Feb 13, 2026
The establishment of an in vitro immunotherapy model based on MTSs
- Guangli Suo1,
- Zhe Zhao1,
- Mengdie Yang1,
- Xuan Xiong1
- 1CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu, 215123, China.
- MTS
Protocol Citation: Guangli Suo, Zhe Zhao, Mengdie Yang, Xuan Xiong 2026. The establishment of an in vitro immunotherapy model based on MTSs. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1kxnygr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 13, 2026
Last Modified: February 13, 2026
Protocol Integer ID: 243169
Keywords: immunotherapy model, characterization of human pbmc, mtss isolation, human pbmc, mt
Funders Acknowledgements:
National Natural Science Foundation of China
Grant ID: 32470814
Abstract
Isolation and characterization of human PBMCs or CD8+ T lymphocytes, followed by their co-culture with MTS in a MWC-chip.
Troubleshooting
The isolation of PBMC
The peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor’s whole blood sample using Ficoll-Paque density gradient medium (GE Healthcare) according to the manufacturer’s instructions. Briefly, the whole blood was diluted with DPBS and layered onto Ficoll-Paque medium, followed by centrifugation at 400 g for 30 minutes. After centrifugation, the mononuclear cells located at the interface were transferred to a new tube, diluted with DPBS, and pelleted at 400 g for 10 minutes. The cells were washed once with DPBS before downstream applications. Human cytotoxic T lymphocytes (CTLs) were isolated from PBMCs using the EasySep Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies) following the manufacturer’s instructions. Briefly, PBMCs derived from BRC patients were incubated with 50 μL/mL of Enrichment Cocktail for 10 minutes at room temperature. Subsequently, the sample was incubated with magnetic particles at a concentration of 150 μL/mL for 5 minutes at room temperature. The sample was then diluted with EasySep Buffer (STEMCELL Technologies) and subjected to cell separation using an EasySep Magnet (STEMCELL Technologies). CTLs, or CD8+ T cells were recovered by transferring the cell suspension into a new tube and centrifuging at 300 g for 5 minutes, and then cultured in T cell medium (STEMCELL Technologies).
The establishment of the co-culture system with MTSs and PBMC
To establish the co-culture system with MTSs and PBMC, the collected MTSs were seeded into a new MWC-chip, then PBMC was stained with DID and seeded in the same MWC-chip at the ratio of PBMC /
MTSs = 5 / 1. After that, 1.5 mL SG-Medium mixed with Annexin V-FITC (Vazyme) was added in the co-cultural system and maintained at 37°C in 5% CO2 for 48 hours and imaged by Confocal (OLYMPUS). To establish the co-culture system with MTSs and CTLs (CD8+ T Cell), the sorted MTSs and CTLs were mixed with the ratio of CTLs / MTSs = 5 / 1 and seeded in a new MWC-chip. Then, 1.5 mL SG-Medium mixed with Annexin V-FITC was added in the co-cultural system and maintained at 37°C in 5% CO2 for 48 hours and imaged by Confocal (OLYMPUS). Before assessing the anti-cancer efficacy of Nivolumab (PD-1 antibody, an immune checkpoint inhibitor) on MTSs, PD-L1 expression in MTSs was assessed by staining them with a PD-L1 antibody on a MWC-chip. In the CTLs + MTSs co-culture system for immune cell potency assay, Nivolumab and annexin V-FITC were added to the medium, followed by incubation at 37°C in 5% CO2 for 48 hours. Confocal imaging (OLYMPUS) was performed to visualize the results. Control groups included MWC-chips seeded with MTSs or MTS + CTLs without Nivolumab treatment.