High-Throughput Live and Fixed Cell Imaging Method to Screen Matrigel-Embedded Organoids
, Robert Vary 1, Twishi Gulati 1,2, Jennii Luu 1, Karla J. Cowley 1, Michael S. Janes 3, Nicholas Radio 3 and Kaylene J. Simpson 1,2,4,*Round 1
Reviewer 1 Report
Overall, you need to make corrections, tone down claims, and focus on the automated pipeline that can be extended in future.
1. The title is somewhat misleading. Since samples are fixed, there is no live cell imaging. Initially, it seems that live cell imaging is for routine monitoring of the growth properties using bright field imaging. Later, mitotracker is used. I suggest to organize the manuscript upfront in the Introduction on what to be expected.
2) Per your protocol on page 3, you are using the ontop method and not the embedded protocol. Please review the differences between the embedded and ontop methods from Bissell's Lab (Nature Protocol 2007). The title needs to be corrected as well.
3) Matrigel is a type of hydrogel. In our experience, we have not seen FA dissolve ECM!! You need to re-evaluate the claims made on page 2.
3a) 10uL of Mitrogel is not economical at all for 384-well plates. Typically, 6-10uL is used in 96 well-plates.
3b) Top row of Figure 1 could be to poor pipetting.
4. The experimental design is limited to one cell line. MCF7 is a Luminal A subtype and forms solid spheroids. But other cell lines such as MDA_MB-231 do not form spheroids. The choice of only one cell line doesn't allow for complete validation! In addition, there needs to be a control cell line such as MCF10A that represents a non-malignant cell line. I suggest that you extend the discussion in this direction for completeness!
5) On page 5, concentrations of drugs seem too high, e.g., 10uM. Do you have references for them? Also, why add DMSO (although small) to the medium for negative control?
6. The image analysis is limited to the colony shape and average molecular endpoints. This should be fine as the "first step" in the screening process. But sometimes detailed analysis is needed, which will require cell-by-cell analysis within the organoid (e.g., PMID 27383056) or related manuscripts that deal with detailed segmentation of organoids. For example, as a result of treatment, MCF7 may acquire an organization similar to MCF10A, i.e., phenotypic reversion. You may want to be including this direction in your Discussion section. Besides, cells on the periphery of the organoids may respond differently than those in the center.
(7) Minor: It is also not clear how the number of nuclei per field (Figure 4) is computed.
Author Response
see attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This article by Ramm et al. discusses imaging and analyzing methods for extracting high content information from spheroids or Matrigel embedded cell aggregates. The paper has potential to be of interest in the field of in situ high content imaging. However, the authors are advised to address the following concerns before it could be considered for publication.
1. In the introduction, the authors describe difficulties with using organoids or patient derived tissues for high content imaging applications. However, they use an immortalized cell line MCF7. MCF7 cultures cannot be called patient derived organoids in theory because cell line cultures are usually referred to as spheroids. Besides, establishing organoid cultures and using them for high content imaging might come with its added set of difficulties that cell lines do not present such as irregular sizes, diameter, less organoid growth, heterotypic populations etc. Can they comment on the expected differences in culture that a researcher would face while using patient derived organoids instead of spheroids? What methods need to be changed if someone has to follow their protocol for organoids?
2. During aspiration of media/solution from the wells using automation, what is the percentage loss of spheroids or Matrigel cultures?
3. Fixation with Glutaraldehyde might destroy some epitopes. Can the authors comment on how they will overcome that?
4. Are they using DMSO as a vehicle for the drug study? The concentration of DMSO needs to be mentioned in the figure (fig 3).
Author Response
see attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Authors have addressed most of my concerns.
