Nonunion of fractures is a major challenge in orthopedics and traumatology, especially with increasing high-energy injuries. Adipose-derived mesenchymal stem cells (ASCs) are a readily accessible source with strong osteogenic potential. This study compared the bone regenerative efficacy of undifferentiated ASCs versus their osteogenically pre-differentiated derivatives in a critical-size femoral nonunion model. Eighteen male New Zealand White rabbits (n = 18), aged 3 months and weighing 2.94 ± 0.10kg, were included. MSCs were isolated enzymatically from abdominal adipose tissue of three donor rabbits. At passage 3, cells were cultured either in basal medium (undifferentiated cohort) or osteogenic medium containing β-glycerophosphate, dexamethasone, and ascorbic acid for 4 weeks (differentiated cohort). 5-bromo-2'-deoxyuridine solution (BrdU) labeling was performed. In this randomized controlled in vivo study, a 5-mm mid-femoral defect was created and stabilized with angular-stable plating. In this randomized, controlled in vivo experimental study, on postoperative day 7 the animals were allocated to three groups (n = 5 per group): control (defect alone), undifferentiated mesenchymal stem cells, and differentiated mesenchymal stem cells. Radiographic assessment of bridging callus and cortical thickness index was performed at 4 and 6 weeks; serum total protein, calcium, and alkaline phosphatase were measured. In vitro, osteogenic differentiation induced progressive mineralization (Alizarin Red staining) and a statistically significant 34% reduction in BrdU corrected total cell fluorescence (CTCF) (p = 0.0473). In vivo, both cell-augmented groups accelerated fracture consolidation versus controls. The differentiated cohort achieved 88% bridging callus at 6 weeks (versus 80% in the undifferentiated group), with superior improvements in bridged cortices (+ 9.4%), bridging callus coverage (+ 10.0%), and distal gap closure (-31.0%) at 6 weeks, plus more pronounced cortical remodeling (average CTI decrease - 6.2% at 4 weeks). Serum alkaline phosphatase (ALP) declined by 53.4% (undifferentiated) and 63.6% (differentiated) versus control (both p < 0.001); plasma calcium was 16.8% higher in the undifferentiated group. Immunofluorescence showed 19% higher BrdU-positive cell density and 12% higher CTCF in undifferentiated cells. Osteogenically pre-differentiated ASCs demonstrated enhanced bone regenerative capacity compared with undifferentiated ASCs. These findings indicate that osteogenic pre-differentiation augments the therapeutic potential of ASCs and support their further evaluation in multimodal strategies for fracture nonunion.

Leptolegnia chapmanii is an oomycete pathogen of mosquito larvae. We investigated whether nutritional factors promoting cyst germination in vitro are associated with early infection events and instar-specific susceptibility in Aedes aegypti. Cysts of the Brazilian isolate ARSEF 12829 germinated rapidly in soybean seed extract, sunflower seed extract and minimal medium supplemented with yeast extract, whereas basal minimal medium did not promote germination. In sunflower seed extract, germination increased significantly with incubation time; in minimal medium, germination at 24 h was much higher with ≥0.2% yeast extract than with 0.1%. In third-instar larvae, a few cysts attached to the cuticle during the first 30-60 min, with no external germ tubes observed. At 3 h, melanized hyphal structures were detected in the midgut, and histological sections showed germinated and ungerminated cysts in the endoperitrophic space, with hyphae crossing the peritrophic matrix and midgut epithelium toward the hemocoel. Mortality increased with cyst concentration and exposure time and decreased with larval instar. At 3.3 × 103 cysts/mL, final mortality reached 100% in L1-L3 and 91.2% in L4 larvae. These results link rapid cyst germination with early midgut invasion and high larvicidal activity.

