Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

Plant Tissue Culture and Metabolite Profiling for High-Value Natural Product Synthesis.

The engineering of plant cell cultures to produce high-value natural products is suggested to be a safe, low-cost, and environmentally friendly route to produce a wide range of chemicals. Given that the expression of heterologous biosynthetic pathways in plant tissue culture is limited by a lack of detailed protocols, the biosynthesis of high-value metabolites in plant cell culture is constrained compared with that in microbes. However, both Arabidopsis thaliana and Nicotiana benthamiana can be efficiently transformed with multigene constructs to produce high-value natural products in stable plant cell cultures. This chapter provides a detailed protocol as to how to engineer the plant cell culture as bio-factories for metabolite biosynthesis.

First Gynogenesis of Vanilla planifolia for Haploid Production and Ploidy Verification Protocol.

Vanilla orchids are members of the Vanilloideae orchid subfamily, and they hold significant economic value as a spice crop in tropical regions. Despite the presence of 180 known species within this subfamily, commercial production focuses on only three species (Vanilla planifolia, V. odorata, and V. pompona) and one hybrid (V. × tahitensis), prized for their aromatic qualities and bioactive compounds. Limited modern breeding initiatives have been undertaken with vanilla orchids, although recent advancements in genomic research are shedding light on this crop's potential. The protracted breeding cycle of vanilla, coupled with increasing demand for germplasm, underscores the importance of research and breeding efforts in vanilla. This paper outlines a protocol for haploid production in V. planifolia using unfertilized ovaries in tissue culture conditions. Additionally, we present a methodology to confirm the haploid nature of putative haploid lines through stomatal size comparison, chromosome counting, and flow cytometry analysis, proving the successful development of haploid vanilla plants. These findings contribute to the advancement of breeding programs and genetic improvement strategies for the vanilla industry.

Functional Analysis of Seed Dormancy Genes by Biolistic Transient Gene Expression in Immature Embryos of Wheat.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.

Enhancing growth and bioactive metabolites characteristics in Mentha pulegium L. via silicon nanoparticles during in vitro drought stress.

The development and production of secondary metabolites from priceless medicinal plants are restricted by drought stress. Mentha pulegium L. belongs to the Lamiaceae family and is a significant plant grown in the Mediterranean region for its medicinal and aesthetic properties. This study investigated the effects of three polyethylene glycol (PEG) (0, 5, and 10%) as a drought stress inducer and four silicon nanoparticle (SiNP) (0, 25, 50, and 100 ppm) concentrations as an elicitor to overcome the adverse effect of drought stress, on the growth parameters and bioactive chemical composition of M. pulegium L. plants grown in vitro. The experiment was performed as a factorial experiment using a completely randomized design (CRD) consisting of 12 treatments with two factors (3 PEG × 4 SiNPs concentrations), 6 replicates were used for each treatment for a total of 72 experimental units.The percentage of shoot formation was inversely proportional to the PEG concentration; for the highest PEG concentration, the lowest percentage of shoot formation (70.26%) was achieved at 10% PEG. SiNPs at 50 ppm enhanced shoot formation, the number of shoots, shoot height, fresh and dry weight, rosmarinic acid, total phenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. The methanol extract from M. pulegium revealed the presence of significant secondary metabolites using gas chromatography‒mass spectrometry (GC-MS). The principal constituents of the extract were limonene (2.51, 2.99%), linalool (3.84, 4.64%), geraniol (6.49, 8.77%), menthol (59.73, 65.43%), pulegone (3.76, 2.76%) and hexadecanoic acid methyl ester or methyl palmitate (3.2, 4.71%) for the 0 ppm SiNPs, PEG 0% and 50 ppm SiNPs, and PEG 10%, respectively. Most of the chemical components identified by GC‒MS in the methanol extract were greater in the 50 ppm SiNP and 10% PEG treatment groups than in the control group. SiNP improves drought tolerance by regulating biosynthesis and accumulating some osmolytes and lessens the negative effects of polyethylene glycol-induced drought stress.Based on the results, the best treatment for most of the parameters was 50 ppm SiNPs combined with 10% PEG, the morphological and chemical characteristics were inversely proportional to the PEG concentration, as the highest PEG concentration (10%) had the lowest results. Most parameters decreased at the highest SiNP concentration (100 ppm), except for the DPPH scavenging percentage, as there was no significant difference between the 50 and 100 ppm SiNPs.

Comparative restriction enzyme analysis of methylation (CREAM) reveals methylome variability within a clonal in vitro cannabis population.

The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically uniform products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid multiplication of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to study a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

The Effect of Combined Elicitation with Light and Temperature on the Chlorogenic Acid Content, Total Phenolic Content and Antioxidant Activity of Berula erecta in Tissue Culture.

