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CN113106065B - Colorectal cancer organoid and liver organoid co-culture model and construction method thereof - Google Patents

  • ️Tue Feb 27 2024
Colorectal cancer organoid and liver organoid co-culture model and construction method thereof Download PDF

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CN113106065B
CN113106065B CN202110287770.9A CN202110287770A CN113106065B CN 113106065 B CN113106065 B CN 113106065B CN 202110287770 A CN202110287770 A CN 202110287770A CN 113106065 B CN113106065 B CN 113106065B Authority
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colorectal cancer
liver
organoid
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organoids
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CN113106065A (en
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秦环龙
蔚青
李曼
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Shanghai Tenth Peoples Hospital
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Abstract

The invention relates to a method for constructing a colorectal cancer organoid and liver organoid co-culture model, which comprises the following steps: collecting colorectal cancer organoids and liver organoids respectively, centrifuging, discarding supernatant, and sucking matrigel as much as possible; respectively adding digestive juice, centrifuging and discarding supernatant after digestion at room temperature, and performing cell count; mixing the colorectal cancer organoids and the liver organoids according to a cell number ratio of 1:1, re-suspending by using matrigel, and dripping onto a preheated 24-pore plate, wherein the gel is quickly transformed into jelly shape after being heated; and (3) placing the 24-pore plate in an incubator for incubation, adding colorectal cancer organoid culture medium and liver organoid culture medium into each pore after the incubation is completed, and then placing the culture medium in the incubator for culture. The colorectal cancer organoid and liver organoid co-culture model can simulate in-vivo 3D growth environment in vitro, and can reflect organism functions more truly, so that pathological processes and occurrence mechanisms of intestinal cancer liver metastasis are studied.

Description

Colorectal cancer organoid and liver organoid co-culture model and construction method thereof

Technical Field

The invention relates to the technical field of organoids, in particular to a colorectal cancer organoid and liver organoid co-culture model and a construction method thereof.

Background

Colorectal cancer (colorectal cancer, CRC) is the third most common cancer species worldwide, with about 100 tens of thousands of people diagnosed with colorectal cancer annually. Of these, colorectal liver metastasis occurs in more than 50% of patients (celorectal liver metastases, CLM). At present, surgical excision metastasis is the main treatment means of CLM, and postoperative chemotherapy can prolong the survival time of several months, and the survival time without any treatment is only 6-9 months. Research on pathological processes and occurrence mechanisms of liver metastasis of intestinal cancer is a research hotspot in the field, however, the traditional research on occurrence and development processes of liver metastasis based on intestinal cancer tumor cell lines cannot faithfully reflect the in-vivo environment. The organoid technology can simulate in-vivo 3D growth environment in vitro, can reflect organism functions more truly compared with a traditional two-dimensional culture system, and is highly consistent with the mutation type of the primary tumor focus gene.

Therefore, there is a need for a colorectal cancer organoid and liver organoid co-culture model and method of constructing the same.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and provides a colorectal cancer organoid and liver organoid co-culture model and a construction method thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the present invention provides a method for constructing a colorectal cancer organoid and liver organoid co-culture model, comprising the steps of:

s1, respectively collecting colorectal cancer organoids and liver organoids, centrifuging, discarding supernatant, and sucking matrigel as much as possible; respectively adding digestive juice, centrifuging and discarding supernatant after digestion at room temperature, and performing cell count;

s2, mixing the colorectal cancer organoids and the liver organoids according to the cell number ratio of 1:1, re-suspending by adopting matrigel, and dripping onto a preheated 24-pore plate, wherein the gel is quickly transformed into jelly shape after being heated;

s3, placing the 24-pore plate in an incubator for incubation, adding colorectal cancer organoid culture medium and liver organoid culture medium into each pore after the incubation is completed, and then placing the culture medium in the incubator for culture.

