Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation

Abstract

A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle a-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

“…Another successful example is the magnetic nanobeads-mediated spheroid formation [41][42][43][44][45][46][47][48][49] . This method (also known as 'magnetic levitation' when the cells are collected on top of the fluid in a tissue culture well rather than on its bottom 41 ), is extremely versatile.…”

“…This method (also known as 'magnetic levitation' when the cells are collected on top of the fluid in a tissue culture well rather than on its bottom 41 ), is extremely versatile. It has been used in several high-profile studies, being applied for 'bioprinting' of complex structures, such as valves 48 , bronchioles 45 , or adipose tissue 46 . Similar methods are being actively developed in other settings as well 50,51 .…”

“…However, among their common limitations is the difficulty to place and maintain the spheroids in a pre-determined position, or to use spheroids of different compositions and to scale-up to surgically meaningful constructs. For this, the authors introduced the use of additional tools, such as a magnetic 'bio-pen' which manually could bring and keep the spheroids in place 45,48 .…”

Progress in scaffold‐free bioprinting for cardiovascular medicine

“…Therefore, the method is said to be universal regarding the phenotype of the involved cells, although the stability of the spheroids and thus of the resulting constructs may vary. Using this regent, a series of high‐profile papers have been published, with examples in vascular,41, 42 pulmonary43 and tumour,44, 45 biology.…”

Perspectives of Constant Gradient Magnetic Fields Applications in Biotechnology

“…Constant magnetic fields are considered to be used for the control 3D cell culture growth. It has been shown in [1], the cell differentiation process may be controlled by magnetic field using Fe 3 O 4 nanoparticles based magnetic gel by generation of weightlessness environment.…”