Sources of Rapid and Delayed New Lower Jaw Input in the Forepaw Barrel Subfield (FBS) in Rat Primary Somatosensory Cortex (SI) Following Forelimb Deafferentation.

Previously, we reported an immediate emergence of new lower jaw input to the anterior forepaw barrel subfield (FBS) in primary somatosensory cortex (SI) following forelimb deafferentation. However, a delay of 7 weeks or more post-amputation results in the presence of this new input to both anterior and posterior FBS. The immediate change suggests pre-existing latent lower jaw input in the FBS, whereas the delayed alteration implies the involvement of alternative sources. One possible source for immediate lower jaw responses is the neighboring lower jaw barrel subfield (LJBSF). We used anatomical tracers to investigate the possible projection of LJBSF to the FBS in normal and forelimb-amputated rats. Our findings are as follows: (1) anterograde tracer injection into LJBSF in normal and amputated rats labeled fibers and terminals exclusively in the anterior FBS; (2) retrograde tracer injection in the anterior FBS in normal and forelimb-amputated rats, heavily labeled cell bodies predominantly in the posterior LJBSF, with fewer in the anterior LJBSF; (3) retrograde tracer injection in the posterior FBS in normal and forelimb-amputated rats, sparsely labeled cell bodies in the posterior LJBSF; (4) retrograde tracer injection in anterior and posterior FBS in normal and forelimb-amputated rats, labeled cells exclusively in ventral posterior lateral (VPL) nucleus and posterior thalamus (PO); (5) retrograde tracer injection in LJBSF-labeled cell bodies exclusively in ventral posterior medial thalamic nucleus and PO. These findings suggest that LJBSF facilitates rapid lower jaw reorganization in the anterior FBS, whereas VPL and/or other subcortical sites provide a likely substrate for delayed reorganization observed in the posterior FBS.

The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea.

BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

Effect of silymarin on lipid profile and glycemic control in patients with type 2 diabetes mellitus.

This randomized clinical trial was conducted to evaluate the effects of silymarin supplementation on glycemic indices and serum lipid profile in type 2 diabetes mellitus (T2DM) patients. In this open-label randomized clinical trial study, 48 patients with T2DM were eligible to participate for 12 weeks and were divided into two groups randomly: 24 subjects in the intervention (received three 140 mg silymarin capsules daily and diet plan) and 24 in control (received a diet plan). Fasting blood samples and anthropometric data were collected, and glycemic indices and lipid profiles were determined at baseline and at the end of the study. Out of 60 patients included in the clinical trial, 48 people completed the study. In comparing silymarin and control groups before and after the study, a significant reduction was observed in weight and body mass index. However, after adjustment, no significant difference was seen between the two groups. Furthermore, daily consumption of three capsules of 140 mg silymarin for 12 weeks did not show any significant difference on the level of fasting blood sugar (p = 0.789), HbA1c (p = 0.719), and lipid profile. The findings of the present study show that silymarin did not lead to changes in the level of glycemic index and lipid profile in patients with T2DM.

Structural basis of sugar recognition by SCFFBS2 ubiquitin ligase involved in NGLY1 deficiency.

The cytosolic peptide:N-glycanase (PNGase) is involved in the quality control of N-glycoproteins via the endoplasmic reticulum-associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F-box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man3GlcNAc2, which corresponds to the core pentasaccharide of N-glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar-binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency.

Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation.

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-ß-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

The Impact of Low-Volume High-Intensity Interval Training (LV-HIIT) on Fatty Liver Index (FLI) and Estimated Glomerular Filtration Rate (eGFR) in Patients with Type 2 Diabetes Mellitus (T2DM).

BACKGROUND: Prevention and reduction of liver fat accumulation and maintenance of Glomerular Filtration Rate (GFR) have been proposed as important therapeutic goals in patients with Type 2 Diabetes Mellitus (T2DM). AIM: This study aimed to determine the effect of Low-Volume High-Intensity Interval Training (LV-HIIT) on fatty liver index (FLI) and GFR estimation in patients with T2DM. METHODS: This randomized controlled trial included 80 patients with T2DM and a sedentary lifestyle, randomly divided into HIIT (n=40) and a control group (n=40). Patients with a history of T2DM for at least one year and HbA1C levels between 6.4% and 10% were selected. The intervention group underwent a 4-week LV-HIIT course, comprising 3 sessions per week, while the control group did not receive any intervention. FLI, eGFR, anthropometric measurements, and laboratory variables were assessed in all participants before and after the intervention. RESULTS: FLI (62.0 at baseline, 53.0 at follow-up) significantly decreased in the LV-HIIT group after the intervention, while eGFR (71.0 at baseline, 73.6 at follow-up) significantly increased (P<0.001). However, the control group showed a significant reduction only in Fasting Blood Sugar (FBS) (P<0.05). After the intervention, the LV-HIIT group had significantly lower FBS (129.0 at baseline, 121.0 at follow-up), Alanine Aminotransferase (ALT) (24.0 at baseline, 18.0 at follow-up), and Gamma-Glutamyl Transferase (GGT) (22.0 at baseline, 19.0 at follow-up), as well as higher eGFR, compared to the control group (P<0.05). CONCLUSIONS: LV-HIIT exercise appears to be a promising and effective training method for improving FLI and eGFR in patients diagnosed with T2DM.

