Maximum Viable Cell Research Articles

Piezoelectric barium titanate/hydroxyapatite composite coatings on Ti-6Al-4V alloy via electrophoretic deposition

Electrophoretic deposition (EPD) is a widely accepted, cost-effective and simple method to obtain conformal coatings of dense nanoparticles via application of a DC voltage. This paper reports the physical and mechanical properties of nanostructured barium titanate/hydroxyapatite (BT/HA) ceramic composites coated onto Ti-6Al-4V alloy via cathodic EPD in various ratios of 40:60, 50:50 and 60:40 (HB4, HB5 and HB6) respectively. Homogenous BT/HA coatings were obtained at a notably low voltage of 10 V. The crystallinity and phase analysis confirmed the formation of tetragonal phase of BT indicating the piezoelectric property. HB6 exhibits maximum piezoelectric coefficient (d33) of 159 pC/N and Vicker's hardness of 242.31 HV. The cross-sectional electron micrographs show well connected and homogeneous coatings with increasing amounts of BT. Human mesenchymal stem cell lines were utilized in biocompatibility experiments, which revealed that HB composites had greater viability of HW-MSCs cells than pure BT, with HB6 exhibiting a maximum cell viability of more than 90 %.

Effect of Tetrapropylammonium Chloride Quaternary Ammonium Salt on Characterization, Cytotoxicity, and Antibacterial Properties of PLA/PEG Electrospun Mat.

In this study, poly(lactic acid) (PLA)-tetrapropylammonium chloride (TCL)-poly(ethylene glycol) (PEG) nonwoven networks were produced using PLA, PEG with different concentrations (3, 5, 7, and 9 wt%), and TCL. PEG is included as a plasticizer in PLA polymer, which has high biocompatibility but a brittle structure. The importance of this study is to investigate the effect of TCL salt on the characterization of PLA-PEG nanofibers. For this research, the cytotoxicity test system responsible for the fibroblast cell line (L929) was evaluated with the liquid absorption capacity (LAC) and drying time tests for its use in wound dressings. The addition of TCL salt reduced bead formation in PLA-PEG nanofibers and increased the homogeneity of fiber dispersion. The smoothest and most homogeneous nonwoven networks were obtained as PLA-5TCL-PEG. It was also reported that this nonwoven network exhibited liquid absorption behavior with a maximum increase of 150% compared to the PLA-PEG nonwoven network and had the highest Young's modulus value of 12.97 MPa. In addition to these tests, evaluations were made with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), drying time test, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and mechanical tests. In addition, high cell viability was observed in L292 mouse fibroblast cells at the end of the 24th hour, again with the effect of TCL salt. In addition, antibacterial activity was tested against gram-negative E. coli and gram-positive S. aureus bacteria, and it was observed that there was no antibacterial activity. Since PLA-TCL-PEG nonwoven webs have a maximum cell viability of 133.27%, they are recommended as a potential dermal wound dressing.

Study on the Bioactivity Response of the Newly Developed Zn-Cu-Mn/Mg Alloys for Biodegradable Implant Application.

