Generation and Characterization of Rat Uterus Organoids from Rat Endometrial Epithelial Stem Cells

Our research focus on developing a novel reproducible system for cultivating and expanding rat endometrial organoids from glial cells. By addressing limitations in current endometrial tissue device methods, we aim to enhance the pace of research in women's reproductive biology. Our research has proposed a method for culturing rat endometrial epithelial stem cells. Based on this method, we can opt-in rat endometrial organoids, overcoming the previous challenge of having to construct and measure blocks. We will explore potential applications including drug testing and gene editing that could contribute to better understanding and treatment of female reproductive system disease.

[Narrator] To begin, position the anesthetized rat on the surgical platform. Perform longitudinal incisions on both sides of the uterus to expose the inner endometrium. With a T10 scalpel blade, scrape the endometrium until the surface is rough. Place all uterine endometrium fragments in a 3.5 centimeter dish and wash them three times with PBS. Now, mix the uterine endometrium fragments with three milliliters of 1% collagenase type one and incubate at 37 degrees Celsius for 60 minutes. Filter the undigested tissue using a 100 micrometer cell strainer and collect the filtrate for further culturing. Calculate the volume of the cell suspension needed to seed the cells in each well of the 12 well plate to achieve the desired cell density. Seed the collected cells into 12 well plates in REEM medium for further culturing. Take cultured REESCs with 70% confluency and digest the cells with 0.25% trypsin and one millimolar EDTA for five to six minutes. Re-suspend the REESCs in 500 microliters of FOD matrigel and place in 12 well plates. Incubate the plates at 37 degrees Celsius for 20 minutes to allow gellation. Once the cell matrigel mixture solidifies, add 1.5 milliliters per well REEM to cover the matrigel. For the long-term culture of the organoids, aspirate the REEM and add one milliliter of organoid harvesting solution for 40 minutes to dissociate the organoids. Incubate on ice to maintain organoid integrity. After incubation, collect the organoids using a pasture pipette and centrifuge them at 300 G for three minutes at four degrees Celsius to pellet them for further processing. Re-suspend the pelleted organoids in matrigel in a ratio of one to two or one to three for embedding and subsequent culture in 12 well plates. To thaw the frozen organoids, prewarm the REEM in a 37 degrees Celsius water bath and immerse the frozen stock tube in the water bath to gently thaw the organoids. After thawing, re-suspend the organoids in 500 microliters of matrigel for re-embedding and culture in a 12 well plate. Take the cultured rat endometrial organoids, aspirate the REEM from the organoids, and add one milliliter of organoid harvesting solution to dissociate the organoids. Incubate on ice to maintain organoid integrity. After incubation, collect the organoids using a pasture pipette and centrifuge them at 300 G for three minutes at four degrees Celsius to pellet them for further processing. Re-suspend the organoids in three milliliters of prewarmed REEM and transfer the re-suspended organoids to a 3.5 centimeter dish. When the cells reach 70% confluency, digest them with 0.25% trypsin and one millimolar EDTA for five to six minutes.