Investigating Cancerous Exosomes' Effects on CD8+ T-Cell IL-2 Production in a 3D Unidirectional Flow Bioreactor Using 3D Printed, RGD-Functionalized PLLA Scaffolds

Daniel Karami; Akhil Srivastava; Rajagopal Ramesh; Vassilios I Sikavitsas

doi:10.3390/jfb13010030

Investigating Cancerous Exosomes' Effects on CD8+ T-Cell IL-2 Production in a 3D Unidirectional Flow Bioreactor Using 3D Printed, RGD-Functionalized PLLA Scaffolds

J Funct Biomater. 2022 Mar 11;13(1):30. doi: 10.3390/jfb13010030.

Authors

Daniel Karami^{1

2}, Akhil Srivastava^{3

4}, Rajagopal Ramesh^{3

4}, Vassilios I Sikavitsas^{1

4}

Affiliations

¹ School of Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, OK 73019, USA.
² Stephenson School of Biomedical Engineering, University of Oklahoma, Norman, OK 73019, USA.
³ Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
⁴ Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

Abstract

Exosomes from cancer cells are implicated in cancer progression and metastasis, carrying immunosuppressive factors that limit the antitumor abilities of immune cells. The development of a real-time, 3D cell/scaffold construct flow perfusion system has been explored as a novel tool in the study of T-cells and exosomes from cancer cells. Exosomes from human lung cancer (H1299 and A549) cells were co-cultured in a unidirectional flow bioreactor with CD8+ T-cells immobilized onto 3D-printed RGD-functionalized poly(L-lactic) acid (PLLA) scaffolds and assessed for IL-2 production. The IL-2 production was investigated for a wide range of T-cell to exosome ratios. With the successful incorporation of the RGD binding motif onto the PLLA surface at controllable densities, CD8+ T-cells were successfully attached onto 2D disks and 3D printed porous PLLA scaffolds. T-cell attachment increased with increasing RGD surface density. The diameter of the attached T-cells was 7.2 ± 0.2 µm for RGD densities below 0.5 nmoles/mm² but dropped to 5.1 ± 0.3 µm when the RGD density was 2 nmoles/mm² due to overcrowding. The higher the number of cancer exosomes, the less the IL-2 production by the surface-attached T-cells. In 2D disks, the IL-2 production was silenced for T-cell to exosome ratios higher than 1:10 in static conditions. IL-2 production silencing in static 3D porous scaffolds required ratios higher than 1:20. The incorporation of flow resulted in moderate to significant T-cell detachment. The portions of T-cells retained on the 3D scaffolds after exposure for 4 h to 0.15 or 1.5 mL/min of perfusion flow were 89 ± 11% and 30 ± 8%, respectively. On 3D scaffolds and in the presence of flow at 0.15 ml/min, both H1299 and A549 cancerous exosomes significantly suppressed IL-2 production for T-cell to exosome ratios of 1:1000. The much higher level of exosomes needed to silence the IL-2 production from T-cells cultured under unidirectional flow, compared to static conditions, denotes the importance of the culturing conditions and the hydrodynamic environment, on the interactions between CD8+ T-cells and cancer exosomes.

Keywords: bioreactor; cancer; exosome; fluid flow; surface modification.

Abstract

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