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Article: Preparation of a universally usable, animal product free, defined medium for 2D and 3D culturing of normal and cancer cells.

Weber, Tilo / Bajramovic, Jeffrey / Oredsson, Stina

2024 Volume 12, Page(s) 102592

Abstract: Since 1958, cell culture media supplemented with fetal bovine serum is used, despite the well-known concerns about animal welfare, reproducibility, reliability, relevance, and safety. To obliterate these concerns and increase scientific accuracy, we ... ...

Abstract	Since 1958, cell culture media supplemented with fetal bovine serum is used, despite the well-known concerns about animal welfare, reproducibility, reliability, relevance, and safety. To obliterate these concerns and increase scientific accuracy, we recently published an open access, publicly available paper on a defined medium composition to make it possible for any lab to prepare this medium. The medium supports routine culturing and cell banking as well as investigations of growth curves, dose response testing of compounds of cells in 2D and 3D, and cell migration; all important aspects for research and toxicology. Here we give a detailed description of how to mix the defined universal cell culture medium in 14 simple steps to support any entity that wishes to make it. We also list different normal and cancer cell lines that have been cultured in the defined medium.•Open source composition of animal product free universal cell culture medium•Protocols for mixing solutions of small xeno free molecules for supplementation•Protocols for mixing solutions of human proteins for supplementation.
Language	English
Publishing date	2024-02-03
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2830212-6
ISSN	2215-0161
ISSN	2215-0161
DOI	10.1016/j.mex.2024.102592
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Article ; Online: P2Y6 receptor-mediated signaling amplifies TLR-induced pro-inflammatory responses in microglia.

Timmerman, Raissa / Zuiderwijk-Sick, Ella A / Bajramovic, Jeffrey J

Frontiers in immunology

2022 Volume 13, Page(s) 967951

Abstract: TLR-induced signaling initiates inflammatory responses in cells of the innate immune system. These responses are amongst others characterized by the secretion of high levels of pro-inflammatory cytokines, which are tightly regulated and adapted to the ... ...

Abstract	TLR-induced signaling initiates inflammatory responses in cells of the innate immune system. These responses are amongst others characterized by the secretion of high levels of pro-inflammatory cytokines, which are tightly regulated and adapted to the microenvironment. Purinergic receptors are powerful modulators of TLR-induced responses, and we here characterized the effects of P2Y6 receptor (P2RY6)-mediated signaling on TLR responses of rhesus macaque primary bone marrow-derived macrophages (BMDM) and microglia, using the selective P2RY6 antagonist MRS2578. We demonstrate that P2RY6-mediated signaling enhances the levels of TLR-induced pro-inflammatory cytokines in microglia in particular. TLR1, 2, 4, 5 and 8-induced responses were all enhanced in microglia, whereas such effects were much less pronounced in BMDM from the same donors. Transcriptome analysis revealed that the overall contribution of P2RY6-mediated signaling to TLR-induced responses in microglia leads to an amplification of pro-inflammatory responses. Detailed target gene analysis predicts that P2RY6-mediated signaling regulates the expression of these genes
MeSH term(s)	Animals ; Cytokines/metabolism ; Heat-Shock Proteins/metabolism ; Macaca mulatta ; Microglia ; NF-kappa B/metabolism ; Receptors, Purinergic P2 ; Toll-Like Receptor 1/metabolism
Chemical Substances	Cytokines ; Heat-Shock Proteins ; NF-kappa B ; Receptors, Purinergic P2 ; Toll-Like Receptor 1 ; purinoceptor P2Y6
Language	English
Publishing date	2022-09-20
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2022.967951
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Article: In vitro

Sharaf, Ahmed / Timmerman, Raissa / Bajramovic, Jeffrey / Accardo, Angelo

Neural regeneration research

2022 Volume 18, Issue 8, Page(s) 1709–1710

Language	English
Publishing date	2022-11-26
Publishing country	India
Document type	Journal Article
ZDB-ID	2388460-5
ISSN	1876-7958 ; 1673-5374
ISSN (online)	1876-7958
ISSN	1673-5374
DOI	10.4103/1673-5374.363828
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Article: The nearest neighbor nuclei method to objectify analysis of pertussis toxin-induced clustering.

Hoonakker, Marieke E / Remarque, Ed / Veth, Jennifer / Sloots, Arjen / Bajramovic, Jeffrey J

ALTEX

2021 Volume 39, Issue 1, Page(s) 140–148

Abstract: The in vivo histamine sensitization test (HIST) has historically been performed to guarantee the safety of acellular pertussis vaccine batches. Non-compliance of batches is primarily associated with the presence of low levels of pertussis toxin (PTx). ... ...