Scaffolds play a crucial role in bone tissue engineering to support the defect area through bone regeneration and defect reconstruction. Promising tissue regeneration without negative repercussions and avoidance of the lifelong presence inside the body make bioresorbable metals prosper in the field of regenerative medicine. Recently, Zn and its alloys have emerged as promising biodegradable materials for their moderate degradation rate and satisfactory biocompatibility. Nevertheless, it is very challenging for cells to adhere and grow over the Zn surface alone, which influences the tissue-implant integration. In this study, an attempt has been made to systematically investigate the bioactivity responses in terms of in vitro hemocompatibility, cytotoxicity, antibacterial activity, and in vivo biocompatibility of newly developed Zn-2Cu-0.5Mn/Mg alloy scaffolds with different surface roughness. The rough surface of Zn-2Cu-0.5Mg shows the highest degradation rate of 0.16 mm/yr. The rough surface exhibits a prominent role in the adsorption of protein, further enhancing cell adhesion. Concentration-dependent alloy extract shows the highest cell proliferation for 12.5% of the extract with a maximum cell viability of 101% in Zn-2Cu-0.5Mn and 108% in Zn-2Cu-0.5Mg after 3 d. Acceptable hemolysis percentages (less than 5%) with promising anticoagulation properties are observed for all of the conditions. Enhanced antibacterial (Staphylococcus aureus and Escherichia coli) activity due to a significant effect of ions illustrates the maximum killing effect on the bacterial colony for the rough Zn-2Cu-0.5Mg alloy. In addition, it is observed that for rough Zn-2Cu-0.5Mn/Mg alloys, the inflammatory response is minimal after subcutaneous implantation, and neo-bone tissue forms in the defect areas of the rat femur with satisfactory biosafety response. The osseointegration property of the Zn-2Cu-0.5Mg alloy is comparable to that of the Zn-2Cu-0.5Mn alloy. Therefore, the rough surface of the Zn-2Cu-0.5Mg alloy has the potential to enhance biocompatibility and promote better osseointegration activity with host tissues for various biomedical applications.

High-density perfusion cultures of the marine bacterium Rhodovulum sulfidophilum for the biomanufacturing of oligonucleotides

Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50 mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44 % compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCDmax), allowing the determination of the minimum cell-specific perfusion rate (CSPRmin) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes.

Disaccharide-assisted inkjet freezing for improved cell viability

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing.

Sustainable carbon dots from Borreria hispida: enhanced colorimetric sensing of Fe3+ ions and biological applications in live cell imaging.

This study presents the synthesis of advanced nanomaterials derived from the hedge-grown herbal plant, Borreria hispida, and explores their environmental and biological applications. Using a one-step hydrothermal synthesis method, carbon dots derived from Borreria hispida (BHCD) were fabricated and thoroughly characterized through XRD, TEM, FTIR, CHNS, UV-visible, and PL spectroscopy analyses. Under UV illumination, these plant-based carbon dots demonstrated exceptional water solubility, notable photo stability, and a high quantum yield of 40.8%. The average particle size of BHCD was absorbed around 0.5 to 3.5 nm, contributing to superior selectivity and sensitivity in detecting Fe3+ ions, with a limit of detection of 1.2 × 10-6 M. Investigation into the sensing mechanism revealed a binding model wherein two carbon atom molecules bind to one Fe3+ atom in a 2 : 1 ratio for BHCDs and Fe3+ interactions. Additionally, the effectiveness of the developed fluorescent probe for Fe3+ detection was validated using real water samples from ponds and lakes, highlighting its potential for environmental monitoring applications. Furthermore, the biological effects of BHCD were evaluated through cytotoxic assays, demonstrating significant inhibitory effects on MCF7 breast cancer cell lines, with a maximum cell viability of 60%. This research underscores the multifaceted potential of BHCD in environmental monitoring and biomedical applications.

In vitro bioactivity of biodegradable films based on chitosan/quince seed mucilage reinforced with ZnO-nanoparticle

In this study, biodegradable active films were designed by adding ZnO-NPs to the quince seed mucilage/chitosan matrix. The films were investigated for characterization and in vitro bioactivity. According to the results, a significant decrease in moisture content, water holding capacity and light (L*) occurred with the addition of ZnO-NP. (p < 0.05). FT-IR spectra showed the interaction between ZnO-NPs and N-H quince seed mucilage and chitosan groups. Nanocomposite films containing ZnO-NP showed improved thermal stability. The developed bionanocomposite films were easily buried in soil and subjected to degradation. The minimum degradation of the film in soil after 20 days was 62.02%. The maximum cell viability (%) of C/ZnO-NP and C/QSM/ZnO-NP films were determined as 86.81% and 91.22%, respectively, and the films were found to be non-toxic. Moreover, while the quince seed mucilage film did not show antibacterial performance, chitosan and ZnO NPs showed antibacterial effects against the tested bacteria Listeria monocytogenes (ATCC 7644) and Escherichia coli O157:H7 (ATCC 35,150). In this context, the design bionanocomposite films can be used as an active food packaging material for food preservation by controlling food-borne pathogens. In addition, the developed films do not pose a threat to the environment and therefore have great potential for the sustainable food packaging industry.

Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture.

Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity. In this study, we have investigated the feasibility of improving the volumetric productivity by infecting fed-batch cultures at high cell densities. Four commercial and one in-house developed serum-free media were first tested for supporting growth of HEK 293 cells and production of adenovirus type 5 (Ad5) in batch culture. Two best media were then selected for development of fed-batch culture to improve cell growth and virus productivity. A maximum viable cell density up to 16 × 106 cells/mL was achieved in shake flask fed-batch cultures using the selected media and commercial or in-house developed feeds. The volumetric virus productivity was improved by up to six folds, reaching 3.0 × 1010 total viral particles/mL in the fed-batch culture cultivated with the media and feeds developed in house and infected at a cell density of 5 × 106 cells/mL. Additional rounds of optimization of media and feed were required to maintain the improved titer when the fed-batch culture was scaled up in a bench scale (3 L) bioreactor. Overall, the results suggested that fed-batch culture is a simple and feasible process to significantly improve the volumetric productivity of Ad5 through optimization and balance of nutrients in culture media and feeds.

Feasibility Insights of the Green-Assisted Calcium-Phosphate Coating on Biodegradable Zinc Alloys for Biomedical Application: In Vitro and In Vivo Studies.

In the field of bone tissue engineering, recently developed Zn alloy scaffolds are considered potential candidates for biodegradable implants for bone regeneration and defect reconstruction. However, the clinical success of these alloys is limited due to their insufficient surface bioactivities. Further, the higher concentration of Zn2+ produced during degradation promotes antibacterial activity, but deteriorates osteogenic properties. This study fabricated an Azadirachta indica (neem)-assisted brushite-hydroxyapatite (HAp) coating on the recently developed Zn-2Cu-0.5Mg alloy to tackle the above dilemma. The microstructure, degradation behavior, antibacterial activity, and hemocompatibility, along with in vitro and in vivo cytocompatibility of the coated alloys, are systematically investigated. Microstructural analysis reveals flower-like morphology with uniformly grown flakes for neem-assisted deposition. The neem-assisted deposition significantly improves the adhesion strength from 12.7 to 18.8 MPa, enhancing the mechanical integrity. The potentiodynamic polarization study shows that the neem-assisted deposition decreases the degradation rate, with the lowest degradation rate of 0.027 mm/yr for the ZHN2 sample. In addition, the biomineralization process shows the apatite formation on the deposited coating after 21 days of immersion. In vitro cytotoxicity assay exhibits the maximum cell viability of 117% for neem-assisted coated alloy in 30% extract after 5d and the improved cytocompatibility which is due to the controlled release of Zn2+ ions. Meanwhile, neem-assisted coated alloy increases the ZOI by 32 and 24% for Gram-positive and Gram-negative bacteria, respectively. Acceptable hemolysis (<5%) and anticoagulation parameters demonstrate a promising hemocompatibility of the coated alloy. In vivo implantation illustrates a slight inflammatory response and vascularization after 2 weeks of subcutaneous implantation, and neo-bone formation in the defect areas of the rat femur. Micro-CT and histology studies demonstrate better osseointegration with satisfactory biosafety response for the neem-assisted coated alloy as compared to that without neem-assisted deposition. Hence, this neem-assisted brushite-Hap coating strategy elucidates a new perspective on the surface modification of biodegradable implants for the treatment of bone defects.