Plant-based hydrolysates as building blocks for cellular agriculture.

Cellular agriculture, an emerging technology, aims to produce animal-based products such as meat through scalable tissue culture methods. Traditional techniques rely on chemically undefined media using fetal bovine serum (FBS) or chemically defined media utilizing specific growth factors. To be a viable alternative to conventional meat production, cellular agriculture requires cost-effective materials with established supply chains for growth media. Here, we investigate hydrolysates from Kikuyu grass, Alfalfa grass, and cattle rearing pellets. We identified conditions that promote C2C12 myoblast cell growth in media containing 0.1% and 0% serum. These effects are more pronounced in combination with existing growth promoters such as insulin, transferrin, and selenium. Overall, the rearing pellet hydrolysates were most effective in promoting growth particularly when in combination with the growth promoters. Our findings suggest that leveraging these materials, along with known growth factors, can facilitate the development of improved, scalable, and commercially viable media for cellular agriculture.

A Systematic Review and Meta-Analysis on the Role of Bempedoic Acid in Cardiovascular Outcomes for Patients With Statin Intolerance.

Atherosclerosis, a multifaceted pathogenic process affecting the arteries and aorta, poses a significant threat because of its potential to impede or entirely obstruct blood flow by narrowing blood vessels. This intricate progression involves various factors such as dyslipidemia, immunological responses, inflammation, and endothelial dysfunction. The initial phase manifests as the formation of fatty streaks, considered a pivotal hallmark in the inception of atherosclerotic plaques, a process that can commence as early as childhood. Over time, this process evolves, characterized by the thickening of the arterial inner layer (intima) and accumulation of lipid-laden macrophages, commonly known as foam cells, along with the buildup of the extracellular matrix. Subsequent stages witness the proliferation and aggregation of smooth muscle cells, culminating in the formation of atheroma plaques. As these lesions progress, apoptosis can occur in the deeper layers, further recruiting macrophages, which may undergo calcification and transform into atherosclerotic plaques. Notably, mechanisms such as arterial remodeling and intraplaque hemorrhage also contribute significantly to the progression of atherosclerotic cardiovascular disease, although these facets fall beyond the scope of this article. This study aimed to systematically review and conduct a meta-analysis of randomized controlled trials investigating the efficacy and safety of bempedoic acid in statin-intolerant patients with hyperlipidemia and to provide conclusions and recommendations accordingly. A systematic search of databases, such as PubMed, Web of Science, and Embase, will be performed. Only randomized trials will be included comparing bempedoic acid with placebo in statin-intolerant patients. This study aimed to provide a comprehensive understanding of the role of bempedoic acid in managing hyperlipidemia in statin-intolerant patients. In primary prevention, for patients unable to tolerate recommended statins, bempedoic acid was associated with a significant reduction in major adverse cardiovascular events (MACE) as the primary endpoint.

Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances.

The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

Potential of animal-welfare compliant and sustainably sourced serum from pig slaughter blood.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

