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Three-dimensional spheroid culture method for maintaining stemness of breast cancer stem cells in vitro

A technology of breast cancer stem cells and spheroids, applied in the field of maintaining the stemness of breast cancer stem cells in vitro, and the three-dimensional spheroid culture, can solve the problems of being unable to maintain the stemness of breast cancer stem cells for a long time, so as to prolong the stemness maintenance time and improve the growth rate. ball rate, reducing the effect of reagent consumables and human resources

Active Publication Date: 2019-11-01
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome the deficiency in the prior art that the stemness of breast cancer stem cells cannot be maintained for a long time, and to provide a three-dimensional spheroid culture method based on a rotating bioreactor to maintain the stemness of breast cancer stem cells in vitro

Method used

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  • Three-dimensional spheroid culture method for maintaining stemness of breast cancer stem cells in vitro
  • Three-dimensional spheroid culture method for maintaining stemness of breast cancer stem cells in vitro
  • Three-dimensional spheroid culture method for maintaining stemness of breast cancer stem cells in vitro

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Isolation of breast tissue:

[0032] 1) Put fresh mammary gland tissue into pre-cooled EpiCult-B medium containing 5% fetal bovine serum;

[0033] 2) Transfer the tissue to a sterile glass petri dish, break it with a spatula, and transfer it to a tissue digestion bottle;

[0034] 3) Configure EpiCult-B Medium digestion solution containing collagenase / hyaluronidase, add it to the digestion bottle, ensure that the tissue block is completely submerged and suspended, and seal it with sterile tin foil;

[0035] 4) Digest with horizontal shaking at 37°C until large tissue pieces disappear;

[0036] 5) Transfer the digestion solution to a sterile 50mL centrifuge tube, centrifuge at 80g for 30 seconds;

[0037] 6) Discard the uppermost fat layer;

[0038] 7) Take another sterile 50mL centrifuge tube, centrifuge at 200g for 3 minutes, and mammary gland epithelial cells are precipitated.

[0039] 2. Isolate a single cell suspension:

[0040] 1) Add 3 mL of pre-warmed trypsi...

Embodiment 2

[0063] 1. Digestion of mammary spheroids cultured in vitro for 14 days:

[0064] 1) Collect mammary spheroids that have been cultured for 14 days into a 50mL centrifuge tube, centrifuge at 350g for 5 minutes, and discard the supernatant as much as possible;

[0065] 2) Add 1 mL of pre-warmed trypsin containing EDTA, slightly reduce the volume of the sampling gun, and wet the tip of the pipette to avoid foaming;

[0066] 3) Press the gun head against the tube wall, gently blow up and down the mammary spheroid, and rinse the tube wall;

[0067] 4) Add 5 mL of pre-cooled Hanks solution containing 2% fetal bovine serum, centrifuge at 350 g for 5 minutes;

[0068] 5) Discard the supernatant, add 1mL PBS to resuspend the pellet, and adjust the cell concentration to 1×10 5 Individuals / mL, some of them were used for follow-up experiments, and the other were subcultured and continued to be cultured.

[0069] 2. Prepare ALDH1 detection samples:

[0070] 1) Take out 2 EP tubes and ma...

Embodiment 3

[0088] Subcutaneous tumors in nude mice:

[0089] 1) Divide the same batch of 4-week-old NOD / SCID mice into three groups, 2 mice / group;

[0090] 2) Pour out the breast cancer stem cell spheroids obtained by in vitro culture in a rotating bioreactor for 21 days from the rotating bioreactor to a 10cm cell culture dish, take three small, medium and large ones, and mix them with Matrigel respectively , to inoculate the middle and rear part of the right armpit;

[0091] 3) After three weeks, the mice were sacrificed, and the tumors were taken out to measure the size.

[0092] The result is as image 3 As shown, breast cancer stem cell spheroids expanded in vitro can form tumors subcutaneously in nude mice, indicating that stemness is maintained.

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Abstract

The invention discloses a three-dimensional spheroid culture method for maintaining stemness of breast cancer stem cells in vitro. Three-dimensional spheroid culture of human breast cancer stem cellsis performed in a rotating bioreactor, and a breast cancer stem cell spheroid is directly obtained. The obtained cell is an ALDH1<+> / CD44<+> / CD24<-> phenotype, and is recognized as the breast cancerstem cell phenotype. The three-dimensional spheroid culture method is a reliable method for breast cancer stem cell culture in vitro. Compared with the prior art, such as a low adhesion material method, a hanging drop method and a gel method, the three-dimensional spheroid culture method is easy to operate, the ball forming rate of stem cells is greatly improved, the maintenance time of the stemness is prolonged to at least 21 days, and the culture can be expanded on a large scale, and the cost of reagent consumables, human resources and the like is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a three-dimensional spheroid culture method for maintaining the stemness of breast cancer stem cells in vitro. Background technique [0002] For early breast cancer (I, II, IIIA and operable IIIC grades), radical surgery combined with radiotherapy is given priority. The adjuvant chemotherapy regimen represented by hormone preparations, trastuzumab and cytotoxic chemotherapy drugs is used to prevent the formation of postoperative micrometastases. Class IIIB and inoperable IIIC generally use systemic chemotherapy or hormonal therapy to downgrade and control the primary tumor before surgery or radiotherapy. Although the above methods can effectively reduce the tumor volume in the short term, if the cancer stem cells cannot be completely eliminated, the drug effect will not be maintained for a long time, and the tumor is very likely to be resistant to drugs and relapse. Therefore, breast...

Claims

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Application Information

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IPC IPC(8): C12N5/095
CPCC12N5/0695C12N2501/11C12N2501/115C12N2501/33C12N2501/39C12N2513/00
Inventor 张洁心张世昌查小明
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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