Forward-reverse mutation cycles in cancer cell lines under chemical treatments.

Spontaneous forward-reverse mutations were reported by us earlier in clinical samples from various types of cancers and in HeLa cells under normal culture conditions. To investigate the effects of chemical stimulations on such mutation cycles, the present study examined single nucleotide variations (SNVs) and copy number variations (CNVs) in HeLa and A549 cells exposed to wogonin-containing or acidic medium. In wogonin, both cell lines showed a mutation cycle during days 16-18. In acidic medium, both cell lines displayed multiple mutation cycles of different magnitudes. Genomic feature colocalization analysis suggests that CNVs tend to occur in expanded and unstable regions, and near promoters, histones, and non-coding transcription sites. Moreover, phenotypic variations in cell morphology occurred during the forward-reverse mutation cycles under both types of chemical treatments. In conclusion, chemical stresses imposed by wogonin or acidity promoted cyclic forward-reverse mutations in both HeLa and A549 cells to different extents.

Lactate modulates zygotic genome activation through H3K18 lactylation rather than H3K27 acetylation.

In spite of its essential role in culture media, the precise influence of lactate on early mouse embryonic development remains elusive. Previous studies have implicated lactate accumulation in medium affecting histone acetylation. Recent research has underscored lactate-derived histone lactylation as a novel epigenetic modification in diverse cellular processes and diseases. Our investigation demonstrated that the absence of sodium lactate in the medium resulted in a pronounced 2-cell arrest at the late G2 phase in embryos. RNA-seq analysis revealed that the absence of sodium lactate significantly impaired the maternal-to-zygotic transition (MZT), particularly in zygotic gene activation (ZGA). Investigations were conducted employing Cut&Tag assays targeting the well-studied histone acetylation and lactylation sites, H3K18la and H3K27ac, respectively. The findings revealed a noticeable reduction in H3K18la modification under lactate deficiency, and this alteration showed a significant correlation with changes in gene expression. In contrast, H3K27ac exhibited minimal correlation. These results suggest that lactate may preferentially influence early embryonic development through H3K18la rather than H3K27ac modifications.

Cell Culture Differentiation and Proliferation Conditions Influence the In Vitro Regeneration of the Human Airway Epithelium.

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an air-liquid interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist among media used for ALI-cultured human airway epithelial cells, the aim of our study was to evaluate the impact of several media (BEGM, PneumaCult, Half & Half, and Clancy) on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling, and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCult-ALI and Half & Half, stronger EGF signaling from basal cells in BEGM-ALI, differential expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry factor ACE2, and distinct secretome transcripts depending on the media used. We also established that proliferation in PneumaCult-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will aid in choosing the most relevant medium according to the processes to be investigated, such as cilia, mucus biology, or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.

The role of lipids in genome integrity and pluripotency.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research.

Selection of the Most Suitable Culture Medium for Patient-Derived Lung Cancer Organoids.

INTRODUCTION: Patient-derived organoids have emerged as a promising in vitro model for precision medicine, particularly in cancer, but also in noncancer-related diseases. However, the optimal culture medium for culturing patient-derived lung organoids has not yet been agreed upon. This study aimed to shed light on the optimal selection of a culture media for developing studies using patient-derived lung organoids. METHODS: Tumor and normal paired tissue from 71 resected non-small cell lung cancer patients were processed for organoid culture. Lung cancer organoids (LCOs) were derived from tumor tissue and normal lung organoids (LNOs) from nonneoplastic lung tissue. Three different culture media were compared: permissive culture medium (PCM), limited culture medium (LCM), and minimum basal medium (MBM). We assessed their effectiveness in establishing organoid cultures, promoting organoid growth and viability, and compared their differential phenotypic characteristics. RESULTS: While PCM was associated with the highest success rate and useful for long-term expansion, MBM was the best option to avoid normal organoid overgrowth in the organoid culture. The density, size, and viability of LNOs were reduced using LCM and severely affected with MBM. LNOs cultured in PCM tend to differentiate to bronchospheres, while alveolosphere differentiation can be observed in those cultured with LCM. The morphological phenotype of LCO was influenced by the culture media of election. Mesenchymal cell overgrowth was observed when LCM was used. CONCLUSION: This work highlights the importance of considering the research objectives when selecting the most suitable culture medium for growing patient-derived lung organoids.

The unbearable ignorance of the composition of IVF culture media.

