CN111849904B

CN111849904B - Culture medium and culture method for neuroblastoma organs and transplant

Info

Publication number: CN111849904B
Application number: CN202010760982.XA
Authority: CN
Inventors: 李程; 肖金平; 孙志坚; 康平
Original assignee: Zhejiang K2oncology Co ltd
Current assignee: ZHEJIANG K2ONCOLOGY Co.,Ltd.
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2021-05-04
Anticipated expiration: 2040-07-31
Also published as: CN111849904A

Abstract

The present disclosure relates to a culture medium for neuroblastoma organoid culture comprising alpha-aminoisocaproic acid and platelet-derived growth factor. By utilizing the culture medium for culturing neuroblastoma organoids provided by the disclosure, the success rate of culturing neuroblastoma organoids can be effectively improved.

Description

Culture medium and culture method for neuroblastoma organs and transplant

Technical Field

The disclosure relates to the technical field of biomedicine, in particular to a culture medium for culturing neuroblastoma organs, a method for culturing neuroblastoma organs, a transplant for constructing a neuroblastoma xenograft model and application thereof.

Background

Neuroblastoma is a common extracranial tumor, the incidence population of neuroblastoma is mainly infants and children aged 1-5 years, and the incidence rate is about one hundred thousand. In the population with neuroblastoma, infants under 2 years of age account for about 60%, and infants under 10 years of age account for about 96%. At present, the treatment method for neuroblastoma mainly comprises operations, radiotherapy, chemotherapy, comprehensive treatment and the like, can relieve the pain of a patient and slow down the growth of tumor within a period of time, but still leads to the final death of children patients due to the recurrence and metastasis of the tumor.

The pathogenesis of neuroblastoma is complex, and the treatment difficulty is large. Therefore, more realistic in vitro biological models are needed to explore new treatment protocols. In the related art, a neuroblastoma cell line has been used as a biological model for neuroblastoma research. However, the variety of established human neuroblastoma cell lines is limited, and in vitro culture is difficult; xenograft tumor (PDX) mouse models derived from neuroblastoma patients were constructed with a power of only 30%. Therefore, the establishment of a neuroblastoma organoid model closer to the pathological state and genetic molecular information of a human can provide an ideal tool for the mechanism research and drug development of the tumor and the in-vitro drug sensitivity test of a patient.

Disclosure of Invention

The present disclosure provides a culture medium for neuroblastoma organoid culture, and a method for culturing neuroblastoma organoids using the same.

In order to achieve the above objects, in a first aspect, the present disclosure provides a culture medium for neuroblastoma organoid culture, the culture medium comprising α -aminoisocaproic acid and platelet-derived growth factor.

Optionally, the concentration of the alpha-aminoisocaproic acid is 0.5 to 1.5mmol/L and the concentration of the platelet-derived growth factor is 5 to 15ng/mL based on the culture medium.

Optionally, the medium further comprises at least one of DMEM/F12 medium, FBS, EGF, bFGF and B27.

Optionally, based on the culture medium, the FBS content is 5-10 vol%, the EGF concentration is 15-20 μ M, the bFGF concentration is 15-20 μ M, the B27 content is 1-5 vol%, and the balance is the DMEM/F12 culture medium.

In a second aspect, the present disclosure provides a method of culturing a neuroblastoma organoid, the method comprising:

mixing neuroblastoma tissue cells, a culture medium and a temperature-sensitive hydrogel to obtain a to-be-cultured substance, wherein the culture medium is the culture medium of any one of the first aspect;

and culturing and amplifying the to-be-cultured substance to obtain the neuroblastoma organs.

Optionally, the mixing the neuroblastoma tissue cells, the culture medium and the temperature-sensitive hydrogel to obtain a culture to be cultured includes:

mixing the neuroblastoma tissue cells with the culture medium to obtain a premix, wherein the concentration of the neuroblastoma tissue cells in the premix is 1 × 10⁵～5×10⁵Per mL;

and mixing the premix with the temperature-sensitive hydrogel to obtain the culture to be cultured, wherein the usage amount of the temperature-sensitive hydrogel is 1-4 parts by volume relative to 1 part by volume of the premix.

In a third aspect, the present disclosure provides a transplant for constructing a neuroblastoma xenograft model, the transplant comprising a neuroblastoma organoid and a coating layer coated on the surface of the neuroblastoma organoid, wherein the neuroblastoma organoid is prepared by the method of any of the second aspects.

