Higher Cell Metabolic Activity Research Articles

Model-predicted effect of radial flux distribution on oxygen and glucose pericellular concentration in constructs cultured in axisymmetric radial-flow packed-bed bioreactors

Radial flow packed-bed bioreactors (rPBBs) overcome the transport limitations of static and axial-flow perfusion bioreactors and enable development of clinical-scale bioengineered tissues. We developed criteria to design rPBBs with uniform medium radial flux distribution along bioreactor length ensuring uniform construct perfusion. We report a model-based analysis of the effect of non-uniform axial distribution of medium radial flux on pericellular concentration of oxygen and glucose. Albeit pseudo-homogeneous, the model predicts how medium flux, solutes transport and cellular consumption interact and determine the pericellular oxygen and glucose concentrations in the presence of pore transport resistance to design optimal axisymmetric rPBBs and enable control of pericellular environment. Thus, oxygen and glucose supply may match cell requirements as tissue matures. Flow and solute transport in bioreactor empty spaces and construct was described with Navier-Stokes and Darcy-Brinkman equations, and with convection–diffusion and convection–diffusion-reaction equations, respectively. Solute transport in construct accounted for Michaelian cellular consumption and bulk medium-to-cell surface oxygen transport resistance in terms of a transport-equivalent bed of Raschig rings. The effect of relevant dimensionless groups on pericellular and bulk solute concentrations was predicted under typical tissue engineering operation and evaluated against literature data for bone tissue engineering. Axial distribution of medium radial flux influenced the distribution of pericellular solutes concentration, more so at high cell metabolic activity. Increasing medium feed flow rates relieved non-uniform solute concentration distribution and decayed at cell surface for metabolic consumption, also starting from axially non-uniform radial flux distribution. Model predictions were obtained in runtimes compatible with on-line control strategies.

Human gingival mesenchymal stem cells-lyosecretome attenuates adverse effect of hydrogen peroxide-induced oxidative stress on osteoblast cells

ObjectiveTo determine total protein content, antioxidant activity, and protective ability of lyophilized human gingival mesenchymal stem cells (hGMSCs)-secretome in hydrogen peroxide (H2O2) induced oxidative stress model. MethodsHuman GMSCs were cultured to obtain a conditioned medium (secretome), then lyophilized to produce lyosecretome. Total protein was determined by bicinchoninic acid assay (BCA) and SDS-PAGE to improve protein measurements. Antioxidant concentration was measured by ABTS assay, while the protective ability of lyosecretome against oxidative stress was determined by the metabolic activity of osteoblast cells. The study group was divided into a control group (culture medium) and a lyosecretome treatment group (0.0; 0.157, 0.313, 0.625, 1.25, 2.5, 5, and 10 mg/mL + H2O2). ResultsLyosecretome had a protein concentration of 2086.00 ± 0.20 μg/ml, with a molecular weight of 174, 74, 61, 55, and 26 kDa, which are thought to facilitate cell migration, as well as bind cytokines and growth factors. Lyosecretome also provided the highest antioxidant activity of 93.51% at a concentration of 4.8 mg/ml, with an IC50 value of 2.08 mg/ml. The highest cell metabolic activity (79.53 ± 2.41%) was shown in the 1.25 mg/ml lyosecretome treatment group. All concentrations of hGMSC-lyosecretome attenuate the adverse effect of H2O2-induced oxidative stress. ConclusionLyosecretome obtained from hGMSCs can maintain metabolic activity in osteoblast cells as protection against H2O2 oxidative stress.

