Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 ( PKP2 ). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells’ contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction–mediated diseases.

Polar body biopsy has been widely applied in preimplantation genetic diagnosis for assisted reproductive technology. The key step in the polar body biopsy is picking up the polar body from the oocyte/embryo using a micropipette. Unfortunately, the almost transparent appearance of the polar body as well as its dynamic drift when the micropipette approaches it inside the cell makes it a challenging task to pick it up with less cytoplasm loss for the cell. The unnecessary cytoplasm loss in the picking up process of the polar body easily causes damage to the development competence of the cell and may lead to disturbances to the biopsy results of the polar body. This paper proposes a precise robotic picking up method of polar bodies with less cytoplasm loss for biopsy purposes. First, a defocus imaging method is proposed to locate the polar body with an almost transparent appearance. Then, the dynamic drift of the polar body with the micropipette moving inside the cell is modeled online based on force analysis to determine an appropriate trajectory for the micropipette to approach the polar body. Further, an Active Disturbance Rejection Controller (ADRC) is designed to move the micropipette along the desired trajectory to approach the polar body and then aspirate it into the micropipette. The experimental results on porcine oocytes demonstrate that our system is capable of localizing the polar body with a success rate of 95 $\%$ and an average error of 1.12 $\pm$ 0.14 $\mu$ m. Moving along the determined trajectory, the micropipette is capable of approaching the edge of the polar body with an average error of 1.84 $\pm$ 0.31 $\mu$ m (n $=$ 20), which is only 11 $\%$ of the results obtained without dynamic drift estimation of the polar body. With this advantage, our system picks up the polar body with a close 60 $\%$ improvement in success rate (95 $\%$ vs 60 $\%$ ) and only half of the average cytoplasm loss (5 $\%$ vs 10 $\%$ ) in comparison to operation results without dynamic drift estimation. Note to Practitioners —Picking up of polar body from the oocyte/embryo using a micropipette is a vital operation in the polar body biopsy. Precisely picking up the polar body with less cytoplasm is vital to maintaining the developmental competence of the embryo/oocyte and reducing disturbances to biopsy result. This article presented a precise robotic picking up process of polar body. This process introduced defocus imaging method for polar body localization, dynamic drift estimation of polar body, and micropipette trajectory design and motion control by Active Disturbance Rejection Controller (ADRC). Experimental results have demonstrated the efficiency of the proposed robotic picking up process. Application of this process may provide an economical and practical method to carry polar body biopsy for practitioners.

Objective: A robotic fast dual-arm patch clamp system with controllable mechanical stimulation is proposed in this paper for mechanosensitive excitability research of neurons in brain slice. Methods: First, a kinematic model of a dual-arm patch clamp system combined with Monte Carlo method is developed to calculate the workspaces of recording micropipette and stimulation micropipette, and optimize the length of end effector for reducing collision incidences during operation. Then, a quantitative stimulation method to cells using one micropipette is developed based on pressing depth control. Finally, a fast robotic dual-arm patch clamp operation process is proposed based on a three-stage motion control of dual micropipettes to approach target cells and form whole-cell recording with quantitative mechanical stimulation. Results: Experimental results on 50 pyramidal neurons in the primary visual cortex of mouse brain slices demonstrate that this system achieves a threefold throughput with a 37% improvement in the success rate of the contact process and a 42% improvement in the success rate of whole-cell recording in comparison to manual operation. With these advantages, a mechanical stimulation-regulated increase in neuron excitability is observed in primary visual cortex. The experimental results also show that the sodium ion current may be more sensitive to mechanical stimulation than potassium ion current. Conclusion: Our system significantly improves the efficiency of mechanical stimulation induced excitability research of neurons in brain slices. Significance: Our methods have the potential to investigate pathological and pathogenic mechanisms of mechanosensitive ion channel dysfunction-induced diseases in the future.

