Biotechnology Progress最新文献_第5页

Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media 在制备 CHO 细胞培养基过程中使用光谱过程分析技术进行快速质量评估

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-05-03 DOI: 10.1002/btpr.3477

Jianfa Ou, Wanyue Cui, Yuxiang Zhao, Yawen Tang, Alexander Williams, Dhanuka Wasalathanthri, Jianlin Xu, Jongchan Lee, Michael C. Borys, Anurag Khetan

{"title":"Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media","authors":"Jianfa Ou, Wanyue Cui, Yuxiang Zhao, Yawen Tang, Alexander Williams, Dhanuka Wasalathanthri, Jianlin Xu, Jongchan Lee, Michael C. Borys, Anurag Khetan","doi":"10.1002/btpr.3477","DOIUrl":"10.1002/btpr.3477","url":null,"abstract":"Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO cell culture process. Process analytical technology (PAT) enables unique ways to quantify assessments and improve media quality. Here, cell culture media were prepared under a wide range of temperatures (40–80°C) and pH (7.6–10.0). Media quality profiles were compared using three real-time PATs: Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and excitation-emission matrix (EEM) spectroscopy. FTIR and Raman spectroscopies identified shifts in media quality under high preparation temperature (80°C) and at differing preparation pH which negatively impacted monoclonal antibody (mAb) production. In fed-batch processes for production of three different mAbs, viable cell density (VCD) and cell viability were mostly unaffected under all media preparation temperatures, while titer and cell specific productivity of mAb decreased when cultured in basal and feed media prepared at 80°C. High feed preparation pH alone was tolerated but cell growth and productivity profiles deviated from the control condition. Further, charge variants (main, acidic, basic species) and glycosylation (G0F, afucosylation, and high mannose) were examined. Statistically significant differences were observed for one or more of these quality attributes with any shifts in media preparation. In this study, we demonstrated strong associations between media preparation conditions and cell growth, productivity, and product quality. The rapid evaluation of media by PAT implementation enabled more comprehensive understanding of different parameters on media quality and consequential effects on CHO cell culture.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140831900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Integrated micro-scale protein a chromatography and Low pH viral inactivation unit operations on an automated platform 在自动化平台上集成微尺度蛋白质层析和低 pH 病毒灭活单元操作

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-30 DOI: 10.1002/btpr.3476

Paras Sharma, Lars Robbel, Michael Schmitt, Duygu Dikicioglu, Daniel G. Bracewell

{"title":"Integrated micro-scale protein a chromatography and Low pH viral inactivation unit operations on an automated platform","authors":"Paras Sharma, Lars Robbel, Michael Schmitt, Duygu Dikicioglu, Daniel G. Bracewell","doi":"10.1002/btpr.3476","DOIUrl":"10.1002/btpr.3476","url":null,"abstract":"High throughput process development (HTPD) is established for time- and resource- efficient chromatographic process development. However, integration with non-chromatographic operations within a monoclonal antibody (mAb) purification train is less developed. An area of importance is the development of low pH viral inactivation (VI) that follows protein A chromatography. However, the lack of pH measurement devices at the micro-scale represents a barrier to implementation, which prevents integration with the surrounding unit operations, limiting overall process knowledge. This study is based upon the design and testing of a HTPD platform for integration of the protein A and low pH VI operations. This was achieved by using a design and simulation software before execution on an automated liquid handler. The operations were successfully translated to the micro-scale, as assessed by analysis of recoveries and molecular weight content. The integrated platform was then used as a tool to assess the effect of pH on HMWC during low pH hold. The laboratory-scale and micro-scale elution pools showed comparable HMWC across the pH range 3.2–3.7. The investigative power of the platform is highlighted by evaluating the resources required to conduct a hypothetical experiment. This results in lower resource demands and increased labor efficiency relative to the laboratory-scale. For example, the experiment can be conducted in 7 h, compared to 105 h, translating to labor hours, 3 h and 28 h for the micro-scale and laboratory-scale, respectively. This presents the opportunity for further integration beyond chromatographic operations within the purification sequence, to establish a fit-to-platform assessment tool for mAb process development.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3476","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140832083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Surface plasmon resonance aptasensing and computational analysis of Staphylococcus aureus IsdA surface protein 金黄色葡萄球菌 IsdA 表面蛋白的表面等离子共振诱导和计算分析

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-29 DOI: 10.1002/btpr.3475

Tracy Ann Bruce-Tagoe, Michael T. Harnish, Shokoufeh Soleimani, Najeeb Ullah, Tongye Shen, Michael K. Danquah

