Clinostat and Bioreactor

On Earth, plant scientists have conducted many experiments to try to unravel the apparently simple responses of plants to the Earth's gravity. The basic problem is how to modify the amplitude of gravity so that developmental responses under different gravitational force levels can be studied. On of the earliest experimental devices for approaching this problem was to rotate material as Knight (1806) has done, but oriented horizontally on the periphery of a disc rotating in a vertical plane (see Figure 1-19C). The apparatus is called a clinostat, and the objective is to compensate the effect of the directional component of the gravitational force vector by having each part of the plant experience a multidirectional gravitational pull.

Plant organs can perceive small changes in orientation of the gravity vector. The length of time required to detect a response to a change in orientation is called the presentation time and ranges from 0.3 to 10+ minutes. The clinostat continuously changes the direction of the gravity field and the plant does not have sufficient time to respond to a change in direction. It is important that the number of revolutions per minute is great enough to assure that the gravitational force exerted upon the tissue from any direction is not sustained for long enough to permit an asymmetric growth response. As the disk in Figure 1-19C revolves, the plant will also rotate, causing the gravitational force vector to move relative to the plant with its direction always perpendicular to the long axis of the plant, but continually progressing

Clinostats

Figure 3-07. The three-dimensional clinostat, also called Random Positioning Machine (RPM), creates a condition in which the weight vector is continually reoriented in all directions. Photo courtesy o f Gilbert Gasset, GSBMS, Toulouse.

As seen above, botanists have used the slow clinostat for many years to study mechanisms of gravity perception. Recently animal investigators have begun to use the clinostat to study possible gravity responsiveness in embryonic structures. Clinostats have also been used for cultured cells: these

Figure 3-07. The three-dimensional clinostat, also called Random Positioning Machine (RPM), creates a condition in which the weight vector is continually reoriented in all directions. Photo courtesy o f Gilbert Gasset, GSBMS, Toulouse.

Shoot Clinostat

around it. In this way, a small seedling originally placed horizontally will continue to grow in a horizontal direction despite that its long axis is oriented at right angles to the gravitational force vector (Figure 3-06). If the rotational rate is too slow, however, the seedling will again show gravity-evoked bending of root and shoot towards its preferred positions.

It is clear that once a seedling has developed beyond the state of a single root and shoot and produces lateral roots, leaves or branches, a simple clinostat can no longer provide directional compensation of the force field to all parts of the plant. It is for this reason that some very sophisticated tumbling clinostats have been developed in attempts to maintain the fully compensated condition in growing plants over prolonged periods of time. A three-dimensional clinostat, for example, is a combination of two or three clinostats that keeps the specimen tumbling in the middle (Figure 3-07). Also, the fast clinostat, which rotates anywhere from 55-120 rpm, can be very useful for very small systems, such as a few cells, because it prevents the internal components of the cell from settling out or continually tumbling about. However, the organism must be kept in the very center of the clinostat stage, or there will be centrifugal forces. Some scientists have also used a tilted clinostat. If the clinostat is at a slight angle off horizontal, say at 9┬░, that will create roughly 1/6 g or 0.18 g, which is the level of gravity on the Moon. If it's tilted at about 17.5 deg, that will give 1/3 g, the level of gravity on Mars (Morey-Holton 2004).

are called rotating wall vessels or bioreactors (see Figure 4-12). These devices are also based on the concept that the g vector must act for at least a few seconds in a constant direction in order to generate an effect in cells. The bioreactor allows cells or bacteria to be cultured in a continuous free-fall state, simulating microgravity and providing a unique cell culture environment on the ground. Shaped like a cylinder, the growth medium-filled cylindrical vessel rotates about a horizontal axis, suspending the cells in a low-shear' culturing environment. Specimens in the bioreactor never hit bottom, they hang suspended in their liquid growth medium, much as they would in Earth's orbit. This allows for cell aggregation, differentiation, and growth.

The bioreactor provides a low-turbulence culture environment that promotes the formation of large, three-dimensional cell clusters. Cell constructs grown in the bioreactor more closely resemble tumors or tissues found in the body. Cell constructs grown in a rotating bioreactor on Earth eventually become too large to stay suspended in the nutrient medium. In the microgravity of orbit, however, the cells can stay suspended. But because there is a liquid medium, streaming during slow rotation generates Coriolis forces that may confound the experiment results. Also, coexisting objects of varying density will not be equally balanced within a rotating system as they are in a state of true freefall. The parameters affecting sedimentation and buoyancy differ for a bioreactor and actual free fall (Klaus 2001). These differences may contribute or be responsible for the altered behavior of biological systems observed to occur in space or under clinorotation.

Clinostats therefore provide "gravity compensation", not "zero gravity". Even the best clinostat technology on Earth cannot provide a facility to either investigate if there is an absolute dependence of plants or cells upon a gravitational force, or determine with certainty the mechanisms by which this force is perceived and responded to. In fact, experiments of 20-day clinostat rotation have identified problems with this method, indicating that the clinostat is a questionable simulator of weightlessness for long-duration studies. Some experiments on plant development showed that inflight plants did not appear significantly different from clinostat controls but the flight specimens took several hours to revert to normal gravitropism, whereas the Earth-clinostat controls rebounded immediately when removed from the rotating clinostat. Furthermore, clinostat rotation increased nuclear volume in some wheat seedling roots, but did not duplicate flight results at the cellular level. These results suggest that some spaceflight effects can be predicted with clinorotation, while others cannot.

For these reasons, many critical experiments have remained undone, and the ISS now offers the opportunity for tests in the 0-g environment with the proper inflight controls.

' Shear is the force caused by the cells sliding against one another.

Three Dimensional Clinostat For Humans

Figure 3-08. The ground-based gondola centrifuge used in the University Paul Sabatier in Toulouse to assess the long-term effects of microgravity on mice development. Four groups of animals, born and raised under conditions of normal gravity, are placed inside the gondolas to live for 1-2 months under 2 g. The light/dark cycle is provided inside the box. Food and water is available at will. However, the centrifuge rotation is stopped briefly every other day for housekeeping and maintenance. Photo courtesy of Gilbert Gasset, Groupement Scientifique en Biologie et M├ędecine Spatiales, Toulouse.

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Responses

  • asmarina
    How do you use a clinostat?
    2 years ago
  • ariam
    Why was the wound up clinostat used in the control in an experiment?
    9 months ago
  • abela piccio
    Why was a wound clinostast used in the control?
    8 months ago
  • semhar
    What will be happen if plants placed horizontally in arotating klinostat?
    2 months ago

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