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Emma Maillard In Announcement, Media, new tools

Introducing Glucose-free PlasmaxTM

To further support cell growth in natural and a physiologically relevant environment, we at CancerTools.org are introducing a glucose-free version of Plasmax to complement the original Plasmax formulation.

This defined medium is unique in offering a glucose-free formulation that opens up possibilities for tailored experimental conditions in cancer and cell biology research. It can support a applications including glucose starvation experiments, tumour microenvironment and cancer metabolism studies, metabolic tracing of glucose incorporating stable isotopes (e.g. 13C), etc. which could shed light on critical pathways and mechanisms underlying cancer cell metabolism and glucose utilisation.

While this glucose-free Plasmax^TM eliminates glucose from its formulation, it still preserves the original optimised composition of Plasmax^TM derived from over 80 components. These constituents encompass amino acids and derivatives, inorganic salts, trace elements, and vitamins, with more than 50 components present at levels mirroring those found in human plasma. Hence, the formulation ensures cultured cells closely mimic physiological and metabolic profiles of their in vivo counterparts.

Proven to provide tailored experimental conditions in a physiologically relevant environment, glucose-free Plasmax^TM can support research projects aimed at advancing cancer research and associated metabolic studies.

Access Plasmax glucose free

About CancerTools.org / Cancer Research UK

CancerTools.org, the research tools arm of CRUK, is a non-profit, global community of cancer researchers, academic institutes and societies, with a shared mission to accelerate cancer research. In this collaborative, researchers contribute research tools and share knowledge to deepen our understanding of cancer, and drive innovation within cancer research.

Cancer Research UK (CRUK) is the world’s leading charity dedicated to beating cancer through research. It invests more than £400 million annually into cancer research through funding schemes, conferences, initiatives, resources, and a UK-wide network of research infrastructure across basic, translational, clinical and population research.

Emma Maillard In Case-study, Cell culture, Media

PlasmaxTM vs DMEM the impact of physiologically relevant cell culture media

The research tool: Plasmax

Choosing an appropriate cell culture medium is a crucial step in in vitro cell biology research. With a wide variety of media currently available, finding the correct one for your cell type and particular experiment can be challenging.

Sunada Khadka, a PhD Candidate at MD Anderson Cancer Center, studies cancer metabolism in glioma cells. Glioma is an intra-axial brain tumour which originates in the glial cells that surround and support neurons in the brains. During her latest research on anaplerosis in glioma cells, Sunada’s initial results obtained in vitro using traditional medium were not reproduced in her in vivo experiments. This led to additional time and resources being used to try and understand the discrepancy.

Here, we explore Sunada’s latest research, and the role Plasmax^TM, a physiologically relevant media, played in resolving the discrepancy between her in vitro and in vivo experimental results.

The researcher

Sunada Khadka

PhD candidate, MD Anderson Cancer Center

Initial in vitro results

Sunada’s research explored the possibility of synergistically killing tumour cells through the inhibition of glycolysis and glutaminolysis, two metabolic pathways that feed The Citric Acid (TCA) cycle.

A novel enolase inhibitor, HEX, was used as a glycolysis inhibitor in this study. HEX was developed through the concept of collateral lethality wherein the passenger deletion of the glycolytic gene ENO1 within a subset of gliomas, selectively renders cancer cells sensitive to inhibition of the redundant isoform ENO2. HEX was tested in combination with CB-839. CB-839 is a glutaminase inhibitor which targets glutamine metabolism and is currently being investigated in randomised clinical trials against a range of malignancies. This made CB-839 of primary interest to extend the metabolism-targeted therapy.

Initially, a pyruvate-free traditional media (DMEM) was used for the in vitro experiments which suggested a very strong effect of CB-839 on ENO1-deleted cancer cells. The combination of CB-839 and HEX provided a dramatic synergetic effect that seemed specific to ENO1-deleted cells.

Difficulty recapitulating in vitro results in an in vivo setting

However, when it was attempted to recapitulate the in vitro results in vivo, within an intracranial tumour model, no effect with CB-839 alone and no additive effects with HEX could be seen.

As CB-839 is known to be very poorly permeable across the brain, a subcutaneous in vivo tumour model was used, where Blood Brain Barrier penetration is not an issue. In this case some delay in tumour growth was observed after using CB-839 alone and when used in combination with HEX, but not to the extent seen in the in vitro research.

”It was disappointing as we did not see any effect at all after the glutaminase inhibitor and that was very surprising because we saw a very dramatic effect - the complete wipe-out of cells - in vitro.
Sunada Khadka

Figure 1. ENO1-deleted glioma cells (D423) were implanted intracranially in immunocompromised nude mice and tumor growth was monitored weekly across different treatment groups by T2 MRI (indicated by dashed yellow outlines) 20-30 days after tumor implantation. Khadka et al. 2021.

