Mechanistic insights of cells in porous scaffolds via integrated culture technologies

This research aimed to combine 3 cell and tissue culture technologies to obtain mechanistic insights of cells in porous scaffolds. When cultivated on 2D (2-dimensional) surfaces, HDFs (human dermal fibroblasts) behaved individually and had no strict requirement on seeding density for proliferation; while HaCat cells relied heavily on initial densities for proliferation and colony formation, which was facilitated when co-cultured with HDFs. Experiments using a 3D CCIS (3-dimensional cell culture and imaging system) indicated that HDFs colonised open pores of varying sizes (125-420 μm) on modular substrates via bridge structures; while HaCat cells formed aperture structures and only colonised small pores (125 μm). When co-cultured, HDFs not only facilitated HaCat attachment on the substrates, but also coordinated with HaCat cells to colonise open pores of varying sizes via bridge and aperture structures. Based on these observations, a 2-stage strategy for the culture of HDFs and HaCat cells on porous scaffolds was proposed and applied successfully on a cellulosic scaffold. This research demonstrated that cell colonisation in scaffolds was dependent on multiple factors; while the integrated 2D&3D culture technologies and the 3D CCIS was an effective and efficient approach to obtain mechanistic insights of their influences on tissue regeneration

Comparison of human dermal fibroblasts and HaCat cells cultured in medium with or without serum via a generic tissue engineering research platform

A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) monoor co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative in medium without serum than with serum. However, both cell types were successfully co-cultured in 2D using serum-free medium if the initial cell seeding density was higher than 80,000 cells/cm2 (with 1:1 ratio). Based on the results from 2D cultures, co-culture of both cell types on modular substrates with small open pores (125 µm) and cellulosic scaffolds with open pores of varying sizes (50–300 µm) were then conducted successfully in serum-free medium. This study demonstrated that the generic research platform had great potential for in-depth understanding of HDFs and HaCat cells cultivated in serum-free medium, which could inform the processes for manufacturing skin cells or tissues for clinical applications

A novel scale-down cell culture and imaging design for the mechanistic insight of cell colonization within porous substrate

At the core of translational challenges in Tissue Engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale-down strategy to deconstruct complex bio-matrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonization within porous substrate in this proof-of-concept study, TEM specimen supporters (10-30µm thick) with fine controlled open pores (100~600µm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Non-invasive imaging of human dermal fibroblasts cultured on these free-standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high quality micrographs of cell-cell and cell-scaffold interactions at micro-scale, depicted cytoskeletal structures in stretched and relaxed areas at nano-scale. Thus this novel scaled-down design was able to improve our mechanistic understanding of tissue formation not only at single- and multiple-cell levels, but also at micro- and nano-scales, which could be difficult to obtain using other methods

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture

Evaluation of genipin-crosslinked chitosan hydrogels as a potential carrier for silver sulfadiazine nanocrystals

In the present study genipin crosslinked chitosan (CHI) hydrogels, which had been constructed and reported in our previous studies (Lei Gao, et al. Colloids Surf. B Biointerfaces. 2014, 117: 398), were further evaluated for their advantage as a carrier for silver sulfadiazine (AgSD) nanocrystal systems. Firstly, AgSD nanocrystals with a mean particle size of 289 nm were prepared by wet milling method and encapsulated into genipin crosslinked CHI hydrogels. AgSD nanocrystals displayed a uniform distribution and very good physical stability in the hydrogel network. Swelling-dependent release pattern was found for AgSD nanocrystals from hydrogels and the release profile could be well fitted with Peppas equation. When AgSD nanocrystals were encapsulated in hydrogels their fibroblast cytotoxicity decreased markedly, and their antibacterial effects against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were still comparable to unencapsulated AgSD nanocrystals. In vivo evaluation in excision and burn cutaneous wound models in mice showed that AgSD nanocrystal hydrogels markedly decreased the expression of inflammatory cytokine IL-6, but increased the levels of growth factors VEGF-A and TGF-β1. Histopathologically, the wounds treated by hydrogels containing AgSD nanocrystals showed the best healing state compared with commercial AgSD cream, hydrogels containing AgSD bulk powders and blank hydrogels. The wounds treated by AgSD nanocrystal hydrogels were dominated by marked fibroblast proliferation, new blood vessels and thick regenerated epithelial layer. Sirius Red staining assay indicated that AgSD nanocrystal hydrogels resulted in more collagen deposition characterized by a large proportion of type I fibers. Our study suggested that genipin-crosslinked CHI hydrogel was a potential carrier for local antibacterial nanomedicines

