{"title":"Effective removal of host cell-derived nucleic acids bound to hepatitis B core antigen virus-like particles by heparin chromatography.","authors":"Angela Valentic, Jürgen Hubbuch","doi":"10.3389/fbioe.2024.1475918","DOIUrl":"10.3389/fbioe.2024.1475918","url":null,"abstract":"<p><p>Virus-like particles (VLPs) show considerable potential for a wide array of therapeutic applications, spanning from vaccines targeting infectious diseases to applications in cancer immunotherapy and drug delivery. In the context of hepatitis B core antigen (HBcAg) VLPs, a promising candidate for gene delivery approaches, the naturally occurring nucleic acid (NA) binding region is commonly utilized for effective binding of various types of therapeutic nucleic acids (NA<sub>ther</sub>). During formation of the HBcAg VLPs, host cell-derived nucleic acids (NA<sub>hc</sub>) might be associated to the NA binding region, and are thus encapsulated into the VLPs. Following a VLP harvest, the NA<sub>hc</sub> need to be removed effectively before loading the VLP with NA<sub>ther</sub>. Various techniques reported in literature for this NA<sub>hc</sub> removal, including enzymatic treatments, alkaline treatment, and lithium chloride precipitation, lack quantitative evidence of sufficient NA<sub>hc</sub> removal accompanied by a subsequent high VLP protein recovery. In this study, we present a novel heparin chromatography-based process for effective NA<sub>hc</sub> removal from HBcAg VLPs. Six HBcAg VLP constructs with varying lengths of the NA binding region and diverse NA<sub>hc</sub> loadings were subjected to evaluation. Process performance was thoroughly examined through NA<sub>hc</sub> removal and VLP protein recovery analyses. Hereby, reversed phase chromatography combined with UV/Vis spectroscopy, as well as silica spin column-based chromatography coupled with dye-based fluorescence assay were employed. Additionally, alternative process variants, comprising sulfate chromatography and additional nuclease treatments, were investigated. Comparative analyses were conducted with LiCl precipitation and alkaline treatment procedures to ascertain the efficacy of the newly developed chromatography-based methods. Results revealed the superior performance of the heparin chromatography procedure in achieving high NA<sub>hc</sub> removal and concurrent VLP protein recovery. Furthermore, nuanced relationships between NA binding region length and NA<sub>hc</sub> removal efficiency were elucidated. Hereby, the construct Cp157 surpassed the other constructs in the heparin process by demonstrating high NA<sub>hc</sub> removal and VLP protein recovery. Among the other process variants minimal performance variations were observed for the selected constructs Cp157 and Cp183. However, the heparin chromatography-based process consistently outperformed other methods, underscoring its superiority in NA<sub>hc</sub> removal and VLP protein recovery.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1475918"},"PeriodicalIF":4.3,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11487522/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461837","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hema Dinesh Barnana, Syed A M Tofail, Krittish Roy, Charlie O'Mahony, Veronika Hidaši Turiničová, Maroš Gregor, Ehtsham Ul Haq
{"title":"Biodielectrics: old wine in a new bottle?","authors":"Hema Dinesh Barnana, Syed A M Tofail, Krittish Roy, Charlie O'Mahony, Veronika Hidaši Turiničová, Maroš Gregor, Ehtsham Ul Haq","doi":"10.3389/fbioe.2024.1458668","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1458668","url":null,"abstract":"<p><p>Biodielectrics is a subset of biological and/or bioinspired materials that has brought a huge transformation in the advancement of medical science, such as localized drug delivery in cancer therapeutics, health monitoring, bone and nerve repair, tissue engineering and use in other nanoelectromechanical systems (NEMS). While biodielectrics has long been used in the field of electrical insulation for over a century, polar dielectric properties of biological building blocks have not been well understood at the fundamental building block level. In this review article, we provide a brief overview of dielectric properties of biological building blocks and its hierarchical organisations to include polar dielectric properties such as piezo, pyro, and ferroelectricity. This review article also discusses recent trends, scope, and potential applications of these dielectrics in science and technology. We highlight electromechanical properties embedded in rationally designed organic assemblies, and the challenges and opportunities inherent in mapping from molecular amino acid building blocks to macroscopic analogs of biological fibers and tissues, in pursuit of sustainable materials for next-generation technologies.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1458668"},"PeriodicalIF":4.3,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11483890/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yan Cheng, Xiaoxue Zhao, Ruolin Li, Jili Liao, Caoxing Huang
