Biotechnology and Bioengineering最新文献

Cost-Efficient Autotrophic High-Cell-Density Cultivation of Cupriavidus necator Enabled by Model-Based Gas Supply. 基于模型的供气系统支持的高效自养高细胞密度培养赤铜鱼。

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-10-09 DOI: 10.1002/bit.70079

Vera Lambauer,Clemens Hagenbuchner,Maximilian Graber,Helmar Wiltsche,Vanja Subotić,Christoph Hochenauer,Markus Reichhartinger,Regina Kratzer

{"title":"Cost-Efficient Autotrophic High-Cell-Density Cultivation of Cupriavidus necator Enabled by Model-Based Gas Supply.","authors":"Vera Lambauer,Clemens Hagenbuchner,Maximilian Graber,Helmar Wiltsche,Vanja Subotić,Christoph Hochenauer,Markus Reichhartinger,Regina Kratzer","doi":"10.1002/bit.70079","DOIUrl":"https://doi.org/10.1002/bit.70079","url":null,"abstract":"Cultivating hydrogen-oxidizing bacteria (HOB), such as Cupriavidus necator, using H 2 ${{rm{H}}}_{2}$ , CO 2 ${text{CO}}_{2}$ , and O 2 ${{rm{O}}}_{2}$ offers a promising route for CO 2 ${text{CO}}_{2}$ valorization into chemicals and materials. To enhance cultivation efficiency in a lab-scale gas fermenter lacking a gas recycling system, an automated gas supply strategy based on real-time CO 2 ${text{CO}}_{2}$ and O 2 ${{rm{O}}}_{2}$ monitoring was developed. Fine-tuning gas delivery is essential to ensure an adequate supply for cellular growth while minimizing excess gas, particularly H 2 ${{rm{H}}}_{2}$ , that leaves the bioreactor unused, to improve process economics. In the absence of ATEX-compliant H 2 ${{rm{H}}}_{2}$ sensors, a soft sensor was implemented to estimate dissolved H 2 ${{rm{H}}}_{2}$ concentrations from O 2 ${{rm{O}}}_{2}$ uptake rates and growth phase identification. Total gas flow was controlled according to the O 2 ${{rm{O}}}_{2}$ requirements of the cells. This strategy reduced overall gas and H 2 ${{rm{H}}}_{2}$ consumption by 67%. In addition, a high-cell-density medium was formulated by integrating published recipes with Inductively Coupled Plasma Optical Emission Spectroscopy and nutrient inhibition testing. The optimized medium increased biomass yield from 15 g/L to 53 g/L, with 75% of the dry weight consisting of the bioplastic poly(3-hydroxybutyrate), without requiring nutrient addition or pH control. Together, these strategies improve the scalability, efficiency, and sustainability of CO 2 ${text{CO}}_{2}$ -based cultivation of hydrogen-oxidizing bacteria.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"12 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145247000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Investigation Into the Role of Reductants and Cosubstrates in Lytic Polysaccharide Monooxygenase Thermothielavioides terrestris AA9E Binding to Cellulose by Single-Molecule Imaging. 还原剂和辅底物在热thielavioides terrestris AA9E与纤维素结合过程中的作用

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-10-09 DOI: 10.1002/bit.70080

Benedikt M Blossom,Peter M Goodwin,Camilla Fløien Angeltveit,Svein Jarle Horn,Alex O Hitomi,Tina Jeoh

