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 the weightless environment of space on astronauts during space flight missions⁵. However, in recent years, these 3D bioreactor technologies originally developed by NASA to simulate microgravity are becoming increasingly relevant as novel approaches for the culture of cell populations that are otherwise not amenable to 2D culture systems.

3D Bioreactors simulate microgravity by growing cells in suspension and thus creating a constant "free-fall" effect. Other advantages of the rotating bioreactors include the lack of air exposure encountered in organ culture systems, a reduction in shear stress and turbulence, and a continuous exposure to a changing supply of nutrients. These dynamic conditions provided by a Rotary Cell Culture System (RCCS) bioreactor favor spatial co-localization and three-dimensional assembly of single cells into aggregates^6,7.

Previous studies have demonstrated the advantages of a rotary bioreactor for bone regeneration⁸, tooth germ culture⁹, and for the culture of human dental follicle cells¹⁰. There has also been a report suggesting that RCCS enhances EOE cell proliferation and differentiation into ameloblasts¹¹. However, differentiated cells were considered ameloblasts based on ameloblastin immunofluorescence and/or amelogenin expression alone¹¹ without considering their elongated morphology or polarized cell shape.

In addition to the NASA-developed rotating wall vessels (RWV) bioreactor, other technologies to generate 3D aggregates from cells include magnetic levitation, the random positioning machine (RPM) and the clinostat¹². To achieve magnetic levitation, cells labeled with magnetic nanoparticles are levitated using an external magnetic force, resulting in the formation of scaffold-free 3D structures that have been used for the biofabrication of adipocyte structures^13,14,15. Another approach to simulate microgravity is the generation of multidirectional G forces by controlling simultaneous rotation about two axes resulting in a cancellation of the cumulative gravity vector at the center of a device called clinostat¹⁶. When bone marrow stem cells were cultured in a clinostat, new bone formation was inhibited through the suppression of osteoblast differentiation, illustrating one of the dedifferentiating effects of microgravity¹⁶.

In vitro systems to facilitate the faithful culture of ameloblasts would provide a major step forward toward tooth enamel tissue engineering¹⁷. Unfortunately, to this date, the culture of ameloblasts has been a challenging undertaking^18,19. So far, five different ameloblast-like cell lines have been reported, including the mouse ameloblast-lineage cell line (ALC), the rat dental epithelial cell line (HAT-7), the mouse LS8 cell line²⁰, the porcine PABSo-E cell line²¹ and the rat SF2-24 cell line²². However, the majority of these cells have lost their distinctive polarized cell shape in 2D culture.

In the present study we have turned to a Rotary Cell Culture Bioreactor System (RCCS) to facilitate the growth of ameloblast-like cells from cervical loop epithelia co-cultured with mesenchymal progenitors and to overcome the challenges of 2D culture systems, including reduced flow of nutrients and cytoskeletal changes due to gravity. In addition, the RCCS has provided novel avenues for the study of cell/scaffold interactions related to periodontal tissue engineering and to examine the effects of inflammatory mediators on lung alveolar tissues in vitro. Together, results from these studies highlight the benefits of microgravity-based rotatory culture systems for the propagation of differentiated epithelia and for the assessment of environmental effects on cells grown in vitro, including cell/scaffold interactions and the tissue response to inflammatory conditions.

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

1. Incubate the scaffolds together with the cells to provide support for cell attachment necessary for further growth.
2. Choose among PGA based scaffolds, hydroxyapatite scaffolds, graphene sheets, gelatin discs, and collagen based scaffolds for co-culture studies.

3. Cervical Loop (CL) dissection and single cell preparation

1. Sacrifice mice by decapitation following respiratory arrest by CO₂ overdose.
   NOTE: All animal studies are in compliance with TAMU institutional animal care guidelines.
2. Dissect cervical loops from 6 days postnatal (DPN) mice following a modified version of a previously published protocol²³.
   NOTE: In our study, the distal portion of the cervical loop not shown in the previously published protocol is included (Figure 3).
   1. Wash the dissected tissue with cold sterile PBS prior to digestion with collagenase dispase at 37 °C for 30 minutes.
3. Pass the solution through a 70 µm sterile cell strainer and further centrifuge at 800 x g for 10 minutes.
4. Discard the supernatant. Centrifuge the pellet and wash twice with cold PBS at 800 x g. Discard the supernatant.
5. Count the cells using a hemocytometer and add 1 X 10⁵ cells per vessel.

4. Dental pulp cell culture and cell line expansion

1. Plate dental pulp cells in a 100 mm tissue culture plate at passage 4 at a concentration of 1 x 10⁵.
2. Prepare media for the culture consisting of DMEM high glucose media, supplemented with 10% fetal bovine serum (FBS), and 1% antibiotic/antimycotic (P/S).
3. When the cells reach 85-90% confluence, aspirate the medium and wash the plates twice with pre-warmed PBS.
4. After the PBS wash, add a pre-warmed 0.25% Trypsin-EDTA solution to the plate, and incubate the dental pulp cell-containing plates at 37 °C for 3 minutes.
5. Confirm cell detachment by visual inspection using a microscope.
6. Collect the media along with the cells from the culture plate and transfer via a 25 mL pipette to a 50 mL sterile conical tube.
7. Centrifuge the cells at 800 x g for 8 minutes and discard the supernatant. Resuspend the cells in fresh media and count using a hemocytometer.
8. For co-culture with cervical loop cells, seed the dental pulp cells in the bioreactor at a concentration of 1 x 10⁴ per vessel.

