Exploring the application of poly(1,2-ethanediol citrate)/polylactide nonwovens in cell culturing.

Biomaterials containing citric acid as a building unit show potential for use as blood vessel and skin tissue substitutes. The success in commercializing implants containing a polymer matrix of poly(1,8-octanediol citrate) provides a rationale for exploring polycitrates based on other diols. Changing the aliphatic chain length of the diol allows functional design strategies to control the implant's mechanical properties, degradation profile and surface energy. In the present work, poly(1,2-ethanediol citrate) was synthesized and used as an additive to polylactide in the electrospinning process. It was established that the content of polycitrate greatly influences the nonwovens' properties: an equal mass ratio of polymers resulted in the best morphology. The obtained nonwovens were characterized by surface hydrophilicity, tensile strength, and thermal properties. L929 cell cultures were carried out on their surface. The materials were found to be non-cytotoxic and the degree of porosity was suitable for cell colonization. On the basis of the most important parameters for assessing the condition of cultured cells (cell density and viability, cell metabolic activity and lactate dehydrogenase activity), the potential of PLLA + PECit nonwovens for application in tissue engineering was established.

Effect of Silica Xerogel Functionalization on Intensification of Rindera graeca Transgenic Roots Proliferation and Boosting Naphthoquinone Production.

Secondary metabolites derived from plants are recognized as valuable products with several successful applications in the pharmaceutical, cosmetic, and food industries. The major limitation to the broader implementation of these compounds is their low manufacturing efficiency. Current efforts to overcome unprofitability depend mainly on biotechnological methods, especially through the application of plant in vitro cultures. This concept allows unprecedented bioengineering opportunities for culture system modifications with in situ product removal. The silica-based xerogels can be used as a novel, porous biomaterial characterized by a large surface area and high affinity to lipophilic secondary metabolites produced by plant tissue. This study aimed to investigate the influence of xerogel-based biomaterials functionalized with methyl, hydroxyl, carboxylic, and amine groups on Rindera graeca transgenic root growth and the production of naphthoquinone derivatives. The application of xerogel-based scaffolds functionalized with the methyl group resulted in more than 1.5 times higher biomass proliferation than for reference untreated culture. The naphthoquinone derivatives' production was noted exclusively in culture systems supplemented with xerogel functionalized with methyl and hydroxyl groups. Applying chemically functionalized xerogels as in situ adsorbents allowed for the enhanced growth and productivity of in vitro cultured R. graeca transgenic roots, facilitating product isolation due to their selective and efficient accumulation.

Disposable rocking bioreactors: recent applications and progressive perspectives.

Disposable rocking bioreactors facilitate scaling up animal and plant cell biomass propagation and developing specified bioprocesses like manufacturing vaccines or chimeric antigen receptor (CAR) T cells. Future contexts for these bioreactors include supporting regenerative medicine, recognising metabolic responses of biochemically or mechanically stressed cells, continuously performing in vitro bioprocesses, or cell-free protein synthesis systems.

Biological Activity of Poly(1,3-propanediol citrate) Films and Nonwovens: Mechanical, Thermal, Antimicrobial, and Cytotoxicity Studies.

Polymers are of great interest for medical and cosmeceutical applications. The current trend is to combine materials of natural and synthetic origin in order to obtain products with appropriate mechanical strength and good biocompatibility, additionally biodegradable and bioresorbable. Citric acid, being an important metabolite, is an interesting substance for the synthesis of materials for biomedical applications. Due to the high functionality of the molecule, it is commonly used in biomaterials chemistry as a crosslinking agent. Among citric acid-based biopolyesters, poly(1,8-octanediol citrate) is the best known. It shows application potential in soft tissue engineering. This work focuses on a much less studied polyester, poly(1,3-propanediol citrate). Porous and non-porous materials based on the synthesized polyesters are prepared and characterized, including mechanical, thermal, and surface properties, morphology, and degradation. The main focus is on assessing the biocompatibility and antimicrobial properties of the materials.

