Trigonella foenum-graecum L. (family Leguminosae) seeds, are typically used as food supplements to increase postnatal lactation. Fenugreek extract displays antioxidative and anti-inflammatory properties, but its mechanisms against skin aging have not been exploited. In this research, we are the first to define an in vitro collagenase inhibitory activity of fenugreek extract (IC50 = 0.57 ± 0.02 mg/mL), which is 2.6 times more potent than vitamin C (IC50 = 1.46 mg/mL). Nanoencapsulation has been applied to improve the extract stability, and subsequently enhanced its bioactivities. Liponiosome encapsulating fenugreek extract (LNF) was prepared using a high-speed homogenizer, resulting in homogeneous spherical nanoparticles with sizes in the range of 174.7 ± 49.2 nm, 0.26 ± 0.04 in PdI, and 46.6 ± 7.4% of entrapment efficiency. LNF formulation significantly facilitated a sustained release and significantly enhanced skin penetration over the extracts, suggesting a potential use of LNF for transdermal delivery. The formulated LNF was highly stable, not toxic to human fibroblast, and was able to enhance cell viability, collagen production, and inhibit MMP1, MMP9, IL-6, and IL-8 secretions compared to the extract in the co-cultured skin model. Therefore, ethanolic fenugreek extract and its developed LNF display molecular mechanisms against skin aging and could potentially be used as an innovative ingredient for the prevention of skin aging.Fenugreek, or  Trigonella foenum-graecum L. (fenugreek), belonging to the family Leguminosae, is a herbaceous plant that typically grows in western Asia, northern India, northern Africa, and the Mediterranean -Leu-Gly-Pro-Ala), direct red 80, picric acid, and dimethyl sulfoxide were purchased from Sigma-Aldrich . Acetonitrile, ethyl acetate, absolute ethanol, formic acid, sodium dehydrate phosphate, disodium hydrogen phosphate, hydrochloric acid, and sodium hydroxide were purchased from Carlo Erba . Commercial grade ethanol was purchased from Italmar Co., Ltd. . Cholesterol was purchased from Cosmeplus Co., Ltd. . Propylene glycol and solubilizer mixture were purchased from S. Tong Chemicals Co., Ltd. . Sorbitan oleate was purchased from Croda Co., Ltd. . Phospholipid (Phospholipon 90G) was purchased from Cargill Siam Ltd. . Tocopherol acetate was purchased from Namsiang Co., Ltd. . The preservative was purchased from Forecus Co., Ltd. . Paraformaldehyde was purchased from Himadia Laboratories  and 3--2,5-diphenyltetrazolium bromide was purchased from Calbiochem .w/w) and it was kept at 4 °C until use.Fenugreek seed powder (500 g) was macerated with 95% ethanol (1:5) for 3 days. The extraction was repeated three times (3 × 2500 mL). The whole extract solution was filtered, with ethanol evaporated under 100 mbar at 40 °C using a rotary evaporator . The obtained extract was the oily yellowish–brown paste with 5% yield (v/v of formic acid solution in ultrapure water (A) and 0.05% v/v of formic acid in acetonitrile (B) was prepared with a flow of 0.25 mL/min in gradient mode: the starting condition was 10% B for 1.0 min, increased to 75% B for 3.5 min, decreased to 10% B for 0.75 min, and held for 0.25 min.Rutin trihydrate, as a standard marker, was used to analyze fenugreek extract. The UHPLC method was developed and validated in terms of linearity, accuracy, precision, and sensitivity ,32 by a 50 values were calculated by plotting a linear regression curve showing sample concentrations on the x-axis and percentage inhibition on the y-axis. The percentage inhibition was calculated using the following equation:A collagenase assay was performed to determine anti-collagenase activity . The mixHuman dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493). Cells were cultivated in Dulbecco’s modified Eagle’s media (DMEM)  supplemented with 10% FBS , 1% penicillin (100 units/mL), and streptomycin (100 µg/mL) . Cells were incubated at 37 °C under a 5% carbon dioxide environment.4 cells/well into 48 well-plates and incubated for 24 h. The cells were then treated with various concentrations of liponiosome without extracts (indicated as blank), liponiosome encapsulating fenugreek extract (LNF), and fenugreek extract for 7 and 14 days, with 0.005% DMSO used as a vehicle control. The media was changed every 2 days. The cells were then washed with PBS and fixed with 100 µL of 4% PFA for 10 min. Cells were washed with PBS (twice) and stained with 100 µL of 0.1% direct red 80 solutions for 10 min. After staining, 0.01 N HCl in 70% ethanol was added to wash the excess dye. Stained collagen was dissolved with 100 µL of 0.5 N NaOH and the absorbance was measured at 540 nm using a microplate reader (Synergy H1 microplate reader). The percentage of collagen content was calculated using the following equation:Human dermal fibroblast cells were seeded at 2.5 × 104 cells/well into 96 well-plates and incubated for 24 h. After incubation, different concentrations (0–1000 µg/mL) of the liponiosomes without extracts (blank), LNF, and fenugreek extract were added and cultivated for 24 h. Then, 100 µL of MTT (1 mg/mL) was added into each well and incubated for 4 h. DMSO was added to dissolve formazan product. The absorbance was measured at 570 nm using a microplate reader (Synergy H1 microplate reader). The percentage of cell viability was calculated using the following equation:Human dermal fibroblast cells were seeded at 2 × 10Pre-emulsification following homogenization was employed for liponiosome formulation. A phospholipid and emulsifier mixture was used to form a spherical lipid bilayer and cholesterol was used to enhance the rigidity of particles . BrieflyTo evaluate the stability of LNF, the particles were kept at 4 °C, 25 °C, and 40 °C for 3 months and the changes in particle size and morphology were examined.Liposome and niosome encapsulation fenugreek extract (LF and NF) were formulated using a similar method as LNF. LF was prepared without the addition of sorbitan oleate into the oil phase, whereas niosome (NF) was prepared without the addition of the phospholipid component. Their physicochemical characterizations were reported in the Encapsulation efficiency (%EE) and bioactive loading (%BL) of LNF were calculated by determining the amount of encapsulated drug using an ultrafiltration technique. The amount of rutin was used as a standard marker. After 20 mg/mL of LNF was dissolved in DI water, it was centrifuged at 80,000 rpm for 4 h. The supernatant was collected and then the unencapsulated rutin was evaluated using an ultra-high-performance liquid chromatography technique (UHPLC) . The % EAn in vitro permeable study of 7 mg/mL of fenugreek extract and LNF was investigated in a Franz diffusion cell system . The syn2. The rutin, fenugreek extract, and LNF were applied onto the cut porcine skin and stored at 4 °C for 24 h. The samples were cut using a cryostat, placed onto an indium tin oxide-coated glass slide, and sprayed by 9-aminoacridine matrix substance (9-AA). Samples were stored at −20 °C prior to analysis. An overlay of the optical images of the rutin was analyzed at 609.15 m/z.The permeation of LNF through porcine skin was visualized by imaging mass microscope . The porcine skin was cut into 1.5 × 1.5 cmDifferential scanning calorimetry (DSC) analysis was performed to determine the melting point of LNF using a differential scanning calorimeter  . After a4 cells/well into 12 well culture inserts  and cultured for 24 h. The second and third layers of dermal fibroblast were repeatedly added onto the first layer on consecutive days. After 24 h of adding the third layer, HaCaT was then seeded at 8.5 × 105 cells/well onto the formed fibroblast layers and cultured for another 24 h, prior to the experiment.Human dermal fibroblast cells were seeded at 8.5 × 102 of UVA and 30 mJ/cm2 of UVB irradiation . After UV irradiation, co-cultured skin cells were further incubated with tested samples for 24 h. Supernatant was then collected for MMP1 and MMP9 quantifications. The levels of UV-induced MMP1 and MMP9 secretions in human co-cultured skin cells were measured by enzyme-linked immunosorbent assay kits  according to manufacturing protocols.Human co-cultured skin cells were pretreated with 7 µg/mL of extract and 100 µg/mL of LNF , together with 100 µg/mL of blank and 10 µg/mL of resveratrol as a positive control, for 24 h. Co-cultured skin cells were then washed twice with PBS and subjected to 5 J/cmThe viability of human co-cultured skin cells after UV exposure was evaluated using a CellTiter-Glo luminescent cell viability assay kit . After 24 h of UV exposure, co-cultured skin cells were washed twice with PBS and 100 μL of Glo Lysis Buffer  was added into each well. After incubation for 10 min, 50 µL of CellTiter-Glo reagent was added and incubated at room temperature for 10 min. The luminescent signal was then measured using microplate luminometer .t-test or one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (GraphPad Prism 9), with p values < 0.05 considered statistically significant.Results were represented as mean ± standard deviation (SD). Significant differences were analyzed by a Student’s Collectively, this research produced two alternative suggestions. Firstly, rutin was not only observed in terms of ultraviolet radiation protection and antioxidative substance, but was also used as a standard marker of chromatogram fingerprints and a key reference of anti-aging activity from fenugreek seed powder extract. Secondly, molecular mechanisms underlying ethanolic extract against aging were demonstrated. The extract showed new biological properties, such as anti-collagenase activity, inducing collagen production, and inhibiting collagen degradation, which refers to a potential alternative natural anti-aging agent for anti-aging products. Encapsulation techniques can be used to protect the bioactive compounds, chemical and physical degradation processes, and prolong their biological activity until use. This technique also reduces the toxicity of herbal extracts. In this study, formulated liponiosomes are highly stable and have a prolonged release. Nano-formulation can also enhance the potency of fenugreek extract on anti-aging properties. On the basis of this study, we suggest that LNF could potentially be applied as a promising active ingredient in the prevention of skin aging.


Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions. Three-dimensional cell cultures have emerged as a novel platform to study cell functions in a microenvironment resembling native tissue, with particular focus on producing transplants for regenerative medicine and screening drug compounds in biorealistic human in vitro tissue. The advances in bioprinting multiple cell types on biomaterial scaffolds , and genHuman brain organoids recapitulate the tissue architecture of the developing cortex to a remarkable degree of fidelity, even discriminating species differences in the development dynamics of neurons between humans and gorilla . Since tThe accumulating body of scientific reports portray brain organoids as a human 3D in vitro platform that achieves remarkable complexity, even resembling its in vivo counterpart, the embryonic human brain ,36,37,38The first such report showed microglia, derived from induced pluripotent stem cells, to migrate spontaneously from culture medium to the brain organoid. Therein, they assumed their native functionality by migrating to the site of tissue injury, induced by a needle prick . RecentlEarlier work in characterising the fundamental neurophysiological properties of organoids in absence of microglia have addressed the maturation of sodium/potassium currents ,30, sustHowever, none of previous studies above focused specifically on the emerging, functional, single-cell electrophysiological properties of neuronal entities grown in organoids and the role of microglial enrichment in modulating those functional properties over developmental time in vitro. Thus, while iPCS-derived microglia in organoids have recently been increasingly well characterised, their role in emergance of neuronal functions merits further exploration. Herein, we carry out a detailed single-neuron interrogation of electrophysiological functions that arise in brain organoids in response to incorporation of microglia-like cells. By evaluating neuronal functions at multiple developmental time points, we show, for the first time, that organoids enriched with microglia-like populations develop neurons that respond to a depolarising stimulus with sustained repetitive firing, while neurons without enrichment are more prone to single spiking and adaptation. Remarkably, neurons co-maturing with microglia-like cells showed a greater diversity in the expression of distinct neuronal currents, including a prominent post-inhibitory low-threshold current leading to post-inhibitory spiking, an action attributed to T-type calcium channels, which have been implicated in a variety of functions, including synaptic plasticity and neuronal differentiation ,67,68. Wg 5 min, resuspended and filtered through 35 µm strainer. Cell viability was assessed by Zombie Aqua , cells fixed in 1% formaldehyde, permeabilised in 0.2% TritonX + 5% FBS, and incubated with fluorophore-conjugated antibodies  in Ca2+/Mg2+-free DPBS (Thermo Fisher Scientific). The medium was supplemented with 5 µM of Y-27632 , a Rho-Associated Coil Kinase (ROCK) inhibitor , for the first 24 h to prevent apoptosis after passaging. Cultures were tested for mycoplasma using a MycoAlert Kit ), and for bacteria, mould, fungi and yeast using solid and liquid lysogeny broth  and sabouraud dextrose agar  at 30 °C and 37 °C. iPSCs were maintained in serum- and feeder-free conditions in Essential 8 Medium  supplemented with 0.5% penicillin-streptomycin  on growth factor reduced Matrigel (Corning) at 5% COCerebral brain organoids were differentiated according to a protocol published earlier , with so2, 5% CO2 and 37 °C. During the differentiation, medium was changed every 24 h. On day 1, the mesodermal medium was replaced but with lower 1 μM Y-27632. On day 2, within 44–46 h from seeding, hemogenic endothelial differentiation was induced by replacing medium with Dif-base medium consisting of DMEM/F-12, 0.5% P/S, 1% GlutaMAX, 0.0543% sodium bicarbonate , 64 mg/L L-ascorbic acid and 14 μg/L sodium selenite (both from Sigma), and supplemented with 100 ng/mL FGF2, 50 ng/mL VEGF (both from PeproTech), 10 μM SB431542 (Selleckchem), and 5 μg/mL insulin (Merck). On day 4, erythromyeloid differentiation was furthered by replacing the media by Dif-base supplemented with 5 μg/mL insulin, 50 ng/mL FGF2, vascular endothelial growth factor (VEGF), interleukin 6 (IL-6) and thrombopoietin (TPO), 10 ng/mL IL-3 and stem cell factor . From now on, cells were maintained at atmospheric 19% O2, 5% CO2 and 37 °C. On day 8, the erythromyeloid progenitors (EMPs) that were blooming into suspension were collected by gentle pipetting through a 100 µm strainer (VWR). The cells were counted and centrifuged at 160× g for 5 min to be ready for incorporation into organoids.To initiate microglial differentiation on day 0, iPSCs were detached with 0.5 mM EDTA at 70–80% confluency, and 60,000–80,000 single cells/dish were seeded on 3.5 cm matrigel coated dish (Sarstedt) in mesodermal differentiation medium consisting of E8, 0.5% P/S, 5 ng/mL BMP4 , 25 ng/mL Activin A (PeproTech), 1 μM CHIR 99021  and 10 μM ROCK inhibitor (Selleckchem #S1049). Cultures were maintained in a low oxygen incubator at 5% O2 for 6 h to allow the progenitors to sediment and attach to the surface of the organoid. Then organoids were transferred back to 6-well format and the culture continued according to organoid maintenance protocol. For Matrigel-droplet incorporation method, day 30 organoids were transferred to parafilm cups and day 8 microglial progenitors (EMPs) were resuspended in Matrigel at 10,000 cells/µL density. Then 2 µL of the suspension was added on an organoid, giving 20,000 EMPs/organoid. The assemblies were then incubated at 37 °C, 5% CO2 to allow the Matrigel to solidify and settle on the organoids. The organoids were transferred to Diff2 the same way as on day 11 and the culture was then continued according to organoid maintenance protocol.To incorporate exogenous microglia into the organoids, day 8 microglial progenitors (EMPs) were allowed to spontaneously migrate into day 30 organoids using two comparable methods: either as free-floating cells or inside Matrigel droplet. For free-floating incorporation, single organoids were transferred to ultra-low attachment 96-well U-bottom wells  in carryover medium. Then 500,000 EMPs/organoid, in 100 µL of Diff2, were seeded on the wells. For control, Diff2 was added without progenitors. Organoids with and without microglia, denoted (+)ORG and (−)ORG, respectively, were incubated at 37 °C, 5% COOrganoids were washed thrice with 0.1 M phosphate buffer and fixed in freshly thawn 4% paraformaldehyde (Merck in phosphate buffer for 4 h at 4 °C. Fixed organoids were cryopreserved in 30% saccharose (VWR) in phosphate buffer at +4 °C overnight. After which, they were embedded in O.C.T. Compound  in 1 × 1 × 0.5 cm plastic moulds  by incubating at RT for 20 min and freezing on a metal block at −70 °C. Organoids were then cryosectioned to 20 µm thickness on cryotome , sections were mounted on microscope glasses (Thermo Scientific) and stored in −70 °C. For heat-induced antigen retrieval, 10 mM sodium citrate  was heated to 92 °C, the slides were submerged and incubated for one hour, as the solution was allowed to cooled down in RT. Sections were blocked and permeabilized in 10% normal goat serum (Merck) and 0.05% Tween20 (Merck) in PBS for 1 h. Primary and secondary antibodies were diluted and incubated in the 5% normal goat serum in 0.05% Tween20 in PBS. Sections were incubated with primary antibodies  at 4 °C All confocal images were obtained with a Zeiss Axio Observer inverted microscope  or 63× (oil) -objectives) equipped with LSM800 confocal module (Carl Zeiss Microimaging GmbH). DAPI and secondary antibodies were imaged with 405 nm (λex 353 nm/λem 465 nm), 488 nm (λex 495 nm/λem 519 nm) and 561 nm (λex 543 nm/λem 567 nm) lasers, respectively.For imaging of representative figures, we used Leica Thunder 3D tissue imager, Figure 1b, , 10× objective using the Leica X application suite version 3.7.2.22383. The Zeiss confocal with 20× objective for Figure 1c–f, 40× for Figure 1g and 63× for Figures 1i, 2, For quantification of morphology, we employed two imaging devices to acquire whole slice Z-stack tiles, capturing the entire Z-dimensional morphology of the microglia. We imaged the day 120 organoids at the Zeiss confocal, with seven focus planes , using the EC Plan-Neofluar 10×/0.30 M27 objective. Day 35 and day 66 organoids we imaged using 3D HISTECH Pannoramic 250 Flash III with 20× objective  and the extended view images exported to TIF for downstream analysis using the Caseviever version 2.4.The analysis on microglia morphology was done with the original CZI files , or the TIF files (3D HISTECH Pannoramic 250 Flash III). Skeletal analysis was performed using Fiji ,74. For For quantification of PSD95 puncta, we manually sampled four optical fields per organoid, four organoids per (+)/(−)ORG group, and captured a Z-stack of ten 109.38 µm × 109.38 µm (1580 px × 1580 px) focus planes with 0.28 µm interval, using the Plan-Apochromat 63×/1.40 oil DIC M27 objective. We performed counting of the PSD95 puncta with a set of 12 prominence thresholds, using the Find Maxima algorithm in Fiji . We appl2/CO2) aCSF of the following composition (in mM). 92 N-methyl-D-Glucamine, 2.5 KCl, 20 HEPES, 25 NaHCO3, 1.25 NaH2PO4, 3 Na-Pyruvate, 2 Thiourea, 5 Na-Ascorbate, 7 MgCl2, 0.5 CaCl2, 25 Glucose (pH adjusted to 7.3 with HCl 10 M). After cutting, slices were placed in a custom-made chamber and allowed first to recover at 34 °C for 30 min in the recording solution  and then for 60 min in the same solution at room temperature (20–22 °C) before use for electrophysiology.Brain organoids (107–213 days in culture) were embedded in 2% low melting point agarose and sliced  using a vibratome  in chilled (4 °C), fully carbonated  using 5–8 MOhm glass electrodes filled with an internal solution containing (in mM) 135 Potassium-gluconate, 5 NaCl, 10 HEPES, 2 MgCl2, 1 EGTA, 2 Mg-ATP, 0.25 Na-GTP and 0.5% Biocytin (pH adjusted to 7.3 with osmolarity at 275–285 mOsm/L). Electrophysiological data were low pass filtered at 1 KHz (4-pole Bessel filter) then captured at 10 KHz via a Digidata 1440A A/D board to a personal computer, displayed in Clampex software  and stored to disk for further analysis. Single-cell electrophysiological current and voltage-clamp data were analysed in Clampfit (Molecular Devices). Synaptic data were detected and analysed with Mini Analysis software .A single slice (350 μm) was transferred and secured with a slice anchor in a large volume bath under an Olympus BX50WI microscope  equipped with differential interference contrast (DIC) optics, an ×40 water immersion objective and a charge-coupled device (CCD) camera ). Slices were continuously perfused at a rate of 2.5–3 mL/min with a recording solution of the following composition (in mM): 120 NaCl, 2.5 KCl, 25 NaHCOA single slice (500 μm) was transferred and secured with a slice anchor in the chamber of a 60-3D-multielectrode recording array  with an 8 × 8 configuration  made of titanium nitride (TiN) and insulated with a thin layer of silicon nitride. Recordings were made with a MEA2100-Mini headstage (MCS) under a Leica S APO microscope equipped with a transillumination base and a digital camera . Slices were continuously perfused at a rate of 3–3.5 mL/min at 32–33 °C