5. Co-culture of cervical loop/dental pulp cells on a scaffold within the RCCS bioreactor

1. Add both cell types, the cervical loop, and the dental pulp to the culture medium containing keratinocyte SFM media with supplemented growth factors, extracellular matrix proteins and scaffolds.
   NOTE: Growth factors are added at a concentration of 0.03 µg/mL bone morphogenetic protein 2 (BMP 2), 0.03 µg/mL bone morphogenetic protein 4 (BMP-4), 10 ng/mL human recombinant fibroblast growth factor (hFGF) and 15 ng/mL epidermal growth factor (hEGF), while ECM components included 200 µg/mL Matrigel, 5 µg/mL laminin and 5 µg/mL fibronectin.
2. Coat scaffolds with a mixture of 500 µL of collagen gel enriched with 1 mg of LRAP (Leucine-rich amelogenin peptide) peptide, 1 mg of lyophilized early stage enamel matrix prepared from porcine teeth²⁴, 5 µg/mL laminin and 5 µg/mL of fibronectin.
   NOTE: This worked best for our experiments.
3. Following scaffold coating, add both cells and scaffold to the bioreactor, and fill the bioreactor with 10 mL medium per vessel.
4. Close the fill port cap of the bioreactor vessel after addition of media, cells, and scaffolds.
5. Attach the sterile valves to the syringe ports at the beginning of the experiment and keep them in place with a cover until the end of the experiment.
6. Once the vessel is 90% full and the fill port caps are closed and tightened, open up the sterile valves.
7. Use two sterile syringes (3 mL) every time a media change is required. Fill one syringe with fresh medium while the other syringe is left empty.
8. Open up both syringe valves and gently maneuver the bubbles towards the empty syringe port.
9. Once the fresh medium from the full syringe is carefully injected in the vessel, remove the bubbles through the empty syringe port.
   NOTE: This procedure is performed until all the microbubbles are removed from the vessels.
10. Close the syringe ports with caps and discard the syringes in a sharps waste container.
11. Attach each vessel to the rotator base and place the rotator base in an incubator with 5% CO₂ and 37 °C temperature.
12. Turn on the power and set the rotational speed to 10.1 rpm.
    NOTE: Adjust the speed to ensure that the scaffolds are in suspension without touching the vessel wall.
13. For media change, place the vessels in an upright position to ensure that the cells settle at the bottom. Open the syringe ports and connect the sterile syringes to the ports.
14. Follow the same procedure by aspirating 75% of the nutrition-depleted medium and injecting fresh medium through the other port.

6. Lung organ preparation and bioreactor based culture

NOTE: E15 wild-type mouse pups were used for the preparation of lung organs.

1. Dissect the lung tissue after euthanizing a pregnant female mouse with CO₂ asphyxiation.
   1. Once the death is confirmed following asphyxiation by cervical dislocation, use a scalpel to expose the thoracic cavity. Thereafter, remove the pups from the uterus and euthanize by decapitation.
2. After euthanization, prepare the thoracic cavity by opening it with a scalpel to locate the lungs. Wash tiny segments of lung tissue with sterile PBS and place it on a pre-sterilized membrane for 2 hours with organ culture medium in an incubator containing 5% CO₂ and 37 °C temperature.
3. Once the lung tissues attach to the membrane in a 2D organ culture plate, transfer the samples to the bioreactor vessel.
4. Prepare the media for culture.
   NOTE: The control DMEM high glucose media contains 10% fetal bovine serum (FBS), 1% antibiotic solution (P/S) (penicillin, 100 U/mL, streptomycin, 50 µg/mL) and 100 µg/mL ascorbic acid (AA) and for the induction of inflammatory conditions, control medium is supplemented with 5 ng/mL IL-6 protein.
5. Carry out the lung culture experiment for 10 days with a media change every 48 hours in the bioreactor as described above.

7. Coated scaffold with Human periodontal ligament (hPDL) cell culture

1. Culture human periodontal ligament cells.
   NOTE: The cells are isolated and cultured in our lab as per the protocol previously published²⁵.
2. Coat the collagen scaffold discs with 30 µL of PBS for the control group and the neuropeptide galanin (GAL) at a concentration of 10^-8 overnight.
3. Seed human PDL cells on the control and GAL coated scaffold for 2 hours at 37 °C in a culture plate, and transfer the cell seeded scaffolds to a bioreactor vessel as mentioned above.
4. Culture the cell seeded scaffolds for 14 days in the bioreactor prior to harvesting the scaffolds for analysis.

8. Bioreactor cleaning and maintenance

1. After the scaffolds/cells are harvested from the bioreactor, remove the syringe ports from the vessel.
   ​NOTE: The screws around the vessels are taken off and the vessel is gently washed with soap water.
2. Rinse the vessels with clean water and tighten the screws once more.
3. Store the vessels after autoclaving until next use.

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