Preferably, the colorectal cancer organoid construction step comprises:

a1, placing colorectal cancer tissues into PBS buffer solution containing penicillin or/and streptomycin for cleaning, cutting the colorectal cancer tissues into fragments by adopting a sterile instrument, placing the fragments into a centrifuge tube containing ice PBS buffer solution, centrifuging, and discarding the supernatant;

a2, adding tissue digestion liquid for resuspension, shaking vigorously, placing on ice for incubation, filtering supernatant liquid in the tissue digestion liquid to a centrifuge tube through a filter screen to obtain filtrate, and enriching gland fragments;

a3, taking the filtrate, centrifuging, discarding the supernatant, and adopting colorectal cancer organoid culture medium to re-suspend and then counting cells;

a4, transferring the cell suspension into an EP tube, centrifuging, discarding the supernatant, and placing the EP tube on ice; adopting a precooled gun head to absorb a proper amount of liquid temperature-sensitive matrigel into the EP pipe, and blowing and sucking for ten times, wherein bubbles should be avoided in the process;

a5, dripping the mixture of the cells and the matrigel onto a preheated 24-pore plate, and rapidly converting the gel into jelly shape after the gel is heated;

a6, after the 24-pore plate is placed in an incubator for incubation, colorectal cancer organoid culture medium is added into each pore respectively, and then the 24-pore plate is placed in the incubator for culture.

Preferably, the constructing step of the liver organoid comprises:

b1, placing liver tissues in PBS buffer solution containing penicillin or/and streptomycin, cleaning, cutting the liver tissues into fragments by adopting a sterile instrument, placing the fragments in a centrifuge tube containing ice PBS buffer solution, centrifuging, and discarding the supernatant;

b2, adding tissue digestion liquid for resuspension, shaking vigorously, placing on ice for incubation, and filtering supernatant liquid in the suspension liquid to a centrifuge tube through a filter screen to obtain filtrate;

b3, taking the filtrate, centrifuging, discarding the supernatant, and adopting a liver organoid culture medium to re-suspend and then counting cells;

b4, transferring the cell suspension into an EP tube, centrifuging, discarding the supernatant, and placing the EP tube on ice; adopting a precooled gun head to absorb a proper amount of liquid temperature-sensitive matrigel into the EP pipe, and blowing and sucking for ten times, wherein bubbles should be avoided in the process;

b5, dripping the mixture of the cells and the matrigel onto a preheated 24-pore plate, and rapidly converting the gel into jelly shape after the gel is heated;

b6, after the 24-pore plate is placed in an incubator for incubation, each pore is added with liver organoid culture medium respectively, and then the mixture is placed in the incubator for culture.

Preferably, 350. Mu.L of colorectal cancer organoid medium and 350. Mu.L of liver organoid medium are added separately to each well.

Preferably, the fragments are 2-4mm 3 Fragments of size.

Preferably, the pieces are 0.5-1mm 3 Fragments of size.

Preferably, the sieve is a 100 μm sieve.

Preferably, the liquid temperature-sensitive matrigel is added in a proportion of 50 mu L of the liquid temperature-sensitive matrigel for every 20000 cells.

In a second aspect, the present invention provides a colorectal cancer organoid and liver organoid co-culture model obtained by the construction method as described above.

Compared with the prior art, the invention has the following technical effects:

the colorectal cancer organoid and liver organoid co-culture model can simulate in-vivo 3D growth environment in vitro, and can reflect organism functions more truly, so that pathological processes and occurrence mechanisms of intestinal cancer liver metastasis are studied.

Drawings

FIG. 1 is a co-culture of colorectal cancer organoids with normal liver organoids, where a is the microscopic morphology of the individual liver organoids; b is the co-culture light-microscopic morphology of liver organoids and colorectal organoids; c is individual liver organoid HE staining; d is HE staining of liver organoids and colorectal organoids in co-culture; e is a separate liver organoid Ki-67 stain; f is the co-culture Ki-67 staining of liver organoids and colorectal organoids;

FIG. 2 shows CDX-2 expression after one week of co-culture of colorectal and liver organoids, wherein the left arrow indicates normal liver organoids, the right arrow indicates colorectal organoids, and the particles represent CDX-2 protein expression;

FIG. 3 shows the expression of CDX-2 protein in liver organoids alone, colorectal organoids alone, liver organoids after co-culture and colorectal organoids after co-culture.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.