Incorporating silver nanoparticles into electrospun nanofibers of casein/polyvinyl alcohol to develop scaffolds for tissue engineering

Developing novel antimicrobial wound dressings that have the potential to address the challenges associated with chronic wounds is highly imperative in providing effective infection control and wound healing support. Biocompatible electrospun nanofibers with their high porosity and surface area enabling efficient drug loading and delivery have been investigated in this regard as viable candidates for chronic wound care. Here, we design Casein/Polyvinyl alcohol (CAN/PVA) nanofibers reinforced with silver nanoparticles (Ag NPs) by the electrospinning technique to develop diabetic wound healing scaffolds. The prepared samples were characterized using spectroscopic and electron microscopic techniques. The biocompatibility of the polymer samples were assessed using 3 T3 fibroblast cell lines and the maximum cell viability was found to 95 % at a concentration of 50 μg/mL for the prepared nanofibers. Scratch assay tests were also performed to analyze the wound healing activity of the nanofibers wherein they demonstrated increased migration and proliferation of fibroblast 3 T3 cells. Moreover, these nanofibers also exhibit antibacterial efficiency against Gram-negative bacteria, Escherichia coli (E.coli). Therefore, the antimicrobial nature of the electrospun nanofibers coupled with their moisture absorption properties and wound healing ability render them as effective materials for wound dressing applications.

Abstract 2930: Recovery of limited viable cells from low-quality precious samples for scRNA-seq

Abstract Biomarker discovery is a critical milestone in pursuing personalized and targeted cancer treatments, yet it faces formidable challenges hindering success rates. Limited access to high-quality samples restricts in-depth biological investigation, with scarce and compromised viable cells acting as barriers to progress. Levitation Technology™ addresses these challenges by significantly improving sample quality and preserving maximum viable cells for single-cell RNA sequencing (scRNA-seq). This innovative approach overcomes limitations in sample quality and availability and heralds a new era of possibilities, propelling significant advancements in biomarker discovery for personalized cancer treatment. To assess the performance of Levitation Technology using the LeviCell® 1.0 system, 105 cryopreserved samples derived from diseased human tissues were processed by Discovery Life Sciences™. This diverse sample set covered 20 disease indications, including 17 types of cancer. A staggering 70% of the samples (n=74) exhibited cell viability ranging between 14-69%, underscoring the need for enhanced viability for effective scRNA-seq analysis. Notably, 45 of these samples presented fewer than 500,000 cells, a scenario marked by low quality and limited viable cells. Traditional methods fail to recover viable cells from such samples without considerable or complete viable cell loss. After processing the samples with the LeviCell 1.0 system, all samples had improved quality, with an impressive 90% of the most difficult samples (n=40) presenting a viability increase to 70-94%, meeting the minimum quality criteria for scRNA-seq. A notable success story was a particular breast cancer sample with only 31,000 cells and 14% viability. Processing with the LeviCell 1.0 system yielded 13,300 viable cells and a substantial 4.5-fold increase in viability to 64%. Such enhancements in sample quality translate into significant improvements in scRNA-seq data quality, enabling more single-cell data extraction from each invaluable sample. Levitation Technology represents a paradigm shift in sample processing, rescuing even the most challenging samples. This groundbreaking technology maximizes the retention of viable cells while efficiently eliminating dead cells and debris, factors that have traditionally compromised scRNA-seq data quality. By revolutionizing this aspect of sample preparation, Levitation Technology opens up unparalleled opportunities to explore deeper into disease biology, providing a leap forward in biomarker discovery. Citation Format: Camila Egidio, Dinesh Bhosale, Lanxin Shen, Susana Jett, Amir Malek, Teddy de Groot, Suzanne Weaver, Gang Sun, Kevin Travers, Geoff Facer. Recovery of limited viable cells from low-quality precious samples for scRNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2930.