(Invited) Engineering Single Wall Carbon Nanotubes for Sub-Cellular Drug Delivery

Single wall carbon nanotubes (SWCNTs) have been rigorously studied and engineered for a myriad of applications including drug delivery due to their unique, desirable properties. Since SWCNTs bundle in aqueous media and the bundling substantially diminishes their desirable properties along with inducing cytotoxicity, strategies to disperse individually SWCNTs with therapeutic molecules and biologic excipients have been heavily investigated. However, a handful of studies has been performed to understand the interactions between therapeutic molecules and excipients with SWCNT interface as well as the mechanism of intracellular release for therapeutic effects. Additionally, little attention has been paid to how cell type and cell metabolic activity level influence internalization of SWCNTs with different lengths. In this talk, I will discuss cell type, cell metabolic activity, and SWCNT length dependent cellular processing of SWCNTs dispersed with diverse biocompatible dispersing agents. Our results demonstrate that proper choice of dispersing agent can allow for interaction with F-actin, accumulation in the endoplasmic reticulum for eventual expulsion, or accumulation in cell nucleus. Further, macrophages with relatively high cell metabolic activity level compared to other cell types such as fibroblasts have shown to internalize significantly higher amount of SWCNTs per cell. The total mass of SWCNTs internalized per cell increases with a decrease in SWCNT length. Lastly, I will discuss our recent effort to create a ternary complex of SWCNTs, drug, and protein for intracellular delivery and release of model drugs. To overcome the limitation of the existing methods being only plausible for model drugs that are soluble in aqueous solutions, we developed a facile schema to assemble stepwise various combinations of therapeutic molecules and excipients on SWCNTs in different solvents using a preformed SWCNT network. These ternary complexes allow dialing in delivery of therapeutic molecules and excipients to achieve desired cell viability reduction. These findings allow for the development of new SWCNT-based biological applications as they establish important parameters of biomolecule adsorption on SWCNTs, SWCNT internalization and subcellular processing, which are crucial for applications ranging from drug delivery to cell type-specific modulation.

A Novel 3D‐Bioprinting Technology of Orderly Extruded Multi‐Materials via Photopolymerization

AbstractAs a 3D bioprinting technique, digital light processing (DLP) has become popular due to its capability to provide high‐throughput and high‐resolution constructs with precise chemical and biological factor distributions. However, despite the advancements in DLP technology, several hurdles remain, including phototoxicity, extensive printing time, and the limited portfolio of biocompatible/photo‐cross‐linkable materials. Recently, few works have attended to resolve some of these issues. However, state‐of‐the‐art techniques bear on complex imaging processing, require highly skilled personnel, and operate with non‐biocompatible/photo‐cross‐linkable materials. Additionally, they are not yet capable of multi‐layer and multi‐material printing of biocompatible/photo‐cross‐linkable materials to fabricate physiologically relevant cell‐laden structures. Herein, a novel DLP‐based 3D‐bioprinting technology called photopolymerization of orderly extruded multi‐materials (POEM), is proposed, developed, and fully characterized. The utility of the POEM technique for rapid and high‐resolution 3D‐printing of multi‐material, multi‐layer, and cell‐laden structures is demonstrated. The printed configurations show high cell viability (≈80%) and metabolic activity for more than 5 days. As a study model, a 3D‐structure representing the esophagus is also successfully printed and characterized. It is envisioned that the reported light‐based POEM technique here enables the fabrication of 3D‐cell‐laden structures in a multi‐material and multi‐layer printing manner in biocompatible/photo‐cross‐linkable materials essential to construct complex heterogeneous tissues/organs.

Production of food-grade microcarriers based on by-products from the food industry to facilitate the expansion of bovine skeletal muscle satellite cells for cultured meat production

A major challenge for successful cultured meat production is the requirement for large quantities of skeletal muscle satellite cells (MuSCs). Commercial microcarriers (MCs), such as Cytodex®1, enable extensive cell expansion by offering a large surface-to-volume ratio. However, the cell-dissociation step post cell expansion makes the cell expansion less efficient. A solution is using food-grade MCs made of sustainable raw materials that do not require a dissociation step and can be included in the final meat product. This study aimed to produce food-grade MCs from food industry by-products (i.e., turkey collagen and eggshell membrane) and testing their ability to expand bovine MuSCs in spinner flask systems for eight days. The MCs’ physical properties were characterized, followed by analyzing the cell adhesion, growth, and metabolic activity. All MCs had an interconnected porous structure. Hybrid MCs composed of eggshell membrane and collagen increased the mechanical hardness and stabilized the buoyancy compared to pure collagen MCs. The MuSCs successively attached and covered the entire surface of all MCs while expressing high cell proliferation, metabolic activity, and low cell cytotoxicity. Cytodex®1 MCs were included in the study. Relative gene expression of skeletal muscle markers showed reduced PAX7 and increased MYF5, which together with augmented proliferation marker MKI67 indicated activated and proliferating MuSCs on all MCs. Furthermore, the expression pattern of cell adhesion receptors (ITGb5 and SDC4) and focal adhesion marker VCL varied between the distinct MCs, indicating different specific cell receptor interactions with the various biomaterials. Altogether, our results demonstrate that these biomaterials are promising prospects to produce custom-fabricated food-grade MCs intended to expand MuSCs.

