The Effect of Microgravity on Stem Cell Mediated Recellularization (Lung Tissue) - 07.12.17

Overview | Description | Applications | Operations | Results | Publications | Imagery

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The Effect of Microgravity on Stem Cell Mediated Recellularization (Lung Tissue) uses the microgravity environment of space to test strategies for growing new lung tissue. Using the latest bioengineering techniques, the Lung Tissue experiment cultures different types of lung cells in controlled conditions aboard the International Space Station (ISS). The cells are grown in a specialized framework that supplies them with critical growth factors so that scientists can observe how gravity affects growth and specialization as cells become new lung tissue.
Science Results for Everyone
Information Pending

The following content was provided by Stefanie Countryman, M.B.A., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: Lung Tissue

Principal Investigator(s)
Joan E. Nichols, Ph.D., Galveston National Laboratory, UTMB, Galveston, TX, United States
Joaquin Cortiella, M.D., Department of Anesthesiology, UTMB, Galveston, TX, United States
Alessandro Grattoni, Ph.D., Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX, United States

Co-Investigator(s)/Collaborator(s)
Information Pending

Developer(s)
BioServe Space Technologies, University of Colorado, Boulder, CO, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Earth Benefits, Scientific Discovery, Space Exploration

ISS Expedition Duration
April 2017 - September 2017

Expeditions Assigned
51/52

Previous Missions
Information Pending

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Experiment Description

Research Overview

  • Recent work has focused on development of decellularized human natural lung as a scaffold to support the growth of lung tissue.
  • The ultimate goal of The Effect of Microgravity on Stem Cell Mediated Recellularization (Lung Tissue) is to produce bioengineered human lung tissue that can be used as a predictive model of human responses allowing for the study of lung development, lung physiology or disease pathology.
  • Lung Tissue studies what happens to lung function and repair during long term spaceflight. A human lung mimic is used to study the effects of spaceflight (gravity loss, hyperoxia and radiation exposure) on human lungs.
  • Development of decellularized whole human lung scaffolds from human lungs allows the production of numerous human lung scaffold pieces (see Image 2) that can be used to tissue-engineer lung tissue.
  • Recellularization of these scaffold pieces with selected lung cell populations results in the formation of individual pieces of viable lung tissue that can be used to examine lung response to a variety of test conditions including the influence of long-term spaceflight.
  • Human lung tissue mimics can be used to focus on a few cell populations at a time and simplify complex mechanisms of pathogenesis in order to better understand the processes involved in development of lung disease.
  • Tissue mimic models such as this also have the potential to be used for assessing drug or chemical toxicity by biotechnology and pharmaceutical companies and could allow for rapid testing of new chemicals and compounds, considerably lowering the overall costs for research and development of new drugs.

Description

The Effect of Microgravity on Stem Cell Mediated Recellularization (Lung Tissue) research team has developed a tissue engineered microphysiologic human organ culture model (MHOC) of lung epithelium and endothelium which can be used to study lung physiology, response to trauma and development of lung disease in vitro. This system allows for deconstruction of complex mechanisms of pathogenesis in order to simplify the study of individual components involved in lung disease such as in development. Human lung stem and progenitor cells are seeded onto an acellular lung scaffold. A bottom-up management strategy is used for the process of production of adequately formed bioengineered tissues. A bottom up synthesis method implies that the first step in the process is to produce the micro- or nano-structures, which gives rise to appropriate cell-to-cell interactions leading to tissue formation. Nano or micro-particles are loaded with selected growth factors to facilitate cell-scaffold attachment, cell viability and tissue development. Because of this, efforts are focused on development of the alveolar-capillary junction which includes both alveolar epithelial cells and endothelial cells. In fetal development these junctions form in the lungs due to the interplay between both cell types. Early studies to examine the role of extracellular matrix (ECM) in embryonic stem cell differentiation were done using acellular (AC) rat lungs as a model system. Later we designed a simple bioreactor system to support the development of small (2.5-3.5cm3) pieces of lung tissue. This allows for the modeling of key cellular responses to environmental changes or growth factors on a small scale. The fluidics system and culture chamber that supports the MHOC lung cultures are shown are used in this approach in order to study the processes that influence the development of alveolar tissue formation. The research team has worked to understand the steps that are required to fully recellularize the acellular lung scaffold by modeling processes and growth factor delivery systems to induce sustainable cell expansion, angiogenesis and lung alveolar development in MHOC model. Lung tissue grown in this system is very similar to normal lung. This small-scale model allows for cheap and efficient testing of the ability of environmental changes or pathogen exposure that alters cell expansion and lung tissue formation. The same stepwise approach is then applied to formation of whole pediatric sized bioengineered lungs and allows for identifying critical growth factor and physical cues that drive maturation of the tissues in whole organ bioengineering. Initial pilot transplantation studies have proved the suitability of the stepwise approach to production of bioengineered lung tissue.
 