{"title":"Surface plasmon resonance aptasensing and computational analysis of Staphylococcus aureus IsdA surface protein","authors":"Tracy Ann Bruce-Tagoe, Michael T. Harnish, Shokoufeh Soleimani, Najeeb Ullah, Tongye Shen, Michael K. Danquah","doi":"10.1002/btpr.3475","DOIUrl":"10.1002/btpr.3475","url":null,"abstract":"Staphylococcus aureus (S. aureus), a common foodborne pathogen, poses significant public health challenges due to its association with various infectious diseases. A key player in its pathogenicity, which is the IsdA protein, is an essential virulence factor in S. aureus infections. In this work, we present an integrated in-silico and experimental approach using MD simulations and surface plasmon resonance (SPR)-based aptasensing measurements to investigate S. aureus biorecognition via IsdA surface protein binding. SPR, a powerful real-time and label-free technique, was utilized to characterize interaction dynamics between the aptamer and IsdA protein, and MD simulations was used to characterize the stable and dynamic binding regions. By characterizing and optimizing pivotal parameters such as aptamer concentration and buffer conditions, we determined the aptamer's binding performance. Under optimal conditions of pH 7.4 and 150 mM NaCl concentration, the kinetic parameters were determined; ka = 3.789 × 104/Ms, kd = 1.798 × 103/s, and KD = 4.745 × 10−8 M. The simulations revealed regions of interest in the IsdA-aptamer complex. Region I, which includes interactions between amino acid residues H106 and R107 and nucleotide residues 9G, 10U, 11G and 12U of the aptamer, had the strongest interaction, based on ΔG and B-factor values, and hence contributed the most to the stability of the interaction. Region II, which covers residue 37A reflects the dynamic nature of the interaction due to frequent contacts. The approach presents a rigorous characterization of aptamer-IsdA binding behavior, supporting the potential application of the IsdA-binding aptamer system for S. aureus biosensing.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140832418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development 对影响表达作为疫苗抗原的 SARS-CoV-2 棘突蛋白的稳定中国仓鼠卵巢细胞池在早期工艺开发中的生长和生产率的工艺参数进行多变量数据分析。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-25 DOI: 10.1002/btpr.3467

Sebastian-Juan Reyes, Lucas Lemire, Raul-Santiago Molina, Marjolaine Roy, Helene L'Ecuyer-Coelho, Yuliya Martynova, Brian Cass, Robert Voyer, Yves Durocher, Olivier Henry, Phuong Lan Pham

{"title":"Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development","authors":"Sebastian-Juan Reyes, Lucas Lemire, Raul-Santiago Molina, Marjolaine Roy, Helene L'Ecuyer-Coelho, Yuliya Martynova, Brian Cass, Robert Voyer, Yves Durocher, Olivier Henry, Phuong Lan Pham","doi":"10.1002/btpr.3467","DOIUrl":"10.1002/btpr.3467","url":null,"abstract":"The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200–2000 L bioreactor.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3467","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140655622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Improved sieving coefficient in perfusion cell culture with reduced effective filtration length of hollow fibers 减少中空纤维的有效过滤长度，提高灌流细胞培养的筛分系数。

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-24 DOI: 10.1002/btpr.3472

Jimmy Vu, J. Alex Gadberry, Jon Coffman, Ken Lee

{"title":"Improved sieving coefficient in perfusion cell culture with reduced effective filtration length of hollow fibers","authors":"Jimmy Vu, J. Alex Gadberry, Jon Coffman, Ken Lee","doi":"10.1002/btpr.3472","DOIUrl":"10.1002/btpr.3472","url":null,"abstract":"The hollow fiber filter is the primary cell-retention device used in high-density perfusion cell culture and often used in an alternating tangential flow (ATF) configuration. The limited commercially available diaphragm pumps for ATF prevent utilization of vertical space when scaling beyond 500 L. Stacking hollow fiber filters coupled with viscous cell culture imposes vacuum pressure exceeding facility capabilities. Additionally, the longer filter assembly increases the hold-up volume and exceeds the diaphragm pump's fluid exchange capacity. The conventional tangential flow filtration (TFF) configuration circumvents this issue by exchanging culture from the bioreactor and cell-retention device in a unidirectional recirculation loop; however, the increased filter length when scaled up exacerbates the TFF's inherent issue with product retention from Starling flow. Stacking commercially available 20 cm TFF filters to make up the similar single-module length TFF used for the platform 3 and 50 L perfusion process at 41.5 and 65 cm, respectively, attempts to reduce fouling caused by Starling flow. The permeate of a single-module filter is partitioned into short independent segments through serially stacked filters, each harvested separately. By partitioning the permeate, the sieving coefficient increased for both 3 and 50 L scales. Reduction of Starling flow was confirmed with lower total hydraulic membrane resistance throughout the culture. This work demonstrates a method for increasing sieving coefficient and filter capacity by stacking TFF filters with independent permeate streams.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3472","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140665270","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pharmaceutical proteins at the interfaces and the role of albumin 界面上的药用蛋白质和白蛋白的作用

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-22 DOI: 10.1002/btpr.3474

Ketki Y. Velankar, Ellen S. Gawalt, Yi Wen, Wilson S. Meng

引用次数: 0

Effects of green light supplementation with red and blue combinations of LED light spectrums on the growth and transcriptional response of Haematococcus pluvialis 绿光补充红光和蓝光组合的 LED 光光谱对血球藻生长和转录反应的影响

