Counting of Patient-Derived IDC Organoids

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Purpose To image and count whole-well populations of organoids in a rapid manner without having to take multiple, time consuming Z-stacked images
Current Method(s) Manually, or Confocal Microscopy
Target Cell Type Patient-derived intestinal differentiated cells (IDC) organoids
Experiment Plan Organoids are cultivated in 6-well plates embedded in Matrigel and imaged in bright field
Hypothesis Celigo will be able to perform rapid, whole-well imaging and analysis of organoids in a high throughput manner

Celigo Setup

Plate Type Corning 12-well microplate
Scan Channels Bright field
Resolution 1 µm/pixel
Scan Area Whole well
Analysis Method Tumorsphere 1
Scan Frequency Once
Scan Time ~8 min

Assay Protocol and Plate Setup

Goal:

To image and count whole-well populations of organoids in a rapid manner without having to take multiple, time consuming Z-stacked images

Protocol:

IDC Organoids Preparation

  1. IDC organoids were cultivated in 12-well microplates and embedded in Matrigel following the plate map below
  2. Next, the organoids were grown for 20 days from IDC progenitor cells to form the organoids
  3. The organoids were also treated with H2O2 at time = 0, to inhibit the growth of the organoids

organoid preperation

Data Collection

  1. The organoids images were captured using bright field imaging
    1. One of the Control wells was used to focus the organoids to set the focus Z-location
    2. The Celigo scan required approximately 5 min

Data Analysis

  • The organoids were counted using Celigo application Tumorsphere 1
  • The IDC organoids counts were generated, as well as the size of the organoids

Results

1. Celigo-captured bright field images of organoids

  • Celigo captured bright field images in 12-well plates containing organoids in matrigel
  • Below is an example whole plate image of the captured data

Celigo-captured bright field images of organoids

  • Celigo captured high-resolution images in bright field that can be zoomed-in on from whole well (below)

zoomed-in image of organoids

  • The Celigo software accurately counted organoids and declustered them into individual organoids

declustered organoids

2. PDO Counting Results

  • Celigo was used to count all the organoids in the 12-well plate
  • The counted results and the size analysis of organoids are shown below

organoid histogram

Conclusion

  • Celigo was able to count the number of organoids directly in the wells and measured the diameter of each organoid
  • Overall, there was no clear difference between the control and H2O2 treated samples
  • Celigo software was able to decluster organoids in close proximity, to improve the counting accuracy of the current method
2018-10-11T19:13:22+00:00