This study aimed to evaluate the efficacy of cinnabarinic acid (CA) in regulating the barrier function and inflammatory responses of primary intestinal epithelial cells (IECs) derived from chicken embryos subjected to hyperthermia, so as to provide a theoretical basis for alleviating heat stress in broilers. The IECs were randomly divided into 4 groups, a thermoneutral group (37°C), a hyperthermia group (43°C), a hyperthermia + CA group and a hyperthermia + CA + aryl hydrocarbon receptor (AhR) inhibitor group. A hyperthermia cell model was established by exposing IECs to 43°C for 6h, and the experimental treatments consisted of 1 µmol/L CA and 1 µmol/L AhR inhibitor. According to the results, the supplementation of 1 µmol/L CA into the basal culture medium markedly improved the relative cell viability of hyperthermia-treated IECs, decreased the levels of lactate dehydrogenase (LDH), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-1β in cell culture supernatants, downregulated the mRNA expression of heat shock protein (HSP)70, HSP90, claudin2, toll-like receptor (TLR)4, TLR5 and TLR21, increased AhR protein expression and the mRNA expression of occludin, increased the secretion of IL-22 of IECs treated with hyperthermia. Notably, the protective and regulatory effects of CA were markedly abolished by the AhR inhibitor under hyperthermic conditions. In conclusion, CA activated AhR in IECs, promoted IL-22 secretion, inhibited inflammatory responses, increased tight junction proteins genes expression, enhanced epithelial barrier integrity.

Klebsiella pneumoniae is a prominent pathogen causing life-threatening bloodstream infections. Although biofilm formation and resistance to human serum are well-recognized virulence traits, their interrelatedness during K. pneumoniae bloodstream infections remains unclear. Here, we hypothesize that biofilm production is related to K. pneumoniae's ability to thrive in human serum and, therefore, may predict the strains' ability for serum survival. We analyzed 57 clinical, genetically diverse classical K. pneumoniae strains and characterized their survival and biofilm-producing ability in human serum. Serum survival patterns revealed three serum resistance categories-Low, Mid, and High. In addition, the biofilm biomass produced by the strains correlated with their serum resistance level (P < 0.001), and 3D biofilm visualization using confocal microscopy further confirmed that biofilm extracellular polysaccharide substances and biomass patterns were consistent with the serum resistance categories. Moreover, we revealed a direct correlation between the level of biofilm formation and the strain's serum survival level (R2 = 0.696), a prerequisite for systemic K. pneumoniae dissemination. As biofilm formation in serum reflects both survival and biofilm-forming ability, we assessed biofilm formation in defined modified basal medium (BM2), to rule out serum-mediated killing, and discovered a strong and significant association between the serum resistance category and BM2 biofilm biomass (P < 0.0001). By applying regression models, we discovered that biofilm formation serves as a significant predictor for bacterial survival in serum. Overall, our findings establish biofilm production in K. pneumoniae as a biomarker of serum survival and may open a new avenue for predicting bloodstream infection risk in clinical settings.IMPORTANCEBloodstream infections caused by Klebsiella pneumoniae are devastating life-threatening infections worldwide. Understanding the survival strategies of K. pneumoniae in the bloodstream is critical for elucidating key aspects of bacterial pathogenicity and developing new diagnostic and therapeutic modalities. Although serum survival is a recognized virulence trait necessary to thrive in the bloodstream, the relationship between serum resistance and biofilm formation, a multicellular organization that may protect bacteria from bloodstream stressors, remains poorly understood. In this article, we demonstrate biofilm production in human serum by clinical classical K. pneumoniae strains for the first time and discovered a direct correlation between the level of biofilm biomass formation and the degree of serum survival in human serum and in defined modified basal medium. These findings offer insights into the importance of biofilm production in K. pneumoniae serum resistance and may be used to develop future therapeutic strategies targeting bloodstream infections.

Establishment of autoclave conditions for disaccharide-containing liquid media using spectrophotometry and high-performance liquid chromatography.

