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Method for individually culturing tumor organoid by using hydrogel prepared from autologous chest and ascites of tumor patient

A tumor patient, hydrogel technology, applied in the field of biomedicine, can solve the problems affecting the accuracy and objectivity of cell culture results, limit wide application, low culture success rate, etc., achieve objective and accurate culture results, good water permeability, good biocompatibility

Pending Publication Date: 2022-03-25
SUZHOU GENOARRAY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the synthetic gel material has good reproducibility, its biocompatibility with cells is poor, and the success rate of culture is low, while the animal-derived scaffold material contains many uncertain components, and there may be batch-to-batch differences, and there will be Affects the accuracy and objectivity of cell culture results; finally, animal-derived gels are generally very expensive, limiting their widespread use

Method used

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  • Method for individually culturing tumor organoid by using hydrogel prepared from autologous chest and ascites of tumor patient
  • Method for individually culturing tumor organoid by using hydrogel prepared from autologous chest and ascites of tumor patient
  • Method for individually culturing tumor organoid by using hydrogel prepared from autologous chest and ascites of tumor patient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the optimization of hydrogel preparation method

[0053]Take 100 mL of malignant pleural and ascites fluid from 10 tumor patients, centrifuge at 400 g for 5 min at room temperature, and collect the supernatant. Various concentrations of glutaraldehyde were added. 293T cells (ATCC, USA) were cultured two-dimensionally to the logarithmic growth phase, and then digested with 0.25% (% expressed in g / 100ml) trypsin (Sigma) to obtain a single cell suspension with a final concentration of 5 × 10 3 / mL mixed with the above groups (glutaraldehyde of different concentrations added to the supernatant of pleural and ascites fluid) and then spread on the bottom of the well of the cell culture plate to observe the coagulation time of each group. DMEM (Thermo Scientific) containing 10% (v / v) fetal calf serum (Thermo Scientific) and 1% (m / v) penicillin / streptomycin (Gibco) was added on top of the gel. At 37°C, 5% CO 2 Observe the state of cells in each group after cult...

Embodiment 2

[0054] Embodiment 2, the preparation method of autologous hydrogel

[0055] 1. Prepare 100× glutaraldehyde storage solution

[0056] Measure 20-80 mL of glutaraldehyde, add it to 100 mL with deionized water, the concentration of glutaraldehyde is 20-80% (v / v), filter and sterilize with a filter membrane with a pore size of 0.22 μm.

[0057] 2. Separation of thoracic and ascites in tumor patients

[0058] Take 10 mL of malignant pleural and ascites fluid from tumor patients, centrifuge at 400 g for 5 min at room temperature, and collect the supernatant.

[0059] 3. Preparation of autologous hydrogel scaffold

[0060] Mix 990 μL of pleural effusion (or ascites) supernatant with 10 μL of 100× glutaraldehyde stock solution (50%, v / v) thoroughly and add to the wells of the cell culture plate, and place it flat at 37°C for 25 minutes until it solidifies into a jelly shape( figure 1 ), under the microscope, it can be seen that a loose network structure is formed inside the hydrog...

Embodiment 3

[0061] Example 3. Individualized culture of tumor organoids

[0062] 1. Organoid culture of autologous tumor cells in thoracic and ascites fluid

[0063] In this example, the effect of personalized organoid culture of autologous tumor cells in pleural effusion of patients with lung cancer and breast cancer and ascites of patients with ovarian cancer and liver cancer was tested.

[0064] 1. Centrifuge all the pleural fluid and ascites collected from the patient's first pleural and peritoneal effusion drainage at room temperature for 10 minutes at 400 g. Keep the supernatant for later use.

[0065] 2. The cell pellet at the bottom of the centrifuge tube was adjusted to a cell concentration of 10 with RPMI-1640 medium (Gibco). 7 individual / mL. Add Ficoll-Paque TM PLUS (GE Healthcare) mononuclear cell separation medium to the centrifuge tube, then slowly add the cell suspension at a volume ratio of 1:1, and centrifuge at 400g room temperature for 25min. The cells at the interf...

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Abstract

The invention discloses a method for individually culturing tumor organs by using hydrogel prepared from autologous chest and ascites of a tumor patient. The invention provides a method for culturing autologous tumor organoid of a tumor patient, which comprises the following steps: mixing tumor cells to be cultured from the tumor patient with a matrix to enable the final concentration of the cells to be (5-10) * 10 < 3 > / mL; the matrix is in-vitro autologous body cavity effusion of a tumor patient without cell components; adding a protein cross-linking agent, then adding a hydrogel solution obtained by mixing to the bottom of a cell culture container, and flatly standing at 37 DEG C until the hydrogel solution is solidified; and adding a tumor organoid culture solution to the upper part of the hydrogel, and culturing to obtain the tumor organoid. The method has the advantages of good biocompatibility, avoidance of heterologous component pollution and low culture cost.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing hydrogels and culturing individualized tumor organoids using autologous thoracic and ascites fluids of tumor patients. Background technique [0002] Organoid culture is the use of three-dimensional cell culture technology to co-cultivate three-dimensional structure scaffold materials and cells in vitro, so that cells can migrate, grow and form a three-dimensional structure in a three-dimensional space structure. Three-dimensional cell culture is a technology between monolayer cell culture and animal experiments. It can not only simulate the in vivo environment to the greatest extent, but also has the advantages of intuitive cell culture and controllable culture conditions. Organoid models have broad application prospects in new drug development, stem cell research, and tumor research. [0003] Synthetic or natural materials such as alginate, gelatin, hyaluronic ...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/071
CPCC12N5/0693C12N5/0688C12N5/0631C12N5/0682C12N5/0671C12N2509/00C12N2513/00C12N2533/90
Inventor 张函槊刘勇
Owner SUZHOU GENOARRAY
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