A Study of the Properties of Scaffolds for Bone Regeneration Modified with Gel-like Coatings of Chitosan and Folic Acid.

The research has been conducted to obtain scaffolds for cancellous bone regeneration. Polylactide scaffolds were made by the phase inversion method with a freeze-extraction variant, including gelling polylactide in its non-solvent. Substitutes made of polylactide are hydrophobic, which limits cell adhesion. For this reason, the scaffolds were modified using chitosan and folic acid by forming gel-like coatings on the surface. The modification aimed to improve the material's surface properties and increase cell adhesion. Analyses of obtained scaffolds confirmed the effectiveness of performed changes. The presence of chitosan and folic acid was confirmed in the modified scaffolds, while all scaffolds retained high open porosity, which is essential for proper cell growth inside the scaffold and the free flow of nutrients. Hydrostatic weighing showed that the scaffolds have high mass absorbability, allowing them to be saturated with biological fluids. There were also cytotoxicity tests performed on 24 h extracts of the materials obtained, which indicated a lack of cytotoxic effect.

From Poly(glycerol itaconate) Gels to Novel Nonwoven Materials for Biomedical Applications.

Electrospinning is a process that has attracted significant interest in recent years. It provides the opportunity to produce nanofibers that mimic the extracellular matrix. As a result, it is possible to use the nonwovens as scaffolds characterized by high cellular adhesion. This work focused on the synthesis of poly(glycerol itaconate) (PGItc) and preparation of nonwovens based on PGItc gels and polylactide. PGItc gels were synthesized by a reaction between itaconic anhydride and glycerol. The use of a mixture of PGItc and PLA allowed us to obtain a material with different properties than with stand-alone polymers. In this study, we present the influence of the chosen ratios of polymers and the OH/COOH ratio in the synthesized PGItc on the properties of the obtained materials. The addition of PGItc results in hydrophilization of the nonwovens' surface without disrupting the high porosity of the fibrous structure. Spectral and thermal analyzes are presented, along with SEM imagining. The preliminary cytotoxicity research showed that nonwovens were non-cytotoxic materials. It also helped to pre-determine the potential application of PGItc + PLA nonwovens as subcutaneous tissue fillers or drug delivery systems.

Stress-Induced Intensification of Deoxyshikonin Production in Rindera graeca Hairy Root Cultures with Ester-Based Scaffolds.

In vitro plant cell and tissue culture systems allow for controlling a wide range of culture environmental factors selectively influencing biomass growth and the yield of secondary metabolites. Among the most efficient methods, complex supplementation of the culture medium with elicitors, precursors, and other functional substances may significantly enhance valuable metabolite productivity through a stress induction mechanism. In the search for novel techniques in plant experimental biotechnology, the goal of the study was to evaluate stress-inducing properties of novel biodegradable ester-based scaffolds made of poly(glycerol sebacate) (PGS) and poly(lactic acid) (PLA) influencing on the growth and deoxyshikonin productivity of Rindera graeca hairy roots immobilized on the experimental constructs. Rindera graeca hairy roots were maintained under the dark condition for 28 days in three independent systems, i.e., (i) non-immobilized biomass (a reference system), (ii) biomass immobilized on PGS scaffolds, and (iii) biomass immobilized on PLA scaffolds. The stress-inducing properties of the applied polymerized esters selectively impacted R. graeca hairy roots. The PGS scaffolds caused the production of deoxyshikonin, which does not occur in other culture systems, and PLA promoted biomass proliferation by doubling its increase compared to the reference system.

Selective Impact of MTMS-Based Xerogel Morphology on Boosted Proliferation and Enhanced Naphthoquinone Production in Cultures of Rindera graeca Transgenic Roots.

