Model based human ovarian cancer organoid culture medium kit

Human Ovarian Cancer Organoid Kit

Human ovarian cancer organoid culture medium kit

1 Product Description

Model based human ovarian cancer organoid culture medium kit（Human Ovarian Cancer Organoid Kit）It is a chemically defined cell culture medium used to establish and maintain human ovarian cancer organoids. The tumor organoids derived from patients summarize the genomic and pathological characteristics of the original tumor, thus having great potential in medical research and precision medicine.

2 Product Information

3 Other self provided materials and reagents

4 Preparation of human ovarian cancer organoid culture medium

Due to the multiple molecular subtypes of ovarian cancer, preparing a complete culture medium will also involve using different components based on its pathological subtypes. It is important to use aseptic techniques in order to prepare a complete culture medium for ovarian cancer organoids. The following is the preparation10mLExample of complete culture medium. If you need to prepare different volumes, please adjust the quantity accordingly.

Due to the high heterogeneity of serous ovarian cancer, it is necessary to test different formulations and select formulations with growth effects for subsequent cultivation. According to the following list, prepare the complete culture medium involved based on its subtypes.

Due to the high heterogeneity of serous ovarian cancer, it is necessary to test different formulations and select formulations with growth effects for subsequent cultivation. According to the following list, prepare the complete culture involved based on its subtypes

a) formula1：

Cultivate ovarian cancer organoid factorsB（50x）Ovarian cancer organoid culture factorsC（250x）Ovarian cancer organoid culture factorsE（250x）Thaw on ice and mix thoroughly. Translate into English200μLOvarian cancer organoid culture factorB（50x），40μLOvarian cancer organoid culture factorC（250x）And40μLOvarian cancer organoid culture factorE（250x）Join9.72mLIn the basic culture medium of ovarian cancer organoids. Fully mixed.

b) formula2：

Cultivate ovarian cancer organoid factorsB（50x）Ovarian cancer organoid culture factorsC（250x）Ovarian cancer organoid culture factorsD（250x）Ovarian cancer organoid culture factorsF（2000x）Thaw on ice. Fully mixed. Translate into English200μLOvarian cancer organoid culture factorB（50x）The40μLOvarian cancer organoid culture factorC（250x）The40μLOvarian cancer organoid culture factorD（250x）And5μLOvarian cancer organoid culture factorF（2000x）Join9.715mLIn the basic culture medium of ovarian cancer organoids. Fully mixed.

Attention: If not used immediately, please2-8°CStore complete culture medium without exceeding2week Ovarian cancer organoid culture factorBContains fungicides and antibiotics（50x）.

c) formula3：

in“formula2”Add to complete culture medium20μLOvarian cancer organoid culture factorG（500x）

Attention: The prepared ovarian cancer organoid culture medium can be used in2-8°CStorage, recommended for use within two weeks. Ovarian cancer organoid culture factorB (50x)Contains antibiotics for bacteria and fungi.

5 Primary culture of ovarian cancer organoids derived from patients

Attention: Research involving major human tissue materials must comply with all relevant institutions and government regulations. Prior to collecting primary human tissue materials, informed consent must be obtained from all subjects.

a) Establishment of primary human ovarian cancer organoids

1 Collect primary ovarian cancer tissue fragments in a cold primary tissue storage solution using a centrifuge tube. Maintain the organizational samples in4℃Until the separation begins.

2 Evaluate whether the obtained tissue fragments are composed of epithelium. If there is fat or muscle tissue present, use a knife or forceps under an dissecting microscope to remove as much of these non epithelial components as possible. If there is no fat or muscle tissue, please proceed to the next step immediately.

3 Using epithelial organ basic culture medium orDPBSRinse the tissue twice.

4 Use a knife or chop the tissue into small pieces1-3mm³Place the small fragments in a cell culture dish.

5 in37℃Use below10mLTumor tissue digestion solution15mLDigesting tissue fragments in centrifuge tubes, with variable digestion time, from30Minutes to arrive1.5The hours vary. Carefully monitor the digestion process and observe the degree of digestion by blowing and tapping the supernatant appropriately. When most tissue fragments can pass through1mLWhen the pipette tip is used, the digestion process is completed.

6 Translate into EnglishFBSAdd to the tissue digestion mixture, with a final concentration of2%And use it100μmCell filter filtration.

