Peggy Pui Yik Chan

A liquid marble micro‐bioreactor is prepared by placing a drop of murine embryonic stem cell (ESC) (Oct4B2‐ESC) suspension onto a polytetrafluoroethylene (PTFE) particle bed. The Oct4B2‐ESC aggregates to form embryoid bodies (EBs) with relatively uniform size and shape in a liquid marble within 3 d. For the first time, the feasibility of differentiating ESC into cardiac lineages within liquid marbles is being investigated. Without the addition of growth factors, suspended EBs from liquid marbles express various precardiac mesoderm markers including Flk‐1, Gata4, and Nkx2.5. Some of the suspended EBs exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac mesoderm differentiation. Some of the EBs are subsequently plated onto gelatin‐coated tissue culture dishes. Plated EBs express mature cardiac markers atrial myosin light chain 2a (MLC2a) and ventricular myosin light chain (MLC2v), and the cardiac structural marker α‐actinin. More than 60% of the plated EBs exhibit spontaneous contraction and express mature cardiomyocyte marker cardiac troponin T (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. Together, these results demonstrate that the liquid‐marble technique is an easily employed, cost effective, and efficient approach to generate EBs and facilitating their cardiogenesis.

Compared with preformed anisotropic matrices, an anisotropic matrix that allows users to alter its properties and structure in situ after synthesis offers the important advantage of being able to mimic dynamic in vivo microenvironments, such as in tissues undergoing morphogenesis or in wounds undergoing tissue repair. In this study, porous gradients are generated in situ in a hydrogel comprising enzymatically crosslinked gelatin hydroxyphenylpropionic acid (GTN–HPA) conjugate and carboxylmethyl cellulose tyramine (CMC–TYR) conjugate. The GTN–HPA component acts as the backbone of the hydrogel, while CMC–TYR acts as a biocompatible sacrificial polymer. The hydrogel is then used to immobilize HT1080 human fibrosarcoma cells in a microfluidic chamber. After diffusion of a biocompatible cellulase enzyme through the hydrogel in a spatially controlled manner, selective digestion of the CMC component of the hydrogel by the cellulase gives rise to a porosity gradient in situ instead of requiring its formation during hydrogel synthesis as with other methods. The influence of this in situ tunable porosity gradient on the chemotactic response of cancer cells is subsequently studied both in the absence and presence of chemoattractant. This platform illustrates the potential of hydrogel‐based microfluidics to mimic the 3D in vivo microenvironment for tissue engineering and diagnostic applications.

Since rates of tissue growth vary significantly between tissue types, and also between individuals due to differences in age, dietary intake, and lifestyle‐related factors, engineering a scaffold system that is appropriate for personalized tissue engineering remains a significant challenge. In this study, a gelatin‐hydroxyphenylpropionic acid/carboxylmethylcellulose‐tyramine (Gtn‐HPA/CMC‐Tyr) porous hydrogel system that allows the pore structure of scaffolds to be altered in vivo after implantation is developed. Cross‐linking of Gtn‐HPA/CMC‐Tyr hydrogels via horseradish peroxidase oxidative coupling is examined both in vitro and in vivo. Post‐implantation, further alteration of the hydrogel structure is achieved by injecting cellulase enzyme to digest the CMC component of the scaffold; this treatment yields a structure with larger pores and higher porosity than hydrogels without cellulase injection. Using this approach, the pore sizes of scaffolds are altered in vivo from 32–87 μm to 74–181 μm in a user‐controled manner. The hydrogel is biocompatible to COS‐7 cells and has mechanical properties similar to those of soft tissues. The new hydrogel system developed in this work provides clinicians with the ability to tailor the structure of scaffolds post‐implantation depending on the growth rate of a tissue or an individual's recovery rate, and could thus be ideal for personalized tissue engineering.

