Studies were generously supported by grants from the National Institute of Dental and Craniofacial Research (UG3-DE028869 and R01-DE027930).

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
<ol>
	<li>Sterilize four high aspect ratio vessels (HARV) for the bioreactor in an autoclave on the plastic instrument cycle for 20 minutes at 121 &#176;C as recommended by the manufacturer.</li>
	<li>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.</li>
</ol>
2. Scaffolds used for the bioreactor co-culture experiment
<ol>
	<li>Incubate the scaffolds together with the cells to provide support for cell attachment necessary for further growth.</li>
	<li>Choose among PGA based scaffolds, hydroxyapatite scaffolds, graphene sheets, gelatin discs, and collagen based scaffolds for co-culture studies.</li>
</ol>
3. Cervical Loop (CL) dissection and single cell preparation
<ol>
	<li>Sacrifice mice by decapitation following respiratory arrest by CO2 overdose. 
	NOTE: All animal studies are in compliance with TAMU institutional animal care guidelines.</li>
	<li>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).
	<ol>
		<li>Wash the dissected tissue with cold sterile PBS prior to digestion with collagenase dispase at 37 &#176;C for 30 minutes.</li>
	</ol>
	</li>
	<li>Pass the solution through a 70 &#181;m sterile cell strainer and further centrifuge at 800 x g for 10 minutes.</li>
	<li>Discard the supernatant. Centrifuge the pellet and wash twice with cold PBS at 800 x g. Discard the supernatant.</li>
	<li>Count the cells using a hemocytometer and add 1 X 105 cells per vessel.</li>
</ol>
4. Dental pulp cell culture and cell line expansion
<ol>
	<li>Plate dental pulp cells in a 100 mm tissue culture plate at passage 4 at a concentration of 1 x 105.</li>
	<li>Prepare media for the culture consisting of DMEM high glucose media, supplemented with 10% fetal bovine serum (FBS), and 1% antibiotic/antimycotic (P/S).</li>
	<li>When the cells reach 85-90% confluence, aspirate the medium and wash the plates twice with pre-warmed PBS.</li>
	<li>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 &#176;C for 3 minutes.</li>
	<li>Confirm cell detachment by visual inspection using a microscope.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For co-culture with cervical loop cells, seed the dental pulp cells in the bioreactor at a concentration of 1 x 104 per vessel.</li>
</ol>
5. Co-culture of cervical loop/dental pulp cells on a scaffold within the RCCS bioreactor
<ol>
	<li>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 &#181;g/mL bone morphogenetic protein 2 (BMP 2), 0.03 &#181;g/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 &#181;g/mL Matrigel, 5 &#181;g/mL laminin and 5 &#181;g/mL fibronectin.</li>
	<li>Coat scaffolds with a mixture of 500 &#181;L 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 &#181;g/mL laminin and 5 &#181;g/mL of fibronectin. 
	NOTE: This worked best for our experiments.</li>
	<li>Following scaffold coating, add both cells and scaffold to the bioreactor, and fill the bioreactor with 10 mL medium per vessel.</li>
	<li>Close the fill port cap of the bioreactor vessel after addition of media, cells, and scaffolds.</li>
	<li>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.</li>
	<li>Once the vessel is 90% full and the fill port caps are closed and tightened, open up the sterile valves.</li>
	<li>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.</li>
	<li>Open up both syringe valves and gently maneuver the bubbles towards the empty syringe port.</li>
	<li>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.</li>
	<li>Close the syringe ports with caps and discard the syringes in a sharps waste container.</li>
	<li>Attach each vessel to the rotator base and place the rotator base in an incubator with 5% CO2 and 37 &#176;C temperature.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Follow the same procedure by aspirating 75% of the nutrition-depleted medium and injecting fresh medium through the other port.</li>
</ol>
6. Lung organ preparation and bioreactor based culture
NOTE: E15 wild-type mouse pups were used for the preparation of lung organs.
<ol>
	<li>Dissect the lung tissue after euthanizing a pregnant female mouse with CO2 asphyxiation.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C temperature.</li>
	<li>Once the lung tissues attach to the membrane in a 2D organ culture plate, transfer the samples to the bioreactor vessel.</li>
	<li>Prepare the media for culture. 
	NOTE: The control DMEM high glucose&#160;media contains 10% fetal bovine serum (FBS), 1% antibiotic solution (P/S) (penicillin, 100 U/mL, streptomycin, 50 &#181;g/mL) and 100 &#181;g/mL ascorbic acid (AA) and for the induction of inflammatory conditions, control medium is supplemented with 5 ng/mL IL-6 protein.</li>
	<li>Carry out the lung culture experiment for 10 days with a media change every 48 hours in the bioreactor as described above.</li>
</ol>
7. Coated scaffold with Human periodontal ligament (hPDL) cell culture
<ol>
	<li>Culture human periodontal ligament cells. 
	NOTE: The cells are isolated and cultured in our lab as per the protocol previously published25.</li>
	<li>Coat the collagen scaffold discs with 30 &#181;L of PBS for the control group and the neuropeptide galanin (GAL) at a concentration of 10-8 overnight.</li>
	<li>Seed human PDL cells on the control and GAL coated scaffold for 2 hours at 37 &#176;C in a culture plate, and transfer the cell seeded scaffolds to a bioreactor vessel as mentioned above.</li>
	<li>Culture the cell seeded scaffolds for 14 days in the bioreactor prior to harvesting the scaffolds for analysis.</li>
</ol>
8. Bioreactor cleaning and maintenance
<ol>
	<li>After the scaffolds/cells are harvested from the bioreactor, remove the syringe ports from the vessel. 
	&#8203;NOTE: The screws around the vessels are taken off and the vessel is gently washed with soap water.</li>
	<li>Rinse the vessels with clean water and tighten the screws once more.</li>
	<li>Store the vessels after autoclaving until next use.</li>
</ol>