{"title":"Study on the effect of phenoxyethanol-citric acid pretreatment for the enzymatic hydrolysis of bamboo residues.","authors":"Yan Cheng, Xiaoxue Zhao, Ruolin Li, Jili Liao, Caoxing Huang","doi":"10.3389/fbioe.2024.1483025","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1483025","url":null,"abstract":"<p><p>This study investigated the biphasic phenoxyethanol-citric acid (PECA) pretreatment for bamboo residues (BRs) and its corresponding effects on the enzymatic hydrolysis performance. It is found that increasing the concentration of citric acid in the pretreatment system from 2.5% to 15% greatly enhanced the delignification and xylan removal for BRs. Consequently, the enzymatic hydrolysis yield of pretreated BRs significantly enhanced, increasing from 12.4% to 58.2% and 28.0%72.4% when the concentration of citric acid was increased from 2.5% to 15.0% at 160°C and 170°C, respectively. The characterization results from cellulose crystallinity, accessibility, and hydrophobicity of pretreated bamboo residues indicated that their changes possessed a beneficial performance on the enzymatic hydrolysis yield, which could result from the synergistic removal of lignin and xylan. The Chrastil model analysis showed that pretreatment at higher conditions resulted in the pretreated BRs possessing weaker diffusion resistance for cellulase, which is attributed to its higher enzymatic hydrolysis yield.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1483025"},"PeriodicalIF":4.3,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11484043/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Diana Herrera-Valenzuela, Isabel Sinovas-Alonso, Ana de Los Reyes, Ángel Gil-Agudo, Antonio J Del-Ama
{"title":"Improvement of the gait deviation index for spinal cord injury to broaden its applicability: the reduced gait deviation index for spinal cord injury (rSCI-GDI).","authors":"Diana Herrera-Valenzuela, Isabel Sinovas-Alonso, Ana de Los Reyes, Ángel Gil-Agudo, Antonio J Del-Ama","doi":"10.3389/fbioe.2024.1431596","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1431596","url":null,"abstract":"<p><strong>Background: </strong>The SCI-GDI is an accurate and effective metric to summarize gait kinematics in adults with SCI. It is usually computed with the information registered with a photogrammetry system because it requires accurate information of pelvic and hip movement in the three anatomic planes, which is hard to record with simpler systems. Additionally, due to being developed from the GDI, the SCI-GDI is built upon nine joint movements selected for a pediatric population with cerebral palsy, for which the GDI was originally developed, but those nine movements are not necessarily as meaningful for adults with SCI. Nevertheless, pelvic movement and hip rotation have been proven to have low reliability even when acquired with gold-standard photogrammetry systems. Additionally, the use of photogrammetry is limited in real-life scenarios and when used with rehabilitation technologies, which limits the use of the SCI-GDI to evaluate gait in alternative scenarios to gait laboratories and to evaluate technologies for gait assistance. This research aimed to improve the SCI-GDI to broaden its applicability beyond the use of photogrammetry.</p><p><strong>Methods: </strong>An exploration of the mathematical relevance of each joint movement included in the original GDI for the performance of the metric is performed. Considering the results obtained and the clinical relevance of each of the 9 joints used to compute the SCI-GDI in the gait pattern of the SCI population, a more adaptable SCI-GDI is proposed using four joint movements that can be precisely captured with simpler systems than photogrammetry: sagittal planes of hip, knee and ankle and hip abduction/adduction.</p><p><strong>Results: </strong>The reduced SCI-GDI (rSCI-GDI) effectively represents gait variability of adults with SCI as does the SCI-GDI, while providing more generalizable results and equivalent or stronger correlations with clinical tests validated in the population. During the derivation of the improved index, it was demonstrated that pelvic movements, hip rotation, and foot progression angle introduce high variability to the dataset of gait patterns of the adult population with SCI, but they have low relevance to characterize gait kinematics of this population. The rSCI-GDI can be calculated using the 14-feature vectorial basis included in the electronic addendum provided.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1431596"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480198/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Arnau Gasset, Joeri Van Wijngaarden, Ferran Mirabent, Albert Sales-Vallverdú, Xavier Garcia-Ortega, José Luis Montesinos-Seguí, Toni Manzano, Francisco Valero
