Cytotechnology最新文献

Adipose-derived stem cells (ASCs) and their small extracellular vesicles (sEVs) hold significant potential for regenerative medicine due to their tissue repair capabilities. The microRNA (miRNA) content in sEVs varies depending on ASC status; however, the effects of aging and cell passage on miRNA profiles remain unclear. In this study, we examined the effects of donor age and cell expansion on ASC characteristics and transcriptome using ASCs obtained from three young and three old donors. Cell expansion significantly impaired stem cell properties, notably reducing proliferation and differentiation capacities. In contrast, donor age had minimal effects on ASCs. RNA sequencing (RNA-seq) revealed differences in gene expression related to stemness, phagocytosis, and metabolic processes influenced by cell expansion. To investigate miRNA variability, we performed small RNA-seq on sEVs collected from ASCs of all six donors. The miRNA profiles were influenced by donor age and cell passage. Interestingly, functional enrichment analysis indicated that advanced donor age and increased cell passage may enhance the production of miRNAs associated with organ development through various pathways. These findings suggest that donor age and cell expansion differentially influence ASC characteristics and sEV miRNA content, highlighting the need for disease-specific conditioning of ASCs to optimize the therapeutic effects of sEVs in clinical applications.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00675-6.

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) can be isolated from umbilical cords which is abundant and easy to obtain. Due to their potent immunosuppressive properties, multilineage differentiation potential, and lack of ethical issues, WJ-MSCs are considered a promising candidate for therapeutic applications. However, large-scale in vitro expansion is necessary to obtain enough cells for therapeutic purposes. Therefore, this study aimed to optimize cell culture conditions and determine the characteristics of expanded WJ-MSCs. WJ-MSCs were seeded in 6-well plates at a density of 5000 cells/cm² and cultured with different mediums, including DMEM-LG+10% FBS, DMEM-LG+10% HPL, serum-free commercial medium 1, serum-free GMP grade commercial medium 2, and HPL supplemented commercial medium 3. The cell morphology and growth kinetics were compared, and the three most suitable mediums were selected for further experiments. WJ-MSCs were then cultured in the selected mediums at seeding densities ranging from 1000 to 5000 cells/cm², and cell growth kinetics were analysed. WJ-MSCs cultured in the selected mediums were characterized by their immunophenotype, tri-lineage differentiation potential and immunosuppression property. WJ-MSCs cultured with DMEM-LG+10% HPL, commercial medium 1 and commercial medium 2 showed smaller size, significantly higher cell yield, and shorter population doubling time than those cultured in other mediums. Hence, these three mediums were selected for further experiments. Only DMEM-LG + 10% HPL medium maintained high cell yields (1.48 ± 0.14 × 10⁶ with bFGF and 1.56 ± 0.17 × 10⁶ without bFGF) at the lowest seeding density tested. However, WJ-MSCs cultured in all three mediums expressed the MSC surface markers, were able to suppress PBMC proliferation, and could differentiate into adipogenic, chondrogenic and osteogenic lineages. In summary, DMEM-LG+10% HPL is the best medium for WJ-MSC expansion, as it provides the highest cell yield without compromising cell characteristics and functionality. The potential of this medium for large-scale expansion using a bioreactor or multilayered flask should be investigated in the future.

Culturing fish myogenic cells in vitro holds significant potential to revolutionize aquaculture practices and support sustainable food production. However, advancement in in vitro culture technologies for skeletal muscle-derived myogenic cells have predominantly focused on mammals, with limited studies on fish. Scaffold-based three-dimensional (3D) culture systems for fish myogenic cells remain underexplored, highlighting a critical research gap compared to mammalian systems. This study evaluated the effects of scaffold composition and manufacturing methods on cellular growth in the 3D culture of black sea bream (Acanthopagrus schlegelii) myogenic cells. Scaffolds were manufactured using three natural polymers: black sea bream-derived extracellular matrix (ECM), sodium alginate, and gelatin. Two scaffold types were tested: "cell-laden scaffolds" prepared by mixing cells into the pre-scaffold solution followed by gelation, and "cell-seeding scaffolds" produced by freezing, gelation, and lyophilization before cell inoculation. Scaffold characteristics, including pore size, porosity, swelling ratio, and degradation rate, were assessed. Cell-seeding scaffolds exhibited relatively larger pore size, higher porosity, and higher degradation rate, while cell-laden scaffolds had higher swelling ratios. When black sea bream myogenic cells were cultured in these scaffolds, cell-seeding scaffolds supported cellular growth, particularly when composed of 3% sodium alginate and 4% gelatin with any concentration of ECM. In contrast, cell-laden scaffolds did not support cellular growth regardless of their composition. These findings provide fundamental insights for optimizing scaffold properties to develop more optimized conditions for 3D culture of fish muscle lineage cells.