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-20 DOI: 10.1002/btpr.3462

G. Karagülle, M. Telli

{"title":"Effects of green light supplementation with red and blue combinations of LED light spectrums on the growth and transcriptional response of Haematococcus pluvialis","authors":"G. Karagülle, M. Telli","doi":"10.1002/btpr.3462","DOIUrl":"10.1002/btpr.3462","url":null,"abstract":"Light management strategy is crucial for improving microalgal production in terms of higher biomass and economically valuable bioactive molecules. However, green light has received less attention in developing light managements for algae and higher plant due to its low absorption rate by chlorophyll. In this study, the effects of green light supplementation, in the combination with red and blue light were investigated in Haematococcus pluvialis. 10% and 20% of green light supplementations were applied in 3:2 ratios of red and blue LED light combinations as an expense of red-light. Growth rates, chlorophyll concentration, and dry weight were measured to assess the growth kinetics of H. pluvialis along with the relative transcript accumulations of four mRNAs: Rubisco, PTOX2, PsaB, and PsbS. Growth rates, chlorophyll concentrations and dry weight were found significantly higher in presence of 10% green light supplementation compared to red and blue light combinations. The relative transcript accumulations of Rubisco and PsbS genes showed significant upregulation at the end of the experiments (with the fold change of 42.91 ± 12.08 and 98.57 ± 27.38, respectively, relative to the beginning of the experiments) compared to combinations of red and blue light (fold change of 19.09 ± 3.0 and 47.77 ± 14.21, respectively, relative to beginning of the experiments). PsaB and PTOX2 transcripts did not show significant accumulation differences between treatments. It seems that green light has a dose dependent additive effect on the growth rate of H. pluvialis. The upregulation of Rubisco and PsbS may indicate green light dependent carbon assimilation and light-harvesting response in H. pluvialis.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140626511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation 提高 CHO 细胞培养性能的策略：靶向删除氨基酸分解和凋亡基因并补充生长抑制剂

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-17 DOI: 10.1002/btpr.3471

Cynthia Lam, Alyssa Sargon, Camil Diaz, Zijuan Lai, Dewakar Sangaraju, Inn Yuk, Gavin Barnard, Shahram Misaghi

{"title":"Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation","authors":"Cynthia Lam, Alyssa Sargon, Camil Diaz, Zijuan Lai, Dewakar Sangaraju, Inn Yuk, Gavin Barnard, Shahram Misaghi","doi":"10.1002/btpr.3471","DOIUrl":"10.1002/btpr.3471","url":null,"abstract":"Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140614566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system 利用新型动态成像系统对生物加工过程中的中国仓鼠卵巢细胞进行单细胞分析

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-13 DOI: 10.1002/btpr.3469

Laura Breen, James Flynn, Adam Bergin, Evangelia Flampouri, Michael Butler

{"title":"Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system","authors":"Laura Breen, James Flynn, Adam Bergin, Evangelia Flampouri, Michael Butler","doi":"10.1002/btpr.3469","DOIUrl":"10.1002/btpr.3469","url":null,"abstract":"Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/btpr.3469","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140566577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Monte Carlo simulation-guided design for size-tuned tumor spheroid formation in 3D printed microwells 在蒙特卡洛模拟指导下设计三维打印微孔中大小可调的肿瘤小球的形成

IF 2.5 3区生物学

Biotechnology Progress Pub Date : 2024-04-13 DOI: 10.1002/btpr.3470

Ismail Eş, Ana-Maria Theodora Ionescu, Burak M. Görmüş, Fatih Inci, Marco P. C. Marques, Nicolas Szita, Lucimara Gaziola de la Torre

{"title":"Monte Carlo simulation-guided design for size-tuned tumor spheroid formation in 3D printed microwells","authors":"Ismail Eş, Ana-Maria Theodora Ionescu, Burak M. Görmüş, Fatih Inci, Marco P. C. Marques, Nicolas Szita, Lucimara Gaziola de la Torre","doi":"10.1002/btpr.3470","DOIUrl":"10.1002/btpr.3470","url":null,"abstract":"Tumor spheroid models have garnered significant attention in recent years as they can efficiently mimic in vivo models, and in addition, they offer a more controlled and reproducible environment for evaluating the efficacy of cancer drugs. In this study, we present the design and fabrication of a micromold template to form multicellular spheroids in a high-throughput and controlled-sized fashion. Briefly, polydimethylsiloxane-based micromolds at varying sizes and geometry were fabricated via soft lithography using 3D-printed molds as negative templates. The efficiency of spheroid formation was assessed using GFP-expressing human embryonic kidney 293 cells (HEK-293). After 7 days of culturing, circularity and cell viability of spheroids were >0.8 and 90%, respectively. At 1500 cells/microwell of cell seeding concentration, the spheroids were 454 ± 15 μm, 459 ± 7 μm, and 451 ± 18 μm when cultured in microwells with the diameters of 0.4, 0.6, and 0.8 μm, respectively. Moreover, the distance between each microwell and surfactant treatment before cell seeding notably impacted the uniform spheroid formation. The centrifugation was the key step to collect cells on the bottom of the microwells. Our findings were further verified using a commercial microplate. Furthermore, Monte Carlo simulation confirmed the seeding conditions where the spheroids could be formed. This study showed prominent steps in investigating spheroid formation, thereby leveraging the current know-how on the mechanism of tumor growth.","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140566487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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