1. Production of Medicinal Plant Compounds in a Microgravity-based Hydrodynamic Focusing Bioreactor (HFB)

Dr. Valluri holding up cultured plant cells in front of an incubator housing his HFB

Cell culture conditions in the simulated microgravity environment of Hydrodynamic Focusing Bioreactor (HFB) combine two beneficial factors: low shear stress, which promotes the assembly of tissue-like 3-D constructs; and randomized gravitational vectors, which affect production of medicinal compounds. Close apposition of the plant cells in the absence of shear forces presumably promotes cell-cell contacts, cell aggregation and cell differentiation. This process then might lead to the rapid establishment and expansion of aggregate cultures, which unlike cells cultured in conventional fermentors, are not disrupted by shear forces. The main direction of our research is to investigate the effects of microgravity on medicinal plant cell cultures, in particular the events occurring at the membrane level and providing the transduction of primary microgravity effects in the production of medicinal secondary metabolites. This project evaluates the 3-D in vitro growth and biomass characteristics and production of medicinal compounds from various plant cell suspension cultures.

150 ml Manual Hydrodynamic Focusing Bioreactor inside a temperature and light controlled incubator.

150 ml Perfused Hydrodynamic Focusing Bioreactor inside a carbon dioxide incubator.

 

Madagascar periwinkle, and it’s callus grown on a modified MS medium (20g/L Sucrose) (left 2 images).  Proliferation of Sandalwood callus three weeks after explantation on MS medium (20g/L Sucrose).  Picture 2 shows the creation of single cell suspension from the callus.  This occurs at 120 rpm on an orbital shaker (right 2 images).

 

This project represents a unique, cooperative effort between our laboratory and the Biotechnology Program at the NASA - Johnson Space Center. This proposed collaboration combines the expertise and experience in three-dimensional growth and differentiation of plant cells and production of high value medicinal compounds from plant cells at Marshall University with the microgravity-based, three-dimensional culture technology and processes at the Johnson Space Center. This project utilizes NASA bioreactor technology to address the role of gravity in the formation and differentiation of three-dimensional plant tissue and in regulating their cellular physiology for the production of medicinal compounds.

 

Products of primary and secondary metabolism in medicinal plants (left).  The chemical structures of the anticancer compounds vinblastine and vincristine derived from Catharanthus roseus (Madagascar periwinkle), (right).

 

 

Confocal Microscopy reveals the cell-to-cell connections of Sandalwood 3-D aggregates generated by a low shear stress environment of the HFB (left)...  and the viability of the Sandalwood cell aggregates (right).  The green auto-fluorescence of the chloroplasts shows that the cells are maintaining photosynthesis.

 

  

Periwinkle 3-D aggregate inside the manual HFB on day three (20rpm) (left).  Sandalwood 3-D aggregates inside the perfusion HFB on day three (20rpm) (right).

 

 

An HFB onboard the KC-135 aircraft used to simulate low-gravity in Earth's atmosphere.

Photo: from an article by Dr. Charles E. Niederhaus of NASA

(http://www.grc.nasa.gov/WWW/RT/2003/6000/6712niederhaus.html)