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© Cranfield University 2016 David Cullen

BAMMsat - A platform for beyond LEO space environments studies on biological systems in

CubeSats and CubeSat-like payloads

David C. Cullen1, Jack Longley1, Jennifer Kingston1, David Lee2, Martin Black2, David Pearson2, Christopher Waring3,

Ryan C. Pink3

1Space Group, School of Aerospace, Transport & Manufacturing, Cranfield University, UK 2The UK Astronomy Technology Centre, Royal Observatory, Edinburgh, UK

3Department of Biological and Medical Sciences, Oxford Brookes University, UK

5th Interplanetary CubeSat workshop (iCubeSat) University of Oxford, UK

24th to 25th May 2016

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© Cranfield University 2016 David Cullen

Outline

• Why “bio-CubeSats”?

– Using CubeSats for bioscience research

• Bio-CubeSats flown to date … and in the near future

– USA only (NASA Ames)

• What is BAMMsat?

– A UK / European bio-CubeSat activity

• History of BAMMsat initiation and development

• BAMMsat current status and baseline

– Fluidics and sensor suite breadboard

– Compact fluorescence optical microscope breadboard

• Routes forward … including beyond LEO flights

Why Bio-CubeSats?

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© Cranfield University 2016 David Cullen

A problem with access to space environments for bioscience experiments?

• Current situation - infrequent, restricted, costly access to space environments, with long lead-times

– … via ISS & other large spacecraft missions – e.g. Bion-M program

– … and with growing interest in beyond LEO environments (lunar and Mars relevant)

• Desire to have rapid and frequent access to the space environment (µ-gravity & radiation) for a multitude of studies …

• … including Bioscience, Astrobiology, Medical and Materials science

– … hence the “BAMM” topics … and therefore BAMMsat

• Common need to (i) house & maintain, (ii) expose, (iii) perturb & (iv) observe

• … therefore a need for alternative platforms / routes …

• … and with CubeSats / nano-satellites a possible route …

• … but with some compromises?

Bioscience on CubeSats to date

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© Cranfield University 2016 David Cullen

Biology on CubeSats to date and near future

• GeneSat-1

– 2006; 3U/”2U” payload; 410km; 10 microwells; OD/FL reading; ±0.5°C regulation; <1wk duration

– measuring gene expression (GFP) in E. coli • PharmaSat

– 2009; 3U/”2U” format; 460km; 48 microwells, 3-color optical absorbance; ±0.5°C reg; <1wk duration

– dose response to drugs in S. cerevisiae cultures • O/OREOS - organism/organic exposure to orbital stresses

– 2010; 3U/2x1U (dual) payload; 650km – SEVO – Space Environment Viability of Organics

• 24 cells; UV-visible-NIR spectrometer; >10mth duration • radiation degradation of thin films of PAH, amino acids, metalloporphyrin,

and quinone – SESLO - Space Environment Survivability of Live Organisms

• 12 microwells; 3-color optical absorbance; >6mth duration • effect of extended space flight on B. subtilis (various strains) survivability,

germination and growth • SporeSat

– 2014; 3U; 32 well; “CD” centrifuge format; Ca+ ion-selective electrodes – Ceratopteris richardii fern spores – study of single-cell gravi-response – NOTE - SporeSat 2 to be deployed from ISS (2016)

• EcAMSat (E. coli AntiMicrobial Satellite)

– Selected (2013) by NASA as future CubeSat mission – launch June 2016 (Falcon -9)? – 6U; 48 microwells; (?)wks duration – Effect on the susceptibility of E. coli to antimicrobial agents

• BioSentinel – 2018 launch on NASA SLS (Space Launch System) with up to 18mth mission – 6U; effect of space radiation beyond LEO on three strains of yeast cells – Lunar fly-by then heliocentric orbit between 0.98 to 0.92 AU

GeneSat-1

PharmaSat

SESLO

O/OREOS

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© Cranfield University 2016 David Cullen

Building upon current CubeSat bioscience experimentation: Current capability and what next?

• Summary of bioscience relevant CubeSat features flown to date

– General spacecraft operations for “bioscience” proven … in LEO

– Handling of multiple samples

– Thermal control demonstrated – to approx. ±1°C

– Fluidics – reservoirs, valving, pumping

– Sensing / observation … to some extent (non-imaging optical absorbance, fluorescence, spectroscopy + house-keeping sensors inc. radiation)

– In situ gravity / acceleration controls – limited / emerging

– Experiment duration – days to multiple months

• What next?

– Beyond LEO flight

– Extension to mammalian cell culture systems … plus?

