Mechanoporation enables rapid and efficient radiolabeling of stem cells for PET imaging

Abstract

Regenerative medicine uses the patient own stem cells to regenerate damaged tissues. Molecular imaging techniques are commonly used to image the transplanted cells, either right after surgery or at a later time. However, few techniques are fast or straightforward enough to label cells intraoperatively. Adipose tissue-derived stem cells (ADSCs) were harvested from knee joints of minipigs. The cells were labeled with PET contrast agent by flowing mechanoporation using a microfluidic device. While flowing through a series of microchannels, cells are compressed repeatedly by micro-ridges, which open transient pores in their membranes and induce convective transport, intended to facilitate the transport of ⁶⁸Ga-labeled and lipid-coated mesoporous nanoparticles (MSNs) into the cells. This process enables cells to be labeled in a matter of seconds. Cells labeled with this approach were then implanted into cartilage defects, and the implant was imaged using positron emission tomography (PET) post-surgery. The microfluidic device can efficiently label millions of cells with ⁶⁸Ga-labeled MSNs in as little as 15 min. The method achieved labeling efficiency greater than 5 Bq/cell on average, comparable to 30 min-long passive co-incubation with ⁶⁸Ga-MSNs, but with improved biocompatibility due to the reduced exposure to ionizing radiation. Labeling time could also be accelerated by increasing throughput through more parallel channels. Finally, as a proof of concept, ADSCs were labeled with ⁶⁸Ga-MSNs and quantitatively assessed using clinical PET/MR in a mock transplant operation in pig knee joints. MSN-assisted mechanoporation is a rapid, effective and straightforward approach to label cells with ⁶⁸Ga. Given its high efficiency, this labeling method can be used to track small cells populations without significant effects on viability. The system is applicable to a variety of cell tracking studies for cancer therapy, regenerative therapy, and immunotherapy.

Figure 1

Experimental scheme. Adipose-derived stem cells (ADSCs) were harvested from knee joints of Goettingen minipigs. For PET imaging, ADSCs were labeled by radiolabeled MSNs with ⁶⁸Ga. Through a microfluidic device, approximately 40% compression of ADSCs was achieved to promote transport of radiolabeled MSNs into cells. Finally, the engraftment of labeled cell in knee joints was imaged using PET.

Figure 2

Demonstration of cell labeling using mechanoporation. (a) TEM showing spherical morphology of MSNs. (b) Fluorescence microscopy showing labeling of ADSCs by FITC-MSNs inside mechanoporation device (green:FITC; blue:nucleus; red: cell membrane). (c) FITC-MSNs were efficiently transported into ADSCs. (d) FACS analysis showing that labeled cells have higher green fluorescence than unlabeled cells. (e) CCK viability found no significant difference between unlabeled and labeled cells. (f) TEM images showing cellular uptake of MSNs in ADSCs after passive incubation (60 min) and microfluidic mechanoporation (0.5 ml/min).

Figure 3

Cell radiolabeling using mechanoporation device. (a) Thin-layer chromatography showing efficient ⁶⁸Ga-labeling of MSNs. (b) PET images showing comparing cell labeling efficiency for passive transport (10 min, 30 min, and 60 min incubation) and microfluidic mechanoporation and (c) region-of-interest quantification of the images. (d) Gamma counting of the cell samples.

Figure 4

Cell biocompatibility assays. (a) γH2AX staining showing DNA damage (double strand breaks) in labeled ADSCs after 30 min, 60 min passive incubation or microfluidic device. (b) Ki-67 staining showed no significant difference in proliferation between control, unlabeled and labeled cells.

Figure 5

Ex vivo PET/MRI imaging of ADSCs implantation in pig knee. (a) MR image showing significant signal loss near labeled ADSC implant (red arrows) compared to unlabeled implant (white arrows). (b) Clear PET signal is also observed near the labeled ADSCs (red arrows) but not for the unlabeled implant (white arrows). (c) PET quantification showing significantly higher radioactivity in the labeled implant. (d) Fused PET/MR images showed co-localization of PET and MR signals (red arrows).