with the same recording solution as with whole-cell recordings and were allowed at least 20 min to settle in the MEA before any recordings or pharmacology was attempted. Organoids from both phenotypes were examined on a single experimental day with a blind protocol to the experimenter. Electrophysiological data were band passed between 300 and 3000 Hz (2nd order Butterworth) and were captured at 20 KHz via MCS-IFB 3.0 multiboot (MCS) to a personal computer, displayed in MCS Experimenter software (version 2.15) and stored to disk for further analysis. We used a firing algorithm based on standard deviation (5.5 times the baseline) of the unsmoothed basal firing rate histogram (in 0.1 s bins) and a minimum definition of 2 spikes per burst to detect bursts of spikes in spontaneously firing and NMDA responding channels. Local field potentials (LFPs) from NMDA responding channels of interest (COIs), were extracted from the raw signal using a 200 Hz low pass filter (2nd order Butterworth), were stored in a separate file and were subjected to fast Fourier transformation (FFT) and spectrogram visualisation  and power spectral density  analysis for different frequency bands  for the duration of the recording (in 10 s bins). PSD was also computed in 2-min epochs for the frequency range between 1 to 100 Hz (frequency bins of 0.61 Hz) at baseline, during NMDA application and during wash-out and the mean relative power induced by drug application was calculated by subtraction from the baseline epoch for each individual electrode. A three-bin wide (each 10 s) Boxcar and Gaussian smoothing routine was used for firing/bursting rate and spectral power determinations, respectively. Firing, bursting and LFP PSD analysis of data was conducted in Neuroexplorer software  while spike sorting for single unit analysis  was conducted in spike2 software .t-test between (+)ORG and (−)ORG + NMDA in Figure 4h). The particulars of appropriate statistical tests used are detailed in text.All data represent mean  ±  s.e.m. The morphologies of microglia were analysed on Origin  with significance tested using the Tukey test. All other statistical tests were performed on Prism , with two-way-ANOVA for time plot comparisons of electrophysiological data, Figure 5j,k, and non-parametric Mann–Whitney unpaired tests for PSD95 puncta and electrophysiology results  would migrate spontaneously to brain organoids at an early state, as had earlier been shown for more mature induced pluripotent stem cell (iPSC)-derived microglia ,69. To tAs the microglia take increasingly complex morphologies during brain development in vivo , and givImmunohistochemical staining against postsynaptic density protein 95 (PSD95)  showed aInspired by the IHC staining against IBA1 and B-tubulin showing the IBA1+ cells to be intensively intertwined within neurons g, we furNext, to analyse if the interaction between microglia and synapses was reflected in the numbers of PSD95 puncta, we quantified the numbers of PSD95 puncta in the (+)/(−) ORGs at 120 DIV using the FindMaxima algorithm in Fiji . Given tTo assess whether enrichment of organoids with IBA1+ cells has the capacity to alter single neuron properties in the developing organoids, we performed whole-cell recordings in acute organoid slices at days 107–120 and days 150–165. The recordings were carried out under voltage-clamp conditions to measure the maximal sodium and potassium  and passive leak current density  as markers of neuronal maturation and stage of development. Currents recorded were normalised to cell size and expressed as pA current per pF of cell capacitance. We detected a statistically larger potassium current density in (+)ORG at the early time point and a significantly larger mean sodium current density in neurons in (+)ORG at both recorded time points a,b. No cNext, we looked if microglia would boost the emergence of network activity, previously reported in organoids . 3D-MEA n = 44 vs. 66 responding channels for (+)ORG and (−)ORG phenotypes, To further elaborate on our functional findings and to ascertain whether the striking differences seen would persist in time, we examined four organoid-slices per group (taken from two different batches) at DIV 200–213 using 3D-MEA techniques coupled to NMDA pharmacology to induce brief periods of persistent neuronal bursting and local field potential (LFPs) oscillations. Unlike our previous results at DIV 150–165, we found a similar number of spontaneously active channels per organoid-slice under both baseline and under induced conditions via NMDA receptor activation ORGs compared to (−)ORGs consistent with the presumed role of microglia in the development of neuronal properties and network formation ,90. Our The striking differences in single-cell electrophysiological properties between (+) and (−)ORGs prompt for dissecting the temporal dynamics of the co-development by depletion of microglia. This might help in directing further microglia-neuron interaction studies to the most relevant stages of organoid development. In addition, single-cell sequencing studies will reveal the neuronal populations that are emerging in response to incorporation of microglia-like cells, similarly as previous sequencing work has revealed enrichment of GABAergic interneurons as the organoids mature .Detailed interrogation of the microglia-neuron interaction is mandatory for understanding the aspects of neuronal development, and more importantly, the aspects of neurodevelopmental disorders most amenable to modelling by using the current organoid approaches . HoweverWhile our work here focuses solely on analysing neuronal functions arising due to presence of microglia-like cells, future key aspects in characterising microglia-containing organoids would be to interrogate the identity and behaviour of the microglia-like cells within. CD11b sorting and single-cell sequencing allow for determining how closely the iPSC-EMP-derived microglia-like cells correspond to the in vivo microglia or to the primary microglia maturing in organoids . While p


P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tβ4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tβ4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tβ4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tβ4 alone and adjunctive Tβ4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tβ4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study. Our previous work has shown that topical thymosin beta 4 (Tβ4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of  Pseudomonas aeruginosa is one of the most common pathogens identified in bacterial keratitis with an increasing frequency, especially among contact lens wearers and immunocompromised individuals [P. aeruginosa-infected corneas [Bacterial keratitis is a rapidly progressing, sight-threatening infection of the cornea that can lead to corneal opacification, perforation, and endophthalmitis. ividuals ,2,3. Kerividuals ,5. Curre corneas ,7. Altho corneas . Cortico corneas ,10. TherTβ4 is a small, naturally occurring 43-amino acid protein that is highly conserved across species and is endogenously expressed in all tissues and cell types, except red blood cells, with a high concentration detected within platelets and wound fluid ,12. InitP. aeruginosa-induced keratitis [Pseudomonas keratitis model. The inflammatory process is usually well-regulated by the signals that initiate and maintain inflammation and signals that shut down or resolve the process. An imbalance between the two signals leaves inflammation poorly modulated, resulting in chronicity and tissue destruction . MΦ are eratitis . Little Firstly, clinical scores were recorded to assess the disease response at 3 days post-infection (p.i.), as shown in P. aeruginosa-infected corneas [+ leukocytic cells were present in infected corneas of Tβ4-, ciprofloxacin-, and combination-treated mice when compared to PBS controls  single-cell populations, as shown in + cells revealed that all three treatment groups trended downward, but only ciprofloxacin and adjunctive Tβ4 treatment groups displayed statistical differences when compared to the PBS control (+ cells and MΦ infiltration into corneas of P. aeruginosa-infected B6 mice. From the CD45 control D. By assp < 0.05). No differences were detected between ciprofloxacin and PBS. These results together reflect that Tβ4 influences the activation state of MΦ in the corneas of P. aeruginosa-infected B6 mice, where decreased pro-inflammatory markers paired with increased anti-inflammatory markers within the MΦ subpopulation in the adjunctive treatment group enhanced the resolution of the host inflammatory response.To next examine whether there were any phenotypic differences in the MΦ populations detected in each treatment group, select cell surface molecules that are considered either pro-inflammatory or anti-inflammatory in nature were detected using flow cytometry. As presented in NO levels were measured to investigate the mechanism(s) by which Tβ4 may influence MΦ cellular function. It is well-known that MΦ produce free radicals, mainly NO, one of the key mediators in response to inflammation . As an iIn the infected cornea, many different cell types function together in response to the invading pathogen. In vitro studies were carried out to complement the in vivo work regarding how Tβ4 may be influencing MΦ activation and function without the confounding factors of other inflammatory cells or the bacteria. Two time points (6 and 24 h) were assessed to evaluate the effect of Tβ4 on LPS-induced NO production and iNOS levels using RAW 264.7 cells . After 6MΦ engulfment of apoptotic PMN has been demonstrated as an essential event in the resolution of inflammation ,31. To fThe efferocytotic activity of MΦ was also analyzed in vitro to further confirm the regulatory influence of adjunctive Tβ4 treatment on MΦ cellular function in response to LPS stimulation. Levels of Tim4 (A), BAI-1 (B), and Rac-1 (C) in LPS-stimulated MΦ were detected after 6 and 24 h, as shown in Pseudomonas aeruginosa is a common virulent, opportunistic pathogen associated with rapid progressing liquefactive necrosis of the cornea, often resulting in opacification and blindness [P. aeruginosa-induced corneal infection in the susceptible B6 mouse model. lindness . Howeverlindness . Developlindness . Additiolindness ,17,33. Olindness . The curWhile the exact mechanisms whereby Tβ4 exerts its inflammatory influence are only superficially understood, we provide evidence suggesting that Tβ4 carries out its profound effects, in large part, by affecting MΦ infiltration, activation, and function in the infected cornea, thus contributing to markedly improved disease outcome when combined with ciprofloxacin. MΦ are essential first-line defenders of the innate immune system , but neeP. aeruginosa-induced keratitis. These findings correlate with our previous work showing that Tβ4 reduces corneal iNOS mRNA expression at both 3 and 5 days p.i. [While increased cellular infiltration and excessive generation of pro-inflammatory mediators are more evident components leading to worsened disease, RNS produced by MΦ also play an important role in the pathogenesis of microbial keratitis. In fact, controlled production of RNS is essential in inhibiting inflammation, promoting resolution, and restoring tissue homeostasis ,29. A reays p.i. . It is ways p.i. , stimulaays p.i. . As