– … and therefore need for microscopic imaging (inc. fluorescence imaging)

BAMMsat: Objectives and early work

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© Cranfield University 2016 David Cullen

BAMMsat (Biology, Astrobiology, Medical and Materials science experiments on Cubesat platforms) … plus activities to date

• Initial thoughts in late 2011 in response to funding call by UK Space Agency

• UK Space Agency funding (2012) for initial desk-based study of BAMMsat concept

• Key objectives

– Develop a reusable CubeSat platform / payload design for “BAMM” applications (2U format)

– To have a UK (and European) led activity / capability

– Initial focus – biomedical applications … but also …

– … with extension of current state-of-the-art to include mammalian cell cultures & fluorescence microscopy

• Cranfield investment (2013-2014)

– Early phase payload breadboard development and demonstration of cell growth – PhD project

– Initial mission and spacecraft design – MSc projects

• UK Space Agency NSTP-2 funding (2015-2016) for BAMMsat payload breadboard development

– Cranfield – fluidic and sensor suite breadboard

– ATC* Edinburgh – fluorescence microscope breadboard development

• Cranfield / Oxford Brookes (2015 – on-going)

– Extension of human cell types grown in BAMMsat test format – BSc / MSc projects *ATC = The UK Astronomy Technology Centre

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© Cranfield University 2016 David Cullen

BAMMsat breadboard design requirements • Compatible with 2U CubeSat payload – for both free-flying and hosted opportunities

• Number of (independent) cell culture samples – baseline 40 – Compatible with common high density (384) microwell plate format wells

– 3.63mm diameter cylinders with nominal depth of 2.00mm (20.7µl vol.) … gives 105 cells as confluent layer on cylindrical base

• Environmental control – 1 atm, +37°C ± 1°C in an isolated (hermetically sealed) system – 2U pressure vessel (nom. 200mm x 100mm x 100mm)

• Duration – baseline 1 month

• Maintain and perturb biological samples – fluidic system to replenish growth media, enable cell passaging, dose other reagents

• Observe – house keeping sensors – temperature, pressure, acceleration, radiation, humidity, pH, solution conductivity,

dissolved CO2, dissolved O2

– optical microscopy with fluorescence capability

• Budgetary and iterative development context – initially pursue low-cost rapid prototyping – laser cutting and lamination, 3D printing, “hobby” electronics

– … then evolve towards FM build quality

Status (in early 2015) of the BAMMsat breadboard - hardware • Fabrication

– Combination of 3D additive printing (structure) and laser cutting and lamination of sheet materials (fluidics and cell culture wells)

• Fluidics – Rotary valve to package 20 (currently 40)

independently addressable cell culture wells … with motor / Geneva drive mechanism

– Miniature peristaltic pump • Environmental control

– Standard laboratory laminar-flow hood and incubator

• Imaging – Initially standard optical microscopy

• Control – Exploit COTS “hobby” components / systems – Arduino micro-controller, stepper / DC motor

controllers, …

disc underside with rotary valve interface

bottom layer top layer (gas permeable)

double-sided adhesive coated film

BAMMsat rotary valve – approach to allow 120 valves in a compact format … and optical addressing of microscope and spectrometer

• For 40 samples / chambers, need for 3 fluidic connections per chamber (inc. for flushing) – 120 connections

• Individual chambers to be addresses fluidically

• Based on PTFE-PTFE interface pressed together

• Sealing force provided by torque on nut

• Increasing force provides better sealing, but harder to rotate

• Should not impede microscope access to disk chambers

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© Cranfield University 2016 David Cullen

Status (in early 2015) of the BAMMsat breadboard – cell culture • Choice of representative mammalian cell line(s)

– Baseline of 3 cell types encompassing a broad range of growth characteristics

– Initially human lung epithelial carcinoma (A549) cell line (recently other - HeLa and C2C12 cell lines)

• Baseline culture conditions

– Mainstream choice – Ham's F-12K medium

– CO2 independence – addition of HEPES buffer salts

– Environment +37°C, ambient atmosphere

• Compatibility of cell culture with breadboard materials and microwell format

– Standard laboratory laminar-flow hoods, incubators

• Initial demonstration of cell culture in BAMMsat breadboard

– Parallel growth in BAMMsat disc & “lozenge” – media change every 24h

– At 72h - disc 50% confluence; lozenge 80% confluence

– Preliminary de-risking of cell-growth in BAMMsat breadboard achieved

• … and passaging … to follow

BAMMsat: fluidics and sensor breadboard for 40

independent samples

BAMMsat NSTP-2 breadboard fluidics and sensor system

Waste reservoir

Growth media

reservoir

Reagent 1 reservoir

Reagent 2 reservoir

P

O2 sensor pH sensor

Selector valves

L

L

P P

Rotary valve

Disk

Cell Culture Chamber (1 of 40)