Examples

The embodiment provides a colorectal cancer organoid and liver organoid co-culture model, and a construction method thereof comprises the following steps:

(1) Construction of colorectal cancer organoids:

a1, after colorectal cancer tissues are washed 2 times in 5mL PBS buffer solution containing 1% penicillin or/and streptomycin, the colorectal cancer tissues are cut into 2-4mm by adopting a sterile instrument 3 Fragments of the size were placed in a 15mL centrifuge tube containing 10mL of ice PBS buffer, centrifuged at 1500rpm for 3min and the supernatant discarded;

a2, adding 5mL of tissue digestion solution (stemcell, cat: 07174), resuspending, shaking vigorously, placing on ice, incubating for 30min, filtering the supernatant to a 50mL centrifuge tube through a 100 μm filter screen to obtain filtrate, and enriching glandular fragments;

a3, taking 1mL of the filtrate, centrifuging 190g for 5min, discarding the supernatant, and re-suspending by adopting 1mL of human colorectal cancer organoid medium (Stemcell, 06010) for cell count;

a4, transferring the cell suspension into an EP tube, centrifuging 190g for 5min, discarding the supernatant, and placing the EP tube on ice; sucking a proper amount (namely 50 mu L of liquid temperature-sensitive Matrigel/20000 cells) of liquid temperature-sensitive Matrigel at 4 ℃ into the EP pipe by adopting a precooled gun head, and blowing and sucking for ten times, wherein bubbles are avoided in the process;

a5, dripping 50 mu L of the mixed solution of the cells and the matrigel into a preheated 24-pore plate, and quickly converting the gel into jelly shape after the gel is heated;

a6, placing the 24-pore plate at 37 ℃ and 5% CO 2 After incubation in the incubator for 30min, 700. Mu.L of human colorectal cancer organoid medium (Stemcell, 06010) was added to each well, followed by incubation at 37℃with 5% CO 2 Culturing in incubator for 5-7 days.

(2) Construction of liver organoids:

b1, washing liver tissue in 5mL PBS buffer solution containing 1% penicillin or/and streptomycin for 2 times, and cutting the liver tissue into 0.5-1mm with sterile instrument 3 Fragments of the size were placed in a 15mL centrifuge tube containing 10mL of ice PBS buffer, and after centrifugation at 300g for 5min, the supernatant was discarded;

b2, adding 5mL of tissue digestion solution (stemcell, cat: 07174), re-suspending, vigorously shaking, placing on ice, incubating for 30min, and filtering the supernatant to a 50mL centrifuge tube through a 100 μm filter screen to obtain filtrate;

b3, taking 1mL of the filtrate, centrifuging 190g for 5min, discarding the supernatant, and adopting 1mL of human liver organoid culture medium (M201 of Chuangxin International Biotech Co.) for cell counting after resuspension;

b4, transferring the cell suspension into an EP tube, centrifuging 190g for 5min, discarding the supernatant, and placing the EP tube on ice; sucking a proper amount (namely 50 mu L of liquid temperature-sensitive Matrigel/20000 cells) of liquid temperature-sensitive Matrigel at 4 ℃ into the EP pipe by adopting a precooled gun head, and blowing and sucking for ten times, wherein bubbles are avoided in the process;

b5, dripping 50 mu L of the mixed solution of the cells and the matrigel into a preheated 24-pore plate, and quickly converting the gel into jelly shape after the gel is heated;

b6, placing the 24-well plate at 37 ℃ and 5% CO 2 After incubation in incubator for 30min, 700. Mu.L of human liver organoid medium (Innovative core International Biotechnology Co., M201) was added to each well, and the mixture was placed at 37℃with 5% CO 2 Culturing in incubator for 5-7 days.

