Magnetic 3D Cell Culture: Engineering innervated secretory epithelial organoids for stimulating epithelial growth in salivary glands.

Published Abstract

Current saliva-based stimulation therapies for radiotherapy-induced xerostomia are not fully effective due to the presence of damaged secretory epithelia and nerves in the salivary gland (SG). Hence, three-dimensional bio-engineered organoids are essential to regenerate the damaged SG. Herein, a recently validated three-dimensional (3D) biofabrication system, the magnetic 3D bioprinting (M3D), is tested to generate innervated secretory epithelial organoids from a neural crest derived mesenchymal stem cell, the human dental pulp stem cell (hDPSC). Cells are tagged with magnetic nanoparticles (MNP) and spatially arranged with magnet dots to generate 3D spheroids. Next, a SG epithelial differentiation stage was completed with fibroblast growth factor 10 (4e400 ng/ml) to recapitulate SG epithelial morphogenesis and neurogenesis.

The SG organoids were then transplanted into ex vivo model to evaluate their epithelial growth and innervation. M3DB-formed spheroids exhibited both high cell viability rate (>90%) and stable ATP intracellular activity compared to MNP-free spheroids. After differentiation, spheroids expressed SG epithelial compartments including secretory epithelial, ductal, myoepithelial, and neuronal. Fabricated organoids also produced salivary a-amylase upon FGF10 stimulation, and intracellular calcium mobilization and transepithelial resistance was elicited upon neuro stimulation with different neurotransmitters.

After transplantation, the SG-like organoids significantly stimulated epithelial and neuronal growth in damaged SG. It is the first-time bio functional innervated SG-like organoids are bioprinted. Thus, this is an important step towards SG regeneration and the treatment of radiotherapy-induced xerostomia.

M3D and Nanoshuttle:

Magnetic 3D Cell Culture relies on the magnetization of cells with biocompatible NanoShuttle™. The reproducible formation of spheroids in the well of flat bottom plates is enabled by magnetic force from permanent magnets, and this can be accomplished by magnetic levitation or bioprinting. Magnetic 3D bioprinting (M3D) uses one magnet below each well and applies mild magnetic forces to induce cell aggregation at the bottom of each well anywhere within 15 minutes to a few hours. After bioprinting, the spheroids can be cultured long-term without the presence of magnetic force. This system overcomes the limitations of other platforms by enabling rapid formation of spheroids, reproducible and scalable in size for high-throughput formats (96, 384 and 1536 well) and without limitation to cell types.

The NanoshuttleTM  is a biocompatible magnetic nanoparticle assembly used to promote spheroid formation. The Nanoshuttle solution has been comprehensively described in the literature.

Study Conclusions

These results show, for the first time, the process of Magnetic 3D Bioprinting (M3D) to reproducibly fabricate robust organoids and enables the formation of functional tissue with distinct salivary gland-like (SG-like) cellular compartments. After transplantation, these SG-like organoids significantly stimulated the epithelial and neuronal growth in irradiation damaged and healthy salivary gland.

Future Direction:

This SG-like organoid model can be used as tool for drug cytotoxicity screening and mechanistic studies. More importantly, further studies will test the regenerative potential of these organoids in long-term/chronic radiotherapy-induced xerostomia models in both small and large animal models.

Cell Types Listed:

• Human dental pulp stem cell (hDPSC)
• Human dental pulp stem cells (hDPSCs) neural crest derived
• Mesenchymal stem cell

M3D System Used

• 96-well bioprinting

Topics

• Stem cells
• Salivary gland
• Regenerative Medicine
• Cancer

Key Findings

• First report of a bio-functional, innervated, salivary gland-like organoid produced through bioprinting
• Applies Magnetic 3D Cell Culturing to create a three-dimensional, innervated, secretory epithelial organoid model from neural crest-derived, mesenchymal stem cells
• Bioprinted organoids were transplanted into animals where they significantly stimulated epithelial and neuronal growth in damaged salivary glands
• Show flexibility of technique for transplantation of SG-like organoids into the excretory ductal area of developing SG glands ex-vivo
• Organoids significantly rescued epithelial growth on this acute SG fetal models of irradiation
• Magnetic 3D Cell culture was superior to controls/other techniques
• Validation of Magnetic 3D Cell Culture technology for the creation of innervated secretory epithelial organoids
• Important step in using 3D bioprinted cells to help advance tissue engineering towards translational applications in regenerative medicine
• This SG-like organoid model can be used as tool for drug cytotoxicity screening and mechanistic studies.
• Bioprinting tool that can consistently control the size of the spheroids by tuning the concentration of MNP, cell number, and the size of the magnet dots

Academic Institutions and Industry (very respected in the field)

• National University of Singapore, Singapore
• Mahidol University, Nakhon Pathom, 73170, Thailand
• University of Texas Health Sciences Center at Houston, Houston, TX, USA
• Nano3D Biosciences Inc., Houston, TX, USA
• Chulalongkorn University, Bangkok, Thailand
• National Institutes of Health, Bethesda, MD, USA

 
Academic Authors (very respected in the field):

• Christabella Adine
• Kiaw K. Ng
• Sasitorn Rungarunlert
• Glauco R. Souza
• Joao N. Ferreira