Optimizing the viability of microencapsulated Lactiplantibacillus plantarum using response surface methodology for dietary probiotic delivery

The study aimed to develop a microencapsulated probiotic (Lactiplantibacillus plantarum) delivery method, cryoprotected by chitosan-inulin complex microcapsules and freeze-dried to obtain maximum cell viability. Probiotic viability was assessed using simulated gastrointestinal conditions and storage at different temperatures. The L. plantarum microcapsules were developed using a Box-Behnken experimental design with varied concentrations of CaCl2, chitosan, and Inulin as an encapsulant agents. In addition, the current study used the response surface approach to explore the influence of various formulations and storage capsulate viabilities to verify an optimized L plantarum formulation. Under optimum conditions, small microbeads with the highest cell encapsulation efficiency were produced and the highest cell viability (68.33 %) was obtained. The results showed that the intelligent microcapsule of L. plantarum with SM significantly increased the viability of cells (p < 0.05) (approx. 108 log CFU/mL) and the release of the extract (85 %) in gastric simulation and intestinal simulation fluids, respectively when compared with free bacteria. In general, microencapsulated L. plantarum under previously optimized freeze-drying conditions showed promising efficiency that depends on cell viability during drying, gastrointestinal treatment, and refrigerated storage conditions and on the physicochemical properties of the powders.

Biomass-derived phosphorous-doped carbon quantum dots (P-CQD): An excellent biocompatible material for in-vitro cell imaging

In this work, a simple hydrothermal process was employed to create phosphorous-doped fluorescent carbon quantum dots (P-CQD) from biomass banana stems. The formation of the P-CQDs and the surface functional group was confirmed through a variety of characterization methods, including X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), (transmission electron microscopy) TEM, (field emission scanning electron microscopy) FESEM, Fourier transform infrared spectroscopy (FTIR), and (ultraviolet–visible) UV–vis spectroscopy. The quantum yield of the synthesized P-CQDs was found to be 15 %. The P-CQDs showed excitation-dependent photoluminescence (PL) emission behaviour possessing a blue fluorescence under UV light. The P-CQDs was utilized for in vitro cytotoxicity effect on Muscle (L6) and Kidney cells (HEK293). The minimum cell viability for the P-CQD was 96.24 %, while the maximum cell viability was approximately 100 % at a concentration of 400 μg/mL. This study suggests that P-CQDs were non-toxic and could be employed for fluorescence-based cell imaging.

Response surface methodology optimizes selenium inhibition of prostate cancer PC-3 cell viability

BackgroundThe rising incidence of prostate cancer in the U.S. necessitates innovative therapeutic approaches to this disease. Though extensive research has studied Selenium as an anticarcinogen against prostate cancer, results have varied due to overlooked experimental confounds. Recent studies have identified differential effects of various selenium compounds on prostate cancer cells. This study leverages Mixture Design Response Surface Methodology to characterize the ideal combination of select Se forms against the PC-3 prostate cancer cell line. MethodsThe PC-3 cell line was chosen as a model for its representation of advanced-stage malignancy. Three Se compounds—sodium selenite, methylseleninic acid, and nano-selenium—were selected for their promising antineoplastic potential. Nano-Se particles were synthesized and subsequently characterized by transmission electron microscopy. Cells were cultured, treated with Se compounds, and assessed for viability using an Alamar Blue Assay. IC50 values of individual Se compounds were determined, and treatment combinations evaluated. In collaboration with statical modeling experts, MDRSM was utilized to optimize Se compound combinations. ResultsAbsolute IC50 values were identified for methylseleninic acid (5.01 μmol/L), sodium selenite (13.8 μmol/L), and nano-selenium (14.6 μmol/L). Combining methylseleninic acid and sodium selenite resulted in only 5% PC-3 cell viability, whereas individual treatments reduced viability by approximately 45%. Among the tested mixtures, the 50:50 combination of MSA and sodium selenite most effectively decreased PC-3 cell viability. Regression analysis indicated the special cubic model had a strong fit (multiple r² = 0.9853), predicting maximum cell viability reduction from the methylseleninic acid and selenite mixture. ConclusionThe specific form of Selenium plays a pivotal role in determining its physiological effects and therapeutic potential against prostate cancer. All three selenium compounds showed variable antineoplastic effects, with a 50:50 mixture of methylseleninic acid and selenite exhibiting optimal results. Nano-selenium, when combined with selenite, showed no additive effect, implying a shared mechanism of action. Our research underscores the critical need to consider Se compound forms as distinct entities in prostate cancer treatment and encourages further exploration of Se compounds against prostate cancer.

