Nexcelom Bioscience

978-327-5340

Embryoid Bodies & Patient Derived Organoids
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Measure Size and Number of Embryoid Bodies and Patient Derived Organoids

  1. Directly image tumor spheroids in various microwell formats
  2. Non-invasive bight field imaging allows the user to image the same plate over multiple days
  3. Perform a two-color fluorescence viability assay

Introduction

The Celigo imaging cytometer has been developed to fully automate imaging and analysis of tumorspheres. This automated morphometric analysis tool significantly reduces the time and effort needed to quantify key aspects of 3D spheres including size, growth, growth tracking over time, and response to chemotherapeutics.

Celigo imaging process

Identification and Counting of Embryoid Bodies from Spinner Flask

Experiment 1 procedure:

  • One mL of cultured embryoid bodies was removed a 250 mL spinner flask and plated onto a 24 well plate
  • The embryoid bodies were analyzed for number and average diameter

Whole well image of embryoid bodies from a 24-well plate

Whole well image of embryoid bodies from a 24-well plate

Bright field image of embryoid bodies

Bright field image of embryoid bodies

Bright field image of counted embryoid bodies

Bright field image of counted embryoid bodies

  Number of EBs Avg. Diameter SD Diameter Min Diameter Max Diameter
Celigo 643 187.1 microns 63.9 microns 82.5 microns 514.6 microns
Manual Count 81 200 microns 61.2 microns 99.6 microns 434.4 microns

The area/size of the embryoid bodies was plotted in a histogram

embryoid bodies was plotted in a histogram
  • A gate was set to show the software identification of embryoid bodies sizes.
  • In this example the red EBs have an average area of 11,577 square microns compared to the green embryoid bodies that have an average area of 38,196 square microns.
  • Also, this distribution shows that most embryoid bodies fall within 10 – 50k square microns with very few very large embryoid bodies that are 100k or more.

Celigo software can also plot the equivalent diameter of embryoid bodies

plot the equivalent diameter of embryoid bodies
  • In this example the size gate was set at 120 microns. Therefore, all EBs circled in red have a diameter of less than 120 microns (which is 181 EBs)
  • All EBs circled in blue are larger than 120 microns and are 462 in number

Experiment 2 Procedure: Identification and counting of embryoid bodies

  • Whole well images from a 6-well plate were acquired and analyzed.
  • Below are representative raw and analyzed images of the forming embryoid bodies.
  • Green outline indicates counted stem cell aggregates.

Whole well image of embryoid bodies from a 6-well plate

Whole well image of embryoid bodies from a 6-well plate

Bright field image of embryoid bodies

Bright field image of embryoid bodies

Bright field image of counted embryoid bodies

Bright field image of counted embryoid bodies

Size gating

Histogram: Plotted sizes of embryoid bodies

Plotted sizes of embryoid bodies
  • Three gates were created based on the sizes of the embryoid bodies.
  • In this example the size gate was set at 0-75 microns for small cells, 75-150 microns for medium cells, and 150-550 for large cells.
  • The EBs outlined in Blue fall into the small size category
  • The EBs outlined in Yellow fall into the medium size category
  • The EBs outlined in Red fall into the large size category

Table 1

EBs Number Min size (µM) Max size (µM) Avg size (µM) STDEV
Total 3362 51.9 517.8 96.5 40.8
Small 1181 51.9 75.1 63.9 6.7
Medium 1863 75.0 149.0 100.47 19.0
Large 323 149.3 517.8 191.9 44.8

Cell Sources for Generation of Patient Derived Organoids (PDOs)

1. Isolated adult stem/ progenitor cells

Isolated adult stem/ progenitor cells

2. HESC/ iPSC differentiation

HESC/ iPSC differentiation

3. Isolated fragments of tissues from the corresponding organ. (e.g. intestinal crypts or liver and pancreatic ducts)

isolate fragments of organ tissues

Mouse pancreatic organoids plated and imaged in 24-well plate

Mouse pancreatic organoids plated and imaged in 24-well plate
  • PDO’s generated from 6000 mouse pancreatic cells seeded in Matrigel® and imaged 3 day post seeding.
  • Whole well bright field images were acquired and organoid sizes automatically determined by Celigo software.
  • Green fill pseudo-color is used to easily identify Celigo counted PDOs

Mouse pancreatic organoids plated and imaged in a 96-well plate

Mouse pancreatic organoids plated and imaged in a 96-well plate
  • PDO’s generated from 2000 mouse pancreatic cells seeded in Matrigel® and imaged 3 day post seeding.
  • Whole well bright field images were acquired and organoid sizes automatically determined by Celigo software.
  • Green fill pseudo-color is used to easily identify Celigo counted PDOs

RFP-labeled mouse pancreatic organoids plated and imaged in a 96-well plate

RFP-labeled mouse pancreatic organoids plated and imaged in a 96 well plate
  • PDO’s generated from 2000 mouse pancreatic cells seeded in Matrigel® and imaged 3 day post seeding.
  • Whole well bright field images were acquired and organoid sizes automatically determined by Celigo software. The inset shows a zoomed in bright field image.
  • Green fill pseudo-color is used to easily identify Celigo counted PDOs

Mouse pancreatic organoids plated and imaged in a 384-well plate

Mouse pancreatic organoids plated and imaged in a 384-well plate
  • PDO’s generated from 500 mouse pancreatic cells seeded in Matrigel® and imaged 3 day post seeding.
  • Whole well bright field images were acquired and organoid sizes automatically determined by Celigo software.
  • Green fill pseudo-color is used to easily identify Celigo counted PDOs
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