Shake flasks are widely used in early-stage bioprocess development but are limited by their inability to monitor and control key gas-transfer variables such as dissolved oxygen and carbon dioxide. In this study, we present a jacketed breathable flask system that enables real-time gas control in a standard shaking environment. Across multiple media formulations and fill volumes, this system consistently deferred oxygen limitation and enhanced culture performance, achieving > 150% higher biomass and 140% greater recombinant protein yield compared to conventional flasks. Time-resolved analysis of pH and extracellular metabolites revealed reduced accumulation of oxygen-sensitive byproducts, including acetate, pyruvate, and succinate, indicating a shift toward more efficient respiratory metabolism. The jacketed breathable flask also enabled continuous monitoring and regulation of critical process parameters, creating a bioreactor-like environment in a high-throughput, low-cost format. The biomass accumulation and specific growth rate observed in jacketed breathable flask are comparable to those reported for Escherichia coli cultures in stirred tank bioreactor application notes for Eppendorf BioBLU 3f. These findings establish breathable flasks as a scalable and accessible platform with bioreactor-like performance for upstream process optimization and accelerate biomanufacturing development at the lab scale.
{"title":"Development of a Jacketed Breathable Shake Flask With Process Monitoring, Control, and Bioreactor-Like Performance.","authors":"Vikash Kumar,Chad Sundberg,Venkatesh Srinivasan,Aaron Thole,Michael Tolosa,Govind Rao","doi":"10.1002/bit.70157","DOIUrl":"https://doi.org/10.1002/bit.70157","url":null,"abstract":"Shake flasks are widely used in early-stage bioprocess development but are limited by their inability to monitor and control key gas-transfer variables such as dissolved oxygen and carbon dioxide. In this study, we present a jacketed breathable flask system that enables real-time gas control in a standard shaking environment. Across multiple media formulations and fill volumes, this system consistently deferred oxygen limitation and enhanced culture performance, achieving > 150% higher biomass and 140% greater recombinant protein yield compared to conventional flasks. Time-resolved analysis of pH and extracellular metabolites revealed reduced accumulation of oxygen-sensitive byproducts, including acetate, pyruvate, and succinate, indicating a shift toward more efficient respiratory metabolism. The jacketed breathable flask also enabled continuous monitoring and regulation of critical process parameters, creating a bioreactor-like environment in a high-throughput, low-cost format. The biomass accumulation and specific growth rate observed in jacketed breathable flask are comparable to those reported for Escherichia coli cultures in stirred tank bioreactor application notes for Eppendorf BioBLU 3f. These findings establish breathable flasks as a scalable and accessible platform with bioreactor-like performance for upstream process optimization and accelerate biomanufacturing development at the lab scale.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"15 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937763","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}
Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.
{"title":"Engineering of 2-ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae.","authors":"Joshua J Dietrich,Maelia Dziedzic,Jia Sun,Sri Harsha Adusumilli,Carla Gonçalves,Chris Todd Hittinger,Brian F Pfleger","doi":"10.1002/bit.70150","DOIUrl":"https://doi.org/10.1002/bit.70150","url":null,"abstract":"Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"265 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937764","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}
Jessica Graham,Sathanandam S Anand,Joel Bercu,Lauren Besenhofer,Christina de Zafra,Yu Feng,Susanne Glowienke,Jedd Hillegass,Richard Hutchinson,Robert Jolly,Melisa Masuda-Herrera,Tyler Nicholas,Daniela Olszova,Matthew Schmitz,Florian Semmelmann,Eric Tien
Host cell proteins (HCPs) are important process-related impurities produced by the host organism during the manufacturing of biotherapeutics. Even trace amounts of these contaminants can be considered significant during drug development due to their potential impact on the quality, safety, and/or efficacy of the therapeutic. This article summarizes the findings of a survey conducted by the IQ DruSafe Impurities Safety Working Group (Biologics Impurities Subteam) concerning industry practices and challenges related to HCPs in biologic therapeutics. The survey addressed four key areas: the scope of HCP control challenges, practices for HCP control and monitoring, methods for qualification of HCP levels, and regulatory interactions. Results revealed both perceived risks and experienced impact from HCP impurities as well as analytical strategies for their identification and quantification. The article also presents current default limits being employed for total and individual HCP impurities, approaches for assessing the safety and immunogenicity risk of HCPs, and a summary of feedback from global health authorities. Overall, the survey results illustrate progress in HCP management across biologic drug development while underscoring persistent challenges. The findings point to emerging best practices informed by historical knowledge and also reveal areas where a harmonized approach may be justified. Identifying and addressing challenges will require sustained industry collaboration and ongoing engagement with regulatory authorities to ensure the continued advancement of safe, effective biologic therapeutics.
