CN110607279A

CN110607279A - 3D culture system of primary tumor cells and culture method and application thereof

Info

Publication number: CN110607279A
Application number: CN201910994591.1A
Authority: CN
Inventors: 喻德华; 肖艳归
Original assignee: Shenzhen Excellent St Kang Medical Laboratory Laboratory
Current assignee: Shenzhen Excellent St Kang Medical Laboratory Laboratory
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2019-12-24
Anticipated expiration: 2039-10-18
Also published as: CN110607279B

Abstract

The invention provides a 3D culture system of primary tumor cells and application thereof, wherein the 3D culture system comprises a lower layer gel, a middle layer cell matrix and an upper layer culture medium; the middle layer cell matrix comprises a modified agarose gel, a conditioned medium containing added factors and tumor cells, wherein the modified agarose gel comprises low-melting-point agarose, polylysine and extracellular matrix; the lower gel comprises agarose gel and conditioned medium; the upper layer culture medium is a conditioned medium containing an additive factor. The 3D culture system provided by the invention can screen tumor cells and normal epithelial cells, realizes the dominant growth of the tumor cells, has high culture success rate, can reflect the characteristics of the whole tumor cell population more truly, accurately represents the growth condition of tumors in vivo, has high reliability of the obtained result when being applied to drug sensitivity detection, and is beneficial to clinical popularization.

Description

3D culture system of primary tumor cells and culture method and application thereof

Technical Field

The invention relates to the field of tumor personalized medicine functional drug sensitivity detection, in particular to a 3D culture method of primary tumor cells, and particularly relates to a 3D culture system of the primary tumor cells, a culture method and application thereof.

Background

Malignant tumor is a disease with very high morbidity and mortality, seriously harms the life and health of human beings, is one of the main diseases causing disability and early death, and is the first cause of death in the age group of 35-59 years. Current treatments for malignant tumors include surgery, radiation therapy, chemotherapy, endocrine therapy and immunotherapy.

Chemotherapy is a systemic treatment method, and plays a very important role in treatment because it can widely kill tumor cells in patients and improve the survival rate and life cycle of patients. Although chemotherapy plays a very important role in tumors, chemotherapy is often accompanied by significant toxic and side effects, and the efficacy of chemotherapy on most tumors, especially solid tumors, is still not ideal. Among them, the development of drug resistance of tumor cells to chemotherapeutic drugs is a common factor causing failure of tumor chemotherapy, and is also a key problem which troubles tumor treatment.

A tumor is a heterogeneous, polymorphic, and differentiated population of cells. The tumor has obvious individual difference to various chemotherapy drugs. That is, different tumor types or patients of the same type, even the same patient in different disease stages, are not completely sensitive to chemotherapy, and the treatment effect is very different. So far, one chemotherapeutic medicament or the combined application of several chemotherapeutic medicaments is not available, and the chemotherapeutic medicament can be 100 percent effective on a certain tumor. The effectiveness of the currently used anti-tumor chemotherapeutic drugs for patient treatment is less than 50%, and about 20% to 35% of patients receive inappropriate drug therapy.

Therefore, a relatively reliable sensitivity test method like a bacterial sensitivity test is established, sensitive chemotherapeutic drugs are accurately screened for different patients, the dosage of the drugs is determined, clinical individualized medication is really realized, the curative effect is greatly improved, over-treatment is avoided, the economic burden of the patients is reduced, and the waste of medical resources is reduced.

Tumor drug sensitivity detection belongs to the precise diagnosis category, and the American society for oncology performs comparative analysis on papers on tumor drug sensitivity tests and drug resistance tests published before 2004, and considers that the application of the drug sensitivity tests has a positive effect on realizing individuation of chemotherapeutic drugs/schemes. The tumor drug sensitivity test is combined with the molecular detection of the genetic background of a patient and applied to clinical tumor treatment, and has important significance for improving the curative effect of a targeted drug and reducing the expense of expensive targeted drugs, but the existing tumor drug sensitivity detection technology is difficult to realize wide clinical application and popularization.

At present, the tumor drug sensitivity test mainly comprises the following three types: in vivo drug sensitivity detection technology, 2D in vitro drug sensitivity detection based on CRC conditional reprogramming culture and conventional 3D drug sensitivity detection technology. The in vivo drug sensitivity detection technology is carried out by animal experiments, and has the advantages that the original tissue morphology and biological characteristics of human tumors can be kept, and the drug sensitivity detection accuracy reaches 90 percent; however, animal experiments are limited by various uncontrollable factors in vivo, so that the inoculation success rate is low, the period is as long as 3-4 months, and the in vivo and in vitro environments influence each other, so that the research on a specific mechanism is not facilitated, and the animal experiments also have the defects of high requirements, high cost, complex operation and the like. Although the 2D in vitro drug sensitivity detection technology based on CRC conditional reprogramming culture has high culture success rate, low cost, simple operation and short period, the 2D in vitro culture has unstable 2D drug sensitivity result and low clinical compliance rate due to mixed amplification of tumor epithelium and normal epithelial cells. The conventional 3D drug sensitive detection technology has low cost and can simulate the environment of solid tumors in vivo, but the obtained primary tumor cells are few, the success rate of tumor microsphere culture is low, the tumor cells and normal cells cannot be distinguished, the period is long, and the large-scale popularization is difficult.

At present, the 3D drug sensitive detection technology is based on a 3D cell culture (Three dimensional cell culture) technology, and the 3D cell culture mainly includes hanging drop culture, hydrogel culture, Matrigel culture, and conventional low melting point agar culture. The 3D cell culture can not only keep the material structure basis of the natural cell microenvironment, but also better simulate the microenvironment for the in vivo cell growth, thus providing a simpler, safer and more reliable method for the research of the cell level. However, none of the hanging drop culture, hydrogel culture and Matrigel culture can achieve the function of screening tumor cells and normal epithelial cells.

The cultured cells within the first to tenth generations are generally collectively referred to as primary cell culture. The low melting point agar culture is a good anchoring independent growth system for screening tumor cells, is a sign of cell malignant transformation, and normal epithelial cells do not proliferate in the agar culture, but researches show that most primary tumor cells can not proliferate in the conventional agar culture system well except that a small part of tumor cell lines can proliferate in the agar well due to the harsh culture conditions of the conventional low melting point agar.