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The authors have no conflicts of interest to disclose.

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...

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 division1,2,3. 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 &#34;free-fall&#34; 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 aggregates6,7.
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 structures13,14,15. 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 undertaking18,19. 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.

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			<td>Antibiotic-antimycotic</td>
			<td>ThermoFisher Scientfic</td>
			<td>15240096</td>
			<td></td>
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		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A4544</td>
			<td></td>
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		<tr>
			<td>BGJb Fitton-Jackson Modification media</td>
			<td>ThermoFisher Scientfic</td>
			<td>12591</td>
			<td></td>
		</tr>
		<tr>
			<td>BIOST PGA scaffold</td>
			<td>Synthecon</td>
			<td>Custom</td>
			<td>Available from the company through a custom order</td>
		</tr>
		<tr>
			<td>BMP-2</td>
			<td>R&#38;D Systems</td>
			<td>355-BM</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP</td>
			<td></td>
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			<td>DMEM Media</td>
			<td>Sigma Aldrich</td>
			<td>D6429-500mL</td>
			<td></td>
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		<tr>
			<td>FBS</td>
			<td>ThermoFisher Scientfic</td>
			<td>16140071</td>
			<td></td>
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		<tr>
			<td>Fibricol</td>
			<td>Advanced Biomatrix</td>
			<td>5133-20mL</td>
			<td></td>
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			<td>Fibronectin</td>
			<td>Corning</td>
			<td>354008</td>
			<td></td>
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		<tr>
			<td>Galanin</td>
			<td>Sigma Aldrich</td>
			<td>G-0278</td>
			<td></td>
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			<td>Gelatin disc</td>
			<td>Advanced Biomatrix</td>
			<td>CytoForm 500</td>
			<td></td>
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			<td>Graphene sheets</td>
			<td>Advanced Biomatrix</td>
			<td>CytoForm 300</td>
			<td></td>
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			<td>hEGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
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			<td>hFGF</td>
			<td>ThermoFisher Scientfic</td>
			<td>AA1-155</td>
			<td></td>
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		<tr>
			<td>Hydroxyapatite disc</td>
			<td>Advanced Biomatrix</td>
			<td>CytoForm 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Il-6 protein</td>
			<td>PeproTech</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte SFM media (1X)</td>
			<td>ThermoFisher Scientfic</td>
			<td>17005042</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Corning</td>
			<td>354259</td>
			<td></td>
		</tr>
		<tr>
			<td>LRAP peptide</td>
			<td>Peptide 2.0</td>
			<td>Custom made sequence: MPLPPHPGSPGYINLSYEVLT 
			PLKWYQSMIRQPPLSPILPEL 
			PLEAWPATDKTKREEVD</td>
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			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Nitrocellulose membrane</td>
			<td>Merck Millipore</td>
			<td>AABP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>RCCS Bioreactor</td>
			<td>Synthecon</td>
			<td>RCCS 4HD</td>
			<td></td>
		</tr>
		<tr>
			<td>SpongeCol</td>
			<td>Advanced Biomatrix</td>
			<td>5135-25EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Syring valve one way stopcock w/swivel male luer lock</td>
			<td>Smiths Medical</td>
			<td>MX5-61L</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes with needle 3cc</td>
			<td>McKESSON</td>
			<td>16-SN3C211</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.25%)</td>
			<td>ThermoFisher Scientfic</td>
			<td>25200056</td>
			<td></td>
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propagation of dental and respiratory cells and organs in microgravity

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.

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...

Watch this Scientific Journal Video about Propagation of Dental and Respiratory Cells and Organs in Microgravity at JoVE.com

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.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

propagation-of-dental-and-respiratory-cells-and-organs-in-microgravity

Research

JoVE Journal

Bioengineering

1.9K Views.  Texas A&M College of Dentistry. 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. 

Video: Propagation of Dental and Respiratory Cells and Organs in Microgravity

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.