{"title":"Investigation Into the Role of Reductants and Cosubstrates in Lytic Polysaccharide Monooxygenase Thermothielavioides terrestris AA9E Binding to Cellulose by Single-Molecule Imaging.","authors":"Benedikt M Blossom,Peter M Goodwin,Camilla Fløien Angeltveit,Svein Jarle Horn,Alex O Hitomi,Tina Jeoh","doi":"10.1002/bit.70080","DOIUrl":"https://doi.org/10.1002/bit.70080","url":null,"abstract":"Cellulose-active Lytic Polysaccharide Monooxygenases (LPMO) facilitate plant cell wall deconstruction by attacking ordered regions of cellulose. In vitro, reductants (e.g., ascorbic acid) reduce LPMOs to Cu(I)-LPMO, and hydrogen peroxide (H2O2) serves as co-substrate for oxidative cleavage of cellulose glycosidic bonds. Super-resolution single-molecule imaging by total internal reflection fluorescence microscopy was used to visualize and enumerate binding events of fluorescently-labeled Thermothielavioides terrestris AA9E (TtAA9E) on highly ordered cellulose fibrils in oxygen-scavenging buffer systems. In the glucose oxidase/catalase (GODCAT) system, oxygen is converted to H2O2, then removed by catalase. Adding ascorbic acid to the GODCAT system promoted rapid binding to cellulose by TtAA9E. In contrast, absent both oxygen and H2O2 in the protocatechuic acid/protocatechuate 3,4-dioxygenase (PCA/PCD) oxygen-scavenging system, adding ascorbic acid nearly eliminated cellulose binding by TtAA9E. Our results suggest that in the GODCAT system, TtAA9Es are reduced by ascorbic acid and activated by H2O2, facilitating binding to cellulose. In the PCA/PCD system, reduced TtAA9Es are not activated due to the lack of H2O2, suggesting that reduced Cu(I)-TtAA9E cannot bind to cellulose without H2O2. Notably, in the PCA/PCD system with ascorbic acid, oxidized sugar release initially lagged but was observed at longer reaction times, suggesting that H2O2 could be a limiting reactant generated in situ as oxygen becomes absorbed into solution. Binding durations of LPMO to cellulose were independent of experimental conditions: ( 82% ± 6%) of cellulose-bound LPMOs resided briefly for 14 ± 2.5 s, while 16% ± 5% of the bound enzymes remained for 60 ± 9 s.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"30 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145254765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biotechnology and Bioengineering: Volume 122, Number 11, November 2025 生物技术和生物工程：第122卷，第11号，2025年11月

IF 3.6 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-10-07 DOI: 10.1002/bit.28751

引用次数: 0

Comprehensive Mapping of Functional Enhancers in Chinese Hamster Ovary Cells. 中国仓鼠卵巢细胞功能增强因子的全面定位。

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-10-03 DOI: 10.1002/bit.70076

María Santos,Yusuf B Johari,Laura Biggins,Natalie C Elliott,Stefan Schoenfelder,Mounika Boddireddy,Daniel K Fabian,Michael Anbar,Peter M O'Callaghan,Peter J Rugg-Gunn

{"title":"Comprehensive Mapping of Functional Enhancers in Chinese Hamster Ovary Cells.","authors":"María Santos,Yusuf B Johari,Laura Biggins,Natalie C Elliott,Stefan Schoenfelder,Mounika Boddireddy,Daniel K Fabian,Michael Anbar,Peter M O'Callaghan,Peter J Rugg-Gunn","doi":"10.1002/bit.70076","DOIUrl":"https://doi.org/10.1002/bit.70076","url":null,"abstract":"Chinese hamster ovary (CHO) cells are the leading mammalian system for recombinant therapeutic protein production. However, optimizing transgene expression remains challenging due to the limited understanding of the regulatory mechanisms controlling gene expression in CHO cells. Towards overcoming this barrier, here we provide a systematic characterization of cis-regulatory elements in CHO cells. Using genome-wide STARR-seq, a high-throughput method for quantifying enhancer strength, we identified regions with enhancer activity in the CHO cell genome. By integrating these data with ATAC-seq and histone modification profiles, we were able to characterize the chromatin state of these regions. Our analysis revealed thousands of newly identified enhancer sequences. The most active sequences could drive transgene expression at levels similar to or higher than strong viral enhancers. Notably, half of the regions found to have enhancer activity were within inaccessible chromatin in their native context. We observed that accessible enhancers were primarily near to transcriptional start sites and associated with ubiquitously-expressed genes, whereas inaccessible enhancers were predominantly intergenic and associated with tissue-specific genes. Additionally, through a deep-learning-based approach ETS and YY1 transcription factor (TF) binding motifs were identified as key determinants of enhancer identity and strength. Disrupting YY1 binding motifs led to reduced enhancer activity, thereby highlighting the importance of YY1 as a transcriptional activator in CHO cells. Our study demonstrates the first comprehensive map of functionally-validated enhancers in CHO cells and generates new insights into gene regulation and the role of TFs in determining enhancer strength. This study helps to lay the foundation for strategic engineering of CHO cell transcriptional networks to achieve enhanced biopharmaceutical production.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"131 1 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145209296","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Porin-Independent Uptake of Small Molecule Antibiotics Facilitated by Escherichia coli Outer Membrane Vesicles. 大肠杆菌外膜囊泡促进小分子抗生素的非孔蛋白摄取。

