Organoid/Cell Recovery Protocol Workflows

Application Notes

Data and References

Figure 1.  Organoids recovered from Matrigel using VitroGel Organoid Recovery Solution with two methods.
Method 1: Re-suspend organoids in VitroGel Organoid Recovery Solution by pipetting to break organoids into small fragments for sub-culture/expansion.
Method 2: Rocking the tube with organoids and VitroGel Organoid Recovery Solution mixture without using a pipette to harvest the intact organoids.
In both, VitroGel Organoid Recover Solution was kept in a 4 °C refrigerator to maintain a low temperature before use. The organoids/Matrigel and VitroGel Organoid Recovery Solution mixture were incubated at room temperature for 2 min before centrifuging. Day 0 images show the morphology of organoids right after harvesting with two different methods.

Figure 2.  Comparison of VitroGel Organoid Recovery Solution and VitroGel Cell Recovery Solution for organoids recovered from Matrigel.
Organoids (Passage 19) were cultured in Matrigel for three days before harvesting. 1 mL cold (4 °C) VitroGel Organoid Recovery Solution (MS04) or VitroGel Cell Recovery Solution (MS03) was added to a well of a 24-well plate to resuspend the organoid-Matrigel dome. The mixture was genteelly pipetted, transferred to a centrifuge tube, and incubated at room temperature for 2 min (MS04) and 10 min (MS03), respectively. After centrifuging to collect the organoids, the Matrigel in MS04 solution was completely dissolved. For the MS03 solution, there was a small amount of soft gel remaining on top of the organoids. The organoids harvested from both conditions were sub-cultured in Matrigel. The images above (Passage 20, Days 1&2) show the healthy organoid expansion from both cell recovery solutions. VitroGel ORGANOID Recover Solution shows a higher recovery efficiency than the VitroGel Cell Recover Solution in this study.

Figure 3.  Comparison of VitroGel Organoid Recovery Solution with other organoid recovery solutions.
The data above shows the comparison study of VitroGel Organoid Recovery Solution (MS04) with other organoid recovery solutions (Solution RLR from Company SC and Solution 3ODR from Company SA). All solutions were used directly from a 4 °C refrigerator. After mixing with organoid/Matrigel, the mixture with MS04 was incubated at room temperature for 2 min, while the mixture with Solution RLR or Solution 3ODR was incubated in an ice bucket for at least 15 min. After centrifuging, all Matrigel in the MS04 mixture was dissolved, but most Matrigel remained in both Solution RLR and Solution 3ODR conditions. Besides recovering intact organoids, the organoids from the MS04 solution can be easily resuspended in a cell culture medium and broken into small fragments for a subculture (MS04 images above). However, the organoid/Matrigel mixture was hard to break in Solution RLR or Solution 3ODR.  VitroGel Organoid Recovery Solution delivers a higher cell recovery rate for organoid expansion.

Figure 4.  Use VitroGel Organoid Recovery Solution for iPSC harvesting from 2D Matrigel coating plate

VitroGel Organoid Recovery Solution can be used to harvest iPSC cells from a 2D Matrigel coating plate.  The solution was warmed up to room temperature before use.  A) Morphology of cells detaching from the Matrigel coating plate. (3 min after adding VitroGel Organoid Recovery Solution), B) Image of the well plate after cell harvesting. (Shows all cells were removed from the Matrigel coating plate), C) Morphology of cells after re-seeding to a new Matrigel coating plate (Day 3).

References/Publications

• De Donato, M., Babini, G., Mozzetti, S., Buttarelli, M., Ciucci, A., Arduini, G., De Rosa, M. C., Scambia, G., & Gallo, D. (2020). KLF7: a new candidate biomarker and therapeutic target for high-grade serous ovarian cancer. Journal of Experimental & Clinical Cancer Research, 39(1). https://doi.org/10.1186/s13046-020-01775-9
• Lan, T., Guo, J., Bai, X., Huang, Z., Wei, Z., Du, G., Yan, G., Weng, L., & Yi, X. (2020). RGD-modified injectable hydrogel maintains islet beta-cell survival and function. Journal of Applied Biomaterials & Functional Materials, 18, 228080002096347. https://doi.org/10.1177/2280800020963473
• Powell K. Adding depth to cell culture. Science, 356(6333), 96–98. https://doi.org/10.1126/science.356.6333.96

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