Abstract	The in vivo histamine sensitization test (HIST) has historically been performed to guarantee the safety of acellular pertussis vaccine batches. Non-compliance of batches is primarily associated with the presence of low levels of pertussis toxin (PTx). Because of ethical, standardization and scientific reasons, a variety of alternative in vitro approaches have been studied to replace the lethal HIST. A broadly applied and partially accepted method is the CHO cell clustering test, which is based on the clustered growth pattern of CHO cells when exposed to minute amounts of PTx. One of the major hurdles for global application of the CHO clustering test is the manual assessment of the clusters, which is associated with suboptimal reproducibility of test outcomes and is time-consuming. Here, various parameters of CHO cell nuclei were evaluated in search for a reliable, objective read-out parameter. We demonstrate that the distance between each nucleus and its nearest neighbor (3N method) is the most suitable parameter to assess clustered cell growth. This method detects 2.8 mIU PTx/mL and thereby complies with the requirement set for the sensitivity of the CHO clustering test based on visual reading. In commercial acellular pertussis vaccines spiked with PTx, the method detects 45 mIU/mL PTx, which is substantially lower than the 181-725 mIU/mL PTx detected by visual interpretation. The 3N method thus allows objective and sensitive assessment of CHO clustering and thereby encourages broad and global implementation of the in vitro test as an alternative to the HIST.
MeSH term(s)	Animal Testing Alternatives ; Animals ; Cell Nucleus ; Cluster Analysis ; Cricetinae ; Cricetulus ; Pertussis Toxin ; Reproducibility of Results ; Vaccines, Acellular
Chemical Substances	Vaccines, Acellular ; Pertussis Toxin (EC 2.4.2.31)
Language	English
Publishing date	2021-10-15
Publishing country	Germany
Document type	Journal Article
ZDB-ID	165707-0
ISSN	1868-596X ; 1018-4562 ; 0946-7785
ISSN	1868-596X ; 1018-4562 ; 0946-7785
DOI	10.14573/altex.2012171
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Article: An Overview of

Timmerman, Raissa / Burm, Saskia M / Bajramovic, Jeffrey J

Frontiers in cellular neuroscience

2018 Volume 12, Page(s) 242

Abstract: Neuroinflammation is a common feature in neurodegenerative diseases and strategies to modulate neuroinflammatory processes are increasingly considered as therapeutic options. In such strategies, glia cells rather than neurons represent the cellular ... ...

Abstract	Neuroinflammation is a common feature in neurodegenerative diseases and strategies to modulate neuroinflammatory processes are increasingly considered as therapeutic options. In such strategies, glia cells rather than neurons represent the cellular targets. Microglia, the resident macrophages of the central nervous system, are principal players in neuroinflammation and detailed cellular biological knowledge of this particular cell type is therefore of pivotal importance. The last decade has shed new light on the origin, characteristics and functions of microglia, underlining the need for specific
Language	English
Publishing date	2018-08-06
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2452963-1
ISSN	1662-5102
ISSN	1662-5102
DOI	10.3389/fncel.2018.00242
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Article ; Online: Regulation of innate immune responses in the central nervous system.

Bajramovic, Jeffrey J

CNS & neurological disorders drug targets

2010 Volume 10, Issue 1, Page(s) 4–24

Abstract: Innate immune responses in the central nervous system must be tightly regulated as unrestrained activation generates a chronic inflammatory environment that can contribute to neurodegeneration and autoimmunity. Microglia express a wide variety of ... ...

Abstract	Innate immune responses in the central nervous system must be tightly regulated as unrestrained activation generates a chronic inflammatory environment that can contribute to neurodegeneration and autoimmunity. Microglia express a wide variety of receptors of the innate immune system and are competent responders to danger. Toll-like receptor-, NOD-like receptor- and RIG1-like receptor-mediated activation of microglia leads to the production of pro-inflammatory cytokines and to the upregulation of molecules implicated in activation of the adaptive immune system. Activated microglia are a characteristic feature of many neuroinflammatory disorders and they represent an attractive therapeutic target. This review describes the mechanisms that are at play to restrain microglia activation under homeostatic conditions, such as CD172a, CD200R, SIGIRR and TREM2-mediated signaling, as well as dynamic inhibitory mechanisms that are at play during inflammatory conditions, such as adenosine receptor-mediated signaling. In addition, intracellular activating and inhibitory signaling cascades are summarized in detail and their therapeutic potential is analyzed.
MeSH term(s)	Adaptive Immunity ; Animals ; Autoimmunity ; Central Nervous System/cytology ; Central Nervous System/immunology ; Central Nervous System/metabolism ; Cytokines/biosynthesis ; Cytokines/genetics ; Cytokines/immunology ; Humans ; Immunity, Innate ; Inflammation/genetics ; Inflammation/immunology ; Inflammation/physiopathology ; Microglia/immunology ; Microglia/physiology ; Molecular Targeted Therapy ; Nerve Degeneration/drug therapy ; Nerve Degeneration/genetics ; Nerve Degeneration/immunology ; Signal Transduction ; Time Factors ; Toll-Like Receptors/genetics ; Toll-Like Receptors/immunology ; Toll-Like Receptors/metabolism ; Up-Regulation
Chemical Substances	Cytokines ; Toll-Like Receptors
Language	English
Publishing date	2010-11-10
Publishing country	United Arab Emirates
Document type	Journal Article ; Review
ZDB-ID	2228394-8
ISSN	1996-3181 ; 1871-5273
ISSN (online)	1996-3181
ISSN	1871-5273
DOI	10.2174/187152711794488610
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Zs.A 5892: Show issues

