Whitepaper

InSphero White Paper
GravityPlus™ and GravityTRAP™ – A Perfect Match
An Integrated Platform Solution for Microtissue Culture and Assay

Introduction

Biology can be thought of as the complex and coordinated interplay of a large
assembly 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, many
laboratories are evaluating 3-dimensional cell culture technologies with a view to
gaining much greater data relevance when assessing the efficacy and toxicity of
compounds. This extra effort is undertaken not only with a view to eliminating poor
candidates but also to discover promising leads that assays in non-physiological
monolayer culture models would not be capable of detecting.

InSphero’s GravityPlus™ hanging drop culture plate (patent pending) has fundamentally
simplified 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 permits
convenient high throughput processing. Culturing cells in hanging drops of media provides a
major advantage over other methods of producing 3D tissue models: contact with artificial
substrates is completely avoided. In hanging drop culture the subtle, intrinsic cues inherent to
natural cellular self-assembly processes are expressed, and this leads to the rapid, reliable
formation 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-well
plate 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 microtissues
transferred from the GravityPLUS™ hanging drop plate. The GravityTRAP™ plate is used for
long term microtissue maintenance and its design prevents the inadvertent aspiration of
microtissues during media changes. GravityTRAP™ renders microtissues compatible with all
routine treatment and assay procedures commonly applied to 2D cell culture models.

GravityTRAP™ features

GravityTRAP™ adheres to the standard microtiter plate definitions as introduced by the Society
for Biomolecular Screening (SBS). 96-wells are arranged by 8 rows and 12 columns, identified by
alphanumeric labels. The plate fulfils several functions relevant to microtissue culture and
handling. The individual wells show a regular wide opening at the top ultimately narrowing
down to a small cavity at the well bottom, which is large enough to harbour microtissues up to
a size of 1 mm in diameter (Figure 1).

InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 1


Figure 1: (a) GravityTRAP™ shown in top angled view, (b) as a schematic drawing and (c) a lower angled
detail view. (d) Close-up view of a single well from the top and (e) the bottom. (f) Detailed schematic
drawing of the well design, showing the wide well opening at the top with its v-shape form and the small
cavity directly above the well bottom. (g) One HCT-116 colon carcinoma microtissue cultured in a
GravityTRAP™ well. Note: The diameter of the transparent well bottom measures exactly 1mm which
serves as an internal scale for the estimation of the microtissue size. (h) Fluorescence image of HCT-116
microtissue (initially seeded at 500 cells per drop, 12 days old) expressing GFP, stained with DAPI. Image
aquisition performed in GravityTRAP™ with Zeiss microscope, 10x objective.

The GravityTRAP™ plate was specifically designed for use with the GravityPlus™ microtissue
production plate. With the three guidance pins located on the underside of the GravityPlus™
frame the plate can be precisely superimposed onto the GravityTRAP™ for subsequent
microtissue transfer into the GravityTRAP™ wells (Figure 2).



Figure 2: Schematic illustration of microtissue
transfer from the production platform
GravityPlus™ to GravityTRAP™ for long term
culture, assay performance and microscopic
analysis.

Microtissues are transferred by adding sufficient additional medium to the GravityPlus™ inlets to
increase drop size to the point where the drops simultaneosuly fall into the GravityTRAP™ wells
beneath. The specific non-adhesive coating inside the wells prevents attachment and spreading
of 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 placed
for safe medium removal and dispense. This allows for almost complete medium removal while
avoiding any loss of microtissues (Figure 3).

Figure 3: Medium exchange is
made easy by the presence of the
ledge on the inside wall of the
GravityTRAP™ well. This ledge
can be easily identified by the
operator during manual
pipetting. The remnant volume
after aspiration corresponds to
approx. 4-7% of the total
medium volume and can be
considered as negligible.

The ability to perform frequent medium exchange without the loss of microtissues permits
reliable long-term culture to study, for example, tumor growth over time or toxic effects in
chronic treatment experiments. One important and easy read out of such experiments is
microtissue morphology and size. The GravityPlus™ plate is perfectly suited for automated
imaging 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).



Figure 4: Example work flow for size analysis of microtissues cultured in GravityTRAP™: (a) The
transparent well bottom with good optical properties allows image acquisition to be performed by
inverted bright field or fluorescence microscopy. (b) Images of the resulting 96-well panel can be
transformed into grey scale pictures and upon correct threshold setting can be converted into
black&white snapshots. (c) Particle analysis algorithms with defined parameter settings (size range, form
factor, edge exclusion) will give the size distribution of the entire microtissue panel within the
GravityTRAP™.

Aside from regular bright field microscopy, the GravityTRAP™ format can be used in
conventional plate readers for fluorescence or luminescence based bioassays. Figure 5 nicely
illustrates the combined measurement of microtissue size and the correlation to the fluorescent
signal of differently sized microtissues composed of GFP expressing HCT-116 colon carcinoma
cells. The perfect linear relationship demonstrates that the GravityTRAP™ system is optimally
suited not only for long term microtissue cultures, but also for optical read outs e.g. for
assessing microtissue viability and function.

Figure 5: GFP fluorescence and microtissue size show
excellent correlation: GFP expressing HCT-116 cells were
seeded at a density of 100-2000 cells/drop into
GravityPlus™ plate and microtissues were then
transferred into GravityTRAP™ on day 4. 2 days later
microtissue size was analysed by morphometric
measurements based on bright field microscopic images
and GFP fluorescence was measured at
Ex480nm/Em514nm.



With GravityTRAP™ InSphero introduces the first commercially available microtiter plate format
for maintaining and assaying scaffold-free 3-dimensional cell cultures. The plate assures
straightforward and safe handling of microtissues comparable to standard procedures with 2D
cell culture. This new plate format opens a completely new range of applications in a field where
improved 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 process


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