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Method for preparing matrigel by using animal carcass

A technology of animal carcasses and matrigel, which is applied in the field of biochemistry, can solve the problems of labor and labor, and achieve the effect of convenient material collection and high output

Pending Publication Date: 2022-07-29
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many animal experiments only need to take part of the animal's tissue or blood, and the rest of the animal body needs to be harmlessly treated, which is laborious and laborious. Therefore, it is of great significance to find a better way to deal with the animal body

Method used

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  • Method for preparing matrigel by using animal carcass
  • Method for preparing matrigel by using animal carcass

Examples

Experimental program
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Effect test

Embodiment 1

[0022] An 8-week-old Balb / c mouse was taken and weighed 23 grams. After carbon dioxide asphyxiation treatment, the fur was stripped and the internal organs were cleaned. The remaining part was about 16 grams. Mice carcasses were minced with surgical scissors, snap-frozen in liquid nitrogen, and carefully ground into powder using a mortar. After the liquid nitrogen was evaporated, 32 mL of pre-cooled PBS solution was added and stirred at 4°C overnight to extract cell matrix proteins. After centrifugation at 8000g, the pellet was discarded. Saturated ammonium sulfate was slowly added to the supernatant to 20% saturation, the precipitate was left to stand at 4°C for 30 minutes, and the precipitate was collected by centrifugation at 8000 g. The pellet was dissolved with 8 mL of PBS, and dialyzed overnight using a 7 kD dialysis bag. The dialysate was changed, and dialysis was continued overnight using DMEM high-glucose medium. Place the matrigel on ice in an ultra-clean bench an...

Embodiment 2

[0024] An 8-week-old SD rat was taken and weighed 232 grams. After carbon dioxide asphyxiation treatment, the fur was peeled off and the internal organs were cleaned. The remaining part was about 137 grams. The mouse carcasses were pulverized using a liquid nitrogen pulverizer. After the liquid nitrogen evaporated, 270 mL of pre-cooled PBS solution was added and stirred at 4°C overnight to extract cell matrix proteins. After centrifugation at 8000g, the pellet was discarded. Saturated ammonium sulfate was slowly added to the supernatant to 20% saturation, the precipitate was left to stand at 4°C for 30 minutes, and the precipitate was collected by centrifugation at 8000 g. The precipitate was dissolved with 40 mL of PBS, and dialyzed overnight using a 7 kD dialysis bag. The dialysate was changed, and dialysis was continued overnight using DMEM / F12 medium. Place the matrigel on ice in a clean bench and dispense the matrigel into centrifuge tubes. The protein concentration me...

Embodiment 3

[0026] HepG2 cells were grown to a confluency of 60% to 80%, gently rinsed with PBS for 3 times, replaced with serum-free medium, and placed in a cell incubator at 37°C and 5% CO2-saturated humidity for 24 hours (serum starvation); Example 1 Thaw the prepared mouse-derived Matrigel at 4 degrees. Add 70 μl of 1:8 diluted Matrigel (diluted with serum-free DMEM medium) to the upper chamber of the Transwell, and place it at 37°C, 5% CO. 2 After incubating in a humidified cell incubator for 8 hours, discard the supernatant and gently rinse with PBS twice; add 100 μl of serum-free DMEM to each well and incubate at 37°C for 3-4 hours; discard the medium for HepG2 cells and gently rinse with PBS for 3 After digestion, cells were digested with trypsin; after the digestion was terminated, single cell suspension was prepared and collected, and centrifuged at 1500 rpm for 3 min; supernatant was discarded, cells were resuspended in serum-free DMEM medium, cells were counted on a hemocytome...

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Abstract

The invention discloses a method for preparing matrigel from animal carcasses. The method comprises the following steps: disinfecting the animal carcasses; removing the skin and viscera of the animal carcasses, freezing the remaining animal tissues with liquid nitrogen, and crushing; extracting an extracellular matrix component, and precipitating the extracellular matrix component by using ammonium sulfate; and re-dissolving the extracellular matrix component, centrifuging, and taking the supernate, namely the matrigel. According to the method disclosed by the invention, the matrigel can be prepared from the remaining animal carcasses in animal experiments, so that the reutilization of the animal carcasses is realized. When the requirement for the yield of the matrigel is high, animals can be fed to prepare the matrigel. According to the method, materials are convenient to obtain, the yield of the matrigel is high, and the performance of the matrigel meets 3D cell culture requirements. By using the method disclosed by the invention, about 10mL of matrigel can be obtained by one mouse, and 50mL of matrigel can be prepared by one rat.

Description

technical field [0001] The present application relates to a method for preparing matrigel by using animal carcasses, and belongs to the technical field of biochemistry. Background technique [0002] Matrigel is widely used in biological experiments and can be used to treat cell culture plates to promote cell adhesion; it can be used to plate Transwell chambers to study the invasive ability of tumor cells; it can be used for 3D culture of cell lines or patient-derived cells , to generate organoids for antitumor drug screening. [0003] Matrigel is a complex composed of soluble extracellular matrix, mainly including collagen, laminin, and nestin. At present, the main source of matrigel is EHS (Engelbreth-Holm-Swarm) matrigel. EHS Matrigel is extracted from EHS cells. EHS cells cannot be cultured in vitro, so it is not convenient for large-scale expansion, which brings great inconvenience to the production of Matrigel. At the same time, the preparation of EHS matrigel requi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
CPCC12N5/0062C12N2533/90
Inventor 滕以刚杜君瑶李晓迪
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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