Biotechnology Journal最新文献

The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

Skin plays a crucial role in human physiological functions, however, it was vulnerable to bacterial infection which delayed wound healing. Nowadays, designing an individual wound dressing with good biocompatibility and sustaining anti-infection capability for healing of chronic wounds are still challenging. In this study, various concentrations of the ciprofloxacin (CIP) were mixed with gelatine (Gel)/sodium alginate (SA) solution to prepare Gel/SA/CIP (GAC) bioinks, following the fabrication of GAC scaffold by an extrusion 3D bioprinting technology. The results showed that the GAC bioinks had good printability and the printed GAC scaffolds double-crosslinked by EDC/NHS and CaCl₂ had rich porous structure with appropriate pore size, which were conducive to drug release and cell growth. It demonstrated that the CIP could be rapidly released by 70% in 5 min, which endowed the GAC composite scaffolds with an excellent antibacterial ability. Especially, the antibacterial activities of GAC7.5 against Escherichia coli and Staphylococcus aureus within 24 h were even close to 100%, and the inhibition zones were still maintained 14.78 ± 0.40 mm and 14.78 ± 0.40 mm, respectively, after 24 h. Meanwhile, GAC7.5 also demonstrated impressive biocompatibility which can promote the growth and migration of L929 and accelerate wound healing. Overall, the GAC7.5 3D bioprinting scaffold could be used as a potential skin dressing for susceptible wounds with excellent antibacterial activity and good biocompatibility to meet urgent clinical needs.

Selecting the optimal microalgal strain for carbon capture and biomass production is crucial for ensuring the commercial viability of microalgae-based biorefinery processes. This study aimed to evaluate the impact of varying bicarbonate concentrations on the growth rates, inorganic carbon (IC) utilization, and biochemical composition of three freshwater and two marine microalgal species. Parachlorella kessleri, Vischeria cf. stellata, and Porphyridium purpureum achieved the highest carbon removal efficiency (>85%) and biomass production at 6 g L⁻¹ sodium bicarbonate (NaHCO₃), while Phaeodactylum tricornutum showed optimal performance at 1 g L⁻¹ NaHCO₃. The growth and carbon removal rate of Scenedesmus quadricauda increased with increasing NaHCO₃ concentrations, although its highest carbon removal efficiency (∼70%) was lower than the other species. Varying NaHCO₃ levels significantly impacted the biochemical composition of P. kessleri, S. quadricauda, and P. purpureum but did not affect the composition of the remaining species. The fatty acid profiles of the microalgae were dominated by C16 and C18 fatty acids, with P. purpureum and P. tricornutum yielding relatively high polyunsaturated fatty acid content ranging between 14% and 30%. Furthermore, bicarbonate concentration had a species-specific effect on the fatty acid and chlorophyll-a content. This study demonstrates the potential of bicarbonate as an effective IC source for microalgal cultivation, highlighting its ability to select microalgal species for various applications based on their carbon capture efficiency and biochemical composition.

The mutual interactions of endoplasmic reticulum (ER) resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease system for Protein-Protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology, and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER-resident proteins, most being identified molecular chaperones, of S. cerevisiae were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER-resident proteins, except the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER-resident proteins perform their functions in a cooperative way and a multimer complex might be formed during the process.

Rapidly expanding biopharmaceutical market demands more cost-effective platforms to produce protein therapeutics. To this end, novel approaches, such as perfusion culture or concentrated fed-batch, have been explored for higher yields and lower manufacturing costs. Although these new approaches produced promising results, but their wide-spread use in the industry is still limited. In this study, a dialysis rolled scaffold bioreactor was presented for long-term production of monoclonal antibodies with reduced media consumption. Media dialysis can selectively remove cellular bio-wastes without losing cells or produced recombinant proteins. The dialysis process was streamlined to significantly improve its efficiency. Then, extended culture of recombinant CHO cells for 41 days was successfully demonstrated with consistent production rate and minimal media consumption. The unique configuration of the developed bioreactor allows efficient dialysis for media management, as well as rapid media exchange to harvest produced recombinant proteins before they degrade. Taken together, it was envisioned that the developed bioreactor will enable cost-effective and long-term large-scale culture of various cells for biopharmaceutical production.

