CelVivo Clinostar Applications
The technology created by CelVivo allows generation of uniform, reproducible and functional spheroids and organoids. These spheroids mimic the function, structure and architecture of in vivo cells. The ClinoStar technology isn’t merely “3D cell culture”, it enables research using cell models with an unprecedented correlation between in vivo and in vitro conditions, and closes the gap between 2D cell culture and cell function.
Cancer research – Ideal 3D models
What is a cancerous tumoroid?
As cancer cells accumulate changes in their genetic makeup (mutations) and how genes are regulated (epigenetic alterations, red), they gradually relinquish the intricate controls that usually manage their behaviour. This loss of control sets the stage for cells to multiply uncontrollably and adopt invasive characteristics, ultimately coalescing into tumoroids. These tumoroids are a significant menace to the body’s overall balance.
In the evolving landscape of oncology, the concept of “cancerous tumoroids” is gaining recognition, denoting irregular growths distinguished by their unregulated cell division and tendency to invade surrounding tissues. These growths prominently display the key indicators of malignancy, showcasing the capability to spread to distant sites through metastasis, thereby perturbing the usual arrangement of healthy tissues.
Understanding disease and finding novel treatments for triple-negative breast cancer with 3D cell culture
In 2D culture, breast cancer cell lines are usually indistinguishable from non-transformed primary mammary epithelial cells. In a 3D microenvironment it was observed that while primary cells developed polarised, lumen containing structures in 3D, the tumour cell line clusters were disorganised (and that stellate or grape-like ‘cluster patterns’ depended on the particular cell line being used).
- • This application note details a protocol using sodium alginate to create 3D constructs for culturing MDA-MB-231 cells in the ClinoStar system
- • Sodium alginate embedding provides a scaffold and protection of cells, which facilitates dynamic 3d culture without clumping
- • Sodium alginate can be used as a reproducible and easy gel embedding technique.
Learn more by downloading the full Application Note below.
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3D multicellular models to study cancer – from spheroids to microfluidics
Dr. Dominika Czaplinska & Dr. Roxane Crouigneau
University of Copenhagen, Department of Biology
The development of three-dimensional (3D) cell cultures has provided new tools for basic research and pre-clinical approaches, allowing the culture of cancer cells under conditions that closely resemble tumour growth in a living organism. We will provide an overview of the main 3D techniques used in our lab, including spheroids, cysts, organoids and microfluidics technology. We will discuss the advantages and limitations of the classical 3D models as well as recent advances in 3D culture techniques, focusing on how these culture methods have been used to study cancer progression.
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Establishing dynamic spheroid cultures as mini-tumour models for treatment evaluation
Prof. Chrisna Gouws
North West Uni. center of excellent for pharmaceutical sciences
In this presentation, the process to establish three-dimensional spheroid models in rotating bioreactors, and benchmarking their suitability to study cancer treatments will be discussed. These mini-tumour models aim to bridge the gap between in vitro studies and the human in vivo system, by mimicking cancer cell behaviour in vivo better. Once established, each model is characterized in terms of growth and viability characteristics, after which it is benchmarked through treatment with a standard chemotherapeutic drug.
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Growth Rate
Most of the key techniques for cell culture were established during the 1950’s. The focus at that time was to make the cells proliferate as quickly as possible. This was done by allowing the cells to grow as monolayer cultures on essentially flat glass or plastic surfaces (i.e. in ‘2D’).
In contrast, cells in normal tissues, cancers and in 3D cultures usually double their numbers in months.
Architecture and Structure
Cells grown 3D can spontaneously form structures which resemble their parental tissue. Key to this is that spheroids develop oxygen, nutrient and waste product gradients similar to those seen in tumours.
Function – Uniformity and Reproducibility
For a 3D cancer model to be relevant, it must successfully replicate the functionality demonstrated by the active tumour.
Reproducibility is inversely proportional to heterogeneity. Patients are heterogeneous. Their tumours are heterogeneous. The cells within a tumour are heterogeneous. It therefore makes no sense to increase this heterogeneity even further by growing the cells in conditions which force them to adapt.
Longevity
Once cells have repaired the damage to their ECM (from 2D passaging), they enter a ‘dynamic equilibrium’ state: a state that resembles a functioning organ. Unperturbed, the cells execute their functions at a steady rate. Treated with a drug (or other biologically active molecules) they respond and when the drug is metabolised (or removed) the cell clusters return to their original dynamic equilibrium. In the ClinoReactors, cell clusters can remain in this dynamic equilibrium for weeks or months and this is an ideal starting point for experimentation.
Biomarkers found using 3D culture
Efficient and timely identification of malignant tumours forms the basis of cancer treatment. Recently, accurately detecting cancer-specific biomarkers have become the pathologist’s chosen tool to study biopsies. To build this toolkit with biomarkers specific to the myriad of cancer types, it is necessary to conduct gene expression profiling of the appropriate tissue. One way to do this is to employ 3D culture to study tumour extracts and cancer cell lines.