Effects of licorice extract in combination with a low-calorie diet on obesity indices, glycemic indices, and lipid profiles in overweight/obese women with polycystic ovary syndrome (PCOS): a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common ovarian dysfunction. Recent studies showed the effectiveness of licorice on metabolic profiles with inconsistent findings. So, we investigated the effect of licorice on obesity indices, glycemic indices, and lipid profiles in women with PCOS. METHODS: This randomized, double-blind, placebo-controlled trial was performed on 66 overweight/obese women with PCOS. The participants were randomly assigned to receive either 1.5 gr/day licorice extract plus a low-calorie diet (n = 33) or placebo plus a low-calorie diet (n = 33) for 8 weeks. Participants' anthropometric indices and body composition were assessed using standard protocols. Fasting blood sugar (FBS), insulin levels, low-density lipoprotein-cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and high-density lipoprotein-cholesterol (HDL-C) were measured using enzymatic kits. The homeostasis model assessment-insulin resistance (HOMA-IR) and HOMA of ß-cell function (HOMA-B) were calculated using valid formulas. RESULTS: Between-group comparisons demonstrated significant differences between the groups in terms of obesity indices (body weight, BMI, and body fat), lipid profiles (TG, TC, LDL-C, and HDL-C), FBS and insulin levels, HOMA-IR, and HOMA-B at the end of the study (P < 0.05). Supplementation with licorice plus a low-calorie diet was also more effective in improving all parameters than a low-calorie diet alone after adjusting for confounders (baseline values, age, weight changes, and physical activity changes) (P < 0.05). CONCLUSION: The findings showed that licorice consumption leads to improvements in obesity indices, glucose homeostasis, and lipid profiles compared to placebo. Due to possible limitations of the study, further research is needed to confirm these findings.

Point prevalence of metabolic syndrome in HIV positive patients.

Introduction: Human immunodeficiency virus (HIV)-related morbidity and mortality have declined over time, but this increased longevity may lead to the development of other diseases, which may further manifest as the metabolic syndrome (MS). Method: To find out the point prevalence of MS in HIV positive patients, a cross-sectional prospective observational study was conducted on 200 patients who approached ART plus Centre of Government Medical College and Hospital Jammu, including 50 symptomatic patients HIV negative as controls. Results: The mean age group in MS was 37.85 ± 6.61. Males consisted of 55% (110) and females consisted of 45% (90). The overall prevalence of MS was 13.5%, with prevalence in males being 16.3% and in females 10%. Patients receiving first line highly active antiretroviral therapy (HAART) showed a 24% prevalence, while that of second line HAART showed a 14% prevalence. Central obesity (47.3%) was the most common component of MS followed by hyperglycemia (43.3%), hypertriglyceridemia (38.6%), and low high density cholesterol (HDL-C) level (38.6%). Out of 84 males with MS, 94% (79) males were having hypertriglyceridemia, 88% (74) were hypertensive, and 72% (60) were having FBS >=100. Out of 66 females with MS, 100% (66) females had central obesity and 88% (58) had hypertriglyceridemia and low HDL-C levels. Conclusion: The metabolic complications as a result of treatment with HAART leave HIV patients at a risk of developing cardiovascular disease and diabetes in spite of improvements in morbidity and mortality. Risk factors like central obesity, hypertension, hyperglycemia, and hypertriglyceridemia should be taken into consideration well before to prevent the add-on effect of developing MS.

The effect of psyllium on fasting blood sugar, HbA1c, HOMA IR, and insulin control: a GRADE-assessed systematic review and meta-analysis of randomized controlled trials.

There is equivocal evidence that psyllium can prevent or attenuate increases in fasting blood sugar. Therefore, this systematic review and meta-analysis sought to investigate the influence of psyllium on hemoglobin A1C (HbA1c), fasting blood sugar (FBS), insulin, and Homeostatic Model Assessment of Insulin Resistance (HOMA IR). We searched PubMed, ISI Web of Science (WOS), and Scopus for eligible publications, up to 15 July 2022, including randomized controlled trials (RCT) assessing the effect of psyllium on HbA1c, FBS, insulin, and HOMA IR levels in adults. Using a random effects model, we report the weighted mean differences (WMD) with 95% confidence intervals (CI). In this article, 19 RCT studies, consisting of 962 participants, were included. Psyllium significantly decreased FBS, HbA1c, and HOMA IR levels, but not insulin levels, as compared to placebo (FBS: WMD): -6.89; 95% CI: -10.62, -3.16; p < .001), HbA1c: (WMD: -0.75; 95% CI: -1.21, -0.29; p < .001), HOMA IR: (WMD: -1.17; 95% CI: -2.11, -0.23; p < .05), and insulin: (WMD: -2.08; 95% CI: -4.21, -0.035; p > .05)). Subgroup analyses illustrated differences in the effects of psyllium on FBS: dosages less than and more than 10 g/d showed significant differences (p value < 0.05). However, it was not significant in intervention durations less than 50 days (p value > 0.05). For HbA1c: psyllium consumption less than 10 g/d (p value > 0.05) was non-significant. For HOMA IR and insulin: no significant changes were noted with psyllium consumption less than vs. more than 10 g/d. In conclusion, we found that psyllium could significantly decrease FBS, HbA1c, and HOMA IR levels, but not insulin levels, as compared to placebo.

Evaluation of Human Platelet Lysate as an Alternative to Fetal Bovine Serum for Potential Clinical Applications of Stem Cells from Human Exfoliated Deciduous Teeth.

