Regenerative Medicine

In the recent protocol of tissue regeneration, stem cells are differentiated into typical cell types through culturing as 3-D multicellular spheroids called embryonic bodies (EBs). There are, however, no conventional methods to efficiently mass-produce EBs with high uniformity. For rapid promotion of regenerative medicine, there is an urgent need to develop more efficient experimental system for large-scale and uniform production of EBs at low cost. To solve this problem, we have developed TASCL (Tapered Stencil for Cluster Culture) which is an array of tapered micro-apertures made of PDMS to enable mass production of EBs. By placing a TASCL on a conventional permeable cell culture insert, microwells are formed on the insert. The surface of TASCL is modified with hydrophilic polymer to prevent cell adhesion. There is no flat surface between microwells so that each cell falls into any one of the microwells, and unexpected intrusion of cells after initial aggregation can be prevented. Therefore, EB formation can be initiated under precisely controlled conditions by just dropping cell suspension on the device [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.

In regenerative medicine, stem cells are differentiated into a typical cell type through culturing as a multicellular spheroid of cells called an embryonic body (EB), which promotes differentiation. There are several conventional ways to make EBs, but formation, evaluation and collection of EBs cannot be done in one device, making EB study costly and time-consuming work. To solve this problem, we developed an innovative device called PASMA (Pressure Actuated Shapable Microwell Array) [1-3]．It has an array of microwells and EBs are formed in each microwell. There are independent fluid lines and independent pressure actuation lines, making orthogonal coordinate system. The fluid lines are composed of a thin elastomer membrane, and pressure actuation lines are placed under the membrane. The membrane is bent downward to form microwells by applying negative pressure to pressure actuation lines. Applying positive pressures to the pressure actuation line, the membrane returns to be flat. This design enables selective collection, differentiation induction to multiple cell types, and on-chip analysis of EBs.

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.

Embryonic body (EB) formation has become a routine step in differentiation of stem cells. The size of the EB is one of the major factor in determining the direction and efficiency of differentiation. To induce differentiation with high reproducibility in mass-production, it is very important to exclude irregular EBs not satisfying the specification at an early stage. In this paper, we propose a system that cultivates many EBs at one time and predicts success ratio of EB formation by machine learning [1]. In the proof-of-concept experiment, EBs were cultured by using a TASCL, and time-lapse image of each well was took every 30 minutes. Then, we input a total of 6 images up to 3 hours after seeding as one training data to the several neural networks and predicted whether EBs were formed 1 day after seeding. As a result, the highest prediction accuracy was >95% with the test data by using three-dimensional convolutional network (3DCNN). Furthermore, we predicted the diameter of the EBs 3 days after seeding by inputting 12 images up to 6 hours just after seeding into the 3DCNN. As a result, we have succeeded in predicting the size of EBs with an error of ±7.1μm.

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.