Quality and Quantity Control of Mitochondria Injection Into Single Cells With Robot-Aided Micro-Manipulation System

Mitochondrial dysfunction plays a significant role in the development of fatal diseases such as aging, cancer, and Alzheimer's. Transferring mitochondria to cells is a new and potential treatment for mitochondrial DNA (mtDNA)-related illnesses. This article describes a novel technique to control the quality and quantity of mitochondria injected into single live cells using a robot-aided microneedle and optical tweezers (OTs)-based micromanipulation system. Isolated mitochondria and cells are patterned in a 1-dimensional (1-D) array in a microfluidic device, and a robot-aided microneedle collects the predefined number of functional mitochondria with the help of OTs. Then, the microneedle precisely and non-invasively injects these collected mitochondria into single live cells. Given that the two manipulation tools of OTs and microneedle were used, a switch controller strategy is developed to enable mitochondria trapping and injection with OTs and microneedle-based micromanipulator, respectively. The effectiveness of the developed robotic system is experimentally demonstrated with automated injections of isolated mitochondria into HeLa and mesenchymal stem cells (aMSCs). A precise and efficient quality and quantity control of mitochondria injection was possible by using Ots in conjunction with microneedle and microfluidics technologies. Quality- and quantity-controlled ability is analyzed and compared with the traditional mitochondria transfer method (co-culture). Biological tests are further conducted to assess the viability of mitochondria recipient cells. Experimental results demonstrate that the developed system can non-invasively transfer healthy mitochondria into single live cells while precisely controlling the quantity and quality of injected mitochondria. The proposed mitochondrial transfer method has the potential to advance precision medicine methods, particularly for cellular therapy of (mtDNA)-related diseases. Note to Practitioners-This study was motivated by the problem of inefficient methods of mitochondria injection into single, small, living cells. Due to the issue of microneedle clogging, existing mitochondria injection methods can only inject mitochondria into cells with a 0.3 to 2% efficiency. To address this issue, we have developed an automated method of mitochondria injection into cells that can inject mitochondria into single cells non-invasively with an efficiency of up to 80 %. Existing methods of mitochondria injection also ignored