The osteopontin released from mesenchymal stem cells (MSC) undergoing lineage differentiation can negatively influence the expansion of hematopoietic stem cells (HSCs) in co-culture systems developed for expanding HSCs. Therefore, minimising the amount of osteopontin in the co-culture system is important for the successful ex vivo expansion of HSCs. Towards this goal, a bioengineered 3D-microfibrous matrix that can maintain MSCs in less osteopontin releasing condition has been developed and its influence on the expansion of HSCs has been studied. The newly developed 3D-matrix significantly decreased the release of osteopontin, depending on the MSC culture conditions used during the priming period before HSC seeding. The culture system with the lowest amount of osteopontin facilitated more than 40-fold increase in HSC number in 1 weeks’ time period. Interestingly, the viability of expanded cells and the CD34+ pure population of HSCs found to be the highest in the low osteopontin containing system. Therefore, bioengineered microfibrous 3D-matrices seeded with MSCs, primed under suitable culture conditions can be an improved ex vivo expansion system for HSC culture.
Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately removed to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taken into consideration of residual HCP profile in the product, the dose, dosage form, and administration route etc. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of a Merck investigational stimulator of interferon genes protein (STING) agonist, MK-1454, which is a cyclic dinucleotide synthesized using E. coli cell lysate overexpressing cyclic GMP-AMP synthase (cGAS) as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 API.
The Ocean covers two-third of our planet and has great biological heterogeneity. Marine organisms like algae, vertebrates, invertebrates, and microbes are known to provide many natural products with biological activities as well potent sources of biomaterials for therapeutic, biomedical, biosensors, and climate stabilization. Over the years, the field of biosensors have gained huge attention due to their extraordinary ability in providing early diseases diagnosis and treatment as well as environmental pollutants. This review focuses on various biomaterials (Carbohydrtae polymers, proteins, polyacids etc) of marine origin such as Alginate, Chitin, Chitosan, Fucoidan, Carrageenan, Chondroitin Sulfate (CS), Hyaluronic acid (HA), Collagen, marine pigments, marine nanoparticles, Hydroxyapatite (HAp), Biosilica, lectins, and marine whole cell. Further, it mentions the source of such marine biomaterials and their promising evolution for the development of biosensors that are potent to be employed in the biomedical, environmental science and agricultural sciences domains.
In the manufacture of therapeutic monoclonal antibodies (mAbs), the clarified cell culture fluid is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of IgG loading to avoid wastage. Therefore, continuous real-time monitoring of IgG flowthrough is of great interest. We previously developed a fluorescence-based monitoring technology that allows mix-and-read mAb detection in cell culture fluid. Here we report the use of reporters immobilized on CNBr-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands to produce an immediately detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified cell culture fluid emerging from a Protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 2 minutes at a flow rate of 0.5 mL/min.
A novel fermentation process was developed in which renewable electricity is indirectly used as a fermentation substrate, synergistically decreasing both the consumption of sugar as a first generation carbon source and emission of the greenhouse gas CO2. To achieve this, a glucose-based process is co-fed with formic acid, which can be generated by capturing CO2 from fermentation offgas followed by electrochemical reduction with renewable electricity. This ‘closed carbon loop’ concept is demonstrated by a case study in which co-feeding formic acid is shown to significantly increase the yield of biomass on glucose of the industrially relevant yeast species Yarrowia lipolytica. First, the optimal feed ratio of formic acid to glucose is established using chemostat cultivations. Subsequently, guided by a dynamic fermentation process model, a fed-batch protocol is developed and demonstrated on laboratory scale. Finally, the developed fed-batch process is proven to be scalable to pilot scale. An extension of this proven concept to also recycle the O2 that is co-generated with the formic acid to the fermentation process for intensification purposes, and a potential further application of the concept to anaerobic fermentations are discussed.
The deformation and detachment of bacterial biofilm are related to the structural and mechanical properties of the biofilm itself. Extracellular polymeric substances (EPS) play an important role on keeping the mechanical stability of biofilms. The understanding of biofilm mechanics and detachment can help to reveal biofilm survival mechanisms under fluid shear and provide insight about what flows might be needed to remove biofilm in a cleaning cycle or for a ship to remove biofilms. However, how the EPS may affect biofilm mechanics and its deformation in flow conditions remains elusive. To address this, a coupled computational fluid dynamic – discrete element method (CFD-DEM) model was developed. The mechanisms of biofilm detachment, such as erosion and sloughing have been revealed by imposing hydrodynamic fluid flow at different velocities and loading rates. The model, which also allows adjustment of the proportion of different functional group of microorganisms in the biofilm, enables the study of the contribution of EPS towards biofilm resistance to fluid shear stress. Furthermore, the stress-strain curves during biofilm deformation have been captured by loading and unloading fluid shear stress to study the viscoelastic properties of the biofilm.
