Scalable units for building cardiac tissue.

Scalable units for building cardiac tissue are fabricated from biodegradable elastomeric polymers by pairwise stacking of heart-cell scaffolds with sinusoidal internal pore architectures and dedicated perfusable microvessels with rapidly degrading porous interfaces in a parallel flow configuration. This platform supports viable heart cells in vitro and, if validated in vivo, may aid in the regenerative repair of vascularized tissues.

Biomimetic scaffold combined with electrical stimulation and growth factor promotes tissue engineered cardiac development.

Toward developing biologically sound models for the study of heart regeneration and disease, we cultured heart cells on a biodegradable, microfabricated poly(glycerol sebacate) (PGS) scaffold designed with micro-structural features and anisotropic mechanical properties to promote cardiac-like tissue architecture. Using this biomimetic system, we studied individual and combined effects of supplemental insulin-like growth factor-1 (IGF-1) and electrical stimulation (ES). On culture day 8, all tissue constructs could be paced and expressed the cardiac protein troponin-T.

A biodegradable microvessel scaffold as a framework to enable vascular support of engineered tissues.

A biodegradable microvessel scaffold comprised of distinct parenchymal and vascular compartments separated by a permeable membrane interface was conceptualized, fabricated, cellularized, and implanted. The device was designed with perfusable microfluidic channels on the order of 100 μm to mimic small blood vessels, and high interfacial area to an adjacent parenchymal space to enable transport between the compartments. Poly(glycerol sebacate) (PGS) elastomer was used to construct the microvessel framework, and various assembly methods were evaluated to ensure robust mechanical integrity.

3D structural patterns in scalable, elastomeric scaffolds guide engineered tissue architecture.

Microfabricated elastomeric scaffolds with 3D structural patterns are created by semiautomated layer-by-layer assembly of planar polymer sheets with through-pores. The mesoscale interconnected pore architectures governed by the relative alignment of layers are shown to direct cell and muscle-like fiber orientation in both skeletal and cardiac muscle, enabling scale up of tissue constructs towards clinically relevant dimensions.

Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures.

Plant cell cultures provide a renewable source for synthesis and supply of commercially valuable plant-derived products, particularly for secondary metabolites. However, instability in product yields over multiple passages has hampered the efficient and sustainable use of this technology. Paclitaxel accumulation in Taxus cell suspension culture was quantified over multiple passages and correlated to mean aggregate size, extracellular sugar level, ploidy, and cell cycle distribution.

Paclitaxel uptake and transport in Taxus cell suspension cultures.

The transport of paclitaxel in Taxus canadensis suspension cultures was studied with a fluorescence analogue of paclitaxel (Flutax-2(®)) in combination with flow cytometry detection. Experiments were carried out using both isolated protoplasts and aggregated suspension cell cultures. Flutax-2(®) was shown to be greater than 90% stable in Taxus suspension cultures over the required incubation time (24 hours).

Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures.

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures.

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures.

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture.

Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures.

Plant cell aggregates have long been implicated in affecting cellular metabolism in suspension culture, yet the rigorous characterization of aggregate size as a process variable and its effect on bioprocess performance has not been demonstrated. Aggregate fractionation and analysis of biomass-associated product is commonly used to assess the effect of aggregation, but we establish that this method is flawed under certain conditions and does not necessarily agree with comprehensive studies of total culture performance. Leveraging recent advances to routinely measure aggregate size distributions, we developed a simple method to manipulate aggregate size and evaluate its effect on the culture as a whole, and found that Taxus suspension cultures with smaller aggregates produced significantly more paclitaxel than cultures with larger aggregates in two cell lines over a range of aggregate sizes, and where biomass accumulation was equivalent before elicitation with methyl jasmonate.

Flow cytometric methods to investigate culture heterogeneities for plant metabolic engineering.

Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated, and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism, and productivity of key metabolites.

Characterization of aggregate size in Taxus suspension cell culture.

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 microm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight.

Pharmaceutically active natural product synthesis and supply via plant cell culture technology.

The chemical diversity of plant-derived natural products allows them to function in a multitude of ways including flavor enhancers, agricultural chemicals, and importantly, human medicinals. Supply of pharmaceutically active natural products is often a challenge due to the slow growing nature of some species, low yields found in nature, and unpredictable variability in accumulation. Several production options are available including natural harvestation, total chemical synthesis, semisynthesis from isolated precursors, and expression of plant pathways in microbial systems.