The MHOC model of the lung used in these experiments was developed by Joan Nichols and was supported by NCATS U18 grant, Grant No. 1-U18-TR-000560. Methods for use of the MHOC lung culture for this type of testing have already been defined. Assessment of MHOC tissues includes analysis by transmission electron microscopy, histological examination of tissue following immunostaining and isolation of cells from the engineered tissue by flow cytometry. Immortalized lung and vascular cells and primary lung and vascular cells isolated from discarded human lungs are used to examine the samples sent to the ISS in these preliminary experiments. The phenotype of the primary lung cells isolated and the subsequent immortalized lung cells created is primarily alveolar epithelial type II cells (AECII) (78%) but also containes alveolar epithelial type I cells (AECI), smooth muscle cells, fibroblasts and mesemchymal stem cells (MSC) as well as other progenitor cells. Acellular human lung scaffold in previous studies was cut to appropriate sized pieces for use in either microfluidic supported 6-well plates or in a specialized culture chamber developed by Joan Nichols with Synthecon, Houston, Texas. For spaceflight the scaffold is cut to fit into bioreactor bags. Using this system numbers of cells required are estimated to support tissue formation, cell phenotypes required to produce functional alveolar-capillary junctions and length of culture time required. In previous studies the MHOC lung model system was used to examine the influence of PRP on cell attachment and tissue formation. In this study platelet-rich plasma (PRP) or AB serum was delivered in a hydrogel carrier. Data supported use of PRP to promote vascular tissue growth in the small MHOC lung model and was then adjusted for use in production of full sized single adult human lungs with good results supporting the concept of testing key factors and delivery platform prior to application in large whole organ culture. In past studies the group used hydrogel colloidal crystal scaffolds to promote cell expansion and differentiation in a bone marrow analog. In this situation layer-by–layer modification of inverse colloidal crystal scaffolds was accomplished by coating with sequential layers of negatively charged 0.5% (w/w) clay platelets (average dimension of 1nm thickness and 70–150 nm in diameter, Southern Clay Products) and positively charged 0.5% (w/w) poly (diallyldimethylammonium chloride) (PDDA, MW=200,000) (Sigma) solution utilizing a layer-by-layer (LBL) surface coating technique. Growth factors to drive cell responses were layered onto the hydrogel scaffold using this method. Similar layer-by-layer methodologies can be used to attach growth factors to the AC scaffold and it is the intention to develop this technology further as part of this proposal. In recent work, a porous silicon nanoparticle that was developed to be easily loaded with chemotherapeutics for cancer treatment as a delivery vehicle for growth factors within the scaffold was used.
 
In order to promote endothelial cell attachment and spreading PF-127 hydrogel was used to coat the inner portion of acellular vascular scaffolds in order to determine what factors/delivery methods enhanced cell attachment. Vascular endothelial growth factor -2 (VEGF2) release along the length of the vascular ECM of the AC scaffold combined with nanoparticle release of VEGF in smaller blood vessels and capillaries provided for site localized vascular tissue formation. Porous silicon nanoparticles were used in these studies. Endothelial CD31+ cells preferentially attached to the ECM of the acellular vascular matrix of the scaffold pretreated with VEGF nanoparticles. Development of endothelial cells in the model is critical to the production of a robust model of the lung.
 