Preliminary Characterization of a Polycaprolactone-SurgihoneyRO Electrospun Mesh for Skin Tissue Engineering.

Skin is a hierarchical and multi-cellular organ exposed to the external environment with a key protective and regulatory role. Wounds caused by disease and trauma can lead to a loss of function, which can be debilitating and even cause death. Accelerating the natural skin healing process and minimizing the risk of infection is a clinical challenge. Electrospinning is a key technology in the development of wound dressings and skin substitutes as it enables extracellular matrix-mimicking fibrous structures and delivery of bioactive materials. Honey is a promising biomaterial for use in skin tissue engineering applications and has antimicrobial properties and potential tissue regenerative properties. This preliminary study investigates a solution electrospun composite nanofibrous mesh based on polycaprolactone and a medical grade honey, SurgihoneyRO. The processing conditions were optimized and assessed by scanning electron microscopy to fabricate meshes with uniform fiber diameters and minimal presence of beads. The chemistry of the composite meshes was examined using Fourier transform infrared spectroscopy and X-ray photon spectroscopy showing incorporation of honey into the polymer matrix. Meshes incorporating honey had lower mechanical properties due to lower polymer content but were more hydrophilic, resulting in an increase in swelling and an accelerated degradation profile. The biocompatibility of the meshes was assessed using human dermal fibroblasts and adipose-derived stem cells, which showed comparable or higher cell metabolic activity and viability for SurgihoneyRO-containing meshes compared to polycaprolactone only meshes. The meshes showed no antibacterial properties in a disk diffusion test due to a lack of hydrogen peroxide production and release. The developed polycaprolactone-honey nanofibrous meshes have potential for use in skin applications.

Microfluidic bioprinting towards a renal in vitro model

It is estimated that 10% of the worldwide population suffers from chronic kidney disease (CKD) with a rising tendency. Patients with CKD have limited treatment options and novel therapies that could halt or even reverse the progression of CKD are urgently needed.Bioprinting is considered one of the most promising approaches to generate novel 3D in vitro models and organ-like constructs to investigate underlying pathomechanism of kidney diseases. This study aimed at establishing a method to isolate primary renal cells in an easy and reproducible way. These cells were used in a new bioprinting platform laying the foundation for the development of a 3D renal tubulointerstitium model for in vitro studies. Primary murine tubular (pmTECs), endothelial and fibroblast cells were successfully isolated, but further optimization is required for the culture and expansion of primary endothelial cells. Therefore, an endothelial cell line (HUVECs) and pmTECs were combined with polysaccharide biomaterial ink solutions and processed with a microfluidic 3D bioprinter, leading to high cell viability and metabolic activity. Core-shell bioprinted constructs with HUVECs and pmTECs were manufactured mimicking tubules.In conclusion, microfluidic bioprinting strategy could be used to build a novel 3D kidney in vitro model.

