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.

<table><tbody><tr><td>Antibiotic-antimycotic</td><td>ThermoFisher Scientfic</td><td>15240096</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>A4544</td><td></td></tr><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&amp;amp;D Systems</td><td>355-BM</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D Systems</td><td>314-BP</td><td></td></tr><tr><td>DMEM Media</td><td>Sigma Aldrich</td><td>D6429-500mL</td><td></td></tr><tr><td>FBS</td><td>ThermoFisher Scientfic</td><td>16140071</td><td></td></tr><tr><td>Fibricol</td><td>Advanced Biomatrix</td><td>5133-20mL</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>354008</td><td></td></tr><tr><td>Galanin</td><td>Sigma Aldrich</td><td>G-0278</td><td></td></tr><tr><td>Gelatin disc</td><td>Advanced Biomatrix</td><td>CytoForm 500</td><td></td></tr><tr><td>Graphene sheets</td><td>Advanced Biomatrix</td><td>CytoForm 300</td><td></td></tr><tr><td>hEGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>hFGF</td><td>ThermoFisher Scientfic</td><td>AA1-155</td><td></td></tr><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&lt;br/&gt; PLKWYQSMIRQPPLSPILPEL&lt;br/&gt; PLEAWPATDKTKREEVD</td><td></td></tr><tr><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></tr></tbody></table>

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...

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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

2.0K Views.  Texas A&M College of Dentistry. Langwerpig en gepolariseerd de amelogenine-afscheidende ameloblast in de cultuur. Dit is ook het eerste protocol voor het kweken van ameloblastcellen in microzwaartekracht. De groei van ameloblasten in de cultuur is een van de grootste uitdagingen op het gebied van glazuur geweest.En voor ons is het gebruik van dit bioreactormodel, het is de eerste keer dat we dit kenmerk in glazuuronderzoek hebben bereikt. Dit model kan worden gebruikt om de biologie van ameloblastcellen te begrijpen....

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Developmental Biology

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

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., ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Biology

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) ...

Immunology and Infection

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

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