{"title":"Continuous Process Verification 4.0 application in upstream: adaptiveness implementation managed by AI in the hypoxic bioprocess of the <i>Pichia pastoris</i> cell factory.","authors":"Arnau Gasset, Joeri Van Wijngaarden, Ferran Mirabent, Albert Sales-Vallverdú, Xavier Garcia-Ortega, José Luis Montesinos-Seguí, Toni Manzano, Francisco Valero","doi":"10.3389/fbioe.2024.1439638","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1439638","url":null,"abstract":"<p><p>The experimental approach developed in this research demonstrated how the cloud, the Internet of Things (IoT), edge computing, and Artificial Intelligence (AI), considered key technologies in Industry 4.0, provide the expected horizon for adaptive vision in Continued Process Verification (CPV), the final stage of Process Validation (PV). <i>Pichia pastoris</i> producing <i>Candida rugosa</i> lipase 1 under the regulation of the constitutive <i>GAP</i> promoter was selected as an experimental bioprocess. The bioprocess worked under hypoxic conditions in carbon-limited fed-batch cultures through a physiological control based on the respiratory quotient (<i>RQ</i>). In this novel bioprocess, a digital twin (DT) was built and successfully tested. The implementation of online sensors worked as a bridge between the microorganism and AI models, to provide predictions from the edge and the cloud. AI models emulated the metabolism of <i>Pichia</i> based on critical process parameters and actionable factors to achieve the expected quality attributes. This innovative AI-aided Adaptive-Proportional Control strategy (AI-APC) improved the reproducibility comparing to a Manual-Heuristic Control strategy (MHC), showing better performance than the Boolean-Logic-Controller (BLC) tested. The accuracy, indicated by the Mean Relative Error (MRE), was for the AI-APC lower than 4%, better than the obtained for MHC (10%) and BLC (5%). Moreover, in terms of precision, the same trend was observed when comparing the Root Mean Square Deviation (RMSD) values, becoming lower as the complexity of the controller increases. The successful automatic real time control of the bioprocess orchestrated by AI models proved the 4.0 capabilities brought by the adaptive concept and its validity in biopharmaceutical upstream operations.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1439638"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480048/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Stephan S W Ende, Albert S Beyer, Reham Ebaid, Mostafa Elshobary, Mafalda C Almeida, Cynthia Couto, Kit W Chew, Tamara Schwenkler, Joachim Henjes
{"title":"Potential of a novel brine-struvite-based growth medium for sustainable biomass and phycocyanin production by <i>Arthrospira platensis</i>.","authors":"Stephan S W Ende, Albert S Beyer, Reham Ebaid, Mostafa Elshobary, Mafalda C Almeida, Cynthia Couto, Kit W Chew, Tamara Schwenkler, Joachim Henjes","doi":"10.3389/fbioe.2024.1466978","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1466978","url":null,"abstract":"<p><p>Nutrient recovery is crucial for sustainability as it helps to recycle valuable resources, reduce environmental pollution, and promote the efficient use of natural materials in various agricultural and industrial processes. The present study investigated the impact of using brine and struvite as sustainable nutrient sources on the growth and c-phycocyanin (C-PC) production by the cyanobacterium <i>Arthrospira platensis</i>. Three modified growth media were compared to the standard SAG-spirul medium under yellow-white light [YLT], and blue-white light [BLT]. In the modified medium BSI, a struvite solution was utilized to replace dipotassium phosphate, while diluted brine was used to replace NaCl and de-ionized H<sub>2</sub>O. For BSII, struvite and brine were used as in BSI, with elimination of the micronutrient from the solution. In BSIII, no other nutrient sources than bicarbonate-buffer were used in addition to struvite and brine. For each medium, <i>A. platensis</i> was cultivated and incubated under YLT or BLT till the stationary phase. The results showed that the combinations of brine and struvite did not have any significant negative impact on the growth rates in BSIII. However, adding struvite as a phosphorus source boosted C-PC production just as effectively as YLT, with boosting biomass yield, unlike when only BLT was used. In conclusion, the brine/struvite-based media resulted in high biomass productivity with higher C-PC yields, making it an ideal growth medium for commercial sustainable C-PC production.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1466978"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11479874/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zakir Husain, Zafar Iqbal Warsi, Sana Khan, Ganesan Mahendran, Shama Afroz, Ashish Chandran, Praveen Kumar Kashyap, Kahkashan Khatoon, Gazala Parween, Sudeep Tandon, Laiq Ur Rahman