Culture media play an essential role in the success of IVF. Their composition has undergone major modifications over the 45 years since the birth of Louise Brown. Most IVF programmes now rely on commercially produced media, which they buy in small vials, guaranteed to be sterile and non-embryotoxic. Unfortunately, information about the components of the culture media and their concentrations is no longer available. Arguing that culture media recipes are proprietary, relevant commercial interests have stopped labelling their products with this vital information. Given the critical role that is played by culture media in the success of IVF, as well as the subsequent health of the children who are born after IVF, this information should not remain a 'company secret'. Clinicians and scientists working in IVF must insist that the labelling of culture media includes all of the constituents and their concentrations. Only in this way can we monitor the influence of culture media on IVF outcomes, innovate and continue to advance the field of IVF.

Vertical transmission of microbiomes into embryo culture media and its association with assisted reproductive outcomes.

RESEARCH QUESTION: Can microbes vertically transmit from semen and follicular fluid to embryo culture media during assisted reproductive technology (ART) treatment? DESIGN: Spent embryo culture media (SECM), seminal fluid and follicular fluid samples were collected from 61 couples with infertility undergoing ART treatment at the Prince of Wales Hospital, Hong Kong SAR, China. Metagenomic analysis was conducted using 16s rRNA sequencing to identify the source of microbes in SECM, correlation between the semen microbiome and male infertility, and correlation between the follicular fluid microbiome and female infertility. RESULTS: Microbial vertical transmission into SECM was reported in 82.5% of cases, and semen was the main source of contamination in conventional IVF cases. The increased abundances of Staphylococcus spp. and Streptococcus anginosus in semen had negative impacts on total motility and sperm count, respectively (P < 0.001). Significant increases in abundance of the genera Prophyromonas, Neisseria and Facklamia were observed in follicular fluid in women with anovulation, uterine factor infertility and unexplained infertility, respectively (P < 0.01). No significant correlation was found between the bacteria identified in all sample types and ART outcomes, including fertilization rate, embryo development, number of available embryos, and clinical pregnancy rate. CONCLUSION: Embryo culture media can be contaminated during ART treatment, not only by seminal microbes but also by follicular fluid and other sources of microbes. Strong correlations were found between specific microbial taxa in semen and sperm quality, and between the follicular fluid microbiome and the aetiology of female infertility. However, no significant association was found between the microbiomes of SECM, semen and follicular fluid and ART outcomes.

Up-regulation of miR-126 via DNA methylation in hypoxia-preconditioned endothelial cells may contribute to hypoxic tolerance of neuronal cells.

BACKGROUND: Endothelial cells (ECs) can confer neuroprotection by secreting molecules. This study aimed to investigate whether DNA methylation contributes to the neuroprotective gene expression induced by hypoxia preconditioning (HPC) in ECs and to clarify that the secretion of molecules from HPC ECs may be one of the molecular mechanisms of neuroprotection. METHODS: Human microvascular endothelial cell-1 (HMEC-1) was cultured under normal conditions (C), hypoxia(H), and hypoxia preconditioning (HPC), followed by the isolation of culture medium (CM). SY5Y cell incubated with the isolated CM from HMEC-1 was exposed to oxygen-glucose deprivation (OGD). The DNA methyltransferases (DNMTs), global methylation level, miR-126 and its promotor DNA methylation level in HMEC-1 were measured. The cell viability and cell injury in SY5Y were detected. RESULTS: HPC decreased DNMTs level and global methylation level as well as increased miR-126 expression in HMEC-1. CM from HPC treated HMEC-1 also relieved SY5Y cell damage, while CM from HMEC-1 which over-expression of miR-126 can reduce injury in SY5Y under OGD condition. CONCLUSIONS: These findings indicate EC may secrete molecules, such as miR-126, to execute neuroprotection induced by HPC through regulating the expression of DNMTs.

Development of an analytical method to quantify N-acyl-homoserine lactones in bacterial cultures, river water, and treated wastewater.

N-Acyl-homoserine lactones (AHL) play a major role in the communication of Gram-negative bacteria. They influence processes such as biofilm formation, swarming motility, and bioluminescence in the aquatic environment. A comprehensive analytical method was developed to elucidate the "chemical communication" in pure bacterial cultures as well as in the aquatic environment and engineered environments with biofilms. Due to the high diversity of AHLs and their low concentrations in water, a sensitive and selective LC-ESI-MS/MS method combined with solid-phase extraction was developed for 34 AHLs, optimized and validated to quantify AHLs in bacterial conditioned medium, river water, and treated wastewater. Furthermore, the developed method was optimized in terms of enrichment volume, internal standards, limits of detection, and limits of quantification in several matrices. An unanticipated variety of AHLs was detected in the culture media of Pseudomonas aeruginosa (in total 8 AHLs), Phaeobacter gallaeciensis (in total 6 AHLs), and Methylobacterium mesophilicum (in total 15 AHLs), which to our knowledge have not been described for these bacterial cultures so far. Furthermore, AHLs were detected in river water (in total 5 AHLs) and treated wastewater (in total 3 AHLs). Several detected AHLs were quantified (in total 24) using a standard addition method up to 7.3±1.0 µg/L 3-Oxo-C12-AHL (culture media of P. aeruginosa).