Therefore, there is an urgent need for a 3D culture system and method that can successfully proliferate primary tumor cells while inhibiting the growth of normal epithelial cells, thereby rapidly culturing tumor cells, accurately reflecting the characteristics of tumor cell populations and the conditions of tumors in vivo, and helping to select appropriate therapeutic drugs in clinical treatment.

Disclosure of Invention

In order to solve the technical problems, the invention provides a primary tumor cell 3D culture system and a culture method thereof, which optimize an agar culture system in the prior art, in the 3D optimized culture system, the primary tumor cells can grow dominantly, and simultaneously inhibit the proliferation of normal epithelial cells, improve the defect that the CRC culture amplification cannot distinguish the tumor cells from the normal epithelial cells, screen out the tumor epithelial cells, accurately reflect the condition of tumors in vivo, ensure that the subsequent drug sensitivity detection result is more accurate, and ensure that tumor patients are treated by more proper drugs.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, a 3D culture system for primary tumor cells comprises a lower layer of gel, a middle layer of cell matrix, and an upper layer of culture medium; the middle layer cell matrix comprises a modified agarose gel, a conditioned medium containing an added factor and a tumor cell suspension, wherein the modified agarose gel comprises low-melting-point agarose, polylysine and extracellular matrix; the lower gel comprises agarose gel and conditioned medium; the upper layer culture medium is a conditioned medium containing an additive factor.

The lower layer gel of the 3D culture system can prevent tumor cells from contacting the bottom and the side wall of a culture dish or a 96-well plate to cause adherent growth; the middle layer cell matrix can enable primary tumor cells to grow dominantly in the 3D culture system; the upper layer culture medium is arranged above the 3D culture system, so that the wettability of the middle layer cell matrix can be maintained, the tumor cells are prevented from being dehydrated and dead, and meanwhile, the regular replacement can also provide nutrition for the tumor cells and promote the rapid proliferation of the 3D microspheres. The normal epithelial cells in the 3D culture system provided by the invention cannot be proliferated in the system, the defect that the tumor cells and the normal epithelial cells cannot be distinguished by CRC (cyclic redundancy check) culture amplification is overcome, the tumor epithelial cells are screened with advantages, and the growth condition of the tumor cells in vivo can be better reflected.

In a preferred embodiment of the present invention, the extracellular matrix comprises a combination of any three or more of mucopolysaccharide, proteoglycan, laminin, fibrin, collagen, and hyaluronic acid, preferably a combination of collagen, laminin, and hyaluronic acid.

Preferably, the collagen is type I collagen.

Preferably, the concentration of the collagen in the modified agarose gel is 10-100U/mL, for example, 10U/mL, 20U/mL, 30U/mL, 35U/mL, 40U/mL, 50U/mL, 60U/mL, 70U/mL, 80U/mL, 90U/mL, or 100U/mL.

Preferably, the concentration of the laminin in the modified agarose gel is 10-20 μ g/mL, and may be, for example, 10 μ g/mL, 12 μ g/mL, 14 μ g/mL, 15 μ g/mL, 18 μ g/mL, or 20 μ g/mL.

Preferably, the concentration of the hyaluronic acid in the modified agarose gel is 10-100U/mL, and may be, for example, 10U/mL, 20U/mL, 30U/mL, 35U/mL, 40U/mL, 50U/mL, 55U/mL, 60U/mL, 70U/mL, 80U/mL, 90U/mL, or 100U/mL.

Preferably, the concentration of polylysine in the modified agarose gel is 0.1 to 1mg/L, and may be, for example, 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.8mg/L, 0.9mg/L, or 1 mg/L.

In a preferred embodiment of the present invention, the mass concentration of the low-melting agarose in the modified agarose gel is 0.1 to 0.4%, and may be, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, or 0.4%.

Preferably, the seeding density of the tumor cells in the middle layer cell culture substrate is (1-2) multiplied by 10⁵/mL, for example, may be 1X 10⁵/mL、1.2×10⁵/mL、1.3×10⁵/mL、1.5×10⁵/mL、1.7×10⁵/mL、1.8×10⁵Per mL or 2X 10⁵/mL。

In a preferred embodiment of the present invention, the agarose mass concentration in the agarose gel of the lower layer medium is 0.4 to 0.8%, and may be, for example, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, or 0.8%.

Preferably, the additional factor comprises any one or a combination of two or more of amino acids, growth factors, reducing agents or transferrin.

Preferably, the amino acid is L-glutamine.

Preferably, the growth factor is any one or a combination of two or more of EGF, FGF and bFGF, preferably bFGF.

Preferably, the reducing agent is beta-mercaptoethanol and/or ascorbic acid.

In a preferred embodiment of the present invention, the molar concentration of L-glutamine in the conditioned medium is 20 to 100. mu.M, and may be, for example, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 60. mu.M, 70. mu.M, 80. mu.M, 90. mu.M or 100. mu.M.

Preferably, the concentration of the growth factor in the conditioned medium is 10-100 ng/mL, for example, 10ng/mL, 15ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.

Preferably, the concentration of the beta-mercaptoethanol in the conditioned medium is 0.05 to 0.15mM, and may be, for example, 0.05mM, 0.06mM, 0.08mM, 0.1mM, 0.12mM, or 0.15 mM.

Preferably, the concentration of the ascorbic acid in the conditioned medium is 40-80. mu.g/mL, for example, 40. mu.g/mL, 45. mu.g/mL, 50. mu.g/mL, 55. mu.g/mL, 60. mu.g/mL, 65. mu.g/mL, 70. mu.g/mL, or 80. mu.g/mL.

Preferably, the concentration of the transferrin in the conditioned medium is 5-20. mu.g/mL, such as 5. mu.g/mL, 8. mu.g/mL, 10. mu.g/mL, 13. mu.g/mL, 15. mu.g/mL, 18. mu.g/mL or 20. mu.g/mL.

