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The study was designed to monitor the cleavage rate (CR) and in-vitro cultivation rate (IVC) after addition of energy sources, non-essential amino acids, and antioxidants to the Synthetic oviductal fluid (SOF) and FertiCult. After in-vitro maturation and in-vitro fertilization, presumptive zygotes were cultured in one of two culture media: FertiCult media and SOF medium, supplemented with pyruvate, glucose, and sodium lactate as energy sources, as well as 10, 20, 250, 500, and 750 mg non-essential amino acids, and antioxidants. All stages of cleavage rate (CR), and in-vitro cultivation rate (IVC) of embryonic development including morula stage (MOR) and blastocyst (BLAS) have been assessed. The findings revealed that there were no significant differences in the CR between the control and other treated groups with sources of energy when added to SOF media (P > 0.05), while there were significant differences (P < 0.05) in the IVC of embryonic development between groups (The percentages of MOR stage in the control, pyruvate, glucose and mixture of source of energy (MIX) were at 50%, 62.5%, 60%, and 63.6%, respectively). The highest percentage of the BLAS was recorded after SOF supplementation with glucose (40%). Similarly, there were no significant differences (P > 0.05) in the CR between control and FertiCult supplemented with sources of energy, while the IVC stages increased significantly (P < 0.05) in the FertiCult media supplemented with glucose, pyruvate, sodium lactate, and MIX. The percentages of the MOR stage in the control, pyruvate, glucose and mix media were at 50%, 55.6%, 55.6%, 54.5%, 57.1% respectively. The lowest percentage of the BLAS was recorded after FertiCult supplementation with pyruvate (11.1%). Replenishing the SOF maturation media with 20 mg of non-essential amino acids significantly (P < 0.05) enhanced the MOR stage (100%). There was also an improvement in the development of BLAS stage, where it reached 31.2% and 47.4% in the SOF maturation media supplemented with 10, and 750 mg non-essential amino acids, respectively. There were no significant differences (P > 0.05) in neither CR nor IVC between control and FertiCult supplemented with antioxidants. There were significant differences (P < 0.05) in the MOR stages (control, 42.9% & treated, 57.9%) and BLAS stages (control, 21.4% & treated, 42.1%) in antioxidant supplemented SOF maturation media compared to control. In conclusion, supplementation of SOF cultivation medium with energy sources, 20 mg of non-essential amino acids and antioxidant addition may improve the cleavage rate (CR) and in vitro cultivation rate (IVC) of buffalos' embryonic development., Competing Interests: Declarations Ethics approval and consent to participate The Department of Theriogenology, Faculty of Veterinary Medicine at Aswan University in Egypt discussed and approved this study. The authors certify that they have followed the journal’s ethical principles, which are listed on the author guidelines page. The manner in which this investigation was carried out the Ethical Committee of the Faculty of Veterinary Medicine, with authorization number 06/2023/0095, ensured that institutional and national guidelines for the care and use of animals were adhered to in accordance with OIE standards. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors have read and agreed that this version of the manuscript could be published. Study approval and animal Accordance Statement This study was conducted in accordance with Institutional and National Guidelines for the care and use of animals were followed according to the OIE standards, the Ethical Committee of the Faculty of Veterinary Medicine, with the permission number (06/2023/0095)., (© 2024. The Author(s).)

Botryococcus braunii is a colonial alga recognized for its slow growth but high hydrocarbon accumulation. Although using genetic engineering to increase the growth rate and hydrocarbon yield of B. braunii is desirable, the presence of an extracellular matrix (ECM) significantly hinders the emergence of a homogeneous colony from a single DNA-transformed cell. Previously, we developed a method to isolate single cells without ECM from colonies. However, following the isolation of single cells, several months are required to regenerate colonies with a sufficient cell mass for subsequent analysis. To shorten the colony regeneration period, we investigated basal media and medium components, along with growth-promoting additives, in a series of single-factor experiments and optimized the concentrations of the medium constituents via response surface methodology (RSM). The results of the single-factor experiments revealed that the nitrogen source (a mixture of NaNO 3 and NH 4 NO 3 ), 1-naphthylacetic acid (NAA) and Fe(III)-citrate significantly increased the growth of B. braunii single cells into colonies. The optimal medium composition identified by RSM included 151.6 mg/L nitrogen source, 2.419 mg/L NAA and 15.3 mg/L Fe(III)-citrate. Verification experiments showed that the optimized medium resulted in a 1.75-fold increase in colony size compared with that of colonies grown in nonoptimized AF6 medium. This is the first report of the optimal medium composition for the regeneration of B. braunii colonies from single cells., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Takashi Ohtsuki reports financial support was provided by University of Yamanashi. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall in vitro maturation efficiency and early embryonic stages., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Mammalian embryos often suffer from oxidative stress in vitro, as the oxygen in the atmosphere is higher than that in the oviductal environment. Vitamin C (Vc) has been proven to enhance early embryonic development in vitro , but the underlying mechanism remains unclear. In this study, we investigated the pathways of action by which Vc promotes the in vitro development of porcine embryos. Comparative analysis of in vitro and in vivo gene expression profiles of morula found that most of the differentially expressed genes were enriched in pathways related to mitochondrial function. The addition of 12.5 μg/mL Vc to the culture medium significantly increased blastocyst production in a dose- and duration-dependent manner. Moreover, ROS levels were significantly higher in embryos cultured in the air (21% oxygen) than cultured in a hypoxic condition (5% oxygen) and were reduced by Vc supplementation. Vc also significantly increased the mitochondrial membrane potential levels and the expression levels of mitochondrial function-related genes ( MFN1 and OPA1 ) and TCA cycle-related genes ( PDHA1 and OGDH ) in embryos cultured in vitro . These results suggest that the addition of Vc to the in vitro culture medium can increase the developmental potential and improve the mitochondrial function of early porcine embryos.