In addition to the choice of appropriate material properties of the tissue construct to be used, such as its biocompatibility, biodegradability, cytocompatibility, and mechanical rigidity, the ability to incorporate microarchitectural patterns in the construct to mimic that found in the cellular microenvironment is an important consideration in tissue engineering and regenerative medicine. Both these issues are addressed by demonstrating a method for preparing biodegradable and photo‐patternable constructs, where modified cellulose is cross‐linked to form an insoluble structure in an aqueous environment. Specifically, hydroxypropyl cellulose (HPC) is rendered photocrosslinkable by grafting with methylacrylic anhydride, whose linkages also render the cross‐linked construct hydrolytically degradable. The HPC is then cross‐linked via a photolithography‐based fabrication process. The feasibility of functionalizing these HPC structures with biochemical cues is verified post‐fabrication, and shown to facilitate the adhesion of mesenchymal progenitor cells. The HPC constructs are shown to be biocompatible and hydrolytically degradable, thus enabling cell proliferation and cell migration, and therefore constituting an ideal candidate for long‐term cell culture and implantable tissue scaffold applications. In addition, the potential of the HPC structure is demonstrated as an alternative substrate to paper microfluidic diagnostic devices for protein and cell assays.

Here we report the preparation and characterization of thermoresponsive cellulosic hydrogels with cell-releasing behavior. Hydroxypropyl cellulose (HPC) was modified with methacrylic anhydride (MA). The resultant macromonomer, HPC-MA, retains the characteristic thermoresponsive phase behavior of HPC, with an onset temperature of 36 °C and a lower critical solution temperature (LCST) of 37–38 °C, as determined by turbidity measurement. Homogenous HPC-MA hydrogels were prepared by UV-cross-linking the aqueous solutions of the macromonomer at room temperature, and characterized by water contact angle and swelling ratio measurements, and dynamic mechanical analysis. These hydrogels exhibit temperature-dependent surface hydrophilicity and hydrophobicity, equilibrium water content as well as mechanical properties. Cell-releasing characteristics were demonstrated using African green monkey kidney cell line (COS-7 cells) and murine-derived embryonic stem cell line (Oct4b2). By reducing temperature to 4 °C, the cultivated cells spontaneously detached from the hydrogels without the need of trypsin treatment. These unique properties make our HPC-MA hydrogels potential substrates for cell sheet engineering.

This study describes the preparation and characterization of a biodegradable 3D hydrogel constructed from hydroxypropyl cellulose (HPC), modified with bifunctional methacrylic anhydride (MA) to form hydroxypropyl cellulose methacrylate (HPC-MA), for adipose tissue engineering applications. The hydrogels were prepared from three different concentrations (10 wt%, 15 wt% and 20 wt%) of HPC-MA with 0.35 degree of substitution. HPC-MA hydrogel scaffolds with open biphasic features were prepared by exploiting the thermal responsive phase behavior of HPC and temperature mediated phase separation of HPC-MA. The resulting scaffolds exhibited pore sizes ranging from 30 to 300 μm and an interconnected porosity of ∼90%. The swelling ratio (SR) and storage modulus of HPC-MA scaffolds were in the range of 12.94 to 35.83 and 0.75 to 4.28 kPa, respectively. The swelling ratio and storage modulus suggested that the scaffold exhibits high water retention, allowing medium exchange during cell culturing and that it is suitable for adipose tissue regeneration. The HPC-MA scaffolds were found to be biocompatible to human adipose-derived stem cells (ASCs). ASCs were successfully differentiated into the adipocytes inside the scaffolds, and therefore demonstrated the potential application of these HPC-MA scaffolds for adipose tissue engineering.

This study presents a novel and inexpensive method to prepare a disposable micro-bioreactor for stem cell expansion. The micro-bioreactor was fabricated in the form of a fixed bed bioreactor with a microchannel reactor bed. The micro-bioreactor was constructed from polydimethylsiloxane (PDMS), and the microchannel was functionalized to enable cell adhesion and resistance to bovine serum albumin protein adsorption. The PDMS reactor bed surface was activated by oxygen plasma, then aminized with trimethoxysilylpropyl(polyethyleneimine), followed by grafting with carboxylmethyl cellulose (CMC) and gelatin in sequence. The functionalized PDMS surface demonstrated improved hydrophilicity and antifouling properties. The grafting of gelatin promoted cell adhesion. The functionalized surface was found to be biocompatible with MDA-MB-231 and Oct4b2 cells and was demonstrated to facilitate cell proliferation. The expanded Oct4b2 cells retained their proliferation potential, undifferentiated phenotype and pluripotency.

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water‐soluble metabolites through their high water‐content matrix. The ability to image three‐dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell‐biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB‐tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well. Biotechnol. Bioeng. 2013; 110: 318–326.