In a preferred embodiment of the present invention, the method for isolating and culturing tumor cells comprises: collecting primary tumor single cells or cell microspheres from tumor tissues or pleural effusion of tumor patients, and performing amplification culture by using a Conditioned reprogramming cell culture (CRC) method to obtain the tumor cells.

Preferably, the step of collecting comprises digestion and separation.

Preferably, the digestive juice used for digestion comprises any one or a combination of two or more of collagenase, neutral dispase, dnase or hyaluronidase.

Preferably, the CRC method is to resuspend the primary tumor cell pellet obtained from collection with F-medium, aspirate the tumor cell suspension, and count the tumor cells using trypan blue staining after appropriate dilution; inoculating the tumor cells in feeder cells which are inoculated with irradiation in advance for co-culture, and performing rapid amplification within one week for subculturing primary tumor cells of P0 generation.

Preferably, the concentration of the collagenase in the digestive juice is 100-200U/mL, for example, 100U/mL, 110U/mL, 130U/mL, 140U/mL, 150U/mL, 160U/mL, 170U/mL, 180U/mL, 190U/mL or 200U/mL.

Preferably, the neutral dispase is 0.6-2.4U/mL in the digestive juice, for example, 0.6U/mL, 0.8U/mL, 1U/mL, 1.2U/mL, 1.5U/mL, 2U/mL, 2.2U/mL or 2.4U/mL.

Preferably, the concentration of the DNase in the digestive juice is 1-10U/mL, for example, 1U/mL, 2U/mL, 3U/mL, 4U/mL, 5U/mL, 6U/mL, 7U/mL, 8U/mL, 9U/mL or 10U/mL.

Preferably, the concentration of the hyaluronidase in the digestive juice is 100-200U/mL, for example, 100U/mL, 110U/mL, 130U/mL, 140U/mL, 150U/mL, 160U/mL, 170U/mL, 180U/mL, 190U/mL or 200U/mL.

AsAccording to the preferable technical scheme, the 3D culture system comprises a lower layer gel, a middle layer cell matrix and an upper layer culture medium; the lower layer gel comprises agarose gel and a conditioned medium, and the mass concentration of the low-melting-point agarose in the agarose gel is 0.4-0.8%; the middle-layer culture medium comprises an improved agarose gel, a condition culture medium containing an additive factor and a tumor cell suspension, the mass concentration of low-melting-point agarose in the improved agarose gel is 0.1-0.4%, the improved agarose gel also comprises hyaluronic acid with the concentration of 10-100U/mL, polylysine with the concentration of 0.1-1mg/mL, collagen of 10-100U/mLI and laminin with the concentration of 10-20 mu g/mL, and the inoculation density of the tumor cell suspension is (1-2) multiplied by 10⁵Volume of media cell matrix/mL; the upper layer culture medium is a condition culture medium containing an additive factor, and the condition culture medium containing the additive factor comprises 20-100 mu M L-glutamine, 10-100 ng/mL bFGF, 0.05-0.15 mM beta-mercaptoethanol, 40-80 mu g/mL ascorbic acid and 5-20 mu g/mL transferrin.

In a second aspect, the present invention also provides the use of a 3D culture system according to the first aspect for the preparation of a drug sensitive detection reagent or a drug sensitive detection kit.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) the primary tumor cell 3D culture system provided by the invention can screen tumor cells and normal epithelial cells, and meanwhile realize the advantageous growth of the tumor cells, so that the characteristics of the whole tumor cell population are reflected more truly, and the in-vivo condition is represented accurately;

(2) the primary tumor cell culture method provided by the invention has the advantages that the sample material is wide, only a small amount of cells are needed, the rapid amplification of the primary tumor cells in vitro within one week can be realized by using the CRC culture technology and the 3D culture system, the culture period is short, the culture success rate is up to more than 90%, the biological characters are not changed greatly because the tumor cells are just separated from the body, the reliability of the drug sensitive result can be reflected truly, and the clinical popularization is facilitated.

Drawings

FIG. 1 is a graph showing the results of culturing non-small cell lung cancer primary tumor cells on day 8 in a 3D culture system (scale: 50 μm).

FIG. 2 is a bar graph of cell expansion of non-small cell lung cancer primary tumor cells cultured on days 1, 4, and 8 in a 3D culture system.

FIG. 3 is a graph showing the results of culturing non-small cell lung cancer paranormal epithelial cells on day 8 in a 3D culture system (scale: 50 μm).

FIG. 4 is a bar graph of the cell expansion fold of non-small cell lung cancer paranormal epithelial cells cultured on days 1, 4, and 8 in a 3D culture system.

Figure 5 is a graph of the sensitivity of different non-small cell lung cancer samples to the same chemotherapeutic drug docetaxel.

FIG. 6(a) is a graph of the sensitivity of NSCLC PCB66 to carboplatin; FIG. 6(b) is a graph of the sensitivity of NSCLC PCB66 to pemetrexed; FIG. 6(c) is a graph of the sensitivity of NSCLC PCB66 to vinorelbine; FIG. 6(d) is a graph of the sensitivity of NSCLCPCB66 to paclitaxel; FIG. 6(e) is a graph of the sensitivity of NSCLC PCB66 to gemcitabine; figure 6(f) is a graph of the sensitivity of NSCLC PCB66 to docetaxel.

FIG. 7(a) is a graph showing the results of a combination drug sensitivity test of NSCLC sample 1; FIG. 7(b) is a graph showing the results of the combination susceptibility testing of NSCLC sample 2; FIG. 7(c) is a graph showing the results of the combination drug sensitivity test of NSCLC sample 3.

FIG. 8 is a graph showing the result of culturing non-small cell lung cancer primary tumor cells on day 1 in a common agar culture system (scale 20 μm).

FIG. 9 is a graph showing the culture results of non-small cell lung cancer primary tumor cells cultured on the 8 th day in a common agar culture system (scale 20 μm).

Detailed Description

For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The formulation of the F-medium in the following examples is as follows:

ham's F-12 medium and complete DMEM medium were mixed at a volume ratio of 3:1 and supplemented as described in Table 1.

