Varun Sai Tadimarri, Marc Blanch-Asensio, Ketaki Deshpande, Jonas Baumann, Carole Baumann, Rolf Mueller, Sara Trujillo, Shrikrishnan Sankaran
{"title":"PEARL: Protein Eluting Alginate with Recombinant Lactobacilli","authors":"Varun Sai Tadimarri, Marc Blanch-Asensio, Ketaki Deshpande, Jonas Baumann, Carole Baumann, Rolf Mueller, Sara Trujillo, Shrikrishnan Sankaran","doi":"10.1101/2024.09.12.612671","DOIUrl":"https://doi.org/10.1101/2024.09.12.612671","url":null,"abstract":"Engineered living materials (ELMs) made of bacteria in hydrogels have shown considerable promise for therapeutic applications since they offer the possibility to achieve controlled and prolonged release of complex biopharmaceuticals at low costs and with reduced wastage. While most therapeutic ELMs use E. coli as the living component due to its large genetic toolbox, most live biotherapeutic bacteria in clinical trials are lactic acid bacteria due to the native health benefits they offer. Among these, lactobacilli are the largest family of probiotic bacteria that are being investigated for their therapeutic potential in almost all sites of the body that host a microbiome. A major factor limiting the use of lactobacilli in ELMs is their limited genetic toolbox. In this study, we build upon our recent work to expand the genetic programmability of a probiotic lactobacillus strain (Lactiplantibacillus plantarum WCFS1) for protein secretion and integrate it into a simple, cost-effective, and biocompatible alginate bead encapsulation format to develop an ELM. We demonstrate the controlled release of a recombinant protein for up to 14 days from this ELM, thereby terming it PEARL - Protein Eluting Alginate with Recombinant Lactobacilli. Notably, encapsulation of the lactobacilli offered multiple benefits such as preventing bacterial outgrowth, stabilizing protein release profiles over time, and preventing potential cytotoxicity caused by bacterial metabolites. These findings demonstrate the mutual benefits of combining recombinant lactobacilli with alginate for the controlled release of proteins for biomedical applications.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
O Aung, Peter Rossi, Mitchell Dyer, Austin Stellpflug, Yingnan Zhai, Allen Kenneth, Xiaolong Wang, Jackie Chang, Yiliang Chen, Brandon James Tefft, Rongxue Wu, Lingxia Gu, Bo Wang
{"title":"Biofabrication of Small Vascular Graft with Acellular Human Amniotic Membrane: A Proof-of-Concept Study in Pig","authors":"O Aung, Peter Rossi, Mitchell Dyer, Austin Stellpflug, Yingnan Zhai, Allen Kenneth, Xiaolong Wang, Jackie Chang, Yiliang Chen, Brandon James Tefft, Rongxue Wu, Lingxia Gu, Bo Wang","doi":"10.1101/2024.09.11.612466","DOIUrl":"https://doi.org/10.1101/2024.09.11.612466","url":null,"abstract":"Synthetic vascular grafts, such as expanded polytetrafluoroethylene (ePTFE), are commonly used for large vessel surgeries [internal diameter (ID) ≥ 10 mm] but present significant challenges in medium to small vessels (ID < 10 mm) due to increased risks of thrombosis, stenosis, and infection. In this study, we developed a small-diameter vascular graft using decellularized human amniotic membrane (DAM graft) (ID = 6 mm) and transplanted it into porcine carotid arteries, comparing it with ePTFE grafts to assess inflammation, biocompatibility, patency, and overall function. One-week post-implantation, ultrasound imaging confirmed blood patency in both graft types. However, after one-month, gross examination revealed pronounced neointimal hyperplasia in ePTFE grafts, while DAM grafts maintained open lumens without signs of stenosis or thrombosis. Histological analysis showed extensive fibrous tissue formation in ePTFE grafts, resulting in luminal narrowing, whereas DAM grafts displayed sustained lumen patency and vascular integration. Immunofluorescence confirmed reduced inflammation and improved tissue organization in DAM grafts, characterized by lower macrophage infiltration and better cellular architecture. These findings suggest that DAM grafts offer superior biocompatibility and significantly lower risks of neointimal hyperplasia, making them a promising alternative for small-diameter vascular surgeries compared to ePTFE grafts.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jairo Lumpuy-Castillo, Yujie Fu, Alan Avila, Kateryna Solodka, Jiantong Li, Oscar Lorenzo, Erica Zeglio, Leonardo Daniel Garma