{"title":"Assessment and Control of Host Cell Proteins in Biologics: Survey of Industry Practices and a Vision for Harmonization.","authors":"Jessica Graham,Sathanandam S Anand,Joel Bercu,Lauren Besenhofer,Christina de Zafra,Yu Feng,Susanne Glowienke,Jedd Hillegass,Richard Hutchinson,Robert Jolly,Melisa Masuda-Herrera,Tyler Nicholas,Daniela Olszova,Matthew Schmitz,Florian Semmelmann,Eric Tien","doi":"10.1002/bit.70154","DOIUrl":"https://doi.org/10.1002/bit.70154","url":null,"abstract":"Host cell proteins (HCPs) are important process-related impurities produced by the host organism during the manufacturing of biotherapeutics. Even trace amounts of these contaminants can be considered significant during drug development due to their potential impact on the quality, safety, and/or efficacy of the therapeutic. This article summarizes the findings of a survey conducted by the IQ DruSafe Impurities Safety Working Group (Biologics Impurities Subteam) concerning industry practices and challenges related to HCPs in biologic therapeutics. The survey addressed four key areas: the scope of HCP control challenges, practices for HCP control and monitoring, methods for qualification of HCP levels, and regulatory interactions. Results revealed both perceived risks and experienced impact from HCP impurities as well as analytical strategies for their identification and quantification. The article also presents current default limits being employed for total and individual HCP impurities, approaches for assessing the safety and immunogenicity risk of HCPs, and a summary of feedback from global health authorities. Overall, the survey results illustrate progress in HCP management across biologic drug development while underscoring persistent challenges. The findings point to emerging best practices informed by historical knowledge and also reveal areas where a harmonized approach may be justified. Identifying and addressing challenges will require sustained industry collaboration and ongoing engagement with regulatory authorities to ensure the continued advancement of safe, effective biologic therapeutics.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"397 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937762","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}
Salidroside, a major bioactive component of Rhodiola rosea, exhibits diverse pharmacological activities and broad applications, but faces biosynthesis challenges. Herein, we developed a synergistic strategy to construct a high-level plasmid-free salidroside production strain W6U4. First, multi-copy genomic integration of the phenylpyruvate decarboxylase mutant ARO10D331C and glycosyltransferase UGT85A1 was performed to enhance precursor tyrosol synthesis and glycosylation efficiency. Subsequently, systematic metabolic engineering was applied to redirect metabolic flux: reinforcing the pentose phosphate and shikimate pathways and eliminating competing pathways, which boosted salidroside titer to 2.63 g/L in shake flasks. Finally, optimized fed-batch fermentation in a 5-L bioreactor with two-stage temperature control (37°C for growth, 30°C for induction) and early log-phase induction (OD600 = 15) resulted in 33.68 g/L salidroside, the highest reported titer to date, with only 0.40 g/L residual tyrosol. This integrated strategy establishes an efficient, stable microbial platform for salidroside production, highlighting the synergistic effect in advancing industrial-scale biosynthesis.