TABLE 1 additives and final concentrations in F-medium

The conditioned media were prepared as follows in the following examples:

the cultured J2 cells are firstly passaged for 3-5 generations, then gamma-ray irradiation treatment or mitomycin C treatment is carried out according to the method, and (1.0-1.5) multiplied by 10 is collected⁷The J2 cells were resuspended in 30mL of F-medium and plated in a T175 flask, and the culture supernatant suspension was collected after 3 days of culture;

centrifuging at 1000g/min for 5min, collecting supernatant, filtering with 0.22 μm filter membrane for sterilization, and transferring into new 50mL centrifuge tube;

the supernatant was mixed with fresh F-medium at a volume ratio of 3:1, and added with ROCK1 inhibitor Y-27632 at a final concentration of 5. mu.M to obtain the final conditioned medium.

The prepared conditioned medium can be directly used, or can be frozen and stored in an ultra-low temperature refrigerator at-80 ℃, when in use, the conditioned medium is unfrozen and melted at 4 ℃ from-80 ℃, and then is placed in a water bath kettle at 37 ℃ for preheating for 30min, and the culture medium preheated in the water bath kettle at 37 ℃ is worth noting that repeated freezing and thawing can be avoided.

Example 1

This example provides a 3D culture system and a method for preparing the 3D culture system, and uses the 3D culture system to culture primary tumor cells.

(1) Isolated culture of tumor cells

First, primary tumor single cells were obtained from tumor tissue samples isolated post-operatively from patients:

1. tumor tissues isolated after a Non-small cell lung cancer (NSCLC) patient operation are subjected to single cell isolation treatment in a sterile environment. In order to obtain enough number of living tumor cells, a tissue block with more tumor tissues at the central part is taken, and the size of a surgical sample is collected to be 5mm multiplied by 5 mm; and immediately immersing the fresh tissue specimen in an aseptic centrifuge tube filled with a specimen preservation solution after the fresh tissue specimen is separated from the specimen, storing at 4 ℃, and refrigerating and transporting. The formula of the sample preservation solution is as follows: basal medium DMEM without serum addition, 10U/mL penicillin and 100. mu.g/mL mitomycin.

2. Sample pretreatment: the samples were first placed in Hank's balanced salt solution (calcium magnesium containing) containing 500U/mL penicillin, 500. mu.g/mL streptomycin (sterile HBSS (Ca)²⁺&Mg²⁺),Hank's Balanced Salt Solution with Ca²⁺and Mg²⁺) Standing for 30min, and cutting into 1mm with sharp sterile scalpel³Small pieces of size, then sterile HBSS (Ca) containing 100U/mL penicillin and 100. mu.g/mL streptomycin²⁺&Mg²⁺) Washing for 4 times to remove blood clots in the tissue sample;

enzyme digestion: transferring the minced tumor tissue sample small block into a new 50mL centrifuge tube, adding 5mL of digestive juice, and placing the mixture into a 37 ℃ oscillation water bath kettle for digestion for 2 h; the formula of the digestive juice is shown in table 2.

TABLE 2 digestive juice formula

Reagent	Final concentration
		Collagenase I (collagen Type I)	100U/mL
Neutral dispase II (DispaseII)	1.2U/mL
		Deoxyribonuclease I (DNase I)	2U/mL
Hyaluronidase (Hyaluronidase)	200U/mL
		Sterile HBSS (Ca)²⁺&Mg²⁺)	Complement

Separation: digesting the tissue mass to remove dispersed cells, filtering the cell slurry through a 100 μm cell filter to remove tissue matrix and other tissue components, filtering the filtrate through a 40 μm cell filter to collect the microspheroidal cells on the 40 μm filter, and precipitating the collected microspheroidal cells with sterile HBSS (Ca)²⁺&Mg²⁺) After 3 washes, resuspension was performed with F-medium and counted.

3. Performing primary tumor cell amplification culture according to a condition reprogramming cell culture method: after the obtained pellet of the microballon cells is resuspended in an F-culture medium, a certain amount of cell suspension is sucked, and after proper dilution, counting is carried out by trypan blue staining.

According to 2X 10⁵Concentration inoculation per well 5X 10 at 2h in advance⁶Co-culture was performed in 96-well plates with 32Gy irradiated feeder cells, Swiss 3T 3J 2 mouse fibroblasts, and P0 primary tumor cells were collected by rapid expansion over a one week period.

(2) Preparation of the lower layer Medium

Preparing 3% of low-melting-point agarose gel, weighing 3g of low-melting-point agarose, putting the low-melting-point agarose gel into a 250mL sterile blue-covered bottle, adding 100mL of sterile ultrapure water, sterilizing at high pressure, and putting the sterilized low-melting-point agarose gel into a 45 ℃ water bath kettle to keep the low-melting-point agarose gel in a molten state;

mixing and diluting 3% agarose gel preheated in 45 deg.C water bath with conditioned medium preheated at 37 deg.C in plating tank to dilute the agarose gel to 0.6%, adding into 96-well plate with 0.05mL agarose gel per well, standing at 37 deg.C and 5% CO₂The incubator (2) is left to solidify for 15 min.

(3) Preparation of the Medium layer cell matrix

Preparation of the improved agarose gel: preheating 3% agarose gel in a water bath kettle at 45 ℃, mixing 3% agarose gel with hyaluronic acid, type I collagen and laminin, and mixing to obtain the components with the concentrations shown in Table 3:

TABLE 3 ingredients of modified agarose gels

Composition (I)	Final concentration
		Low melting point agarose	0.4％(w/v)
Polylysines	0.5mg/L
		Hyaluronic acid	100U/mL
Collagen concentration	50U/mL
		Laminin	10μg/mL

Tumor cell suspension and conditioned medium containing added factors were prepared:

adding an additive factor into the conditioned medium, wherein the type and concentration of the additive factor are shown in Table 4, and preparing the cell sediment with the concentration of 1 × 10 by using the conditioned medium containing the additive factor to resuspend and centrifugally collect⁶Tumor cell suspension/mL, at a final primary tumor cell suspension concentration of 1X 10⁵Inoculation Density 1X 10/mL⁶mixing/mL primary tumor cell suspension with a conditioned medium containing an additive factor, placing the mixture in a water bath kettle at 37 ℃, and preheating for later use, wherein each well of a 96-well plate contains the conditioned medium containing the additive factor and 1 × 10⁶The total volume of the/mL primary tumor cell suspension and the modified agarose gel was 50. mu.L.