Skeletal muscle satellite cells (SCs) are essential for muscle regeneration. Their proliferation and differentiation are influenced by fibroblast growth factor (FGF)-2. In this study, we screened for FGF-2-derived peptides that promote SC proliferation. Utilizing photocleavable peptide array technology, a library of 7-residue peptides was synthesized, and its effect on SC proliferation was examined using a mixture of five peptides. The results showed that peptides 1–5 (136%), 21–25 (136%), 26–30 (141%), 31–35 (159%), 71–75 (135%), 76–80 (144%), and 126–130 (137%) significantly increased SC proliferation. Further experiments revealed that peptide 33, CKNGGFF, enhanced SC proliferation. Furthermore, its extended form, peptide 33-13, CKNGGFFLRIHPD, promoted SC proliferation and increased the percentage of Pax7-positive cells, indicating that SCs were maintained in an undifferentiated state. The addition of FGF-2 and peptide 33-13 further induced cell proliferation but did not increase the percentage of Pax7-positive cells. A proliferation assay using an FGF receptor (FGFR) inhibitor suggested that peptide 33-13 acts through the FGFR-mediated and other pathways. Although further research is necessary to explore the mechanisms of action of these peptides and their potential for in vivo and in vitro use, the high sequence conservation of peptides 33 and 33-13 in FGF-2 across multiple species suggests their broad application prospects in biomedical engineering and biotechnology.

The demand for the essential commodity chemical 1,2-propanediol (1,2-PDO) is on the rise, as its microbial production has emerged as a promising method for a sustainable chemical supply. However, the reliance of 1,2-PDO production in Escherichia coli on anaerobic conditions, as enhancing cell growth to augment precursor availability remains a substantial challenge. This study presents glucose-based aerobic production of 1,2-PDO, with xylose utilization facilitating cell growth. An engineered strain was constructed capable of exclusively producing 1,2-PDO from glucose while utilizing xylose to support cell growth. This was accomplished by deleting the gloA, eno, eda, sdaA, sdaB, and tdcG genes for 1,2-PDO production from glucose and introducing the Weimberg pathway for cell growth using xylose. Enhanced 1,2-PDO production was achieved via yagF overexpression and disruption of the ghrA gene involved in the 1,2-PDO-competing pathway. The resultant strain, PD72, produced 2.48 ± 0.15 g L⁻¹ 1,2-PDO with a 0.27 ± 0.02 g g⁻¹-glucose yield after 72 h cultivation. Overall, this study demonstrates aerobic 1,2-PDO synthesis through the isolation of the 1,2-PDO synthetic pathway from the tricarboxylic acid cycle.

Microalgae are a group of microorganisms containing chlorophyll A, which are highly photosynthetic and rich in nutrients. And they can produce multiple bioactive substances (peptides, proteins, polysaccharides, and fatty acids) for biomedical applications. Despite the unique advantages of microalgae-based biotherapy, the insufficient treatment efficiency limits its further application. With the development of nanotechnology, the combination of microalgae and biomaterials can improve therapeutic efficacies, which has attracted increasing attention. In this microalgal-biomaterials hybrid system, biomaterials with excellent optical and magnetic properties play an important role in biological therapy. Microalgae, as a natural vehicle, can increase oxygen content and alleviate hypoxia in diseased areas, further enhancing therapeutic effects. In this review, the synergistic therapeutic effects of microalgal-biomaterials hybrid system in different diseases (cancer, myocardial infarction, ischemia stroke, chronic infection, and intestinal diseases) are comprehensively summarized.

Scalable single-use adherent cell-based biomanufacturing platforms are essential for unlocking the full potential of cell and gene therapies. The primary objective of this study is to design and develop a novel fixed bed bioreactor platform tailored specifically for scaling up adherent cell culture. The bioreactor comprises a packed bed of vertically stacked woven polyethylene terephthalate mesh discs, sandwiched between two-fluid guide plates. Leveraging computational fluid dynamics modeling, we optimized bioreactor design to achieve uniform flow with minimal shear stress. Residence time distribution measurements demonstrated excellent flow uniformity with plug flow characteristics. Periodic media sampling coupled with offline analysis revealed minimal gradients of crucial metabolites (glucose, glutamine, lactate, and ammonia) across the bioreactor during cell growth. Furthermore, the bioreactor platform demonstrated high performance in automated cell harvesting, with ≈96% efficiency and ≈98% viability. It also exhibited linear scalability in both operational parameters and performance for cell culture and adeno-associated virus vector production. We developed mathematical models based on oxygen uptake rates to accurately predict cell growth curves and estimate biomass in real-time. This study demonstrates the effectiveness of the developed fixed-bed bioreactor platform in enabling scalable adherent cell-based biomanufacturing with high productivity and process control.