(3) Construction of colorectal cancer organoids and liver organoids co-culture model

S1, respectively collecting the cultured colorectal cancer organoids and liver organoids,centrifuging, discarding the supernatant, and sucking matrigel as much as possible; respectively adding 2mL of TrypLE TM Express digest (Gibco, 12604-013), after digestion for 5min at room temperature, centrifuged and the supernatant discarded for cell counting;

s2, mixing the colorectal cancer organoids and the liver organoids according to a cell number ratio of 1:1, re-suspending by using matrigel, and dripping 50 mu L of matrigel per hole onto a preheated 24-hole plate, wherein the gel is quickly changed into jelly shape after being heated;

s3, placing the 24-pore plate at 37 ℃ and 5% CO 2 After incubation in incubator for 30min, 350. Mu.L of colorectal cancer organoid medium and 350. Mu.L of liver organoid medium were added to each well, and the mixture was placed at 37℃in 5% CO 2 The incubator was cultured for 1 week.

As shown in FIG. 1, normal liver organoids increased in volume and increased levels of Ki-67 expression that labeled cell proliferation after 1 week of co-culture with colorectal organoids; as shown in fig. 2, the colorectal cancer organoids highly expressed CDX-2 protein, and after co-culturing with the colorectal cancer organoids for 1 week, the normal liver organoids also began to significantly express CDX-2, suggesting that the liver organoids were affected by the colorectal cancer organoids; as shown in FIG. 3, normal liver organoids alone did not express CDX-2, and after 1 week of co-culture with colorectal organoids, CDX-2 was also highly expressed under the influence of intestinal organoids, further validating the results of FIG. 2.

The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the present invention, and are intended to be included within the scope of the present invention.

Claims (7)

1. A method for constructing a colorectal cancer organoid and liver organoid co-culture model, comprising the steps of:

s1, respectively collecting colorectal cancer organoids and liver organoids, centrifuging, discarding supernatant, and sucking matrigel as much as possible; respectively adding digestive juice, centrifuging and discarding supernatant after digestion at room temperature, and performing cell count;

s2, mixing the colorectal cancer organoids and the liver organoids according to the cell number ratio of 1:1, re-suspending by adopting matrigel, and dripping onto a preheated 24-pore plate, wherein the gel is quickly transformed into jelly shape after being heated;

s3, placing the 24-pore plate in an incubator for incubation, adding colorectal cancer organoid culture medium and liver organoid culture medium into each pore after the incubation is completed, and then placing the culture medium in the incubator for culture;

the colorectal cancer organoids are constructed by the steps of:

a1, placing colorectal cancer tissues into PBS buffer solution containing penicillin or/and streptomycin for cleaning, cutting the colorectal cancer tissues into fragments by adopting a sterile instrument, placing the fragments into a centrifuge tube containing ice PBS buffer solution, centrifuging, and discarding the supernatant;

a2, adding tissue digestion liquid for resuspension, shaking vigorously, placing on ice for incubation, filtering supernatant liquid in the tissue digestion liquid to a centrifuge tube through a filter screen to obtain filtrate, and enriching gland fragments;

a3, taking the filtrate, centrifuging, discarding the supernatant, and adopting colorectal cancer organoid culture medium to re-suspend and then counting cells;

a4, transferring the cell suspension into an EP tube, centrifuging, discarding the supernatant, and placing the EP tube on ice; adopting a precooled gun head to absorb a proper amount of liquid temperature-sensitive matrigel into the EP pipe, and blowing and sucking for ten times, wherein bubbles should be avoided in the process;

a5, dripping the mixture of the cells and the matrigel onto a preheated 24-pore plate, and rapidly converting the gel into jelly shape after the gel is heated;

a6, after the 24-pore plate is placed in an incubator for incubation, colorectal cancer organoid culture medium is added into each pore respectively, and then the 24-pore plate is placed in the incubator for culture;

the construction steps of the liver organoid comprise:

b1, placing liver tissues in PBS buffer solution containing penicillin or/and streptomycin, cleaning, cutting the liver tissues into fragments by adopting a sterile instrument, placing the fragments in a centrifuge tube containing ice PBS buffer solution, centrifuging, and discarding the supernatant;

b2, adding tissue digestion liquid for resuspension, shaking vigorously, placing on ice for incubation, and filtering supernatant liquid in the suspension liquid to a centrifuge tube through a filter screen to obtain filtrate;

b3, taking the filtrate, centrifuging, discarding the supernatant, and adopting a liver organoid culture medium to re-suspend and then counting cells;

b4, transferring the cell suspension into an EP tube, centrifuging, discarding the supernatant, and placing the EP tube on ice; adopting a precooled gun head to absorb a proper amount of liquid temperature-sensitive matrigel into the EP pipe, and blowing and sucking for ten times, wherein bubbles should be avoided in the process;

b5, dripping the mixture of the cells and the matrigel onto a preheated 24-pore plate, and rapidly converting the gel into jelly shape after the gel is heated;

b6, after the 24-pore plate is placed in an incubator for incubation, each pore is added with liver organoid culture medium respectively, and then the mixture is placed in the incubator for culture.

2. The method of claim 1, wherein 350 μl of colorectal cancer organoid medium and 350 μl of liver organoid medium are added per well, respectively.

3. The method of claim 1, wherein the fragments obtained by cutting the colorectal cancer tissue into pieces of 2-4mm 3 Fragments of size.

4. The method of claim 1, wherein the pieces of liver tissue cut into pieces of 0.5-1mm 3 Fragments of size.

5. The method of claim 1, wherein the screen is a 100 μm screen.

6. The construction method according to claim 1, wherein the liquid temperature-sensitive matrigel is added in a proportion of 50 μl per 20000 cells.

7. A colorectal cancer organoid and liver organoid co-culture model obtained by the construction method according to any one of claims 1-6.

CN202110287770.9A 2021-03-17 2021-03-17 Colorectal cancer organoid and liver organoid co-culture model and construction method thereof Active CN113106065B (en)

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Families Citing this family (4)

* Cited by examiner, † Cited by third party
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CN114015643A (en) * 2021-10-28 2022-02-08 南京市第一医院 Liver and intestine cell co-culture method applied to prodrug pharmacokinetics research
CN115537395A (en) * 2021-11-04 2022-12-30 上海万何圆生物科技有限公司 A treatment method and application of co-cultivation of liver cancer organoids and TILs cells
CN114058588A (en) * 2021-11-12 2022-02-18 复旦大学附属中山医院 Preparation method of human-derived intestinal adenoma organoid
CN116539862B (en) * 2022-07-20 2024-03-12 成都诺医德医学检验实验室有限公司 Method for testing tumor metastasis capability based on organoid symbiotic long platform

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108148811A (en) * 2018-01-16 2018-06-12 复旦大学附属中山医院 A kind of method of the xenograft tumor models based on temperature sensitive type biogel dimensional culture Establishing colorectal cancer patients source
CN112143699A (en) * 2020-09-11 2020-12-29 上海市第十人民医院 Method for reconstructing immune microenvironment of colorectal cancer organoid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108148811A (en) * 2018-01-16 2018-06-12 复旦大学附属中山医院 A kind of method of the xenograft tumor models based on temperature sensitive type biogel dimensional culture Establishing colorectal cancer patients source
CN112143699A (en) * 2020-09-11 2020-12-29 上海市第十人民医院 Method for reconstructing immune microenvironment of colorectal cancer organoid

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Matrigel uses in cell biology and for the identification of thymosin b4, a mediator of tissue regeneration;Hynda K. Kleinman等;Appl Biol Chem;第61卷(第6期);703–708 *
体外不同比例再生肝细胞与结肠癌细胞的共培养;徐波等;中华生物医学工程杂志;第14卷(第6期);432-435 *
体外与肝细胞共培养对结肠癌细胞的移行和增殖的影响;吴国洋等;中华普通外科杂志;第22卷(第8期);609-612 *

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