Three-dimensional (3D) bioprinting shows great potential for autologous vascular grafts due to its simplicity, accuracy, and flexibility. 6mm diameter vascular grafts are used in clinic. However, producing small-diameter vascular grafts are still an enormous challenge. Normally, sacrificial hydrogels are used as temporary lumen support to mold tubular structure which will affect the structure’s stability. In this study, we develop a new bioprinting approach to fabricating small-diameter vessel using two-step crosslinking process. ¼ lumen wall of bioprinted gelatin mechacrylate (GelMA) flat structure is exposed to ultraviolet (UV) light briefly for having certain strength, while ¾ lumen wall shows as concave structure remained uncrosslinked. Pre-crosslinked flat structure is merged towards the uncrosslinked concave structure. Two individual structures will be combined tightly into an intact tubular structure by receiving more UV exposure time. Complicated tubular structures are constructed by these method. Notably, the GelMA-based bioink loaded with smooth muscle cells (SMCs) are bioprinted as the outer layer and human umbilical vein endothelial cells (HUVECs) are seeded onto the inner surface. A bionic vascular vessel with dual layers is fabricated successfully and keeps good viability, and functionality. This study may provide a novel idea for fabricating biomimetic vascular network or other more complicated organs.
Transition of rapid, ready-to-use, and low-cost nucleic acid-based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling, through nucleic acid isolation and detection in the field. Here, we developed a simple, portable, low-cost, colorimetric, and remotely controllable platform for reliable, high-throughput, and rapid diagnosis using loop mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low-cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady-state temperature error of the system is less than 1%. We performed LAMP, Colony-LAMP, and Colony PCR reactions using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65˚C and 30 min. We detected the end-point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony-LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.
Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.
High volumetric productivities can be achieved when perfusion processes are operated at high cell densities. Yet it is fairly challenging to keep high cell density cultures in a steady state over an extended period. Aiming for robust processes, in this study cultures were operated at a constant biomass specific perfusion rate (BSPR). The cell density was monitored with a capacitance probe and a continuous bleed maintained the cell density at the targeted viable cell volume (VCV). Despite our tightly controlled BSPR, a gradual accumulation of ammonium and changes in cell diameter were observed during the production phase for the three different monoclonal antibodies (mAbs). Although a lot of efforts in media optimization have been made to reduce ammonium in fed-batch process, less examples are known about how media components impact the cellular metabolism and thus the quality of monoclonal antibodies in continuous processes. In this work, we show that a continuous Na-pyruvate fed at 2 g/L/day strongly reduced ammonium production and stabilized fucosylation, sialylation and high mannose content for three different mAbs.
Taste is one of the most basic and important sensations that is able to monitor the food quality and avoid intake of potential danger materials. Whether as an inevitable symptom of aging or a complication of cancer treatment, taste loss so seriously affects the patient’s life quality. Taste bud organoids provide a great convenience for the research of taste functions and the underlying mechanisms due to their characteristics of availability, strong maneuverability, and high similarity to the in-vivo taste buds. This review gives a systemic and comprehensive introduction to the preparation and application of taste bud organoids towards chemical sensing mechanisms. For the first, the basic structure and function of taste buds in biomedicine will be brief introduced. Then, the currently available approaches for the preparation of taste bud organoids are summarized and discussed, which are mainly divided into two categories, i.e. stem/progenitor cell-derived approach and tissue-derived approach. For the next, different applications of taste bud organoids in biomedicine are outlined based on their central roles such as disease modeling, biological sensing, gene regulation, and signal transduction. Finally, the current challenges, future development trends and prospects of research in taste bud organoids are proposed and discussed.
Bioconversion process with a single target product often lacks economic competitiveness owing to incomplete use of raw material and high costs of downstream processing (DSP). Here, we show with the microbial conversion of crude glycerol that an integrated strain engineering and catalytic conversion of the so-called byproducts can greatly improve DSP and the process economy. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72 and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The co-production of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for a complete use of the raw material with the simultaneous production of three appealing products which greatly improve the process economy and ecology.
Silk is a fibrous protein, has been a part of human lives for centuries and was used as suture and textile material. Silk is mainly produced by members of certain arthropods such as spiders, butterflies, mites, and moths. However, recent biotechnological advances have revolutionized silk as a biomaterial for various applications ranging from diverse sensors to robust fibers. The biocompatibility, mechanical resilience, and biodegradability of the material make it a suitable candidate for biomaterials. Silk can also be easily converted into several morphological forms, including fibers, films, sponges, and hydrogels. Provided these abilities, silk has received excellent traction from scientists worldwide for various developments, one of them being its use as a bio-sensor. The diversity of silk materials offers various options, giving scientists the freedom to choose from and personalize them as per their needs. In this review, we foremost look upon the composition, production, properties, and various morphologies of silk. The numerous applications of silk and its derivatives for fabricating biosensors to detect small molecules, macromolecules, and cells have been explored comprehensively. Also, the data from various globally developed sensors using silk have been described into organized tables for each category of molecules, along with their important analytical details.
Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively towards their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells (ASCs), towards their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans (sGAGs) compared to cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.
In vitro systems serve as compact and manipulate models to investigate interactions between different cell types. A homogeneous population of cells predictably and uniformly responds to external factors. In a heterogeneous cell population, the effect of external growth factors is perceived in the context of intercellular interactions. Indirect cell co-cultivation allows one to observe the paracrine effects of cells and separately analyze cell populations. The article describes an application of custom-made cell co-cultivation systems based on protein membranes separated from the bottom of the vessel by the 3d printed holder or kept afloat by a magnetic field. Using the proposed co-cultivation system, we analyzed the interaction of A549 cells and fibroblasts, in the presence and absence of growth factors. During co-cultivation of cells, the expression of genes of the activation for epithelial and mesenchymal transitions decreases. The article proposes the application of a newly available system for the co-cultivation of different cell types.
Biofilms are typically heterogeneous in morphology, structure, and composition, resulting in non-uniform mechanical properties. The distribution of mechanical properties, in turn, determines the biofilm mechanical behavior, such as deformation and detachment. Most past studies neglected heterogeneity of biofilms. In this study, an image-based modeling approach was developed to transform two-dimensional optical coherence tomography biofilm images to a pixel-scale non-Newtonian viscosity map of the biofilm. The spatial distribution of non-Newtonian viscosity was applied in an established Oldroyd-B constitutive model and implemented using the phase-field continuum approach for the deformation and stress analysis. The heterogeneous model was able to predict deformations and stresses more accurately than a homogenous one. This is the first time, to the best of our knowledge, that an image-based approach is used to map the mechanical heterogeneity of biofilms for computational studies. It provides an efficient method to characterize biofilm mechanical behavior.
Omics approaches have been applied to understand the boosted productivity of natural products by industrial high-producing microorganisms. Here, with the updated genome sequence and transcriptomic profiles derived from high-throughput sequencing, we exploited comparative omics analysis to further enhance the biosynthesis of erythromycin in an industrial overproducer, Saccharopolyspora erythraea HL3168 E3. By comparing the genome of E3 with the wild type NRRL23338, we identified fragment deletions inside 56 coding sequences and 255 single nucleotide polymorphisms over the genome of E3. Substantial numbers of genomic variations were observed in genes responsible for pathways which were interconnected to the biosynthesis of erythromycin by supplying precursors/cofactors or by signal transduction. Through comparative transcriptomic analysis, L-glutamine/L-glutamate and 2-oxoglutarate were identified as reporter metabolites. Around the node of 2-oxoglutarate, genomic mutations were also observed. Furthermore, the transcriptomic data suggested that genes involved in the biosynthesis of erythromycin were significantly up-regulated constantly, whereas some genes in biosynthesis clusters of other secondary metabolites contained nonsense mutations and were expressed at extremely low levels. Based on the omics association analysis, readily available strategies were proposed to engineer E3 by simultaneously overexpressing sucB (coding for 2-oxoglutarate dehydrogenase E2 component) and sucA (coding for 2-oxoglutarate dehydrogenase E1 component), which increased the erythromycin titer by 71% compared to E3 in batch culture. This work provides more promising molecular targets to engineer for enhanced production of erythromycin by the overproducer.
Detergent-mediated virus inactivation (VI) provides a valuable orthogonal strategy for viral clearance particularly for next generation continuous manufacturing. Furthermore, there exists an industry-wide need to replace the conventionally employed detergent, Triton X-100, with eco-friendly alternatives. This study provides a systematic approach to screen detergents as VI agents through the study of VI of three different enveloped viruses for monoclonal antibodies and fusion proteins. We investigated three major aspects of VI namely, the impact of VI agent on the therapeutic quality attributes, clearance of the VI agent and other impurities through subsequent chromatographic steps and lastly the efficacy of VI for the said detergent. Several quality attributes such as charge variance, oxidation, deamidation, glycosylation and aggregation were investigated. Aggregation was a key indicator of stability. Experimental and modeling data was used to decipher the mechanism and kinetics of aggregation for pH sensitive molecules by exploring worst case VI conditions. We found product aggregation and its kinetics to be driven by extrinsic factors such as detergent and protein concentration. Aggregation was also impacted by initial aggregation level as well as intrinsic factors such as the protein sequence and detergent hydrophobicity and critical micelle concentration (CMC). VI efficiency was dependent on the virus tested, duration of incubation as well as detergent CMC and concentration. Dodecyl maltopyranoside (DDM) was found to be a promising candidate for potential application in VI. Knowledge gained here on factors driving product stability and VI provides valuable insight to design, standardize and optimize conditions (concentration, duration of inactivation) for screening of detergent-mediated VI.