Plasmax impact

This inconsistency in data led to a return to in vitro experimental conditions and a closer examination of the cell culture media used. Plasmax^TM was selected as a cell culture media that better reflected the in vivo nutrient profile. Plasmax^TM is a ready-to-use, physiologically relevant cell culture medium, consisting of >80 components, of which >50 have been optimised to levels found within human plasma.

By comparing the in vitro results from Plasmax^TM to DMEM, it was observed that the toxicity of CB-839 in ENO1-deleted cells is significantly reduced in Plasmax^TM compared to DMEM. These results confirmed the in vivo data and demonstrated that the ENO1-deleted gliomas microenvironment may not be conducive to glutamine addiction.

”We decided to try something that matches the physiological profile. And again we saw what we did not expect, which is that the effect of CB-839 seem to be completely diminished in PlasmaxTM medium compared to pyruvate-free DMEM.
Sunada Khadka

Figure 2. Sensitivity of glioma cells to CB-839 is attenuated in physiological Plasmax medium. ENO1 homozygously deleted (D423), ENO1-isogenic rescue (D423 ENO1), and ENO1 wild type (LN319) cells were grown in pyruvate free DMEM or Plasmax medium with or without 5 mM pyruvate supplementation. Khadka et al. 2021.

Conclusion:

Sunada’s results emphasize the importance of triaging your cell culture media with physiologically relevant media like Plasmax^TM to better recapitulate the in vivo environment.

As an extension of the paper she recently published, Sunada is now studying the effect of the glycolysis inhibitor in combination with an angiogenesis inhibitor. The restriction of oxygen and nutrient flow into the tumour should have a profound effect. For these experiments she plans on using an intracranial in vivo tumour model and together with Plasmax^TM in her in vitro experiments.

”In the future, whatever metabolism related work I do, I'll make sure to compare DMEM to PlasmaxTM to ensure that the nutrient profile is not effecting the certain phenotype that I’m seeing. It doesn't hurt - if you are already doing one experiment in one certain media condition, just make another plate with PlasmaxTM for side-by-side comparison. So, I actually don't see why one wouldn’t try it. Especially before you jump into big in vivo experiments, which involve a lot of time and money. Using physiologically relevant media is a time saver and will make you more confident in your data.
Sunada Khadka

Plasmax^TM is already being repeatedly purchased by various cancer researchers across different academic institutes worldwide.

Discover how PlasmaxTM could benefit your research:

Access PlasmaxTM

About CancerTools.org

CancerTools.org, the research tools arm of Cancer Research UK, is a non-profit, global community of cancer researchers, academic institutes and societies, with a shared mission to accelerate cancer research discoveries. In this collaborative, researchers contribute research tools and share knowledge to deepen our understanding of cancer, and drive innovation within cancer research.

About Dr. Saverio Tardito

Dr. Saverio Tardito is the group leader for the oncometabolism research group at the CRUK Glasgow Beatson Institute and senior lecturer for the School of Cancer Sciences at the University of Glasgow.

About Cancer Research UK Glasgow: The Beatson Institute

One of Cancer Research UK’s core-funded institutes, The Beatson Institute have built an excellent reputation for basic cancer research, including world-class metabolism studies and renowned in vivo modelling of tumour growth and metastasis. Learn more at: https://www.beatson.gla.ac.uk/About/about-beatson.html

Emma Maillard In Case-study, Cell culture, Media

PlasmaxTM: A physiologically relevant cell culture media

The research tool: PlasmaxTM

Plasmax^TM is a physiologically relevant cell culture medium that closely resembles the metabolic and nutritional profile of human plasma. Unlike traditional media designed to supply excessive levels of a few nutrients, it provides unmatched metabolic fidelity.

CancerTools.org spoke with Dr. Tardito about Plasmax^TM to explore the importance of its development and what its contribution to the CancerTools.org initiative can do for cancer research.

The contributor

Dr. Saverio Tardito

Cancer Research UK Glasgow: The Beatson Institute

The future of cell culture media

Cell culture media is a critical component of cell-based assays, but its contribution to results is often overlooked. The right cell culture is critical in order to comprise the correct energy and compounds to regulate and support the cell cycle. For in vitro experiments, scientists typically use media like Dulbecco’s Modified Eagle Medium (DMEM) – a mixture of vitamins, selected amino acids, sugars and salts which sustain cellular growth. Yet, such types of mediums typically focus on cell proliferation rather than the nutritional environment that cells withstand in tumours. Dr. Tardito, an oncometabolism expert from the CRUK Beatson Institute, required a cell culture medium which better reflected human physiological conditions in order to study cancer biology.

Overcoming in vitro and in vivo variance

Traditional cell culture media was originally produced to rapidly and successfully increase cell proliferation in an in vitro environment (Eagle, 1955). This was made possible by adding nutrients in excessive concentrations to avoid nutrient depletion and simultaneously promote cell growth. Such disproportionate nutrient composition, in comparison to in vivo conditions like human plasma, affects both phenotypic and genotypic behaviour of cells (Schug et al, 2015 and Tardito et al, 2015). Usage of traditional media for cell culture can therefore lead to unrepresentative in vitro conditions and variance between in vitro and in vivo cancer cell metabolism. This becomes particularly important in research relating to cancer cell biology and related metabolic pathways.