Protocol for preparing fiber-defined simulated media for growth profiling of diverse human gut bacterial isolates

Antimicrobial postbiotic compounds have attracted increasing interest due to their potential applications in the food industry, particularly as candidates for biopreservation strategies. From a technological perspective, process optimization represents a valuable strategy for improving the production of postbiotic compounds and supporting the development of efficient fermentation bioprocesses. This study aimed to optimize the culture medium to enhance the antibacterial activity of a postbiotic derived from Pediococcus acidilactici CECT 9879. Plackett-Burman design (PBD) was first used to screen carbon and nitrogen sources, followed by one-factor-at-a-time (OFAT) experiments to evaluate yeast-derived ingredients and MRS-derived basal medium components, and response surface methodology based on a central composite design (CCD-RSM) for final medium optimization. Significant linear, quadratic, and interaction effects of key medium components, particularly yeast-derived ingredients and sodium acetate, were identified, indicating well-defined optimal concentration ranges. Under optimized composition, the minimum inhibitory concentration (MIC) against Escherichia coli decreased from 48.33 ± 2.89% (v/v) in the non-optimized medium to 31.67 ± 2.89% (v/v). The results highlight the role of nutrient optimization in modulating postbiotic antibacterial activity during fermentation and provide insights that may contribute to improving process efficiency in the agri-food sector.

Bovine embryonic stem cells (bESCs) are a cornerstone for next-generation applications such as cell-cultured meat, large-animal gene editing, and embryo-stem cell breeding systems. Although primed bESCs and bovine expanded potential stem cells have recently been established, their routine maintenance commonly relies on mouse embryonic fibroblast or bovine fetal fibroblast feeder layers, which are complex, labor-intensive, poorly standardized, and incompatible with scalable manufacturing. Here, we report a simplified feeder-free culture platform for bESCs in which a commercially available basal medium is combined with Growth Factor Reduced Matrigel to generate a three-dimensional, niche-mimetic substrate. Using this approach, we successfully derived a novel feeder-free bESCs (FF-bESCs) that can be robustly expanded for more than 60 passages without ROCK inhibitors, while maintaining a normal karyotype, high proliferation rates, and stable expression of core pluripotency markers. Functional assays confirmed that FF-bESCs possess bona fide pluripotency, as evidenced by their ability to form embryoid bodies in vitro and generate teratomas containing derivatives of all three germ layers in vivo. Transcriptomic profiling further revealed that FF-bESCs align with a formative pluripotent state. Notably, these cells can be directed to differentiate into primordial germ cell-like cells. Our feeder-free culture system provides a scalable, reproducible foundation for advancing bESC-based technologies in bovine precision genome editing and germ cell differentiation for embryo-stem cell breeding systems.

Human mesenchymal stromal cells (MSCs) possess extensive therapeutic potential, and their biological characteristics and functions can be modulated by culture media. This study aimed to systematically evaluate the effects of two basal media, DMEM/F12(DF12) and low-glucose Dulbecco's Modified Eagle Medium (LG-DMEM), on the biological characteristics and functions of umbilical cord-derived MSCs. The influences of basal medium on crucial quality parameters of MSCs, including identity, differentiation potential, proliferation, replicative senescence, genetic stability, gene expression profile, and immunomodulatory capacity, were assessed. MSCs cultured in DF12 exhibited smaller cell size, faster proliferation, higher expression levels of genes associated with growth factor activity, cytokine activity, and extracellular matrix binding, but showed more notable replicative senescence with serial passages. MSCs cultured in LG-DMEM expressed higher levels of indoleamine 2,3-dioxygenase (IDO), had a stronger inhibitory effect on T cell proliferation in vitro, especially on Th17 cells, and showed a better therapeutic effect on alleviating skin lesions in imiquimod (IMQ)-induced psoriasis-like mice. These results suggest that DF12 is more suitable for the early passages of MSCs, potentially favoring applications in injury repair, while LG-DMEM is more suitable for the culture of higher passages of MSCs and enhances efficacy in immune-related diseases. These findings provide insights for selecting the appropriate basal media, a critical process parameter during expansion, contributing to the optimization of MSCs manufacturing bioprocesses to meet the requisite quantity and quality for therapeutic applications.