TABLE 4 additional factors and final concentrations contained in conditioned Medium

In order to prevent the modified agarose gel from solidifying, after the modified agarose gel is prepared, the modified agarose gel is quickly mixed with a tumor cell suspension which is preheated in a water bath kettle at 37 ℃ in advance and a conditioned medium containing an additive factor; adding into a 96-well plate with 50 μ L of gel at 37 deg.C in the lower layer, and standing with 5% CO₂The incubator is left for 15min until it solidifies.

(4) Preparation of the supernatant Medium

mu.L of conditioned medium containing L-glutamic acid at a final concentration of 20. mu.M, 50ng/mL bFGF, 0.1mM beta-mercaptoethanol, 10. mu.g/mL transferrin and 50. mu.g/mL ascorbic acid was overlaid on the mesocellular matrix.

(5) 3D optimized culture of primary tumor cells

Placing the 3D culture system prepared above into 37 deg.C, 5% CO₂Culturing in incubator with upper layer culture medium replaced every 2 days, and using inverted phase contrast microscopeThe growth of the cells was observed by a mirror (CKX53, Olympus, Japan), FIG. 1 is a graph showing the result of culturing NSCLC primary tumor cells on day 8 in a 3D culture system, wherein NSCLC primary tumor cells normally proliferate, FIG. 2 is a histogram showing the cell expansion rate on days 1, 4 and 8, wherein the cell density is 1.5 times the initial density on day 4 and 1.8 times the initial density on day 8.

Example 2

The difference from example 1 is that the tumor cells in the media matrix of the 3D culture system were replaced with normal epithelial cells, which were primary cells extracted from the normal paracancerous tissues as identified by non-small cell lung cancer pathology. As a result, as shown in fig. 3 and 4, normal epithelial cells failed to grow normally in the 3D tumor cells.

Comparing example 1 with example 2, it can be seen that the 3D culture system provided by the present invention can screen out tumor cells, achieve advantageous growth of tumor cells, and inhibit growth of normal epithelial cells.

Example 3

This example provides a 3D culture system and a method for preparing the 3D culture system, and also provides a method for culturing tumor cells using the 3D culture system.

(1) The isolation and culture procedure of tumor cells was different from that of example 1 in that the composition of the digestion solution used was as shown in Table 5;

TABLE 5 digestive juice formula

Reagent	Final concentration
		Collagenase I	100U/mL
Neutral dispase II	0.6U/mL
		DNase I	1U/mL
Hyaluronidase	100U/mL
		Sterile HBSS (Ca)²⁺&Mg²⁺)	Complement

(2) Preparation of the lower layer culture medium: preparing 2% low-melting-point agarose gel, weighing 2g of low-melting-point agarose, putting the low-melting-point agarose gel into a 250mL sterile blue-covered bottle, adding 100mL of ultrapure water for dissolving, placing the sterilized product into a 45 ℃ water bath kettle after autoclaving, and keeping the sterilized product in a molten state;

mixing and diluting 2% agarose gel preheated in a water bath kettle at 45 ℃ and a conditioned medium preheated at 37 ℃ in a plating tank to dilute the agarose gel to 0.5%, adding the agarose gel into a 384-pore plate, wherein each pore is 15 mu L, and waiting for solidification at 25 ℃;

(3) preparing a middle layer cell matrix: preheating 2% agarose gel in 45 deg.C water bath, mixing 2% agarose gel with hyaluronic acid, type I collagen and laminin, and mixing to obtain the final product with the concentrations shown in Table 6.

TABLE 6 ingredients of modified agarose gels

Composition (I)	Final concentration
		Low melting point agarose	0.1％(w/v)
Polylysines	0.1mg/L
		Hyaluronic acid	50U/mL
Collagen concentration	100U/mL
		Laminin	20μg/mL

The tumor cell suspension and the conditioned medium containing the additional factors were prepared in the same manner as in example 1, wherein the additional factors contained in the conditioned medium are shown in Table 7.

TABLE 7 additional factors and final concentrations contained in conditioned Medium

Composition (I)	Final concentration
		L-Glutamine	100μM
bFGF	100ng/mL
		Beta-mercaptoethanol	0.15mM
Transferrin	20μg/mL
		Ascorbic acid	80μg/mL

In order to prevent the modified agar gel from solidifying, after the modified agar gel is prepared, the modified agar gel is quickly mixed with a tumor cell suspension which is preheated in a water bath kettle at 37 ℃ in advance and a conditioned medium containing an additive factor; adding to a 384-well plate at a volume of 15 μ L/well, placing at 37 deg.C and 5% CO₂The incubator is left for 20min until it solidifies.

(4) Preparation of the supernatant Medium

mu.L of F-medium containing L-glutamic acid at a final concentration of 10. mu.M, 10ng/mL bFGF, 0.05mM beta-mercaptoethanol, 5. mu.g/mL transferrin and 40. mu.g/mL ascorbic acid was applied to the mesocellular matrix.

(5) 3D optimized culture of primary tumor cells

Placing the 3D culture system prepared above into 37 deg.C, 5% CO₂Culturing in an incubator, wherein the culture time interval of the upper layer culture medium is changed once every 2 days, observing the growth condition of the cells, and NSCLC primary tumor cells are normally proliferated in a 3D culture system, the cell density is 1.2 times of the initial density after culturing for 4 days, and the cell density is 2.0 times of the initial density after culturing for 8 days.

