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Product Manual
GravityTRAP™ ULA Plate
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ISP-09-001
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GravityTRAP™ ULA Plate Manual
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Contents
Introduction 3
GravityTRAP™ ULA Components 5
Generating 3D microtissues 8
Additional materials required 8
Preparation 9
Pre-wetting 9
Microtissue seeding 10
Sedimentation/Spheroid maturation 11
Medium exchange in the GravityTRAP™ ULA Plate 12
Analysis and assays in the GravityTRAP™ ULA Plate 13
Annex 1: Microtissue harvest from GravityTRAP™ ULA Plates 14
Annex 2: Troubleshooting guide 16
Annex 3: Step-by-step protocol for HCT116 & HEY microtissues 17
Version 2.0, July, 2015
451-0009-01-B
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Introduction
The GravityTRAP™ Ultra-Low Attachment
(ULA) plate1represents a simple, exible,
and automation-compatible platform for the
generation, long-term cultivation, observation
and testing of scaold-free 3D microtissue
spheroids in 96-well format. Each plate
consists of a special non-adhesively coated
96-well, sterile-packaged GravityTRAP™ ULA Plate and lid.
InSphero recommends GravityTRAP™ ULA plates for the generation of
spheroids using immortalized or modied cell lines that are known to
readily form microtissues, or as a starting point for investigating whether or
not a cell line can form self-aggregating, scaold-free spheroids. InSphero
recommends our patented GravityPLUS™ Hanging Drop System (ISP-06-
001, ISP-06-010) if generating spheroids in more complex 3D cell culture
scenarios, such as when using primary cells, cell lines that are sensitive to
self-assembly, or when generating co-culture microtissues (e.g., tumor/
stroma). In such cases, the GravityPLUS™ Hanging Drop System provides
the greatest opportunity for success.
1 The GravityTRAP™ ULA Plate and GravityPLUS™ Plate and related technology are protected by several granted and pending patents
world-wide.
GravityTRAP™ ULA Plate Manual
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Figure 1: Spheroid formation in the GravityTRAP™ ULA Plate begins with initial seeding
of cells in suspension, followed by a brief spin to concentrate cells. Following microtissue
maturation,the SureXchange™ ledge of the tapered well facilitates medium exchange and
compound dosing without disturbing or losing the microtissue.
Advantages of the GravityTRAP™ ULA Plate:
1. Convenient scaold-free formation of spheroids via cellular self-assembly in
ultra-low attachment (ULA-treated) plates
2. SureXchange™ tapered ledge and culture chamber facilitates easy medium
exchange and prevents microtissue loss during long-term spheroid growth and
analysis
3. 1 mm diameter at bottom observation chamber enables simple spheroid
observation, and greater working eld-to-eld distance reduces well-to-well
imaging cross-talk compared to standard 96-well plates
4. 3D-optimized protocols available for analysis in GravityTRAP™ ULA Plate
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GravityTRAP™ ULA Plate Components
The complete GravityTRAP™ ULA Plate assembly consists of the following
components:
1. Bottom GravityTRAP™ ULA Plate (96-well) (A)
2. Lid (B)
Figure 2:
Components of
the GravityTRAP™
ULA Plate
GravityTRAP™ ULA Plate Manual
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The GravityTRAP™ ULA Plate
The GravityTRAP™ (Tissue Re-aggregation and Assay Plate) ULA Plate
is a special non-adhesively coated 96-well microtiter plate. It is designed
to accomodate production of 3D microtissues (spheroids) for convenient long-
term cultivation and analysis. GravityTRAP™ tapered wells feature a
SureXchange™ ledge to prevent inadvertent microtissue aspiration and disruption
during medium exchange and compound dosing (Fig. 3). Microtissues are posi-
tioned in a 1.0 mm observation chamber at the bottom of each well, which enables
automated imaging processes (Fig. 4). Biochemical assays as well as optical
analytical methods such as inverse bright eld and uorescence microscopy can
be performed.
