Cultures of Rindera graeca hairy roots on chemically functionalized xerogels

Data used in the publication: Wierzchowski, K.; Nowak, B.; Kawka, M.; Sykłowska-Baranek, K.; Pilarek, M. Effect of Silica Xerogel Functionalization on Intensification of Rindera graeca Transgenic Roots Proliferation and Boosting Naphthoquinone Production. Life 2024, 14, 159. https://doi.org/10.3390/life14010159 This research was funded by National Science Centre, Poland, grant no. 2021/41/N/ST8/00958

Oxygen Transfer Effects in a Two-Phase System of an Aqueous Phase and Liquid Perfluorochemical Subjected to Continuous Wave-Assisted Agitation in Disposable Bioreactor

Systems of two immiscible liquid phases—aqueous phase (i.e., distilled water (dH2O) or phosphate-buffered saline (PBS)) and liquid perfluorochemical (i.e., perfluorodecalin (PFD))—were subjected to wave-assisted agitation, i.e., oscillatory rocked, in a disposable bag-like container in a ReadyToProcess WAVETM25 bioreactor, to recognize oxygen transfer effects and effectivity of the surface aeration. According to the DoE methodology, values of the volumetric liquid-side mass transfer (kLa) coefficient for dH2O, PBS, dH2O-PFD, and PBS-PFD systems were determined for the whole range of operating parameters of the WAVE 25 bioreactor. A significantly higher maximal value of kLa was found for waving dH2O than for dH2O-PFD (i.e., 0.00460 s−1 vs. 0.00331 s−1, respectively) compared to more equal maximal values of kLa reached for PBS and PBS-PFD (0.00355 s−1 vs. 0.00341 s−1, respectively). The interface development factor (f) depended on the interfacial area a, and the enhancement factor (EPFD), depending on kLa, was introduced to quantitatively identify the mass transfer effects in the systems of waving two immiscible liquids. The phase of PFD was identified as the reservoir of oxygen. Dimensional correlations were proposed for the prediction of the kLa coefficient, in addition to the f and EPFD factors. The presented correlations, and the set of kLa values, can be directly applied to predict oxygen transfer effects reached under continuous oscillatory rocked systems containing aqueous phase and liquid perfluorochemical

Propagation of non-adherent HL-60 cells in batch cultures maintained in staticand wave-type agitated systems

Typically applied static (i.e. non-agitated) cultures do not provide sufficient conditions for efficient propagation of suspended non-adherent cells, in general. Feasibility of small-scale wave-type agitated single-use bioreactors for gentle agitation underlies applicability of such systems for scaling-up of fragile biomass of animal cells. The basic aim of the study was to compare the results of non-adherent HL-60 cell propagation performed referentially as the batch culture in typical static (i.e. non-agitated) disposable culture flasks (50 cm3 of culture medium) and in ReadyToProcess WAVETM25 bioreactor system (GE Healthcare) equipped with disposable culture bag (300 cm3 of culture medium) subjected to continuous wave-type agitation. The density and viability of HL-60 cells were significantly higher for the bioprocess subjected to wave-type agitation, than in the reference static culture. The values of the specific rate of glucose consumption per cell (rglc=cell) exhibited by HL-60 cells maintained in the system with continuous wave-type agitation was significantly lower (i.e. up to more than 42%) than the values noted for the static culture, for exactly the same time-points of two compared cultures. The results of the studies undoubtedly and comprehensively confirmed the applicability of the studied disposable bioreactor with wave-induced agitation as the right platform for proceeding the propagation of non- adherent HL-60 cells and for providing the culture conditions required by HL-60 cells for sustainable metabolism

MTMS-Based Aerogel Constructs for Immobilization of Plant Hairy Roots: Effects on Proliferation of <i>Rindera graeca</i> 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

Stress-Induced Intensification of Deoxyshikonin Production in <i>Rindera graeca</i> 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 &micro;g) 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 &micro;g 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

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