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Evaluating the Impact of Intensifying Treatment from Human to Analogue Insulin on Glycaemic Control and Insulin Expenditure in Patients with Type 2 Diabetes: A Retrospective Cohort Study.

Background: Achieving good glycaemic control is essential to reducing the risk of diabetes complications. Insulin is the most effective therapy for achieving good glycaemic control; however, it is associated with a higher risk of hypoglycaemia, especially with human insulin. This study aimed to evaluate the efficacy of intensification from human to analogue insulin and its added cost. Methods: This retrospective study was conducted at the Hospital Universiti Sains Malaysia (HUSM). Patients with type 2 diabetes mellitus (T2DM) who underwent intensification for at least 3 months from human to analogue insulin were included in this study. The patients' medical records, haemoglobin A1c (Hba1c) and fasting blood sugar (FBS) were retrieved. The total cost pre- and post-intensification of insulin was obtained from the pharmacy database. Differences in HbA1c, FBS and total insulin cost pre- and post-intensification were analysed. Results: A total of 163 patients with T2DM who had intensification from human to analogue insulin were included in this study. HbA1c and FBS levels were significantly lower in analogue insulin. However, the differences were not clinically significant, as the mean reduction in HbA1c was less than 0.5%. Meanwhile, the total costs of analogue insulin for 3 months were higher. Conclusion: There were no clinically significant improvements in patients' HbA1c and FBS after the intensification of insulin, despite the extra costs spent. Hence, it is vital to choose the right group of patients to receive an insulin analogue to maximise its benefit but at the most optimal cost.

1D and 2D NMR for KRAS:Ligand Binding.

Fragment-based screening by ligand-observed 1D NMR and binding interface mapping by protein-observed 2D NMR are popular methods used in drug discovery. These methods allow researchers to detect compound binding over a wide range of affinities and offer a simultaneous assessment of solubility, purity, and chemical formula accuracy of the target compounds and the 15N-labeled protein when examined by 1D and 2D NMR, respectively. These methods can be applied for screening fragment binding to the active (GMPPNP-bound) and inactive (GDP-bound) states of oncogenic KRAS mutants.

Preliminary study on comparison of egg extraction methods for development of fetal bovine serum substitutes in cultured meat.

Fetal bovine serum (FBS) substitution remains one of the challenges to the realization of cultured meat production in the marketplace. In this study, three methods were developed to extract a substitute for FBS using egg white extract (EWE): using 25 mM CaCl2/2.5 % ammonium sulfate/citric acid (A); ethyl alcohol (B); and 5 % ammonium sulfate/citric acid (C). B EWE can effectively replace up to 50 % of FBS in growth media (10 % of the total). Ovalbumin in the extracts can promote cell proliferation, and components along the 12 kDa protein band have the potential to inhibit cell proliferation. Chick primary muscle cells applied with B EWE, an edible material that improved the cost and time efficiency of cultured meat production, effectively proliferated/differentiated. Therefore, EWE extracted using ethyl alcohol may be used as an FBS substitute to reduce animal sacrifices and should be considered a viable alternative to FBS for cultured meat.

Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.

Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.

Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Comparison between Platelet Lysate, Platelet Lysate Serum, and Fetal Bovine Serum as Supplements for Cell Culture, Expansion, and Cryopreservation.

As cell culture supplements, human platelet lysate (PL) and human platelet lysate serum (PLS) are alternatives to fetal bovine serum (FBS) due to FBS-related issues such as ethical concerns, variability between batches, and the possible introduction of xenogenic contaminants. This study compared the composition and efficacy of PL, PLS, and FBS as supplements in the culture and cryopreservation of human dermal fibroblasts, Wharton's jelly-derived mesenchymal stem cells (WJ-MCS), and adipose tissue (AdMSC). Biochemical components, some growth factors, and cytokines present in each of them were analyzed; in addition, the cells were cultured in media supplemented with 5% PL, 5% PLS, and 10% FBS and exposed to different freezing and thawing solutions with the supplements under study. Biochemical parameters were found to be similar in PL and PLS compared to FBS, with some differences in fibrinogen and calcium concentration. Growth factors and cytokines were higher in PL and PLS compared to FBS. Cell proliferation and morphology showed no significant differences between the three culture media. Regarding the cryopreservation and thawing of cells, better results were obtained with PLS and FBS. In conclusion, PL and PLS are an excellent choice to replace the standard supplement of animal origin (FBS) in the media used for the culture and cryopreservation of fibroblasts, WJ-MSC, and AdMSC.