{"title":"Engineering Plasmid-Free Escherichia coli via Synergistic Metabolic Tuning and Fermentation Optimization for High-Titer Salidroside Biosynthesis","authors":"Man Zhao, Kerui Liu, Xingyu Chen, Guofei Zheng, Zhiqiang Liu, Yuguo Zheng","doi":"10.1002/bit.70153","DOIUrl":"https://doi.org/10.1002/bit.70153","url":null,"abstract":"Salidroside, a major bioactive component of <i>Rhodiola rosea</i>, exhibits diverse pharmacological activities and broad applications, but faces biosynthesis challenges. Herein, we developed a synergistic strategy to construct a high-level plasmid-free salidroside production strain W6U4. First, multi-copy genomic integration of the phenylpyruvate decarboxylase mutant <i>ARO10</i><sup><i>D331C</i></sup> and glycosyltransferase <i>UGT85A1</i> was performed to enhance precursor tyrosol synthesis and glycosylation efficiency. Subsequently, systematic metabolic engineering was applied to redirect metabolic flux: reinforcing the pentose phosphate and shikimate pathways and eliminating competing pathways, which boosted salidroside titer to 2.63 g/L in shake flasks. Finally, optimized fed-batch fermentation in a 5-L bioreactor with two-stage temperature control (37°C for growth, 30°C for induction) and early log-phase induction (OD<sub>600</sub> = 15) resulted in 33.68 g/L salidroside, the highest reported titer to date, with only 0.40 g/L residual tyrosol. This integrated strategy establishes an efficient, stable microbial platform for salidroside production, highlighting the synergistic effect in advancing industrial-scale biosynthesis.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"131 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145919883","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}
Maximilian Siska, Emma Pajak, Katrin Rosenthal, Antonio del Rio Chanona, Eric von Lieres, Laura M. Helleckes
Bayesian optimization has become widely popular across various experimental sciences due to its favorable attributes: it can handle noisy data, perform well with relatively small data sets, and provide adaptive suggestions for sequential experimentation. While still in its infancy, Bayesian optimization has recently gained traction in bioprocess engineering. However, experimentation with biological systems is highly complex and the resulting experimental uncertainty requires specific extensions to classical Bayesian optimization. Moreover, current literature often targets readers with a strong statistical background, limiting its accessibility for practitioners. In light of these developments, this review has two aims: first, to provide an intuitive and practical introduction to Bayesian optimization; and second, to outline promising application areas and open algorithmic challenges, thereby highlighting opportunities for future research in machine learning.
{"title":"A Guide to Bayesian Optimization in Bioprocess Engineering","authors":"Maximilian Siska, Emma Pajak, Katrin Rosenthal, Antonio del Rio Chanona, Eric von Lieres, Laura M. Helleckes","doi":"10.1002/bit.70129","DOIUrl":"https://doi.org/10.1002/bit.70129","url":null,"abstract":"Bayesian optimization has become widely popular across various experimental sciences due to its favorable attributes: it can handle noisy data, perform well with relatively small data sets, and provide adaptive suggestions for sequential experimentation. While still in its infancy, Bayesian optimization has recently gained traction in bioprocess engineering. However, experimentation with biological systems is highly complex and the resulting experimental uncertainty requires specific extensions to classical Bayesian optimization. Moreover, current literature often targets readers with a strong statistical background, limiting its accessibility for practitioners. In light of these developments, this review has two aims: first, to provide an intuitive and practical introduction to Bayesian optimization; and second, to outline promising application areas and open algorithmic challenges, thereby highlighting opportunities for future research in machine learning.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"43 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145919809","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}
{"title":"Biotechnology and Bioengineering: Volume 123, Number 2, February 2026","authors":"","doi":"10.1002/bit.70152","DOIUrl":"10.1002/bit.70152","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"123 2","pages":"269-272"},"PeriodicalIF":3.6,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70152","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145920295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ting Huang,Hongmei Ge,Zhenbing Wu,Yibo Zhang,Lifeng Wang,Chenyuan Dang,Jie Fu
Nano-Ag is increasingly detected in WWTP due to its widespread application, posing a significant threat to microbial communities responsible for wastewater treatment efficiency. Prior studies have demonstrated that quorum sensing (QS) can modulate bacterial tolerance to various environmental stressors in sludge systems. However, the feasibility and mechanisms of N-acyl homoserine lactones (AHLs)-mediated QS regulation to improve the resistance of microorganisms in WWTPs to nano-Ag shocks have been unexplored. Hence, we conducted sequencing batch reactor experiments, and as expected, nano-Ag significantly reduced the treatment performance of bioreactors. However, with the addition of AHLs (C6-HSL, C10-HSL, and C14-HSL) in the bioreactors, the microbial resistance in activated sludge to nano-Ag stress had been evidently enhanced, including the restoration of the sludge morphology, settleability, biomass and extracellular polymeric substances (EPS), as well as the treatment performance of bioreactors on removals of ammonium nitrogen (NH4 +-N), chemical oxygen demand (COD), and suspended solids. The joint analysis of metagenomics, metatranscriptomics, and metametabolomics indicated the multifunctional bacteria (e.g., Amaricoccus, Hydrogenophaga, and Brevundimonas) played a very important role during the regulation of AHLs-mediated QS, which harbored functional genes associated with nitrogen metabolism, carbon metabolism, silver resistance, and AHLs response. The upregulation on glutathione-dependent metabolisms (e.g., glutathione-oxidized glutathione redox cycle) and biosynthesis of EPS (e.g., poly-N-acetylglucosamine) were beneficial for the enhancement of microbial resistance to nano-Ag. This study provided a potentially feasible strategy and important theoretical basis to enhance the robustness and restore the function of microorganisms in wastewater treatment systems by using AHLs-mediated QS regulation.
