Dispensing of very low volumes of ultra high viscosity alginate gels: a new tool for encapsulation of adherent cells and rapid prototyping of scaffolds and implants.

We present a tool for dispensing very low volumes (20 nL or more) of ultra high viscosity (UHV) medical-grade alginate hydrogels. It uses a modified piezo-driven micrometering valve, integrated into a versatile system that allows fast prototyping of encapsulation procedures and scaffold production. Valves show excellent dispensing properties for UHV alginate in concentrations of 0.4% and 0.7% and also for aqueous liquids. An optimized process flow provides excellent handling of biological samples under sterile conditions. This technique allows the encapsulation of adherent cells and structuring of substrates for biotechnology and regenerative medicine. A variety of cell lines showed at least 70% viability after encapsulation (including cell lines that are relevant in regenerative medicine like Hep G2), and time-lapse analysis revealed cells proliferating and showing limited motility under alginate spots. Cells show metabolic activity, gene product expression, and physiological function. Encapsulated cells have contact with the substrate and can exchange metabolites while being isolated from macromolecules in the environment. Contactless dispensing allows structuring of substrates with alginate, isolation and transfer of cell-alginate complexes, and the dispensing of biological active hydrogels like extracellular matrix-derived gels.

Physical and biological properties of barium cross-linked alginate membranes.

We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the "crystal gun" [Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L(p), from PEG-induced flows ranges between 2.4x10(-12) and 6.5x10(-12) m Pa(-1)s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3x10(-10) m Pa(-1)s(-1) at 4kPa. Mechanical introduction of 200 microm-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes.

Physicochemical features of ultra-high viscosity alginates.

The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHV(N)) and Lessonia trabeculata (UHV(T)) were analyzed. Fluorescence and (1)H NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHV(T), but low for UHV(N). Intrinsic viscosity [eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas [eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M(w) up to 1.1x10(6) g/mol) and of the z-average gyration radius (R(G)(z) up to 191 nm). The M(w) and R(G)(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M(w) dependency of [eta] and R(G)(z) could be described by [eta]=0.059xM(w)(0.78) and R(G)(z)=0.103xM(w)(x). (UHV(N): x=0.52; UHV(T): x=0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M(w) and R(G)(z) values of UHV(T)- and UHV(N)-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates.

Alginate-based encapsulation of cells: past, present, and future.

Replacing dysfunctional endocrine tissues (eg, islets) with healthy, nonautologous material protected against the immune defense of the patient could soon become a reality. Recent advances have resulted in the development of alginate-based microcapsules that meet the demands of biocompatibility, long-term integrity, and function. Focus on the development of good manufacturing practice-conforming microfluidic chip technology for generation of immunoisolated transplants and on cryopreservation technology will bring the cell-based therapy to the market and clinics.