scholarly journals Adeno-associated and lentiviral vector production in 2D and 3D formats with adherent cells in chemically defined, blood-free media

2021 ◽  
Author(s):  
Sofia Pezoa ◽  
Randall Alfano ◽  
Atherly Pennybaker ◽  
Nathan Hazi ◽  
Andrew Laskowski
Keyword(s):  
Bovine Serum ◽  
Viral Vectors ◽  
Large Scale ◽  
Viral Vector ◽  
Fetal Bovine Serum ◽  
Adherent Cells ◽  
Scale Production ◽  
Fetal Bovine ◽  
Free Media ◽  
Doubling Times

Large scale manufacturing of viral vectors or vaccines with adherent cells still relies heavily on the inclusion of fetal bovine serum for the growth and production phases. The inclusion of serum presents numerous problems with the undefined chemical makeup, the undesirable safety profile, and the constraints and limitations on the global supply. Despite these challenges, alternatives to serum for adherent cells have been limited; however, advances in large-scale production of recombinant human proteins have enabled the advancement of blood-free media that can support adherent cell growth. In order to circumvent the need for serum in adherent platforms, we developed a serum and blood-free, chemically defined medium specific for adherent human epithelial kidney cells and evaluated growth kinetics as well as viral vector production with associated adenovirus and lentivirus. We observed doubling times equal to or faster than doubling times observed in serum containing medium. We also demonstrate transfection efficiencies and viral titers that are equivalent to or higher than that of serum. Our results demonstrate that fetal bovine serum is not required for culture of adherent HEK cells, and that a serum-free, blood-free, chemically defined approach can be reliably implemented in the production of viral vectors for gene therapy.

scholarly journals Isolation and culture of endothelial cells from embryonic rat yolk sac

2017 ◽  
Vol 1 (2) ◽  
pp. 149-154
Author(s):  
Harun Ülger ◽  
Ahmet K. Karabulut ◽  
Margaret K. Pratten
Keyword(s):  
Endothelial Cells ◽  
Bovine Serum ◽  
Cultured Cells ◽  
Fetal Bovine Serum ◽  
Wistar Rat ◽  
Yolk Sac ◽  
Immunofluorescent Staining ◽  
Adherent Cells ◽  
Growth Supplement ◽  
Fetal Bovine

Abstract Yolk sac blood islands are the first morphologic evidence of hematopoietic development during mammalian embryogenesis, and visseral yolk sac mesoderm gives rise to the first embryonic blood cells within a rich endothelial network. Present study reports the isolation and culture of endothelial cells from 11.5 days old embryonic rat yolk sac. The embryos were dissected from 11.5 days pregnant Wistar rat (Rattus norvegicus) and the external yolk sac membrane and embryos were removed under aseptic condition. After washing three times with Calcium-Magnesium free Hank’s balanced salt solution (CMF-HBSS), the tissue was minced, and fragments were incubated in CMF-HBSS containing 2mg/ml Trypsin, 100mg/ml collagenase I and 40mg/ml DNAse at 37°C until the tissue was completely dispersed. The digestion effect was then neutralized by fetal bovine serum at 1:3 (v/v). The cell suspension was centrifuged at 1000 rpm for 10 min., the supernatants were discarded and the cell pellets resuspended in Dulbecco modified Eagle medium containing 15% fetal bovine serum, 1.25mg/ml amphotericin B, 25mg/ml gentamycin sulphate and 100mg/ml endothelial cell growth supplement. The resuspended cells were plated in two diverse 25cm2 culture flasks for overnight differential adherence at 37°C. The non-adherent cells were removed by gentle aspiration and adherent cells refed with fresh medium. The cells were transferred using 1ml of 0.2% Trypsin when cultures reached near-confluence. The cultured yolk sac endothelial cells had characteristic cobblestone appearence and positive immunofluorescent staining for von Willebrand Factor (vWF). Weibel-Palade bodies, the major ultrastructural marker for endothelium, were also detected in cultured cells by electron microscopy.

scholarly journals Multivalent and Multipathogen Viral Vector Vaccines

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Katharina B. Lauer ◽  
Ray Borrow ◽  
Thomas J. Blanchard
Keyword(s):  
Immune Response ◽  
Measles Virus ◽  
Viral Vectors ◽  
Large Scale ◽  
Viral Vector ◽  
Natural Infection ◽  
Scale Production ◽  
Veterinary Vaccine ◽  
Large Scale Production ◽  
Foreign Proteins