a reThere is abundant evidence that apoptotic cells can suppress inflammation and that failure to clear apoptotic cells exacerbates the response, indicating that efferocytosis plays a crucial role in modulating the inflammatory response of MΦ to promote resolution ,30,31,392+ influx and trigger phosphorylation of ERK1/2, resulting in proliferation and migration of human corneal epithelial cells [The differences observed between in vitro and in vivo studies emphasize our previous findings that Tβ4 does not appear to function as an antibacterial and, as a result, fails to exert its regulatory effects to full capacity in the presence of bacteria. By omitting the bacteria from the in vitro studies, Tβ4 alone was demonstrated to regulate the MΦ response to LPS-induced inflammation, as shown by reduced RNS generation and efferocytosis marker expression. In fact, our findings show that Tβ4 acts as a transcription modulator, as indicated by changes at both the mRNA and protein levels . Our preal cells . In addial cells . P2X7R sal cells , suggestal cells .Collectively, these findings suggest that Tβ4 treatment has a minimal effect when used alone in vivo. However, adjunctive Tβ4 with ciprofloxacin substantially enhances and regulates the host defense against inflammation, resulting in markedly improved disease outcome. As Tβ4 has no reported adverse effects, this work strongly supports its use as an adjunct treatment for bacterial keratitis to alleviate the drawbacks of the current standard of care. 6 CFU/mL of the cytotoxic Pseudomonas aeruginosa strain ATCC 19,660  was immediately applied to the corneal surface, as previously described [Eight-week-old female C57BL/6 (B6) mice were purchased from The Jackson Laboratory . Following wounding of the cornea using established methods, a 5 μL aliquot of bacterial suspension containing 10escribed . Mice wen = 11 per group per treatment) to express disease severity and presented as mean clinical score ± SEM for each experimental group: 0 = clear or slight opacity, partially or fully covering the pupil; +1 = slight opacity, fully covering the anterior segment; +2 = dense opacity, partially or fully covering the pupil; +3 = dense opacity, covering the entire anterior segment; +4 = corneal perforation [After infection, the corneas were observed daily in a blinded fashion and were graded using an established grading scale . A cliniforation . 2. Before treatment, cells were seeded in 6-well plates at a density of 0.2 × 106 cells/well  and divided into four different groups: media only/no treatment (positive control), Tβ4 , ciprofloxacin , and combination . Once cells became confluent, all four groups were stimulated with lipopolysaccharide  for up to 24 h. Media only/no treatment without LPS stimulation served as the negative control. At both 6 and 24 h, cell supernatants and lysates from each group were collected for analysis.Murine monocyte/MΦ-like RAW 264.7  cells, initially derived from BALB/c mice, were cultured in DMEM containing 10% heat-inactivated FBS  and penicillin (100 U/mL) at 37 °C and 5% COTM Fixable Aqua dead cell stain (L34965) , BV785-conjugated rat anti-mouse CD192 , APC-R700-conjugated rat anti-mouse F4/80 , and APC-Fire 750-conjugated rat anti-mouse Ly-6G  .Individual corneas from each treatment group were harvested at 3 days p.i. in sterile tubes containing 250 µL of RPMI 1640 without serum. Single-cell suspensions were obtained as previously described . Cell pe3PO4) in the corneas of PBS-, Tβ4-, ciprofloxacin-, and Tβ4 + ciprofloxacin-treated B6 mice. In brief, corneas were homogenized in 500 µL of degassed PBS and micro-centrifuged at 3500 rpm for 5 min. Next, 100 µL of supernatant was added to an equal volume of Griess reagent in triplicate in a 96-well microtiter plate and incubated at room temperature for 15 min. Absorbance at 570 nm was measured and nitrite concentrations were estimated against a sodium nitrite standard curve. Data were reported as the mean μM/cornea concentration of nitrite ± SD.NO production was measured (as previously described ) as its Infected corneas were excised at 3 days p.i. from all treatment groups and homogenized in 200 µL of RIPA buffer . For in vitro studies, RAW 264.7 cells were collected at both 6 and 24 h after treatment and lysed in M-PER buffer . A protease inhibitor cocktail  was added to both the corneal samples and cell lysates. Then, the samples were sonicated to achieve complete lysis and centrifuged at 12,000 RPM for 20 min. Supernatants were collected and normalized for equal amounts of protein as determined by BCA methods, then separated onto 4–20% tris-glycine gels  and transferred to PVDF membranes. After blocking the membranes in 5% non-fat milk dissolved in TBST  at room temperature for 60 min, membranes were incubated overnight at 4 °C with antigen-specific primary antibodies. The primary antibodies were used as follows: anti-iNOS , anti-Tim4 , anti-BAI1 , anti-RAC-1 , and anti-β-actin . They were then followed by incubation with species-specific horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized by incubation with a chemiluminescence substrate kit . Western blot images were collected , and target protein expression was quantified using Image Studio Lite software version 5.2 , normalizing to β-actin. All the antibodies were repeated at least three times, and one representative blot was shown for each molecule.® Green Master Mix , per the manufacturer’s instruction, with the reaction mixture previously described [Total RNA was isolated from individual whole corneas for gene expression analysis using RNA-STAT 60 , according to the manufacturer’s recommendations, and quantified by spectrophotometric determination (260 nm). cDNA templates were constructed by reverse transcribing 100 ng of total RNA, then amplified using SYBRescribed . All priescribed . Resultsp < 0.05.Sample sizes were determined statistically before experimentation based on previous work. All experiments were carried out from a minimum of three independent experiments, and representative data from a typical experiment are shown. Data are presented as mean ± SD unless otherwise noted. All data were analyzed by the one-way ANOVA followed by Bonferroni’s multiple comparison test . Data were considered significant at


Alternatively, hASCs were cultured on plastic, under 20% O2 and without cytokines for 14 days (standard cell culture). After two days, the bone construct model decreased total DNA (3.9-fold), COL1 (9.8-fold), and RUNX2 expression (19.6-fold) and metabolic activity (4.6-fold), but increased VEGF165 expression (38.6-fold) in hASCs compared to standard cultures. After seven days, the bone construct model decreased RUNX2 expression (64-fold) and metabolic activity (2.3-fold), but increased VEGF165 (54.5-fold) and KI67 expression (5.7-fold) in hASCs compared to standard cultures. The effect of the bone construct model on hASC proliferation and metabolic activity could be largely mimicked by culturing on BCP alone . The effect of the bone construct model on VEGF165 expression could be mimicked by culturing hASCs under hypoxia alone . In conclusion, we developed a new, biologically relevant in vitro 3D-model to study the bone regeneration potential of MSCs. Our model is likely more suitable for the screening of novel factors to enhance bone regeneration than standard cell cultures.Standard cell cultures may not predict the proliferation and differentiation potential of human mesenchymal stromal cells (MSCs) after seeding on a scaffold and implanting this construct in a bone defect. We aimed to develop a more biologically relevant in vitro 3D-model for preclinical studies on the bone regeneration potential of MSCs. Human adipose tissue-derived mesenchymal stromal cells  were seeded on biphasic calcium phosphate (BCP) granules and cultured under hypoxia (1% O Bone tissue engineering strategies developed to treat large bone defects are generally based on the incorporation of mesenchymal stromal cells (MSCs) and bioactive factors into a scaffold, which together form a bioactive bone construct . This bo2 (PGE2) production by monocytes, and bone resorption by osteoclasts [Upon implantation of a tissue engineered bone construct, an inflammatory response reminiscent of the early stage of fracture repair occurs in the bone microenvironment, where inflammation and hypoxia trigger the healing process ,6,7,8,9.eoclasts ,17. Boneeoclasts , while Ieoclasts . This su2) occurs at the periphery of a bone defect due to the disruption of blood vessels, and may be maintained by inflammation [Hypoxia  for 14 days with a cytokine-mix of TNFα, IL4, IL6, and IL17F (10 mg/mL each) added during the first three days of culture (bone construct model). Alternatively, hASCs were cultured on plastic, without cytokines, and in the presence of 20% O2 for 14 days (standard cell culture). hASC proliferation and osteogenic differentiation were assessed by quantifying DNA content, metabolic activity, osteogenic gene expression, total protein, and expression of VEGF165 at up to seven days of culture. Mineralization by hASCs was assessed after 14 days. Our bone construct model, which mimics conditions inside a tissue engineered construct for bone healing after implantation, strongly affected proliferation, osteogenic differentiation, total protein, metabolic activity, and VEGF165 expression by hASCs compared to standard tissue culture conditions. Our preclinical 3D-model may therefore be more suitable for predicting the success, or lack thereof, of novel bone tissue engineering constructs in a later stage of development than standard practices, while being relatively easy to implement in the workflow and still allowing medium-to-high throughput analysis of the bone regeneration potential of human adipose stem cells.To address our hypothesis, human MSCs derived from adipose tissue (hASCs) were used as model for MSCs. hASCs were used in this study as adipose tissue is more easily accessible and contains more MSCs, resulting in larger amounts of hASCs available for (pre)clinical studies and for clinical applications compared to bone marrow-derived MSCs . Further9 platelets/mL [g for 10 min to eliminate remaining platelet fragments. The supernatant was added to the medium at 2% (v/v) or stored at 4 °C and used up to 1 week.Pooled platelet products from five donors were obtained from the Bloodbank Sanquin  and contained approximately 1 × 10elets/mL . Platele2 in air at 37 °C. The medium was refreshed two times a week. After reaching confluence, cells were harvested by incubation with 0.25% trypsin (Gibco) and 0.1% ethylenediaminetetraacetic acid  in phosphate buffered saline (PBS) at 37 °C, counted, and stored in liquid nitrogen until further use. For experiments, hASCs from the 5 different donors were thawed and individually cultured at 0.5 × 106 cells in T-175 cm2 culture flasks  in α-MEM containing 1% PSF, 10 IU/mL heparin, and 2% PL in 5% CO2 in air at 37 °C. Only hASCs at passage 2 (P2) were used. Medium was changed every 3 days.Subcutaneous adipose tissue samples were harvested from abdominal wall resections of five healthy female donors (age range: 31–56 years) who underwent elective plastic surgery at the Tergooi Hospital Hilversum and a clinic in Bilthoven, The Netherlands. The Ethical Review Board of the VU University Medical Center, Amsterdam, The Netherlands, approved the protocol (Protocol number 2016/105) on 17 March 2016, and informed consent was obtained from all patients. Isolation, characterization, and osteogenic differentiation capacity of hASCs as a model for MSCs has been previously reported by our group ,31. Cryo3 cells/cm2) containing α-MEM with 1% PSF, 10 IU/mL heparin, and 2% human PL in a humidified atmosphere containing 20% O2 (normoxia) at 37 °C. Alternatively, hASCs from different donors were seeded on scaffolds. Straumann® BoneCeramic 60/40 , a porous BCP scaffold composed of 60% HA and 40%  β-TCP (BCP60/40) with particle size 500–1000 μm and 90% porosity, was used. BCP scaffolds were prepared using 20 mg of BCP60/40 pre-hydrated in PBS for 30 min. After PBS removal, 1 × 105 hASCs in a volume of 100 µL α-MEM without PL were allowed to attach to BCP for 30 min at room temperature. Earlier, we determined that more than 90% of the hASCs within the freshly isolated stromal vascular fraction rapidly (within 10–30 min) adhere to various scaffold types [One day before the start of the experiment (day –1), hASCs from different donors were seeded in 12-well plates  for the entire duration of the experiment, whereas cells cultured on BCP were supplemented with 50 µM vitamin C and 10 nM vitamin D3. BCP is commonly used to induce the osteogenic differentiation of MSCs, providing a source of phosphate for bone matrix calcification [2, as described earlier [2 test tubes . To maintain hypoxia, the medium was pre-incubated for 3 h under hypoxia before use. Hypoxia was defined as 1% O2 in 5% CO2, and 94% N2. Every 3 days, the medium was replaced by fresh medium without cytokines for all groups. hASCs were harvested after 2, 7, and 14 days of culture to analyze proliferation and osteogenic differentiation.At the start of the experiment (day 0), the medium of hASCs was replaced by osteogenic medium supplemented with 1% PSF, 10 IU/mL heparin, and 2% human PL. The medium of cells cultured on tissue culture plastic was further supplemented with 50 µM ascorbic acid-2-phosphate , 5 mM β-glycerophosphate (Sigma), and 10 nM 1,25-dihydroxyvitamin Dfication , similarfication . Therefo earlier . The oxy® spectrophotometer . DNA was isolated using Trizol® reagent  according to the manufacturer’s instructions. DNA concentration and quality were determined by reading the absorbance at 260 nm and 280 nm respectively using a Synergy HT® spectrophotometer (BioTek Instruments). Total protein was assessed by measuring the protein/DNA (µg/ng) ratio.The amount of protein was measured with a bicinchoninic acid (BCA) Protein Assay Kit  and the absorbance was read at 540 nm with a Synergy HTTM Cell viability reagent (Invitrogen). Cells were incubated with medium containing 10% AlamarBlue for 4 h at 37 °C. Then, 100 μL supernatant was transferred into a black 96-well microtiter plate, and fluorescence was measured at 530–560 nm wavelength in a Synergy HT® spectrophotometer (BioTek Instruments). Medium without cells containing 10% AlamarBlue was placed in an autoclave container and used as positive control of 100% chemically reduced AlamarBlue solution.The metabolic activity of hASCs cultured on BCP or plastic, with or without cytokines, and under normoxia or hypoxia, was assessed using AlamarBlue® reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA concentration and quality were determined using a Synergy HT® spectrophotometer. RNA was reverse-transcribed to cDNA using a RevertAid™ First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. The obtained cDNA was diluted to a final concentration of 2 ng/μL. Real-time PCR was performed using the SYBR® Green I Mastermix  in a LightCycler® 480 (Roche Diagnostics). Every PCR reaction was prepared with 4 μL cDNA, 0.5 μL forward primer (1 μM), 0.5 μL reverse primer (1 μM), and 5 μL LightCycler® 480 SYBR® Green I Mastermix (Roche Diagnostics) in a final volume of 10 μL. Based on BestKeeper, values were normalized to TBP and GUSB [KI67, RUNX2, COL1, and VEGF165. The primer sequences are listed in Total RNA was isolated from hASCs using TRIzoland GUSB . Real-ti® spectrophotometer. ALP activity was expressed as µmol/µg protein.hASCs cultured for 0, 2, or 7 days without or with cytokines under hypoxia were lysed with 250 µL Milli-Q water and stored at −20 °C prior to use. The 4-nitrophenyl phosphate disodium salt (Merck) at pH 10.3 was used as a substrate for ALP, according to the method described by Lowry . The absMatrix mineralization was analyzed in cells cultured on plastic without or with cytokines under hypoxia or normoxia for 14 days by using 2% Alizarin Red (Sigma) in water at pH 4.3, as described earlier . Brieflyn = 6, 1 donor used twice on separate occasions). Mann–Whitney U tests were performed to assess differences between the bone construct model and standard cultures after day 0, 2, 7, and 14. All data are depicted as box–whisker plots . A p-value < 0.05 was considered significant. All analyses were performed with RStudio 4.0.3 . All graphs were created with GraphPad Prism 9 .Data were obtained from duplicate cultures of 6 independent experiments, performed with cells from 5 independent donors  activity. The bone construct model decreased COL1 expression by hASCs compared to standard cultures after two days  were quantified to assess hASC proliferation and metabolic activity respectively. The bone construct model decreased total DNA of hASCs compared to standard cultures after two days . Neither the bone construct model nor hypoxia alone modulated VEGF165 expression by hASCs compared to standard cultures after day 0  and after seven days , BCP alone differentially affected MSC proliferation and differentiation, but affected metabolic activity and total protein in a similar manner as the complete bone construct model. Hypoxic conditions stimulated VEGF165 expression by MSCs on plastic without cytokines, which was similar to the effect of the bone construct model.hASCs are often used for bone regenerative surgery as they are easier to collect (via liposuction) and more abundant than other stem/stromal cells. hASC incorporation in a carrier material such as BCP has already proven effective. For example, hASCs seeded on β-tricalcium and BCP increase bone and osteoid formation compared to ceramic only in patients after six months . The in 2) due to blood vessel disruption, while platelet-derived factors orchestrate the migration of inflammatory cells, such as neutrophils and macrophages, to the site of damage [2 and 5% CO2 atmosphere [RUNX2 and bone morphogenetic protein-2 (BMP2) expression [The microenvironment of a bone defect is hypoxic , affected IL17 enhances ALP activity in MSCs after 10 days of culture . IL7 alsRUNX2 expression [RUNX2 was strongly decreased in our bone construct model. Taken together, hypoxia alone might be the critical factor that decreases RUNX2 expression in MSCs residing in a bone construct after implantation in vivo. Hypoxia increased VEGF expression in MSCs on plastic without cytokines, and so did the complete bone construct model. These results are in accordance with data obtained by others showing that hypoxia induces VEGF expression in primary human osteoblasts [VEGF expression in endothelial cells [r = 0.8) between VEGF protein expression and vessel density was found by Lund et al. [Hypoxia has been shown to inhibit osteogenic differentiation of MSCs by impairing pression . We founeoblasts . Hypoxiaal cells ,59. Blooal cells ,61,62. Hd et al. , while Vd et al. . TherefoThis study has a number of limitations. When mimicking a bone defect in vitro, the local acidity level (pH) may play a role. Fluctuating pH levels have been observed during various stages of bone repair in rats . In the VEGF165 expression compared to standard cultures. Our preclinical 3D-model may therefore be more suitable for predicting the success, or lack thereof, of novel bone tissue engineering constructs in a later stage of development than standard practices, while being relatively easy to implement in the workflow, and still allowing medium-to-high throughput analysis of the bone regeneration potential of human adipose stem cells.We conclude that the bone construct model differentially affects MSC proliferation, differentiation, metabolic activity, total protein, and


Five configurations of these insulin-microPlates (INS-μPLs)were realized with different heights  andPLGA contents . After detailed physicochemicaland biopharmacological characterizations, the tissue-compliant 10HINS-μPL, realized with 10 mg of PLGA, presented the most effectiverelease profile with ∼50% of the loaded insulin delivered at4 weeks. In diabetic mice, a single 10H INS-μPL intraperitonealdeposition reduced BGLs to that of healthy mice within 1 h post-implantation and supportednormoglycemic conditions for about 2 weeks. Furthermore, followingthe glucose challenge, diabetic mice implanted with 10H INS-μPLsuccessfully regained glycemic control with a significant reductionin AUC0–120min (799.9 ± 134.83 vs 2234.60 ±82.72 mg/dL) and increased insulin levels at 7 days post-implantation(1.14 ± 0.11 vs 0.38 ± 0.02 ng/mL), as compared to untreateddiabetic mice. Collectively, these results demonstrate that INS-μPLsare a promising platform for the treatment of T1DM to be further optimizedwith the integration of smart glucose sensors.Type-1 diabetes (T1DM)is a chronic metabolic disorder resultingfrom the autoimmune destruction of β cells. The current standardof care requires multiple, daily injections of insulin and accuratemonitoring of blood glucose levels (BGLs); in some cases, this resultsin diminished patient compliance and increased risk of hypoglycemia.Herein, we engineered hierarchically structured particles comprisinga poly(lactic- Furthermore, the improper dosages ofinsulin can also result in excessive fluctuations of BGLs that, ifchronic, result in severe complications , in addition to many othercomorbidities.10Type-1 diabetes (T1DM)is a chronic metabolic disorder characterizedby elevated blood glucose levels (BGLs).12 nanoparticles,14 and hydrogels14) and lipid-based systems  have been suggested and investigatedin preclinical and clinical trials to achieve non-invasive administrationincluding oral or nasal or pulmonary or transdermal and a controlledrelease of insulin.21 The suggested platforms havebeen found to be beneficial in many aspects, such as protecting drugsfrom enzymatic degradation, improving their stability, enhancing thehalf-life in the body circulation, overcoming different physical,chemical, and biological barriers in vivo, and increasing bioavailabilityand therapeutic efficacy. However, during the preparation of theseplatforms, insulin can be easily damaged. For example, the use ofchemicals , dehydration ,high shear forces , organic–aqueous interfaces,and hydrophobic contacts between insulin and the polymer can causealterations in the structure and a loss of its bioactivity.25 Moreover, often these techniques are difficult to scale-up for industrialproduction. Despite these findings, the inability to obtain an optimalformulation that can match the needs of individual diabetic patientshas led to innovative self-regulated insulin delivery systems thatmimic native insulin production from a healthy pancreas.27 These pancreas-like systems areable to release insulin in response to glucose changes and includeinsulin pumps or systems that have glucose-sensing elements and areable to trigger insulin release .31 However, many challenges remain unaddressed including slow responserate, lack of glucose specificity, instability, acute and long-termtoxicity, and short-term efficacy.30To improve the life quality of diabetics, differentapproacheshave been studied to control and facilitate insulin delivery. Forinstance, polymeric drug delivery systems  acid (PLGA). Spherical,200 nm insulin granules are uniformly dispersed within the polymericmatrix that protects them from exposure to biological fluids and consequentrapid dissolution. We propose five different configurations of microparticlesmade with different thicknesses and PLGA concentrations, namely, 5μm thick with 10 mg of PLGA (5H INS-μPL); 10 μmthick with 10 mg of PLGA (10H INS-μPL); and 20 μm thickwith 10, 40, and 60 mg of PLGA . These five different microparticles are characterizedfor their physicochemical and biopharmacological properties to identifythe configuration with the most suitable properties that is, eventuallytested in murine models of T1DM [streptozotocin (STZ)-treated C57BL/6].In thiswork, hierarchically structured polymeric particles carryinginsulin granules, called insulin-microPlates (INS-μPLs), arefirst introduced with their unique geometrical attributes. INS-μPLsare obtained via a multi-step, top-down fabrication process and resultin prismatic microparticles made out of the FDA-approved, biodegradablepolymer poly(lactic-The two main components in the INS-μPLare the insulin granules (INS) 1 and the34 insulin granules wereprepared using a crystallization process.32 Briefly, insulin (5 mg/mL) was dissolved in acidified water , whereupon zinc acetate (0.05 M), trisodium citrate(0.05 M), and acetone 15% were added at room temperature under magneticstirring (250 rpm) for 90 min. After the evaporation of the organicsolvent, INS were collected by centrifugation   1a. Under 30 min) 1b. These 30 min) 1c,d. INS 30 min) 1e. When  30 min) 1c,d.36 Briefly, a direct laser writing processwas adopted to realize silicon master templates with an array of 20× 20 μm squared wells whose depth can range from a fewmicrons to several tens of microns to modulate the thickness of theresulting microparticles. These master templates were replicated intopolydimethylsiloxane (PDMS) templates which were then replicated intoa sacrificial PVA templates .The wells in the PVA template were carefully loaded with a mix ofPLGA and INS (160 μg) (see Table S1 for details). The PLGA content in the paste can be modified to realizeμPLs with different levels of compactness. Eventually, the resultingprismatic INS-μPLs were recovered via centrifugation after dissolutionin water of the PVA templates. To accurately control and modulatethe dissolution of the INS and the consequent release of insulin,five different configurations of INS-μPLs were realized withdifferent thicknesses and PLGA concentrations. Specifically, theseinclude 5 μm thick μPL with 10 mg of PLGA (5H INS-μPL),10 μm thick μPL with 10 mg of PLGA (10H INS-μPL),and 20 μm thick μPL with either 10, 40, or 60 mg of PLGA(20H INS-μPL).In orderto protect the granules from an excessively rapid dissolutionand thus provide a sustained insulin release, INS were entrapped inmicroscopic, biodegradable PLGA porous matrices [microPlates (μPLs)]to realize the INS-loaded μPLs . INS-μPLswere obtained using a replica molding multi-step, top-down fabricationprocess.36 Furthermore, representative confocalimages (The morphology of the μPL is documentedby scanning electronmicroscopy (SEM) images and Multisizer Coulter Counter analysis, asshown in limages 3 ofthe limages 3, and thlimages 3. The to36 Thesefactors are the viscosity of the polymeric paste, the INS initialinputs, the spreading technique, the separation distance among thewells in the PVA template, the environmental conditions (humidityand temperature), and the PVA concentration in the sacrificial template.However, this systematic analysis transcends the objective of thiswork which provided an initial proof of the INS-μPL efficacyin managing T1DM.To assess the insulin encapsulation efficiency (EE) and loading,INS-μPLs were loaded with 160 μg of the INS initial inputper PVA template. As reported in 39 it was here hypothesized that insulin could be releasedfrom the μPL upon dissolution of INS and the progressive diffusionof the molecular insulin out of the PLGA matrix. Based on this, weselected the 10H INS-μPL as the best configuration for the functionaland in vivo studies as it provides an intermediate release profile:sufficiently faster than the 20H INS-μPL to possibly preventhyperglycemic conditions within the first hours of the application;sufficiently slower than the 5H INS-μPL to guarantee a sustainedinsulin release for at least 30 days .More importantly, the insulin release profile was evaluated upto 1 month under physiologically relevant conditions , mimicking the volume associatedwith an intra-tissue deposition of the μPL 4c. The m 30 days 4c. For t37 It is just important to notethat differently from several other conventional PLGA microparticles,the μPLs exhibit a less compact structure that would allow watermolecules to slowly permeate into the matrix without requiring extensivehydrolysis of the constituting polymer. Indeed, μPL stabilityanalysis  to assess any significantmorphological change 4d. SEM aanalysis 4ddid shp < 0.05) (Table S2). Notably, 10H INS-μPLs were more effective at lowconcentrations (0.5 μM), as compared to the molecular commercialinsulin formulation (Insulin Rapid) returning, respectively, a 5.00± 1.3 and 2.52 ± 0.1-fold increase in AKT phosphorylation,as compared to the control (Table S2). Furthermore, 10H INS-μPLshowed no significant cytotoxic effect for the tested concentrations(0.01–100 μM) (Table S3), while confocaland SEM images confirmed no visible effects on the cell morphologyfor up to 24 h  without any alterationin the function of the insulin, as was readily demonstrated by quantifyingthe amount of AKT phosphorylation induced upon exposure to INS  and day 7  (0–120min (Table S6). A significant increase in AUC0–120min was observed when comparing healthy and INS-μPL-treatedmice at day 14  and day 21  post-implantation.Consistently with registered euglycemic levels, the serum insulinmeasurement by ELISA exhibited a release of insulin in mice treatedwith INS-μPL at day 1, 7, and 14, significantly higher comparedto diabetic mice , followed by a reduction in serum insulinat day 21 (0.52 ± 0.07 vs 0.41 ± 0.14 ng/mL) (Table S7). Furthermore, mice treated with INS-μPL did not experienceany significant change in the body weight, as compared to healthymice for at least 18 days of treatment. In contrast, diabetic miceand mice treated with INS did undergo a significant weight loss startingalready on day 5 (p < 0.05) (Table S8).An ip glucose tolerance test (IPGTT)was performed administeringglucose at dosage 2 g/kg body weight in fasted mice at 1, 7, 14, and21 days post-ip deposition of 10H INS-μPL to assess the BGLregulation capacity. At day 1 and 7, the treated diabetic mice efficaciouslyrecovered the glycemic control after an initial increase in BGLs,and the BGLs were maintained normal (70–200 mg/dL) followingthe same temporal trend of the healthy mice 6d. Also,ctively) 6e. Howev47Collectively, in vivo data show that mice treatedwith 10H INS-μPLmaintain a full normoglycemic state for up to 7 days (<200 mg/dL)and return to a hyperglycemic state only at 13 days post-implantation(>350 mg/dL). Also, after ip infusion of glucose, 10H INS-μPLsare also able to successfully regain the glycemic control after aninitial spike in BGLs at day 1 and 7 post-implantation, and this effectis slightly maintained at 14 days, and then, it disappears at 21 dayspost-implantation. The basal insulin release from 10H INS-μPLsis therefore not only sufficient to control the BGLs overtime in diabeticmice but somehow can also respond to glucose changes without provokinghypoglycemic crisis. Notably, this is occurring without using anyglucose sensor but simply relying on the progressive degradation ofthe insulin granules entrapped within the PLGA matrix of μPL.It is important also to note that the effect of 10H INS-μPLin vivo appeared to be exerted over a shorter time scale, as comparedto the in vitro release rates (only 30% of insulin released in 30days) and particle degradation 4. This cHierarchically—structured polymeric microparticles carryinginsulin granules were realized and pre-clinically validated in diabeticmice for the controlled and long-term release of insulin. 10H INS-μPLsare prismatic particles with a square base of 20 μm and a heightof 10 μm capable of controlling the release of insulin for severalweeks. A single ip administration of 10H INS-μPLs in STZ-diabeticmice reduced, already at 1 h post-implantation, the BGLs returnedalmost to the same value as that of healthy mice. Normoglycemic conditionswere restored for about 2 weeks based on the solo-controlled degradationof the INS and the consequent sustained release of molecular insulin.Furthermore, mice implanted with 10H INS-μPL successfully regainglycemic control following the ip administration of a glucose bolus.In summary, these data show the potential of 10H INS-μPL asa promising platform for the treatment of T1DM with high and durableefficacy. Future studies will have to modulate the risk of hypoglycemiaand extend further the temporal window of action of 10H INS-μPLthrough the addition of a highly specific biochemical sensor for BGLcontrol.l-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl)  (Lip-RhB)was purchased from Avanti Polar, while Cy5-conjugated 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine lipid chain (Lip-Cy5)was synthetized in our laboratory.PDMS(Sylgard 184 and RTV615) was obtainedfrom Dow Corning (USA). Poly (PVA), PLGA (50:50), MTTassay, trifluoroacetic acid, acetonitrile (ACN), STZ, zinc acetate,trisodium citrate, and paraformaldehyde were bought from Sigma-Aldrich(USA). Polycarbonate membrane filters were obtained from SterlitechCorporation (USA). PBS, high-glucose Dulbecco’s modified Eagle’sminimal essential medium (DMEM), and fetal bovine serum (FBS) wereobtained from GIBCO . Curcumin (CURC)was purchased from Alfa Aesar (USA). 