{"title":"Resistance of Microbial Community in Activated Sludge to Nano-Ag Stress Through Regulation of N-Acyl Homoserine Lactones-Mediated Quorum Sensing.","authors":"Ting Huang,Hongmei Ge,Zhenbing Wu,Yibo Zhang,Lifeng Wang,Chenyuan Dang,Jie Fu","doi":"10.1002/bit.70155","DOIUrl":"https://doi.org/10.1002/bit.70155","url":null,"abstract":"Nano-Ag is increasingly detected in WWTP due to its widespread application, posing a significant threat to microbial communities responsible for wastewater treatment efficiency. Prior studies have demonstrated that quorum sensing (QS) can modulate bacterial tolerance to various environmental stressors in sludge systems. However, the feasibility and mechanisms of N-acyl homoserine lactones (AHLs)-mediated QS regulation to improve the resistance of microorganisms in WWTPs to nano-Ag shocks have been unexplored. Hence, we conducted sequencing batch reactor experiments, and as expected, nano-Ag significantly reduced the treatment performance of bioreactors. However, with the addition of AHLs (C6-HSL, C10-HSL, and C14-HSL) in the bioreactors, the microbial resistance in activated sludge to nano-Ag stress had been evidently enhanced, including the restoration of the sludge morphology, settleability, biomass and extracellular polymeric substances (EPS), as well as the treatment performance of bioreactors on removals of ammonium nitrogen (NH4 +-N), chemical oxygen demand (COD), and suspended solids. The joint analysis of metagenomics, metatranscriptomics, and metametabolomics indicated the multifunctional bacteria (e.g., Amaricoccus, Hydrogenophaga, and Brevundimonas) played a very important role during the regulation of AHLs-mediated QS, which harbored functional genes associated with nitrogen metabolism, carbon metabolism, silver resistance, and AHLs response. The upregulation on glutathione-dependent metabolisms (e.g., glutathione-oxidized glutathione redox cycle) and biosynthesis of EPS (e.g., poly-N-acetylglucosamine) were beneficial for the enhancement of microbial resistance to nano-Ag. This study provided a potentially feasible strategy and important theoretical basis to enhance the robustness and restore the function of microorganisms in wastewater treatment systems by using AHLs-mediated QS regulation.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"23 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145907709","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}
Yuanheng Zhao, Kaitlyn Wingnean, Nishaka William, Jason P. Acker
Red blood cells (RBCs) are needed for life‐saving blood transfusions, but they undergo continuous degradation during storage. Preserving RBCs for clinical transfusion remains a challenge due to storage‐induced damage and limitations of traditional freezing methods. This study investigates isochoric freezing—a constant‐volume, high‐pressure cryopreservation technique that suppresses ice formation—as an alternative approach for RBC cryopreservation. RBC samples were preserved under isochoric freezing conditions at −2.5°C to −15°C with corresponding pressures of 31–156 MPa and a comparator supercooled control group. Hemolysis, cell count, morphology, and membrane integrity were assessed using hematological analyses, imaging flow cytometry, and dextran permeability assays. It is reported that hemolysis and morphological deterioration increased with decreasing temperature and rising pressure, with higher membrane damage compared to supercooling. Lower‐temperature isochoric freezing resulted in loss of membrane integrity that was irreversible. While isochoric freezing minimized ice formation, elevated pressures adversely affected RBC viability. These findings highlight critical pressure‐temperature thresholds necessary for optimizing isochoric freezing protocols for RBC preservation and inform future development of safer, long‐term blood storage strategies.
