.33%, P < 0.001). The VM formation ability of the sorted cells was stronger than that of the parental SKOV3 cells in the three-dimensional cell culture system. RT-PCR and Western blotting showed that the expression levels of MMP-2 and MMP-9 were significantly higher in the 7th passage cells than in the parental cells (P < 0.05). The sorted cells from SKOV3 cells cultured in serum-free medium exhibit biological properties of tumor stem cells with strong VM formation ability, suggesting their role in VM formation.

Hematopoietic Developmental Potential of Human Pluripotent Stem Cell Lines Is Accompanied by the Morphology of Embryoid Bodies and the Expression of Endodermal and Hematopoietic Markers.

The potential clinical applications of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs) are limited by the difficulty of recapitulating embryoid hematopoiesis and by the unknown differentiation potential of hPSC lines. To evaluate their hematopoietic developmental potential, available hPSC lines were differentiated by an embryoid body (EB) suspension culture in serum-free medium supplemented with three different cytokine mixes (CMs). The hPSC differentiation status was investigated by the flow cytometry expression profiles of cell surface molecules, and the gene expression of pluripotency and differentiation markers over time was evaluated by real-time reverse transcription polymerase chain reaction (qRT-PCR). hPSC lines differed in several aspects of the differentiation process, including the absolute yield of hematopoietic progenitors, the proportion of hematopoietic progenitor populations, and the effect of various CMs. The ability to generate hematopoietic progenitors was then associated with the morphology of the developing EBs, the expression of the endodermal markers AFP and SOX17, and the hematopoietic transcription factor RUNX1. These findings deepen the knowledge about the hematopoietic propensity of hPSCs and identify its variability as an aspect that must be taken into account before the usage of hPSC-derived HSCs in downstream applications.

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150–200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.

Construction and expression of human scFv-Fc against interleukin-33

Interleukin-33 (IL-33) is a member of the IL-1 family and the ligand of orphan ST2 molecules. IL-33 is widely expressed in multiple tissues and cells, and mainly involved in regulating Th2 immune and inflammatory responses. Inhibiting IL-33 signaling pathways relieves the symptoms of allergic inflammation, indicating that IL-33 is a potential target for the treatment of allergic diseases. In this study, the recombinant vectors SP-scFv-Fc/pcDNA3.1 and SP-scFv-Fc/PMH3EN were constructed to express a human scFv-Fcs against IL-33. The size of the inserted SP-scFv-Fc was approximately 1540bp. The RT-PCR results showed that SP-scFv-Fcs were successfully transfected into CHO K1 cells. Western blot analysis indicated specific binding of the expressed scFv-Fcs fusion protein (approximately 60kDa under reduced condition) with a goat anti-human IgG1 Fc antibody. The expression level of the scFv-Fcs from SP-scFv-Fc/PMH3EN was higher than that from SP-scFv-Fc/pcDNA3.1. A single high-expressing cell line was selected after three rounds of screening and the fusion protein was expressed in a suspension culture in serum-free medium. The level of expression products reached 20mg/L and the expressed and purified scFvs was further characterized and analyzed for bioactivity and functionality. The recombinant vectors for eukaryotic expression of scFv-Fcs against IL-33 were successfully constructed and the expressed scFv-Fcs was shown to be a suitable candidate for the development of a new therapy for allergic and autoimmune diseases.

Adaptation of glucocerebrosidase-producing CHO cells to serum-free suspension culture

Background Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher disease, the most common inherited storage disorder [1]. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive [2]. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. With this purpose, we have previously developed a cell line producing recombinant GCR using anchorage-dependent CHO cells [3]. Recent advances in cell culture technology have allowed the elimination of serum along with the growth of cells in suspension mode. This mode is preferred in industrial production due to the well-understood principles of scaling parameters and the ease of process control. Additionally, the serum is expensive and a source of contaminants [4]. Here, we describe the adaptation of a CHO cell line producing human GCR to suspension culture in serumfree medium, and evaluate its effects on protein expression, glycosylation and enzymatic activity.