Efficacy of non-invasive chromosome screening, preimplantation genetic testing for aneuploidy, and morphological grading in selecting embryos of patients with advanced maternal age: a three-armed prospective cohort study.

BACKGROUND: Non-invasive chromosome screening (NICS) and trophectoderm biopsy preimplantation genetic testing for aneuploidy (TE-PGT) were both applied for embryo ploidy detection, However, the cumulative live birth rates (CLBR) of NICS and TE-PGT in older age groups have yet to be reported. This study aimed to ascertain whether NICS and TE-PGT could enhance the cumulative live birth rates among patients of advanced maternal age. METHODS: A total of 384 couples aged 35-40 years were recruited. The patients were assigned to three groups: NICS, TE-PGT, and intracytoplasmic sperm injection (ICSI). All patients received frozen single blastocyst transfer. Patients in the NICS and TE-PGT groups underwent aneuploidy screening. RESULTS: When compared to the ICSI group, the CLBR was significantly higher in the NICS and TE-PGT groups (27.9% vs. 44.9% vs. 51.0%, p = 0.003 for NICS vs. ICSI, p < 0.001 for TE-PGT vs. ICSI). There were no significant differences in the clinical outcomes between the NICS and TE-PGT groups. Adjusting for confounding factors, the NICS and TE-PGT groups still showed a higher CLBR than the ICSI group (adjusted odds ratio (OR) 3.847, 95% confidence interval (CI) 1.939 to 7.634; adjusted OR 3.795, 95% CI 1.981 to 7.270). Additionally, the cumulative pregnancy loss rates of the NICS and TE-PGT groups were significantly lower than that of the ICSI group (adjusted OR 0.277, 95% CI 0.087 to 0.885; adjusted OR 0.182, 95% CI 0.048 to 0.693). There was no significant difference in the birth weights of the three groups (p = 0.108). CONCLUSIONS: In women 35-40 years old, the CLBR can be increased by selecting euploid embryos using NICS and TE-PGT. For elderly women at high risk of embryonic aneuploidy, NICS, characterized by its safety and non-invasive nature, may emerge as an alternative option for preimplantation genetic testing.

A rapid and simple sterility test method based on solid culture medium containing blood.

Current sterility test performed for most biological products takes 14 days. We evaluated solid medium, containing 5% blood for use in the membrane filtration (MF) and direct inoculation (DI) sterility test. Representative microorganisms prepared in a sample matrix at approximately 0.1, 1, 10 and 100 colony forming units were tested for growth by compendial MF sterility test using fluid thioglycolate medium and tryptic soy broth and also on the Schaedler blood agar (SBA). Sterility test performed on SBA was significantly more sensitive and faster in detecting various microorganisms than the compendial method, particularly for sample matrix containing 0.01% thimerosal (p < 0.05). SBA detected all microorganisms within 7 days. To implement solid medium in the DI sterility test, multiple BA plates were inoculated with the sample. All representative microorganisms were detected within 5 days. The sterility test using solid medium required 3 different incubation conditions, 30-35 °C aerobically and anaerobically to detect bacteria, and at 20-25 °C aerobically to detect mold and yeast. To eliminate aerobic incubation of solid medium at 20-25 °C, we evaluated representative species of mold and yeast for their growth at 30-35 °C and 20-25 °C in the sterility test performed on solid medium. Penicillium chrysogenum could not be detected at 30-35 °C consistently within 7 days. Sterility test performed on solid medium without any additional technology could be completed in 7 days, as compared to the 14 days required for the current compendial method.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Differential expression of tsRNAs and miRNAs in embryo culture medium: potential impact on embryo implantation.