32 Briefly, insulin (5 mg/mL)was dissolved in acidified water , whereuponzinc acetate (0.05 M), trisodium citrate (0.05 M), and acetone 15%were added at room temperature under magnetic stirring (250 rpm) for90 min. After the evaporation of the organic solvent, particles werecentrifuged at 18,000g for 30 min, washed in water,and stored at 4 °C. The fluorescent insulin granules (INS) wereobtained by adding 20 μL of a solution containing the fluorescentprobe of interest, either Lipid-RhB (Lip-RhB) or Lipid-Cy5 (Lip-Cy5),to the mix comprising insulin (5 mg/mL) in acidified water, zinc acetate(0.05 M), trisodium citrate (0.05 M), and acetone 15% in the ambervial. After the evaporation of the organic solvent, the so-formedINS  were collected by centrifugationat 18,000g for 30 min, washed in water, and storedat 4 °C.Insulin granules (INS) were prepared usinga crystallization process, as reported in the literature with somemodifications.48 However, the measurement ofthe ζ-potential was performed using a Smoluchowski constant F (ka) of 1.5 as a function of the electrophoretic mobility.Average size (nm), size distribution, and zetapotential (mV) of insulin crystals were analyzed using a DLS, as previouslyreported.High-resolution scanning electron microscopy  analysis was also performed to confirm the averagesize and evaluate the shape of the crystals. Briefly, a drop of insulingranules was put on a silica support, dried, and sputtered with gold/palladiumfor increasing the contrast and reducing the damage of the sample.SEM images were obtained with an acceleration voltage of 50 kV.2) of 50 L/h, respectively. Data were obtained by MassLynxsoftware and quantified by QuantLynx software.The insulin crystals yielding was quantified using ultra performanceliquid chromatograph tandem mass spectrometry (Waters ACQUITY UPLC/MS).The mobile phase consisted of deionized water acidified with formicacid (FA) (0.1% v/v) (phase A) and ACN acidified with FA (0.1% v/v)(phase B), and the analysis was performed using an ACQUITY UPLC BEHC8 , and the followinglinear gradient was applied: 0–0.5 min: 20% phase B, 0.5–3.5min: 20–100% phase B, 3.5–4.5 min: 100% phase B, 4.5–4.6min: 100–20% phase B, 4.6–6.0 min: 20% phase B witha run time of 6 min and injection volumes of 2 μL. The massspectrometer was run in the positive ESI mode, and insulin was measuredby the single ion recording acquisition mode. The capillary and thecone voltages were set at 2.80 kV and 40 V, respectively. The sourcetemperature was set at 125 °C, while the desolvation was setat 800 L/h with a temperature of 400 °C and a cone gas flow  and deionized water acidified with FA (0.1% v/v) ata ratio of 2:1 v/v], and the detection wavelength was fixed at 214nm.Furthermore, the stability of INS granules was assessedin waterat room temperature for 32 days by DLS analysis of the samples atpredetermined time points .36 First, three silicon master templates with a specific geometricalfeature were made up via direct laser writing. These silicon mastertemplates had wells squared in shape with an edge length of 20 μm,separated by 10 μm gap and a depth of 5, 10, and 20 μmfor 5H INS-μPL, 10H INS-μPL, and 20H INS-μPL, respectively(Table S1). Then, the original master templateswere replicated into PDMS templates using a mixture containing PDMSand a silicone elastomer curing agent . The replicas werethen left under vacuum to remove bubbles, which were formed whilemixing the PDMS with the curing agent, and polymerized at 60 °Cfor 4 h. The PDMS templates were peeled off the silica stub and usedto obtain PVA templates by putting a PVA solution (10 w/v %) on theirpatterned surface. The resulting PVA films were dried at 60 °Cto create the same arrays of wells as the original master templates.In the last step, the PVA templates were loaded with PLGA and INS(160 μg) dissolved in ACN, as reported in Table S1, to generate INS-μPLs. Then, the loaded PVAtemplates were dissolved in deionized water at room temperature inan ultrasonic bath, filtered in polycarbonate membrane filters (50μm pore size), and the resultant INS-μPLs were collectedvia sequential centrifugation  and kept at 4 °C.INS-loaded PLGA microPlates(INS-μPLs) were obtained via a multi-step replica molding process.INS-μPLs were characterized usingdifferent methods. INS-μPLnumber, average size, and size distribution were analyzed using aMultisizer 4 Coulter particle counter . However,the square shape and size of INS-μPLs were confirmed after SEManalysis . Specifically, a drop of the INS-μPLsuspension was placed on a silica stub, dried, and homogeneously sputteredwith gold to protect the sample from degradation and enhance the contrast.The images were obtained using an acceleration voltage of 5–15kV.Finally, confocal microscopy  analysiswas performed to assess the size, shape of INS-μPL, and alsothe distribution of INS through the μPL polymeric matrix. Briefly,Lip-Cy5 has been used to stain the INS (Lip-Cy5-INS), while Curcumin(CURC) has been used to stain the PLGA matrix of the particles.g for 5 min at 4 °C), and the supernatant was analyzedby UPLC/MS, according to the chromatographic method reported in theabove paragraph. The amount of insulin encapsulated in INS-μPLwas calculated using a standard calibration curve obtained with sampleshaving known insulin concentrations (0.1–50 μg/mL). EEand loadings were defined using the following equationsg for 5 minat 4 °C), supernatants were removed, and pellets were destroyedwith the mixture ACN acidified with FA (0.1% v/v) and deionized wateracidified with FA (0.1% v/v) in a ratio of 2/1 v/v and analyzed viaUPLC/MS.To evaluate INS EE and loading, INS-μPLs were dissolved inACN with FA (0.1% v/v) and deionized water with FA (0.1% v/v) in aratio of 2:1 v/v. PLGA debris was removed after centrifugation  and SEM analysis . EmptyμPLs (500 μL) were incubated in PBS (pH 7.4) under mechanicalstirring at 37 °C for 1 month. Samples were analyzed to monitorchanges in the shape and structure of the particles at predeterminedtime points .2, in high-glucoseDMEM, supplemented with 15% FBS, 1% l-glutamine, and 1% penicillin/streptomycin.First, a cytotoxicity study was carried out on L6 cells at 24 h afterincubation with INS and 10H INS-μPL at different concentrations. Empty-μPL and acommercial rapid acting insulin form  have beenused as control groups. Specifically, empty-μPLs were used inamounts corresponding to the number of μPLs used for 10H INS-μPL,while Insulin Rapid was used at the same concentrations used for INSand 10H INS-μPL. L6 cells (1 × 104 cells/well)were seeded into 96-well plates, and cells were treated with empty-μPL,Insulin Rapid, INS, and 10H INS-μPLs at the previous definedconcentrations for 24 h. After 24 h of incubation, MTT assay was performed,as previously reported.36 The cell viability(%) was measured, as reported belowT is the absorbance ofcells incubated with 10H INS-μPLs and AbsC is theabsorbance of cells used as the control group (non-treated cells).The biocompatibilityand the biological activity of INS-μPLshave been tested on skeletal muscle cells (L6 cells). In detail, L6cells were cultured at 37 °C in 5% CO39 However, for confocal microscopy imaging, the samples were washedthree times with PBS, fixed with 4% paraformaldehyde, and stainedwith 4',6-diamidin-2-fenilindolo (DAPI) and phalloidin .The safety of 10H INS-μPLs was also confirmed through a microscopeobservation  of the interaction of L6cells with 10H INS-μPL. Briefly, L6 cells were seeded on a glassslides pre-treated with fibronectin . Theday after, the cells were incubated with 10H INS-μPL (1 μM)for 24 h and treated, as previously reported.alloidin 3 or whea4 cells/well) were seeded in 96-well platesand allowed to grow for 24 h. Cell serum starved overnight were treatedwith empty-μPL, Insulin Rapid, INS, and INS-μPL at differentconcentrations  for 1 h. After treatment,cells were lysed, and phosphorylated AKT at Ser473 was assayed inaccordance with the manufacturer’s protocol. The kit is designedspecifically to quantify the activated (phosphorylated) AKT at Ser473and/or total AKT.Furthermore, the biological activity of INS-μPLwas assessedto prove the activation of the insulin receptor. Briefly, L6 cells. STZ was dissolvedin disodium citrate buffer (pH = 4.5) at the concentration of 40 mg/mL.After STZ ip injection, the BGL of mice was measured daily for 7 days,and animals were considered diabetic when their non-fasting BGLs were>350 mg/dL. Briefly, the BGL was monitored daily by collectingblood(∼3 μL) from the tail vein of the mouse and measuringthe BGLs using a Clarity GL2Plus glucose monitor.Diabetic micewere intraperitoneally injected with a single administration of 10HINS-μPL or INS at an insulin dosage of 0.5 IU/g body weightand 0.05 IU/g body weight, respectively. In addition, two differentgroups of animals, no treated diabetic mice and healthy mice havebeen used as control groups. The non-fasting BGL was monitored atdefined time points  right after the particleinjection and daily in the following 21 days.0–120min) was alsocalculated.IPGTTs were assessedto evaluate the glucose responsiveness ofINS-μPLs at predetermined time points .Mice were fasted overnight with free access to water before the ipadministration of a bolus of glucose (2 g/kg). Briefly, BGLs werechecked each 30 min for 2 h  after injection,and the area under the curve (AUCg for 5 min), and serum insulin levels weredetermined using a mouse insulin ELISA kit (Mercodia), according tothe manufacturer’s protocol.Blood was collected from the tail vein of the fastedmice at thepredetermined time points  for the measurementof serum insulin levels; serum was obtained by centrifugation of blood.The statistical significance was determined by one/two-way ANOVA posthoc Tukey test, and any difference was considered statistically significantwhen p < 0.05.Data were expressed as means ±standard error of the mean, and all the in vitro and in vivo studieswere performed in


P<0.05). Additionally, the immunostaining score of LGALS8, LGALS10 and LGALS13 expression were lower but LGALS1 was higher in caner tissues than those in normal tissues (P<0.001). In conclusion, LGALS10 possibly is a valuable biomarker for predicting a favorable prognosis in patients with ovarian cancer, especially with serous, all stages and grade III cancer.To explore the prognosis of Galectins (LGALS) expression on patients with ovarian cancer, the prognosis of LGALS members in ovarian cancer was retrieved and analyzed by using 'Kaplan-Meier plotter' database. The relation of LGALS to overall survival (OS) was evaluated according to histological subtypes, clinical stages and pathological grade. Quantitative real-time polymerase chain reaction and western blot were used to detect the mRNA and protein expression of LGALS in ovarian cancer and normal ovarian cells. Immunohistochemistry was applied to evaluate the different expression of LGALS between cancer and normal tissues. In total patients with ovarian cancer, LGALS4, LGALS8, LGALS10 and LGALS13 mRNA levels were related to a better OS, and LGALS1 to a worse OS. LGALS1 predicted a worse OS in women with serous, stages III+IV or grade II ovarian cancer. LGALS4 predicted a better OS in patients with endometrioid, stages I+II or grade III ovarian cancer. LGALS10 predicted a longer OS in females with serous, all stages, or grade III cancer. LGALS8 overexpression was related to a better OS in all stages. Notably, mRNA and protein expressions of LGALS4, LGALS10 and LGALS13 were decreased in cancer cells than those in normal cells ( Ovarian cancer has been the most frequent gynecological cancer and the dominating cause of death from gynecological malignant tumors worldwide Galectins (LGALS) are the S-type lectins bound to galactosidase-containing glycoproteins that have a conserved carbohydrate-recognition domain (CRD) with increasing attention as possible regulators in cellular physiology, such as invasion, inflammation, cell adhesion, migration, survival and synthesis Hence, in the present research we performed comprehensive exploration on the prognostic role of LGALS family members in patients with ovarian cancer along with its relations with clinical stages, pathological grades, and histological subtypes. Furthermore, in order to select a more valuable prognostic biomarker for predicting the prognosis of patients with ovarian cancer, the different expression of LGALS members between ovarian cancer cell lines and normal ovarian cell lines was observed, and the different expression between ovarian cancer tissues and normal ovarian tissues was also explored.http://kmplot.com/analysis) An online