The development of new compounds or materials for medicine suggests a study of their cytotoxicity. The effect of a material on cell viability can be evaluated by several methods based on DNA content, DNA synthesis, plasma membrane integrity, cellular enzyme activity, cellular reducing potential, and ATP level. Sometimes it is impossible to apply widely used commercially available reagents, e.g., when cells are cultured on materials, that interfere with the chemicals used or resulting in the course of enzymatic reaction. Here, we offer a method for monitoring the viability of cells proliferating on different supports in vitro. The method is based on the measurement of lactate dehydrogenase (LDH) activity in cellular lysates. After cells were lysed in 1% Nonidet P40 and supernatants were transferred into fresh tubes, LDH activity was measured in the supernatants using colorimetric method. The usefulness of the test was studied using human cervical adenocarcinoma HeLa cells and human gingival fibroblasts cultivated on different materials, including activated carbon-loaded scaffolds. The comparison with widely used AlamarBlue® assay confirms the LDH-based method as an appropriate alternative for measuring the living cell number in vitro in a quick, simple, and cost-effective manner when widespread methods for the evaluation of cell viability could not be used.

MicroRNA profiling in human cartilage is necessary for chondrogenesis. The study aimed to compare microRNA 127-5p (miR-127-5p) and TGF-β signaling pathway gene expressions of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and synovial fluid-derived stem cells (hSF-MSCs) after induced chondrogenesis. MSCs induced into chondrogenic differentiation. Alcian Blue and Safranin O staining were performed to determine chondrogenic differentiation. The RT-qPCR determined the expression levels of miR-127-5p and TGF-β signaling pathway genes. miR-127-5p expression was significantly higher in chondrogenic differentiated hSF-MSCs (dhSF-MSCs) (p < 0.05). TGF-β, SMAD2, and SMAD3 expressions were substantially higher in dhSF-MSCs (all p < 0.001), while SMAD4, and ACAN expressions were downregulated (all p < 0.001). No difference was detected between COL1A2 expression levels. This study suggests that miR-127-5p derived from hSF-MSCs may regulate chondrogenesis, thereby inducing the TGF-β pathway activation, and also presents, for the first time, a comparative analysis of the expression of miR-127-5p and the TGF-β signaling pathway genes of hSF-MSCs and hAT-MSCs concerning differences in chondrogenic potential.

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Osteoarthritis is a degenerative disease of cartilage, and exosome derived from mesenchymal stem cells (MSCs) are considered promising for treating inflammatory musculoskeletal disorders, although their mechanisms are not fully understood. This study aimed to investigate the effects of exosomes derived from canine bone marrow mesenchymal stem cells (cBMSCs-Exos) on the expression of inflammatory factors and genes related cartilage matrix metabolism in IL-1β-induced canine chondrocytes. Canine BMSCs were isolated and characterized for surface markers and trilineage differentiation. Exosomes were then extracted and performed surface labeling detection. Canine chondrocytes were exposed to IL-1β to mimic osteoarthritis in vitro. Subsequently, the chondrocytes were treated with exosomes from BMSCs, and the expression levels of related genes and IL-6 protein were assessed. The mesenchymal stem cells isolated from bone marrow and cultured exhibited positive CD44 and CD90, negative expression of CD45 and HLA, and demonstrated potential to differentiate into adipocytes, osteoblasts and chondrocytes. Exosomes from BMSCs exhibited positivity expression of CD9, CD63 and CD81. Treatment with exosomes significantly reduced IL-6 and TNF-α mRNA levels induced by IL-1β, as well as IL-6 protein expression. Additionally, a significant decrease was observed in the mRNA levels catabolic marker genes MMP-13, ADAMTS-5, and COX2. Conversely, there was a significant increase in the mRNA levels of anti-inflammatory cytokines IL-4, IL-10, and anabolic marker genes, such as COL2A1, ACAN, and SOX9. cBMSCs-Exos play a vital role in cartilage protection by suppressing the expression of pro-inflammatory and anabolic genes while simultaneously enhancing the expression of genes involved in synthesis metabolism.

Osimertinib has been demonstrated to be effective for improving the prognosis of patients with epidermal growth factor receptor mutation-positive lung cancer. However, osimertinib resistance inevitably emerges throughout the treatment course. This study explored the function and mechanism of long noncoding RNA LINC01278 in osimertinib-resistant NSCLC cells. Osimertinib-resistant non-small cell lung cancer (NSCLC) cells were established by treating PC9 and HCC827 cells with increasing doses of osimertinib for over 6 months. LINC01278 expression in parental and drug-resistant cells (PC9-OR and HCC827-OR) was measured by polymerase chain reaction. Cell counting kit 8 assays were used to examine cell viability and half-maximal inhibitory concentration values. The effects of LINC01278 knockdown on cell proliferation and apoptosis were measured by colony formation assays and flow cytometry. A luciferase reporter assay was performed to verify the interaction between LINC01278 and miR-324-3p or the binding ability between miR-324-3p and ZFX. Protein levels of ZFX and apoptotic markers in NSCLC cells were measured by western blotting. As shown by experimental results, LINC01278 was highly expressed in osimertinib-resistant NSCLC cells compared to its expression in parental cells. The silencing of LINC01278 improved the sensitivity of drug-resistant cells towards osimertinib. LINC1278 depletion inhibited osimertinib-resistant cell proliferation while promoting cell apoptosis. LINC01278 interacted with miR-324-3p to regulate ZFX expression. ZFX could be targeted by miR-324-3p in PC9-OR and HCC827-OR cells. ZFX overexpression counteracted the suppressive impact of LINC01278 silencing on the malignant behavior of PC9-OR and HCC827-OR cells. In conclusion, LINC01278 knockdown alleviates osimertinib resistance of NSCLC cells by regulating downstream miR-324-3p and ZFX.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00673-8.

