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Development of an inexpensive 3D clinostat and comparison with other microgravity simulators using Mycobacterium marinum

Year: 2022
Type: article
Abstract: 2D and 3D Clinostats are used to simulate microgravity on Earth. These machines continuously alter the sample’s orientation, so the acceleration vector changes faster than the biological endpoint being monitored. Two commercially available microgravity simulators are the Rotary Cell Culture System (Synthecon Inc.), which is a 2D clinostat, and the RPM 2.0 (Yuri), which is a 3D clinostat that can operate as a random positioning machine or in constant frame velocity mode. We have developed an inexpensive 3D clinostat that can be 3D printed and assembled easily. To determine the optimal combination of inner (I) and outer (O) frame velocities to simulate microgravity, two factors were considered: the time-averaged magnitude and the distribution of the acceleration vector. A computer model was developed to predict the acceleration vector for combinations of frame velocities between 0.125 revolutions per minute (rpm) and 4 rpm, and a combination of I = 1.5 rpm and O = 3.875 rpm was predicted to produce the best microgravity simulation. Two other frame velocity combinations were also used in further tests: I = 0.75 rpm and O = 3.625 rpm, and I = 2 rpm and O = 1.125 rpm. By operating the RPM 2.0 in constant velocity mode at these three velocity combinations, the RPM 2.0 algorithm data confirmed that these operating conditions simulated microgravity. Mycobacterium marinum was selected for biological comparison experiments as this bacterium can grow as a biofilm or a planktonic culture. Biofilm experiments revealed that the RPM 2.0 and the 3D clinostat with I = 1.5 rpm and O = 3.825 rpm produced similar structures in attached biofilm, and similar changes in transcriptome for the bacteria in suspension compared to the normal gravity transcriptome. Operating the 3D clinostat at I = 2 rpm and O = 1.125 rpm, and the Synthecon 2D clinostat in simulated microgravity orientation at 25 rpm resulted in the same decreased planktonic growth and increased rifampicin survival compared to normal gravity. This study validates the inexpensive 3D clinostat and demonstrates the importance of testing the operating conditions of lab-developed clinostats with biological experiments.
DOI: 10.3389/frspt.2022.1032610
Authors: Joseph L. Clary, Creighton S. France, Kara Lind, Runhua Shi, J.Steven Alexander, Jeffrey T. Richards, Rona S. Scott, Jian Wang, Xiao-Hong Lu, Lynn Harrison
Institutions: Louisiana State University Health Sciences Center Shreveport, Louisiana State University, Feist-Weiller Cancer Center, Kennedy Space Center, National Aeronautics and Space Administration, Amentum (United States)
Language: English
Subfield: Physiology
Field: Medicine
Open Access status: gold
Estimated APC paid: $1,900
Award: THE OBJECTIVE OF THIS PROPOSAL IS TO UNDERSTAND THE MECHANISMS USED BY MYCOBACTERIA TO ADAPT TO LIFE UNDER LSMMG AND TO DETERMINE WHETHER THEIR ABILITY TO FORM BIOFILM IS ALTERED UNDER LSMMG. M. MARINUM WILL BE THE MODEL ORGANISM. IT IS A GOOD MODEL FOR THE MORE POTENT HUMAN PATHOGENIC MYCOBACTERIA AS IT IS A BSL2 ORGANISM. M. MARINUM WILL BE GROWN IN HIGH ASPECT RATIO VESSELS USING THE ROTATING CELL CULTURE SYSTEM TO SIMULATE MICROGRAVITY. PREVIOUSLY WE WERE FUNDED BY NASA FOR 1 YEAR TO EXAMINE HOW M. MARINUM ALTERED GROWTH AND GENE EXPRESSION UNDER LSMMG. OUR PREVIOUS TRANSCRIPTOME ANALYSIS SUGGESTS THAT THE REL AND SIGMA FACTOR H (SIGH) STRESS RESPONSE PATHWAYS ARE ACTIVATED WHEN M. MARINUM ARE SUBJECTED TO LSMMG. WE PLAN TO EXTEND THESE STUDIES WITH THIS PROPOSAL. AIM 1 WILL DECREASE TRANSCRIPTION FROM THE REL GENE AND DETERMINE HOW THE LOSS OF THIS PATHWAY ALTERS M. MARINUM SURVIVAL AND ANTIBIOTIC RESISTANCE UNDER LSMMG. WE WILL USE CRISPR INTERFERENCE (CRISPRI) TO DISRUPT REL TRANSCRIPTION AND RNASEQ TO EXAMINE THE TRANSCRIPTOME. THIS WILL CONFIRM OUR HYPOTHESIS THAT THE REL STRINGENT RESPONSE IS NECESSARY FOR THE SURVIVAL OF LSMMG-GROWN M. MARINUM. WE ARE CURRENTLY STUDYING LSMMG AND THE SIGH TRANSCRIPTOME USING A SMALL GRANT FROM THE LASPACE CONSORTIUM. AIM 2 WILL EXAMINE BIOFILM FORMATION UNDER LSMMG USING MICROCARRIERS FOR BACTERIA ATTACHMENT IN THE HARVS. WE WILL ALSO EXAMINE THE TRANSCRIPTOME RESPONSE OF THE BACTERIA IN BIOFILMS. WE WILL USE WILD-TYPE AND CRISPRI-MODIFIED M. MARINUM WITH DECREASED SIGH OR REL TRANSCRIPTS IN AIM 2. WE HYPOTHESIZE THAT BIOFILM FORMATION WILL BE ENHANCED UNDER LSMMG AND THAT THE REL AS WELL AS SIGH STRESS RESPONSE PATHWAYS WILL BE REQUIRED FOR BIOFILM FORMATION. THIS WILL EXTEND OUR UNDERSTANDING OF MYCOBACTERIA SUBJECTED TO SPACE TRAVEL AND POTENTIALLY REVEAL MECHANISMS TO TARGET MYCOBACTERIUM INFECTIONS IN SPACE AND ON EARTH. THE PROPOSED WORK IS THEREFORE CONSISTENT WITH THE OBJECTIVES OF THE NASA SPACE BIOLOGY PROGRAM.
OpenAlex ID: W4307701815
FWCI: 1.867
Cites: 59
Cited by: 20
Related to: 10