{"title":"Inkjet-printed graphene multielectrode arrays: an accessible platform for in vitro cardiac electrophysiology","authors":"Jairo Lumpuy-Castillo, Yujie Fu, Alan Avila, Kateryna Solodka, Jiantong Li, Oscar Lorenzo, Erica Zeglio, Leonardo Daniel Garma","doi":"10.1101/2024.09.09.611887","DOIUrl":"https://doi.org/10.1101/2024.09.09.611887","url":null,"abstract":"In vitro models have now become a realistic alternative to animal models for cardiotoxicity assessment. However, the cost and expertise required to implement in vitro electrophysiology systems to study cardiac cells poses a strong obstacle to their widespread use. This study presents a novel, cost-effective approach for in vitro cardiac electrophysiology using fully-printed graphene-based microelectrode arrays (pGMEAs) coupled with an open-source signal acquisition system. We characterized the pGMEAs' electrical properties and biocompatibility, observing low impedance values and cell viability. We demonstrated the platform's capability to record spontaneous electrophysiological activity from HL-1 cell cultures, and we monitored and quantified their responses to chemical stimulation with noradrenaline. This study demonstrates the feasibility of producing fully-printed, graphene-based devices for in vitro electrophysiology. The accessible and versatile platform we present here represents a step further in the development of alternative methods for cardiac safety screening.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"73 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yubin Lin, Alexander Silverman-Dultz, Madeline Bailey, Daniel J. Cohen
{"title":"SCRATCH: A programmable, open-hardware, benchtop robot that automatically scratches cultured tissues to investigate cell migration, healing, and tissue sculpting.","authors":"Yubin Lin, Alexander Silverman-Dultz, Madeline Bailey, Daniel J. Cohen","doi":"10.1101/2024.08.27.609782","DOIUrl":"https://doi.org/10.1101/2024.08.27.609782","url":null,"abstract":"Despite the widespread popularity of the \"scratch assay\", where a pipette is dragged through cultured tissue to create an injury gap to study cell migration and healing, the manual nature of the assay carries significant drawbacks. So much of the process depends on individual manual technique, which can complicate quantification, reduce throughput, and limit the versatility and reproducibility of the approach. Here, we present a truly open-source, low-cost, accessible, and robotic scratching platform that addresses all of the core issues. Compatible with nearly all standard cell culture dishes and usable directly in a sterile culture hood, our robot makes highly reproducible scratches in a variety of complex cultured tissues with high throughput. Moreover, we demonstrate how scratching can be programmed to precisely remove areas of tissue to sculpt arbitrary tissue and wound shapes, as well as enable truly complex co-culture experiments. This system significantly improves the usefulness of the conventional scratch assay, and opens up new possibilities in complex tissue engineering and cell biological assays for realistic wound healing and migration research.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"271 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Biofabrication of engineered tissues by 3D bioprinting of tissue specific high cell-density bioinks","authors":"Oju Jeon, Hyoeun Park, Kent Leach, Eben Alsberg","doi":"10.1101/2024.09.11.612457","DOIUrl":"https://doi.org/10.1101/2024.09.11.612457","url":null,"abstract":"Bioprinting of high cell-density bioinks is a promising technique for cellular condensation-based tissue engineering and regeneration medicine. However, it remains difficult to create precisely controlled complex structures and organization of tissues with high cell-density bioink-based bioprinting for tissue specific condensation. In this study, we present newly biofabricated tissues from directly assembled, tissue specific, high cell-density bioinks which have been three-dimensionally printed into a photocrosslinkable and biodegradable hydrogel microparticle supporting bath. Three types of tissue specific high cell-density bioinks have been prepared with individual stem cells or stem cell aggregates by incorporation of growth factor-loaded gelatin microparticles. The bioprinted tissue specific high cell-density bioinks in the photocrosslinked microgel supporting bath condense together and differentiate down tissue-specific lineages to form multi-phase tissues (e.g., osteochondral tissues). By changing the growth factors and cell types, these tissue specific high cell-density bioinks enable engineering of various functional tissues with controlled architecture and organization of cells.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255980","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Samantha G Zambuto, Samyuktha S Kolluru, Abir Hamdaoui, Annabella M Mascot, Siobhan S Sutcliffe, Jerry L Lowder, Michelle L Oyen
