Microgravity and Cells:  Morphotype and Phenotype Correlation (Cell Shape and Expression) - 11.22.16

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

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Microgravity causes many changes to living organisms, beginning at the cellular level. Microgravity and Cells: Morphotype and Phenotype Correlation (Cell Shape and Expression) studies how microgravity changes the physical structure of cells, and whether this affects the way they function. Results aim to provide an experimental model that highlights the relationships among microgravity, cell shape and gene expression, and whether new drugs may be able to counteract these microgravity-induced changes.
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The following content was provided by Marco Vukich, and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: Cell Shape And Expression

Principal Investigator(s)
Marco Vukich, Kayser Italia Srl., Livorno, Italy

Co-Investigator(s)/Collaborator(s)
Alessandro Palombo, Ph.D., University of Rome, Roma, Italy

Developer(s)
Kayser Italia Srl., Livorno, Italy
University of Rome - Department of Clinical and Molecular Medicine - "Sapienza", Italy

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
Italian Space Agency (ASI)

Research Benefits
Scientific Discovery

ISS Expedition Duration
March 2015 - September 2015

Expeditions Assigned
43/44

Previous Missions

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Experiment Description

Research Overview

  • On Earth, it is well known that modification of the cytoskeleton can be related to a pathological state of the cell. Cells exposed to microgravity display architectural and cytoskeleton change, as well as profound biochemical and genetic modifications. Those changes ultimately result in biological functions impairment and physiological failure. The Cell Shape and Expression investigation provides a look into the results of those changes.
  • It has been suggested that microgravity-induced modification on cytoskeleton (CSK) structure results in shape and subsequent gene-expression changes. According to that hypothesis, wide alteration in gene expression profile might be considered as secondary consequence of the overall structural reconfiguration of cells fostered by microgravity.
  • However, such correlation has not been clearly yet established. Beside the theoretical relevance of such a relationships, the dependence of genetic changes on shape structural stability implies that by stabilizing the CSK some microgravity effects could be neutralized.
  • Given that experimental and pharmacological/nutritional manipulation of microenvironment has proven to preserve CSK and cell shape conformation, it is tempting to speculate if such measure could efficiently counteract microgravity-related changes in CSK and cell shape.

Description

Morphogenesis and phenotypic differentiation are time and space-dependent processes:  morphological plasticity, rather than being the result of genetic “adaptation”, reflects the influence of external physico-chemical parameters on any material system and is therefore an inherent, inevitable property of organisms. The physical setting integrating the different chemical as the physical signals that drive cells and tissues towards differentiation is known as the “morphogenetic field”. Within this field, morphogenetic cues exert short and long-range influences by affecting gradients of morphogens and mechanical stresses. This process is strongly dependent from the geometry of the morphogenetic field governing the topology of signaling cues. Within this framework, the geometric form a cell acquires – that is to say, its shape - represents the integrated end point of the morphogenetic cues acting on the living system: morphogenesis is indeed the process through which a population of cells rearranges into a distinctive shape. Microgravity exerts several, relevant effects on living organisms at both molecular and supra-molecular level. Namely, cells exposed to microgravity display architectural and cytoskeleton changes, as well as profound biochemical and genetic modifications. Those changes ultimately result in biological functions impairment and physiological failure. It has been suggested that microgravity-induced modification on cytoskeleton (CSK) structure results in shape and subsequent gene-expression changes. According to that hypothesis, wide alteration in gene expression profile might be considered as secondary consequence of the overall structural reconfiguration of cells fostered by microgravity. However, such correlation has not been clearly yet established. Beside the theoretical relevance of such a relationship, the dependence of genetic changes on shape structural stability implies that by stabilizing the CSK some microgravity effects could be neutralized. Given that experimental and pharmacological/nutritional manipulation of microenvironment has proven to preserve CSK and cell shape conformation, it is tempting to speculate if such measures could efficiently counteract microgravity-related changes in CSK and cell shape. This is the purpose for the Cell Shape and Expression investigation.
 
Breast cancer cells (MCF7), are seeded in both a control culture-medium (CTRL sample) and in a culture medium supplemented with melatonin (treated sample). The latter medium due to the capability of melatonin to counteract the exposure to microgravity (melatonin is a stabilizing agent of the CSK), will represent a countermeasure-like control for the experiment. MCF7 CTRL and MCF7 treated with melatonin is cultured on normal gravity field (ground control), in simulated microgravity on ground (simulated-microgravity control) and on board of the ISS (true microgravity field).
 
To manage the samples as required, each KIC contains one EU, and is powered and the cells are incubated at 37°C by the Kubik.
 
Experiment on ISS: The EU is designed to allow a liquid culture of cells plated on a solid support. Cells are activated, incubated, grown, and fixed in a fully autonomous way. The EU makes possible analyses both on cells (at cytological and molecular level) and on the growth media (being the exhausted growth medium collectable).
 
Preflight
A biological laboratory next to the launching pad is required for the preliminary operations described above. The cells are seeded in conventional flasks before their insertion in the KIC. Twenty-four hours before the launch the cells (3x103 cells/cm2) are seeded in each EU that is, in turn, integrated in the KIC experimental container.
 