White Paper – Myriad Approaches to a Curious Problem: Designing relevant in vitro models for optimal cancer research
Start your journey of growing optimal cancer models in the Clinostar system today.
Read how other researchers are growing organoids in 3D and learn about their protocols.
Simply fill in the form to be emailed your copy.
Neural Organoid Development
What is a brain organoid?
A brain organoid is a 3D structure that is typically grown in vitro from pluripotent stem cells and is engineered to resemble certain aspects of the developing human brain.
By exposing stem cells to specific growth factors and other signalling molecules in a temporal sequence and in a controlled environment, researchers can coax them to differentiate into brain cells and self-organize into brain-like structures.
Brain organoids are small, approximately pea-sized, and contain different types of brain cells, including neurons, astrocytes, and oligodendrocytes. In view of the paucity of available human brain material, organoids can be used as models to study development, test drug efficacy and toxicity, and investigate the underlying mechanisms of neurodegenerative diseases and neurological disorders. While brain organoids are not exact replicas of the human brain, they can provide a readily available tool in a more controlled and ethically acceptable manner than animal models or human embryos.
Human brain organoids were generated from primary dermal fibroblast derived hiPSCs using the CelVivo ClinoStar.
View more details in the pre-print linked below.
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Using a mouse model of infection the team at QIMR clearly demonstrated that the newer Omicron variant BA.5 shows increased neurovirulence, encephalitis, and mortality compared to the older BA.1 variant.
In addition, BA.5 infected human brain organoids (cortical) to a higher extent than both the BA.1 and the original ancestral isolate. Differing from earlier variants, the infection did not appear to be mediated by the transmembrane protease TMPRSS22. The organoid model also demonstrated that the BA.5 variant could effectively suppress the anti-viral type 1 interferon response and suggested that new variants were more capable than old.
So while omicron-infected patients are exhibiting reduced respiratory symptoms, some BA.5 infected patients are experiencing increased loss of the sense of smell (anosmia).
This has been previously linked to long-lasting cognitive problems in COVID-19 patients. Thus, it appears to be critically important to be aware of the risk for acute and long-term neurological complications.
Read more details in the pre-print linked below.
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Neuropathology of Japanese encephalitis virus (JEV)
Researchers at QIMR characterised the neuropathology of JEV in adult mice and found that the virus caused neuronal degeneration, leukocyte infiltrates, and other brain lesions. The virus was also lethal to some mice and destroyed human neuronal cells in vitro.
In the pre-print, the team successfully characterised a new JEV mouse model using IRF7-/- mice, which shows high penetrance of CNS infections, as well as neuropathology that recapitulates many aspects seen in human post mortem brain specimens.
This paper also describes novel behaviour of a recent JEV G4 isolate from the Australian outbreak in mice and in human cortical brain organoids.
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Advanced methods are needed to produce improved cellular therapeutics in order to meet the standard of or improve the efficacy of existing treatments. The use of 3D culture systems to develop spheroids has contributed to the field of regenerative medicine and neural tissue engineering by expanding opportunities to study processes of neuron development and repair.
Neurospheres are spheroids composed of primary neural cells (PNCs), which, as a cell therapy, can contribute to axon growth, myelination, angiogenesis, immunoregulation of the microenvironment, and proliferation and differentiation of the endogenous cells.
In a publication by scientists from the University of Tennessee, they have demonstrated a method to successfully develop spheroids from rat primary neurons using the CelVivo ClinoStar system.
- • Primary neural cells were isolated from hippocampi from 3 day old rat pups
- • Neural cells were incubated for 24 hours in ClinoReactors coated in 2% Pluronic, then seeded on Poly-D lysine coated plates
- • Neurospheres maintained >84% viability
- • Neurospheres expressed neural markers MAP2, beta-tubulin III, and GFAP
Neurosphere cultures maintained high viability (≥80%) across the 7-day culture period. (Left) MAP2 and (Middle) b-Tubulin 3 are expressed by neural cells. (Right) GFAP is expressed by glial cells in day 3 neurospheres.
The use of the ClinoStar incubator and ClinoReactor has provided a high-throughput method that produces a large number of neurospheres in a short period of time. As a result, this method reduces the reagents needed, improves efficiency, and reduces variation. The stress-free environment provides a technique that alleviates the current challenges of neurosphere development.
Read the full publication
White Paper - Breaking the Bias: Addressing the challenges facing optimal neural organoid development
Start your journey of growing brain organoids in the Clinostar system today.
Read how other researchers are growing organoids in 3D and learn about their protocols.
Simply fill in the form to be emailed your copy.