Abstract 2469: ATM inhibitor 55933 abolishes the radiation resistance of CD44+/CD24−or low subsets of breast cancer cells

Abstract CD44+/CD24− subsets of breast cancer cells are enriched in cancer stem-like cells (CSC) and are radiation resistant. We isolated ESA+/CD44+/CD24− or low subsets by flow cytometry from MCF7, MDA-MB231, and HCC1937 breast cancer cell lines and primary cultures of breast cancer cells from two patients. The isolated CD44+/CD24−orlow subsets showed a 50 to 70% increase of sphere formation by in suspension culture in serum-free medium compared to CD44−/CD24high subsets. CD44+/CD24−or low subsets of the cell lines and primary cultures showed a significantly increased radiation resistance compared to the CD44−/CD24high cell line subsets or the unsorted primary cells with > 40% increased survival at 2Gy. Post-radiation survival showed that the radiation resistance was dependent on the CD44+/CD24 −or low cell phenotype. The correlation of CD44/CD24 status with radiation resistance was demonstrated by culture of the subsets. After 2 weeks of culture the following transitions occurred: 1) CD44+/CD24−or low cells changed to CD44−/CD24high cells (MCF-7 and HCC1937) or to CD44+/CD24+ (MDA-MB-231); and 2) CD44−/CD24 high (MCF-7 and HCC1937) and CD44+/CD24+ (MDA-MB-231) cells became CD44+/CD24−or low cells. Cells that lost the CD44+/CD24 −or low phenotype were no longer radiation resistant. The mechanisms of increased radiation resistance in CD44+/CD24−orlow subsets was not a function of non-homologous end joining (NHEJ) as there was no in vivo and in vitro change in NHEJ activity in the resistant cells nor altered expression of the NHEJ related proteins Ku 80, Ku70, PARP-1, and DNA-PK. However, there were differences ATM activity between CD44+/CD24−or low and the CD44−/CD24 high (MCF-7 and primary breast cancer) or CD44+/CD24+ (MDA-MB-231) subsets. Depending on the cell type and radiation dose, ATM phsophorylation increased by 2-5 fold in radiated CD44+/CD24−or low subsets of breast cancer cell lines and primary cultured breast cancer cells compared to the control subsets. The ATM inhibitor 55933 in a dose-dependent manner increased radiation sensitivity of all subsets including significantly abolishing the radiation resistance of CD44+/CD24−or low subsets of cells. After 2Gy of radiation, the survival fractions of cells treated with10μM of 55933 were 8-10 fold lower than that of untreated cells and were now similar between CD44+/CD24−or low subsets and control subsets for the cell lines and primary cultures. In conclusion, ATM is a candidate target for eliminating the radiation resistance of CSC's of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2011-2469

Transfection of HEK293-EBNA1 Cells in Suspension with 293fectin for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin, due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, other methods suitable for efficient transfection of mammalian cells are easier to use. This protocol describes the steps needed for successful transfection of 293-6E cells in suspension culture in serum-free medium using 293fectin.

An attempt to generate neurons from an astrocyte progenitor cell line FBD-104

In the present study, a clonal astrocyte progenitor cell line derived from p53-deficient fetal brains, named FBD-104, was characterized in monolayer and suspension culture. In monolayer culture with medium containing 10% serum, FBD-104 cells expressed some markers of astrocytes, such as glial fibrillary acidic protein (GFAP), S100β, and glutamate aspartate transporter (GLAST). They never expressed any markers of neurons or oligodendrocytes. Thus the cell line appears to be restricted to the astroglial lineage. However, in suspension culture in serum-free medium supplemented with EGF and FGF2, FBD-104 cells proliferated and formed neurospheres expressing mRNAs for Mash1 and Math3, generating cells expressing neuron specific β-III tubulin. Re-plating the spheres onto an adhesive substrate and withdrawal of the growth factors induced the expression of mRNAs for NeuroD and Olig2 and generated more β-III tubulin-positive cells. The present study demonstrated that neurosphere culture is an efficient method to induce neurogenesis from the astrocyte progenitor cell line FBD-104. We also determined that pretreatment with FGF2 caused a significant increase in yield of neurospheres. Thus, the FBD-104 line is an interesting in vitro model to study effect of trophic factors and adhesive substrates on lineage determination of neural progenitor cells.