7 Collect and in4°CBelow is250gCentrifuge filtered cells3minute Inhale the supernatant with visible red sediment and use2mLResuspend and precipitate red blood cell lysate, and lyse red blood cells at room temperature1Minutes, and within4°CBelow is250gcentrifugation3minute

8 Suck out the supernatant and resuspend the precipitate in the basic culture medium4°CBelow is250gcentrifugation3Minute, repeat this step again.

9 Suck out the supernatant and resuspend the precipitate in the matrix gel. The matrix adhesive should be stored on ice to prevent it from solidifying. Carry out the process as soon as possible. The amount of matrix adhesive used depends on the size of the particles. approximately10，000Cells should be seeded in25μLIn the matrix adhesive.

10 Do not excessively dilute the matrix adhesive (the proportion of matrix adhesive should be>70%(Volume of matrix adhesive)/(Total volume), as this may inhibit the correct formation of solid droplets. Lay the matrix adhesive containing organoids on24At the bottom of the well cell culture plate, each droplet surrounds the center of the well approximately30μL.

Attention: Once the organoid is resuspended in the matrix gel, proceed with seeding as soon as possible, as the matrix gel may solidify in the test tube or pipette tip. Do not allow matrix particles to come into contact with the pipe wall.

11 Place the culture plate in37°Cand5%CO₂In the incubator15-25Minutes, allow the matrix gel to cure.

12 Prepare the required amount of ovarian cancer organoid culture medium.

13 Once the matrix gel droplets solidify（15-25Minutes), open the board and carefully add to each hole500μLHuman ovarian cancer organoid culture medium.

Caution: Do not add the culture medium directly to the top of the matrix gel droplets, as this may damage the already solidified structure.

14 Place the culture plate in37℃and5%CO₂In a constant temperature incubator.

15 every3-4Change the culture medium once a day, carefully extract the culture medium from the well, and replace it with fresh preheated organoid culture medium.

16 Closely monitor the growth status of organoids. Ideally, human ovarian cancer organoids should be in7-10Built within the day.

b) Passage culture of human ovarian cancer organoids

1 Use a gun tip that has been moistened with a cleaning solution to blow and scrape off the organoids, and transfer the suspension of organoids and culture medium to the one that has been moistened with the cleaning solution1.5mL EPIn the management.

2 Use a gun tip that has been moistened with a cleaning solution to resuspend the organoid suspension vigorously, separating the organoid from the matrix gel.

3 250gCentrifugal force room temperature centrifugation3min.

4 Discard the supernatant and use organoid digestion solution or mechanical destruction. For cell dissociation using organoid digestion fluid, resuspend the organoid suspension in the organoid digestion fluid, repeatedly blow it up and down with a pipette, and place it in37°CIncubate until the organoids dissociate. Use a nozzle with a filter cartridge every time2Repeatedly blow and suck up and down every minute8To assist in the dissociation of organoids. Closely monitoring the digestion process minimizes the incubation time in organoid dissociation fluid. If a mechanical failure occurs, in1.5mLResuspend organoid suspension in epithelial organoid basic culture medium. Carefully use a pipette to aspirate the suspension of organoids, repeatedly up and down30This will help with digestion.

Caution: Do not dissociate more than in the organoid dissociation solution7Minutes, as this may lead to poor growth or even destruction of organoids. Based on experience, if it is a mixture of small cells, it can be observed that10-50The digestion of a cell cluster composed of individual cells is completed.

5 After digestion is complete, use1mLRinse the basic culture medium of epithelial organs once, and then leave it at room temperature250gcentrifugation3minute

6 Discard the supernatant, resuspend the organoid sediment with an appropriate amount of matrix gel, resuspend it on ice, and resuspend for no more than30sTo avoid premature solidification of the matrix adhesive.

Attention: The dilution ratio of the matrix adhesive should be within70%The above is to ensure the structural stability of the matrix gel during the cultivation process.

7 Point in the mixed suspension of matrix gel and organoids24The bottom of the orifice plate should be aligned to avoid contact between the suspension and the side wall of the orifice plate. Each hole30uLabout. Attention: To prevent the matrix adhesive from solidifying at room temperature, this step should be completed as soon as possible.

8 Place the inoculated culture plate in37℃Incubate in a carbon dioxide constant temperature incubator15minWait for the matrix adhesive to solidify on both sides.

9 Prepare ovarian cancer organoid culture medium.

10 After the matrix gel solidifies, add the prepared human ovarian cancer organoid culture medium,24Add to each hole of the orifice plate250uLCulture medium.

11 Translate into English2The orifice plate is placed37℃Cultivate in a carbon dioxide incubator until the organoids require further experimentation

V2.0edition

Update time:2025/6/21