To obtain accurate results, the requirement of these solvents are that they be biocompatible and non-toxic to cells. The aim of this study was to investigate the biological effects of the three agents most commonly used as drug vehicles, i. Methods : To minimize the errors from the measurement techniques, this study used the xCelligence RTCA system which entails real-time monitoring of cell proliferation without dye labeling.
The concentrations of ethanol and methanol ranging from 2. Depending on the extracts or agents, each should be diluted in the suitable vehicle at the proper concentrations. In biological research, solvents for dilution of therapeutic drugs are necessary, especially for drugs that are weakly soluble in water.
The most common solvents for use as vehicles for drug delivery purpose in biological studies are dimethyl sulfoxide DMSO , ethanol and methanol. DMSO is an organic amphiphilic molecule that is widely used in cell biology; it exhibits a number of capabilities such as vasodilatory, diuretic, anti-inflammatory and bacteriostatic functions 1 , 2.
In vitro, DMSO is routinely used for cryopreservation due to its high freeze temperature. However, its strong interaction with phospholipids makes it efficient in facilitating drug molecule delivery through the cell membrane, which is an area of study with great interest by many researchers. DMSO ranks high in terms of choice of solvent for drug delivery, despite its potential cytotoxicity on cells.
Alternative solvent that have been used in cell biology include ethanol and methanol. In drug screening, besides DMSO, crude plant extract could be dissolved in methanol or ethanol. However, ethanol could interfere with the structure of low cholesterol in the cell membrane and cause disorder of cellular physical activities 3.
This damage in cell membrane could amplify the effect of therapeutic drugs, leading to non-reproducible results. Furthermore, different cell types respond with different behavior to solvents 4. Although solvent control could be used to compare to the drug efficacy in experiment , nonetheless if the solvents are toxic to the cells over treatment, the effect of the drug could be wrongly evaluated.
It is important to evaluate and define the maximum concentration of the solvents that could be used for dissolving drugs in biological assays. Hence, it is necessary to understand the optimal concentration of the solvents on specific cell lines to ensure accurate and reproducible experimental results. Cell proliferation was measured by recording the change in the impedance of electron flow caused by adherent cells. After cells were cultured for 24 hours for cell attachment, the solvents were added; the proliferation curves were slightly perturbed Figure 1.
From that time point onward, the cell proliferation curves dispersed. In contrast to methanol, DMSO induced more than one flattened curve in the 4 cell lines. Ethanol also appeared to induce divergent curves in the 4 cell lines. The concentrations of 1.
The slope of the CI at these concentration were significantly different from that of the lower concentrations of 0. Interestingly, DMSO at the concentration of 0. However, 0. At the concentration of 2. Methanol seemed to have the least impact on the cell lines in this study. Lower concentrations of methanol, such as 2. Only of the 1, genes The induced alterations affected preferentially satellites and simple repeats, as well as CGIs distal to promoters without known regulatory evidence.
The expression of neighboring genes was not significantly correlated to methylation differences. We therefore conclude that transcriptional repression by promoter hypermethylation was not the primary regulatory effect of the altered methylation marks, but the findings indicate a global disruption of DNA methylation mechanisms.
While high doses of DMSO were initially investigated for medical applications, lower doses are now commonly used as solvent for toxicology or for cryopreservation of cell cultures, oocytes and embryos. In the meantime, biological effects of DMSO have been forgotten or considered negligible.
With the evolution of biomedical science towards more sensitive high-throughput techniques and towards new areas of research, including epigenome modifications and microRNA-mediated gene silencing, we evaluated the biological effects of DMSO exposure on the transcriptome and the epigenome. Differences between the investigated tissue types may relate to the fact that the cardiac model contains iPSC-derived human cardiomyocytes that are still maturing, while the hepatic model contains mature human hepatocytes.
Currently, it is not yet possible for iPSC cells to reach an adult-like phenotype 20 , 21 and the concise review on iPSC-derived human cardiomyocytes of Robertson et al. Our observations of DMSO effects, notably the extreme epigenetics modifications in fetal-like cardiac cells, are particularly relevant for cryopreservation during assisted reproductive technology embryos, oocytes and sperm cells.
