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.

Effect of Nanobubble Presence on Murine Fibroblasts and Human Leukemia Cell Cultures.

Nanobubbles can enhance both the proliferation and metabolic activity of microorganisms (mainly bacteria) and the growth of the whole higher organisms such as mice, fish, or plants. The critical fact is that nanobubbles of different gases can affect given cells differently. As animal cell cultures are used in industry and research studies, investigations of their interactions with nanobubbles should be carried out. This study aims to uncover whether the presence of nanobubbles improves the proliferation rate and metabolic activity of L929 fibroblasts and HL60 leukemia cells as exemplary animal cell lines of adherent and non-adherent cells, respectively. The long-term (8-day) cultures of both L929 and HL-60 cells with nanobubble addition to the appropriate medium were carried out. The medium was not exchanged for the whole duration of the culture. Nanobubbles of two gases - oxygen and nitrogen - were dispersed in the appropriate media and then used to culture cells. The density and viability of cells were assessed microscopically while their metabolic activity was determined using PrestoBlue or XTT assays. Additionally, we have performed the analysis of substrate consumption rate during the growth and activity of lactate dehydrogenase. We have shown that nanodispersion of both gases enhances the proliferation rate and metabolic activity of L929. For HL-60 cultures, reference cultures exhibited better viability, cell density, and metabolic activity than those with either oxygen or nitrogen nanobubbles. Obtained results clearly show that nanobubble dispersions of both oxygen and nitrogen positively affect the cultures of L929 while inhibiting the growth of HL-60 cells. We suspect that a similar positive effect would be visible for other adherent cells, similar to L929. Such results are promising for intensifying the growth of animal or human cells in routine cell cultures.

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.