Cell transportation is a key step in many biological applications. Currently, most cell transportation research relies on microscopic vision feedback, which limits its application at places when a microscopic view is unavailable. For the first time, this paper proposed a robotic microscopic view-free cell transportation method based on pressure variation model inside a self-made bilayer micropipette. The bilayer micropipette, made by inserting a thin micropipette into a thicker one, picks the target cell with the outsider micropipette and holds it with the inner micropipette in cell transportation. To achieve this, the appropriate fluidic forces to pick and place the cell were determined based on force analysis. Then, the appropriate inner diameter of the inner thin micropipette was determined based on the micropipette aspiration model. Further, the pressure variation inside the bilayer micropipette during holding and releasing process were modeled to conduct a robotic microscopic vision-fee cell transportation. Experimental results demonstrated that our system was capable of transporting zebrafish embryos at an average speed of 25 s/cell with a success rate of 90%, with a transportation efficiency comparable to the related methods with microscopic view feedback. Besides, ignorable mechanical harm to the development competence of the zebrafish embryos was confirmed through culturing experiments. Note to Practitioners —Cell transportation is an important step in many biomedical researches. Currently, cell transportation usually relies on microscopy for picking up, and releasing cells, limiting its application in special environments without microscopic view feedback. In this paper, a robotic microscopic vision-free cell transportation method is proposed for the first time. In this method, cells are transported within a self-made bilayer micropipette. Pressure variations inside the micropipette are utilized to determine cell picking up and release. This robotic transport method demonstrated a high success rate and low harm to cells in experiments. Our research may contribute to the future development of vision-free and fully automated robotic cell culture techniques. Our research may spike inspiration for future research on microscopic vision-free cell transportation in fully automated cell culture system.

In recent years, the perforated patch clamp technique has been widely applied in cellular electrophysiology research due to its low mechanical disturbance and almost no loss of cellular content in cell membrane perforation process. However, the current passive release process of perforating materials prolongs the perforation process and easily disturbs the gigaseal formation process, significantly lowering the efficiency of perforated patch clamp operation. Addressing this, a robotic perforated patch clamp system was developed based on the active release control of perforating materials in this paper. First, a novel holding module of the patch micropipette integrated with an independently driven transmission channel of the perforating materials was developed for the first time. Then, through release tests of the perforating materials, the appropriate drive mode of transmission channel was determined to be the hydraulic mode with a faster response and higher stability. Further, the concentration gradient field of the perforating materials at the opening of the channel was modeled according to Fick’s law to prevent the false release of them in gigaseal formation process. Furthermore, a cell circuit model was developed to detect perforation degree online for feedback control of the perforating materials release. Experimental results on pyramidal neurons in mouse brain slices demonstrated that, in comparison to the traditional method with passive releases of perforating materials, the proposed system was capable of perforating cell membrane at an almost doubled throughput and with a 57% improvement in the success rate. In comparison to the traditional non-perforated whole-cell patch clamp method, the signal recording duration of neurons operated by our method was doubled due to its fewer negative influences on gigaseal and almost no cellular content loss. Note to Practitioners —The perforated patch clamp technique, utilizes the cell membrane-perforating molecules to drill subnanometer-sized conductive pores in the cell membrane aspirated into a micropipette for the measurement of cellular electrophysiological signals. Unfortunately, the poor controllability of the current passive release of perforating materials in the patch clamp operation easily leads to a long drug diffusion process, and also, disturbs the gigaseal formation between the aspirated cell membrane and micropipette, which is required for the measurement of the extremely weak cellular electrophysiological signals. For the first time, an active release control method of perforating materials was developed in this paper based on the self-developed novel three-channel holding device. With active release control of perforating materials, the proposed method was capable of perforating cell membranes at a doubled speed with a significantly higher success rate, and doubled recording duration in comparison to the traditional perforated patch clamp methods and non-perforated whole-cell patch clamp method, respectively, due to its fewer negative influences on gigaseal and almost no cellular content loss. With the above advantages, our robotic perforated patch clamp method may be applied in cellular electrophysiology research in the future.

For the first time, an improved robotic micropipette aspiration (MA) method considering the effects of the intra-cellular pressure, friction between cell and micropipette, and the micropipette opening size was proposed for improving the measurement accuracy of the cell elasticity properties in this paper. First, an elastic energy model of the aspirated cell was established to analyze the effects of intracellular pressure on MA results. Then a force analysis of MA process was conducted to evaluate the influences of the friction force between the cell surface and micropipette opening as well as the inner diameter of the micropipette opening to MA results. Based on the above work, an improved robotic MA process was developed for reducing the cell elasticity measurement errors through automated release of intracellular pressure, micropipette opening polishing and inner diameter control of the micropipette. The effectiveness of the above work was validated by both finite elemental model (FEM) and MA experiments. The MA results on porcine oocytes demonstrate that our proposed MA method can rectify the 20.11%, 35.08% and 6.80% measure errors of cell Young’s modulus caused by the above three factors, respectively. Our research may provide a more objective evaluation of cell mechanical properties in the related research.