Human Induced Pluripotent Stem Cells
We provide human induced pluripotent stem cell iPSC-based conventional 2D monolayer based and 3-dimensional (3D) neurosphere-based toxicity assays, documented according to the ECVAM DB-ALM system for screening the effect of compounds.
Environmental stressors, such as chemicals or drugs can have a toxic effect on humans which may occur at any stage of their life, including during fetal development, childhood, or adult life. The toxic effects of environmental agents coupled with inherited susceptibility of individuals make the toxicology prediction difficult. Conventional animal-based toxicity and safety tests have high costs, use large numbers of animals (mainly rodents) and in many cases, they do not provide clearly translatable results for humans. Consequently, in line with legislations, there is an increasing need to develop alternative testing methods which could handle large number of drugs or chemicals with affordable time and cost and with human-relevant neurotoxicology (NT) outcomes. Development of new approach methods (NAMs), is important both for NT and developmental neurotoxicology (DNT) tests, providing data on the effect of chemicals and the potential adverse outcomes (AOs).
Major international initiatives have started to convert the traditional animal-based neurotoxicity tests to in vitro assays using both mammalian brain cells and human cells to detect and predict chemical hazards. However, their availability is limited, and the highly complex structure of the human brain makes in vitro modelling very difficult. Human induced pluripotent stem cells (hiPSCs) can fill this niche and offer the advantage that different cell types (e.g. kidney, liver, cardiac, neuronal, intestinal) sharing the same individual genetic background can be created using specific differentiation protocols in a replicable manner. This can provide a very effective in vitro tool for toxicologists for capturing the individual variability in the human population.
hiPSC-derived 21day old neural cultures provided by BioTalentum contain developing neurons and astroglia precursors, while TD42 cultures contain more mature neurons, differentiated astrocytes, where synapses of the neurons are physically built up, and spontaneous firing activity is detectable.
Beside 2D cultures, 3D spheroids are also available that can mimic better the original tissue environment and can promote complex interactions among more matured cell types. Acute and chronic exposure schemes (24 hours, 72 hours, 5 days) are available for compound testing without using animals. Assays are suitable to investigate the DART/DNT/NT nature of tested compounds; moreover, they can also be used for drug-repurposing and post-market safety tests.
- Consultation with customers, surveying their needs (agreement on number of compounds and controls, concentrations to be tested, defining need for 2D or 3D assays and differentiation stages);
- hiPSC-derived neuron differentiation on a 96-well plate format according to the customer’s request;
- Compound treatment, number of dilutions and biological replicates based on customer needs and discussions. A well-known neurotoxic agent is used as positive control.
- Incubation with compounds, generally 72 hours to test acute and 96 hours to test chronic exposures;
- Viability assays (ATP or resazurin-based);
- Data analysis to determine the EC50 and IC10 values of the test compounds;
- Reporting of results including data analysis to customers.
- Replaces animal tests and provides more human-relevant results
- hiPSC-based neurons replace primary human brain cell samples
- The effect of compounds on different differentiation stages of the neuronal tissue can be investigated
- 3D Spheroids represent better the complexity of the neuronal tissue
Human iPSC derived neural precursor cells (NPCs) are differentiated towards TD21 (less mature neurons, cell culture containing astrocytes and dividing NPCs) or TD42 neurons (mature astrocytes, synaptically-active and firing neurons to investigate synaptotoxicity, as well) in a 2D monolayer culture. At the end of differentiation, test compounds in different dilutions are added to cells.
After 24, 72 or 96 hours incubation, cell viability measurements are carried out to examine the acute, or the chronic effect of the test chemicals. After calculation, EC50 and IC10 values are determined. Importantly, from the training set of compounds in EU-ToxRisk, clearly neurotoxic and non-neurotoxic agents are determined that can serve as positive and negative controls.
Our hiPSC-based 3D neurospheres can serve as the basis for a more complex toxicology assay. Spheroids are differentiated for 21 and 42 days before compound treatment; their response to the compound can show distinct patterns compared to 2D monolayers, since 3D spheroids are often more sensitive to toxic compounds.
For viability measurements, we use the following assays:
- ATP assay (CellTiter- Glo®; Promega): the assay is based on the detection of ATP that is generated by metabolically active cells. Assay reagents cause cell lysis and generate luminescent signal proportion to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. ATP assay is present in our already accepted protocols (DB-ALM Protocol No. 207, 208, 209).
- Resazurin-based assay (PrestoBlue®, Thermo Fisher Sci): this assay uses the reducing power of living cells. Therefore, the amount of reduced resazurin resembles the amount of viable cells in the culture. We suggest to use this assay when the test compound might alter ATP efflux transporters.
Kobolak J., et al. Human induced pluripotent stem cell-derived 3D neurospheres are suitable for neurotoxicity screening. Cells. 2020 doi: 10.3390/cells9051122
Krebs A., et al. The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach method. Arch. Toxicol. 2020. doi: 10.1007/s00204-020-02802-6