{"title":"Evaluating Isochoric Freezing as a Strategy for Storage of Red Blood Cells","authors":"Yuanheng Zhao, Kaitlyn Wingnean, Nishaka William, Jason P. Acker","doi":"10.1002/bit.70145","DOIUrl":"https://doi.org/10.1002/bit.70145","url":null,"abstract":"Red blood cells (RBCs) are needed for life‐saving blood transfusions, but they undergo continuous degradation during storage. Preserving RBCs for clinical transfusion remains a challenge due to storage‐induced damage and limitations of traditional freezing methods. This study investigates isochoric freezing—a constant‐volume, high‐pressure cryopreservation technique that suppresses ice formation—as an alternative approach for RBC cryopreservation. RBC samples were preserved under isochoric freezing conditions at −2.5°C to −15°C with corresponding pressures of 31–156 MPa and a comparator supercooled control group. Hemolysis, cell count, morphology, and membrane integrity were assessed using hematological analyses, imaging flow cytometry, and dextran permeability assays. It is reported that hemolysis and morphological deterioration increased with decreasing temperature and rising pressure, with higher membrane damage compared to supercooling. Lower‐temperature isochoric freezing resulted in loss of membrane integrity that was irreversible. While isochoric freezing minimized ice formation, elevated pressures adversely affected RBC viability. These findings highlight critical pressure‐temperature thresholds necessary for optimizing isochoric freezing protocols for RBC preservation and inform future development of safer, long‐term blood storage strategies.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"26 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145897480","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}
Justin P. Lomont, Tracy N. Love, James M. Wagner, Nicole M. Ralbovsky, Joseph P. Smith
In microcarrier (MC) cell culture processes for live virus vaccine (LVV) production, a variety of process phenomena (i.e., cell surface adhesion, cell aggregation, MC aggregation, cell lysis, cell death, surface detachment, and accumulation of cellular debris) exist that significantly underlie the performance of the process itself. Nonetheless, it remains difficult to directly characterize these critical phenomena during a cell culture process. Process analytical technology (PAT) offers a unique opportunity to potentially overcome these challenges in a manner that provides real‐time information via directly interfacing analytical technology with the bioprocess itself. In this work, we propose the utilization of in situ microscopy as a real‐time, in‐line PAT to directly visualize and characterize both cell and microcarrier behavior simultaneously using two commercially available probe‐based technologies. To the best of our knowledge, this is the first report of in situ microscopy applied to an upstream LVV cell culture process, providing direct visualization and characterization of key process phenomena in real‐time. Cell growth, cell death, surface detachment, accumulation of cellular debris, and aggregation of MCs are directly elucidated via our proposed in situ technology. Notably, we observe significant differences in the in situ microscopy data relative to offline microscopy with regards to aggregation of MCs. MC aggregation is observed to be highly prevalent in the LVV‐based process studied herein, particularly during viral replication. Significant MC aggregation is not observed in offline analysis, suggesting that manual sampling may disrupt MC‐aggregate structures present in the bioreactor culture, and as such, highlights the abundant need for in situ observation to enable accurate and representative process analysis and modeling. Our observations of MC aggregation carry implications for cell growth, cell death, and viral infection in MC‐based LVV cell cultures, indicating this elucidated phenomena may be much more prevalent in LVV cell culture processes than previously believed. In situ microscopy thus provides a novel and powerful PAT methodology, with easy to interpret direct visualization for readout, for characterizing upstream LVV processes in real time, in which we can now significantly advance our process understanding beyond what can be achieved using traditional offline characterization methods.
