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Culture method of osteosarcoma organ and bone tumor culture medium thereof

A culture method and organoid technology, applied in 3D culture, culture process, tissue culture, etc., can solve the problem of lack of osteosarcoma organoid culture method, bone tumor culture medium, etc.

Pending Publication Date: 2020-10-23
上海昊佰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, there is a lack of a culture method for osteosarcoma organoids and its bone tumor culture medium

Method used

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  • Culture method of osteosarcoma organ and bone tumor culture medium thereof
  • Culture method of osteosarcoma organ and bone tumor culture medium thereof
  • Culture method of osteosarcoma organ and bone tumor culture medium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Such as Figure 1 to Figure 5 as shown, figure 1 and figure 2 It is a diagram of an osteosarcoma sample of the present invention, it can be seen from the diagram that after a certain period of culture, fibrous organoids can be cultivated; image 3 It is the bone tumor sample figure of the present invention; Figure 4 It is a diagram of a sample of gastric bone tumor lung metastasis of the present invention. It can be seen from the diagram that solid spherical or irregular organoids can be cultured after a certain period of time. Figure 5 It is a bone tumor sample diagram of the present invention.

[0050] A method for preparing an osteosarcoma organoid 3D culture model of the present invention comprises the following steps:

[0051] (1) Tissue cleaning: wash 2 times with 1xPen-Strep Glutamine ADMEM / F12 10ml, add 2 times the amount;

[0052] (2) Cut the tissue into 1mm 3 After size, digest with digestive juice for 30-60 minutes, centrifuge at 1200r for 5 minutes ...

Embodiment 2

[0063] Such as Figure 6 to Figure 8 as shown, Figure 6 It is a sample diagram of bone tumor lymphatic metastasis of the present invention. Figure 7 It is a sample diagram of bone tumor lymphatic metastasis of the present invention. Figure 8 It is a picture of a bone tumor lung metastasis sample of the present invention. It can be seen from the figure that the sample changes significantly with the increase of culture time, and the bone tumor organoid gradually grows and extends around and grows from a solid texture to a cystic structure.

[0064] A method for preparing an air-liquid culture model of osteosarcoma organoids of the present invention comprises the following steps:

[0065] (1) Prepare collagen matrix; the preparation method of Collagen collagen matrix:

[0066] The ratio of Cellmatrix type I-A, 10×Ham’s F-12, and buffer solution is 8:1:1, and 50ug / ml laminin is added for bone tumor culture. The buffer consists of 0.05N NaOH, 200mM HEPES and 2.2g NaHCO 3 co...

Embodiment 3

[0075] The difference between Example 3 and Example 1 is that a method for preparing a 3D culture model of osteosarcoma organoids of the present invention comprises the following steps:

[0076] (1) Tissue cleaning: wash 3 times with 1xPen-Strep Glutamine ADMEM / F12 10ml, add 3 times the amount;

[0077] (2) Cut the tissue into 1mm 3 After size, digest with digestive juice for 30-60 minutes, centrifuge at 1200r for 5 minutes at 4°C after digestion; the digestive juice includes the following components:

[0078]

[0079]

[0080] (3) Wash 3 times with 1x Pen-Strep Glutamine ADMEM / F12 10ml;

[0081] (4) Count and centrifuge after filtering with 100uM cell sieve, resuspend Collagen in 1ug / ml laminin and then plate; Minutes, add medium 500ul / well.

[0082] (5) During the culture period, the bone tumor medium was replaced every 4 days;

[0083] (6) Organoids are generally passaged once every 15 days, and 2-5 times the amount of TrpLE Express is added to each well during pa...

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Abstract

The invention discloses a culture method of an osteosarcoma organ and an osteosarcoma culture medium thereof. The invention uses two culture methods, namely a preparation method of a 3D culture modelof an osteosarcoma organ and a preparation method of a gas-liquid culture model of the osteosarcoma organ. The preparation method of the osteosarcoma organ 3D culture model comprises the following steps: (1) tissue cleaning: performing cleaning for 2 to 5 times by using 10ml of ADMEM / F12 containing 1 x Pen-Strep Glutamine; (2) cutting tissues into pieces with a size of 1mm<3>, performing digestionfor 30-60 minutes by using digestive juice, and performing centrifuging for 5 minutes at a temperature of 4 DEG C and a speed of 1200r after the digestion; (3) performing cleaning for three times byusing 10ml of ADMEM / F12 containing 1 x Pen-Strep Glutamine; (4) performing filtering with a 100 [mu]M cell sieve, performing counting and centrifuging, adding Collagen into 1-100 [mu] g / ml laminin, performing re-suspending, and paving a plate; (5) replacing the bone tumor culture medium once every 3-4 days in the culture period; and (6) performing passage on the organoids once every 15-20 days generally, and adding 2-5 times of TrpLE Express into each hole during passage.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for culturing osteosarcoma organoids and a culture medium for bone tumors. Background technique [0002] Bone tumors are tumors that occur in the bone or its accessory tissues. There are benign and malignant. Benign bone tumors are easy to cure and have a good prognosis. Malignant bone tumors develop rapidly, have poor prognosis and high mortality. Malignant bone tumors are divided into primary and secondary. Secondary malignant bone tumors are secondary malignant tumors that metastasize from other tissues or organs in the body to bones through the blood circulation and lymphatic system. There is another type of lesion called tumor-like lesions. The tissue of tumor-like lesions does not have the characteristics of tumor cell morphology, but its ecology and behavior are destructive to tumors. It is generally limited and easy to cure. [0003] The real cause of the ...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0654C12N2509/00C12N2509/10C12N2513/00C12N2533/54C12N2533/52C12N2500/32C12N2501/345C12N2501/11C12N2501/119C12N2501/15C12N2500/60C12N2500/38C12N2501/727C12N2501/155C12N2501/998
Inventor 程婉莹张登
Owner 上海昊佰生物科技有限公司
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