{"title":"Metabolic engineering of hairy root cultures in <i>Beta vulgaris</i> for enhanced production of vanillin, 4-hydroxybenzoic acid, and vanillyl alcohol.","authors":"Zakir Husain, Zafar Iqbal Warsi, Sana Khan, Ganesan Mahendran, Shama Afroz, Ashish Chandran, Praveen Kumar Kashyap, Kahkashan Khatoon, Gazala Parween, Sudeep Tandon, Laiq Ur Rahman","doi":"10.3389/fbioe.2024.1435190","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1435190","url":null,"abstract":"<p><p>The flavor of vanilla is a complex blend of compounds, with vanillin as the most prominent, along with vanillyl alcohol and 4-hydroxybenzoic acid. Natural vanillin extracted from vanilla beans is expensive, so researchers use heterologous synthesis to produce nature-identical vanillin in plant hosts. Consequently, alternative traditional farming and gathering methods are required to bridge the significant disparity between supply and demand. The current research successfully developed a method to induce hairy root formation from leaves. It integrated the Vanillin synthase (VpVAN) gene into transgenic hairy root lines of <i>Beta vulgaris</i>, synthesizing vanillin-related compounds. The presence of the VpVAN gene in transgenic roots was confirmed using PCR analysis. Additionally, RT-qPCR analysis demonstrated the expression of the VpVAN gene in the transgenic root lines. The transgenic hairy root clones H1, H2, and H5 showed enhanced vanillin production, vanillyl alcohol, and 4-hydroxybenzoic acid. Elicitation with methyl jasmonate (MJ) and salicylic acid (SA) further improved the production of these compounds in <i>B. vulgaris</i> hairy roots. The maximum hairy root biomass was observed after 60 days, with the maximum synthesis of vanillin and 4-hydroxybenzoic acid obtained from hairy root clones H5 and HR2, respectively. Vanillyl alcohol HR2 was obtained on the 45th day of cultivation. Elicitation with wound-associated hormone methyl jasmonate and salicylic acid enhanced the yield of vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid, with a 215-fold increase in vanillin, a 13-fold increase in vanillyl alcohol, and a 21 fold increase in 4-hydroxybenzoic acid. The study results indicate that establishing transgenic hairy root cultures with the VpVAN gene is a promising alternative method for enhancing the production of vanilla flavor compounds such as vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid. A cost-effective protocol has been developed to mass-produce phenolic compounds using a hairy root culture of <i>B. vulgaris</i>. This approach addresses the increasing demand for these substances while reducing the cost of natural vanillin production, making it suitable for industrial-scale applications.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1435190"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480924/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Establishment of a platform based on dual RPA combined with CRISPR/Cas12a for the detection of <i>Klebsiella pneumoniae</i> and its KPC resistance gene.","authors":"Meiying Tan, Xueli Yi, Chuan Liao, Zihan Zhou, Baoyan Ren, Lina Liang, Xuebin Li, Guijiang Wei","doi":"10.3389/fbioe.2024.1447963","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1447963","url":null,"abstract":"<p><p>Carbapenem resistant <i>Klebsiella pneumoniae</i> (CRKP) can cause serious hospital- and community-acquired infections. Treatment for CRKP infection is limited, resulting in prolonged hospitalization and high consultation costs. The KPC genotype has the highest detection rate of CRKP, and its mortality rate is higher than the overall mortality rate of CRKP. However, traditional testing methods have disadvantages such as long time and reliance on complex and sophisticated instruments, which are not conducive to rapid screening for CRKP. Therefore, this study aimed to establish a detection platform for early screening of CRKP so that effective antimicrobial therapy could be administered promptly to prevent the widespread spread of CRKP. We integrated dual RPA with CRISPR/Cas12a to establish a dual platform for the detection of <i>K. pneumoniae</i> (<i>Kp</i>) rcsA-specific gene and KPC resistance gene. Four result reading methods were established, including fluorescence detection (FD), blue light irradiation detection (BLID), ultraviolet irradiation detection (UID), and lateral flow test strips (LFTS). For the rcsA gene, the LOD of FD was 1 × 10 pg/μL, and the other three methods could detect 1 × 10<sup>1</sup> pg/μL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 10<sup>2</sup> pg/μL of bacterial DNA. In 59 clinical strains tested, the dual RPA-CRISPR/Cas12a detection of the rcsA had 100% sensitivity, specificity, and accuracy compared to the culture method. Compared with the drug sensitivity test, the sensitivity of dual RPA-CRISPR/Cas12a detection for the KPC was 85.71%, the specificity was 100%, and the accuracy was 94.92%. In summary, our dual RPA-CRISPR/Cas12a platform proved to be rapid, precise, and convenient for the efficient detection of <i>Kp</i> with KPC in the laboratory or at the point of care.