Another difference to be considered between our two cell models is the number of donors. Indeed, while the cardiac MTs are composed of IPSC-derived cardiomyocytes from a single donor co-cultured with fibroblasts also from 1 donor , hepatic MTs contain mature hepatocytes from 10 donors co-cultured with Kupffer cells of 1 donor. While a single-donor model is more comparable to an in vivo situation then a multi-donor model, single-donor cardiac MTs might be more susceptible for bias due to genotype sensitivity than our multi-donor hepatic MTs that have the ability to even out these effects.
DMSO effects on cellular processes were analyzed using full transcriptome data. This indicates robust actions of DMSO that may possibly be extrapolated to other tissues. Observed changes in cellular processes include changes in mitochondrial pathways linked to ROS production and cellular ATP generation.
DMSO is a known radical scavenger Different downstream effects are relevant depending on the biological application of DMSO. For instance, for cell assays and toxicological research, changes in the amount of ROS or free energy in the cell impact on the capability of the cell to deal with induced stresses, thereby potentially leading to erroneous interpretation of results from in vitro assays. Within assisted reproductive technology, the ATP content is a good predictor of embryo viability.
The DMSO-induced ATP decrease, especially in the cleavage-stage, can induce downstream effects that may disrupt cellular function, implantation ability and fetal development 25 , DMSO-induced reduction in implantation rates and pregnancy losses were already observed from animal models Next to overlap in DMSO-affected processes between the tissue types, pathway analysis also revealed tissue-specific differences. The higher amount of affected processes detected in cardiac MTs may indicate increased susceptibility of cardiac MTs to the effects of DMSO, though there might also be a statistical bias due to different amount of DEGs used in the pathway analysis.
Cardiac MTs also show more effects in other processes that regulate gene expression. Genome-wide methylation effects upon DMSO treatment show large tissue-specific variation. While there is no significant difference with respect to hepatic MTs, strong effects are observed with cardiac MTs, in particular with respect to specific repetitive sequences such as satellites and simple repeats. Changes in the methylation status of repeats after DMSO treatment might destabilize the genome, implying phenotypic and pathological consequences.
Indeed, recently, methylation changes of repetitive sequences, in particular satellite repeat elements, have been found in cardiomyopathic heart tissue compared to normal heart tissue Furthermore, it is also possible that DMSO acts as a methyl donor to induce hypermethylation However, it has to be kept in mind that there is an interplay between DNA methylation and chromatin configuration, which is regulated by more processes than just DNMTs.
Overall, we speculate the effects of DMSO on methylation changes to be a global deregulation characterized by a genome wide hypermethylation. Cell mechanisms in charge of removing detrimental methylations at important regions, such as TFBS, may then have removed the methylation changes with a negative outcome on cell survival, leaving an over-representation of DMR in the repetitive elements.
This is in analogy to mutation rates, which are higher for repetitive elements since they are less subjected to natural selection and DNA repair mechanisms We believe that the methylation difference between mature hepatic and maturing cardiac cells may be detrimental for cryopreserved cells and especially for oocytes and embryos. Previously reported DMSO effects on oocytes and embryos are limited to changes in gene expression in animal models 27 , 33 , 34 , 35 , We now showed, for the first time, the influence of DMSO on the epigenome of human cells in vitro.
It is uncertain if exposed cells can recover from temporary DMSO exposures. While methylation changes have an adaptive nature, they may also be persistent. From research conducted on the Dutch famine cohort, it is known that the periconceptional period before conception until early pregnancy is crucial for establishing and maintaining epigenetic marks. During this period epigenetic marks present on the parental DNA are removed to produce a totipotent zygote which is reprogrammed after implantation Exposures during this time may induce persistent epigenetic differences, which can result in disease later in life or possibly be inherited by the offspring to induce transgenerational effects 38 , 39 , 40 , 41 , It is already well known that infants conceived through assisted reproductive technologies are prone to be born preterm, have low birth weight and even have a significantly increased risk of birth defects 43 , Furthermore, some short-term health outcomes were slightly poorer in children conceived by IVF, though overall the outcomes were positive 42 , Regarding long-term health and development, though available data is limited, cardiovascular and metabolic risk factors that may ultimately result in chronic cardiometabolic disease have been indicated Furthermore, also epigenetic risks have been debated, mostly focusing on methylation and imprinting.