The MHOC model can be constructed to include cell phenotypes of interest such as MSCs or components of the innate immune response such as macrophages. Scientists have known since the early days sending humans into space that living in the microgravity environment results in suppression of the human immune system. Nothing is known regarding the influence of spaceflight on the function of lung stem or progenitor cells or on MSCs which play a major role in modulating human immune responses and providing support for generation of new lung tissue following trauma or disease. MSCs are multipotent stem or progenitor cells that can be isolated from bone marrow, umbilical cord, adipose tissue including peripheral blood. MSCs interact with human macrophages to augment human immune responses in the lung. Along with their self-renewing capacity and ability to differentiate into multiple cell lineages, these cells also have immunomodulatory functions that suppress inflammatory immune responses. MSCs and macrophages in our bodies normally play a significant role in tissue repair after injury. Even in the lung, repair mechanisms rely on the capacity of the lung’s resident MSCs to migrate to sites of injury, modulate immune responses in the lung and help in tissue repair. These qualities have made them attractive candidates for clinical applications in stem cell therapies, allograft rejection in transplantations, regenerative medicine and autoimmune disease.
 
A microphysiologic human organ culture (MHOC) model of the lung is used to examine lung development and repair. Currently the intention is to use this model in a CASIS-supported study to evaluate the effect of spaceflight related microgravity and radiation on stem and/or progenitor cells grown on a novel scaffold of human acellular lung scaffolds treated with nanoparticles containing vascular endothelial growth factor and other critical factors supporting lung tissue formation. In order to modify the model to fit into already existing bioreactor systems modifications have been made to the size and shape of the scaffolds used and the chamber the models are cultured within. The MHOC lung models are produced on earth and then once tissue formation has been established the model is sent to the ISS.
 
This project aids in understanding of the influence of spaceflight on lung tissue development and lung repair. Lung Tissue evaluates human lung tissue responses to microgravity and radiation exposure and in future projects allows for the examination of influence of hyperoxia as well as well as potential for development of oxidative lung damage during long-term spaceflight. The Lung Tissue hypothesis is that spaceflight alters the ability of human lung progenitor cells to function and macrophages to function due to low gravity conditions and radiation levels experienced on the ISS. The conditions of spaceflight may influence the proliferation and self-renewal capacity of MSCs and lung progenitor cells and may alter the capacity of these cell types to differentiate into mature cell lineages necessary for lung tissue repair.
 
In order to understand the impact that spaceflight has on MSC function it is intended to compare the responses of MSCs and lung progenitor cells cultured on acellular human scaffolds on the ISS to similar cultures on earth. Using the space station laboratory of the ISS the capacity of MSCs and lung progenitors to function in space as described in the following aims is examined:
 
Aim 1) The influences of low gravity conditions and radiation levels experienced by astronauts during spaceflight on lung stem and progenitor cell attachment, tissue formation and MSC responses are examined.
 
Aim 2) The influences of the low gravity conditions and radiation levels experienced on the ISS on human macrophages cocultured with our lung model system are examined. Cell activation and production of immunosuppressive factors or cell specific products (neuronal, neuroendocrine, lung epithelial or endothelial) indicative of tissue repair or tissue damage are evaluated.
 
Production of MSC and macrophage cell products that are involved in the normal control of lung inflammation following infection or injury and potentially play a role in suppression of cancer are evaluated. These experiments may even eventually allow for design of strategies to modulate MSC or macrophage responses in space which decrease health risks associated with immune suppression and other health risks of long term spaceflight on humans which may benefit individuals suffering from immunosuppressive conditions on earth.

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Applications

Space Applications
Lung Tissue demonstrates how the low gravity environment of space can serve as a laboratory for making life science discoveries and developing new medical therapies. Experiments like Lung Tissue advance understanding of how stem cells work and pave the way for further utilization of microgravity environments in stem cell research. Bioengineering therapies can also ultimately support long-term missions by providing tissue repair strategies in response to exposure, injuries and other medical emergencies.

