Cell fibers promote proliferation of co-cultured cells on a dish

Abstract

This paper describes a co-culture method using cell fiber technology. Cell fibers are cell-laden hydrogel microfibers, in which cells are cultured three-dimensionally and allowed to reach more mature state than the conventional two-dimensional cell culture. Cells in the cell fibers are encapsulated by alginate shell. Only cellular secretome is released into the surrounding environment through the shell while the cells were retained by the fiber. With their high handleability and retrievability, we propose to use the cell fibers for co-culture to ensure steady supply of cellular secretome. We cultured mouse C2C12 myoblasts with mouse 3T3 fibroblasts encapsulated in the cell fibers for two days. The number of C2C12 cells increased proportionally to the number of co-cultured 3T3 fibers, suggesting that the secretome of 3T3 fibers promoted survival and proliferation of C2C12 cells. We believe that cell fiber technology is a useful tool for co-culturing cells, and it will contribute to both basic cell biology and tissue engineering with its unique features.Co-culture, in which two or more types of cells are cultured together, is a major method to study interactions between different types of cells in vitro. Cells interact with each other both directly (via physical contact) and indirectly (via secreted molecules; for example, cytokines, growth factors and hormones) and these interactions have an impact on cellular survival, proliferation, differentiation and maturation. To investigate the indirect cellular interactions, two major methods have been established; one using culture inserts and the other using conditioned medium. Culture inserts make upper and lower compartments in culture wells, which enables a concurrent co-culture. Two different types of cells are plated and cultured in the upper and lower compartments. Only cellular secretome, but not the cells themselves, is then transferred between those two compartments through the pores on the bottom of culture inserts, when the pore size is smaller than the cells. On the other hand, in the method that employs conditioned medium, a certain type of cells are cultured and the supernatant containing their secretome (conditioned medium) is collected to subsequently culture the other type of cells. These methods are often employed and have been shown to be effective to study various cellular interactions 1-3 . However, neither of them is highly space-efficient; the number of available cells that provides secretome is limited because of the culture area.Cell fiber is a unique tool for culturing cells three-dimensionally for a long period until they differentiate into a mature tissue 4 . Cell fibers, which are cell-laden hydrogel microfibers formed by using a double-coaxial laminar-flow microfluidic device, consist of two parts; the core containing cells and extra cellular matrix (ECM) proteins such as collagen, and the alginate shell. Various types of cells have been shown to form three-dimensional (3D) tissues in cell fibers; for example, cardiomyocytes, vascular endoth...

In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

“…al. has paved the way for this kind of development using spheroids 12 , and we also have already succeeded in developing cell fibers for different types of human-derived cells [23][24][25]42…”

“…Primary hepatocytes were isolated from six-to nine-week-old male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) using standard two-step collagenase perfusion method. [25] Isolated rat hepatocytes with a viability of more than 85% as determined by trypan blue exclusion test were used.…”

“…All the cells in the core can easily access oxygen and nutrients in the culture medium since the thickness of the cell fiber is kept a few hundreds of micrometres over its entire length. Cell fibers have been shown to serve for three-dimensional tissue formation of various types of cells including cardiomyocytes, vascular endothelial cells, nerve cells, smooth muscle cells and adipocytes [22][23][24][25] . The advantage of cell fiber is to be mass-producible and scalable; a cell fiber with long (meter-order) length can be fabricated in a short time (a few minutes) and cell fibers have allowed pluripotent stem cells to increase in number while keeping their original characters 24 .…”

Chips for Biomaterials and Biomaterials for Chips: Recent Advances at the Interface between Microfabrication and Biomaterials Research

“…[87] In addition to these microgels produced by droplet-based techniques, coaxial laminar flow in a microfluidic device can be used for the production of hydrogel fibers. For instance, it has been used to produce hydrogel fibers with encapsulated cells, [88,89] and self-assembled polysaccharide fibers [90] that in turn have been assembled into fibrous hydrogel films incorporating graphene. [91]…”

In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

“…All the cells in the core can easily access oxygen and nutrients in the culture medium since the thickness of the cell ber is kept a few hundreds of micrometres over its entire length. Cell bers have been shown to serve for three-dimensional tissue formation of various types of cells including cardiomyocytes, vascular endothelial cells, nerve cells, smooth muscle cells and adipocytes [22][23][24][25] . The advantage of cell ber is to be mass-producible and scalable; a cell ber with long (meter-order) length can be fabricated in a short time (a few minutes) and cell bers have allowed pluripotent stem cells to increase in number while keeping their original characters 24 .…”