Preparation of a universally usable, animal product free, defined medium for 2D and 3D culturing of normal and cancer cells
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Cited by 4 publications
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An Evaluation of the Replacement of Animal-derived Biomaterials in Human Primary Cell Culture
The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition (‘humanise’) a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.
An Evaluation of the Replacement of Animal-derived Biomaterials in Human Primary Cell Culture
The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition (‘humanise’) a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.
Increasing sustainability and reproducibility of in vitro toxicology applications: serum-free cultivation of HepG2 cells
Fetal Bovine Serum (FBS) is an important ingredient in cell culture media and the current standard for most cells in vitro. However, the use of FBS is controversial for several reasons, including ethical concerns, political, and societal pressure, as well as scientific problems due to the undefined and variable nature of FBS. Nevertheless, scientists hesitate to change the paradigm without solid data de-risking the switch of their assays to alternatives. In this study, HepG2 cells, a human hepatoblastoma cell line commonly used to study drug hepatotoxicity, were adapted to serum-free conditions by using different commercially available media and FBS replacements. After transition to these new culture conditions, the success of adaptation was determined based on cell morphology and growth characteristics. Long-term culturing capacity for each medium was defined as the number of passages HepG2 cells could be cultured without any alterations in morphology or growth behavior. Two media (Advanced DMEM/F12 from ThermoFisher and TCM® Serum Replacement from MP Biomedicals) showed a long-term cultivation capacity comparable to media containing FBS and were selected for further analysis. Both media can be characterized as serum-free, however still contain animal-derived components: bovine serum albumin (both media) and bovine transferrin (only TCM® serum replacement). To assess the functionality of the cells cultivated in either of the two media, HepG2 cells were treated with reference compounds, specifically selected for their known hepatotoxicity characteristics in man. Different toxicological assays focusing on viability, mitochondrial toxicity, oxidative stress, and intracellular drug response were performed. Throughout the different assays, response to reference compounds was comparable, with a slightly higher sensitivity of serum-free cultivated HepG2 cells when assessing viability/cell death and a lower sensitivity towards oxidative stress. Taken together, the two selected media were shown to support growth, morphology, and function of serum-free cultivated HepG2 cells in the early preclinical safety space. Therefore, these results can serve as a starting point to further optimize culture conditions with the goal to remove any remaining animal-derived components.
Use of serum-free media for peripheral blood mononuclear cell culture and the impact on T and B cell readouts
IntroductionAs part of a wider programme of work developing next-generation risk assessment approaches (NGRA) using non-animal methods (NAMs) for safety assessment of materials, Unilever SEAC is exploring the use of a peripheral blood mononuclear cell (PBMC) system to investigate how cells from different arms of the human immune system are impacted by different treatments. To maximise human relevance, the cell cultures are supported by human serum, but this came with some challenges, including an inability to measure induced levels of immunoglobulins due to high background levels. Therefore, a study comparing use of human sera containing media with three different chemically defined serum-free media was undertaken.Materials and MethodsPBMC were isolated from healthy donors and cultured in the absence (media alone) or presence of stimulation reagents (CpG-ODN plus IL-15, Pokeweed Mitogen (PWM) or Cytostim (CS)), in RPMI plus human serum, AIM-V, CTS OpTmizer T cell expansion SFM or X-VIVO 15 media. T cell (CD4+ and CD8+) and B cell proliferation and viability were measured after 6 days, along with levels of total IgG in the cell culture supernatants.ResultsEach of the serum-free media tested supported good levels of viable and proliferating T cells and B cells over the 6 days of culture, with only a few, small differences across the media, when there was no stimulation. They also enabled detection of a stimulation-evoked increase in IgG levels. There were however some differences in the viability and proliferation responses of T and B cells, to different stimuli, across the different media.DiscussionThe serum-free media formulations tested in this study offer defined systems for. measuring B cell IgG responses, in vitro, in either a ‘T cell-independent’ (CpG + IL-15) or “T cell-dependent” (PWM or CS) manner and for assessing B cell proliferation, particularly in response to a “T cell-independent” stimulus. However, there are some characteristics and features endowed by human serum that appear to be missing. Therefore, further work is required to optimise animal-free, chemically defined culture conditions for PBMC based assays for inclusion in tiered safety assessments.
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