(Invited) Engineering Single Wall Carbon Nanotubes for Sub-Cellular Drug Delivery

Single wall carbon nanotubes (SWCNTs) have been rigorously studied and engineered for a myriad of applications including drug delivery due to their unique, desirable properties. Since SWCNTs bundle in aqueous media and the bundling substantially diminishes their desirable properties along with inducing cytotoxicity, strategies to disperse individually SWCNTs with therapeutic molecules and biologic excipients have been heavily investigated. However, a handful of studies has been performed to understand the interactions between therapeutic molecules and excipients with SWCNT interface as well as the mechanism of intracellular release for therapeutic effects. Additionally, little attention has been paid to how cell type and cell metabolic activity level influence internalization of SWCNTs with different lengths. In this talk, I will discuss cell type, cell metabolic activity, and SWCNT length dependent cellular processing of SWCNTs dispersed with diverse biocompatible dispersing agents. Our results demonstrate that proper choice of dispersing agent can allow for interaction with F-actin, accumulation in the endoplasmic reticulum for eventual expulsion, or accumulation in cell nucleus. Further, macrophages with relatively high cell metabolic activity level compared to other cell types such as fibroblasts have shown to internalize significantly higher amount of SWCNTs per cell. The total mass of SWCNTs internalized per cell increases with a decrease in SWCNT length. Lastly, I will discuss our recent effort to create a ternary complex of SWCNTs, drug, and protein for intracellular delivery and release of model drugs. To overcome the limitation of the existing methods being only plausible for model drugs that are soluble in aqueous solutions, we developed a facile schema to assemble stepwise various combinations of therapeutic molecules and excipients on SWCNTs in different solvents using a preformed SWCNT network. These ternary complexes allow dialing in delivery of therapeutic molecules and excipients to achieve desired cell viability reduction. These findings allow for the development of new SWCNT-based biological applications as they establish important parameters of biomolecule adsorption on SWCNTs, SWCNT internalization and subcellular processing, which are crucial for applications ranging from drug delivery to cell type-specific modulation.

Evaluation of the Osteogenic Potential of Stem Cells in the Presence of Growth Hormone under Magnetic Field Stimulation.

The human alveolar bone-derived mesenchymal stem cells (hABMSCs) are considered an attractive source for the development of bone tissues. However, their mechanism of action is still unclear. This work aimed to investigate the potential of the natural human growth hormone (NHGH) derived from stem cells under magnetic field (MF) stimulation for tissue engineering by exploring the paracrine or autocrine effects of hABMSCs in vitro. The secretion of anti-inflammatory cytokines and growth factors from hABMSCs was profoundly affected by the intensity of the applied MFs. The effects of stimulated MFs on vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) production were quantified by an ELISA kit. Notably, higher cell metabolic activity was observed in MF stimulation compared to the control, and this was more prominent in 130 mT strength of MF. An enhancement in the production of VEGF and BMP-2 was noted in MFs compared to the control. Moreover, higher accumulation of osteogenesis-related genes has occurred in MFs than the control. Furthermore, a significant enhancement in cell metabolic activity and mineralized nodule formations was spotted in the presence of NHGH via MF stimulation; vis-à-vis, MF stimulation only through autocrine and paracrine effects demonstrated the better osteogenic potential of NHGH in the presence of MFs for tissue engineering applications.

Dual crosslinking strategy to generate mechanically viable cell-laden printable constructs using methacrylated collagen bioinks.

Photopolymerization of methacrylated collagen (CMA) allows for 3D bioprinting of tissue scaffolds with high resolution and print fidelity. However, photochemically crosslinked CMA constructs are mechanically weak and susceptible to expedited enzymatic degradation in vivo. The goal of the current study was to develop a dual crosslinking scheme for the generation of mechanically viable cell-laden printable constructs for tissue engineering applications. Dual crosslinking was performed by first photochemical crosslinking of CMA hydrogels using VA-086 photoinitiator and UV exposure followed by chemical crosslinking with two different concentrations of genipin (i.e., 0.5 mM (low dual) or 1 mM (high dual)). The effect of dual crosslinking conditions on gel morphology, compressive modulus, stability and print fidelity was evaluated. Additionally, human MSCs were encapsulated within CMA hydrogels and the effect of dual crosslinking conditions on viability and metabolic activity was assessed. Uncrosslinked, photochemically crosslinked, and genipin crosslinked CMA hydrogels were used as controls. SEM results showed that gel morphology was maintained upon dual crosslinking. Further, dual crosslinking significantly improved the compressive modulus and degradation time of cell-laden and acellular CMA hydrogels. Cell viability results showed that high cell viability (i.e., >80%) and metabolic activity in low dual crosslinked CMA hydrogels. On the other hand, cell viability and metabolic activity decreased significantly (p < 0.05) in high dual crosslinked CMA hydrogels. Quantitative fidelity measurements showed the measured parameters (i.e., line widths, pore size) were comparable between photochemically crosslinked and dual crosslinked constructs, suggesting that print fidelity is maintained upon dual crosslinking. In conclusion, application of low dual crosslinking is a viable strategy to yield mechanically superior, cell compatible and printable CMA hydrogels.