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-10-03 DOI: 10.1002/bit.70078

Meishan Wu,Rachael M Harrower,Ziang Li,Angela C Brown

{"title":"Porin-Independent Uptake of Small Molecule Antibiotics Facilitated by Escherichia coli Outer Membrane Vesicles.","authors":"Meishan Wu,Rachael M Harrower,Ziang Li,Angela C Brown","doi":"10.1002/bit.70078","DOIUrl":"https://doi.org/10.1002/bit.70078","url":null,"abstract":"The development of novel antimicrobial agents that are effective against Gram-negative bacteria is hindered by the dual membrane cell envelope of these bacteria. To reach their intracellular targets, most small-molecule antibiotics must first pass through protein channels called porins; however, a common mechanism of acquired resistance is decreased expression of these outer membrane proteins. Additionally, parameters such as size, shape, and charge regulate passage of antibiotics through porins, further limiting the design space of novel antibiotic molecules. Inspired by the ability of bacterial outer membrane vesicles (OMVs) to deliver cargo to the bacterial cytosol, we hypothesized that encapsulation of small molecule antibiotics within OMVs would improve the activity of the drugs by facilitating uptake. To test this hypothesis, we investigated the ability of imipenem-encapsulated OMVs to inhibit the growth of several Gram-negative bacteria, including multidrug-resistant (MDR) clinical isolates. Our results demonstrated that encapsulation within OMVs significantly lowers the effective concentration of imipenem in several MDR isolates. Using a panel of porin knockout strains, we further demonstrated that this mechanism of antibiotic delivery does not require porin expression. Together, our results demonstrate the potential of OMVs as novel antibiotic delivery vehicles to treat antibiotic-resistant bacterial infections by improving drug uptake.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"5 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145209289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Reinforcement Learning for Robust Dynamic Metabolic Control 鲁棒动态代谢控制的强化学习

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-10-02 DOI: 10.1002/bit.70077

Sebastián Espinel‐Ríos, River Walser, Dongda Zhang

{"title":"Reinforcement Learning for Robust Dynamic Metabolic Control","authors":"Sebastián Espinel‐Ríos, River Walser, Dongda Zhang","doi":"10.1002/bit.70077","DOIUrl":"https://doi.org/10.1002/bit.70077","url":null,"abstract":"Dynamic metabolic control allows key metabolic fluxes to be modulated in real time, enhancing bioprocess flexibility and expanding available optimization degrees of freedom. This is achieved, for example, via targeted modulation of metabolic enzyme expression. However, identifying optimal dynamic control policies is challenging due to the generally high‐dimensional solution space and the need to manage metabolic burden and cytotoxic effects arising from inducible enzyme expression. The task is further complicated by stochastic dynamics, which reduce bioprocess reproducibility. We propose a reinforcement learning framework to derive optimal policies by allowing an agent (the controller) to interact with a surrogate dynamic model. To promote robustness, we apply domain randomization, enabling the controller to generalize across uncertainties. When transferred to an experimental system, the agent can in principle continue fine‐tuning the policy. Our framework provides an alternative to conventional model‐based control such as model predictive control, which requires model differentiation with respect to decision variables; often impractical for complex stochastic, nonlinear, stiff, and piecewise‐defined dynamics. In contrast, our approach relies on forward integration of the model, thereby simplifying the task. We demonstrate the framework in two Escherichia coli bioprocesses: dynamic control of acetyl‐CoA carboxylase for fatty‐acid synthesis and of adenosine triphosphatase for lactate synthesis.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"22 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