Fomes fomentarius is a medicinal mushroom from the Northern Hemisphere, recognized for its therapeutic and biotechnological applications. In contrast, tropical Fomes species remain poorly characterized, and information on their properties and cultivation is limited. We evaluated the biotechnological potential of the Neotropical species Fomes fasciatus. Four strains from Paraguay were isolated and assessed for growth on solid media, using both potato dextrose agar and oat seeds, and for indoor basidiomata production using a sawdust-based substrate. The strain showing the best performance on solid media was further evaluated for mycelial growth and exopolysaccharide production in liquid culture. Additionally, the nutritional and mineral composition of wild and cultivated basidiomata and mycelium was analyzed. Fomes fasciatus optimal growth on solid media was observed at approximately 32 °C, with no significant differences in basidiomata yield across strains under indoor cultivation. Successful domestication was achieved for three of the four strains studied. Exopolysaccharide production remained statistically unaffected by elicitor supplementation (i.e., CaCl2 or Tween 40) or variation in carbon and nitrogen sources of the liquid media. In contrast, mycelial biomass was significantly higher in the basal medium than in modified treatments. The wild and domesticated basidiomata and mycelium exhibited high protein content, a balanced amino acid profile, dietary fiber, and unsaturated fatty acids, along with essential macroelements such as zinc. These findings highlight that F. fasciatus is a promising medicinal mushroom species with potential for indoor cultivation, characterized by fast mycelial growth and valuable nutritional and bioactive properties. © 2026 Society of Chemical Industry.

Hyaluronic acid (HA) is a glycosaminoglycan commonly administered orally, and its molecular weight (MW) influences its physicochemical behavior and potential interactions with the gut microbiota. However, MW-dependent effects on community assembly and fermentation-derived metabolites within the low-molecular-weight (LMW) range remain insufficiently resolved. In this study, five HA samples (6.9–35 kDa) were evaluated using an in vitro human fecal fermentation model. Microbial composition was profiled by 16S rRNA gene sequencing, and SCFAs were quantified by UPLC. Compared with the control under the same basal medium, HA supplementation was associated with shifts in community structure and higher alpha diversity. The 6.9 and 9.5 kDa groups were associated with significantly higher total SCFA concentrations, particularly butyrate, than the 13, 17, and 35 kDa groups under the same basal medium. Because soluble starch was present in the fermentation medium, these differences should be interpreted as modulation effects rather than direct evidence of HA-specific fermentation. 16S-based functional prediction suggested MW-dependent differences in predicted central carbohydrate metabolism potential, which were consistent with the observed SCFA patterns but should be interpreted as inferred functional potential rather than direct evidence of pathway activity. These findings indicate that HA molecular weight is associated with differential microbial and SCFA response patterns under the present in vitro conditions. Lower-MW HA within the tested range was associated with higher SCFA output, particularly butyrate, under a shared starch-containing basal medium, highlighting molecular weight as a potential formulation parameter in HA microbiota-centered applications.

Fetal calf serum concentration and sex did not affect the differentiation of human primary osteoblasts, in contrast to dexamethasone.

We investigated the holding condition in collection media to establish transportation system of immature bovine cumulus-oocyte complexes (COCs). We examine holding temperature (38℃ or 20℃), basal media (Lactated Ringer's solution and TCM199) and supplementation of hypoxanthine, an inhibitor of meiotic resumption. As a result, no blastocyst was obtained from COCs held at 38℃ for 6 h or longer, whereas blastocysts were obtained even after 24 h holding in all experimental groups held at 20℃. The blastulation rate was maintained (42%) in TCM199 with hypoxanthine group even after 12 h holding, while reduction of the blastulation rates were observed in all other groups (P < 0.05). After 24 h of storage, Lactated Ringer's solution without hypoxanthine showed the highest blastulation rate (40%). In conclusion, holding temperature of oocytes critically affects the developmental competence of oocytes, and oocytes held at 20°C maintain their developmental competence at least 12 h.