In vitro blastoid model for embryogenesis
In a new breakthrough publication, researchers at UT Southwestern Medical Center in Texas, have developed an efficient method to generate bovine blastocyst-like structures in vitro.
This breakthrough holds great potential to advance animal agriculture by providing an accessible in vitro blastoid model for studying bovine embryogenesis, (including understanding the causes of early embryonic loss).
While further optimisation is needed, this novel approach to constructing bovine blastoids could lead to the development of new artificial reproductive technologies for cattle breeding. This would be a paradigm shift in livestock reproduction.
Phase-contrast images of blastoids grown in the
ClinoReactor (the culture vessel of the ClinoStar System).
The breakthrough was achieved by cultivating several types of recently identified pluripotent stem cells (including extended-pluripotent stem cells) from bovine blastocysts with trophoblast stem cells in newly identified growth conditions which support the derivation and long-term culture of the trophoblast stem cells (3D assembly and culture in suspension).
As such this model eliminates the bottleneck of limited material.
For rotating-culture, blastoids were collected at day 4 post aggregation and placed in ClinoReactors in 10ml culture media. ClinoReactors were then placed in the ClinoStar incubator at 37 °C with a gas mix of 5% CO2, 5% O2 and air. The rotation speed was set between 10 and 12 rpm and was lowered progressively as the blastoids expanded.
Optimal growth conditions were achieved by exchanging media every four days.
Immunofluorescent images of bovine blastoids grown in the ClinoStar.
The bovine blastoids constructed have been shown to closely resemble blastocysts in their morphology, cell composition and single-cell transcriptomes.
The blastoids constructed contain an outer trophectoderm-like layer, a cavity, and an inner cell mass-like compartment. This closely resembles bovine blastocysts produced by in vitro fertilization (IVF). Both the blastoids and IVF-derived blastocysts proliferate and can be expanded for more than 2 weeks. Embryo transfer experiments illustrate that both blastoids and blastocysts induce the anti-luteolytic hormone interferon-tau (INFτ) at the same level, and in the same proportion of cultures. This is significant because INFτ is the signal for maternal recognition of pregnancy in ruminants.
Hepatotoxicity
Determining toxicity is a major need and it is challenging
The ClinoStar in vitro 3D spheroid system that can be used for determining toxicity. As described by Fey et al 2020.
Toxicol Res, Volume 9, Issue 4, July 2020, Pages 379–389, https://doi.org/10.1093/toxres/tfaa033
Toxicity must be assessed using both short-term (single dose, 24–48h; acute) and long-term (multiple doses, weeks to months; chronic) culture conditions. This is because drug induced liver injury (DILI) can develop following a single acute exposure as well as from repeated chronic treatment (e.g. by development of drug tolerance or deposition of metabolites). Long term treatment is especially important because it corresponds to the usual treatment regimen for patients but is difficult to replicate in an in vitro system.
Animals have proven not to be an accurate model and 40–50% of the drug candidates associated with hepatotoxicity in humans did not present the same toxicological concerns in animal models.
One of the reasons for this discrepancy is the differential expression and activity of drug metabolising enzymes between animals and humans that might confound the extrapolation of data derived from model species.
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Acute toxicity of 6 common drugs
Fey et al 2020 has shown that when using a clinostat-based system as the growth environment for an immortal hepatoma cell line, the model constructs responded correctly to treatment with six commonly used drugs.
Results summary
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Accumulated toxicity of Paracetamol
Fey et al also investigated HEPG2/C3A-derived spheroids’ response to multiple doses of paracetamol at 48-h intervals during a 10-day period. Due to the simplicity of the ClinoStar system, it is possible to test repeated drug treatments over extended periods of time.
This study demonstrated that the therapeutic dose is 4 g/day resulting in a blood concentration of 10–25 μg/ml. If the ATP levels are indicators of the therapeutic effect, then treatment is maximised already at very low doses. Thus, a lower therapeutic dose might be sufficient and maintenance doses could be even lower.
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Improved toxicology prediction by utilising hepatic spheroids
This Application Note discusses the method used for the following:
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On-demand webinar(s)
Understanding mechanisms of drugs toxicity using hepatocytes-based spheroids
Prof. Adelina Rogowska-Wrzesinska, University of Southern Denmark
In this presentation, Prof. Adelina Rogowska-Wrzesinska will discuss how she uses HepG2/C3A spheroids to study hepatocytes response to therapeutic-equivalent doses of APAP (5 mg APAP per mg total soluble protein). She will share tips and tricks on how to design experiments to obtain maximum sensitivity and reproducibility. She will also present how advanced mass spectrometric techniques was used to quantify changes in proteins and their S-nitrosylation and S-sulfenylation levels in C3A spheroids treated with APAP, and how this approach is explored to characterise the early-stage drug response that is very often overlooked in rodent based toxicity models.