Considering that follicular development is an energy-dependent process, supplementation of the culture medium with energy substrates, such as lactose, would improve follicle viability and growth. Thus, the aim of this study was to evaluate the effect of lactose on morphology, development, glutathione (GSH) concentration, mitochondrial activity, DNA fragmentation, and meiotic resumption of oocytes from sheep secondary follicles cultured in vitro. Secondary follicles were isolated from the cortex of ovine ovaries and cultured individually for 18 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium and ascorbic acid (control medium: α-MEM + ) or in α-MEM + plus different concentrations of lactose (0.025, 0.05 and 0.1 M). After culture, some of the oocytes were subjected to TUNEL assay and in vitro maturation (IVM). Follicular morphology, glutathione (GSH) concentration and mitochondrial activity were evaluated at the end of the culture. At the day 18, the percentage of morphologically normal follicles was greater (P<0.05) in the treatment of 0.025 M lactose (92.5 %) compared to the control group (75.55 %). In addition, GSH concentrations increased (P<0.05) in treatment containing 0.025 M lactose compared to the other treatments. Furthermore, oocytes cultured in 0.025 M lactose had greater (P<0.05) mitochondrial activity levels than in α-MEM+ and 0.1 M lactose. The group α-MEM + presented a increase of TUNEL-positive oocytes (35.09 %) compared to 0.025 lactose (9.09 %). The percentage of meiotic resumption was greater (P<0.05) in oocytes from secondary follicles cultured in 0.025 M lactose (54.5 %) than in α-MEM + (45.5 %). In conclusion, 0.025 M lactose improved survival, GSH and active mitochondria levels and meiotic resumption of oocytes from in vitro cultured secondary follicles. Supplementation of the culture medium of preantral follicles with lactose can gradually provide energy to follicular cells, potentially enhancing the production of viable oocytes for biotechniques such as IVM and in vitro fertilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

WNT3A is an intestinal ligand triggering the Wnt/β-catenin (Wnt) pathway, which can be enhanced by R-spondin 1 (RSPO1) through the RSPO1-LGR axis or antagonized by the adenomatous polyposis coli (APC) protein supporting β-catenin-degradation. Wnt interplays with several pathways including PI3K/mTOR (mTOR). In this study, we evaluated the influence of WNT3A-commercial and home-made culture media and RSPO1 protein on the Wnt and mTOR interplay in non-APC and APC-mutated intestinal patient-derived organoids (PDOs). Normal mucosa (NM) of sporadic CRC and FAP PDOs were cultured with: WNT3A-lacking/containing commercial (A/A+B) or home-made (BASAL/WNT3A-conditioned medium (CM)±RSPO1) media. In non-APC-mutated-PDOs (CRC-NM), WNT3A-CM, over commercial A+B, strongly activated Wnt-target-genes CCND1 and c-MYC . Most importantly, the addition of RSPO1 to home-made WNT3A-CM or A+B led to the downregulation of the mTOR-downstream-effector phospho-S6 ribosomal protein (p-S6R), highlighting the activation of the RSPO1-pS6R in both non-APC (CRC-NM) and APC-mutated (FAP-NM) PDOs, independently from LGR5 gene expression modulation. Our work demonstrates that home-made WNT3A-CM strongly impacts the crosstalk between Wnt and mTOR over commercial media, and proposes RSPO1 as a key regulator of the RSPO1-p-S6R axis in both non-APC and APC-mutated PDOs. Together, these findings represent an important methodological guide for scientists working in these fields to select the most appropriate intestinal PDO media.

Embryoid bodies (EB) are sensitive to changes in the culture conditions. Recent studies show that the addition of PEG 300 to culture medium affects cell growth and differentiation; however, its effect on the embryoid body is unclear. This study aims to understand the role of PEG 300 in the process of EB formation and germ layer differentiation. EBs formed more efficiently and differentiated toward the mesoderm when cultured in a medium supplemented with appropriate concentrations of PEG 300. The expression of T/Bry, a marker of mesodermal differentiation, increases in EBs in the PEG group, and the expression of TUBB3 generally decreases, showing a quantitative relationship with PEG. Furthermore, further differentiation of PEG-pretreated EB into vascular smooth muscle cells (VSMCs) by directional induction shows that PEG 300-pretreated induced VSMCs have higher expression of phenotypic markers and greater secretory and contractile functions. This study highlights the role of PEG 300 in the culture medium during EB differentiation, which can significantly enhance mesodermal gene expression and the efficiency of subsequent differentiation into smooth muscle cells and other target cells., (© 2024 Wiley‐VCH GmbH.)

Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.

Background: Fetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS)., Methods: Specific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum., Results: Fibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively., Conclusion: This investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted., (© 2024. The Author(s).)

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos., (© 2024. The Author(s).)