Periodic pressure change improves mass transfer

Periodic pressure change improves mass transfe

Adhesive bandage type artificial active tissue constructed by using culture solution without serum or bovine pituitary extracts and construction method of adhesive bandage type artificial active tissue

Abstract：The invention discloses an adhesive bandage type artificial active tissue constructed by using a culture solution without serum or bovine pituitary extracts and a construction method of the adhesive bandage type artificial active tissue. A cell culture solution without the serum or bovine pituitary extracts is adopted, and a biological film carrier is specially treated to ensure that a niche suitable for cell interaction is formed on a biological film, and promote human fibroblasts and epidermal cells to give play to self potentials, proliferate, disintegrate and excrete growth factors, so that an artificial tissue with active cells can be formed on the biological film in 4-7 days. The artificial tissue is high in cell activity and strong in division and migration ability, can home back to wound surfaces, and can promote tissue regeneration by serving as seed cells for curing burns and chronic ulcer. The adhesive bandage type artificial active tissue disclosed by the invention completely avoids immunological rejection of animal derived products including serum, pituitary extracts and collagen and the risk of infection of viruses transmitted to humans from animals, and a thin cell tissue taking the biological film as a scaffold and an infection barrier is formed, so that the adhesive bandage type artificial active tissue with biological safety is truly realized

Enhanced cellulase production in fed-batch solid state fermentation of Trichoderma viride SL-1

A novel fed-batch solid state fermentation process was developed in order to avoid problems associated with high initial nutrient concentration, while retaining advantages from high total effective salt concentration. Cellulase production by Trichoderma viride SL-1 was selected as the experimental model. Ammonium sulphate solution was atomized and fed to the culture by pneumatic transfer. Different feed modes were investigated that resulted in improved enzyme productivity

Cell marbles: a novel cell encapsulation technology by wrapping cell suspension droplets using electrospun nanofibers for developmental engineering

Developmental Engineering aims to imitate natural tissue regeneration processes via an additive manufacturing approach. This research developes a technology to fabricate ready-made cell marbles (CMs) by wrapping cell suspension droplets of (3−15 μl) with electrospun hydrophobic nanofibers, as modular building blocks for developmental engineering. Human dermal fibroblasts and/or immortalised keratinocytes were suspended in the culture media cores of the CMs. The encapsulated cells were observed to precipitate at bottoms or up-inclined inner surfaces of the fibrous shells within 10 min. The CMs were mechanically strong enough to be handled as soft solids, thus easily and accurately delivered using forceps into three distinct culture systems, including tissue culture plastics, cellulosic scaffolds and in vitro fibrin wound models. The release of the cells, culture media and nanofibers into specific delivery points within the investigated culture systems was achieved via the controlled rupture of the CMs triggered by the simple hydrophobic-hydrophilic interaction between the nanofibers and the aqueous surroundings. Further cell and tissue culture studies indicated that the prominent traits of the skin cells were well preserved during cell encapsulation and delivery processes, suggesting the great potential of the CMs for additive tissue manufacturing in developmental engineering

A novel design of solid state fermenter and its evaluation for cellulase production by Trichoderma viride SL-1

A novel design of a large scale solid state fermenter, designated as PPSSF--Periodic Pressure Solid State Fermenter-- was constructed. Due to the respiration created by periodical variations of air pressure, air was uptaken / expelled from the fermenting mass and heat moved from the fermenter easily. It is possible to operate the unit at different capacities simply by adding or removing the bins. The results of its evaluation for enzyme formation by Trichoderma viride SL-1 indicated that a 2-3 fold increased cellulase production can be obtained in this system