Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

Abstract

Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

The effect of electrostatic microencapsulation process on biological properties of tumour cells

“…Almost all the encapsulated cells (93-98%) were viable after encapsulation, which was similar to the other groups ( Figure 3A). The results were consistent with previous studies (Maguire et al, 2006;Thakur et al, 2010). There were no significant differences among all groups during the progressive stages of apoptosis ( Figure 3B and C).…”

“…In combination with the annexin V assay, our results indicated that stimuli from encapsulation process did not destroy ER homeostasis. Moreover, not only tumour cells but also normal cells (Thakur et al, 2010), even stem cells (Maguire et al, 2006), maintained viability when cultured within microencapsulate by electrostatic droplet technology. Overall, encapsulation process might be universal and friendly to the mammalian cells.…”

“…It is clear that several key non-physiological factors, including suspension status, nutrition-free condition, the high-voltage electrostatic field and shear forces, exist in the electrostatic microencapsulation process. Although previous studies have proven that most of the encapsulated cells are alive and retain their proliferation ability after the electrostatic microencapsulation process (Maguire et al, 2006;Thakur et al, 2010), it is still unknown whether the microencapsulated process influences the biological properties of tumour cells because tumour cells undergo significant environmental changes during this process. Studying the change in cellular biological properties during this process will help us to optimise the preparation conditions of microencapsulation.…”

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

“…Our method of analysis defines dead cells as those which have a compromised cell membrane. An apoptotic/ necrotic dead cell with a compromised cell membrane is measured by the loss calcein intracellular staining and the change in its size and granularity, while (Bertho et al 2000;Thakur et al 2010). This is inherently more sensitive than relying on the ability of a second dye such as 7-AAD or ethidium bromide to label cells with a damaged cell membrane.…”

Programmed cell delivery from biodegradable microcapsules for tissue repair

“…[27] The most widely studied alginate-based formulations involve complexation with polycations, poly-L-lysine and chitosan in particular, to control permeability and increase strength and stability. [28][29][30] Nevertheless, other, different alginate-based formulations have been examined over the years to further improve the effectiveness of cell encapsulation, including cross-linked alginate-gelatine, [31] alginate-poly(ethylene glycol), [32] or alginate and hyaluronic acid stabilized with polyethyleneimine. [33] Together with alginate, different biopolymers capable of gelling under mild conditions have attracted attention as cell carriers.…”