Effective lineage-specific differentiation is essential to fulfilling the great potentials of human pluripotent stem cells (hPSCs). In this report, we investigate how modulation of medium pH and associated metabolic changes influence mesendoderm differentiation from hPSCs. We show that daily medium pH fluctuations are critical for the heterogeneity of cell fates in the absence of exogenous inducers. Acidic environment alone leads to cardiomyocyte generation without other signaling modulators. In contrast, medium alkalinization is inhibitory to cardiac fate even in the presence of classic cardiac inducers. We then demonstrate that acidic environment suppresses glycolysis to facilitate cardiac differentiation, while alkaline condition promotes glycolysis and diverts the differentiation toward other cell types. We further show that glycolysis inhibition or AMPK activation can rescue cardiac differentiation under alkalinization, and glycolysis inhibition alone can drive cardiac cell fate. This study highlights that pH changes remodel metabolic patterns and modulate signaling pathways to control cell fate., Competing Interests: Declaration of interests W.L. and G.C. filed a patent application based on cardiomyocyte differentiation that is induced by acidic pH., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We evaluated the influence of different media plus various concentrations of Glial cell line-derived neurotrophic factor (GDNF) during the in vitro culture (IVC) of testicular tissues from prepubertal collared peccary. Testes from 5 individuals were collected, fragmented and cultured for 28 days (34°C and 5% CO 2 ). Culture media were Dulbecco's modified essential medium (DMEM) or stem cell serum free media (StemPro-34™ SFM), both supplemented with various concentrations of GDNF (0, 10, or 20 ng/mL). Fragments were cultured on the flat surface of 0.75% agarose gel and were evaluated every 7 days for fragment area, histomorphology, cellular viability, and proliferative activity. Data were expressed as mean ± standard error and analyzed by Kruskal-Wallis's and Tukey test. Fragments area decreased over the 28 days-culture, regardless of the treatment. For morphology, the StemPro-37 SFM medium plus 10 ng/mL GDNF provided higher scores at all time points in comparison to DMEM using any GDNF concentration (p < .05). After 28 days, similar cellular viability (~70%) was observed in all treatments (p > .05). For proliferating cell nuclear antigen assay, only DMEM plus 10 ng/mL GDNF improved (p < .05) cellular proliferation on Days 14 and 28. Looking at argyrophilic nucleolar organizing regions, after 28 days, there were no differences among treatments regarding cell proliferative capacity for both spermatogonia and Sertoli cells (p > .05). In summary, the DMEM and StemPro-34 SFM are adequate medium for IVC of prepubertal peccary testicular tissue. Supplementation with GDNF, especially at a 10 ng/mL concentration, appears to be essential for the maintenance of cell survival and proliferation., (© 2024 International Federation of Cell Biology.)

The aim of this study was to clarify the transcriptional and metabolic characteristics of C2C12 myoblasts cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20 % chicken serum (CHS) (C2C12-CHS cells) compared with C2C12 myoblasts cultured in DMEM containing 20 % fetal bovine serum (FBS) (C2C12-FBS cells). After 3 days of culture, C2C12-CHS cells showed a marked accumulation of lipid droplets, accompanied by increased expression levels of brown adipocyte-related genes (i.e., Bmp7, Prdm16, Ucp1, Cidea, Pgc1α, Cox7a1, Cox8, and β 3 -adorenoceptor). Furthermore, stimulation of β 3 -adorenoceptor by its selective agonist, mirabegron, increased the mRNA expression of Ucp1 and Pgc1α in C2C12-CHS cells. Wide-targeted metabolomic analysis performed by gas chromatography-tandem mass spectrometry revealed that the metabolic profile of C2C12-CHS cells was obviously different to that of C2C12-FBS cells. Additionally, the metabolomic analysis indicated that β 3 -adrenoceptor stimulation by mirabegron upregulated energy metabolism in C2C12-CHS cells as seen in brown adipocytes. These results suggest that C2C12-CHS cells may differentiate into brown adipocyte-like cells, accompanied by increased functional β 3 -adrenoceptor., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest that would prejudice the impartiality of this scientific work., (Copyright © 2024. Published by Elsevier Inc.)

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) constitute an appealing tool for drug discovery, disease modeling, and cardiotoxicity screening. However, their physiological immaturity, resembling CMs in the late fetal stage, limits their utility. Herein, we have developed a novel, scalable cell culture medium designed to enhance the maturation of hPSC-CMs. This medium facilitates a metabolic shift towards fatty acid utilization and augments mitochondrial function by targeting Acetyl-CoA carboxylase 2 (ACC2) with a specific small molecule inhibitor. Our findings demonstrate that this maturation protocol significantly advances the metabolic, structural, molecular and functional maturity of hPSC-CMs at various stages of differentiation. Furthermore, it enables the creation of cardiac microtissues with superior structural integrity and contractile properties. Notably, hPSC-CMs cultured in this optimized maturation medium display increased accuracy in modeling a hypertrophic cardiac phenotype following acute endothelin-1 induction and show a strong correlation between in vitro and in vivo target engagement in drug screening efforts. This approach holds promise for improving the utility and translatability of hPSC-CMs in cardiac disease modeling and drug discovery.

