Whitepaper

708 views

Published on

Published in: Technology
1 Comment
0 Likes
Statistics
Notes
  • GravityPlus and GravityTRAP - a perfect match for 3D microtissues
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Be the first to like this

No Downloads
Views
Total views
708
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
4
Comments
1
Likes
0
Embeds 0
No embeds

No notes for slide

Whitepaper

  1. 1. InSphero White Paper GravityPlus™ and GravityTRAP™ – A Perfect Match An Integrated Platform Solution for Microtissue Culture and AssayIntroductionBiology can be thought of as the complex and coordinated interplay of a largeassembly of cells. When cultivated on flat surfaces, the same cells are quite "selfish",and abandon native function and morphology. The normal "community behaviour"displayed by cells in tissues and organs is largely determined by the close 3-dimensional proximity of neighbouring cells. Understandably therefore, manylaboratories are evaluating 3-dimensional cell culture technologies with a view togaining much greater data relevance when assessing the efficacy and toxicity ofcompounds. This extra effort is undertaken not only with a view to eliminating poorcandidates but also to discover promising leads that assays in non-physiologicalmonolayer culture models would not be capable of detecting.InSphero’s GravityPlus™ hanging drop culture plate (patent pending) has fundamentallysimplified the process of miniaturizing organs and simulating native cellular functions in vitro.The system minimizes the effort expended on creating and testing 3D tissue models and permitsconvenient high throughput processing. Culturing cells in hanging drops of media provides amajor advantage over other methods of producing 3D tissue models: contact with artificialsubstrates is completely avoided. In hanging drop culture the subtle, intrinsic cues inherent tonatural cellular self-assembly processes are expressed, and this leads to the rapid, reliableformation of small, compact spheroidal tissues that can closely reflect native organ architecture.Once microtissues have been formed in hanging drops their transfer to an appropriate multi-wellplate will result in one microtissue in each well. GravityTRAP™ (Tissue Receiver and Assay Plate),is a 96-well SBS standard microtiter plate designed for failsafe reception of microtissuestransferred from the GravityPLUS™ hanging drop plate. The GravityTRAP™ plate is used forlong term microtissue maintenance and its design prevents the inadvertent aspiration ofmicrotissues during media changes. GravityTRAP™ renders microtissues compatible with allroutine treatment and assay procedures commonly applied to 2D cell culture models.GravityTRAP™ featuresGravityTRAP™ adheres to the standard microtiter plate definitions as introduced by the Societyfor Biomolecular Screening (SBS). 96-wells are arranged by 8 rows and 12 columns, identified byalphanumeric labels. The plate fulfils several functions relevant to microtissue culture andhandling. The individual wells show a regular wide opening at the top ultimately narrowingdown to a small cavity at the well bottom, which is large enough to harbour microtissues up toa size of 1 mm in diameter (Figure 1).InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 1
  2. 2. InSphero White Paper GravityPlus™ and GravityTRAP™ – A Perfect Match An Integrated Platform Solution for Microtissue Culture and AssayFigure 1: (a) GravityTRAP™ shown in top angled view, (b) as a schematic drawing and (c) a lower angleddetail view. (d) Close-up view of a single well from the top and (e) the bottom. (f) Detailed schematicdrawing of the well design, showing the wide well opening at the top with its v-shape form and the smallcavity directly above the well bottom. (g) One HCT-116 colon carcinoma microtissue cultured in aGravityTRAP™ well. Note: The diameter of the transparent well bottom measures exactly 1mm whichserves as an internal scale for the estimation of the microtissue size. (h) Fluorescence image of HCT-116microtissue (initially seeded at 500 cells per drop, 12 days old) expressing GFP, stained with DAPI. Imageaquisition performed in GravityTRAP™ with Zeiss microscope, 10x objective.The GravityTRAP™ plate was specifically designed for use with the GravityPlus™ microtissueproduction plate. With the three guidance pins located on the underside of the GravityPlus™frame the plate can be precisely superimposed onto the GravityTRAP™ for subsequentmicrotissue transfer into the GravityTRAP™ wells (Figure 2).InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 2
