Introduction

The encapsulation of cells and drugs in small hydrogel spheres has been established over the past 10 years. Various techniques have been developed in this area ranging from airflow systems (1) to lab-on-chip microfluidic systems (2).

Encapsulation of insulin-producing islets of Langerhans (1) or metabolically active hepatocytes (3) in an alginate matrix provides implantable microbioreactors for regenerative medicine and drug delivery (4,5). Further applications for encapsulated cells can be found in the food industry (6), bioprocess engineering (7), pharmaceutical industries (8), and in cell culture (9). Alginate hydrogel capsules isolate embedded cells from the immune system or environment while allowing transport of O₂, nutrients, hormones, toxic metabolites, and CO₂ (Figure 1A). Hormones produced by encapsulated cells may pass through a matrix of suitable pore size. A technique capable of directly encapsulating adherent cells on substrates would be of great value, as cells suspended in hydrogel environments are round and not adherent. They cannot, for example, interact with extracellular matrix (ECM)–derived signal molecules, and therefore, intrinsic signal cascades are not triggered. However, these intrinsic pathways are crucial for cell survival, proliferation, migration, differentiation, and apoptosis (10). Proteins immobilized on surfaces like fibronectin, vitronectin, or laminin trigger signal transduction by binding to transmembrane receptors. Such signal cascades are very complex, but in the case of integrin receptors phospholipase Cs (PLCs), Ras proteins and phosphoinositide 3 kinases are phosphorylated and initiate gene transcription and the expression of gene products relevant to cell proliferation, differentiation, migration, or apoptosis (11). Cells that are removed from their natural environment often tend to apoptose (12), and fibroblasts in suspension are arrested in the G phase of the cell cycle and cannot proliferate (13,14,15).

Figure 1. Two-dimensional (2-D) encapsulation and technical details. (Click to enlarge)

In this paper, we present a new method and device for encapsulating cells in clinical-grade alginate while allowing them to adhere to surfaces and receive chemical signals (see Figure 1B). The system dispenses high-viscosity liquids and allows selection of the points of interest to be encapsulated. We show first proof-of-concepts for several applications in biotechnology.

Materials and Methods

Alginate Dispenser System

We designed a completely new system for dispensing ultra high viscosity (UHV) alginates. At the core, a modified micrometering valve (commercially available by DELO Industrial Adhesives GmbH, Windach, Germany, and modified by GeSiM, Rossendorf, Germany) is integrated into an xy-table Nano-Plotter (GeSiM). The heatable micrometering valve is controlled by DELO-MAT (DELO Industrial Adhesives GmbH), which controls the piezo-actuator (stack) of the valve. The alginate reservoir is a luer-lock syringe connected to a precision pressure controller and pressure source. An additional technical industrial miniature video microscope T.I.M.M. 150 (SPI GmbH, Oppenheim, Germany) fixed on the xy-table allows in-process inspection and selection of targets. Figure 1C illustrates the general setup of the system. A dilutor as part of the Nano-Plotter transports fluids to a wash station on the xy-table. The DELO-Dot alginate dispenser and Nano-Plotter are linked to a personal computer with appropriate Nano-Plotter software NPC16 (GeSiM) that allows the control of the system and the editing of individual dispensing procedures. A light source (Fiber Illuminator; Nikon, Düsseldorf, Germany) and pump complete the system and facilitate handling of biological samples during encapsulation procedures. A laminar flow hood keeps the system sterile.

Micrometering Valve

The micrometering valve consists mainly of 034, 301, and 305 stainless steel. The integrated sealing system is a zirconium oxide ceramic component (Chemraz, Greene, Tweed, Hofheim am Taunus, Germany). Microvolumes are dispensed by a piezo-actuated mechanism. Alginate flows in the inner chamber of the valve under pressure from the syringe. Normally, a small ceramic sphere connected by a needle to the piezo-actuator seals the output channel. To dispense one or more spots, the PC sends signals with user-defined parameters (pulsewidth, voltage, and frequency) to the valve and position instructions to the xy-table. The piezoactuator contracts, the needle moves up, and the ceramic sphere opens the output. The piezo-actuator relaxes after a given pulsewidth and moves the ceramic sphere downward. The movement ejects fluid with high velocity. The dispensing of more than one spot can be controlled by the frequency parameter, and the manufacturers claim that fluids with viscosities in the range of 50–200,000 mPas can be dispensed. Figure 1D is a drawing of the valve, which produces small cylindrical jets.