{"title":"Vaginal Tissue Engineering via Gelatin-Elastin Fiber-Reinforced Hydrogels","authors":"Samantha G Zambuto, Samyuktha S Kolluru, Abir Hamdaoui, Annabella M Mascot, Siobhan S Sutcliffe, Jerry L Lowder, Michelle L Oyen","doi":"10.1101/2024.09.09.611932","DOIUrl":"https://doi.org/10.1101/2024.09.09.611932","url":null,"abstract":"The vagina is a fibromuscular tube-shaped organ spanning from the hymenal ring to the cervix that plays critical roles in menstruation, pregnancy, and female sexual health. Vaginal tissue constituents, including cells and extracellular matrix components, contribute to tissue structure, function, and prevention of injury. However, much microstructural function remains unknown, including how the fiber-cell and cell-cell interactions influence macromechanical properties. A deeper understanding of these interactions will provide critical information needed to reduce and prevent vaginal injuries. Our objectives for this work herein are to first engineer a suite of biomaterials for vaginal tissue engineering and second to characterize the performance of these biomaterials in the vaginal microenvironment. We successfully created fiber-reinforced hydrogels of gelatin-elastin electrospun fibers infiltrated with gelatin methacryloyl hydrogels. These composites recapitulate vaginal material properties, including stiffness, and are compatible with the vaginal microenvironment: biocompatible with primary vaginal epithelial cells and in acidic conditions. This work significantly advances progress in vaginal tissue engineering by developing novel materials and developing a state-of-the-art tissue engineered vagina.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yuankai Lu, Yi Hua, Po-Yi Lee, Andrew Theophanous, Shaharoz Tahir, Qi Tian, Ian A Sigal
{"title":"Impact of anatomic variability and other vascular factors on lamina cribrosa hypoxia","authors":"Yuankai Lu, Yi Hua, Po-Yi Lee, Andrew Theophanous, Shaharoz Tahir, Qi Tian, Ian A Sigal","doi":"10.1101/2024.09.12.610282","DOIUrl":"https://doi.org/10.1101/2024.09.12.610282","url":null,"abstract":"Insufficient oxygenation in the lamina cribrosa (LC) may contribute to axonal damage and glaucomatous vision loss. To understand the range of susceptibilities to glaucoma, we aimed to identify key factors influencing LC oxygenation and examine if these factors vary with anatomical differences between eyes. We reconstructed 3D, eye-specific LC vessel networks from histological sections of four healthy monkey eyes. For each network, we generated 125 models varying vessel radius, oxygen consumption rate, and arteriole perfusion pressure. Using hemodynamic and oxygen supply modeling, we predicted blood flow distribution and tissue oxygenation in the LC. ANOVA assessed the significance of each parameter. Our results showed that vessel radius had the greatest influence on LC oxygenation, followed by anatomical variations. Arteriole perfusion pressure and oxygen consumption rate were the third and fourth most influential factors, respectively. The LC regions are well perfused under baseline conditions. These findings highlight the importance of vessel radius and anatomical variation in LC oxygenation, providing insights into LC physiology and pathology. Pathologies affecting vessel radius may increase the risk of LC hypoxia, and anatomical variations could influence susceptibility. Conversely, increased oxygen consumption rates had minimal effects, suggesting that higher metabolic demands, such as those needed to maintain intracellular transport despite elevated intraocular pressure, have limited impact on LC oxygenation.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Melinda Pohle, Edward Curry, Suzanne Gibson, Adam Brown
{"title":"Synthetic G-quadruplex components for predictable, precise two-level control of mammalian recombinant protein expression","authors":"Melinda Pohle, Edward Curry, Suzanne Gibson, Adam Brown","doi":"10.1101/2024.09.10.612233","DOIUrl":"https://doi.org/10.1101/2024.09.10.612233","url":null,"abstract":"Control of mammalian recombinant protein expression underpins the <em>in vitro</em> manufacture and <em>in vivo</em> performance of all biopharmaceutical products. However, routine optimization of protein expression levels in these applications is hampered by a paucity of genetic elements that function predictably across varying molecular formats and host cell contexts. Herein, we describe synthetic genetic components that are specifically built to simplify bioindustrial expression cassette design processes. Synthetic G-quadruplex elements with varying sequence feature compositions were systematically designed to exhibit a wide-range of regulatory