Microcapsule degradation

We implemented microencapsulation of engineered human cells and designed the system for degradation of alginate microcapsules.

Engineered HEK293T cells were succesfully incorporated into alginate microcapsules and extended time viability of the encapsulated cells was demonstrated.

Secretory alginate lyases from Pseudomoalteromonas elyakovii and from Pseudomonas aeruginosa were cloned by replacing the signal peptide of the alginate lyase with the eukaryotic signal peptide.

Alginate lyases were succesfully produced and secreted from HEK293T cells.

Degradation of alginate beads was demonstrated by alginate lyase from Sphingobacterium multivorum.

Degradation of alginate microcapsules

Our biopharmaceutical delivery system is based on microencapsulated mammalian cells which produce the required therapeutics. These cells are safely sealed in the alginate microcapsules, forming an immune-privileged environment for the therapeutic cells, and implanted into the tissue such as e.g. the eye or placenta. The semi-permeable capsule allows free transport of nutrients, signalling molecules, and produced protein therapeutics, while it prevents immune cells from reaching and destroying the implanted therapeutic cells. Therapeutic cells therefore do not need to be immuno-compatible for each individual patient and can threfore be optimized and mass produced to increase their efficiency and affordability of this therapy.

We designed our device to leave no trace after the therapy has been completed, by initiating secretion of an alginate-degrading enzyme to break down the microcapsules followed by the apoptosis of therapeutic cells. This approach should increase the safety and decrease the unwanted effects of treatment and makes the surgical removal of microcapsules or fibrotic tissue around microcapsules obsolete.

Figure 1. Schematic representation of alginate microcapsule and its degradation. Microcapsules serve as a semi-permeable membrane, allowing exchange of therapeutic and inducer molecules as well as nutrients and metabolites between encapsulated cells and the environment. On the other hand the microcapsule prevents the immune cells and immunoglobulin complexes to access the engineered cells while at the same time preventing uncontrolled dissemination of therapeutic cells throughout the body.

Alginate microcapsules

Alginate is the most widely used and clinicaly tested biomaterial for cell encapsulation. It is found in cell walls of brown seaweed. This polysaccharide is made of (1, 4)-linked monomeric units of β-D-mannuronate (M) and α-L-guluronate (G) (Figure 2). Consecutive M or G residues form so called M or G-blocks, whereas the alternating M and G units constitute MG-blocks of alginate (Duan et al., 2009).

Figure 2. Structure of alginate polymer. α-L-guluronate sugar residues is shown on the left and β-D-mannuronate on the right side. Source: http://en.wikipedia.org/wiki/Alginate

The host immune system imposes a great threat on our therapeutic cells as the human defence system could quickly locate and destroy our drug-producing cells since they lack the signatures of the endogenous cells. Alginate microcapsules are an almost ideal solution for this problem: the encapsulated cells are protected against the cytotoxic cells while the material used is biocompatible and allows exchange of smaller molecules, including proteins (Figure 1). It has been demonstrated that encapsulated cells remain viable for several months, therefore many therapeutic programs can be accomplished within this time frame. This sequestration technology also prevents the uncontrolled dissemination of our device throughout the patient's body (Ausländer et al., 2011).

Cell encapsulation is technically achieved by dropping a mixture of cells and liquid alginate into a calcium chloride solution which solidifies the droplets, transforming them into the hydrogel beads whose size in our system can be adjusted within the range of 300-500 μm in diameter. These beads are then coated with a polycation such as poly(L-lysine) (PLL) to form a membrane which further decreases the porosity of the wall of capsules. In the next step of cell encapsulation, an outer alginate layer is applied. This alginate coat also minimizes the attachment of patient's cells to the microcapsules due to the net negative charge of its surface (Vos et al., 2006). To allow cell growth and division within the alginate beads, the core of microcapsules is depolymerized with sodium citrate.

Figure 3. Büchi Encapsulator B-395 Pro was used for cell encapsulation.

References

Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., Spies, T. (1999) Activation of NK Cells and T Cells by NKG2D, a receptor for Stress-Inducible MICA. Science 285, 727-729.

Borrego, F., Kabat, J., Kim, D.K., Lieto, L., Maasho, K., Peña, J., Solana, R., Coligan J.E. (2001) Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol. Immunol. 38, 637-660.

Groh, V., Rhinehart, R., Secrist, H., Bauer., S., Grabstein, K.H., Spies, T. (1999) Broad tumor-associated expression and recognition by tumor-derived gd T cells of MICA and MICB. Proc. Natl. Acad. Sci. 96, 6879–6884.

Salih, H.R., Rammensee, H.G., Steinle, A. (2002) Cutting Edge: Down-Regulation of MICA on Human Tumors by Proteolytic Shedding. J Immunol. 169, 4098-4102.

Stenile, A., Li, P., Morris, D.L., Groh, V., Lanier, L.L., Strong, R.K., Spies, T. (2001) Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. Immunogenet. 53, 279-287.