Earth Applications
Lung Tissue contributes to understanding of how stem cells develop into functional tissue. This research assists Earth-based efforts at developing complex bioengineered tissue that can be used to repair damaged organs or reduce organ rejection.

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Operations

Operational Requirements and Protocols

The cells are flown live in tissue culture bags to the ISS via conditioned stowage assets (37°C) within BioCell Habitat containers that are charged with 5% CO2. Once on board, the BioCell habitats are placed inside SABL with the ACM unit which will provide temperature and CO2 control for the cell cultures. The cells are cultured for approximately 5 weeks with periodic sampling. Fluid manipulations will occur within the MSG, disposable glove bag, or other appropriate location. Necessary accessory hardware and kits for these operations and procedures are included in the suite of support items. Once the cultures have grown for a predetermined amount of time (5 time points with 6 bags being processed per time point) a 4.5 mL sample is pulled from the bag and frozen at -80°C for the remainder of the flight and then -20°C or colder for return. The cells remaining within the bags are fixed with paraformaldehyde (PFA) and stored at <4°C for the remainder of the flight and return and returned within bubble bags. Fixative kits, other than samples, and support kits are discarded.

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Decadal Survey Recommendations

Information Pending

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Results/More Information

Information Pending

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Related Websites

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Imagery

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Human lung scaffold pieces (0.5 mm3) can be made, for use as a scaffold to support growth of bioengineered lung for research studies like Lung Tissue. Image courtesy of Joan Nichols.

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The microphysiologic human organ culture (MHOC) lung platform. (A) acellular human scaffold material and cartoon of cell attachment in the scaffold cushion. (B) image of platform for MHOC produced in collaboration with Synthecon, Houston, Texas. (C) Chamber that houses MHOC (D) Hematoxylin and Eosin stained normal (D) and (E) (MHOC grown lung (F) at the bottom, is the full system with microfluidic pumps, pump control box and reservoirs for media and waste. Image courtesy of Joan Nichols.

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Bioreactor bag for culture of the MHOC lung samples. Image courtesy of Joan Nichols.

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Examination of Scaffold Platelet Rich Plasma (PRP) Pretreatment on Cell Attachment (a, d, g) Influence of cell numbers used or pretreatment of AC scaffolds with AB serum or PRP on cell attachment and tissue formation. 2.5 cm3 pieces of AC scaffold were untreated, treated with AB serum or with PRP and then seeded with (a-c) 100,000 primary human lung cells, (d-f) 500,000 primary human lung cells or (g-i) 1,000,000 primary human lung cells. Averaged values for numbers of recovered cells and viable cells are shown. Significantly higher levels of viable cells were collected after 7 days of culture following treatment with AB serum (*P<.005) compared to untreated scaffolds. Treatment with PRP (**P<0.0005) was always significantly better than untreated or AB serum treated samples. Representative 7µm H & E stained sections of (b, c) untreated, (e, h) human AB serum treated or (f, i) PRP pretreated scaffolds indicated that increased cell attachment occurred in PRP treated pieces of AC scaffold. Bar= 50 mm for b, c, e, f, h, i. Higher cell numbers combined with PRP-pretreatment of scaffolds also produced better tissue formation. Image courtesy of Joan Nichols.

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Comparison of Growth factor delivery methods used in production of the MHOC lung. (A) acellular lung vascular scaffold showing vascular ECM (arrow). (B Hydrogel delivered VEGF stained for presence of VEGF (green) and CD31 (red).) (C and D) Control (C) and (D) VEGF hydrogel treated scaffolds cultured in the MHOC lung system showing DAPI stained cell nuclei indicating cell attachment (E and F) image of nanoparticles developed for use in these studies. (G) Nanoparticles loaded with VEGF2 immediately after injection into vascular scaffold. Section was stained for presence of VEGF2. (H) Untreated control showing DAPI stained and no CD31 (red) indicating endothelial cell attachment and (I) section of tissue piece in G stained for CD31 an endothelial cell marker (red). Unpublished data. Image courtesy of Joan Nichols.

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