AI-Enhanced Continued Process Verification for Ultrafiltration/Diafiltration. 人工智能增强的超滤/滤持续过程验证。

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-09-30 DOI: 10.1002/bit.70075

Naveen G Jesubalan,Nikita Saxena,Vinesh Balakrishnan Yezhuvath,Navnath Deore,Anurag S Rathore

{"title":"AI-Enhanced Continued Process Verification for Ultrafiltration/Diafiltration.","authors":"Naveen G Jesubalan,Nikita Saxena,Vinesh Balakrishnan Yezhuvath,Navnath Deore,Anurag S Rathore","doi":"10.1002/bit.70075","DOIUrl":"https://doi.org/10.1002/bit.70075","url":null,"abstract":"The guidelines from the Food and Drug Administration (FDA) and the European Union Good Manufacturing Practice (EU GMP) Annex 15 necessitate biopharmaceutical manufacturers to uphold continuous control of their processes throughout the product lifecycle, thereby ensuring consistent strength, quality, and purity of the final drug product. As a result, there is enormous interest in continued process verification (CPV) in the biopharmaceutical industry. Typical manufacturing processes generate significant process and analytical data for every manufactured batch. The industry has accepted that manual data collection and statistical trending are labor-intensive and error-prone. In this study, an attempt has been made to streamline CPV for the ultrafiltration-diafiltration unit operation. It entails numerous tasks, including data acquisition using sensors, predictive machine learning models, statistical trending against control limits, process capability assessment (Cpk and Ppk) at defined intervals, fault detection, and a robust process control strategy. We hope the proposed framework will help the biopharmaceutical industry implement CPV and move closer to adopting Industry 4.0.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"7 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145194787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Intrinsically Fluorescent Nano-Scaled Peptide Aggregates Upon Arginine to Citrulline Swap. 本质荧光纳米级肽聚集体精氨酸到瓜氨酸交换。

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-09-29 DOI: 10.1002/bit.70073

Nauman Nazeer,Anupama Ghimire,Jan K Rainey,William D Lubell,Brian D Wagner,Marya Ahmed

{"title":"Intrinsically Fluorescent Nano-Scaled Peptide Aggregates Upon Arginine to Citrulline Swap.","authors":"Nauman Nazeer,Anupama Ghimire,Jan K Rainey,William D Lubell,Brian D Wagner,Marya Ahmed","doi":"10.1002/bit.70073","DOIUrl":"https://doi.org/10.1002/bit.70073","url":null,"abstract":"The supramolecular assembly of short peptides into ordered structures offers promise for developing bio-nanomaterials with diverse applications in drug delivery, electronics, and optical engineering. Intrinsic fluorescence of polypeptide aggregates is typically associated with delocalization of electron densities in dense hydrogen bonding networks, dipolar coupling of aromatic amino acid residues, and possibly by the 'cluster-derived luminescence' associated with supramolecular structures. In a handful of examples, self-assembly of short peptides has provided ordered intrinsically fluorescent nanostructures. In this study, replacement of a single arginine residue with citrulline in a macrocyclic peptide has led to intrinsic fluorescence. The parent arginine-containing peptide macrocycle was previously shown to adopt a β-sheet conformation that aggregated into nonfluorescent spherical particles. The change from the electrostatic positive charge of the guanidine side chain to a hydrogen bonding neutral urea caused the β-sheet peptide to aggregate into larger-sized intrinsically fluorescent rods by a phenomenon that is ascribed to electron delocalization through π-π stacking interactions.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"4 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145182846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Rational Design Strategy for Engineering CH4 Domain of IgE for Heterodimerization. IgE异源二聚化工程CH4结构域的合理设计策略

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-09-27 DOI: 10.1002/bit.70068

Shikha Kumari,Vanessa Siegmund,Achim Doerner,Sanjay Ghosh,Ralf Guenther,Saurabh Joshi,Saravanamuthu Thiyagarajan