Example 4

(1) The isolation and culture procedure of tumor cells was different from that of example 1 in that the composition of the digestion solution used was as shown in Table 8;

TABLE 8 digestive juice formula

Reagent	Final concentration
		Collagenase I	200U/mL
Neutral dispase II	2.4U/mL
		DNase I	10U/mL
Hyaluronidase	200U/mL
		Sterile HBSS (Ca)²⁺&Mg²⁺)	Complement

(2) Preparation of the lower layer culture medium: preparing 3% of low-melting-point agarose gel, weighing 3g of low-melting-point agarose, putting the low-melting-point agarose gel into a 250mL sterile blue-covered bottle, adding 100mL of sterile ultrapure water for dissolving, placing the sterilized product into a 45 ℃ water bath kettle after autoclaving, and keeping the melted product in a molten state;

mixing 3% agarose gel preheated in a water bath kettle at 45 ℃ with a conditioned medium preheated at 37 ℃ in a plating tank, diluting to 0.5% agarose gel, adding into a 24-pore plate with 0.2mL agarose gel per pore, and solidifying at room temperature;

(3) preparation of the Medium layer cell matrix

Preparation of the improved agarose gel: preheating 3% agarose gel in 45 deg.C water bath, mixing 3% agarose gel with hyaluronic acid, type I collagen and laminin, and mixing to obtain the final product with the concentrations shown in Table 9.

TABLE 9 ingredients of modified agarose gels

Composition (I)	Final concentration
		Low melting point agarose	0.5％(w/v)
Polylysines	1mg/L
		Hyaluronic acid	10U/mL
Collagen concentration	10U/mL
		Laminin	15μg/mL

Tumor cell suspensions and conditioned media containing additional factors were prepared as in example 1, wherein the additional factors contained in the conditioned media are shown in Table 10.

TABLE 10 additional factors and final concentrations contained in conditioned Medium

To prevent the modified agar gel from coagulating, the modified agar gel is prepared and then rapidly pre-mixed with the modified agar gel in a water bath kettle at 37 deg.CMixing the hot tumor cell suspension with a conditioned medium containing an additive factor; adding into a 24-well plate with 200 μ L gel at 37 deg.C in a well plate, and standing at 5% CO₂The incubator is left for 15min until it solidifies.

(4) Preparation of the upper medium: 200 μ L of conditioned medium containing L-glutamic acid at a final concentration of 50mM, 100ng/mL bFGF, 0.15mM β -mercaptoethanol, 20 μ g/mL transferrin, and 80 μ g/mL ascorbic acid was overlaid on the supernatant medium.

(5) 3D optimized culture of primary tumor cells

And (3) culturing the prepared 3D culture system in a 5% CO2 incubator at 37 ℃ for 2 days, wherein the culture medium at the upper layer is replaced every other 2 days, the growth condition of the cells is observed, NSCLC primary tumor cells normally proliferate in the 3D culture system, the cell density is 1.6 times of the initial density after culturing for 4 days, and the cell density is 2.5 times of the initial density after culturing for 8 days.

Example 5

This example utilizes the 3D culture system provided in example 1 to culture tumor cells, except that the tumor cells are derived from pleural effusion and ascites of tumor patients.

(1) Isolated culture of tumor cells

First, tumor single cells were obtained from the pleural effusion of tumor patients:

1. collecting ascites and pleural fluid of a patient with advanced non-small cell lung cancer in a sterile environment, and collecting 200mL of ascites and pleural fluid in order to obtain a sufficient number of living tumor cells; and (3) sample preservation and transportation: collected pleural effusion was collected in sterile serum bottles containing 5mL of 2000U/mL heparin sodium and 2mL of 100U/mL penicillin and 100. mu.g/mL mitomycin, stored at 4 ℃ and transported under refrigeration.

2. Separating and preparing a hydrothorax and ascites sample: pouring the chest and abdomen water suspension sample into 50mL centrifuge tubes in sequence, centrifuging for 10min at 200g/min, after centrifuging, using Dulbecco's Phosphate Buffer Solution (DPBS) without calcium and magnesium to resuspend and combine into 1 centrifuge tube, washing twice, after centrifuging for 5min at 180g/min, then using F-culture medium to wash once, adjusting appropriate cell concentration, using separating medium with main components of dextran (dextran) and meglumine diatrizoate, and performing density gradient centrifugation on the separating medium with the density of 1.077 +/-0.001 g/mL at 25 ℃;

and (3) recovering the cell separation solution to 25 ℃, subpackaging the separation solution into a centrifuge tube by using a 10mL pipette, wherein each tube is 15mL, then sucking the cell suspension with the proper concentration by using the 10mL pipette, slowly adding the cell suspension along the tube wall of the centrifuge tube, slowly and naturally sliding the first drop of cell suspension along the tube wall to be paved on the liquid surface of the separation solution, observing the interface between the cell suspension and the separation solution, controlling the flow rate to avoid impacting the interface at a too large flow rate, and adding 20mL of cell suspension into each tube.

After layering is finished, carefully covering the centrifuge tube, taking out, balancing, putting into a centrifuge, and performing gentle action in the whole process to prevent the cell mixed suspension from being mixed with the separation liquid, centrifuging at the temperature of 25 ℃ at 1100g/min for 15min, and paying attention to that the speed rising speed and the speed reducing speed are adjusted to be lower;

after centrifugation is finished, the centrifugal tube is carefully taken out and placed in a super clean bench; the method comprises the following steps of sequentially arranging a red blood cell layer, a separating medium, a white cell layer and a yellow transparent liquid in a centrifuge tube from bottom to top, wherein the white cell layer mainly comprises tumor cells and a small amount of lymphocytes and does not contain interstitial cells, the density of the tumor cells is 1.05g/L, carefully absorbing the upper yellow transparent liquid, then transferring the middle white cell layer into a new 50mL centrifuge tube, paying attention to not absorb the red blood cells, and then carrying out centrifugation to collect cell precipitates; after 2 washes with DPBS, the collected cell pellets were resuspended in F-medium and counted.

(2) Amplification culture according to the CRC method

And (3) after the obtained microsphere cell sediment is resuspended in an F-culture medium, sucking a certain amount of cell suspension, and counting by trypan blue staining after proper dilution. According to 2X 10⁵The concentration of seed/well is 5X 10 in advance 2h⁶32Gy irradiated feeder cells per well were co-cultured for one week for rapid expansion for subculture of primary tumor cells P0.