Rosa canina L. is a medicinal and nutritionally valuable species with increasing industrial demand, yet its conventional propagation is limited by low rooting capacity and high genetic heterogeneity. In this study, a complete and reproducible in vitro micropropagation protocol was established, from explant introduction to plantlet acclimatization. Axillary buds were disinfected and introduced into Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP). Shoot multiplication was achieved using sequential cytokinin treatments, and shoot elongation was promoted by adding liquid MS medium containing activated charcoal (AC). The highest and fastest root induction percentage (up to 75%) was obtained on WPM with 2 mg·L−1 IBA and under a 16 h light/8 h dark photoperiod. Light promoted adventitious root formation depending on the nutrient formulation. Thereafter, shoots developed well-structured root systems in vitro, and plantlets fully survived to ex vitro acclimatization. This protocol provides an efficient platform for the large-scale propagation of R. canina and demonstrates that its auxin-driven adventitious rooting is strongly conditioned by the interaction between basal medium composition and photoperiod.

Freeze-dried (FD) somatic cells serve as a novel method for preserving animal genetic resources; however, the efficiency of cloned mice production from FD somatic cells remains low, owing to severe nuclear damage induced by the freeze-drying. In this study, we aimed to mitigate FD-induced damage by evaluating protectants that have been reported to exert protective effects during freeze-drying of microorganisms, and to identify those most effective for somatic cells. Results showed, that using Tris-EGTA as the basal medium for freeze-drying, together with monosodium glutamate (MSG) as a protective agent, significantly reduced DNA damage in FD somatic cells after injection into oocytes. Subsequent optimization of MSG concentration revealed that the addition of 3% MSG markedly increased the formation rate of premature chromosome condensation from 50.0% to 80.4% compared with the non-MSG condition. Moreover, the rate of normal chromosome segregation at the two-cell stage of cloned embryos increased from 0% to 20.0%. Furthermore, although no blastocysts were obtained in the absence of MSG, the addition of 3% MSG enabled the formation of morphologically good-quality blastocysts, albeit at a low frequency. These findings indicated that the addition of 3% MSG reduces DNA damage in FD somatic cells and improves the developmental competence of somatic cell nuclear transfer embryos, representing the first step toward the practical application of FD somatic cells as genetic resources.

Ubiquitination, a post-translational modification, is a critical regulator of intracellular protein function. Ubiquitination modulates protein functions by promoting proteasomal degradation or altering subcellular localization through ubiquitin chain-dependent signaling. Here, we describe two cell lysis methods for detecting SMAD2 ubiquitination levels in HEK293T cells and compare their effectiveness in analyzing protein ubiquitination levels. Protein overexpression in cells was induced by transient transfection. The plasmids (HA-Ub, FLAG-SMAD2, and MYC-SMURF2) and transfection reagents were separately added to basal medium, mixed, and the mixture was added to the cells. Prior to harvest, MG132 was added to inhibit proteasomal degradation and enhance ubiquitinated protein accumulation. The primary divergence between the two experimental approaches is their cell lysis methods-the ice-bath method (performed at 4 °C) and the heat-treatment method (involving incubation at 95 °C), which substantially affects the efficiency of protein lysis. After cell lysis was completed, the cell lysate, agarose beads, and FLAG antibody were mixed and incubated at 4 °C overnight. Ubiquitinated proteins were then detected by western blot analysis. Before detecting ubiquitinated proteins, a light-chain antibody was used for secondary antibody incubation. Then, ubiquitination bands were detected. The results show that both the ice-bath method and the heat-treatment method can be used to detect ubiquitination levels, while the heat-treatment method may make it easier to detect ubiquitination of SMAD2. This study delineates and compares two cell lysis methods for measuring ubiquitination levels in mammalian cells, using SMURF2/SMAD2 as a model, to assist researchers in selecting more appropriate methods for detecting the ubiquitination levels of substrate proteins.