For the most studied imprinting disorders Beck-with-Wiedemann syndrome and Angelman syndrome, the incidence was higher for children born after assisted reproductive technologies compared to natural conception 41 , 46 , 47 , 48 , 49 , While there are many factors contributing to these abnormalities, decreased infertility, higher age of the parents, technical interference with a biological process, etc.
DMSO-induced effects may be one of them. In the last decade, improvements made in artificial reproductive technology greatly increased the success rate of IVF treatments. Especially, improved cryopreservation techniques transition to vitrification aided greatly. Though not proven yet, increased success rates are reported when implanting previously frozen embryos 51 , 52 , Furthermore, while storing frozen oocytes was initially intended for women threatened by decreased fertility due to disease, in our carrier-focused society, it has also become a method to prevent age-related infertility for women who want to delay motherhood The most recent development in assisted reproductive technology is the emerging of concerns about DMSO effects.
While there is still controversy on this topic, the field is moving towards DMSO-free methods. However, DMSO-free does not automatically mean safe because of the use of other cryoprotectants 55 , 56 , Our study highlights the capability of DMSO to induce changes in cellular processes in both cardiac and hepatic cells, but more severely, induce alterations in miRNA and epigenetic landscape in the 3D maturing cardiac model.
The changes in cellular processes can have consequences for conclusions drawn from cell assays and therefore also in any application of these findings e. Furthermore, the extreme changes in miRNA and alterations in the epigenetic landscape may pose a threat, especially for assisted reproductive technology. Genome-wide hypermethylation induced by global deregulation of methylation mechanisms, especially when it affects genes important in development, may have negative consequences directly, later in life or possibly in later generations.
Overall, use of DMSO should be avoided where possible. However, for the time being, DMSO is indispensable within biotechnological applications. In these cases, the effects that DMSO may have should be considered and the concentration should be kept as low as possible, because even at low concentrations DMSO is not inert. In this study, 3D InSight TM Human Cardiac Microtissues InSphero, for beta-testers were used, containing a co-culture of approximately iPSC-derived human cardiomyocytes from a female Caucasian donor with no known disease phenotype and cardiac fibroblasts from an 18 year old Caucasian male.
Furthermore, 3D InSight TM Human Liver Microtissues InSphero were used, approximately primary human hepatocytes multi-donor pool of 5 males and 5 females between 7—59 years old in co-culture with primary human Kupffer cells from a Caucasian 27 year old of unreported gender.
Medium was changed three times daily, in concordance with the experimental design of our current research Two files per sample were obtained, split in left and right reads, and Lexogen adapter sequences first 12 bases of all reads were removed using Trimmomatic 59 v. The quality of the sequencing data was checked using FastQC 60 v. For cardiac samples, no samples were discarded because of poor quality.
For hepatic samples, no samples were discarded. Here, the design was set according to exposure UNTR or DMSO , other settings were kept to their default parameters, except for minimum count, which was set to an average read count of one across all samples.
Finally, remaining ribosomal genes were filtered out of the dataset to ensure complete ribosomal depletion of the data. Furthermore, principal component analysis Supplementary Fig. MiRNA data analysis was done similarly as described previously 65 , except for the use of mirDeep2, which does not gain additional information when using human samples.
In short, PatMaN 66 v. Proteomics data was also obtained and analyzed. Methods and results are included in Supplementary data. Because of lower DNA yield for cardiac 0. In short, denatured DNA was mixed with antimeC-antibody and captured using magnetic beads. In order to gain exhaustive genome-wide coverage the triplicate samples that have been sequenced individually were merged before alignment.
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