In situ extraction is a method for separating plant secondary metabolites from in vitro systems of plant biomass cultures. The study aimed to investigate the MTMS-based xerogels morphology effect on the growth kinetics and deoxyshikonin productivity in xerogel-supported in vitro culture systems of Rindera graeca hairy root. Cultures were supplemented with three types of xerogel, i.e., mesoporous gel, microporous gel, and agglomerated precipitate, in the disintegrated or monolithic form. Structure, oil sorption capacity, and SEM analyses for xerogel-based additives were performed. Application of monolithic macroporous xerogel resulted in the highest biomass proliferation, i.e., 5.11-fold fresh biomass increase after four weeks of the screening culture. The highest deoxyshikonin production (i.e., 105.03 µg) was noted when hairy roots were maintained with particles of disintegrated mesoporous xerogel. The detailed kinetics investigations (6-week culture) revealed the highest growth of hairy root biomass and secondary metabolite production, equaling 9.46-fold fresh weight biomass and 204.08 µg deoxyshikonin, respectively. MTMS-based xerogels have been recognized as selective biocompatible scaffolds for boosting the proliferation of transgenic roots or for productivity enhancement of naphthoquinones without detrimental effects on biomass growth, and their successful applicability in in situ removal of secondary plant metabolites has been experimentally confirmed.

Microcarrier-Supported Culture of Chondrocytes in Continuously Rocked Disposable Bioreactor.

Disposable wave-assisted bioreactors are devices originally designed for scaling-up cultures of extremely fragile animal cells. In such bioreactors, agitation is achieved by continuous horizontal oscillations of disposable culture bag-like container fixed in a rocker unit. The continuous rocking movement of the container induces waves in the two-phase (i.e., gas-liquid) culture system composed of CO2-enriched air and aqueous culture medium. Such continuously oscillating devices can be utilized for supporting homogeneity in systems for in vitro propagation of animal anchorage-dependent, that is, adherent, cells, like CP5 chondrocytes cells. As most of in vitro cultured cells exhibit anchorage-dependency toward solid surface, the suitable interface can be provided by beads of microcarriers made of polymers and characterized by large surface-to-volume ratio. This chapter describes a methodology for efficient propagation of CP5 chondrocytes on Cytodex 3 microcarriers performed in ReadyToProcess WAVE 25 disposable bioreactor, as well as all useful procedures for daily monitoring the growth of CP5 chondrocytes.

MTMS-Based Aerogel Constructs for Immobilization of Plant Hairy Roots: Effects on Proliferation of Rindera graeca Biomass and Extracellular Secretion of Naphthoquinones.

Unique biosynthetic abilities revealed by plants determine in vitro cultures of hairy roots as a suitable source of pharmaceutically relevant bioactive compounds. The basic aim of the study was to examine the applicability of aerogel composed of methyltrimethoxysilane (MTMS) for immobilization of Rindera graeca hairy roots by identifying quantitative effects of biomass proliferation and naphthoquinones extracellular secretion in the aerogel-supported culture system. R. graeca hairy roots were simultaneously cultured for 28-days, as (i) nonimmobilized biomass (reference system), (ii) biomass immobilized on macroporous polyurethane foam (PUF), (iii) biomass with disintegrated MTMS aerogel, (iv) biomass immobilized on polypropylene (PP) fibers (as control), and (v) biomass immobilized on monolithic PP-reinforced MTMS aerogel. MTMS aerogel exhibited high level of biocompatibility toward R. graeca hairy roots which grew into the structure of monolithic aerogel-based constructs. Monolithic MTMS-based constructs significantly promoted the proliferation of hairy roots, resulting in 55% higher fresh mass than the reference system. The highest level of naphthoquinones productivity, i.e., 653 µg gDW-1, was noted for PUF-supported culture system.

Efficient propagation of suspended HL-60 cells in a disposable bioreactor supporting wave-induced agitation at various Reynolds number.