Microtissue production with GravityTRAP™ ULA Plates is very simple, and
recommended for cell lines that are known to readily form spheroids in ULA
conditions, or as a rst step in characterizing the spheroid-forming capabilities
of a particular cell type of interest. A cell suspension is delivered to the
bottom plate using a multi-channel pipette or a robotic liquid handler. Following
brief centrifugation to concentrate cells near the bottom of the tapered chamber,
microtissues begin forming by gravity-enforced self-assembly. Spheroid maturation
typically occurs within 2-5 days of seeding depending on the cell type and culture
conditions (Figs. 1 & 4).
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Figure 3: Bottom plate of the GravityTRAP™ Tissue Re-aggregation and Assay Plate.
Figure 4: HCT-116 colon carcinoma microtissue cultured in
GravityTRAP™ ULA Plate. Picture acquisition with a Zeiss
Axiovert25 microscope, 5× objective.
GravityTRAP™ ULA Plate Manual
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Generating 3D microtissues
Generating 3D microtissues in the GravityTRAP™ ULA Plate is a straightforward
process, but one that must be optimized for each cell type. Cell type, growth
medium, and intended downstream applications will impact the starting density
and desired culture volume. Optimization is recommended for each cell type and
application. In addition to the process overview in this chapter, Annex 3 illustrates
the formation of spheroids using HCT-116 (human colon carcinoma) and HEY
(human ovarian carcinoma) cell lines as an example for optimizing your own
protocol.
Additional materials required
1. Mammalian cells (primary or cell line) of interest
2. 3D InSight™ Tumor Microtissue Media Kit (InSphero, cat. no. CS-17-001-01) -
includes 3D InSight™ Tumor Re-aggregation Medium (CS-07-111-02) and 3D
InSight™ Tumor Maintenance Medium (CS-07-112-01)
3. Inverted microscope with a 5x/10x objective
4. Cell counter, e.g. Neubauer chamber
5. 8- or 12-channel pipette (e.g. Viao 10.0-300.0 μl, Integra Biosciences,
InSphero, cat. no. IS-001-01)
6. Medium reservoir for multichannel pipettes
7. Microplate centrifuge
8. Humidied CO2incubator 37°C
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Preparation
1. Prior to seeding, pre-warm the 3D InSight™ Tumor Re-aggregation Medium
(CS-07-111-02).
2. Wipe the GravityTRAP™ ULA Plate bag with 70% EtOH before opening.
3. Carefully open the bag under sterile working conditions and take out the
GravityTRAP™ ULA Plate assembly.
Pre-wetting
IMPORTANT – Pre-wetting the wells of the GravityTRAP™ ULA Plate according
to the procedure below is required prior to seeding microtissues to prevent
inclusion of air bubbles.
IMPORTANT - Perform all of the following steps under sterile conditions.
1. Apply 40 µl of 3D InSight™ Tumor Re-aggregation Medium (CS-07-111-02)
to each well by placing the tip far into the wells. It is recommended to use a
multichannel pipette (8- or 12-channel).
2. Remove pre-wetting solution by placing the tip at the ledge of the upper
cavity of the well (Fig. 5). Aspirate until medium is completely removed from
each well. A negligible amount of medium (<5-7 µl) may remain in the bottom
of the chamber.
GravityTRAP™ ULA Plate Manual
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Microtissue seeding
1. Trypsinize cells expanded in cell-culture asks according to your standard pro-
tocol.
2. Count the cells to determine cells per ml of medium.
3. Prepare a cell suspension for seeding, using a nal volume per well of 70 µl.
Recommended cell concentration: For long-term growth proling start with
low cell numbers (250–500 cells per well). If non-proliferating cells or rap-
id production of larger microtissues is required then start with 2500–25000
cells/70 µl. See Annex 3 for a detailed example using HCT-116 (colon
carcinoma) and HEY (ovarian carcinoma) cell lines.
IMPORTANT – For homogeneity of forming microtissues, it is essential to
assure a homgeneous distribution of the cell suspension by gently pipetting
up and down prior to seeding into the GravityTRAP™ ULA Plate.
Figure 5: Medium removal from
GravityTRAP™ ULA well.
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