M S

Pump

Microscope Spectrometer

F

Flow sensor Pressure

sensor Liquid (bubble)

presence sensor

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© Cranfield University 2016 David Cullen

BAMMsat NSTP-2 integrated fluidic and microscope breadboard

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© Cranfield University 2016 David Cullen

BAMMsat NSTP-2 integrated fluidic and microscope breadboard

BAMMsat: compact fluorescence

microscope breadboard for CubeSat applications

Breadboard Prototype Fluorescence Microscope

Parameter Value

Magnification x6

FOV 1.5 mm

Resolution 0.67 µm/pix

Possible Dyes BFP, CFP, WGFP, GFP, FITC, YFP, TRITC, CY3.5,TXRED

Light Source Wavelength

Various available across range 280 – 1550 nm, broadband, warm white, cold white

Breadboard Prototype Microscope

“White light” image with 10µm beads

“Fluorescent” image with 10µm beads

Real-time BAMMsat video capture of a protozoa (adventitious colonisation of BAMMsat fluidics by environmental protozoa)

NOTE – for iCubeSat 2016 presentation – this is compressed, not native resolution due to need to upload presentation

BAMMsat: packaging of breadboard into

2U flight model pressure vessel

EO 63748 IDS UI-5584-LE TL MWWHD1

Preliminary layout of microscope within pressure vessel

Packaging exercise – will it fit into 2U?

• Shown with vessel cover removed

• Fluidic pipework not shown

• Electrical and fluid harness not shown

• LED for spectrometer not shown (known clash)

• Assembly procedure TBC

BAMMsat: options beyond LEO

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© Cranfield University 2016 David Cullen

Bio-CubeSats (and Bio-CubeSat-like payloads) beyond LEO

• Many applications for bio-CubeSats (BAMMsat) in LEO but …

• Why go beyond LEO? – primarily access to non-LEO radiation environments

– Fundamental bioscience questions

• Effect of deep space radiations on biology

– Astrobiology (and astrochemistry) questions – e.g. lithopanspermia, pre-biotic chemistry evolution, etc.

• Life in the Solar System and beyond

– Assess planetary protection issues

• Understanding transport of biology associated with human space activities

– In support of human exploration, exploitation and habitation

• Effect on human biology (cells) – e.g. Mars and long duration lunar human missions

• Effect on other biology – e.g. biological-based life support systems (microalgae, microbiological systems, etc.)

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© Cranfield University 2016 David Cullen

Bio-CubeSats (and Bio-CubeSat-like payloads) beyond LEO

• Possible opportunities – Current example - NASA BioSentinel - launch in 2018 of NASA SLS EM-1

• Lunar fly-by then heliocentric orbit at approx. 1AU

• Future opportunities on SLS EM-2 in …

– ESA proposing beyond LEO CubeSat flight(s)

• Technology demonstration flight for CubeSats beyond LEO … with opportunity to fly appropriate payload(s) … e.g. GTO, etc.

– European Lunar Communications Pathfinder Mission (Ceres) - partnership between SSTL / Goonhilly / ESA (see later talk of Chris Saunders of SSTL)

• Delivering payloads to lunar orbit (e.g. CubeSats, hosted payloads) and provide communications

– ESA’s LUnar Cubesat for Exploration (LUCE) opportunity … coming late 2016

– Team Indus (Indian Lunar X Prize competitors) … and follow-on opportunities

• Lunar lander and other beyond LEO opportunities

• Generic challenges … for free-flying CubeSats – Communications

– Radiation hardness

– Launch opportunities

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© Cranfield University 2016 David Cullen

Outline roadmap for BAMMsat

• Short term tasks

– Continue to refine initial breadboard inc. fluidics and cell growth conditions

– Integrate monitoring / sensors / imaging

– Produce outline FM (inc. packaging) design

• Medium term tasks (technical)

– Demonstrate automated approach to cell passaging

– Demonstrate ability to operate in a closed / isolated system for multiple weeks

– Further mission design (inc. ground and launch operations wrt maintaining cell cultures)

– Further spacecraft / satellite design (inc. thermal modelling)

– Simulation of µ-gravity on cell growth – e.g. Random Positioning Machine (RPM)

– Simulation of µ-gravity on fluidics operations – e.g. parabolic flights

• Medium term tasks (scientific)

– Identify specific science case(s) / partners - mammalian cell culture experiments

– Other applications (bioscience, astrobiological – e.g. BioRock, and materials)

• … and further funding … and flight opportunity?

• Exploring terrestrial applications – initial with biomedical organisations

Questions