Lumina imager

A 1:10 PNP hydrogel was used according to published methods.²⁰ (link) Rheology measurements were performed on a DHR‐2 rheometer (TA Instruments, New Castle, DE) using a 20-mm serrated plate geometry with a gap of 600 µm. Flow sweeps were performed at shear rates from 0.01 to 100 s⁻¹. Frequency sweeps were performed at a constant strain of 1% from 0.1 to 100 rad/s. dibenzocyclooctyne group (DBCO)-functionalized Alexa Fluor 647 dye (DBCO-AF647; Jena Bioscience, Jena, Germany) was conjugated to azide-functionalized PEG–PLA nanoparticles through a copper-free click reaction. A 1.5× molar excess of dye to nanoparticle was added to the nanoparticle solution overnight at room temperature. The nanoparticles were then concentrated through centrifugation to remove any excess dye. To prepare the fluorescent PNP hydrogel, 50% dye-functionalized PEG–PLA nanoparticles were used in combination with 50% non-functionalized PEG–PLA nanoparticles. An in vitro imaging system, the Lumina Imager (PerkinElmer, Waltham, MA), was used to image fluorescent PNP hydrogel retention in the mouse eye. An exposure time of 0.1 second was used to quantify PNP hydrogel retention as the total flux of photons in the region of interest over time.

Cytolytic activity was assessed by co-culturing 1E4 CAR-modified or non-CAR-modified control T cells (mock) and GFP-labeled target cells at an effector-to-target ratio of 1:1 for 24 hours before analyzing the frequency of residual viable target cells by flow cytometry. Cytokine production was analyzed from the same co-culture by harvesting 100 µL supernatant from the 24 hours co-culture and quantification using the human MACSPlex Cytokine 12 Kit (Miltenyi Biotec) according to the manufacturer’s instructions.
All in vivo studies were approved by the Institutional Animal Care and Use Committee of the University of Würzburg. In brief, NSG mice (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ, Charles River) were inoculated with 1E6 firefly luciferase expressing Raji tumor cells by intravenous (i.v.) injection, and 7 days later treated with 4.7E6 T cells from the final cell product (from run #3, containing 2E6 CAR T cells) or 4.7E6 mock T cells (also i.v.). Serial bioluminescence imaging on an in vivo imaging system Lumina imager (Perkin Elmer) was performed to assess tumor burden and distribution.