Progress Report
Creating A Society Whose Citizen’s Health is Monitored by Remote Control of Intracellular Cybernetic Avatars[3] Loading CAs into cells
Progress until FY2024
1. Outline of the project
In this R&D Item, three types of loading technologies have been developed to produce various intracellular CA-loaded cells. To achieve effective loading of varied CAs designed and produce in the R&D item “Intracellular CA Designs” into cells selected in the R&D Item “Evaluating remote control of intracellular CA in vivo”, we also need to design loading methods based on characteristics of both CAs and cells. In terms of different intra/intercellular interactions in the loading processes, there are three types of loading technologies.
In (3-1), a loading technology based on physical stimulations of cells has been developed. To provide moderate stimulations onto cells, electrical and mechanical stimulations are employed in 3-1, and the loading system called “electro-mechanical poration” has been developed.
In (3-2), a loading technology utilizing biocompatible nanoparticles composed of lipid molecules has been developed. To achieve low invasive and non-viral loading of CAs into various cells in vitro and in vivo, appropriate nanoparticles have been designed based on cellular uptake preferences.
In (3-3), a loading technology utilizing fusion of cells has been developed. To load large CAs encoding amount of information into cells with less processes, (3-3) has employed fusion techniques of cells.
2. Outcome so far
- (3-1)
- By utilizing the constructed electro-mechanical poration system, production of gene-based CA loaded immune cells were achieved (Figure 1). In addition, to achieve spatially controlled loading of CAs onto tissues, controlled poration on a commercialized tissue were demonstrated.
- (3-2)
- By utilizing the appropriately designed nanoparticles for immune cells, production of several gene-based CAs loaded immune cells were achieved (Figure 2). In addition, remotely controlled molecular expressions were observed by using the produced CA-loaded cells.
- (3-3)
- Successful fusions of cells and transporting of model molecules were demonstrated (Figure 3). In addition, key insights to control removing process of excess nucleus in the fused cells were founded.
3. Future plans
We will selectively apply three loading technologies not only to established cell lines but also to primary cells. In addition, as a CA loading technology for in vivo use, we will develop loading technologies allowing selective loading of CAs that work as markers for precise inspection into/onto target cells in (3-1) and (3-2).
The remote controllability and other biological functions of CA-loaded cells will be confirmed by image analysis, measurement methods such as flow cytometers, RNA sequencing, etc. Based on detailed evaluations of loading efficiencies of CAs and the cellular functions after the loading process, we aim to construct a more reliable and safe loading systems.
By simultaneously developing several CA loading technologies, we aim to construct a CA-loaded cells production platform that allows selective utilization of suitable CA loading technologies for various cells.