Optionally, the diameter of the neuroblastoma organoid is 1-5 mm, and the thickness of the wrapping layer is 1-4 mm; preferably, the wrapping layer is a temperature-sensitive hydrogel layer.

In a fourth aspect, the present disclosure provides a use of the graft of any one of the third aspects for constructing a neuroblastoma xenograft model.

In a fifth aspect, the present disclosure provides a method of producing a neuroblastoma xenograft mouse model, the method comprising:

implanting the graft of any one of the third aspects into subcutaneous and/or orthotopic tissues of an immunodeficient mouse to obtain the neuroblastoma xenograft mouse model.

By the technical scheme, the culture medium for culturing neuroblastoma organoids provided by the disclosure can effectively improve the success rate of culturing neuroblastoma organoids.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In a first aspect of the present disclosure, there is provided a culture medium for neuroblastoma organoid culture comprising α -aminoisocaproic acid and platelet-derived growth factor.

The inventors of the present disclosure unexpectedly found that the success rate of in vitro culture of neuroblastoma organoids can be effectively increased by using α -aminoisocaproic acid in combination with platelet-derived growth factor.

The culture medium provided by the disclosure contains alpha-aminoisocaproic acid and platelet-derived growth factors, and the culture medium is used for in vitro culture of neuroblastoma organs, so that the culture success rate can be effectively improved.

According to the present disclosure, the concentrations of α -aminoisocaproic acid and platelet-derived growth factor in the above culture medium may vary within a certain range, for example, the concentration of α -aminoisocaproic acid may be 0.5 to 1.5mmol/L and the concentration of platelet-derived growth factor may be 5 to 15ng/mL based on the culture medium.

According to the present disclosure, in order to further increase the success rate of culturing the neuroblastoma organoids, the culture medium may preferably further comprise at least one of DMEM/F12 medium, FBS, EGF, bFGF and B27.

A second aspect of the present disclosure provides a method of culturing a neuroblastoma organoid, the method comprising: mixing neuroblastoma tissue cells, a culture medium and a temperature-sensitive hydrogel to obtain a to-be-cultured substance, wherein the culture medium is the culture medium of any one of the first aspect; and culturing and amplifying the to-be-cultured substance to obtain the neuroblastoma organs.

According to the present disclosure, the neuroblastoma tissue cells are cells obtained by subjecting a neuroblastoma primary tissue to an enzymatic treatment, and include neuroblastoma cells and mesenchymal cells. When the neuroblastoma tissue is subjected to the enzymatic hydrolysis treatment, the neuroblastoma tissue cell can be obtained by subjecting a tissue mass of the neuroblastoma tissue and an enzymatic hydrolysate to a conventional mixed culture, followed by a separation operation such as filtration and centrifugation. Wherein, the enzymolysis liquid that above-mentioned enzymolysis treatment in-process related to can select in the great scope, for example can be 1 ~ 2 mg/mL's I type collagenase solution.

According to the present disclosure, the temperature-sensitive hydrogel may be selected from a wide range, and may be, for example, a 2 × Mebio Gel temperature-sensitive hydrogel.

According to the present disclosure, mixing the neuroblastoma tissue cells, the culture medium and the temperature-sensitive hydrogel to obtain a culture may include: the neuroblastoma tissue cell and the neuroblastoma tissue cellMixing the culture media to obtain a premix, wherein the concentration of the neuroblastoma tissue cells in the premix is 1 × 10⁵～5×10⁵Per mL; and mixing the premix with the temperature-sensitive hydrogel to obtain the culture to be cultured, wherein the usage amount of the temperature-sensitive hydrogel is 1-4 parts by volume relative to 1 part by volume of the premix.

When the neuroblastoma tissue cells are mixed with the culture medium, the neuroblastoma tissue cells may be added to the culture medium, or the culture medium may be added to a container loaded with the neuroblastoma tissue cells. The cell concentration in the premix can be measured by known techniques and will not be described further herein.

Similarly, when the premix is mixed with the temperature-sensitive hydrogel, the premix may be added to the temperature-sensitive hydrogel, or the temperature-sensitive hydrogel may be added to the premix.

