Micro-5 Conceptual Design Review

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Micro-5 Conceptual Design Review
Jake Freeman Jon Beno Luis Zea
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Our Mission Work with partners to conduct space
life sciences research to advance fundamental
science, applied research and commercial
interests.
More Information www.colorado.edu/engineering/Bio
Serve/

• Based at Univ. of Colorado Aerospace
  Engineering Sciences
• Operating since 1987
• Faculty, staff and students with extensive space
  life sciences and payload engineering expertise

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Summary

• Problem Increased virulence in microgravity is
  dangerous for long-term spaceflight.
• Hypothesis Media ion concentration affects
  virulence.
• Experiment Infect C. elegans with S. typhimurium
  in media of differing ion concentrations
• 3 media formulations
• With and without Pi-PEG nutritional
  countermeasure
• Measurements
• Video analysis to determine time until death
  (TD50)
• Gene expression assays of fixed samples
  (RNALaterII)
• Pathogen localization studies of fixed samples
  (4 PFA)

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Separate Studies TD50 and Sampled

• The TD50 cultures will be monitored from start to
  finish of infection process
• The sampled cultures require large volume samples
  taken during infection
• Therefore the two studies must be performed on
  separate cultures

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Experiment Flow TD50
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Experiment Flow - Sampled
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36 Cultures for Each of TD50 and Sampled Studies

• o Uninfected controls 6 media conditions x 3
  technical replicates 18
• C. elegans LB
• C. elegans LB-M9
• C. elegans LBPO4
• C. elegans LB Pi-PEG
• C. elegans LB-M9 Pi-PEG
• C. elegans LBPO4 Pi-PEG
• o Infected with S. Typhimurium 6 media
  conditions x 3 technical replicates 18
• C. elegans S. Typhimurium cultured in LB
• C. elegans S. Typhimurium cultured in LB-M9
• C. elegans S. Typhimurium cultured in LBPO4
• C. elegans S. Typhimurium cultured in LB
  Pi-PEG
• C. elegansS. Typhimurium cultured in LB-M9
  Pi-PEG
• C. elegansS. Typhimurium cultured in LBPO4
  Pi-PEG

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Multiplicity of Infection

• Is the ratio of pathogens to hosts
• Key factor for host-pathogen interaction
• Affected by
• Inoculum concentration
• Inoculum volume
• Number of C. elegans loaded

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Side A
Proposal Concept
Inoculum
To distribution port of 4-way valve
Bacterial media
Side B
C. Elegans Chamber 1
C. Elegans Chamber 2
Nematode media
90 Flow path 4-ports (HV 86731)
C. Elegans Chamber 3
Cell Count Fixative
Distribution flow path 4 ports (HV 86738)
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Proposal Concept Shortcomings

• Does not address sampling of cultures or related
  factors associated with interfaces and levels of
  containment
• Large surface area of OptiCells make them
  inefficient for video analysis
• Poor control of multiplicity of infection (MOI)
• No dilution of inoculum before infection
• Imprecise metering of media addition

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Temperature Reqs

• Under investigation, but our best guesses are
• Ambient before activations
• Controlled to near-ambient in CGBA during C.
  elegan recovery and bacterial incubation
• Fixed inoculum counting sample stored ambient
• Controlled to near-ambient in CGBA for infection
• Gene-expression samples frozen in Melfi _at_ -80C
• Pathogen localization samples cooled in Melfi to
  4C
• Gene-expressionamples transferred to G

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Sampling
Fixative Culture Fixative Ratio Req Sample Volume(culture fixative) Timing of Samples StorageTemp
Inoculum counting 4 PFA 101? 2mL Before infection Ambient
Gene expression RNALater II 11 5mL? During infection -80C
Pathogen quantification and localization 4 PFA 101? 2 mL During infection 4C
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Culture Vessels

• FEP Teflon films separated by 2mm
• Sampled cultures will be in microplate-sized
  10mL vessel (similar to OptiCell)
• TD50 cultures in 6-well microplate-sized plate
• 1mL per well
• 5cm2 per well (e.g. 22mm x 22mm, 16mm x 32mm)

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Integrated Concept
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Modular Concept
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CeMM Integrated Concept
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• Research partnerships
• In-house research expertise
• Musculoskeletal disuse
• Bacterial responses to low gravity
• Flight hardware and software development
• Space flight research
• Planning and execution
• Integration, safety, mission operations
• Education and public outreach

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Operations Timeline
No. Description Facilities Duration
1 Handover hardware KSC?  
2 Transfer from Dragon to ISS n/a  
3 Add media to bacteria to initiate growth, place in CGBA CGBA 30 min
4 Add media to C. elegans to bring out of stasis, place in CGBA CGBA 30 min
5 Dilute bacterial culture to create infection inoculum, fix a sample of each inoculum mixture, and infect C. elegans, place cultures in CGBA CGBA 3 hr
6 Draw samples in MSG, place samples in cold stowage MSG 2 hr sampling, 30 min transfer
7 Add media to bacteria to initiate growth place in CGBA CGBA 30 min
8 Add media to C. elegans to bring out of stasis place in CGBA CGBA 30 min
9 Remove series 1 TD50 cultures and stow. Dilute bacterial culture to create infection inoculum, fix a sample of each inoculum mixture, and infect C. elegans, place cultures in CGBA CGBA 3 hr ops, 15 min stow
10 Draw samples in MSG, place samples in cold stowage MSG 2 hr sampling, 30 min transfer
11 Transfer remaining hardware from CGBA for return CGBA  
12 Transfer samples from MELFI to GLACIER MELFI, GLACIER  
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Sampling Concept

