Cells encapsulated in alginate: a potential system for delivery of recombinant proteins to malignant brain tumours

Abstract

Growth and progression of malignant brain tumours occurs in a micromilieu consisting of both tumour and normal cells. Several proteins have been identified with the potential of interfering directly with tumour cells or with the neovascularisation process, thereby inhibiting tumour growth. A continuous delivery of such inhibitory proteins to the tumour microenvironment by genetically engineered cells could theoretically be of considerable therapeutic importance. In this study we have investigated the growth characteristics of cells encapsulated in alginate, which represents a potential delivery system for recombinant proteins that may have antitumour effects. Three different cell lines, NHI 3T3, 293 and BT4C were encapsulated in alginate, which is an immuno-isolating substance extracted from brown seaweed. The encapsulated cells were observed at specific intervals during a 4-month period after in vitro propagation and as transplants into the cortex of BD-IX rats. Morphological studies showed that encapsulated cells proliferated and formed spheroids within the alginate in the in vitro cultures and after implantation into the brain. Even after 4 months in vivo a substantial amount of living cells were observed within the alginate beads. A vigorous infiltration of mononuclear cells was observed in the brain bordering the alginate beads, one week after implantation. However, there was a gradual decrease of mononuclear cells at the border zone beyond the first week of implantation. The majority of inflammatory cells were reactive microglia and invading monocytes, as verified by immunohistochemistry. The data further shows that alginate encapsulated cells can be frozen in liquid N2 and will retain their viability and proliferative capacity.

Macrophages promote angiogenesis in human breast tumour spheroids in vivo

“…A number of studies have used alginate, derived from brown seaweed, as a means of encapsulating cells for in vivo implantation, thereby isolating them from the host immune cells. Previous studies demonstrated the coating has no effect on cellular growth or proliferation, and indicated that in the first week after implantation in vivo, mononuclear cells migrated to the rim of the alginate spheroid but there was no penetration, and over the following nine weeks, the number of migrating cells gradually decreased (Read et al, 1999(Read et al, , 2001. The permeability to diffusible molecules was demonstrated by Hoescht staining of tumour cells, following addition to the culture medium, and the release of VEGF but not cells into the well suggesting that coating allowed the passage of molecules but not cells into and out of the spheroids.…”

“…At the end of each experiment all tissue from the chamber was processed and stained, and demonstrated that the tumour cells remained viable, that macrophages remained within the spheroid throughout the experimental period and that host macrophages had not permeated the alginate coating. The size of each spheroid was not measured at the end of the experiments, but previous studies have shown that it is possible for cell growth to occur within the confines of the agarose coating (Read et al, 1999(Read et al, , 2001). …”

“…Spheroids were cultured for 14 -18 days and human monocytes (10 5 spheroid À1 well À1 ) were added and allowed to infiltrate the spheroids for 24 h. In order to prevent autologous (i.e. murine) macrophages from infiltrating the spheroid following implantation in vivo, the spheroids were coated with alginate (Read et al, 1999(Read et al, , 2001) by immersion in a 1.25% solution of sodium alginate (Pronova, UK) and transfer to a 0.1 M CaCl 2 solution, prepared from a 0.15 M NaCl stock solution, to initiate gel-formation by cross-linking. The alginate coated spheroids were washed three times with PBS and once in RPMI with 10% FCS (Read et al, 1999(Read et al, , 2001.…”

“…murine) macrophages from infiltrating the spheroid following implantation in vivo, the spheroids were coated with alginate (Read et al, 1999(Read et al, , 2001) by immersion in a 1.25% solution of sodium alginate (Pronova, UK) and transfer to a 0.1 M CaCl 2 solution, prepared from a 0.15 M NaCl stock solution, to initiate gel-formation by cross-linking. The alginate coated spheroids were washed three times with PBS and once in RPMI with 10% FCS (Read et al, 1999(Read et al, , 2001. The coated spheroids were cultured for a further 5 days to allow differentiation of monocytes to macrophages.…”

Inhibition of heparanase activity and heparanase‐induced angiogenesis by suramin analogues

“…32 Briefly, HSPG-bFGF exposed to heparanase in presence or absence of suramin analogues was mixed with alginate gel (1.8%). The mixture was used to prepare beads, at a volume of 10 ml, by dropping them into a hardening solution of 1.5% CaCl 2 .…”