Dear Researchers,
I am reaching out to this esteemed community seeking insights into a recent experiment I conducted involving cell seeding in agarose micro-wells. My aim is to engage with fellow researchers who may shed light on the intriguing outcomes of my experiment.
In my recent experimentation, I seeded cells from the RD cell line into agarose micro-wells at a significantly high concentration. The resulting images closely resembled those depicted in published works. However, I found myself disheartened by the outcome as I had anticipated observing dense spheroids akin to those formed with fibroblasts – distinct, rounded structures with diameters smaller than the wells. Instead, the cells appeared densely packed within the micro-wells.
Upon reflection, I conjectured that the outcome I obtained merely represented physical cell packing under the influence of gravity within a confined space, lacking fidelity to in vivo conditions. However, my perspective shifted upon encountering a publication authored by members of this community. The depicted results closely mirrored my own, with cells spanning the entirety of the micro-wells, discernible to the naked eye. This instilled a sense of hope within me, suggesting that my results may not be as unsatisfactory as initially perceived. Nonetheless, doubts linger.
I am writing to seek your professional opinions on why such constructs are considered spheroids. Do they truly replicate in vivo conditions? Are they formed as a result of intercellular contacts, or are they merely conglomerations of cells within the micro-wells? Have you observed alterations in gene expression and surface protein profiles compared to monolayer cultures?
I believe your insights into these questions will greatly contribute to the advancement of our understanding in this field. Your expertise and guidance would be immensely appreciated.
Thank you for considering my inquiry. I eagerly anticipate your responses and the opportunity for fruitful discussion.
Warm regards.
Dear Miguel Andrés Santos Franco
I hope this message finds you well. As per your request, I am attaching article about a non-canonical nature, specifically cone-like spheroids (DOI:
- 10.1039/C7LC00832E).
Additionally, I would appreciate your insights and expertise in evaluating the spheroids I have captured. Take a look at my experiments and the result I obtained. I was hoping to achieve canonical spheroids, and it seems they do indeed have a round shape. However, upon closer inspection, they appear somewhat porous. I would appreciate your expert assessment and advice on how to improve the quality of these spheroids or any additional experiments that could help achieve more canonical results.
Based on the images you submitted I would classify them as "compact agreggates". They do not seem to be compact enough, nor do they have a regular outline. You can read this article by Froehlich et al., there they describe cualitative categories for "compactness": https://pubmed.ncbi.nlm.nih.gov/27518775/.
Strictly speaking, they are spheroids, but they may not be as useful as a more compact and regular shaped spheroid. You are very close tho! I am sure you will achieve it, try slightly changing culture conditions.
Hello Miguel Andrés Santos Franco
Thank you for your link.
what do you mean by the phrase "but they may not be as useful as a more compact and regular shaped spheroid"?
Do you have a picture of the your spheroids?
Sure,
Usually the "compactness" is a measure utilized to test how reproducible a method for generating spheroids is. Having a regular shaped spheroid would ensure that test ran on those cells are standarized. Cell density, spheroid size, compacteness and cell viability are all important measures when evaluating the "quality" of a spheroid. Depending on what you are measuring this may be even more important, for example in high throuhput assays for drug testing.
Sadly, I don't have a photo of the spheroids, but in the link I provided you can request access to the paper, which has very clear photos.
I hope this helps
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