Graphene oxide containing self-assembling peptide hybrid hydrogels as a potential 3D injectable cell delivery platform for intervertebral disc repair applications

Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP).In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (β-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells. Statement of SignificanceShort self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the ab initio design of cells’ microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs.

Designing bioactive porous titanium interfaces to balance mechanical properties and in vitro cells behavior towards increased osseointegration

Titanium implant failures are mainly related to stress shielding phenomenon and the poor cell interaction with host bone tissue. The development of bioactive and biomimetic Ti scaffolds for bone regeneration remains a challenge which needs the design of Ti implants with enhanced osseointegration. In this context, 4 types of titanium samples were fabricated using conventional powder metallurgy, fully dense, dense etched, porous Ti, and porous etched Ti. Porous samples were manufactured by space holder technique, using ammonium bicarbonate particles as spacer in three different ranges of particle size (100–200 μm, 250–355 μm and 355–500 μm). Substrates were chemically etched by immersion in fluorhydric acid at different times (125 and 625 s) and subsequently, were characterized from a micro-structural, topographical and mechanical point of view. Etched surfaces showed an additional roughness preferentially located inside pores. In vitro tests showed that all substrates were biocompatible (80% of cell viability), confirming cell adhesion of premioblastic cells. Similarly, osteoblast showed similar cell proliferation rates at 4 days, however, higher cell metabolic activity was observed in fully dense and dense etched surfaces at 7 days. In contrast, a significant increase of alkaline phosphatase enzyme expression was observed in porous and porous etched samples compared to control surfaces (dense and dense etched), noticing the suitable surface modification parameters (porosity and roughness) to improve cell differentiation. Furthermore, the presence of pores and rough surfaces of porous Ti substrates remarkably decreased macrophage activation reducing the M1 phenotype polarization as well M1 cell marker expression. Thus, a successful surface modification of porous Ti scaffolds has been performed towards a reduction on stress shielding phenomenon and enhancement of bone osseointegration, achieving a biomechanical and biofunctional equilibrium.

Polyurethane-Polycaprolactone Blend Patches: Scaffold Characterization and Cardiomyoblast Adhesion, Proliferation, and Function.

A remarkable challenge in myocardial tissue engineering is the development of biomimetic constructs that can potentially improve myocardial repair and regeneration. Polyurethane (PU) scaffolds are extensively utilized in the cardiovascular system. We have synthesized a new biodegradable poly(ester-ether urethane urea) (PEEUU) using a new and simple method. To enhance mechanical and physicochemical properties, the PEEUU was blended with polycaprolactone (PCL). We then fabricated a series of new PU-PCL scaffolds. The scaffolds were then characterized using SEM, porosity measurement, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), DSC, water contact angle measurement, swelling measurement, in vitro degradation rate, and mechanical tests. Expression of the cardiac-specific proteins on the scaffolds was investigated using immunofluorescence staining and quantitative real-time PCR. The elasticity of blends increased with an increase of PEEUU. In the blend scaffolds, the size and interconnectivity of pores were in an appropriate range (142-170 μm) as reported in the literature. These blend scaffolds revealed high cell metabolic activity for cardiomyoblasts and also enabled cells to proliferate and express cardiac marker proteins at higher rates. Histological examination of subcutaneously transplanted scaffolds after two months revealed degradation in the blend scaffolds. It is demonstrated that functionality of cells is sensitive to the composition of biomaterials used, and the effective cell-biomaterial interactions are critical in order to create a functional tissue engineered product that allows seeded cells to develop their normal activity. The PEEUU-PCL blends could potentially provide a versatile platform to fabricate functional scaffolds with an effective cell-biomaterial interaction for cardiac tissue regeneration.

