Controlled Delivery of Human Cells by Temperature Responsive Microcapsules

Abstract

Cell therapy is one of the most promising areas within regenerative medicine. However, its full potential is limited by the rapid loss of introduced therapeutic cells before their full effects can be exploited, due in part to anoikis, and in part to the adverse environments often found within the pathologic tissues that the cells have been grafted into. Encapsulation of individual cells has been proposed as a means of increasing cell viability. In this study, we developed a facile, high throughput method for creating temperature responsive microcapsules comprising agarose, gelatin and fibrinogen for delivery and subsequent controlled release of cells. We verified the hypothesis that composite capsules combining agarose and gelatin, which possess different phase transition temperatures from solid to liquid, facilitated the destabilization of the capsules for cell release. Cell encapsulation and controlled release was demonstrated using human fibroblasts as model cells, as well as a therapeutically relevant cell line-human umbilical vein endothelial cells (HUVECs). While such temperature responsive cell microcapsules promise effective, controlled release of potential therapeutic cells at physiological temperatures, further work will be needed to augment the composition of the microcapsules and optimize the numbers of cells per capsule prior to clinical evaluation.

Keywords: cell delivery; cell encapsulation; human fibroblast; human umbilical vein endothelial cells; hydrogel; microcapsules; temperature responsive.

Figure 1

Schematic diagram illustrating (A) the concept of cell delivery and release with temperature responsive microcapsules and (B) the hypothesized mechanism of cell release from a composite temperature responsive hydrogel comprising two materials with different phase transition temperatures.

Figure 2

Light micrographs showing cell capsules prepared with various initial cell concentrations (A) 2,000,000, (B) 4,000,000 and (C) 8,000,000 cells·mL⁻¹, respectively. (D) Size distribution of capsules prepared with various hydrogel formulation. (E) Cell number(s) per single capsule (as a function of various initial cell concentrations).

Figure 3

The relatively cell viability of (A) encapsulated human fibroblast cells and (B) encapsulated HUVECs over time, within the agarose–gelatin–fibrinogen microcapsules. Time points with a connecting line over their bars were not found to be significantly different. Bars not connected by lines were found to be significantly different (GLM using Tukey post hoc, p ≤ 0.05).

Figure 4

Decomposition kinetics of hydrogel microcapsules measured by release of gelatin (A) at 37 °C and (B) control at 4 °C as a function of time. (C,D) Optical images showing the decomposition and release of fluorescent-labeled gelatin into the suspended PBS solution, causing an increase of background fluorescence intensity at 37 °C, but not at 4 °C.

Figure 5

Optical micrographs showing the release of human fibroblast cells onto collagen coated tissue culture dishes from microcapsules composed of (A) agarose–gelatin–fibrinogen, (B) agarose only negative controls and (C) non-encapsulated fibroblast controls. (D) Sequence of images (i) to (v) with duration of ~1.5 h showing the release of a human fibroblast cell from an agarose–gelatin–fibrinogen capsule onto the culture substratum.

Figure 6

Kinetics of cell release from temperature responsive microcapsules comprising 1% low melting agarose, 0.1% to 0.5% gelatin, and 10 mg·mL⁻¹ fibrinogen, showing that 28% of cells were released within 24 h, and 70% cells were released by 48 h.

Figure 7

(A) Micrographs of HUVECs within agarose–gelatin–fibrinogen microcapsules at Day 0 and after their release. On Day 1, released HUVECs are seen attached and spread after their release onto gelatin-coated tissue culture plastic. By Day 2, several cells have migrated and have aligned themselves into a cord-like structure that is typical of HUVEC in vitro tubulogenesis behavior [27]. (B) Non-encapsulated HUVEVs control.

Figure 8

Schematic diagram illustrating the high throughput preparation of cell microcapsules using the Nisco VAR J30 System.