TASCL
Recent tissue regeneration protocols involve culturing stem cells as three-dimensional, multicellular spheroids called embryoid bodies (EBs) and allowing them to differentiate into typical cell types. However, until now, there has been no way to mass-produce EB efficiently and with high uniformity. In order to rapidly advance regenerative medicine, there is an urgent need to develop efficient experimental systems to produce EBs in large quantities, uniformly, and at low cost. To solve this problem, we developed TASCL (Tapered Stencil for Cluster Culture), a tapered microaperture array made of PDMS that enables mass production of EBs.
By placing TASCL on top of a conventional permeable cell culture insert, microwells are formed on the insert. The surface of TASCL is modified with a hydrophilic polymer to prevent cell attachment. Since there is no flat surface between the microwells, each cell will fall into one of the microwells, preventing unexpected invasion of cells after initial aggregation. Therefore, simply dropping a cell suspension can initiate EB formation under precisely controlled conditions [1-3].
1.Yukawa H, Ikeuchi M, Noguchi H, Miyamoto Y, Ikuta K, Hayashi S. Embryonic body formation using the tapered soft stencil for cluster culture device. Biomaterials. 2011 May;32(15):3729-38. doi: 10.1016/j.biomaterials.2011.01.013. Epub 2011 Feb 26. Erratum in: Biomaterials. 2013 Nov;34(33):8531. Ikeuchi, Masashi [added]; Miyamoto, Yoshitaka [added];(5) Ikuta, Koji [added]. PMID: 21354615.
2.Miyamoto Y, Ikeuchi M, Noguchi H, Yagi T, Hayashi S. Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device. Cell Med. 2015 Aug 26;8(1-2):47-56. doi: 10.3727/215517915X689056. PMID: 26858908; PMCID: PMC4733911.
3.Miyamoto Y, Ikeuchi M, Noguchi H, Yagi T, Hayashi S. Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture. Cell Med. 2016 Sep 14;9(1-2):35-44. doi: 10.3727/215517916X693096. PMID: 28174673; PMCID: PMC5225676.
PASMA
In regenerative medicine, stem cells can be cultured as multicellular spheroids called embryoid bodies (EBs) and differentiated into typical cell types by promoting differentiation. Conventionally, there are several methods for creating EBs, but EB formation, evaluation, and recovery cannot be performed with a single device, making EB research a costly and time-consuming task. To solve this problem, we developed an innovative device called PASMA (Pressure Actuated Shapable Microwell Array) [1-3].
The fluid lines and pressure actuation lines are independent and form orthogonal coordinate systems. The fluid line consists of a thin elastomeric membrane, and the pressure actuated line is located below the membrane. When a negative pressure is applied to the pressure actuation line, the membrane bends downward to form a microwell. Applying positive pressure to the pressure actuation line causes the membrane to return to flatness. This mechanism enables selective collection of EBs, induction of differentiation into various types of cells, and on-chip analysis.
1.T. Nishijima, M. Ikeuchi and K. Ikuta, "Pneumatically actuated spheroid culturing Lab-on-a-Chip for combinatorial analysis of embryonic body," 2012 IEEE 25th International Conference on Micro Electro Mechanical Systems (MEMS), 2012, pp. 92-95, doi: 10.1109/MEMSYS.2012.6170101.
2.A. Yasukawa, T. Nishijima, M. Ikeuchi and K. Ikuta, "Integrated micro culture device for fully automated closed culture experiment of embryonic body," 2014 IEEE 27th International Conference on Micro Electro Mechanical Systems (MEMS), 2014, pp. 181-184, doi: 10.1109/MEMSYS.2014.6765604.
3.M. Ikeuchi, M. Shibata and K. Ikuta, "Independently controllable microwell array with fluidic multiplexer for mass production of embryonic bodies," 2017 19th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS), 2017, pp. 277 -280, doi: 10.1109/TRANSDUCERS.2017.7994042.
Machine learning for regenerative medicine
Embryonic body (EB) formation has become a routine step in stem cell differentiation. The size of the embryo body is one of the major factors that determines the direction and efficiency of differentiation. In order to induce differentiation with high reproducibility in mass production, it is extremely important to quickly eliminate amorphous EBs that do not meet specifications. In this paper, we propose a system that cultivates a large number of EBs at once and predicts the success rate of EB formation using machine learning [1]. In the demonstration experiment, EBs were cultured using TASCL, and time-lapse images of each well were taken every 30 minutes. A total of six images taken up to 3 hours after seeding were input into multiple neural networks as one learning data to predict whether EBs had formed one day after seeding. As a result, by using a three-dimensional convolutional network (3DCNN), the highest prediction accuracy on test data was over 95%. Furthermore, by inputting 12 images from immediately after seeding to 6 hours after seeding into a 3D CNN and predicting the diameter of EBs 3 days after seeding, we were able to predict the diameter of EBs 3 days after seeding. As a result, we succeeded in predicting the size of EBs with an error of ±7.1 μm.
1.S. Suda, C. Aoyama and M. Ikeuchi, "Quality Prediction of Embryonic Bodies on Integrated Spheroid Culture Chip by Using 3D Convolutional Neural Network,"2020 IEEE 33rd International Conference on Micro Electro Mechanical Systems (MEMS), 2020, pp. 461 -464, doi: 10.1109/MEMS46641.2020.9056339.
Multi-pattern cell stretching device
Increased hemodynamic load on the heart causes cardiac hypertrophy, which progresses to heart failure. However, many points remain unclear about the mechanobiological mechanisms that lead to heart failure.
To clarify these points, we are investigating the differences in mechanical stress responses between cardiomyocytes differentiated from iPS cells from healthy individuals and those from patients with heart disease. However, it is unclear what strength of stretch stimulation is appropriate. Additionally, when analyzing mechanical stress responses, it is effective to record historical data on the load and morphology of cardiomyocytes in real time and link that data with gene expression information obtained after stretching.
Therefore, we developed a cell stretching system that can apply multiple strain ratios and perform real-time observation. By changing the well width and extensional displacement, multiple strain regions from 4 to 12% are achieved. We demonstrated that it is possible to observe the expansion of iPS cardiomyocytes in real time 3 days after seeding.
Development of a nonfreezing preservation system for cells and organs
The preservation of reproductive cells, such as oocytes, early-stage embryos, and sperm, is a critical and widely used technology in reproductive medicine and livestock production. We are currently developing a nonfreezing preservation system to meet various social needs. To meet our aim of achieving non-freezing preservation, our efforts target (1) seeking natural supercooling-promoting substances and antifreeze proteins effective for cell preservation and (2) developing custom preservation solutions and programs suitable for different types of cells, tissues, and organs. Developing an effective nonfreezing preservation systems for cells and organs is expected to expand opportunities for successful cell-based therapies and help solve the problem of organ transplantation.