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Article ; Online: Adverse immunostimulation caused by impurities: The dark side of biopharmaceuticals.

Reijers, Joannes A A / Malone, Karen E / Bajramovic, Jeffrey J / Verbeek, Richard / Burggraaf, Jacobus / Moerland, Matthijs

British journal of clinical pharmacology

2019 Volume 85, Issue 7, Page(s) 1418–1426

Abstract: Drug safety is an important issue, especially in the experimental phases of development. Adverse immunostimulation (AI) is sometimes encountered following treatment with biopharmaceuticals, which can be life-threatening if it results in a severe systemic ...

Abstract	Drug safety is an important issue, especially in the experimental phases of development. Adverse immunostimulation (AI) is sometimes encountered following treatment with biopharmaceuticals, which can be life-threatening if it results in a severe systemic inflammatory reaction. Biopharmaceuticals that unexpectedly induce an inflammatory response still enter the clinic, even while meeting all regulatory requirements. Impurities (of microbial origin) in biopharmaceuticals are an often-overlooked cause of AI. This demonstrates that the current guidelines for quality control and safety pharmacology testing are not flawless. Here, based on two case examples, several shortcomings of the guidelines are discussed. The most important of these are the lack of sensitivity for impurities, lack of testing for pyrogens other than endotoxin, and the use of insensitive animal species and biomarkers in preclinical investigations. Moreover, testing for the immunotoxicity of biopharmaceuticals is explicitly not recommended by the international guidelines. Publication of cases of AI is pivotal, both to increase awareness and to facilitate scientific discussions on how to prevent AI in the future.
MeSH term(s)	Animals ; Biological Products/adverse effects ; Biological Products/immunology ; Biological Products/standards ; Drug Contamination ; Endotoxins/isolation & purification ; Guidelines as Topic ; Humans ; Immunomodulation/drug effects ; Pyrogens/isolation & purification ; Quality Control
Chemical Substances	Biological Products ; Endotoxins ; Pyrogens
Language	English
Publishing date	2019-05-29
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	188974-6
ISSN	1365-2125 ; 0306-5251 ; 0264-3774
ISSN (online)	1365-2125
ISSN	0306-5251 ; 0264-3774
DOI	10.1111/bcp.13938
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Zs.A 1118: Show issues

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Article ; Online: Transcriptome analysis reveals the contribution of oligodendrocyte and radial glia-derived cues for maintenance of microglia identity.

Timmerman, Raissa / Zuiderwijk-Sick, Ella A / Oosterhof, Nynke / 't Jong, Anke E J / Veth, Jennifer / Burm, Saskia M / van Ham, Tjakko J / Bajramovic, Jeffrey J

Glia

2021 Volume 70, Issue 4, Page(s) 728–747

Abstract: Microglia are increasingly being recognized as druggable targets in neurodegenerative disorders, and good in vitro models are crucial to address cell biological questions. Major challenges are to recapitulate the complex microglial morphology and their ... ...

Abstract	Microglia are increasingly being recognized as druggable targets in neurodegenerative disorders, and good in vitro models are crucial to address cell biological questions. Major challenges are to recapitulate the complex microglial morphology and their in vivo transcriptome. We have therefore exposed primary microglia from adult rhesus macaques to a variety of different culture conditions including exposure to soluble factors as M-CSF, IL-34, and TGF-β as well as serum replacement approaches, and compared their morphologies and transcriptomes to those of mature, homeostatic in vivo microglia. This enabled us to develop a new, partially serum-free, monoculture protocol, that yields high numbers of ramified cells. We also demonstrate that exposure of adult microglia to M-CSF or IL-34 induces similar transcriptomes, and that exposure to TGF-β has much less pronounced effects than it does on rodent microglia. However, regardless of culture conditions, the transcriptomes of in vitro and in vivo microglia remained substantially different. Analysis of differentially expressed genes inspired us to perform 3D-spherical coculture experiments of microglia with oligodendrocytes and radial glia. In such spheres, microglia signature genes were strongly induced, even in the absence of neurons and astrocytes. These data reveal a novel role for oligodendrocyte and radial glia-derived cues in the maintenance of microglial identity, providing new anchor points to study microglia in health and disease.
MeSH term(s)	Animals ; Cues ; Ependymoglial Cells ; Gene Expression Profiling ; Macaca mulatta ; Microglia ; Oligodendroglia ; Transcriptome
Language	English
Publishing date	2021-12-28
Publishing country	United States
Document type	Journal Article
ZDB-ID	639414-0
ISSN	1098-1136 ; 0894-1491
ISSN (online)	1098-1136
ISSN	0894-1491
DOI	10.1002/glia.24136
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Article ; Online: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes.