According to the present disclosure, the expanding the culture to obtain the neuroblastoma organoid may include: adding the culture to be cultured into culture holes of a culture plate so that each culture hole contains 100-300 mu L of the culture to be cultured, and then carrying out liquid changing subculture on the culture to be cultured in each culture hole until the diameter of the culture in the culture hole is not less than 0.5mm to obtain the neuroblastoma organoid.

According to the present disclosure, after successful neuroblastoma culture, conventional freezing treatment may be performed.

In the above technical solution, since the adopted culture medium is the culture medium provided in the first aspect of the present disclosure, the success rate of culturing neuroblastoma by using the above method is high; in addition, in the method, neuroblastoma tissue cells are used as primary cells for culture, and the neuroblastoma tissue cells contain interstitial cells in addition to neuroblastoma cells, so that neuroblastoma organs cultured by the method better retain heterogeneity of neuroblastoma, and have biological characteristics similar to those of neuroblastoma primary tissues, which are beneficial to stable passage of neuroblastoma organs in vitro and stable expression of mutant genes of neuroblastoma in neuroblastoma organs.

A third aspect of the present disclosure provides a transplant for constructing a neuroblastoma xenograft model, the transplant comprising a neuroblastoma organoid and a coating layer coated on the surface of the neuroblastoma organoid, wherein the neuroblastoma organoid is prepared by the method of any of the second aspects.

In the technical scheme, the transplant provided by the disclosure contains neuroblastoma organs, the neuroblastoma organs can be obtained by culturing only a small amount of primary tissues, and the culture process is simple, rapid and high in success rate, so that when the transplant is used for constructing a xenograft model, the number of the finally constructed xenograft model can be effectively increased, and more resources can be provided for clinical treatment or scientific research. Meanwhile, the transplant body also comprises a wrapping layer, and the wrapping layer wraps the surface of the organoid, so that the activity of the organoid in the transplant body can be guaranteed, and the success rate of constructing a xenograft model is effectively improved.

A fourth aspect of the present disclosure provides a use of the graft of any one of the third aspects for constructing a neuroblastoma xenograft model.

A fifth aspect of the present disclosure provides a method of producing a neuroblastoma xenograft mouse model, the method comprising: implanting the graft of any one of the third aspects into subcutaneous and/or orthotopic tissues of an immunodeficient mouse to obtain the neuroblastoma xenograft mouse model.

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

The starting materials, reagents, instruments and equipment referred to in the examples of the present disclosure may be obtained by purchase, unless otherwise specified.

Where specific experimental temperatures are not noted in the examples of the present disclosure, the experimental temperatures are all room temperature (20-25 ℃). The sources of reagents used in the examples of the disclosure are as follows:

RS-I Medium is available from AQIX Liquid; DMEM-F12, EGF, BioTrypsin-EDTA (0.25%) from Gibco; bFGF and B27 were purchased from invitrogen; type i collagenase was purchased from sigma invitrogee; alpha-aminoisocaproic acid from hengdail ao; PDGF was purchased from Beijing Zhongke science and technology, Inc.; mebio Gel temperature sensitive hydrogels were purchased from Cosmo.

The experimental animals and cell sources used in the examples of the disclosure are as follows:

adult nude mice of cleaning grade nu/nu were purchased from Nanjing Junceae biotechnology; SH-SY5Y cell line was purchased from Shanghai Tong mu Wei Biotech.

The culture medium used in the embodiments of the present disclosure is a dedicated culture medium for neuroblastoma organoid culture (hereinafter collectively referred to as "dedicated culture medium") that is configured autonomously, and includes: DMEM/F12 medium, 10 vol.% FBS (fetal bovine serum), EGF (epidermal growth factor) at a concentration of 20. mu.M, bFGF (basic fibroblast growth factor) at a concentration of 20. mu.M, 2 vol.% B27, alpha-aminoisocaproic acid at a concentration of 0.8mmol/L and PDGF (platelet-derived growth factor) at a concentration of 10 ng/mL.

Example 1

This example illustrates the preparation of neuroblastoma organoids.

(1) Preservation and transport of neuroblastoma tissue samples

Obtaining a clinically confirmed surgical tissue from a patient with neuroblastoma, collecting the tissue as a neuroblastoma tissue sample, and storing the tissue sample in a container containing the neuroblastoma tissue sample

In the preservation solution of RS-I Medium for 48 hoursThe inner 4 degree cold chain is transported to the operation room.

