Propagation of Dental and Respiratory Cells and Organs in Microgravity

Summary

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Abstract

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

Introduction

Gravity affects all aspects of life on Earth, including the biology of individual cells and their function within organisms. Cells sense gravity through mechanoreceptors and respond to changes in gravity by reconfiguring cytoskeletal architectures and by altering cell division^1,2,3. Other effects of microgravity include the hydrostatic pressure in fluid filled vesicles, sedimentation of organelles, and buoyancy-driven convection of flow and heat⁴. Studies on the effect of loss of gravity on human cells and organs were originally conducted to simulate t....

Protocol

All necessary institutional approval was obtained to ensure that the study was in compliance with TAMU institutional animal care guidelines.

1. Bioreactor assembly and sterilization

1. Sterilize four high aspect ratio vessels (HARV) for the bioreactor in an autoclave on the plastic instrument cycle for 20 minutes at 121 °C as recommended by the manufacturer.
2. After sterilization, assemble the vessels in a cell culture hood by tightening the screws provided with the bioreactor after which the vessels are ready to use.

2. Scaffolds used for the bioreactor co-culture experiment

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Results

The inside chamber of the bioreactor provides an environment for the cells to proliferate and differentiate, attach to a scaffold or congregate to form tissue like assemblies. Each HARV vessel holds up to 10 mL of medium and facilitates a constant circulation of nutrients so that each cell has an excellent chance to survive. Figure 1A illustrates the attachment of the syringe ports to the front plate of the vessel where sterile one-stop valves are attached. These valves act as doorkeepers to.......

Discussion

Critical steps of the protocol for the growth of cells in microgravity include the bioreactor, the scaffold, the cells used for 3D culture, and the scaffold coating as a means to induce cell differentiation. The type of bioreactor used in our studies comprises the RCCS-4 bioreactor, a recent modification of the original Rotary Cell Culture System (RCCS) rotating cylindrical tissue culture device developed by NASA to grow cells in simulated microgravity. This RCCS-4 environment provides extremely low shear stresses, which.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibiotic-antimycotic	ThermoFisher Scientfic	15240096
Ascorbic Acid	Sigma Aldrich	A4544
BGJb Fitton-Jackson Modification media	ThermoFisher Scientfic	12591
BIOST PGA scaffold	Synthecon	Custom	Available from the company through a custom order
BMP-2	R&D Systems	355-BM
BMP-4	R&D Systems	314-BP
DMEM Media	Sigma Aldrich	D6429-500mL
FBS	ThermoFisher Scientfic	16140071
Fibricol	Advanced Biomatrix	5133-20mL
Fibronectin	Corning	354008
Galanin	Sigma Aldrich	G-0278
Gelatin disc	Advanced Biomatrix	CytoForm 500
Graphene sheets	Advanced Biomatrix	CytoForm 300
hEGF	Peprotech	AF-100-15
hFGF	ThermoFisher Scientfic	AA1-155
Hydroxyapatite disc	Advanced Biomatrix	CytoForm 200
Il-6 protein	PeproTech	200-06
Keratinocyte SFM media (1X)	ThermoFisher Scientfic	17005042
Laminin	Corning	354259
LRAP peptide	Peptide 2.0	Custom made sequence: MPLPPHPGSPGYINLSYEVLT PLKWYQSMIRQPPLSPILPEL PLEAWPATDKTKREEVD
Matrigel	Corning	354234
Millipore Nitrocellulose membrane	Merck Millipore	AABP04700
RCCS Bioreactor	Synthecon	RCCS 4HD
SpongeCol	Advanced Biomatrix	5135-25EA
Syring valve one way stopcock w/swivel male luer lock	Smiths Medical	MX5-61L
Syringes with needle 3cc	McKESSON	16-SN3C211
Trypsin EDTA (0.25%)	ThermoFisher Scientfic	25200056