Growth of human nonadherent HL-60 cell cultures performed in disposable bioreactor under various hydrodynamic conditions of 2-D wave-assisted agitation has been compared and discussed. Influence of Reynolds number for liquid (ReL) and the kLa coefficient, as key parameters characterized the bioprocessing of HL-60 cells in ReadyToProcess WAVETM 25 system, on reached values of the apparent maximal specific growth rate (µmax) and the specific yield of biomass (Y*X/S) has been identified. The values of ReL (i.e., 510-10,208), as well as kLa coefficient (i.e., 2.83-13.55 h-1), have been estimated for the cultures subjected to wave-induced mixing, based on simplified dimensionless correlation for various presents of WAVE 25 system. The highest values of apparent µmax = 0.038 h-1 and Y*X/S = 25.64 × 108 cells gglc-1 have been noted for cultures independently performed at wave-induced agitation characterized by ReL equaled to 5104 and 510, respectively. The presented results have high applicability potential in scale-up of bioprocesses focused on nonadherent animal cells, or in the case of any application of disposable bioreactors presenting similitude.

Design of experiments-based high-throughput strategy for development and optimization of efficient cell disruption protocols.

Efficient and reproducible cell lysis is a crucial step during downstream processing of intracellular products. The composition of an optimal lysis buffer should be chosen depending on the organism, its growth status, the applied detection methods, and even the target molecule. Especially for high-throughput applications, where sample volumes are limited, the adaptation of a lysis buffer to the specific campaign is an urgent need. Here, we present a general design of experiments-based strategy suitable for eight constituents and demonstrate the strength of this approach by the development of an efficient lysis buffer for Gram-negative bacteria, which is applicable in a high-throughput format in a short time. The concentrations of four lysis-inducing chemical agents EDTA, lysozyme, Triton X-100, and polymyxin B were optimized for maximal soluble protein concentration and ß-galactosidase activity in a 96-well format on a Microlab Star liquid handling platform under design of experiments methodology. The resulting lysis buffer showed the same performance as a commercially available lysis buffer. The developed protocol resulted in an optimized buffer within only three runs. The established procedure can be easily applied to adapt the lysis buffer to other strains and target molecules.

Liquid perfluorochemical-supported hybrid cell culture system for proliferation of chondrocytes on fibrous polylactide scaffolds.

CP5 bovine chondrocytes were cultured on biodegradable electrospun fibrous polylactide (PLA) scaffolds placed on a flexible interface formed between two immiscible liquid phases: (1) hydrophobic perfluorochemical (PFC) and (2) aqueous culture medium, as a new way of cartilage implant development. Robust and intensive growth of CP5 cells was achieved in our hybrid liquid-solid-liquid culture system consisting of the fibrous PLA scaffolds in contrast to limited growth of the CP5 cells in traditional culture system with PLA scaffold placed on solid surface. The multicellular aggregates of CP5 cells covered the surface of PLA scaffolds and the chondrocytes migrated through and overgrew internal fibers of the scaffolds. Our hybrid culture system simultaneously allows the adhesion of adherent CP5 cells to fibers of PLA scaffolds as well as, due to use of phase of PFC, enhances the mass transfer in the case of supplying/removing of respiratory gases, i.e., O2 and CO2. Our flexible (independent of vessel shape) system is simple, ready-to-use and may utilize a variety of polymer-based scaffolds traditionally proposed for implant development.

Liquid perfluorodecalin application for in situ extraction and enhanced naphthoquinones production in Arnebia euchroma cell suspension cultures.