{"title":"In Situ Microscopy for Real‐Time Visualization of Microcarrier Cell Cultures for Live Virus Vaccine Process Development","authors":"Justin P. Lomont, Tracy N. Love, James M. Wagner, Nicole M. Ralbovsky, Joseph P. Smith","doi":"10.1002/bit.70127","DOIUrl":"https://doi.org/10.1002/bit.70127","url":null,"abstract":"In microcarrier (MC) cell culture processes for live virus vaccine (LVV) production, a variety of process phenomena (i.e., cell surface adhesion, cell aggregation, MC aggregation, cell lysis, cell death, surface detachment, and accumulation of cellular debris) exist that significantly underlie the performance of the process itself. Nonetheless, it remains difficult to directly characterize these critical phenomena during a cell culture process. Process analytical technology (PAT) offers a unique opportunity to potentially overcome these challenges in a manner that provides real‐time information via directly interfacing analytical technology with the bioprocess itself. In this work, we propose the utilization of in situ microscopy as a real‐time, in‐line PAT to directly visualize and characterize both cell and microcarrier behavior simultaneously using two commercially available probe‐based technologies. To the best of our knowledge, this is the <jats:italic>first</jats:italic> report of in situ microscopy applied to an upstream LVV cell culture process, providing direct visualization and characterization of key process phenomena in real‐time. Cell growth, cell death, surface detachment, accumulation of cellular debris, and aggregation of MCs are directly elucidated via our proposed in situ technology. Notably, we observe significant differences in the in situ microscopy data relative to offline microscopy with regards to aggregation of MCs. MC aggregation is observed to be highly prevalent in the LVV‐based process studied herein, particularly during viral replication. Significant MC aggregation is not observed in offline analysis, suggesting that manual sampling may disrupt MC‐aggregate structures present in the bioreactor culture, and as such, highlights the abundant need for in situ observation to enable accurate and representative process analysis and modeling. Our observations of MC aggregation carry implications for cell growth, cell death, and viral infection in MC‐based LVV cell cultures, indicating this elucidated phenomena may be much more prevalent in LVV cell culture processes than previously believed. In situ microscopy thus provides a novel and powerful PAT methodology, with easy to interpret direct visualization for readout, for characterizing upstream LVV processes in real time, in which we can now significantly advance our process understanding beyond what can be achieved using traditional offline characterization methods.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"29 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145897335","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}
Annually, over 200,000 cancer patients in the United States are diagnosed with brain metastases. Notably, brain metastatic breast cancer (BMBC) is the second most common, accounting for ~30% of all brain metastasis cases. BMBC typically has poor prognosis and is resistant to chemotherapy. In the brain tumor environment, metastatic cells interact with stromal cells, such as astrocytes, influencing tumor growth and protecting them from chemotherapy. Herein, we report a three‐dimensional (3D) co‐culture spheroid model to study astrocyte induced growth and chemoresistance in BMBC cells. We prepared co‐culture spheroids of BMBC cells and human astrocytes (1:1 ratio) or only BMBC cell spheroids, cultured them in suspension for 7 days, and treated them with paclitaxel (PTX). Using proliferation and apoptosis assays our results demonstrate that tumor cells in co‐culture spheroids were non‐responsive to PTX, while the tumor cell spheroids were responsive. Moreover, the chemoprotection of tumor cells by astrocytes in co‐culture spheroids was mediated by connexin 43 (CX43) dependent STAT1 signaling pathway. Accordingly, the inhibition of CX43 alleviated PTX resistance in co‐culture spheroids. Our 3D co‐culture spheroid platform could serve as a tool to study resistance to therapy in BMBC, and to identify combination treatments for therapy resistant BMBC.
{"title":"Astrocytes Protect Brain Metastatic Breast Cancer Cells From Chemotherapy Through CX43 Dependent STAT1 Signaling in Co‐Culture Spheroids","authors":"Venu Yakati, Shreyas S. Rao","doi":"10.1002/bit.70144","DOIUrl":"https://doi.org/10.1002/bit.70144","url":null,"abstract":"Annually, over 200,000 cancer patients in the United States are diagnosed with brain metastases. Notably, brain metastatic breast cancer (BMBC) is the second most common, accounting for ~30% of all brain metastasis cases. BMBC typically has poor prognosis and is resistant to chemotherapy. In the brain tumor environment, metastatic cells interact with stromal cells, such as astrocytes, influencing tumor growth and protecting them from chemotherapy. Herein, we report a three‐dimensional (3D) co‐culture spheroid model to study astrocyte induced growth and chemoresistance in BMBC cells. We prepared co‐culture spheroids of BMBC cells and human astrocytes (1:1 ratio) or only BMBC cell spheroids, cultured them in suspension for 7 days, and treated them with paclitaxel (PTX). Using proliferation and apoptosis assays our results demonstrate that tumor cells in co‐culture spheroids were non‐responsive to PTX, while the tumor cell spheroids were responsive. Moreover, the chemoprotection of tumor cells by astrocytes in co‐culture spheroids was mediated by connexin 43 (CX43) dependent STAT1 signaling pathway. Accordingly, the inhibition of CX43 alleviated PTX resistance in co‐culture spheroids. Our 3D co‐culture spheroid platform could serve as a tool to study resistance to therapy in BMBC, and to identify combination treatments for therapy resistant BMBC.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"38 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145897478","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}
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