Metabolite supplementation during in vitro embryo development improves blastocyst quality, however, our understanding of the incorporation of metabolites during in vitro maturation (IVM) is limited. Two important metabolites, follistatin and choline, have beneficial impacts during in vitro culture; however, effects of supplementation during IVM are unknown. The objective of this study was to investigate combining choline and follistatin during IVM on bovine oocytes and subsequent early embryonic development. We hypothesized that supplementation of choline with follistatin would synergistically improve oocyte quality and subsequent early embryonic development. Small follicles were aspirated from slaughterhouse ovaries to obtain cumulus oocyte complexes for IVM with choline (0, 1.3 or 1.8 mM) and follistatin (0 or 10 ng/mL) supplementation in a 3 × 2 design. A subset of oocytes underwent transcriptomic analysis, the remaining oocytes were used for IVF and in vitro culture (IVC). Transcript abundance of CEPT1 tended to be reduced in oocytes supplemented with 1.8 mM choline and follistatin compared to control oocytes ( P = 0.07). Combination of follistatin with 1.8 mM choline supplementation during maturation, tended ( P = 0.08) to reduce CPEB4 in oocytes. In the blastocysts, HDCA8 , NANOG , SAV1 and SOX2 were increased with choline 1.8 mM supplementation without follistatin ( P < 0.05), while HDCA8 and SOX2 were increased when follistatin was incorporated ( P < 0.05). The combination of choline and follistatin during oocyte maturation may provide a beneficial impact on early embryonic development. Further research is warranted to investigate the interaction between these two metabolites during early embryonic development and long-term influence on fetal development.

In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán et al., Theriogenology, 198, 2023 and 231; Oberlender et al., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo et al., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on in vitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 10 6 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The direct antitumor effect of bevacizumab (BEV) has long been debated. Evidence of the direct antitumor activities of drugs are mainly obtained from in vitro experiments, which are greatly affected by experimental conditions. In this study, we evaluated the effect of BEV-containing medium renewal on the results of in vitro cytotoxicity experiments in A549 and U251 cancer cells. We observed starkly different results between the experiments with and without BEV-containing medium renewal. Specifically, BEV inhibited the tumor cell growth in the timely replacement with a BEV-containing medium but promoted tumor cell growth without medium renewal. Meanwhile, compared with the control, a significant basic fibroblast growth factor (bFGF) accumulation in the supernatant was observed in the group without medium renewal but none in that with replaced medium. Furthermore, bFGF neutralization partially reversed the pro-proliferative effect of BEV in the medium non-renewed group, while exogenous bFGF attenuated the tumor cell growth inhibition of BEV in the medium-renewed group. Our data explain the controversy over the direct antitumor effect of BEV in different studies from the perspective of the compensatory autocrine cytokines in tumor cells., (© 2024. Pleiades Publishing, Ltd.)

Background: While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified., Methods: This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel ® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture., Results: The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology., Conclusions: This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems., (© 2024. The Author(s).)

Background: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA)., Methods and Results: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 10 9 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells., Conclusions: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration., (© 2024. The Author(s).)

The complexification of in vitro models requires the compatibility of cells with the same medium. Since immune cells are the most sensitive to growth conditions, growing intestinal epithelial cells in their usual medium seems to be necessary. This work was aimed at comparing the sensitivity of these epithelial cells to pro-inflammatory stimuli but also to dietary polyphenols in both DMEM and RPMI-1640 media. Co-cultures of Caco-2 and HT29-MTX cells were grown for 21 days in the two media before their stimulation with a cocktail of TNF-α (20 ng/mL), IL-1β (1 ng/mL), and IFN-γ (10 ng/mL) or with LPS (10 ng/mL) from E. coli (O111:B4). The role of catechins (15 µM), a dietary polyphenol, was evaluated after its incubation with the cells before their stimulation for 6 h. The RPMI-1640 medium did not alter the intensity of the inflammatory response observed with the cytokines. By contrast, LPS failed to stimulate the co-culture in inserts regardless of the medium used. Lastly, catechins were unable to prevent the pro-inflammatory response observed with the cytokines in the two media. The preservation of the response of this model of intestinal epithelium in RPMI-1640 medium is promising when considering its complexification to evaluate the complex cellular crosstalk leading to intestinal homeostasis.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model., (© 2024 Wiley‐VCH GmbH.)

Based on our previous finding that polysaccharide peptide (PSP) has substantial antiviral activity, we cultured strawberry plants infected with strawberry mild yellow edge virus (SMYEV) or strawberry vein banding virus (SVBV) in Murashige and Skoog (MS) media supplemented with PSP to test its ability to eliminate these viruses. PSP not only improved the elimination of SMYEV and SVBV but also promoted the growth and rooting of strawberry plants in tissue culture. On the 45th day, the average height of the 'Ningyu' strawberry plants in the 1-mg/ml PSP treatment group was 1.91 cm, whereas that of the plants in the control group was 1.51 cm. After the same time point, the number of new leaves on the tissue culture media supplemented with 1 mg/ml and 500 μg/ml of PSP and without PSP were 4.92, 4.41, and 3.53, respectively. PSP also promoted strawberry rooting and significantly increased both the length and number of roots. In addition, after treatment with the 1-mg/ml PSP treatment in tissue culture for 45 days followed by meristem-shoot-tip culture, the elimination rates of SMYEV and SVBV in regenerated 'Ningyu' strawberry plants ranged from 60 to 100%. This study investigated the use of the antiviral agent PSP for virus elimination. PSP has a low production cost and thus has great application potential for virus elimination in crop plants., Competing Interests: The author(s) declare no conflict of interest.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Stem cells possess the ability to differentiate into different lineages and the ability to self-renew, thus representing an excellent tool for regenerative medicine. They can be isolated from different tissues, including the adipose tissue. Adipose tissue and human adipose-derived stem cells (hADSCs) are privileged candidates for regenerative medicine procedures or other plastic reconstructive surgeries. The cellular environment is able to influence the fate of stem cells residing in the tissue. In a previous study, we exposed hADSCs to an exhausted medium of a breast cancer cell line (MCF-7) recovered at different days (4, 7, and 10 days). In the same paper, we inferred that the medium was able to influence the behaviour of stem cells. Considering these results, in the present study, we evaluated the expression of the major genes related to adipogenic and osteogenic differentiation. To confirm the gene expression data, oil red and alizarin red colorimetric assays were performed. Lastly, we evaluated the expression of miRNAs influencing the differentiation process and the proliferation rate, maintaining a proliferative state. The data obtained confirmed that cells exposed to the medium maintained a stem and proliferative state that could lead to a risky proliferative phenotype.