  3. 3. InSphero White Paper GravityPlus™ and GravityTRAP™ – A Perfect Match An Integrated Platform Solution for Microtissue Culture and AssayFigure 2: Schematic illustration of microtissuetransfer from the production platformGravityPlus™ to GravityTRAP™ for long termculture, assay performance and microscopicanalysis.Microtissues are transferred by adding sufficient additional medium to the GravityPlus™ inlets toincrease drop size to the point where the drops simultaneosuly fall into the GravityTRAP™ wellsbeneath. The specific non-adhesive coating inside the wells prevents attachment and spreadingof cells, leaving the microtissue shape intact even for culture periods up to 4 weeks.Another feature of the GravityTRAP™ wells is a ledge onto which the pipette tip can be placedfor safe medium removal and dispense. This allows for almost complete medium removal whileavoiding any loss of microtissues (Figure 3).Figure 3: Medium exchange ismade easy by the presence of theledge on the inside wall of theGravityTRAP™ well. This ledgecan be easily identified by theoperator during manualpipetting. The remnant volumeafter aspiration corresponds toapprox. 4-7% of the totalmedium volume and can beconsidered as negligible.The ability to perform frequent medium exchange without the loss of microtissues permitsreliable long-term culture to study, for example, tumor growth over time or toxic effects inchronic treatment experiments. One important and easy read out of such experiments ismicrotissue morphology and size. The GravityPlus™ plate is perfectly suited for automatedimaging acquisition by inverted bright field and fluorescence microscopy, generating 96 images,which then can be processed by conventional cell image analysis software (e.g. CellProfiler,ImageJ) (Figure 4).InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 3
  4. 4. InSphero White Paper GravityPlus™ and GravityTRAP™ – A Perfect Match An Integrated Platform Solution for Microtissue Culture and AssayFigure 4: Example work flow for size analysis of microtissues cultured in GravityTRAP™: (a) Thetransparent well bottom with good optical properties allows image acquisition to be performed byinverted bright field or fluorescence microscopy. (b) Images of the resulting 96-well panel can betransformed into grey scale pictures and upon correct threshold setting can be converted intoblack&white snapshots. (c) Particle analysis algorithms with defined parameter settings (size range, formfactor, edge exclusion) will give the size distribution of the entire microtissue panel within theGravityTRAP™.Aside from regular bright field microscopy, the GravityTRAP™ format can be used inconventional plate readers for fluorescence or luminescence based bioassays. Figure 5 nicelyillustrates the combined measurement of microtissue size and the correlation to the fluorescentsignal of differently sized microtissues composed of GFP expressing HCT-116 colon carcinomacells. The perfect linear relationship demonstrates that the GravityTRAP™ system is optimallysuited not only for long term microtissue cultures, but also for optical read outs e.g. forassessing microtissue viability and function.Figure 5: GFP fluorescence and microtissue size showexcellent correlation: GFP expressing HCT-116 cells wereseeded at a density of 100-2000 cells/drop intoGravityPlus™ plate and microtissues were thentransferred into GravityTRAP™ on day 4. 2 days latermicrotissue size was analysed by morphometricmeasurements based on bright field microscopic imagesand GFP fluorescence was measured atEx480nm/Em514nm.InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 4
  5. 5. InSphero White Paper GravityPlus™ and GravityTRAP™ – A Perfect Match An Integrated Platform Solution for Microtissue Culture and AssayWith GravityTRAP™ InSphero introduces the first commercially available microtiter plate formatfor maintaining and assaying scaffold-free 3-dimensional cell cultures. The plate assuresstraightforward and safe handling of microtissues comparable to standard procedures with 2Dcell culture. This new plate format opens a completely new range of applications in a field whereimproved biological in vitro systems are in great demand. GravityTRAP™ is specifically designed for … • scaffold-free microtissue culture and assay performance • frequent safe and reliable medium exchange without the risk of tissue loss • microscopic and spectrometric read outs • easy implementation of 3D cell culture into existing screening processes enabling the broadest application of spheroid cultures throughout the drug development processInSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 5

×