Microencapsulated cell tracking

Abstract

Microencapsulation of therapeutic cells has been widely pursued to achieve cellular immunoprotection following transplantation. Initial clinical studies have shown the potential of microencapsulation using semi-permeable alginate layers, but much needs to be learned about the optimal delivery route, in vivo pattern of engraftment, and microcapsule stability over time. In parallel with noninvasive imaging techniques for 'naked' (i.e. unencapsulated) cell tracking, microcapsules have now been endowed with contrast agents that can be visualized by (1) H MRI, (19) F MRI, X-ray/computed tomography and ultrasound imaging. By placing the contrast agent formulation in the extracellular space of the hydrogel, large amounts of contrast agents can be incorporated with negligible toxicity. This has led to a new generation of imaging biomaterials that can render cells visible with multiple imaging modalities.

Figure 1

Schematic illustration of general microcapsule synthesis. Cells (a) and contrast agents (b) are suspended in low-viscosity guluronate (LVG) alginate solution inside a syringe (c) in step 1. The syringe is mounted on a syringe pump (d) and connected to a high-voltage power supply (e). The gelation bath (f) is connected to the ground. Microcapsules are produced by an electrostatic droplet generator and gelated in the cationic bath in step 2 (g). (h) Schematic drawing showing microencapsulation of cells and contrast agents. The first alginate (LVG) layer of the microcapsule is cross-linked with protamine sulfate (PS) or poly-l-lysine (PLL) as polycationic polymer (i, red) in step 3, and is further cross-linked with low-viscosity mannuronate (LVM) alginate (j, blue) in step 4.

Figure 2

Macroscopic (a, c) and fluoroscopic (b, d) images of bismuth X-caps (a, b) and barium X-caps (c, d). Single capsules can be clearly identified. Reproduced, with permission, from ref. (38).

Figure 3

Post-treatment angiographic analysis of collateral vessel development. (a) Bar graph of the average modified thrombolysis in myocardial infarction (TIMI) frame count (a measure of collateral vessel development) for the mesenchymal stem cell (MSC)–X-cap, empty X-cap, unencapsulated MSC and sham injection-treated animals demonstrates a significant improvement in distal filling only in rabbits with peripheral arterial disease that received microencapsulated cells. (b–g) Representative digital subtraction angiograms (DSA, red) obtained during peak contrast opacification performed at 2 weeks post-injection of encapsulated MSC–X-caps (b) and empty microcapsules (c) with an overlay of microcapsule injections (green) obtained from the mask image of DSA. An increased collateralization can be appreciated in the MSC–X-cap-treated animal DSA (d) relative to the X-cap-treated animal DSA (e). Native mask digital radiographs demonstrate the location of the MSC–X-caps (f) and empty X-caps (g) in the same animals. (h) Box-whisker plot showing the difference between left and right distal deep femoral artery diameters at baseline and 2 weeks after superficial femoral artery occlusion in treated (MSC–X-caps) and untreated (empty X-caps) animals. There was no statistically significant difference in vessel diameter between the treatment groups. Reproduced, with permission, from ref. (23).

Figure 4

Micro-computed tomography (CT) images of 100 phantom barium microcapsules in saline 1 day (a) and 15 months (b) post-synthesis. These capsules are intrinsically radio-opaque, whereas alginate–protamine sulfate–alginate (APSA) capsules gelated with calcium ions (c) and alginate–poly-l-lysine–alginate (APLLA) capsules (d, each with 100 capsules per tube) are not visible under CT imaging. Axial (e), sagittal (f) and three-dimensional volume-rendered (g) micro-CT images of 3000–4000 barium capsules transplanted subcutaneously in mouse abdomen. Yellow arrows, bladder; white arrows, capsules. Reproduced, with permission, from ref. (37).

Figure 5

(a) Using conventional T₂*-weighted images, individual magnetocapsules can be easily identified as hypointensities. (b) Using the inverse recovery with on resonance (IRON) sequence to generate positive contrast, individual magnetocapsules appear as a bright signal with depiction of the capsule surface. (c, d) Magnetocapsules before (c) and after (d) rupture using glass bead treatment. After rupture, a significant loss of hypointensity occurs and the Feridex^®-induced contrast decreases to a pinpoint double-dipole T₂* susceptibility effect. (e) MR image of a mouse following injection of 500 magnetocapsules in the peritoneal cavity. Single capsules can be readily identified (arrows). Reproduced, with permission, from ref. (40).

Figure 6

(a) In vitro proton T₂-weighted MRI (left) and corresponding ¹⁹F MRI (right) scans at 11.7 T of perfluoropolyether fluorocapsules in a standard 5-mm glass tube (the capsules were introduced in a 500-µm capillary tube). (b) In vivo ¹⁹F MR images at 9.4 T of a mouse following intraperitoneal transplantation of 2000 perfluoropolyether fluorocapsules. Shown is the overlay of the ¹⁹F image (pseudo-color) on the ¹H image (gray scale). (c) ¹⁹F MR image of perfluoropolyether fluorocapsules obtained using a 3-T clinical system. Left to right: images of gel phantoms containing 1, 2, 5, 10, 15, 25 and 50 capsules. Reproduced, with permission, from ref. (44).

Figure 7

Positive-contrast 9.4-TMR (a) and micro-computed tomography (CT) (b) images of gadolinium–gold (GadoGold) microcapsules (arrow) engrafted subcutaneously into the mouse abdomen. (c, d) Ultrasound (US) images at 40 MHz of the cavity between the abdominal skin and parietal peritoneum of a mouse with (c) and without (d) injected GadoGold microcapsules. Reproduced, with permission, from ref. (36).

Figure 8

Dual shielding of pancreatic islets and multimodal imaging using capsule-in-capsules (CICs). The semi-permeable outer alginate membrane blocks the penetration of immune cells and antibodies, yet allows unhindered diffusion of nutrients, glucose, oxygen and insulin produced by islets. The inner capsule, containing iron oxide and gold nanoparticle contrast agents, which enable concurrent MRI, computed tomography (CT) and ultrasound (US) imaging, prevents direct exposure of nanoparticles to cells. Islets within CIC exhibit improved insulin secretion compared with single capsules that contain the two types of nanoparticles and islets altogether. NP, nanoparticle. Reproduced, with permission, from ref. (39).