(3) 3D optimized culture of primary tumor cells

The above P0 was collected according to the method of preparing the 3D system described in example 1Subjecting the pooled primary cells to 3D-optimized culture, wherein the primary cells are seeded at a density of 1 × 10 in a 3D system in 96-well plates⁵/mL, 5% CO at 37 ℃₂Culturing in an incubator, wherein the upper layer culture medium in example 1 is replaced every 2 days, and observing the growth condition shows that the tumor cells isolated from the pleural effusion and ascites of the tumor patients can also realize rapid proliferation in the 3D culture system.

Example 6

In this example, the 3D culture system of example 1 was used to perform single drug susceptibility testing of primary tumor cells, and the tumor drug Docetaxel (DOC) was used to perform drug susceptibility testing on different NSCLC samples from the surgical tissues of five different patients, each sample being identified as PCB 55/61/63/68/70.

After the 3D culture system prepared in example 1 is used for culturing for 8 days, the solution is changed directly for drug sensitivity detection after the primary tumor cells start to grow predominantly to form microspheres.

Gradient single drug administration and combined drug administration are carried out according to the concentration converted from the plasma peak concentration of each chemotherapeutic drug by the binding rate, 3 multiple holes are made for each administration concentration, a control group is arranged, drug sensitivity detection is carried out by utilizing Alamar blue (Alamar blue) detection reagent after administration for 72h, and the analysis of the single drug administration result is carried out according to the following modes: the peak plasma concentration and the in vitro binding efficiency of each chemotherapeutic drug are used to determine the appropriate drug concentration for single drug sensitivity detection, and the drug sensitivity detection results are analyzed by comparing the calculated drugs IC50, IC90 with PPC (IC50 represents the peak plasma concentration of the drug inhibiting 50% of tumor growth, IC90 represents the peak plasma concentration of the drug inhibiting 90% of tumor growth, PPC represents the peak plasma concentration of the drug corresponding to a certain clinical drug dose).

Wherein IC50< 25% PPC and IC90< 100% PPC are highly sensitive; IC50< 25% PPC and IC90> 100% PPC are moderately sensitive; IC50> 25% PPC and IC90< 100% PPC were mildly sensitive; resistance was obtained with IC50> 25% PPC and IC90> 100% PPC.

The results are shown in fig. 5, which shows that NSCLC PCB68 and PCB70 are highly sensitive to DOC, PCB61 and PCB63 are moderately sensitive to drug, and PCB55 is not sensitive to drug, i.e., has drug resistance.

Example 7

In this example, the 3D culture system of example 1 was used to perform single drug susceptibility testing of primary tumor cells, and six drugs were used to perform drug susceptibility testing on the same NSCLC sample. The six drugs are Carboplatin (CBP), Pemetrexed (PEM), Vinorelbine (VNR), paclitaxel (TAX), Gemcitabine (GEM) and Docetaxel (DOC), respectively, NSCLC samples were derived from the surgical tissues of patients and were numbered PCB 66.

After the 3D culture system prepared in example 1 is used for culturing for 8 days, the solution is changed after the primary tumor cells start to grow predominantly to form microspheres, and the drug sensitivity detection method is the same as that in example 6.

The results of drug-sensitive tests of NSCLC PCB66 for six drugs are shown in FIGS. 6(a) to 6(f), wherein FIG. 6(a) shows that NSCLC PCB66 is moderately sensitive to CBP, FIG. 6(b) shows that NSCLC PCB66 is not sensitive to PEM, FIG. 6(c) shows that NSCLCPCB66 is highly sensitive to VNR, FIG. 6(d) shows that NSCLC PCB66 is moderately sensitive to TAX, FIG. 6(e) shows that NSCLC PCB66 is highly sensitive to GEM, and FIG. 6(f) shows that NSCLC PCB66 is moderately sensitive to DOC.

Example 8

In this example, primary tumor cells were tested for drug sensitivity using the 3D culture system of example 1.

The combined drug sensitivity detection method comprises the following steps: the IC50 value of each chemotherapeutic drug is used for the combination of two drugs, such as drug A and drug B, wherein the drug A has weak or insensitive activity to the cell line, i.e. IC50 is more than or equal to 10 mu M, and the drug B has strong or sensitive activity to the cell line, i.e. IC50 is less than 10 mu M. The concentration points for single drug administration of drug B were set to 6, the analysis of the results was performed according to the curve translation method and the Combinationonindex (CI) was found according to the CompuSyn software.

Wherein CI <1 is synergistic effect; CI ═ 1 is equivalent; CI >1 is antagonistic.

Tumor drugs DOC and CBP are used in the embodiment, the CBP is used as a drug A, the DOC is used as a drug B, combined drug sensitive detection is carried out on NSCLC samples 1-3, the NSCLC samples 1-3 are from different operation tissue samples of patients with non-small cell lung cancer, the samples 1-3 are numbered respectively, after the 3D culture system prepared in the embodiment 1 is used for culturing for 8 days, the drug sensitive detection is carried out by changing liquid after the primary tumor cells start to grow to form microspheres, and the combined drug use scheme is as follows:

1. DOC is used alone; 2. DOC was used concurrently with CBP IC 50.

The results show that the DOC and the CBP are taken together, as shown in figure 7(a), when the DOC and the CBP are taken together for NSCLC sample 1, the combination index CI of the DOC and the CBP is less than 1, and the DOC and the CBP have synergistic effect; as shown in fig. 7(b), for NSCLC sample 2, the combined index of DOC and CBP, CI ═ 1, both effects were equivalent to single agent effects; as shown in FIG. 7(c), for NSCLC sample 3, the DOC and CBP combined index CI >1, both were antagonistic.