Background: In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed., Results: IVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise, secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs., Conclusions: The addition of secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA)., (© 2024. The Author(s).)

Osteochondral allograft transplantation is a successfully proven method to repair articular cartilage defects and prevent the degenerative effects of osteoarthritis. The number of osteochondral transplantations that can be performed each year is limited by availability of donor cartilage tissue and storage time constraints. Osteochondral transplantation success has been linked to high chondrocyte viability of the donor cartilage tissue at the time of implantation. Determining optimal storage conditions for donor cartilage is essential for tissue banks to safely provide quality cartilage tissue. In this study, we compared three tissue/cell media (DMEM/F12, RPMI-1640 and X-VIVO 10) for their ability to maintain chondrocyte viability during hypothermic storage for 28 days. Porcine osteochondral dowels were stored in each media for 28 days and cell viability was assessed every 7 days. Over the 28 day storage period, the chondrocyte viability of dowels stored in DMEM/F12, RPMI-1640, and X-VIVO 10 media all declined in a similar fashion. Our results show that all three media were equivalent in their ability to maintain cell viability of the cartilage tissue and provides rationale for the use of lower cost cell media (DMEM/F12 and RPMI-1640) for hypothermic storage of articular cartilage tissue., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background Aims: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media., Methods: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center., Results: Center methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01)., Conclusions: Local manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H 2 O 2 . The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Introduction: Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated Supernatant Rich in Growth Factors (SRGF) can replace fetal bovine serum (FBS) to expand ASC by a clinical grade compliant protocol. The therapeutic potential of ASC is based also on their homing capacity toward inflammatory/cancer sites: oriented cell migration is a fundamental process in this scenario. We investigated the impact of SRGF on ASC migration properties., Methods: The motility/migration potential of ASC expanded in 5% SRGF was analyzed, in comparison to 10% FBS, by standard wound healing, bidimensional chemotaxis and transwell assays, and by millifluidic transwell tests. Mechanisms involved in the migration process were investigated by transient protein overexpression., Results: In comparison to standard 10% FBS, supplementation of the cell culture medium with 5% SRGF, strongly increased migration properties of ASC along the chemotactic gradient and toward cancer cell derived soluble factors, both in static and millifluidic conditions. We showed that, independently from applied migratory stimulus, SRGF expanded ASC were characterized by far lower expression of α-smooth muscle actin (αSMA), a protein involved in the cell migration machinery. Overexpression of αSMA induced a significant and marked decrease in migration capacity of SRGF expanded ASC., Discussion: In conclusion, 5% SRGF addition in the cell culture medium increases the migration potential of ASC, reasonably through appropriate downregulation of αSMA. Thus, SRGF could potentially improve the therapeutic impact of ASC, both as modulators of the immune microenviroment or as targeted drug delivery vehicles in oncology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Agostini, Vicinanza, Lombardi, Da Ros, Marangon, Massarut, Mazzucato and Durante.)

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Preparing a suitable cell culture medium that supports the biological needs of the growing cells is crucial to enhancing the success rate of any in vitro and in vivo experiments and minimizing undesirable interferences.  Mesenchymal stem cells ( MSCs) which are powerful regenerative stem cells require being grown in proper culture media to preserve their stemness and therapeutic properties. MSCs are usually grown in Dulbecco's Modified Eagle low glucose Medium (DMEM low glucose) which contains 5.6 mmol/L of glucose and is supplemented with Fetal Bovine Serum (FBS), antibiotics, and 2-Mercaptoethanol. The addition of 2-Mercaptoethanol to the cell culture medium was proposed long ago and has continued to be used until now. Despite the positive effects of adding 2-Mercaptoethanol in the cell culture medium, its use is still controversial and needs continuous updates to limit its interference with experimental treatments. Herein, we found that 2-Mercaptoethanol is beneficial to enhancing the proliferation and survival of MSCs at higher passage numbers while its effect is negligible for earlier passages. This concise study provides updates regarding the suitable time to add 2-Mercaptoethanol which can minimize its intermeddling with the experimental design and treatments.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and β-glycerophosphate (βGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 μg.mL -1 /2 mM Asc/βGP combination on day 21 in OSM, and this was supported by Runx2 , Alpl , Bglap , and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