{"title":"A Rational Design Strategy for Engineering CH4 Domain of IgE for Heterodimerization.","authors":"Shikha Kumari,Vanessa Siegmund,Achim Doerner,Sanjay Ghosh,Ralf Guenther,Saurabh Joshi,Saravanamuthu Thiyagarajan","doi":"10.1002/bit.70068","DOIUrl":"https://doi.org/10.1002/bit.70068","url":null,"abstract":"The use of bispecific antibodies in cancer immunotherapy is a rapidly expanding domain. Growing relevance of diverse bispecific formats mediating multiple novel mechanisms of action is indicated by the approval of nine bispecific antibodies between 2021 and 2023. Likewise, interest is extending to explore different isotypes of monoclonal antibodies comprising IgA and IgE. Compared to IgG, IgE has been reported to elicit superior antitumour activity in ovarian carcinomas, melanomas, and breast carcinomas. Exploring other isotypes, such as IgE, in a bispecific format may enhance therapeutic applicability and efficacy in the case of solid tumors. However, studies investigating IgE antibodies in bispecific formats are limited due to missing technologies for heterodimerization with only one study providing a proof of concept for the development of bispecific IgE. Our study explored a computational approach for designing heterodimeric IgE antibodies on the basis of the conventional IgG knob-into-hole (KiH) strategy. Furthermore, the resulting novel variant of heterodimeric IgE retained target binding and Fc epsilon receptor engagement, thus providing a proof of concept for future development of therapeutic bispecific IgEs.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"23 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145153328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Customizable Stacks for Enhanced Cell Culture in 3D Tissue Engineering Using Alginate Membranes. 使用海藻酸盐膜的3D组织工程中增强细胞培养的可定制堆栈。

IF 3.8 2区生物学

Biotechnology and Bioengineering Pub Date : 2025-09-25 DOI: 10.1002/bit.70074

Patipat Kamdenlek,Komgrit Eawsakul,Chalaisorn Thanapongpibul,Pakorn Sangngam,Kullapop Suttiat,Warit Powcharoen,Nipon Theera-Umpon,Chawan Manaspon

{"title":"Customizable Stacks for Enhanced Cell Culture in 3D Tissue Engineering Using Alginate Membranes.","authors":"Patipat Kamdenlek,Komgrit Eawsakul,Chalaisorn Thanapongpibul,Pakorn Sangngam,Kullapop Suttiat,Warit Powcharoen,Nipon Theera-Umpon,Chawan Manaspon","doi":"10.1002/bit.70074","DOIUrl":"https://doi.org/10.1002/bit.70074","url":null,"abstract":"Stacked cell culture has emerged as a successful 3D technique in tissue engineering, demonstrated by prototypes developed with various scaffold materials. This study aimed to enhance cell culture by integrating an alginate membrane with a 3D-printed stacking platform composed of polylactic acid material. The platform featured an optimized three-layer structure using L929 cells and was evaluated against MG-63 cells. Cells were seeded on alginate membrane layers comprising a mixture of alginate, agarose, gelatin, and glycerol (1.5:0.25:1:1%w/v). Increased alginate content resulted in greater weight loss without significantly affecting the degradation rate. In contrast, reducing agarose content accelerated membrane degradation and halved tensile strength (from ~6 to ~3 MPa). Cell viability, Live/Dead staining, and SEM imaging assessed the performance of the stacked culture across layers. The alginate membrane exhibited biocompatibility with L929 cells over 28 days, as verified by Live/Dead staining and SEM imaging on Days 7 and 14. The stacked culture maintained over 70% cell viability per layer for both L929 and MG-63 cells compared to single-layer culture. MG-63 cells, representing osteoblastic differentiation, showed enhanced outcomes, with bone-related genes (RUNX2, OCN, BMP2, COL1A2) exhibiting 1-2-fold increased expression, particularly in the upper layers, compared to general medium. These findings highlight the potential of stacked cell culture to support cell growth and differentiation across layers. This prototype offers promise for tissue engineering applications, with future studies aiming to utilize it for stem cell culture and large-scale 3D tissue development.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"14 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145140309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0