(2) Neuroblastoma tissue cell harvesting

The neuroblastoma tissue samples obtained above were placed in sterile petri dishes, pretreated to remove blood clots, capsules and necrotic tissue, and then rinsed 3 times with sterile PBS, after which the PBS was aspirated. Then, 1.5mg/mL collagenase type I solution was added to the petri dish, wherein 10mL collagenase type I solution was added for each 1mg neuroblastoma tissue sample. Cutting a neuroblastoma tissue sample to 1mm³The plates were then placed on a shaker at 30rpm and subjected to enzymatic hydrolysis for 1h in a 37 ℃ incubator.

And after the enzymolysis is finished, filtering the collected enzymolysis liquid by using a 100-micron cell sieve, and collecting filtrate to obtain cell suspension. The collected cell suspension was centrifuged at 1500rpm for 5min and then rinsed 3 times with PBS. After the completion of rinsing, the cells were centrifuged at 300g for 3min, and the supernatant was discarded to collect cell pellets, thereby obtaining neuroblastoma tissue cells. If necessary, the neuroblastoma tissue cells obtained as described above may be stored in a DMEM/F12 medium.

(3) Culture of neuroblastoma-like organs

Mixing the neuroblastoma tissue cells obtained in step (2) with a special culture medium so that the concentration of the neuroblastoma tissue cells is 1 × 10⁵one/mL to give a premix. And (3) mixing the premix with 2 multiplied Mebio Gel temperature-sensitive hydrogel in the same volume to obtain the to-be-cultured substance. Adding the culture to be cultured into culture wells of a cell culture plate, wherein the addition amount of each well is 100 mu L, then placing the culture plate in an incubator at 37 ℃ and 5% carbon dioxide for culturing for 1h, then adding 150 mu L of special culture medium into each well, and continuing culturing. During the culture process, the special culture medium is replaced every 3 days, and the subculture is carried out every 7 days. And (4) carrying out microscopic detection in the culture process, and when the diameter of the culture in a single culture hole is larger than 0.5mm, successfully culturing to obtain the neuroblastoma organoid.

Example 2

This example illustrates the preparation of the graft of the present disclosure.

The neuroblastoma organoids cultured in example 1 were cultured until the diameter of the individual neuroblastoma organoids was not less than 1.0mm, and then the organoids in each culture well were removed together with the coating layer having a thickness of not less than 1.0mm around the organoids, to obtain graft a of this example.

Example 3

Neuroblastoma organoids were cultured using the method of example 1, except that: the concentration of alpha-aminoisocaproic acid in the special culture medium used was 0.5mmol/L and the concentration of PDGF was 15 ng/mL.

Example 4

Neuroblastoma organoids were cultured using the method of example 1, except that: the concentration of alpha-aminoisocaproic acid in the special culture medium used was 1.5mmol/L and the concentration of PDGF was 5 ng/mL.

Example 5

Neuroblastoma organoids were cultured according to the method of example 1, except that: the concentration of alpha-aminoisocaproic acid in the special culture medium used was 0.3mmol/L and the concentration of PDGF was 18 ng/mL.

Example 6

Neuroblastoma organoids were cultured according to the method of example 1, except that: the concentration of alpha-aminoisocaproic acid in the special culture medium used was 1.8mmol/L and the concentration of PDGF was 3 ng/mL.

Comparative example 1

Neuroblastoma organoids were cultured according to the method of example 1, except that: the culture medium used in step (3) is a first culture medium which does not contain alpha-aminoisocaproic acid as compared with the special culture medium.

Comparative example 2

Neuroblastoma organoids were cultured according to the method of example 1, except that: the medium used in step (3) is a second medium which does not contain PDGF compared to the special medium described above.

Test example 1

1. Neuroblastoma organoid number detection

On the 14 th day after the start of the culture, the numbers of neuroblastoma organoids cultured in examples 1, 3 to 6 and comparative examples 1 to 2 were counted by using an inverted microscope, and the numbers thereof are expressed as "M. + -. SD", and the results are shown in Table 1.

TABLE 1

Culture method	Neuroblastoma organoid number (× 10)³one/mL)
		Example 1	9.96±1.14
Example 3	8.73±1.03
		Example 4	8.12±1.22
Example 5	6.11±0.94
		Example 6	5.94±1.16
Comparative example 1	0.91±2.32
		Comparative example 2	0.16±1.67

As can be seen from examples 1, 3-6, comparative examples 1-2 and Table 1, under the condition that different culture media are used, the number of successfully cultured neuroblastoma organoids in examples 1 and 3-6 is significantly higher than that in comparative examples 1-2 after 14 days of culture, which shows that the culture medium provided by the present disclosure can effectively improve the success rate of neuroblastoma organoid culture.