MCF7 cells have been used as an experimental model for breast cancer for decades. Typically, a culture medium is designed to supply cells with the nutrients essential for their continuous proliferation. Each medium has a specific nutritional composition. Therefore, cells cultured in different media may exhibit differences in their metabolism. However, only a few studies have investigated the effects of media on cells. In this study, we compared the effects of Dulbecco's modified Eagle medium (DMEM) and minimum essential medium alpha modification (αMEM) on MCF7 cells. The two media differentially affected the morphology, cell cycle, and proliferation of MCF7 cells, but had no effect on cell death. Replacement of DMEM with αMEM led to a decrease in ATP production and an increase in reactive oxygen species production, but did not affect the cell viability. RNA-sequencing and bioinformatic analyses revealed 721 significantly upregulated and 1247 downregulated genes in cells cultured in αMEM for 48 h compared with that in cells cultured in DMEM. The enriched gene ontology terms were related to mitosis and cell proliferation. Kyoto encyclopedia of genes and genomes analysis revealed cell cycle and DNA replication as the top two significant pathways. MCF7 cells were hypoxic when cultured in αMEM. These results show that the culture medium considerably affects cultured cells. Thus, the stability of the culture system in a study is very important to obtain reliable results., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bovine serum albumin (BSA) plays a crucial role in cell culture media, influencing cellular processes such as proliferation and differentiation. Although it is commonly included in chondrogenic differentiation media, its specific function remains unclear. This study explores the effect of different BSA concentrations on the chondrogenic differentiation of human adipose-derived stromal/stem cells (hASCs). hASC pellets from six donors were cultured under chondrogenic conditions with three BSA concentrations. Surprisingly, a lower BSA concentration led to enhanced chondrogenesis. The degree of this effect was donor-dependent, classifying them into two groups: (1) high responders, forming at least 35% larger, differentiated pellets with low BSA in comparison to high BSA; (2) low responders, which benefitted only slightly from low BSA doses with a decrease in pellet size and marginal differentiation, indicative of low intrinsic differentiation potential. In all cases, increased chondrogenesis was accompanied by hypertrophy under low BSA concentrations. To the best of our knowledge, this is the first study showing improved chondrogenicity and the tendency for hypertrophy with low BSA concentration compared to standard levels. Once the tendency for hypertrophy is understood, the determination of BSA concentration might be used to tune hASC chondrogenic or osteogenic differentiation.

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos., (© 2024. The Author(s).)

Objectives: This study aimed to investigate whether adding tissue samples directly into thioglycolate (TG) broth yielded a greater number of anaerobic organisms than freshly sampled tissue in suspected hip and knee prosthetic joint infections (PJIs)., Patients and Methods: Between January 2017 and December 2020, a total of 90 patients (46 males, 44 females; median age: 71.7 years; range, 50.8 and 87.8 years) who underwent revision hip or knee arthroplasty were included. Intraoperative samples were taken, with five placed in TG broth and five in standard containers (PC) with subsequent aerobic and anaerobic culturing conducted. Demographic and baseline data of the patients were recorded. The primary outcome was positive bacterial growth from a PJI specimen inoculated directly into TG broth at the time of collection or standard PJI specimen processing. Secondary outcomes investigated were the presence of Cutibacterium acnes (C. acnes) and the curative success of revision procedure., Results: A total of 900 samples (450 PC and 450 TG) were taken from 90 revision arthroplasty patients (47 knees and 43 hips). There was no statistically significant difference in the number of positive bacterial growth samples between TG broth and standard processing (p=0.742). This was consistent with subgroup analysis analyzing C. acnes (p=0.666)., Conclusion: In hip and knee arthroplasty, there is no benefit in substituting or adding TG broth as a culture medium to better identify both general bacterial species and C. acnes infections specifically. However, the use of TG may be useful in confirming a true positive result for infection.

The culture of preimplantation embryos in vitro is an important method for human and mouse reproductive technology. This study aims to investigate the influence of different conditions of culture media on the preimplantation stage of mouse embryos cultured in vitro, and monitor the post-implantation development of new mice after embryo transfer to surrogate females. We demonstrated here that mouse embryos cultured in vitro in fresh M16, KSOM, Global, and HTF embryo culture media from one cell to the blastocyst stage and the subsequent embryo transfer to surrogate females are able to proceed through post-implantation development and, after birth, develop into healthy mice. However, culture of embryos in differently aged media shows various (often unpredictable) results. To find the optimal storage conditions of culture media, we suggest that the freezing and long-term storage of these media at - 80°C will not influence the quality of the media. To test this hypothesis, we grew embryos from one cell to blastocysts in vitro in the selected media after thawing and subsequently transferring them to surrogate females. Embryo culture in these four media after thawing does not affect preimplantation and postnatal mouse development. Thus, we have shown that storage of embryo culture media at low temperature (- 80°C) does not impact the quality of the media, and subsequently, it can be used for the culture of embryos for the full preimplantation period, the same as in fresh media., (© 2024. The Society for In Vitro Biology.)

In the quest to unravel the mysteries of neurological diseases, comprehending the underlying mechanisms is supreme. The SH-SY5Y human neuroblastoma cell line serves as a crucial tool in this endeavor; however, the cells are known for its sensitivity and slow proliferation rates. Typically, this cell line is cultured with 10% Fetal Bovine Serum (FBS) supplement. Nu-Serum (NuS), a low-protein alternative to FBS, is promising to advance cell culture practices. Herein, we evaluated the substitution of NuS for FBS to test the hypothesis that an alternative serum supplement can aid and promote SH-SY5Y cell proliferation and differentiation. Our findings revealed that the NuS-supplemented group exhibited a notable increase in adhered cells compared to both the FBS and serum-free (SF) groups. Importantly, cell viability remained high in both sera treated groups, with the NuS-supplemented cells displaying significantly larger cell sizes compared to the SF-treated group. Furthermore, cell proliferation rates were higher in the NuS-treated group, and neuroblast-like morphology was observed earlier than FBS group. Notably, both FBS and NuS supported the differentiation of these cells into mature neurons. Our data supports NuS as an alternative for SH-SY5Y cell culture, with the potential to elevate the quality of research in the neuroscience field., (© 2024. The Author(s).)

