Chemically crosslinked alginate porous microcarriers modified with bioactive molecule for expansion of human hepatocellular carcinoma cells

Abstract

Microcarrier is an essential matrix for the large-scale culture of anchorage-dependent cells. In this study, chemical cross-linked alginate porous microcarriers (AMC) were prepared using microemulsion and freeze-drying technology. Moreover, chitosan was coated on the surface of microcarriers (AMC-CS) via electrostatic interactions to improve the mechanical strength. The size of AMC can be modulated through adjusting the concentration of alginate, amount of dispersant and stirring rate. The surface chemical characteristics and morphology of AMC-CS were evaluated by Fourier transformed infrared, X-ray photoelectron spectroscopy, and scanning electron microscope. Fibronectin (Fn) or heparin/basic fibroblast growth factor (bFGF) was then immobilized on the surface of microcarriers via layer-by-layer technology to improve the cytocompatibility. Our data suggested that the size of AMC can be accurately modulated from 90 μm to 900 μm with a narrow size distribution. Micropore structures of AMC-CS were relatively disordered and the pore size ranged between 20 μm and 100 μm. Using AMC after modified with Fn or bFGF as the cell expansion microcarriers, we showed that the proliferation rates of HepG2 cells increased significantly, reaching to more than 30-fold of cell expansion after 10 days of culture, with minor cellular damage caused by the microcarriers. Moreover, the AMC microcarriers modified with Fn or bFGF can increase albumin secretion of HepG2. We suggest that our new modified AMC-based microcarriers will be an attractive candidate for the large-scale cell culture of therapeutic cells.

Alginate/PEG based microcarriers with cleavable crosslinkage for expansion and non-invasive harvest of human umbilical cord blood mesenchymal stem cells

“…Especially for the AL/PEG microcarriers, its mechanical strength decreases when the non-crosslinked PEG molecules release from the microcarriers during the culture process. Our previous study [12] showed that the microcarriers after modified with chitosan have enough mechanical strength to support the adhesion of cells, but this study finds the number of cells in AL/PEG-CS microcarriers keep almost unchanged after 2 days. It is because the high interactions between negative cell and positive chitosan layer.…”

“…According to our previous study, [12] chitosan and Fn were successively immobilized on the surface of porous AL/PEG microcarriers via layer-by-layer (LBL) self-assembly technology before seeding hUCB-MSCs. We proposed that the chitosan coating can improve the mechanical rigidity and bio-adhesive properties of microcarriers, and the Fn layer is beneficial to the expansion of hUCB-MSCs.…”

“…In addition, some proteins, peptides or growth factors can be incorporated into microcarriers to improve the expansion efficiency. [11] In our previous studies, [12] for example, the fibronectin (Fn) or basic fibroblast growth factor (bFGF) was immobilized on the surface of alginate microcarriers via layer-by-layer technology. Until now, few studies focused on the cell harvesting method from the microcarriers.…”

“…They suggested that the POx-based microbeads can support the proliferation of bone marrow derived human MSCs, moreover, the expanded MSCs can be harvested via the degradation of microbeads by glutathione under mild physiological conditions. In our previous studies, alginate scaffolds [21] and porous microcarriers [12] containing disulfide bonds were prepared via chemically cross-linking by a condensation reaction of cystamine or cysteine with di-terminated amine groups. Results demonstrated that the alginate based microcarriers could support the attachment and proliferation of L929 fibroblasts and human hepatocellular carcinoma cell line HepG2 cells.…”

“…Fn was further coated on the AL/PEG-3/CS microcarriers to improve the cell attachment capacity. Specifically, AL/PEG-3/CS microcarriers were immersed in Fnsolution (4 μg/mL, Biological Industries, Israe) according to our previous study [12].…”