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1447963"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480703/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Paolo Ritter, Stefania Oliveto, Chiara Cordiglieri, Alessandra Fasciani, Christian Andrea Di Buduo, Lucrezia Della Volpe, Alberto Bocconi, Claudio Conci, Carolina Paula Miguel, Raffaella Di Micco, Alessandra Balduini, Manuela Teresa Raimondi, Stefano Biffo
{"title":"A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34<sup>+</sup> hematopoietic cells while allowing the detection of tumorigenic expansion.","authors":"Paolo Ritter, Stefania Oliveto, Chiara Cordiglieri, Alessandra Fasciani, Christian Andrea Di Buduo, Lucrezia Della Volpe, Alberto Bocconi, Claudio Conci, Carolina Paula Miguel, Raffaella Di Micco, Alessandra Balduini, Manuela Teresa Raimondi, Stefano Biffo","doi":"10.3389/fbioe.2024.1388312","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1388312","url":null,"abstract":"<p><p>Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient <i>in vitro</i> assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4<sup>+</sup> T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34<sup>+</sup> cells. A fraction of these cells is viable for up to 3 months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4<sup>+</sup> or CD34<sup>+</sup> primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1388312"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11479935/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461928","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abbas Fazel Anvari Yazdi, Kobra Tahermanesh, Maryam Ejlali, Amin Babaei-Ghazvini, Bishnu Acharya, Ildiko Badea, Daniel J MacPhee, Xiongbiao Chen
{"title":"Comparative analysis of porcine-uterine decellularization for bioactive-molecule preservation and DNA removal.","authors":"Abbas Fazel Anvari Yazdi, Kobra Tahermanesh, Maryam Ejlali, Amin Babaei-Ghazvini, Bishnu Acharya, Ildiko Badea, Daniel J MacPhee, Xiongbiao Chen","doi":"10.3389/fbioe.2024.1418034","DOIUrl":"https://doi.org/10.3389/fbioe.2024.1418034","url":null,"abstract":"<p><strong>Introduction: </strong>Decellularized uterine extracellular matrix has emerged as a pivotal focus in the realm of biomaterials, offering a promising source in uterine tissue regeneration, research on disease diagnosis and treatments, and ultimately uterine transplantation. In this study, we examined various protocols for decellularizing porcine uterine tissues, aimed to unravel the intricate dynamics of DNA removal, bioactive molecules preservation, and microstructural alterations.</p><p><strong>Methods: </strong>Porcine uterine tissues were treated with 6 different, yet rigorously selected and designed, protocols with sodium dodecyl sulfate (SDS), Triton<sup>®</sup> X-100, peracetic acid + ethanol, and DNase I. After decellularization, we examined DNA quantification, histological staining (H&E and DAPI), glycosaminoglycans (GAG) assay, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and Thermogravimetric Analysis (TGA).</p><p><strong>Results: </strong>A comparative analysis among all 6 protocols was conducted with the results demonstrating that all protocols achieved decellularization; while 0.1% SDS + 1% Triton<sup>®</sup> X-100, coupled with agitation, demonstrated the highest efficiency in DNA removal. Also, it was found that DNase I played a key role in enhancing the efficiency of the decellularization process by underscoring its significance in digesting cellular contents and eliminating cell debris by 99.79% (19.63 ± 3.92 ng/mg dry weight).</p><p><strong>Conclusions: </strong>Our findings enhance the nuanced understanding of DNA removal, GAG preservation, microstructural alteration, and protein decomposition in decellularized uterine extracellular matrix, while highlighting the importance of decellularization protocols designed for intended applications. This study along with our findings represents meaningful progress for advancing the field of uterine transplantation and related tissue engineering/regenerative medicine.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"12 ","pages":"1418034"},"PeriodicalIF":4.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480021/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142461834","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}