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Chrysanthemum (&lt;em&gt;Chrysanthemum morifolium&lt;/em&gt;) is one of the most economically important ornamental species in Mexico, particularly in the State of Mexico. Optimizing &lt;em&gt;in vitro&lt;/em&gt; propagation protocols is crucial to overcome the limitations of conventional methods and meet the demand for high-quality plants. &lt;strong&gt;Objective:&lt;/strong&gt; To evaluate the effect of different concentrations of benzyladenine (BAP) and sucrose on the &lt;em&gt;in vitro&lt;/em&gt; organogenesis of &lt;em&gt;C. morifolium&lt;/em&gt; var. Indianapolis. &lt;strong&gt;Methodology:&lt;/strong&gt; A completely randomized experimental design with a 3x3 factorial arrangement, plus a control, was established. Three concentrations of BAP (0.0, 0.5, and 1.0 mg L⁻¹) and three of sucrose (30, 45, and 60 g L⁻¹) were evaluated in an MS basal medium. The control consisted of a standardized medium with kinetin (1.0 mg L⁻¹) and sucrose (40 g L⁻¹). Variables of survival, callogenesis frequency, leaf number, main shoot length, and root length were recorded after 70 days of culture. Data were analyzed by ANOVA and Tukey's test (α = 0.05). &lt;strong&gt;Results:&lt;/strong&gt; The combination of 0.5 mg L⁻¹ BAP and 30 g L⁻¹ sucrose (T4) significantly promoted survival (100%), shoot length (15.7 mm), and leaf number (6.2). The highest frequency of callogenesis (95.8%) was observed in the absence of BAP and with the maximum sucrose concentration (T3). Root development was limited across all treatments, observed in only 10% of explants, with no statistically significant differences among the treatments that induced roots. &lt;strong&gt;Implications:&lt;/strong&gt; Optimizing the balance between hormonal and carbon source is fundamental to directing &lt;em&gt;in vitro&lt;/em&gt; morphogenesis. The identified protocol (0.5 mg L⁻¹ BAP and 30 g L⁻¹ sucrose) allows for efficient direct organogenesis, which can lead to faster and more uniform clonal propagation, benefiting the productivity and competitiveness of ornamental growers. &lt;strong&gt;Conclusion:&lt;/strong&gt; The concentration of 0.5 mg L⁻¹ BAP combined with 30 g L⁻¹ sucrose constitutes an optimal stimulus for direct organogenesis in &lt;em&gt;C. morifolium&lt;/em&gt;, maximizing survival and shoot development, while high sucrose concentrations induce a stress response leading to callogenesis.&lt;/p&gt;

To overcome the seasonal constraints of explant availability and facilitate genetic improvement in Catalpa ovata, this study established a dual-pathway in vitro regeneration system (encompassing adventitious shoot organogenesis and somatic embryogenesis) using mature zygotic embryos. We systematically evaluated the synergistic effects of maternal genotypes, plant growth regulators (PGRs), basal media, and the histone deacetylase inhibitor Trichostatin A (TSA). Genotype screening revealed significant divergence in regenerative potential, with the half-sib family 32F17 exhibiting superior responsiveness (84.7% callus induction). A high cytokinin-to-auxin ratio (ZA3 medium) optimally drove direct shoot organogenesis. For adventitious shoot proliferation, the addition of TDZ significantly improved the multiplication coefficient (up to 2.99 on ZB4 medium), although a physiological trade-off with shoot elongation was observed. In parallel, the application of 10 µM TSA significantly enhanced somatic embryogenesis from embryogenic calli, effectively alleviating the inhibitory constraints of exogenous PGRs. For rhizogenesis, the DKW basal medium proved superior to half-strength MS, with the ZE3 treatment (0.1 mg·L-1 NAA + 0.1 mg·L-1 IBA) yielding the highest rooting frequency (69.6%) and robust root architecture. Notably, while somatic embryo conversion remained recalcitrant, plantlets derived exclusively from the adventitious shoot organogenesis pathway were successfully acclimatized ex vitro. These transplanted plantlets exhibited consistently high survival rates (83.1-84.4%) across all tested genotypes, effectively overcoming the initial genotype-dependent recalcitrance. Collectively, this optimized protocol provides a reliable technical platform for the large-scale clonal propagation and biotechnological breeding of C. ovata.