In-flight
At maximum of 24 hours after docking, the samples start incubation in Kubik at 37°C. All the actions required for medium changes and fixation take place automatically.
 
Once incubation starts, 2 samples (i.e. 2 different EUs, respectively containing cells in control culture-medium or culture-medium added with melatonin) are fixed by T0 +2h. Per each EU, it requires a first washing with PBS buffer, fixation in NotoxHisto, and final washing in PBS to avoid over-fixation.
 
These samples are kept at +4°C until the end of the flight by stowing the KICs on board in a cold container and by returning in cold stowage. The other 6 samples are kept in Kubik for further incubation (still at 37°C) for a maximum of 47 hours.
 
Medium refresh takes place in the remaining 6 EUs by T0 +3h (refresh is performed in 3 EUs with control culture-medium and in 3 EUs culture-medium added with melatonin).
 
Fixation occurs in the 6 remaining samples by T0 +51h. Per each EU, it requires a first washing with PBS buffer, fixation in NotoxHisto, final washing in PBS to avoid over-fixation.
 
All these samples are kept at +4°C until the end of the flight by stowing the KIC on board in a cold container and by returning in cold stowage.
 
Postflight
On ground, the condition of cell cultures are assessed by optical microscopy. NOTOXHisto fixed samples are treated specifically for optical and confocal microscopy studies. Acquired images are analyzed by technical methods that measure nuclear and cytoplasmic membrane profiles of both single cells (Normalized Bending Energy, NBE; Fractal Box Counting; Lacunarity) and of clusters of cells (Fractal Analysis and Entropy). On waste mediums, immunoassays are used for detecting growth factors, enzymes, and proteins such as ß-casein (functional hallmark of differentiation).
 
The protocol described above is applied on ground as well, under gravity and under microgravity simulated through a Random Positioning Machine (RPM).

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Applications

Space Applications
A reduced-gravity environment induces major changes in cell structure and function, including changes to the cytoskeleton. On Earth, modified cytoskeletons are related to cell damage. This investigation studies how microgravity changes cell shape, and whether the changes in structure cause secondary genetic changes. Results can shed more light on whether drug treatments designed to preserve cytoskeleton shape could also prevent cell damage.

Earth Applications
The International Space Station (ISS) provides a unique way to study how physical forces affect cells, and how these forces shape the health of living things. Shear stress, stiffness, surface tension, and gravity itself all exert forces on cells, causing physical changes that result in a cascade of reactions. This investigation benefits research for diseases in which the cell cytoskeleton is involved, including cancer, osteoporosis and many others.

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Operations

Operational Requirements and Protocols

Resources required by the experiment include an allocation in a pressurized environment,upload of 8 KICs integrated in the BIOKIT, downloads of 8 KICs (BIOKIT may be disposed after retrieval of KICs). The overall launch mass of the experiment is 1.28 kg (for 8 KICs) + 8.52 kg (BIOKIT). The overall volume at launch is 23100 cm3 (volume of BIOKIT; the 8 KIC contained in the BIOKIT). The volume on board ISS is 1200 cm3 (8 KIC), the BIOKIT may be discarded after arrival on ISS). Crew time required is 80 minutes, it includes extraction of KICs from BIOKIT, insertion in Kubik, removal from Kubik and operations with cold stowage. Data is downloaded after the return to earth.

After docking to the ISS the BIOKIT is transferred as soon as possible to the Columbus Module (within 24 hours) where the KUBIK is, the KIC ECs are extracted from the BIOKIT and inserted into the KUBIK. The temperature of the KUBIK is preset to 37°C in advance. When all the KIC ECs are inside KUBIK, the KUBIK lid is closed and the experiment starts. Within 2 hours of incubation, a subset of the KIC ECs is removed from the KUBIK and transferred to cold stowage at 4°C. After not later of 51 hours from start of incubation, the remaining set of the KIC ECs is removed from the KUBIK and transferred to cold stowage at 4°C.

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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

image NASA Image: ISS043E125592 - ESA astronaut Samantha Cristoforetti is photographed configuring and performing a checkout of Kubik-5 in support of Cell Shape and Expression.
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image NASA Image: ISS043E122392 - Photographic documentation taken of Kubik-5 in support of Cell Shape and Expression experiment.
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Kubik Interface Container (KIC) outside of Kubik. The KIC offers a dedicated environment for the execution of life science experiments in microgravity and provides a Level of Containment. Image courtesy of the Italian Space Agency (ASI).

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Kubik Interface Containers (KIC) inside of Kubik. The KIC offers a dedicated environment for the execution of life science experiments in microgravity and provides a Level of Containment. Image courtesy of the Italian Space Agency (ASI)

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The fluidic scheme of the Cell Shape and Expression Investigation. Image courtesy of the Italian Space Agency (ASI).

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BIOKIT: passive thermal conditioning systems based on phase change materials, which allows to maintain the biological samples at a controlled temperature in the range of 30°C-37°C without the need for power supply. Image courtesy Italian Space Agency (ASI).

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