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What is the Kenzan Method for 3D Bioprinting from Cyfuse’s Regenova System?

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Regenova is a novel robotic system that facilitates the fabrication of three- dimensional cellular structures by placing cellular spheroids in fine needle arrays, the Kenzan method, according to pre-designed 3D data.

Generation of thick and elastic tissue that is like real tissue but grown from cultured cells has proved difficult with conventional technologies.

The Kenzan method was invented by prof. Koich Nakayama (Saga University)and is globally patented intellectual property. Cyfuse Biomedical K.K. is granted exclusive rights of use. Dr. Koichi Nakayama focused on the natural and intrinsic features of cell self-aggregation and invented a novel method to assemble cellular aggregates (spheroids) into any desired three-dimensional macroscopic tissue without collagen or hydrogel materials. 3D Bioprinting materials

With this method, cellular aggregates called spheroids are cultured, with several tens of thousands of cells per spheroid.
Then, spheroids are placed in fine needle arrays and allowed to merge with adjacent spheroids to form a connected structure that is firm enough not to require needles.
With appropriate alignment of needles to form the needle array, cellular spheroids can be positioned in any desired three-dimensional layout. Finally, culturing connected cellular spheroids in a bioreactor promotes self-organization of cells and yields a 3D tissue with the desired function and quality.The benefit of placing cellular spheroids in the needle array is not only freedom of three-dimensional positioning but also easier circulation of culture medium around the assembled spheroids to supply efficient neutralization and oxygen to cells until tissue is matured and no longer requires the support array.
Sphere-like cell
Sphere-like cell aggregates are secured in place by skewers.

The aggregates
The aggregates are allowed to fuse on the skewers for a few days.

The skewers
The skewers are removed and the tissue is allowed to further mature.

The 3D Bioprinting process

First the cells are isolated and proliferated to target levels. In low adhesion multi-well plates, several tens of thousands of cells form a spheroid in a day or two with a diameter of approximately 500 micrometers.
Spheroids photo3D data photo






Then a three-dimensional arrangement of cellular spheroids is designed and and 3D data is prepared using the original software. Multiple types of spheroids can be selected as desired. To design three dimensional structure, Cyfuse offer special software called “B3D”, which was developed for Regenova. You can install B3D into your own computer and design no matter where you are.

Finally Needles are aligned to serve as a temporary scaffold during cellular spheroid assembly. The needles are made of stainless steel with a diameter of 100-200 micrometers and pitch of 300-400 micrometers. Regenova assembles cellular spheroids into a three-dimensional shape by placing spheroids in needle arrays according to the pre-designed 3D data. Further culturing of 3D printed tissue with proper flow of medium in a bioreactor facilitates rearrangement of cells for self-organization to exhibit physical strength and tissue function. The 3D shape is self-sustained without needles.

See the Regenova system at work in this video


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