Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media

AbstractMycoplasma pneumoniae is a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective, and vaccination is required. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation for vaccine production challenging and expensive.To understand factors limiting growth, we developed a genome-scale, constraint-based model of M. pneumoniae called iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict and validate in vitro two serum-free media able to sustain growth.Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth of M. pneumoniae.Altogether, our modelling approach enabled us to optimize medium composition, capacitated growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive vaccine production.

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Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media

npj Systems Biology and Applications ◽

10.1038/s41540-020-00153-7 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Erika Gaspari ◽

Antoni Malachowski ◽

Luis Garcia-Morales ◽

Raul Burgos ◽

Luis Serrano ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Large Scale ◽

Atypical Pneumonia ◽

Metabolic Potential ◽

Membrane Formation ◽

Serum Free ◽

A Genome ◽

Essential Components ◽

Free Media

Abstract Mycoplasma pneumoniae is a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation challenging and expensive. To understand factors limiting growth, we developed a genome-scale, constraint-based model of M. pneumoniae called iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict essential components validated with in vitro serum-free media able to sustain growth. Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth of M. pneumoniae. Altogether, our modelling approach allowed us to optimize medium composition, enabled growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive production.

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Sodium chloride, osmolyte, and osmolarity effects on blastocyst formation in bovine embryos produced by in vitro fertilization (IVF) and cultured in simple serum-free media

Journal of Assisted Reproduction and Genetics ◽

10.1007/bf02066609 ◽

1996 ◽

Vol 13 (7) ◽

pp. 562-568 ◽

Cited By ~ 29

Author(s):

Zishu Liu ◽

Robert H. Foote

Keyword(s):

Sodium Chloride ◽

In Vitro Fertilization ◽

Bovine Embryos ◽

Blastocyst Formation ◽

Vitro Fertilization ◽

Serum Free ◽

Free Media

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TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.882 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi220-vi220

Author(s):

Hasan Alrefai ◽

Andee Beierle ◽

Lauren Nassour ◽

Nicholas Eustace ◽

Zeel Patel ◽

...

Keyword(s):

Cell Types ◽

In Vitro Models ◽

Tumor Initiating Cells ◽

Serum Free ◽

Brain Tumor Initiating Cells ◽

Multiple Cell ◽

Anti Inflammatory ◽

Free Media ◽

Inflammatory Macrophages

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p > 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM.

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Enhancement of In Vitro Growth of Pathogenic Bacteria by Norepinephrine: Importance of Inoculum Density and Role of Transferrin

Applied and Environmental Microbiology ◽

10.1128/aem.00075-06 ◽

2006 ◽

Vol 72 (7) ◽

pp. 5097-5099 ◽

Cited By ~ 55

Author(s):

Phyllis M. O'Donnell ◽

Hernan Aviles ◽

Mark Lyte ◽

Gerald Sonnenfeld

Keyword(s):

Bacterial Growth ◽

Pathogenic Bacteria ◽

Inoculum Density ◽

In Vitro Growth ◽

Stress Hormone ◽

Serum Free ◽

Free Media

ABSTRACT Norepinephrine is a stress hormone that enhances bacterial growth. We examined the effects of a small inoculum on the norepinephrine-induced growth of species previously reported to be unaffected by norepinephrine. The results indicated that a reduced inoculum density is essential for observing norepinephrine-induced effects. Additional studies using serum-free media suggested that transferrin plays a role in norepinephrine-induced growth.

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268 ROLE OF ATPase MOTOR CYTOPLASMIC DYNEIN AND PRIMARY CILIA IN TESTICULAR MORPHOGENESIS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab268 ◽

2015 ◽

Vol 27 (1) ◽

pp. 223

Author(s):

C. Dores ◽

I. Dobrinski

Keyword(s):

Primary Cilia ◽

Somatic Cells ◽

Treated Group ◽

Cytoplasmic Dynein ◽

Control Group ◽

Tubule Formation ◽

Serum Free ◽

Free Media

In vertebrates, the primary cilium is a nearly ubiquitous organelle present in somatic cells, but little is known about its function in the male gonad. We investigated the role of primary cilia in testis cells using in vitro formation of seminiferous tubules and in vitro culture of testicular somatic cells by inhibiting the primary cilium with CiliobrevinD, a cell-permeable, reversible chemical modulator that inhibits the major component of the organelle: ATPase motor cytoplasmic dynein. We analysed in vitro cultures for the presence of primary cilia and the activation of hedgehog signalling through translocation of Gli2 to the nuclei; in vitro tubule formation was evaluated by length and width of tubules formed. Methods: testicular cells were harvested from neonatal pigs by 2-step enzymatic digestion. Cells (50 × 106 mL–1) were plated on 100 mm Petri dishes in 15 mL of DMEM + 5% FBS + 50 U of penicillin and incubated at 37°C in 5% CO2 in air overnight, cells remaining in suspension and those slightly attached were removed and the somatic cells attached were trypsinized to obtain a single cell suspension, and then submitted to two different protocols: in vitro culture (A) or in vitro tubule formation (B), n = 5 replicates each. For A, somatic cells were replated on coverslips in 24-well plates and cultured in serum free media for 48 h, then for the treated group, 10 mM of CiliobrevinD was added for 24 h, attached cells from control and treated groups were fixed in 4% PFA and characterised by immunocytochemistry for ARL13B, Vimentin, and Gli2. For B: 1 × 106 cells were added to 24-well plates coated with 1 : 1 diluted Matrigel, the control group was kept in serum free media and to the treated group was added 20 mM CiliobrevinD at Day 0. Results: A) primary cilia were present in 89.3 ± 2.3% of cells cultured in serum-free media for the control group and Gli2 was located in the nuclei of 90.2 ± 1.2% of cells; in the CiliobrevinD-treated group the percentage of primary cilia decreased (P < 0.05) to 3.1 ± 2.5% and nuclear Gli2 to 3.9 ± 0.7; B) tubules formed in the control group were significantly longer and wider than the ones formed when CiliobrevinD was added (9.91 ± 0.35 v. 5.540 ± 1.08 mm and 339.8 ± 55.78 v. 127.2 ± 11.9 µm, respectively, P < 0.05 by Student's t-test). In conclusion, the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, blocks Hedgehog signalling, and impairs in vitro tubule formation. Therefore, primary cilia on testicular somatic cells appear to be essential for testicular morphogenesis.Research was supported by 5 R01 OD016575-13.

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