activities, and inserted into identified optimal positions within a standardized, bioindustry compatible core promoter-5' UTR control unit. The resulting library tuned protein production rates over two orders of magnitude, where DNA and RNA G-quadruplexes could be deployed individually, or in combination to achieve synergistic two-level regulatory control. We demonstrate these components can predictably and precisely tailor protein expression levels in i) varying gene therapy and biomanufacturing cell hosts, and ii) both plasmid DNA and synthetic mRNA contexts. As an exemplar use-case, a vector design platform was created to facilitate rapid optimization of polypeptide expression ratios for difficult-to-express multichain products. Permitting simple, predictable titration of recombinant protein expression, this technology should prove useful for gene therapy and biopharmaceutical manufacturing applications.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Claire Bridges, Lu Fu, Jonathan Yeow, Xiaojing Huang, Miriam Jackson, Rhiannon Kuchel, James Sterling, Shenda Baker, Megan Lord
{"title":"The level of endothelial glycocalyx maturity modulates interactions with charged nano-materials","authors":"Claire Bridges, Lu Fu, Jonathan Yeow, Xiaojing Huang, Miriam Jackson, Rhiannon Kuchel, James Sterling, Shenda Baker, Megan Lord","doi":"10.1101/2024.09.10.611831","DOIUrl":"https://doi.org/10.1101/2024.09.10.611831","url":null,"abstract":"Nanomaterials have been extensively investigated for their potential in delivering therapeutics to target tissues, but few have advanced to clinical application. The luminal surface of endothelial cells that line blood vessels are covered by a glycocalyx, a complex extracellular matrix rich in anionic glycans. However, the role of this glycocalyx in governing nanomaterial- cell interactions is often overlooked. In this study, we demonstrate that gold nanoparticles functionalized with branched polyethyleneimine (AuNP+) bind to primary human endothelial cells expressing either a developing or mature glycocalyx, with the interaction involving hyaluronan and heparan sulfate. Notably, the mature glycocalyx decreases the toxicity of AuNP+. In contrast, lipoic acid-functionalized gold nanoparticles (AuNP-) bind to endothelial cells with a developing glycocalyx, but not a mature glycocalyx. To further investigate this phenomenon, we studied charged polymers, including poly(arginine) (polyR) and poly(glutamic acid) (polyE). PolyE does not associate with endothelial cells regardless of glycocalyx maturity, but when glycans are enzymatically degraded, it can bind to the cells. Conversely, polyR associates with endothelial cells irrespective of glycocalyx maturity or glycan degradation. These findings highlight the intricate relationship between nanomaterial charge and presentation in interactions with endothelial cells, offering insights for modulating nanomaterial interactions with the blood vessel wall.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ju-Chan Park, Heesoo Uhm, Yong-Woo Kim, Ye Eun Oh, Sangsu Bae
{"title":"AI-generated small binder improves prime editing","authors":"Ju-Chan Park, Heesoo Uhm, Yong-Woo Kim, Ye Eun Oh, Sangsu Bae","doi":"10.1101/2024.09.11.612443","DOIUrl":"https://doi.org/10.1101/2024.09.11.612443","url":null,"abstract":"The prime editing 2 (PE2) system comprises a nickase Cas9 fused to a reverse transcriptase utilizing a prime editing guide RNA (pegRNA) to introduce desired mutations at target genomic sites. However, the PE efficiency is limited by mismatch repair (MMR) that excises the DNA strand containing desired edits. Thus, inhibiting key components of MMR complex through transient expression of a dominant negative MLH1 (MLH1dn) exhibited approximately 7.7-fold increase in PE efficiency over PE2, generating PE4. Herein, by utilizing a generative artificial intelligence (AI) technologies, RFdiffusion and AlphaFold 3, we ultimately generated a de novo MLH1 small binder (named MLH1-SB), which bind to the dimeric interface of MLH1 and PMS2 to disrupt the formation of key MMR components. MLH1-SB's small size (82 amino acids) allowed it to be integrated into pre-existing PE architectures via the 2A system, creating a novel PE-SB platform. Resultantly, by incorporating MLH1-SB into PE7, we have developed an improved PE architecture called PE7-SB, which demonstrates the highest PE efficiency to date (29.4-fold over PE2 and 2.4-fold over PE7 in HeLa cells), providing an insight that generative AI technologies will boost up the improvement of genome editing tools.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255986","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}