BackgroundRecent advances in swine gut culturomics have substantially expanded our understanding of the cultivable repertoire and revealed the compositional complexity of the porcine gut microbiota. Nevertheless, how multiple environmental and cultivation-related factors interact to shape cultured communities under controlled nutrient conditions remains poorly understood. This study evaluated the relative and interactive contributions of intestinal segment, oxygen availability, cultivation mode, and carbon-substrate identity within a standardized nutrient-rich basal medium framework. Using inocula from the ileum and colon of Wuzhishan piglets, a roughage-tolerant Chinese indigenous breed, microbial communities were cultured on modified yeast extract casitone fatty acid (YCFA) media in which the original carbon substrate was replaced with ten alternative carbohydrates representing simple and complex substrates.ResultsCulture-dependent (CD) sequencing showed that intestinal segment was the dominant factor shaping cultured communities (P < 0 0.001), followed by oxygen availability (P < 0 0.001) and donor identity (P < 0 0.001), whereas, when evaluated within a nutrient-rich basal medium, carbon-substrate identity did not function as an independent or strongly additive driver of community differentiation (P > 0 0.05). A total of 1,308 isolates belonging to 67 genera were obtained through an empirical streaking strategy, including several rare taxa such as Culturomica that were below sequencing detection thresholds, revealing a disparity between CD sequencing profiles and actual strain isolation. Integration of community composition with short-chain fatty acid (SCFA) profiles further indicated that metabolic accumulation represents an additional ecological dimension influencing cultured community differentiation.ConclusionsOur findings demonstrate that under multi-factor cultivation frameworks, ecological filters do not operate in a freely additive manner. Instead, intestinal origin, oxygen exposure, and metabolic context collectively constrain culturability, while carbon-substrate variation alone does not independently drive community clustering within nutrient-rich conditions. Together, this study provides a refined conceptual framework for interpreting culture-dependent outcomes and for designing multi-factor cultivation strategies in future swine gut culturomics research.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-026-04955-3.

The global challenge of feeding a growing population while minimizing environmental impacts necessitates novel food production systems. Plant cellular agriculture offers a sustainable alternative for producing food ingredients; however, its commercial viability is hindered by the high costs and regulatory hurdles associated with conventional reagent-grade culture media. In this study, we developed a novel, cost-effective, and food-grade basal culture medium, FG-N6CI, for rice cellular agriculture. By replacing reagent-grade basal-medium components of the N6CI medium with food-grade alternatives, specifically by substituting chemical reagents with yeast extract, kelp powder, manganese yeast, and a boron supplement, we formulated a food-grade basal nutrient composition while retaining reagent-grade phytohormones. Rice (Oryza sativa L. 'Taichung 65') callus cultured on FG-N6CI medium exhibited significantly higher fresh weight (7.1 g) than the conventional N6CI medium (5.8 g) after 35 days (p < 0.05). Gene expression analysis showed no significant differences between the expression of OsHDA710 and OsTIR1, suggesting that FG-N6CI supports normal cellular proliferation and signaling similar to the standard medium. Economically, the cost of FG-N6CI medium was reduced by approximately 72% compared with that of the commercial reagent-grade mixture (219 JPY/L vs. 795 JPY/L). These results demonstrate that FG-N6CI is an economically competitive basal medium for scaling-up plant cellular agriculture.