KM plotter . OVCAR-3 was purchased from American Type Culture Collection (ATCC). OVCAR-3 is a cell line derived from human ovarian epithelial adenocarcinoma, ES2 from ovarian clear cell carcinoma, and A2780 from ovarian undifferentiated carcinoma. A2780, ES2 and IOSE80 cell lines were cultured in DMEM medium (Gibco) plus 10% FBS  in 5% CO2 atmosphere at 37°C, and OVCAR-3 cell was cultured in RPMI-1640 medium with 10% fetal bovine serum.Total RNA was isolated using the TRIzol reagent  according to the manufacturer′s instructions, and cDNAs were synthesized by reverse transcription. qRT‐PCR was conducted with the QuantiNova SYBR Green PCR Kit  under the following reaction conditions: 94.0°C for 30 seconds, followed by 39 cycles of 94.0°C for 5 seconds and 60.0°C for 30 seconds. GAPDH was set as an internal control for gene quantification. The numbers of technical and biological replicates were at least three times for each gene with qRT-PCR analysis. The primers used in this study were displayed as following:Human LGALS1:Forward: 5′-TCTCTCTCGGGTGGAGTCTT-3′Reverse: 5′-GAGATTCAGGTTGCTGGCGA-3′Human LGALS4:Forward: 5′-TCTCACAGGACCAGCCACTA-3′Reverse: 5′-ATCCTGCCCAACCACAAAGT-3′Human LGALS8:Forward: 5′-CCTTGCACTTTCCGGCAATC-3′Reverse: 5′-GGGGGAGGTGTGAGCTACTA-3′Human LGALS10:Forward: 5′-GCGACCACTTGCCTGTTTCT-3′Reverse: 5′-CATGACCACACGACGACCA-3′Human LGALS13:Forward: 5′-AATGTCTTCTTTACCCGTGCCA-3′Reverse: 5′-AGCTGTGGGTCATTGATAAAAGAGT-3′Human GAPDH:Forward: 5′-GACTCATGACCACAGTCCATGC-3′Reverse: 5′-CAGGTCAGGTCCACCACT GA-3′Whole-cell extracts were lysed by lysis buffer, and quantified by the bicinchoninic acid (BCA) method. Protein samples (40 µg) were added to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the membranes of PVDF (Millipore). After blocking with 5% skim milk, blots were incubated with primary antibodies anti‐LGALS1 , anti‐LGALS4 , anti‐LGALS8 , anti‐LGALS10 , anti‐LGALS13  or β-tubulin  at 4°C overnight. The blots were incubated with horseradish peroxidase (HRP)- conjugated secondary antibody at room temperature for 2h, and visualized with chemiluminescence reagent by a Tanon 4100 gel imaging system. The intensities of protein bands were quantified by densitometry analysis using NIH Image J software . Experiments were performed for three times.Upon appropriate approval from the ethics committee at the Second Affiliated Hospital of Wenzhou Medical University, we constructed 10 ovarian cancer tissue specimens obtained from staging surgery or cytoreductive surgery performed for patients with ovarian cancer and 10 normal ovarian tissues samples from patients who receiving surgery because of other gynecological diseases. Inclusion criterion contains nonpregnant, nonbreastfeeding women older than 18 years of age. Immunohistochemistry was performed on the tissue sections (4 μm) from 10 formalin-fixed, paraffin-embedded ovarian tumor tissues and 10 normal ovarian tissues, which have been pathologically confirmed. After dehydrating in xylene and graded ethanol, the slides were incubated with 0.3% hydrogen peroxide and then blocked with 10% normal goat serum. Following these, the sections were incubated with primary antibodies anti‐LGALS1 , anti‐LGALS4 , anti‐LGALS8 , anti‐LGALS10  and anti‐LGALS13  at 4°C overnight. Subsequently, the sections were incubated with secondary antibody, and then detected with 3'3-diaminobenzidine tetrahydrochloride (DAB)  staining and counterstained with hematoxylin. Positive and negative controls were set for each experiment.The staining strength was observed and scored by two authors blindly depending on the positive cell percentage and positive cell staining density. The positive cell percentage was recorded as 0 score with 0% positively stained cells, 1 score with 1%-25% stained cells, 2 score with 26%-50% stained cells, 3 score with 51%-75% stained cells and 4 score with 76%-100% stained cells. The positive cell staining density was graded as 0 with no staining, 1 with light yellow staining, 2 with yellow staining, 3 with brown staining. Finally, the immunoreactivity score (IRS) was evaluated by multiplying the percentage of positively stained cells by the staining intensity (score ranged from 0 to 12). The average value from the two referees was used as the final score.t-test was used for group comparisons. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. Hazard ratio (HR), 95% confidence intervals (95% CI) and log rank P were calculated. P value of <0.05 was considered as statistical significance.Statistical analyses were performed using SPSS 17.0 software . Data was expressed as the mean ± standard deviation of the mean (SD), and Student's http://www.kmplot.com, and no avalibale search data on other members can be found. Therefore, the nine LGALS members were studied and the prognostic values of their mRNA expression in patients with ovarian cancer were presented in Figure  Elevated mRNA expression of LGALS4, LGALS8, LGALS10 and LGALS13 was significantly related to a favorable OS in total patients with ovarian cancer, with HR=0.78 (0.69-0.89), P=0.0003; HR=0.86 (0.76-0.99), P=0.032; HR=0.82 (0.71-0.95), P=0.0065; HR=0.81 (0.71-0.94), P=0.0046; HR=0.87 (0.76-1), P=0.045. In contrast, elevated mRNA level of LGALS1 was significantly associated with a poor OS in total patients with ovarian cancer, with HR=1.35(1.16-1.56), P < 0.001. However, overexpression of LGALS2, LGALS3, LGALS12 and LGALS14 mRNA showed no correlation with OS in total patients with ovarian cancer, with HR=0.89 (0.78-1.03), P=0.11; HR=0.88 (0.76-1.03), P=0.11; HR=1.15 (0.91-1.44), P=0.24; HR=0.89 (0.77-1.02), P=0.096, respectively. These results were displayed in Figure .Among fifteen LGALS family members, merely nine members  could be searched in Table 1). Referring to different tumor histological types, among patients with serous ovarian cancer LGALS10 overexpression predicted a better OS and LGALS1 predicted a worse OS. For patients with endometrioid ovarian cancer, LGALS4 showed a favorable OS. However, LGALS8 and LGALS13 showed a null association with OS either in endometrioid ovarian cancer or in serous ovarian cancer. Furthermore, high expression of LGALS4 and LGALS10 mRNA in patients with grade III was related to a favorable OS, and elevated LGALS1 mRNA expression was correlated with a worse OS in patients with grade II. Nevertheless, high mRNA expression of LGALS8 and LGALS13 showed no correlation with OS among patients with any pathological grades. Regarding with different clinical stages of ovarian cancer, it was observed that elevated LGALS8 and LGALS10 mRNA expression predicted a better OS in patients with stages I+II as well as with stages III+IV, and LGALS4 predicted a better OS in patients with stages I+II. Meanwhile, LGALS1 expression presented a worse OS in patients with stages III+IV, but LGALS13 has no association with OS in patients with any stages.Considering the significant correlation between LGALS level and survival outcomes of patients with ovarian cancer, the association between the prognostic values of LGALS1, LGALS4, LGALS8, LGALS10 and LGALS13 and clinicopathological features was further assessed, including tumor histological types, pathological grades and clinical stages . In addition, LGALS8 mRNA expression in ovarian cancer ES2 and A2780 cell lines was significantly lower than that in normal ovarian cell but its level in OVCAR-3 cell line showed no significant difference with that in normal ovarian cell (P = 0.14). In surprise, LGALS1 mRNA expression in normal ovarian cell was significantly lower than that in ovarian cancer ES2 and A2780 cell lines, but significantly higher than that in ovarian cancer OVCAR-3 cell line (P < 0.05).As displayed in Figure 4, the protein level of LGALS1, LGALS4, LGALS8, LGALS10 and LGALS13 in ovarian cancer cell lines were all decreased compared with those in the normal ovarian cell .As shown in Table 2. The immunohistochemistry results revealed that the staining of LGALS1, LGALS8, LGALS10 and LGALS13 were seen in the cytoplasm of positive cells, but no detectable immunostaining of LGALS4 in cytoplasm, nucleus or membrane was observed either in normal ovarian tissues or in ovarian cancer tissues analyzed . Notably, the staining score of LGALS8, LGALS10 and LGALS13 in ovarian cancer tissues were 3.04±1.24, 1.26± 0.65 and 1.52 ± 0.58, respectively, which were all lower than those in normal ovarian tissues  . Conversely, LGALS1 protein expression in ovarian cancer tissues (8.45±0.63) was significantly higher than that in normal ovarian samples (2.02± 1.05) (P<0.001).The clinical characteristics of patients were displayed in At present, the prognostic role of LGALS family in human cancer has been widely studied, however it is not clear about the roles of LGALS in ovarian cancer. By investigating the prognostic events of nine LGALS family members in patients with ovarian cancer, we found that elevated levels of LGALS4, LGALS8, LGALS10 and LGALS13 were related to a better OS in total patients with ovarian cancer, LGALS1 level was associated with a worse OS in total patients with ovarian cancer, and LGALS2, LGALS3, LGALS12 and LGALS14 had no significant prognostic influence on total patients with ovarian cancer. Hence, in the following discussion we mainly focused on the discussion of LGALS1, LGALS4, LGALS8, LGALS10 and LGALS13.The predictive value of LGALS1 for tumor prognosis has been widely researched in multiple types of cancer. LGALS1 overexpression seems a poor prognostice factor for cancer. For instance, the survival rate of patients with gingival squamous cell carcinoma, lung cancer and colon cancer with strong LGALS1 expression was more significantly associated with a poorer outcome than that of patients with LGALS1 negative or weak expression in vitroLGALS4 has been identified as an adherent junction protein in porcine oral epithelial cells and primarily found in epithelial cells from the tongue to the large intestine but infrequently found in other human tissues Immunohistochemical study reported that LGALS8 expression was elevated in tumor tissues compared with that in normal tissues in bladder, kidney, lung, prostate and stomach Moreover, multiple studies have suggested that LGALS10 was localized mostly in eosinophils and basophils Similarly, LGALS13 has been reviewed highly to have anti-inflammatory functions, which is uniquely expressed more in placental cells Our research was the first study to explore the prognostic fuanction of LGALS family for patients with ovarian cancer. However, there were some limitations in our study that needs to be considered. First, due to no avalibal data of 6 members  in K-M plotter database among these fifteen LGALS members, we did not explore the predictive roles of these 6 LGALS members in ovarian cancer, although previous researches have widely studied their functions. For example, a meta-analysis demonstrated that overexpression of LGALS9 in cancer tissues was related to a longer cancer-specific survival (CSS) and OS in cancer patients In summary, our results showed that LGALS10 is related to a better OS and decreased in ovarian cancer cell and tissues, implying that possibly LGALS10 could be a favorable prognosis predicting marker for patients with ovarian cancer, especially in patients with serous, all clinical stages and grade III cancer. However, future research on biological mechanism is needed to validate the function of LGALS10.