Suspension cultures of Arnebia euchroma supported with liquid perfluorodecalin (PFD) degassed, aerated, or ethylene-saturated were investigated as a novel in situ extraction system for enhanced alkannin/shikonin production. Simultaneously, the effect of PFD applied as the liquid gas carrier on the growth of A. euchroma biomass was studied. The similar dry (4-fold) and fresh (7-fold) biomass increase was observed in the control (without PFD addition) and supplemented with PFD-degassed or PFD-aerated cultures while PFD-ethylene application impeded cell growth. The highest total of alkannin/shikonin production (23.23 mg flask(-1)) was observed when PFD-aerated has been used and it resulted in about 50% higher yield of alkannin/shikonin compared with the control culture. Chiral HPLC analysis revealed that in cultures supported with PFD, both alkannin and shikonin were produced. Their mutual ratio varied depending on culture conditions, and the accumulation of alkannin prevailed under almost all culture conditions. PFD has proved to be exceptionally efficient and cell-safe solvent for the in situ extraction of naphthoquinone red pigments without exerting any detrimental effects on cell growth. Extracellularly secreted red naphthoquinones were easily dissolved and extracted from the PFD phase, which can be regenerated and reused (e.g., in continuous culture system).

Cultivation of marine microorganisms in single-use systems.

: Marine cultures are an important source of novel substances and enzymes. As efforts to isolate strains from (deep) sea environments increase, the demand for methodology platforms to cultivate these organisms is also rising. Due to the high salt concentration and the shear sensitivity exhibited by some heterotrophic microalgae, single-use systems originally designed for the cultivation of mammalian cell lines can be a valuable alternative. Using the cultivation of the heterotrophic marine microalgae Crypthecodinium cohnii as an example, this chapter makes suggestions for experimental design, for improving process development by integrating parallel experiments, and for scaling-up and scaling-down methodologies. It describes how to identify suitable single-use systems and how to integrate a two-layer system with perfluordecalin to improve the gas transfer in deep-well plates. The process is also scaled up in several single-use systems. We also describe challenges in the process development to achieve sufficient oxygen transfer, monitoring, and control, and we discuss limitations such as corrosion, long-term stability, and leachables in single-use systems. Finally, we demonstrate a method for cheap, fast, and consistent process development for marine microorganisms.

Mini-scale cultivation method enables expeditious plasmid production in Escherichia coli.

The standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 µL cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5­4 µg mL(­1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 µg mL(­1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase®). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.

Morphology and growth of mammalian cells in a liquid/liquid culture system supported with oxygenated perfluorodecalin.

Adherent A431, BHK-21, and C2C12 cells were cultured on a flexible interface formed between two immiscible liquid phases: (i) hydrophobic perfluorodecalin (PFD) and (ii) aqueous culture medium (DMEM). BHK-21 cells formed multicellular aggregates characterized by irregular shapes. A431, as well as C2C12 cells, grew as tight multicellular sheets of 3-D cells. Enhanced mass transfer and facilitated access of the cells to the O2 dissolved in PFD/DMEM by approx. 250 % and thereby increased the density of BHK-21 cells. Thus the liquid/liquid system is a simple, ready-to-use, and fully scalable (independent of vessel shapes); consequently it is a method for 3-D cultures of adherent animal cells in which the growth of anchorage-dependent cells is not limited by confluence effect.

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier.

A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase(®)) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 µL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The µL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.

Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures.

BACKGROUND: Liquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase. RESULTS: In EnBase(®) cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 µM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase. CONCLUSIONS: PFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.

Kinetic model of 1,3-specific triacylglycerols alcoholysis catalyzed by lipases.

A new model of enzymatic 1,3-specific alcoholysis of triacylglycerols has been developed. The irreversibility of the acyl bounds cleavage in glycerides, a reversible monoglycerides isomerization and an irreversible enzyme deactivation have been assumed. The Ping Pong Bi Bi mechanism with competitive inhibition by alcohol has been applied to describe rates of acyl bonds cleavage. The enzymatic propanolysis and iso-propanolysis of triacetin and tricaprylin catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) have been investigated to verify the model. Good agreement between experimental data and calculations has been obtained. It was shown that the rate of tricaprylin alcoholysis is higher than the triacetin alcoholysis and that the rate of iso-propanolysis reactions are higher than propanolysis. The irreversible enzyme deactivation affects the conversion of glycerides whereas the competitive alcohol inhibition may be neglected. Empirical correlations of rates for monoglycerides isomerization and enzyme deactivation have been proposed.