PURPOSE: Can small RNA derived from embryos in conditioned embryo culture medium (ECM) influence embryo implantation? METHODS: We employed small RNA sequencing to investigate the expression profiles of transfer RNA-derived small RNA (tsRNA) and microRNA (miRNA) in ECM from high-quality and low-quality embryos. Quantitative real-time PCR was employed to validate the findings of small RNA sequencing. Additionally, we conducted bioinformatics analysis to predict the potential functions of these small RNAs in embryo implantation. To establish the role of tiRNA-1:35-Leu-TAG-2 in embryonic trophoblast cell adhesion, we utilized co-culture systems involving JAR and Ishikawa cells. RESULTS: Our analysis revealed upregulation of nine tsRNAs and four miRNAs in ECM derived from high-quality embryos, whereas 37 tsRNAs and 12 miRNAs exhibited upregulation in ECM from low-quality embryos. The bioinformatics analysis of tsRNA, miRNA, and mRNA pathways indicated that their respective target genes may play pivotal roles in both embryo development and endometrial receptivity. Utilizing tiRNA mimics, we demonstrated that the prominently expressed tiRNA-1:35-Leu-TAG-2 in the low-quality ECM group can be internalized by Ishikawa cells. Notably, transfection of tiRNA-1:35-Leu-TAG-2 into Ishikawa cells reduced the attachment rate of JAR spheroids. CONCLUSION: Our investigation uncovers significant variation in the expression profiles of tsRNAs and miRNAs between ECM derived from high- and low-quality embryos. Intriguingly, the release of tiRNA-1:35-Leu-TAG-2 by low-quality embryos detrimentally affects embryo implantation and endometrial receptivity. These findings provide fresh insights into understanding the molecular foundations of embryo-endometrial communication.

Do IVF culture conditions have an impact on neonatal outcomes? A systematic review and meta-analysis.

PURPOSE: Are embryo culture conditions, including type of incubator, oxygen tension, and culture media, associated with obstetric or neonatal complications following in vitro fertilization (IVF)? METHODS: A systematic search of MEDLINE, EMBASE, and Cochrane Library was performed from January 01, 2008, until October 31, 2021. The studies reporting quantitative data on at least one of the primary outcomes (birthweight and preterm birth) for the exposure group and the control group were included. For oxygen tension, independent meta-analysis was performed using Review Manager, comparing hypoxia/normoxia. For culture media, a network meta-analysis was carried out using R software, allowing the inclusion of articles comparing two or more culture media. RESULTS: After reviewing 182 records, 39 full-text articles were assessed for eligibility. A total of 28 studies were kept for review. Meta-analysis about the impact of incubator type on perinatal outcomes could not be carried out because of a limited number of studies. For oxygen tension, three studies were included. The pairwise meta-analysis comparing hypoxia/normoxia did not show any statistical difference for birthweight and gestational age at birth. For culture media, 18 studies were included. The network meta-analysis failed to reveal any significant impact of different culture media on birthweight or preterm birth. CONCLUSION: No difference was observed for neonatal outcomes according to the embryo culture conditions evaluated in this review. Further research is needed about the safety of IVF culture conditions as far as future children's health is concerned.

Chemical gasification: An alternative approach to in vitro maturation of bovine oocytes.

This study aimed to evaluate the effect of chemical gasification and HEPES as alternative systems to pH control during in vitro maturation on bovine oocytes competence. Groups of 20 bovine cumulus oocytes complexes (COCs) were randomly distributed and cultured for 24 h in one of the following experimental groups: (i) chemical reaction (ChRG) system: CO2 generated from sodium bicarbonate and citric acid reaction (ii) culture media TCM-HEPES (HEPES-G); and (iii) control group (CNTG) in conventional incubator. After in vitro maturation (IVM), the COCs were in vitro fertilized (IVF), and in vitro cultivated (IVC) in a conventional incubator. We evaluated oocyte nuclear maturation, cleavage and blastocyst rates, in addition to the relative mRNA expression of BAX, BMP-15, AREG and EREG genes in oocytes and cumulus cells. The proportion of oocytes in metaphase II was higher in CNTG and ChRG (77.57% and 77.06%) than in the HEPES-G (65.32%; p = .0408 and .0492, respectively). The blastocyst production was similar between CNTG and ChRG (26.20% and 28.47%; p = .4232) and lower (p = .001) in the HEPES-G (18.71%). The relative mRNA expression of BAX gene in cumulus cells was significantly higher (p = .0190) in the HEPES-G compared to the CNTG. Additionally, the relative mRNA expression of BMP-15 gene was lower (p = .03) in oocytes from HEPES-G compared to the CNTG. In conclusion, inadequate atmosphere control has a detrimental effect on oocyte maturation. Yet, the use of chemical gasification can be an efficient alternative to bovine COCs cultivation.