2. Neuroblastoma organoid cell viability assay

The neuroblastoma organs cultured in examples 1, 3 to 6 and 1 to 2 were collected and prepared to have a cell concentration of 1X 10⁴Single cell suspension per mL. Then, each cell suspension was inoculated into a 96-well culture plate for culture in an amount of 180. mu.L per well, and 5 wells were repeatedly inoculated per each group of cell suspension. After completion of inoculation, the 96-well plates inoculated with each cell suspension were placed at 37 ℃ in 5% CO₂Then 10. mu.L of MTT solution at a concentration of 5mg/mL was added to each well, and the incubation was continued for 4 hours. After the culture is finished, centrifuging for 5min at 3000rpm, removing supernatant, adding 150 mu L DMSO to dissolve crystals, after complete dissolution, placing the dissolved product under an enzyme-linked immunosorbent assay (ELISA) instrument to detect the absorbance value at 595nm, calculating and counting the cell activity of each group, and the statistical result is shown in table 2.

TABLE 2

Culture method	Cell viability (%)
		Example 1	93.14±1.21
Example 3	90.59±0.86
		Practice ofExample 4	92.21±0.96
Example 5	79.54±1.04
		Example 6	66.38±1.11
Comparative example 1	36.32±2.14
		Comparative example 2	38.21±1.12

As can be seen from table 2, under the condition that different culture media are used, the cell viability of the successfully cultured neuroblastoma organoids in examples 1 and 3 to 6 is significantly higher than that in comparative examples 1 to 2, which indicates that the culture medium provided by the present disclosure can effectively improve the cell viability of the cultured neuroblastoma organoids.

Test example 2

Human neuroblastoma cell line SH-SY5Y was used as transplant B. Then, using the graft a prepared in example 2 and the graft B, mouse models a and B for neuroblastoma xenografting were constructed.

The construction method of the neuroblastoma xenograft mouse model comprises the following steps: a clean nu/nu adult nude mouse is anesthetized by a 10mg/mL anesthetic (containing 2g/L xylazine and 5g/L ketamine), fixed on a sterile operating table, wiped with 75% alcohol, and then incised with a sterile scalpel on the renal skin on the right side of the back to make an incision of 1cm and expose the right kidney of the mouse. Then, the prepared transplant is quickly implanted into the kidney by using a trocar, a window bleeding part is treated by using sterile gauze, and then the right kidney implanted with the transplant is reset and sutured. And observing the mouse every day, and obtaining a bile duct cancer xenograft model after the transplant forms tumor.

After modeling is finished, the intrarenal tumorigenesis condition of the mice after operation is observed by using an IVIS Spectrum CT system, the success rate is calculated, and the result is shown in Table 3.

TABLE 3

As can be seen from Table 3, the success rate of using the transplant prepared in example 2 to construct a mouse model of neuroblastoma xenograft was significantly higher than that of using the human neuroblastoma cell line SH-SY5Y, thus indicating that the transplant provided by the present disclosure is helpful for increasing the success rate of constructing a xenograft model.

The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. A culture medium for neuroblastoma organoid culture, comprising alpha-aminoisocaproic acid and platelet-derived growth factor, wherein the concentration of the alpha-aminoisocaproic acid is 0.5 to 1.5mmol/L and the concentration of the platelet-derived growth factor is 5 to 15ng/mL, based on the culture medium;

the culture medium further comprises a DMEM/F12 culture medium, FBS, EGF, bFGF and B27, wherein the culture medium is taken as a reference, the FBS content is 5-10 vol%, the EGF concentration is 15-20 mu M, the bFGF concentration is 15-20 mu M, the B27 content is 1-5 vol%, and the balance is the DMEM/F12 culture medium.

2. A method of culturing a neuroblastoma organoid, said method comprising:

mixing neuroblastoma tissue cells, a culture medium and a temperature-sensitive hydrogel to obtain a culture to be cultured, wherein the culture medium is the culture medium according to claim 1;

3. The method according to claim 2, wherein the step of mixing the neuroblastoma tissue cells, the culture medium and the temperature-sensitive hydrogel to obtain a culture comprises: