Propagation of Dental and Respiratory Cells and Organs in Microgravity

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.

Transcript

Elongated and polarized the amelogenin-secreting ameloblast in culture. This is also the first protocol for growing ameloblast cells in microgravity. The growth of ameloblasts in culture has been one of the major challenges in the enamel field.

And for us, it's using this bioreactor model, it's the first time that we have accomplished this hallmark in enamel research. This model can be used to understand the biology of ameloblast cells. This model will be demonstrated by Dr.Mirali Pandya.

Begin by placing the collected tissue from six-day postnatal mice under the dissection microscope and dissected cervical loops. Set up a bioreactor by attaching the sterile valves to the syringe ports. Add both cells, the cervical loop and the dental pulp, along with coated scaffold to the bioreactor and fill the bioreactor vessel with 10 milliliters of keratinocyte SFM media supplemented with growth factors and extracellular matrix proteins.

Close and tighten the fill port cap of the bioreactor vessel, then open the sterile valves. Use two sterile three milliliter syringes for a media change. To do so, fill one syringe with the fresh medium and leave the other syringe empty.

Open both syringe valves and gently maneuver the bubbles towards the empty syringe port. Once the fresh medium from the full syringe is carefully injected into the vessel, remove all the bubbles through the empty syringe port. Close the syringe ports with caps.

Attach each vessel to the rotator base. Turn on the power and set the speed to 10.1 rotations per minute. Adjust the speed to ensure the scaffolds are in suspension and not touching the vessel wall.

For media change every 24 hours, place the vessels upright to ensure the cells settle at the bottom. Open the syringe ports to connect the sterile syringes to the ports. Aspirate 75%of the nutrition-depleted medium and inject the fresh medium through the other port as previously demonstrated.

The macrographs of graphene scaffold and collagen scaffold are shown here. The graphene scaffold had a parallel array of surface embossments, while the collagen scaffold exhibited the porous structure. A skeletonized mouse mandible was dissected and the distal most portion of the lower mouse incisor was exposed.

The precise position of the resected cervical loop is demarcated in the six-day postnatal mouse incisor, while a similar region in an adult skeletonized mouse mandible is provided as a reference. The hemimandibles were comprised of three mandibular molars and a constantly growing incisor, while the cervical loop cell niche contained a varied cell population necessary for enamel formation. The successful differentiation of the cervical loop cells in a tailored microenvironment resulted in the formation of ameloblasts with a typical characteristic of polarized cells with the nucleus at one end and long cellular processes at the other.

The culture of cells in media alone without growth factors and scaffold coating resulted in cervical loop cells that secreted key enamel proteins, but did not elongate or polarize. The galanin-coated scaffold group demonstrated a significantly higher proliferation rate compared to the control group containing uncoated scaffolds. The lung tissue segments harvested from E15 mice were successfully cultured for 10 days in a 3D microgravity environment in the bioreactor.

The addition of interleukin-6 to the culture medium resulted in inflammation-associated changes in alveolar morphology similar to those seen in vivo. In the introduction, I told you how difficult it was for the enamel team to come up with an ameloblast cell culture model. Now we have addressed this challenge by using a highly innovative procedure that focuses on two aspects:sponge collagen scaffold and also the employment of matrix to differentiate the ameloblast cells.

Together, these two innovations resulted in the polarization and growth of ameloblast-like cells in 3D culture. These 3D cultured cells are a wonderful model to understand ameloblast biology. And to understand this biology, we can use a variety of techniques including electron microscopy, proteomics, and genomics.

Ameloblasts are fantastic cells. Yes, they have the ability to secrete prismatic tooth enamel. Now, this bioreactor model puts us in the position to understand how ameloblasts secrete matrix and secrete apatite minerals to grow prismatic tooth enamel.

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Journal (Bioengineering)

Journal (Bioengineering)

Propagation of Dental and Respiratory Cells and Organs in Microgravity

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