To address this challenge, the research team at the Beatson Institute for Cancer Research, Glasgow, UK, under the supervision of Dr. Saverio Tardito, developed a novel cell culture medium, Plasmax^TM, to study the cell metabolism in different tumour types.

”The clear cut differences between experiments performed with Plasmax vs commercial media available at the time become obvious once you realise, they aren’t physiologically relevant
Dr. Tardito

The development of PlasmaxTM

Dr. Tardito and his research team, optimised the concentrations of over 80 compounds typically found in human plasma to achieve the cell growing conditions. The cell culture medium contains all relevant elements to mimic human plasma, consisting of proteinogenic amino acids, vitamins, salts, and sugars, determined through Dr.Tardito’s optimisation experiments. The inclusion of metabolites enhances its physiological relevance and thereby mimics the in vivo environment. Trace elements, while essential for survival and proliferation, are often missing from traditional cell culture media and have to be supplemented before use. Plasmax^TM, is uniquely formulated with trace elements including vanadium, zinc, manganese, copper and selenium. The presence of these increase the antioxidant capacity of cells, which promotes colony growth by preventing ferroptosis-induced cell death (VandeVoorde et al., 2019).

Benefits of PlasmaxTM

It is critical for biomedical research to renew and refine models to improve their relevance to human physiology – which is exactly what the development of Plasmax^TM helps to execute.

Plasmax^TM has been successfully validated across primary cells of different tissue, species, and experimental conditions (see Table 1), and is suitable for both primary and established cell lines. Additional cell lines are successfully cultured using Plasmax^TM regularly, which makes Table 1 a running list of validated cell lines. Plasmax^TM is anticipated to work across a broad range of cancer cell culture models.

Using a physiological relevant medium significantly impacts the results obtained from common cellular assays, including colony formation and gene expression. This has the potential to improve results for cancer cell biology experiments associated with drug discovery and in vitro cancer models.

”Using physiologically relevant media is a time saver and will make you more confident in your data.
Sunada Khadka

Physiologically relevant

Plasmax^TM is optimised to reflect the in vivo profiles of nutrients and metabolites found in human plasma, including essential and non-essential amino acids, amino acid derivatives, organic acids, and other polar metabolites.

Improves in vitro metabolic fidelity

Plasmax^TM can better approximate the overall metabolic phenotype of tumours, with both 2D and 3D cells cultured in Plasmax^TM, better recapitulating the tumours’ metabolic signatures.

By increasing the metabolic fidelity and biological relevance of in vitro cancer models, better drug discovery and improved understanding of cancer at a cellular level can ensure.

Produces a faster proliferation

When compared with traditional mediums, Plasmax^TM produces a faster proliferation, even when aged up to 12 months, in comparison with DMEM when both are supplemented with 2.5% foetal bovine serum.

Better mimics tumour metabolism

Plasmax^TM better mimics tumour metabolism. Breast cancer spheroids grown in Plasmax^TM , have shown to better approximate the metabolic profile of mammary tumours (Vande Voorde et al., 2019).

Uncover role of trace elements

Cancer cells seeded at low densities in the absence of the trace element selenium are unable to form colonies in traditional media due to lipid peroxidation and ferroptosis. The growth-enabling trace elements in addition to vitamins and inorganic salts in Plasmax^TM, prevent ferroptosis-induced cell death, and promote colony growth.

Table 1: A selected list of cultured cell lines successfully validated for growth and viability in Plasmax^TMunder standard conditions

Cell lines grown in Plasmax ^TM	Tissue of origin	Cell line status	Species
HepG2	Liver Cancer	Established line	Human
HuH7	Liver Cancer	Established line	Human
HuH6	Liver Cancer	Established line	Human
BT549	Breast Cancer	Established line	Human
MDA-MB-468	Breast Cancer	Established line	Human
Cal120	Breast Cancer	Established line	Human
A375	Melanoma	Established line	Human
Colo829	Melanoma	Established line	Human
LN18	Brain Cancer	Established line	Human
Naive glioblastoma cell line	Brain Cancer	Low passage lines	Human
Dermal fibroblasts	Epidermis	Primary	Human
Small intestine organoid	Small intestine	Primary	Mouse
Mammospheres	Mammary gland	Primary	Mouse
Mesenchimal stromal cell line	Bone marrow	Primary	Human
Embryonic stem cell line	Embryo	Primary	Human
Trophoblast stem cell line	Placenta	Primary	Human
A549	Lung cancer	Established line	Human
HCT116	Colon Cancer	Established line	Human
SaOS2	Bone tumour	Established line	Human
HT1080	Fibrosarcoma	Established line	Human