Comparative example 1

The difference of comparative example 1 compared to example 1 is that primary tumor cells were cultured using a general agar culture system. The preparation method of the common agar culture system comprises the following steps:

1. preparation of the lower layer culture medium: preparing 2% low-melting-point agarose gel, weighing 2g of low-melting-point agarose, putting the low-melting-point agarose gel into a 250mL sterile blue-covered bottle, adding 100mL of sterile ultrapure water for dissolving, placing the sterilized product into a 45 ℃ water bath kettle after autoclaving, and keeping the melted product in a molten state;

mixing and diluting 2% agarose gel preheated in a water bath kettle at 45 ℃ and a common DMEM culture medium preheated at 37 ℃ in a plating tank to 0.5% agarose gel, adding into a 96-well plate, and solidifying at 25 ℃ and 0.05mL agarose gel per well;

2. preparing an intermediate cell matrix layer: mixing 0.1% upper agar, common DMEM medium and the tumor cells obtained according to the method of example 1; the cells were added to wells plated with the substratum medium at a volume of 50. mu.L/well in a 96-well plate, and incubated at 37 ℃ with 5% CO₂The incubator is left for 30min until it solidifies.

When the prepared agar culture system is used for culturing tumor cells, the growth condition of the cells is observed by using an inverted phase contrast microscope, the primary tumor cells can not grow normally, as shown in fig. 8 and 9, fig. 8 is the culture result of the culture on the 1 st day, a single tumor cell after mixing can be observed in the common agar culture system, and fig. 9 is the culture result of the culture on the 8 th day, the tumor cells can not grow normally in the common agar culture system.

Therefore, as can be seen from the analysis combining example 1 and comparative example 1, the 3D culture system provided by the present invention can allow tumor cells to grow preferentially.

The applicant states that the present invention is illustrated by the above examples of the components of the 3D culture system and the method of culturing primary tumor cells using the same, but the present invention is not limited to the above detailed procedures and reagents used, i.e., it is not meant that the present invention must rely on the above detailed procedures and reagents to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A3D culture system of primary tumor cells is characterized by comprising a lower layer gel, a middle layer cell matrix and an upper layer culture medium;

the middle layer cell matrix comprises a modified agarose gel, a conditioned medium containing added factors and tumor cells, wherein the modified agarose gel comprises low-melting-point agarose, polylysine and extracellular matrix;

the lower gel comprises agarose gel and conditioned medium;

the upper layer culture medium is a conditioned medium containing an additive factor.

2. The 3D culture system of claim 1, wherein the extracellular matrix comprises any one or a combination of two or more of mucopolysaccharide, proteoglycan, laminin, fibrin, collagen, or hyaluronic acid, preferably a combination of collagen, laminin, and hyaluronic acid;

preferably, the collagen is type I collagen.

3. The 3D culture system according to claim 1 or 2, wherein the concentration of the collagen in the modified agarose gel is 10-100U/mL;

preferably, the concentration of the laminin in the modified agarose gel is 10-20 mug/mL;

preferably, the concentration of the hyaluronic acid in the modified agarose gel is 10-100U/mL;

preferably, the concentration of the polylysine in the modified agarose gel is 0.1-1 mg/L.

4. The 3D culture system according to any one of claims 1-3, wherein the mass concentration of the low-melting agarose in the modified agarose gel is 0.1-0.4%;

preferably, the seeding density of the tumor cells in the middle layer cell matrix is (1-2) multiplied by 10⁵/mL；

Preferably, the mass concentration of the low-melting-point agarose in the agarose gel of the lower-layer gel is 0.4-0.8%.

5. The 3D culture system of any one of claims 1-4, wherein the additional factors comprise any one or a combination of two or more of amino acids, growth factors, reducing agents, or transferrin;

preferably, the amino acid is L-glutamine;

preferably, the growth factor is any one or a combination of more than two of EGF, FGF or bFGF, preferably bFGF;

preferably, the reducing agent is beta-mercaptoethanol and/or ascorbic acid.

6. The 3D culture system according to any one of claims 1 to 5, wherein the molar concentration of L-glutamine in the conditioned medium is 20 to 100 μ M;

preferably, the concentration of the growth factor in the conditioned medium is 10-100 ng/mL;

preferably, the concentration of the beta-mercaptoethanol in the conditioned medium is 0.05-0.15 mM;

preferably, the concentration of the ascorbic acid in the conditioned medium is 40-80 mug/mL;

preferably, the concentration of the transferrin in the conditioned medium is 5-20 mug/mL.

7. The 3D culture system according to any one of claims 1 to 6, wherein the tumor cells are isolated and cultured by the method comprising:

collecting primary tumor single cells or cell microspheres from tumor tissues or pleural effusion of a tumor patient, and performing amplification culture by using a conditional reprogramming culture method to obtain the tumor cells;

preferably, the step of collecting comprises digestion and separation.

8. The 3D culture system according to any one of claims 1 to 7, wherein the digestive fluid used for digestion comprises any one or a combination of two or more of collagenase, neutral dispase, DNase, and hyaluronidase;

preferably, the concentration of the collagenase in the digestive juice is 100-200U/mL;

preferably, the content of the neutral dispase in the digestive juice is 0.6-2.4U/mL;

preferably, the concentration of the DNase in the digestive juice is 1-10U/mL;

preferably, the concentration of the hyaluronidase in the digestive juice is 100-200U/mL.

9. The 3D culture system of any one of claims 1-8, comprising a lower gel, a middle cell matrix, and an upper medium;

the lower layer gel comprises agarose gel and a conditioned medium, and the mass concentration of the low-melting-point agarose in the agarose gel is 0.4-0.8%;

the middle-layer cell matrix comprises an improved agarose gel, a conditioned medium containing an additive factor and a tumor cell suspension, the mass concentration of low-melting-point agarose in the improved agarose gel is 0.1-0.4%, the improved agarose gel also comprises hyaluronic acid with the concentration of 10-100U/mL, polylysine with the concentration of 0.1-1mg/mL, I-type collagen with the concentration of 10-100U/mL and laminin with the concentration of 10-20 mu g/mL, and the inoculation concentration of tumor cells is (1-2) multiplied by 10⁵/mL；

The upper layer culture medium is a condition culture medium containing an additive factor, and the condition culture medium containing the additive factor comprises 20-100 mu M L-glutamine, 10-100 ng/mL bFGF, 0.05-0.15 mM beta-mercaptoethanol, 40-80 mu g/mL ascorbic acid and 5-20 mu g/mL transferrin.

10. Use of a 3D culture system according to any of claims 1 to 9 for the preparation of a drug sensitive detection reagent or a drug sensitive detection kit.