3D Printed Silicone-Hydrogel Scaffold with Enhanced Physicochemical Properties.

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds.

The use of biopolymers as a three dimensional (3D) support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface properties to assess whether seeding efficiencies and cell growth on 3D scaffolds were affected. Cell attachment and growth of murine calvarial osteoblast (MC3T3-E1) cells within a melt-electrospun poly-ε-caprolactone scaffold were assessed when cultured in either “low-adhesive” non-treated or corona discharged-treated well-plates. Increased cell adhesion was observed on the scaffold placed in the surface treated culture plates compared to the scaffold in the non-treated plates 24 h after seeding, although it was not significant. However, higher cell metabolic activity was observed on the bases of all well-plates than on the scaffold, except for day 21, well metabolic activity was higher in the scaffold contained in non-treated plate than the base. These results indicate that there is no advantage in using non-treated plates to improve initial cell seeding in 3D polymeric tissue engineering scaffolds, however non-treated plates may provide an improved metabolic environment for long-term studies.

Functionality of freeze-dried L. casei cells immobilized on wheat grains

Abstract Lactobacillus casei cells were immobilized on wheat grains and the effect of nine cryoprotectants during freeze-drying was investigated. Survival and fermentative activity of the freeze-dried immobilized biocatalysts was studied by monitoring pH, lactic acid and lactose content in successive fermentations batches of both synthetic lactose medium and milk. Freeze-dried L. casei cells immobilized on wheat grains without using cryoprotectants resulted in high cell survival and metabolic activity. The same biocatalysts were stored at room temperature for 9 months and at 4 °C and −18 °C for 12 months. Reactivation of the stored biocatalysts was carried out in synthetic lactose medium. Storage at room and low temperatures (4 °C and −18 °C) resulted in about 5.11, 4.9 and 4.3 final pH respectively during fermentations, indicating the suitability of the immobilized biocatalysts for the production of mild and low pH dairy products. The immobilization of a probiotic microorganism, such as L. casei , on boiled wheat which contains prebiotic compounds might provide a potential synbiotic preparation.

Osteoblast activity on collagen‐GAG scaffolds is affected by collagen and GAG concentrations

Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen-glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of approximately 99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering.

438 Mesenchymal progenitor cells as gene delivery systems for cancer and leukemia therapy

A major challenge for successful cultured meat production is the requirement for large quantities of skeletal muscle satellite cells (MuSCs). Commercial microcarriers (MCs), such as Cytodex®1, enable extensive cell expansion by offering a large surface-to-volume ratio. However, the cell-dissociation step post cell expansion makes the cell expansion less efficient. A solution is using food-grade MCs made of sustainable raw materials that do not require a dissociation step and can be included in the final meat product. This study aimed to produce food-grade MCs from food industry by-products (i.e., turkey collagen and eggshell membrane) and testing their ability to expand bovine MuSCs in spinner flask systems for eight days. The MCs’ physical properties were characterized, followed by analyzing the cell adhesion, growth, and metabolic activity. All MCs had an interconnected porous structure. Hybrid MCs composed of eggshell membrane and collagen increased the mechanical hardness and stabilized the buoyancy compared to pure collagen MCs. The MuSCs successively attached and covered the entire surface of all MCs while expressing high cell proliferation, metabolic activity, and low cell cytotoxicity. Cytodex®1 MCs were included in the study. Relative gene expression of skeletal muscle markers showed reduced PAX7 and increased MYF5, which together with augmented proliferation marker MKI67 indicated activated and proliferating MuSCs on all MCs. Furthermore, the expression pattern of cell adhesion receptors (ITGb5 and SDC4) and focal adhesion marker VCL varied between the distinct MCs, indicating different specific cell receptor interactions with the various biomaterials. Altogether, our results demonstrate that these biomaterials are promising prospects to produce custom-fabricated food-grade MCs intended to expand MuSCs.