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs ( ACTB , EF1A , GAPDH , RPLP0 , and TBP ) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability ( GAPDH being best), albeit divergent from FBS expanded products ( EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A / TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study.

The implantation of good-quality embryos to the receptive endometrium is essential for successful live birth through in vitro fertilization (IVF). The higher the quality of embryos, the higher the live birth rate per cycle, and so efforts have been made to obtain as many high-quality embryos as possible after fertilization. In addition to an effective controlled ovarian stimulation process to obtain high-quality embryos, the composition of the embryo culture medium in direct contact with embryos in vitro is also important. During embryonic development, under the control of female sex hormones, the fallopian tubes and endometrium create a microenvironment that supplies the nutrients and substances necessary for embryos at each stage. During this process, the development of the embryo is finely regulated by signaling molecules, such as growth factors and cytokines secreted from the epithelial cells of the fallopian tube and uterine endometrium. The development of embryo culture media has continued since the first successful human birth through IVF in 1978. However, there are still limitations to mimicking a microenvironment similar to the reproductive organs of women suitable for embryo development in vitro. Efforts have been made to overcome the harsh in vitro culture environment and obtain high-quality embryos by adding various supplements, such as antioxidants and growth factors, to the embryo culture medium. Recently, there has been an increase in the number of studies on the effect of supplementation in different clinical situations such as old age, recurrent implantation failure (RIF), and unexplained infertility; in addition, anticipation of the potential benefits from individuation is rising. This article reviews the effects of representative supplements in culture media on embryo development.

Methodologies for culturing muscle tissue are currently lacking in terms of quality and quantity of mature cells produced. We analyse images from in vitro experiments to quantify the effects of culture media composition on mouse-derived myoblast behaviour and myotube quality. Metrics of early indicators of cell quality were defined. Images of muscle cell differentiation reveal that altering culture media significantly affects quality indicators and myoblast migratory behaviours. To study the effects of early-stage cell behaviours on mature cell quality, metrics drawn from experimental images or inferred by approximate Bayesian computation (ABC) were applied as inputs to an agent-based model (ABM) of skeletal muscle cell differentiation with quality indicator metrics as outputs. Computational modelling was used to inform further in vitro experiments to predict the optimum media composition for culturing muscle cells. Our results suggest that myonuclei production in myotubes is inversely related to early-stage nuclei fusion index and that myonuclei density and spatial distribution are correlated with residence time of fusing myoblasts, the age at which myotube-myotube fusion ends and the repulsion force between myonuclei. Culture media with 5% serum was found to produce the optimum cell quality and to make muscle cells cultured in a neuron differentiation medium viable.

There are many facts about the possible role of gamma-aminobutyric acid (GABA) in the development and differentiation of cells not only in nervous but also in muscle tissue. In the present study, a primary culture of rat skeletal muscle myocytes was used to evaluate the correlation between the content of GABA in the cytoplasm and the processes of myocyte division and their fusion into myotubes. The effect of exogenous GABA on the processes of culture development was also estimated. Since the classical protocol for working with myocyte cultures involves the use of fetal bovine serum (FBS) to stimulate cell division (growth medium) and horse serum (HS) to activate the differentiation process (differentiation medium), the studies were carried out both in the medium with FBS and with HS. It was found that cells grown in medium supplemented with FBS contain more GABA compared to cultures growing in medium supplemented with HS. Addition of exogeneous GABA leads to a decrease in the number of myotubes formed in both media, while the addition of an amino acid to the medium supplemented with HS had a more pronounced inhibitory effect. Thus, we have obtained data indicating that GABA is able to participate in the early stages of skeletal muscle myogenesis by modulating the fusion process., (© 2023 S. Karger AG, Basel.)

Follicle-stimulating hormone (FSH) stimulates the proliferation, survival, and estradiol synthesis of granulosa cells by binding to their G protein-coupled receptors. Although FSH activates sphingosine kinase-1 (SPHK1) to induce sphingosine-1-phosphate (S1P) synthesis, which is required to mediate the proliferative and survival effect of this gonadotrophin, the mechanisms, and the role of S1P in estradiol synthesis have not been reported. This study aimed to evaluate the importance of FSH-induced S1P synthesis as a mediator of the effects of this gonadotrophin on granulosa cell viability and steroidogenesis and to determine if FSH-induced S1P synthesis depends on estradiol, cAMP, PKA, or PKC. To achieve these objectives, we tested the effects of FSH, a sphingosine kinase-1 inhibitor (SKI-178), estradiol and inhibitors of aromatase, cAMP, PKA, and PKC (Formestane, MDL-12330A, H-89 dihydrochloride hydrate and Calphostin C respectively), on granulosa cell viability, S1P and estradiol production, and the mRNA expression of CYP19A1 and STAR in four in vitro culture experiments. The addition of FSH (1 ng/mL) increased (P < 0.05) granulosa cells number and S1P concentration in the culture media. Conversely, the addition of SKI-178 (10 μM) reduced (P < 0.05) S1P concentration negating the effect of FSH on cell viability. Inhibition of PKC and PKA, but not cAMP, reduced (P < 0.05) S1P secretion of FSH treated granulosa cells. It is important to note that the reduction in S1P secretion was strong (49 %) with the use of the PKC inhibitor. The use of formestane (10 μg) did not modify (P > 0.05) S1P secretion in FSH-treated cells; however, the addition of 5 or 10 ng/mL of estradiol increased (P < 0.05) S1P secretion. Finally, FSH increased (P < 0.05) estradiol concentration in the culture media, but this effect was not blocked by the inhibition of S1P synthesis. Similarly, FSH, SKI-178 or their combination did not modify the mRNA expression of CYP19A1 and STAR. In conclusion, S1P synthesis is stimulated FSH in granulosa cells and mediated mainly by PKC. S1P in turn promotes the granulosa cell viability, however, this does not influence estradiol synthesis. Additionally, estradiol synthesis induced by FSH is not essential for S1P synthesis, however high estradiol concentration may stimulate S1P production by granulosa cells., (Copyright © 2023. Published by Elsevier Inc.)