• COTS needless injection port mates to COTS
  luer-lock
• Custom syringe based on BioServes fixation tubes
• Two levels of containment

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Sample Concept Before Sampling

• Pre-loaded with fixative
• Short handle precludes accidentally pressurizing
• Cap in place to complete 2nd level of containment

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Sample Concept During Sampling

• Pull syringe to take sample
• After pulling syringe most of the way, turn valve
  to vent to draw in air
• Clears fluid paths at mates
• Adds air for headspace during freezing

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Sample Concept After sampling

• Handle removed, back of plunger on handle oring
  forms second level of containment
• Cap replaced to complete second level

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Concept Rough Solid Model4.7wide x 5long x
4high
Luer sampling interfaces
Inoc valves
Integrated Syringe manifold
Check valves
OptiCells
6 Well Plate
Inoculum counting samples
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Concept Rough Solid Model
6 Well Plate
Handleless valve
Inoculum counting samples
Check valves
OptiCells
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Arrangement InsideCGBA Freezer
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Levels of Containment Inoc and Culture

• House fluidics in sealed box for second level of
  containment
• Must have valve handle and sample port
  penetrations or be easily removable for MSG
  operations

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Levels of Containment Sample Containers
Period Hazardous Fluids HL or BSL Levels of containment
Gene-expression samples Before sampling RNALaterII HL 1 Syringe cap 2
During sampling RNALaterII S. Typhimurium HL 1 BSL 2 Syringe MSG(2) 3
Stowage RNALaterII HL 1 Syringe Cap 2
Pathagen loc. samples Before sampling PFA HL 2 Syringe Cap Bag 3
During sampling PFA S. Typhimurium HL 2 BSL 2 Syringe MSG(2) 3
Stowage PFA HL 2 Syringe Cap Bag 3
Inoculum counting samples Before stowage PFA HL2 Syringe checkvalve box 3
During sampling PFA S. Typhimurium HL 2 BSL 2 Syringe checkvalve box 3
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Cameras

• USB no DIOB necessary
• C/CS Mount for higher-fidelity lenses that
  support higher resolution
• CMOS is showing higher resolutions

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C/CS Mount, CMOS, USB Cameras
Source Model No. Max Res. Size (mm) Sensor size Features Cost
The Imaging Source DMK 72AUC02 2592 x 1944 _at_ 7 fps 36x36x23 1/2.5 Programmable resolution and frame rate. Aluminum housing 359 board only 395 enclosed
Edmund Optics EO-1312M 1280x1024 _at_ 25 fps 44x44x34 1/2" machine vision 975
Edmund Optics EO-3112BL 2048x1536 _at_11 fps 36x36x20 1/2" Board camera, micro lens mount (no C/CS mount) 745
Thor Labs 1280 x 1024 _at_ 25 fps 49x44x26 1/1.8 315
Hilife Tech via alibaba.com H-500-CM 2272x1592 _at_ 4fps 55x55x35 1/3 Programmable everything.
Point Grey FMVU-13S2C 1328x1048 _at_ 23fps 34x34x24 1/3 350
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Camera Resolution

• Assuming
• C. elegans are 0.1mm across
• 74mm x 56mm sample area (shown right)
• 3 pixels across a C. elegan requires 2220x1665
  pixels
• 5 pixels across a C. elegan requires 3700x2775
  pixels

74mm x 56mm Sample Area
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Prototype

• Jon Beno BioServe Engineer

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Physical Prototype - Overall
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Physical Prototype Inoculation Preparation and
Dilution
S2
S1
S3
Out to samples
S4
S5
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Physical Prototype Syringe Pump and metering
assembly
Out to samples
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Physical Prototype Large samples
PTFE vent
Sample Port
Out to TD50 samples
Distribution port - In from metering syringe
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Physical Prototype TD50 array
Fill/Bleed Port
Priming/Overflow reservoir/ Inoculum counting
sample
Distribution port - input
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Fluidics priming

• Luis Zea Aerospace Graduate Student

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Fluidics Loading Procedure
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TD50 Preparation
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TD50 Preparation Well 1 Fill
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TD50 Preparation Well 2 Fill
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TD50 Preparation Well 3 Fill
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TD50 Preparation Well 4 Fill
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TD50 Preparation Well 5 Fill
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TD50 Preparation Well 6 Fill
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OptiCells Preparation
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OptiCells Preparation O1 O2 Fill
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OptiCells Preparation O3 O4 Fill
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OptiCells Preparation O5 O6 Fill
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Inoculum Preparation
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Inoculum Preparation
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Inoculum Preparation (cont.)
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Assembly and Flowpath Priming
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Assembly and Flowpath Priming
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Prime Bacterial Media Checkvalve
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Prime Media Syringe Pump Line
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Prime CeMM Checkvalve
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Prime CeMM Syringe Pump Line
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Assembly and Flowpath Priming
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Inoculum Checkvalve Priming
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Inoculum Syringe Pump Line Priming
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CeMM Checkvalve Priming
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CeMM Syringe Pump
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Completed Assembly