The enhancement of recombinant protein production by polymer nanospheres in cell suspension culture

Recombinant Chinese hamster ovary (rCHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins. Three-dimensional suspension culture is preferred to two-dimensional monolayer culture for the efficient large-scale culture of rCHO cells and subsequent mass production of recombinant proteins. Previously, we have demonstrated that the use of plain polymer nanospheres enhances the growth of anchorage-dependent animal cells (human embryonic kidney 293 cells) in suspension culture in serum-containing medium. Vitronectin and fibronectin were adsorbed onto poly(lactic-co-glycolic acid) (PLGA) nanospheres (696nm in average diameter) by immersing the nanospheres in fetal bovine serum. In this study, we investigated if the use of vitronectin/fibronectin-adsorbed polymer nanospheres enhances recombinant protein production in rCHO cell suspension culture in serum-free medium. Cell aggregate formation may be critical for the survival and growth of anchorage-dependent animal cells in suspension culture, and cells in single cell suspension may result in cell death. The addition of vitronectin/fibronectin-adsorbed nanospheres to rCHO cell suspension culture promoted the rate and efficiency of cell aggregate formation. The nanospheres enhanced cell growth (2.9 folds on day 10) and, importantly, recombinant antibody production (1.8 folds on day 14), compared to suspension culture without nanospheres. The viability of cells in the aggregates in the nanosphere-added culture was high for the entire culture period of 14 days. Apoptotic activity of cells was much lower in the nanosphere-added culture than in the culture without nanospheres on day 5. The nanosphere suspension culture method developed in this study may be useful for the mass production of recombinant proteins through large-scale suspension culture of anchorage-dependent animal cells.

Increased Virus Production in Suspension Culture by a Trichoplusia ni Cell Line in Serum-Free Media

Trichoplusia ni-derived BTI-Tn5B1–4 (Tn-5B1–4) cells were grown in 50 mL spinner flasks in four different formulations of serum-free medium (SFM) . The objectives of this work were to determine the capacity of this cell line to grow and support virus replication in suspension culture in different SFM. Maximum cell densities exceeding 3.0 × 106 cells/mL were attained for all cell/medium combinations. Likewise, cell doubling times were all within 20–22 h. Infection studies with a wild-type isolate of Autographa californicanuclear polyhedrosis virus (AcMNPV) yielded between 156 and 187 occlusion bodies (OBs) /cell. The lethal effect of AcMNPV-OBs produced in this cell line in the various types of media was demonstrated in vivousing T. ni neonate larvae. The percent mortality at a single dose (1.0 × 106 OBs/mL) ranged from 40 to 60% at 4 days post-inoculation (p.i.) . These results compared favorably with mortality rates found for AcMNPV-OBs produced in vivo.DNA quantification analysis, comparing polyhedra-derived viral DNA concentrations from an equal number of cell culture- and larval-derived OBs, was also determined. The larval-derived OB DNA concentration averaged 0.514 μg/108 OBs, whereas the cell culture-derived viral DNA concentrations ranged from 0.347 to 0.710 μg/108 OBs. These results suggest that no significant differences exist in DNA content between larvae- and cell culture-derived samples. Expression of the recombinant protein, secreted alkaline phosphatase (SEAP), ranged from 2.7 to 5.5 IU/mL for each cell/medium combination, which was consistent with previous static culture results for this same cell line in serum-containing TNM-FH medium. Electron microscopic analysis of OBs produced in vivo or in vitro revealed no obvious differences in packaging or number of enveloped nucleocapsids per OB between samples. However, AcMNPV-OBs from infected larvae appeared physically smaller (1.28 ± 0.164 μm) than those produced in cell culture (1.72 ± 0.120) . The Tn-5B1–4 cell line is adaptable to suspension culture in serum-free medium and is suitable for further development in the production of viruses and recombinant proteins on an industrial scale.