Long non-coding RNA LINC01214 is reported to be up-regulated in non-small cell lung cancer (NSCLC), however, its function in NSCLC has not been elucidated yet. In our study, we verified that LINC01214 was aberrantly higher in the tumor tissues and cell lines than that in the normal controls, and was relevant to the severity and prognosis of NSCLC through using real-time quantitative PCR. Then, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay and flow cytometry illustrated that knocking down LINC01214 restrained cell proliferation and promoted apoptosis in A549 and H1299 cells. Additionally, western blot results confirmed that LINC01214 silence reduced the protein expression of CDK2, CDK6, CyclinD1 and Bcl2, but increased the protein expression of Bax and Caspase-3. Of note, compared to normal cells, NSCLC cells had higher enrichment level of N6-methyladenosine (m6A) modification of LINC01214, while reducing m6A modification of LINC01214 weakened the stability of LINC01214 and diminished its level in A549 and H1299 through down-regulating methyltransferase METTL3 or overexpressing demethylase ALKBH5. Subsequently, molecular experiments proved that LINC01214 acted as a sponge for miR-195-5p to elevate ROCK1 expression in NSCLC. Furthermore, data from functional recovery experiments showed that elevating miR-195-5p also exerted tumor-suppressive effects in NSCLC; meanwhile, the effects were reversed by overexpressing ROCK1 or inhibiting miR-195-5p. In short, m6A modification-mediated up-regulation of LINC01214 advances cell proliferation and tumorigenesis to promote NSCLC progression through inhibiting miR-195-5p to up-regulate ROCK1.

β-lactoglobulin (BLG), a major protein in whey, was conjugated with high methoxy pectin (HMP) by the Maillard reaction to improve emulsifying property and reduce allergenicity of BLG. The Maillard reaction was carried out at a weight ratio BLG: HMP = 1:4.15 and 1:6 at 60 °C at a relative humidity of 79% for 5 days. Crude conjugates were purified by dialysis and anion exchange chromatography. These two conjugates were named Conj. LS and Conj. HS, respectively. The results of SDS-PAGE showed that conjugates of various molecular weights were generated, and the weight ratios of protein to saccharide of Conj. LS and Conj. HS were 1:2.15 and 1:6.44 respectively, and the degree of reduction of free amino groups was 6.1 and 6.7 respectively. Emulsifying property of BLG was significantly improved by both conjugation. Both conjugates showed excellent emulsifying property in the acidic pH and in the presence of NaCl. Conjugation with HMP significantly reduced immunogenicity, which was more pronounced in conj. HS. Conjugation with HMP was considered to be an effective method to improve the functionality of BLG. Conjugation method used in this study is a safe method that is considered to be very valuable for food processing.

Despite the importance of finding replacements for fetal bovine serum (FBS), very few studies have focused on this subject. Historically, the use of animals and their derivatives in growth, reproduction, and physiological studies has raised several concerns. The supplementation of culture media with FBS, also known as fetal calf serum, continues to be widespread, despite its limitations in quality, reproducibility, and implications for animal welfare. Moreover, the presence of counterfeit and illegal products can adversely affect cell cultures and treatments, prompting the search for alternative solutions. To reduce reliance on FBS, various substitutes have been introduced, such as plant-derived proteins, bovine eye fluid, sericin protein, human platelet lysate, and inactivated coelomic fluid, which can provide roles similar to that of FBS. Therefore, it is essential to develop serum-free and animal supplement-free environments suitable for therapeutic and clinical applications, tailored to the specific needs of different cell types. Among the alternatives, plant-based options have gained attention as sustainable and ethical solutions. These include plant-derived peptones from sources like soy and wheat, which are rich in amino acids and peptides essential for mammalian cell growth, as well as plant protein hydrolysates from beans and peas that serve as sources of amino acids and growth factors. Plant extracts, especially from soy and various seeds, contain necessary proteins and growth factors, while phytohormones such as cytokinins and plant polysaccharides can help regulate cell growth. While these alternatives offer benefits like reduced costs and lower risks of disease transmission, further research is necessary to refine and align them with the specific requirements of diverse cell types.

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