From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single-cell cloning strategy was established to generate monoclonal suspension Madin-Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine-relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell-specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50-fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high-yield influenza virus production in chemically defined medium starting from a single cell.

Synthesis and characterization of hydroxyapatite self-assembled nanocomposites on graphene oxide sheets from seashell waste: A green process for regenerative medicine

Bone transplantation is the second most common transplantation surgery in the world. Therefore, there is an urgent need for artificial bone transplantation to repair bone defects. In bone tissue engineering, hydroxyapatite (HA) plays a major role in bone graft applications. This study deals with a facile method for synthesizing HA hexagonal nanorods from seashells by a solid-state hydrothermal transition process. The synthesized HA nanorods (∼2.29 nm) were reinforced with carbon nanotube and chitosan on graphene oxide sheets with polymeric support by in-situ synthetic approach. Among the synthesized nanocomposites viz., hydroxyapatite-graphene oxide (HA-GO), hydroxyapatite-graphene oxide-chitosan (HA-GO-CS), hydroxyapatite-graphene oxide-chitosan-carbon nanotube-polylactic acid (HA-GO–CS–CNT-PLA). Among them, the HA-GO–CS–CNT-PLA composite exhibits micro and macro porosity (∼200 to 600 μm), higher mechanical strength, (Hardness ∼90.5 ± 1.33 MPa; Tensile strength 25.62 MPa), and maximum cell viability in MG63 osteoblast-like cells (80%). The self-assembled hybrid-nanocomposite of HA-GO–CS–CNT-PLA is a promising material for bone filler application and could efficiently utilize seashell waste through the green process.

Automated Shape and Process Parameter Optimization for Scaling Up Geometrically Non-Similar Bioreactors

Scaling bioprocesses remains a major challenge. Since it is physically impossible to increase all process parameters equally, a suitable scale-up strategy must be selected for a successful bioprocess. One of the most widely used criteria when scaling up bioprocesses is the specific power input. However, this represents only an average value. This study aims to determine the Kolmogorov length scale distribution by means of computational fluid dynamics (CFD) and to use it as an alternative scale-up criterion for geometrically non-similar bioreactors for the first time. In order to obtain a comparable Kolmogorov length scale distribution, an automated geometry and process parameter optimization was carried out using the open-source tools OpenFOAM and DAKOTA. The Kolmogorov–Smirnov test statistic was used for optimization. A HEK293-F cell expansion (batch mode) from benchtop (Infors Minifors 2 with 4 L working volume) to pilot scale (D-DCU from Sartorius with 30 L working volume) was carried out. As a reference cultivation, the classical scale-up approach with constant specific power input (233 W m−3) was used, where a maximum viable cell density (VCDmax) of 5.02·106 cells mL−1 was achieved (VCDmax at laboratory scale 5.77·106 cells mL−1). Through the automated optimization of the stirrer geometry (three parameters), position and speed, comparable cultivation results were achieved as in the small scale with a maximum VCD of 5.60·106 cells mL−1. In addition, even on the pilot scale, cell aggregate size distribution was seen to strictly follow a geometric distribution and can be predicted with the help of CFD with the previously published correlation.