Chlorogenic acid is one of the most prominent bioactive phenolic acids with great pharmacological, cosmetic and nutritional value. The potential of Berula erecta in tissue culture was investigated for the production of chlorogenic acid and its elicitation combined with light of different wavelengths and low temperature. The content of chlorogenic acid in the samples was determined by HPLC-UV, while the content of total phenolic compounds and the antioxidant activity of their ethanol extracts were evaluated spectrophotometrically. The highest fresh and dry biomasses were obtained in plants grown at 23 °C. This is the first study in which chlorogenic acid has been identified and quantified in Berula erecta. The highest chlorogenic acid content was 4.049 mg/g DW. It was determined in a culture grown for 28 days after the beginning of the experiment at 12 °C and under blue light. The latter also contained the highest content of total phenolic compounds, and its extracts showed the highest antioxidant activity. Berula erecta could, potentially, be suitable for the in vitro production of chlorogenic acid, although many other studies should be conducted before implementation on an industrial scale.

In Vitro Propagation Technology for the Endangered Aquatic Species Nymphoides coronata.

Nymphoides coronata is an endangered aquatic plant species with significant medicinal and ecological importance. To preserve N. coronata from going extinct, we need to provide seedlings and efficient multiplication techniques so that it can be extensively studied. This study aimed to identify the most suitable sterilization treatment, growth medium, and substrate for the cultivation and propagation of N. coronata. Ethanol sterilization, fungicide treatment, and sterile water washing were the most important sterilization steps. A combination of 6-benzylaminopurine (6-BA) and indoleacetic acid (IAA) was the most suitable medium for bud induction and shoot proliferation. The use of α-naphthaleneacetic acid (NAA) increased the rooting rate and rooting time compared to indole-3-butyric acid (IBA). Increasing the concentration of NAA from 0.5 to 1.0 mg/L increased the rooting rate from 78 to 100% and reduced the rooting time from 7 to 5 days. The survival rate of N. coronata seedlings was 100% in a mixture of red soil and sand (1:1, w/w). As a result, the procedure mentioned above could potentially be used to safely propagate this rare species on a large scale. These findings provide valuable insights into the optimal conditions for the successful cultivation and propagation of N. coronata, which can contribute to the conservation and sustainable use of this important rare plant species.

Spontaneous chromosome instability and tissue culture-induced karyotypic alteration in wheat-Thinopyrum intermedium alien addition lines.

MAIN CONCLUSION: Wheat lines harboring wild-relative chromosomes can be karyotypically unstable during long-term maintenance. Tissue culture exacerbates chromosomal instability but appears inefficient to induce somatic homoeologous exchange between alien and wheat chromosomes. We assessed if long-term refrigerator storage with regular renewal via self-fertilization, a widely used practice for crop germplasm maintenance, would ensure genetic fidelity of alien addition lines, and explored the possibility of inducing somatic homoeologues exchange by tissue culture. We cytogenetically characterized sampled stock seeds of originally confirmed 12 distinct wheat-Thinopyrum intermedium alien addition lines (dubbed TAI lines), and subjected immature embryos of the TAI lines to tissue culture. We find eight of the 12 TAI lines were karyotypically departed from their original identity as bona fide disomic alien addition lines due to extensive loss of whole-chromosomes of both Th. intermedium and wheat origins during the ca. 3-decade storage. Rampant numerical chromosome variations (NCVs) involving both alien and wheat chromosomes were detected in regenerated plants of all 12 studied TAI lines, but at variable rates among the wheat sub-genomes and chromosomes. Compared with NCVs, structural chromosome variations (SCVs) occurred at substantially lower rates, and no SCV involving the added alien chromosomes was observed. The NCVs manifested only moderate effects on phenotypes of the regenerated plants under field conditions.

Trusting the forces of our cell lines.

Cells isolated from their native tissues and cultured in vitro face different selection pressures than those cultured in vivo. These pressures induce a profound transformation that reshapes the cell, alters its genome, and transforms the way it senses and generates forces. In this perspective, we focus on the evidence that cells cultured on conventional polystyrene substrates display a fundamentally different mechanobiology than their in vivo counterparts. We explore the role of adhesion reinforcement in this transformation and to what extent it is reversible. We argue that this mechanoadaptation is often understood as a mechanical memory. We propose some strategies to mitigate the effects of on-plastic culture on mechanobiology, such as organoid-inspired protocols or mechanical priming. While isolating cells from their native tissues and culturing them on artificial substrates has revolutionized biomedical research, it has also transformed cellular forces. Only by understanding and controlling them, we can improve their truthfulness and validity.

Human Skin as an Ex Vivo Model for Maintaining Mycobacterium leprae and Leprosy Studies.

The in vitro cultivation of M. leprae has not been possible since it was described as causing leprosy, and the limitation of animal models for clinical aspects makes studies on leprosy and bacteria-human host interaction a challenge. Our aim was to standardize the ex vivo skin model (hOSEC) to maintenance and study of M. leprae as an alternative animal model. Bacillary suspensions were inoculated into human skin explants and sustained in DMEM medium for 60 days. Explants were evaluated by RT-PCR-16SrRNA and cytokine gene expression. The viability and infectivity of bacilli recovered from explants (D28 and D60) were evaluated using the Shepard's model. All explants were RT-PCR-16SrRNA positive. The viability and infectivity of recovered bacilli from explants, analyzed after 5 months of inoculation in mice, showed an average positivity of 31%, with the highest positivity in the D28 groups (80%). Furthermore, our work showed different patterns in cytokine gene expression (TGF-ß, IL-10, IL-8, and TNF-α) in the presence of alive or dead bacilli. Although changes can be made to improve future experiments, our results have demonstrated that it is possible to use the hOSEC to maintain M. leprae for 60 days, interacting with the host system, an important step in the development of experimental models for studies on the biology of the bacillus, its interactions, and drug susceptibility.

Challenges of Diphtheria Toxin Detection.

Diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Moreover, new Corynebacterium species with the potential to produce diphtheria toxin have also been described. Therefore, the detection of the toxin is the most important test in the microbiological diagnosis of diphtheria and other corynebacteria infections. Since the first demonstration in 1888 that DT is a major virulence factor of C. diphtheriae, responsible for the systemic manifestation of the disease, various methods for DT detection have been developed, but the diagnostic usefulness of most of them has not been confirmed on a sufficiently large group of samples. Despite substantial progress in the science and diagnostics of infectious diseases, the Elek test is still the basic recommended diagnostic test for DT detection. The challenge here is the poor availability of an antitoxin and declining experience even in reference laboratories due to the low prevalence of diphtheria in developed countries. However, recent and very promising assays have been developed with the potential for use as rapid point-of-care testing (POCT), such as ICS and LFIA for toxin detection, LAMP for tox gene detection, and biosensors for both.

Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 µg/mL) as compared to controls (0.37 µg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.

Advancing Tissue Culture with Light-Driven 3D-Printed Microfluidic Devices.

Three-dimensional (3D) printing presents a compelling alternative for fabricating microfluidic devices, circumventing certain limitations associated with traditional soft lithography methods. Microfluidics play a crucial role in the biomedical sciences, particularly in the creation of tissue spheroids and pharmaceutical research. Among the various 3D printing techniques, light-driven methods such as stereolithography (SLA), digital light processing (DLP), and photopolymer inkjet printing have gained prominence in microfluidics due to their rapid prototyping capabilities, high-resolution printing, and low processing temperatures. This review offers a comprehensive overview of light-driven 3D printing techniques used in the fabrication of advanced microfluidic devices. It explores biomedical applications for 3D-printed microfluidics and provides insights into their potential impact and functionality within the biomedical field. We further summarize three light-driven 3D printing strategies for producing biomedical microfluidic systems: direct construction of microfluidic devices for cell culture, PDMS-based microfluidic devices for tissue engineering, and a modular SLA-printed microfluidic chip to co-culture and monitor cells.

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.

Expanding science skills: teaching tissue culture, data analysis, and reporting through imaging the actin cytoskeleton.

Within the eukaryotic cell, the actin cytoskeleton is a crucial structural framework that maintains cellular form, regulates cell movement and division, and facilitates the internal transportation of proteins and organelles. External cues induce alterations in the actin cytoskeleton primarily through the activation of Rho GTPases, which then bind to a diverse array of effector proteins to promote the local assembly or disassembly of actin. We have harnessed the extensively studied functions of RhoA in the dynamics of the actin cytoskeleton to craft a practical series for Stage 2 Biology students. This series not only imparts essential tissue culture laboratory skills but also reinforces them through repetition. These activities are presented in a scenario designed for students to explore the function of a hypothetical RhoA family member. Students produce slides from transfected cells, undertake fluorescence microscopy, process the images using ImageJ, and compile their findings in a comprehensive scientific report. The composition of the report requires independent acquisition of new knowledge and synoptic learning. According to student feedback, this early experience greatly aids in solidifying and honing the skills required to report on more extensive and intricate research projects, such as capstone projects.

Secondary metabolite fingerprinting, anti-pathogenic activity, elite chemotype selection and conservation of Curcuma caesia- an ethnomedicinally underutilized species.