ABSTRACT The presentation and delivery of antigens are crucial for inducing immunity and, desirably, lifelong protection. Recombinant viral vectors—proven safe and successful in veterinary vaccine applications—are ideal shuttles to deliver foreign proteins to induce an immune response with protective antibody levels by mimicking natural infection. Some examples of viral vectors are adenoviruses, measles virus, or poxviruses. The required attributes to qualify as a vaccine vector are as follows: stable insertion of coding sequences into the genome, induction of a protective immune response, a proven safety record, and the potential for large-scale production. The need to develop new vaccines for infectious diseases, increase vaccine accessibility, reduce health costs, and simplify overloaded immunization schedules has driven the idea to combine antigens from the same or various pathogens. To protect effectively, some vaccines require multiple antigens of one pathogen or different pathogen serotypes/serogroups in combination (multivalent or polyvalent vaccines). Future multivalent vaccine candidates are likely to be required for complex diseases like malaria and HIV. Other novel strategies propose an antigen combination of different pathogens to protect against several diseases at once (multidisease or multipathogen vaccines).

scholarly journals Publisher Correction: Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bettina Mannerström ◽  
Riku O. Paananen ◽  
Ahmed G. Abu-Shahba ◽  
Jukka Moilanen ◽  
Riitta Seppänen-Kaijansinkko ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Serum Free ◽  
Non Coding Rna ◽  
Fetal Bovine ◽  
Free Media

Effect of different culture media on isolation and differentiation of dendritic cells / Farklı besiyerlerinin dendritik hücre izolasyon ve farklılaşmasına etkisi

2015 ◽  
Vol 40 (6) ◽  
Author(s):  
Ayşe Mine Yılmaz ◽  
Ergül Mutlu Altundağ ◽  
Gülşah Gedik ◽  
Semra Koçtürk ◽  
A. Süha Yalçın ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Autologous Serum ◽  
Serum Free ◽  
Fetal Bovine ◽  
Free Media

AbstractObjective: Dendritic cells (DCs) are members of the mammalian immune system and are considered to be the most powerful antigen presenting cells. They are responsible for the induction of T-cells or T-cell dependent immunity and tolerance. In this study we have investigated the effect of different serum supplements on generation and yield of mature dendritic cells isolated from peripheral blood mononuclear cells.Methods: Three different serum supplements (10% Fetal Bovine Serum, 1% Human Serum Albumin and 1% autologous serum) were compared with serum-free media to identify the role and importance of serum supplements on DC cultivation. Effect of different media on maturation signs (CD40, CD80, CD86, CD209a) and cytokine release (TNF-α, IL-10, IL-12, IL-6) was examined.Results: DCs generated in serum-free media was similar to those of cells in medium with autologous serum. Few dendritic-like cells were observed in fetal bovine serum and human serum albumin. The effect of different media on maturation of DCs was compared phenotypically and increased expression of CD80, CD86 and CD209a identified maturation and yield of DCs.Conclusion: Our results suggest that serum free media can be used to overcome potential drawbacks associated with different serum containing supplements.

scholarly journals Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bettina Mannerström ◽  
Riku O. Paananen ◽  
Ahmed G. Abu-Shahba ◽  
Jukka Moilanen ◽  
Riitta Seppänen-Kaijansinkko ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Serum Free ◽  
Non Coding Rna ◽  
Fetal Bovine ◽  
Free Media

Human platelet lysate is an alternative to fetal bovine serum for large-scale expansion of bone marrow-derived mesenchymal stromal cells

2012 ◽  
Vol 34 (7) ◽  
pp. 1367-1374 ◽  
Author(s):  
Sanjay Gottipamula ◽  
Archana Sharma ◽  
Sagar Krishnamurthy ◽  
Anish Sen Majumdar ◽  
Raviraja N. Seetharam
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bovine Serum ◽  
Large Scale ◽  
Human Platelet ◽  
Fetal Bovine Serum ◽  
Platelet Lysate ◽  
Human Platelet Lysate ◽  
Fetal Bovine

scholarly journals Xeno-Free Strategies for Safe Human Mesenchymal Stem/Stromal Cell Expansion: Supplements and Coatings

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
M. Cimino ◽  
R. M. Gonçalves ◽  
C. C. Barrias ◽  
M. C. L. Martins
Keyword(s):  
Stromal Cells ◽  
Ethical Issues ◽  
Therapeutic Potential ◽  
Fetal Bovine Serum ◽  
Clinical Applications ◽  
Adherent Cells ◽  
Regulatory Guidelines ◽  
Fetal Bovine ◽  
Free Media

Human mesenchymal stem/stromal cells (hMSCs) have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiringin vitroexpansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS) as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

Treatment of fetal bovine serum improves early development of porcine embryos by alleviating oxidative stress

2014 ◽  
Author(s):  
Seon-A Choi ◽  
Seong-Eun Mun ◽  
Pil-Soo Jeong ◽  
Hae-Jun Yang ◽  
Seung-Bin Yoon ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Early Development ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Fetal Bovine
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