Chéneau, Coraline / Eichholz, Karsten / Tran, Tuan Hiep / Tran, Thi Thu Phuong / Paris, Océane / Henriquet, Corinne / Bajramovic, Jeffrey J / Pugniere, Martine / Kremer, Eric J

Frontiers in immunology

2021 Volume 12, Page(s) 685218

Abstract: Despite decades of clinical and preclinical investigations, we still poorly grasp our innate immune response to human adenoviruses (HAdVs) and their vectors. In this study, we explored the impact of lactoferrin on three HAdV types that are being used as ... ...

Abstract	Despite decades of clinical and preclinical investigations, we still poorly grasp our innate immune response to human adenoviruses (HAdVs) and their vectors. In this study, we explored the impact of lactoferrin on three HAdV types that are being used as vectors for vaccines. Lactoferrin is a secreted globular glycoprotein that influences direct and indirect innate immune response against a range of pathogens following a breach in tissue homeostasis. The mechanism by which lactoferrin complexes increases HAdV uptake and induce maturation of human phagocytes is unknown. We show that lactoferrin redirects HAdV types from species B, C, and D to Toll-like receptor 4 (TLR4) cell surface complexes. TLR4-mediated internalization of the HAdV-lactoferrin complex induced an NLRP3-associated response that consisted of cytokine release and transient disruption of plasma membrane integrity, without causing cell death. These data impact our understanding of HAdV immunogenicity and may provide ways to increase the efficacy of HAdV-based vectors/vaccines.
MeSH term(s)	Adenoviridae Infections/immunology ; Adenoviridae Infections/pathology ; Adenoviruses, Human/genetics ; Adenoviruses, Human/immunology ; Cytokines/metabolism ; Flow Cytometry ; Humans ; Immunity, Innate ; Lactoferrin/immunology ; Lactoferrin/metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Phagocytes/virology ; Toll-Like Receptor 4/genetics ; Toll-Like Receptor 4/metabolism
Chemical Substances	Cytokines ; NLR Family, Pyrin Domain-Containing 3 Protein ; NLRP3 protein, human ; TLR4 protein, human ; Toll-Like Receptor 4 ; Lactoferrin (EC 3.4.21.-)
Language	English
Publishing date	2021-05-20
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.685218
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Article ; Online: Exposure of Microglia to Interleukin-4 Represses NF-κB-Dependent Transcription of Toll-Like Receptor-Induced Cytokines.

Zuiderwijk-Sick, Ella A / van der Putten, Céline / Timmerman, Raissa / Veth, Jennifer / Pasini, Erica M / van Straalen, Linda / van der Valk, Paul / Amor, Sandra / Bajramovic, Jeffrey J

Frontiers in immunology

2021 Volume 12, Page(s) 771453

Abstract: Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here ... ...

Abstract	Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here demonstrate that IL-4 exposure induces changes in the cell surface protein expression profile of primary rhesus macaque microglia and enhances their potential to induce proliferation of T cells with a regulatory signature. Moreover, we show that Toll like receptor (TLR)-induced cytokine production is broadly impaired in IL-4-exposed microglia at the transcriptional level. IL-4 type 2 receptor-mediated signaling is shown to be crucial for the inhibition of microglial innate immune responses. TLR-induced nuclear translocalization of NF-κB appeared intact, and we found no evidence for epigenetic modulation of target genes. By contrast, nuclear extracts from IL-4-exposed microglia contained significantly less NF-κB capable of binding to its DNA consensus site. Further identification of the molecular mechanisms that underlie the inhibition of TLR-induced responses in IL-4-exposed microglia may aid the design of strategies that aim to modulate innate immune responses in the brain, for example in gliomas.
MeSH term(s)	Animals ; Cell Proliferation ; Cells, Cultured ; Cytokines/immunology ; Female ; Histone Deacetylases/genetics ; Lipopolysaccharides/pharmacology ; Macaca mulatta ; Male ; Microglia/immunology ; NF-kappa B/immunology ; T-Lymphocytes/immunology ; Toll-Like Receptors/immunology ; Transcription, Genetic
Chemical Substances	Cytokines ; Lipopolysaccharides ; NF-kappa B ; Toll-Like Receptors ; Histone Deacetylases (EC 3.5.1.98)
Language	English
Publishing date	2021-11-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.771453
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