Development of a Robot-Aided Micromanipulation System for Single-Cell Mitochondria Biopsy and Transfer

可用於單細胞線粒體活檢及轉移的機器人輔助微操作系統研究

Student thesis: Doctoral Thesis

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Award date9 Jan 2019

Abstract

High-precision micromanipulation systems and tools that can manipulate and interrogate cell organelles and components have potential applications in clinical settings and therapy development. Although various micromanipulation systems have been developed, some tasks remain challenging. Microneedles and optical tweezers are powerful tools for micromanipulation tasks, such as manipulation or removal of subcellular components or/and organelles from a single cell. In this thesis, several novel approaches based on microneedles and optical tweezers are developed and investigated for their efficiency in minimally invasive single-cell surgery, such as organelle extraction and/or biopsy and organelle transfer. This thesis consists of the following main parts.

First, a robotics surgical system for automated single-cell organelle biopsy and transfer in small cells (<20 µm in diameter) is developed. The system can perform completely automated single-cell biopsy tasks, including cell positioning, organelle detection, aspiration, and organelle release or transfer, at a specific location or inside a cell. A microfluidic single-cell patterning device that can compress and pattern cells inside channels in a 1D array is designed to simplify the automated process while achieving high organelle positioning accuracy. Under a visual-based robust control algorithm, the system can be applied to extract an organelle and transfer it in a broad range of cells with high manipulation accuracy while addressing model uncertainties and external disturbances.

Second, the developed robot-aided microneedle-based manipulation system is applied in the automated extraction of mitochondria and nucleus from human acute promyelocytic leukemia and human fibroblast cells. A microfluidic device is designed to pattern individual cells in a 1D array. A micropipette mounted onto a 3-DOF micromanipulator and an X-Y stage is developed to perform high-precision single-cell mitochondrial biopsy. Biological tests are conducted to indicate the functionality of the biopsied mitochondria and the viability of the cells after the removal of mitochondria. The developed system is further used for the automatic extraction of mitochondria from an A-type cell followed by injection of the extracted mitochondria into a B-type cell. The invasiveness and efficiency of the proposed single-cell mitochondrial transfer approach are investigated.

Third, an optical tweezer-based robot-aided system is developed for high-throughput mitochondrial transfer in single cells. A microfluidic device is developed to pattern cells and mitochondria in a parallel way, and then optical tweezers are used to pick the required number of functional mitochondria. The trapped mitochondria are automatically transported onto the surface of the cell and then transferred inside the cell by endocytosis. In contrast to other mitochondrial transfer methods, the proposed system can control the quality and quantity of mitochondria prior to the mitochondrial transfer. Biological tests are conducted to examine the viability of the transferred cells and the functionality of the mitochondria before and after transfer. The condition of the cell after the transfer of functional and healthy mitochondria by the proposed system is also analyzed and compared with those obtained using a simple co-culture mitochondrial transfer method.

In summary, robotic micromanipulation systems based on microneedles and optical tweezers are developed to simplify single-cell organelle manipulation and transfer. Such high-precision robotic systems can help reduce variable outcomes, human error, and operator fatigue and improve precision, efficiency, and repeatability in complex single-cell surgical tasks. The novel methodologies introduced in this thesis deliver new concepts of manipulation and analysis of single cells and their organelles. Thus, these methods have potential therapeutic applications in medicine.