Synthesis of Cu/Co-hybrid MOF as a multifunctional porous compound in catalytic applications, synthesis of new nanofibers, and antimicrobial and cytotoxicity agents

Several biological properties of metal–organic frameworks (MOFs) and fiber compounds have been reported, and combinations of these structures can have unique properties. In this study, copper-containing and cobalt-containing MOF nanostructures were synthesized by the ultrasonic technique. Then, novel Cu/Co-hybrid MOF nanostructures were synthesized using the ultrasonic method. Synthesized Cu/Co-hybrid MOF nanostructures were used as a new and efficient recyclable catalyst in the synthesis of pyrano[2,3-c]pyrazole derivatives using the four-component reaction of phenylhydrazine, ethyl acetoacetate, malononitrile, and aldehyde. In the following, novel Cu/Co-hybrid MOF/PVA (poly vinyl alcohol) fiber nanostructures were synthesized by electrospinning and using Cu/Co-hybrid MOF nanostructures and PVA. The structures of the Cu/Co-hybrid MOF nanostructures and the Cu/Co-hybrid MOF/PVA fiber nanostructures were identified and confirmed using BET, TGA, FTIR, SEM, and XRD. In biological studies, the antibacterial, antifungal, and cytotoxicity activities of Cu/Co-hybrid MOF and Cu/Co-hybrid MOF/PVA fiber nanostructures were evaluated. In investigating the catalytic activity of Cu/Co-hybrid MOF, pyrano[2,3-c]pyrazole derivatives were synthesized with higher efficiency and less time than previously reported methods. High antibacterial (against gram-negative and gram-positive strains) and antifungal properties of synthesized Cu/Co-hybrid MOF nanostructures and Cu/Co-hybrid MOF/PVA fiber nanostructures were observed (MIC between 16 and 256 μg/mL), which were higher than some commercial drugs. In the investigation of cytotoxicity activity, the effectiveness on breast cancer cells was studied. The maximum cell proliferation and viability for Cu/Co-hybrid MOF and Cu/Co-hybrid MOF/PVA fiber nanostructures were 38% and 38% higher than the control in a concentration of 200 μg/mL after 48 h. The high catalytic and biological properties of the synthesized nanoparticles can be attributed to the presence of nano-sized bioactive metals and their high specific surface area. The significant physical-chemical properties obtained for synthesized nanoparticles in this study can be related to the desirable synthesis methods, the development of materials with high purity, and the incorporation of hybrid compounds into the nanostructures.

Influence of mechanochemically fabricated nano-hardystonite reinforcement in polycaprolactone scaffold for potential use in bone tissue engineering: Synthesis and characterization

Bone tissue engineering (BTE) has gained significant attention for the regeneration of bone tissue, particularly for critical-size bone defects. The aim of this research was first to synthesize nanopowders of hardystonite (HT) through ball milling and then to manufacture composite scaffolds for BTE use out of polycaprolactone (PCL) containing 0, 3, 5, and 10 wt% HT by electrospinning method. The crystallite size of the synthesized HT nanopowders was 42.8 nm. including up to 5 wt% HT into PCL scaffolds resulted in significant improvements, such as a reduction in the fiber diameter from 186.457±15.74 to 150.021±21.99 nm, a decrease in porosity volume from 85.2±2.5 to 80.3±3.3 %, an improvement in the mechanical properties (ultimate tensile strength: 5.7±0.2 MPa, elongation: 47.5±3.5 %, tensile modulus: 32.7±0.9 MPa), an improvement in the hydrophilicity, and biodegradability. Notably, PCL/5%HT exhibited the maximum cell viability (194±14 %). Additionally, following a 4-week of submersion in simulated body fluid (SBF), the constructed PCL/HT composite scaffolds showed a remarkable capacity to stimulate the development of hydroxyapatite (HA), which increased significantly for the 5 wt% HT scaffolds. However, at 10 wt% HT, nanopowder agglomeration led to an increase in the fiber diameter and a decrease in the mechanical characteristics. Collectively, the PCL/5%HT composite scaffolds can therefore help with the regeneration of the critical-size bone defects and offer tremendous potential for BTE applications.

Production of retroviral vectors in continuous high cell density culture

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.Key points• A continuous high cell density process for MLV vector production was established• The tubular cell retention membrane allowed for continuous vector harvesting• The established process had a 18-fold higher space time yield compared to a batch