Development of porcine embryos cultured in media irradiated with ultraviolet-C.

Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.

Upcycling food waste as a low-cost cultivation medium for Chlorella sp. microalgae.

BACKGROUND: Global food loss and waste have raised environmental concerns regarding the generation of greenhouse gases (e.g., carbon dioxide and methane gas), which directly contribute to climate change. To address these concerns, the present research aims to upcycle food waste into an alternative culture medium for the cultivation of microalgae. Various parameters including pretreatment of food waste (i.e., autoclave and non-autoclave), concentration of food waste culture medium (i.e., 10%, 30%, 50%, 70%, 90% and 100%), harvesting efficiency and biochemical compounds of Chlorella sp. microalgae were carried out. RESULTS: Based on the preliminary findings, the highest biomass concentration obtained from 10% food waste culture medium in the autoclave for Chlorella sp., including strains FSP-E, ESP-31 and CY-1, were 2.869 ± 0.022, 2.385 ± 0.018 and 0.985 ± 0.0026 g L-1, respectively. Since Chlorella vulgaris FSP-E exhibited the highest biomass concentration, this microalgal strain was selected to examine the subsequent parameters. Cultivation of C. vulgaris FSP-E in 100FW achieves a biomass concentration of 4.465 ± 0.008 g L-1 with biochemical compounds of 6.94 ± 1.396, 248.24 ± 0.976 and 406.23 ± 0.593 mg g-1 for lipids, carbohydrates and proteins, respectively. CONCLUSION: This study shows that using food waste as an alternative culture medium for C. vulgaris FSP-E can achieve substantial biomass productivity and biochemical content. This research work would contribute to the concept of net zero emission and transitioning toward a circular bioeconomy by upcycling food waste as an alternative culture medium for the cultivation of microalgae. © 2024 Society of Chemical Industry.

[Thoughts on Evaluation of Culture Medium for Human Assisted Reproduction Technology].

As the direct microenvironment of assisted reproductive technology, the evaluation of culture medium for human assisted reproduction technology is particularly important. This paper discussed the classification management, technical review points, clinical evaluation and review thinking, focusing on the basic requirements, raw material control, biological evaluation, sterilization process of culture medium for human assisted reproduction technology, combined with some material limit, new added material and quality system control to thoroughly assess management risk of the whole life cycle of culture medium for human assisted reproduction technology.

Physiologically relevant culture medium Plasmax improves human placental trophoblast stem cell function.

Human trophoblast cultures provide powerful tools to model key processes of placental development. In vitro trophoblast studies to date have relied on commercial media that contains nonphysiological levels of nutrients, and the impact of these conditions on trophoblast metabolism and function is unknown. Here, we show that the physiological medium (Plasmax) with nutrient and metabolite concentrations recapitulating human plasma improves human trophoblast stem cell (hTSC) proliferation and differentiation compared with standard medium (DMEM-F12). hTSCs cultured in Plasmax-based medium also show altered glycolytic and mitochondrial metabolism, as well as reduced S-adenosylmethionine/S-adenosyl-homocysteine ratio compared with DMEM-F12-based medium. These findings demonstrate the importance of the nutritional environment for phenotyping cultured human trophoblasts.

Medium supplementation with human, but not fetal calf serum facilitates endocytosis of PLGA nanoparticles by human primary B-lymphocytes via complement opsonization.

The "biological identity" of nanoparticles (NPs) is governed by a shell consisting of various biomolecules that is formed upon exposure to biological media, the so-called biomolecule corona. Consequently, supplementation of cell culture media with e.g. different sera is likely to affect interactions between cells and NPs ex-vivo, especially endocytosis. We aimed to investigate the differential impact of human and fetal-bovine serum on the endocytosis of poly (lactic-co-glycolic acid) NPs by human peripheral blood mononuclear cells via flow cytometry. Furthermore, we employed different methods to inhibit endocytosis, providing mechanistic insights. The resulting biomolecule corona was characterized via denaturing gel electrophoresis. We found profound differences between human and fetal bovine serum regarding the endocytosis of fluorescently labeled PLGA nanoparticles by different classes of human leukocytes. Uptake by B-lymphocytes was particularly sensitive. We further present evidence, that these effects are mediated by a biomolecule corona. We demonstrate to our knowledge for the first time that the complement is an important contributor to the endocytosis of non-surface-engineered PLGA-nanoparticles prepared via emulsion solvent evaporation by human immune cells. Our data demonstrates that results obtained with xenogeneic culture supplements such as fetal bovine serum may have to be interpreted with caution.