Curcuma caesia Roxb. is an ethnomedicinally important, essential oil (EO) yielding aromatic plant. A total of twelve accessions of this plant rhizome were collected from six different agro-climatic zones of West Bengal, India and evaluated for their antimicrobial activities against eight disease-causing, multi-drug-resistant pathogenic strains of urinary-tract infection and respiratory-tract infection. The EO and extracts demonstrated antibacterial activity, with the highest inhibition zone of 18.00 ± 0.08 and 17.50 ± 0.14 mm against Klebsiella pneumoniae by accession 06, even where all the broad-spectrum antibiotics failed to respond. In this study, we employed high-performance thin-layer chromatography (HPTLC) to quantify curcumin, the primary secondary metabolite of C. caesia, and the highest 0.228 mg/gm of curcumin resulted from accession 06. Hence, on the basis of all aspects, accession 06 was identified as the elite chemotype among all twelve accessions. The chemical profiling of EO from accession 06 was done using gas chromatography-mass spectroscopy (GC-MS). Conceivably, about 13 medicinally significant compounds were detected. As this plant species is seasonal and has difficulties in conventional breeding due to dormancy, it must be conserved through in vitro tissue culture for a steady supply throughout the year in massive amounts for agricultural demand. A maximum number of 19.28 ± 0.37 shoots has been obtained in MS medium fortified with 6-Benzylaminopurine, Kinetin, and Naphthalene acetic acid. The genetic uniformity of the plants has been studied through Start Codon Targeted Polymorphism. Therefore, this study must help meet the need for essential phytoactive compounds through a simple, validated, and reproducible plant tissue culture protocol throughout the year.

Development of an iPSC-derived tissue-resident macrophage-based platform for the in vitro immunocompatibility assessment of human tissue engineered matrices.

Upon implanting tissue-engineered heart valves (TEHVs), blood-derived macrophages are believed to orchestrate the remodeling process. They initiate the immune response and mediate the remodeling of the TEHV, essential for the valve's functionality. The exact role of another macrophage type, the tissue-resident macrophages (TRMs), has not been yet elucidated even though they maintain the homeostasis of native tissues. Here, we characterized the response of hTRM-like cells in contact with a human tissue engineered matrix (hTEM). HTEMs comprised intracellular peptides with potentially immunogenic properties in their ECM proteome. Human iPSC-derived macrophages (iMφs) could represent hTRM-like cells in vitro and circumvent the scarcity of human donor material. iMφs were derived and after stimulation they demonstrated polarization towards non-/inflammatory states. Next, they responded with increased IL-6/IL-1ß secretion in separate 3/7-day cultures with longer production-time-hTEMs. We demonstrated that iMφs are a potential model for TRM-like cells for the assessment of hTEM immunocompatibility. They adopt distinct pro- and anti-inflammatory phenotypes, and both IL-6 and IL-1ß secretion depends on hTEM composition. IL-6 provided the highest sensitivity to measure iMφs pro-inflammatory response. This platform could facilitate the in vitro immunocompatibility assessment of hTEMs and thereby showcase a potential way to achieve safer clinical translation of TEHVs.

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Persistent organic pollutants dysregulate energy homeostasis in human ovaries in vitro.

Exposure to persistent organic pollutants (POPs), such as dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), has historically been linked to population collapses in wildlife. Despite international regulations, these legacy chemicals are still currently detected in women of reproductive age, and their levels correlate with reduced ovarian reserve, longer time-to-pregnancy, and higher risk of infertility. However, the specific modes of action underlying these associations remain unclear. Here, we examined the effects of five commonly occurring POPs - hexachlorobenzene (HCB), p,p'-dichlorodiphenyldichloroethylene (DDE), 2,3,3',4,4',5-hexachlorobiphenyl (PCB156), 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB180), perfluorooctane sulfonate (PFOS) - and their mixture on human ovaries in vitro. We exposed human ovarian cancer cell lines COV434, KGN, and PA1 as well as primary ovarian cells for 24 h, and ovarian tissue containing unilaminar follicles for 6 days. RNA-sequencing of samples exposed to concentrations covering epidemiologically relevant levels revealed significant gene expression changes related to central energy metabolism in the exposed cells, indicating glycolysis, oxidative phosphorylation, fatty acid metabolism, and reactive oxygen species as potential shared targets of POP exposures in ovarian cells. Alpha-enolase (ENO1), lactate dehydrogenase A (LDHA), cytochrome C oxidase subunit 4I1 (COX4I1), ATP synthase F1 subunit alpha (ATP5A), and glutathione peroxidase 4 (GPX4) were validated as targets through qPCR in additional cell culture experiments in KGN. In ovarian tissue cultures, we observed significant effects of exposure on follicle growth and atresia as well as protein expression. All POP exposures, except PCB180, decreased unilaminar follicle proportion and increased follicle atresia. Immunostaining confirmed altered expression of LDHA, ATP5A, and GPX4 in the exposed tissues. Moreover, POP exposures modified ATP production in KGN and tissue culture. In conclusion, our results demonstrate the disruption of cellular energy metabolism as a novel mode of action underlying POP-mediated interference of follicle growth in human ovaries.