This study proposes the use of vinasse, an inexpensive and readily available waste biopolymer, as a fundamental component of a waste culture medium that can enhance the effectiveness and cost-efficiency of the microbial-induced calcite precipitation (MICP) method for sustainable soil improvement. Vinasse enriched with urea, sodium caseinate, or whey protein concentrate is employed to optimize bacterial growth and urease activity of Sporosarcina pasteurii (S. pasteurii) bacterium. The best culture medium is analyzed using Taguchi design of experiments (TDOE) and statistical analysis, considering the concentration of vinasse and urea as effective parameters during growth time. To test the best culture medium for bio-treated soil, direct shear tests were performed on loose and bio-treated sand. The results demonstrate a substantial cost reduction from $0.455 to $0.005 per liter when using the new culture medium (vinasse and urea) compared to the conventional Nutrient Broth (NB) culture medium. Additionally, the new medium enhances soil shear strength, increasing the friction angle by 2.5 degrees and cohesion to 20.7 kPa compared to the conventional medium. Furthermore, the recycling of vinasse as a waste product can promote the progress of a circular economy and reduce environmental pollution. As ground improvement is essential for many construction projects, especially those that require high shear strength or are built on loose soil, this study provides a promising approach to achieving cost-effective and sustainable soil microbial improvement using enriched vinasse., (© 2023. The Author(s).)

Background: Despite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action., Methods: This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers., Results: Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media., Conclusions: These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release., (© 2023. The Author(s).)

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination., Competing Interests: NO, (Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Traditionally, in vitro oocyte and embryo culture progresses through a series of varying culture medium. To investigate simplifying the in vitro production of bovine cumulus-oocyte complexes (COCs), this study used synthetic oviductal fluid (SOF) supplemented with conjugated linoleic acid (CLA). Special interest was placed on gene expression linked to lipid metabolism and oocyte maturation. COCs were matured in different media: Medium 199 (M199 group), M199 with 100 μM CLA (M199 + CLA group), SOF (SOF group), and SOF with 100 μM CLA (SOF + CLA group). COCs matured with SOF showed a higher relative abundance of mRNA of quality indicators gremlin 1 ( GREM1 ) and prostaglandin-endoperoxide synthase 2 ( PTGS2 ) in oocytes, and GREM1 in cumulus cells compared with in the M199 group. SOF medium COCs had a higher relative abundance of fatty acid desaturase 2 ( FADS2 ) compared with the M199 group, which is essential for lipid metabolism in oocytes. Furthermore, the abundance of stearoyl-coenzyme A desaturase 1 ( SCD1 ) in oocytes matured with SOF was not influenced by the addition of CLA, whereas the relative abundance of SCD1 was reduced in M199 medium with CLA. We concluded that maturation in SOF medium results in a greater abundance of genes linked to quality and lipidic metabolism in oocytes, regardless of the addition of CLA.

The role of microalgae in the production of bioproducts and biofuels, along with their ability to provide a sustainable pathway for wastewater treatment, makes them promising alternatives to conventional processes. Nevertheless, large-scale downstream processing requires an understanding of biomass rheology that needs to be addressed further. This study aimed to characterize microalgal concentrates rheologically in different culture media. The presence of bacteria was quantified by photorespirometry and plate counting techniques. The culture medium was found to significantly influence viscosity, with primary wastewater exhibiting the highest viscosity and seawater plus pig slurry the lowest. The concentration of heterotrophic bacteria was directly related to the viscosity. Extracellular polysaccharides (EPS) in supernatant exhibited an inverse viscosity trend compared to biomass concentrates, with pig slurry cultures having higher concentrations. These findings emphasize the profound influence of culture medium and EPS on the rheology of microalgal biomass, underscoring the need for continued research aimed at facilitating and optimizing large-scale downstream processes within the framework of a circular economy and the attainment of the Sustainable Development Goals (6,8, and 12)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to investigate whether antimicrobial blue light (aBL) can cause the death of Aggregatibacter actinomycetemcomitans (A.a) and to determine the influence of different culture media, specifically brain heart infusion and blood agar, on bacterial survival fraction. An LED emitting at 403 ± 15 nm, with a radiant power of 1W, irradiance of 588.2 mW/cm 2 , and an irradiation time of 0 min, 1 min, 5 min, 10 min, 30 min, and 60 min, was used. The plates were incubated in microaerophilic conditions at 37 °C for 48 h, and the colony-forming units were counted. The photosensitizers were investigated using spectroscopy and fluorescence microscopy. There was no significant difference between the culture media (p > 0.05). However, a statistical reduction in both media was observed at 30 min (1058 J/cm 2 ) (p < 0.05). The findings of this study suggest that aBL has the potential to kill bacteria regardless of the culture media used. Light therapy could be a promising and cost-effective strategy for preventing periodontal disease when used in combination with mechanical plaque control., (© 2023. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)