CN107988161A - A kind of method for promoting tumour cell reprogramming - Google Patents

Abstract

The invention discloses a method for promoting the reprogramming of tumor cells. The tumor cells are cultured in vitro by using a serum-free medium, and serum albumin with a concentration of 1-55 g/L is added to the serum-free medium. The medium is based on DMEM/F12, and β-mercaptoethanol with a final concentration of 0.52-0.88mg/L, transferrin with a concentration of 3.6-5.8mg/L, and selenium with a final concentration of 0.0017-0.002mg/L sodium nitrite, 1.6-12.8 mg/L insulin and 0.48-1.1 mg/L aminoethanol. Compared with the traditional serum-free suspension method, the present invention has higher efficiency of CSCs obtained by promoting tumor cell reprogramming, which is reflected in shorter time used, larger proportion of CSCs obtained, higher success rate of CSCs obtained, and obtained CSCs Stable in nature. By adjusting different concentrations of serum albumin, the present invention can greatly simulate the changes in the tumor vascular microenvironment in the process of tumor cell transfer from the primary focus to the circulatory system. Research provides more suitable tumor cell models.

Description

A method for promoting tumor cell reprogramming

technical field

The invention relates to the field of cell biology research, in particular to a method for promoting cell reprogramming.

Background technique

Tumor is one of the common diseases that seriously threaten human health, and the reason for the high mortality rate of tumor is the complexity of tumor itself. A variety of tumor cells with different degrees of differentiation and different phenotypes are often associated with clinical phenomena such as tumor resistance to chemotherapy drugs and radiation therapy, and postoperative recurrence. In recent years, with the deepening of tumor research, the theory of cancer stem cells (CSC) has brought new ideas to the treatment of tumors. Cancer stem cells are a group of cells in a tumor population that are extremely small in number and have self-renewal and self-replication capabilities. This group of cells in a quiescent state in the body can differentiate into tumor cells of different phenotypes under the induction of a specific microenvironment. Therefore, the research on CSC can help to fundamentally explore the molecular mechanisms of tumor development, metastasis, and poor prognosis, which is of great significance to the clinical treatment of tumors.

Currently, the authoritative cell banks on the market can only provide established lines of non-stem tumor cell lines or only a few types of tumor stem cell lines. However, if CSCs are isolated from tumor cell lines or primary tumor cells by immunological sorting of CSC surface molecular markers or side population (sidepopulation, SP) sorting, only a very small amount of CSCs can be obtained, which is difficult to meet the increasing demands. The number of cells that needs to be increased, in addition, the process of sorting cells by flow cytometry will cause inevitable damage to the cells, and the operation is not convenient enough. Therefore, on this basis, a serum-free medium that does not contain animal serum and other animal-derived components was developed. Through the serum-free medium, most common malignant tumors can be enriched to stem cells, and through stem cell surface markers, Drug resistance, suspension culture and stable passage prove that these enriched tumor balloon cells are indeed CSCs. However, in most reports, these CSCs obtained by serum-free medium have the following problems: the success rate is unstable, the required reprogramming time is long, and the continuous passage is rarely more than 10 passages. The commercial serum-free medium is added with a large number of growth factors of various components, which is not conducive to the subsequent CSC-related research on these pathways.

In order to obtain the largest proportion of CSCs for research in the adherent tumor cells, in addition to enriching the very few CSCs in the cell population, reprogram the adherent tumor cells in vitro to obtain a large number of CSCs, then It can guarantee the amount of each enrichment, ensure the repeatability and reliability of the experiment, and save a lot of enrichment time. The vascular microenvironment plays an important role in the initiation and maintenance of tumor stem cells, and the blood leakage involved in the tumor vascular microenvironment and the plasma microenvironment contain a large amount of serum albumin, which suggests that serum albumin plays an important role in The reprogramming of tumor cells into CSCs plays an important role.

Serum albumin is the most abundant protein in vertebrate plasma. It is synthesized by liver cells and released into the portal circulation. The concentration of serum albumin in adults is about 35g/L-50g/L, while in children The protein concentration is about 29g/L-55g/L. The amino acid sequence and spatial structure of serum albumin from different sources are very conserved. It has many functions in the body such as maintaining blood colloid osmotic pressure, binding and transporting endogenous and exogenous substances, scavenging free radicals and enhancing enzyme activity. . In the culture medium reported in the past, serum albumin is often added as a function of providing nutrition and maintaining cell activity, but most of them are low concentration (1-30g/L) serum albumin.

Publication No. CN102634482A, title of invention "a serum-free complete medium for mesenchymal stem cells" and publication number CN104694470A, title of invention "a serum-free culture medium for stem cells" are all mentioned in the Chinese invention patent application A complete serum-free medium for culturing mesenchymal stem cells in vitro, which respectively contains human serum albumin 1-30g/L and 5-30g/L. The two patent applications focus on the cultivation and massive expansion of ready-made mesenchymal stem cells, and the role of serum albumin is to maintain osmotic pressure and provide nutrition and other basic physiological functions, and it is derived from human serum albumin It is difficult to obtain in large quantities, and the price is expensive, and it is difficult to realize experiments that require a large amount of CSCs; the publication number is CN103898056B, the invention name is "cell culture medium and its application in culturing human primary tumor cells" and the publication number is CN101984051A, The Chinese invention patent application titled "Serum-free Cell Culture Medium Suitable for Enrichment and Culture of Tumor Stem Cells" all involve a method for enrichment and in vitro culture of tumor stem cells. By placing tumor cell lines or human primary tumor cells in the serum-free medium mentioned in the invention, the reprogrammed tumor stem cells can be enriched to a certain extent, respectively containing plant-derived recombinant human serum albumin 3-5g/ L and bovine serum albumin 0.45-0.6g/L, both of which are low concentrations of serum albumin. Moreover, in addition to serum albumin, the former also added additives such as various hormones such as hydrocortisone, growth factors such as human epidermal growth factor EFG, etc., which greatly increased the cost of obtaining CSCs, and for those with such hormones or growth factors Factor-related research has caused great interference.

In the existing serum-free cell culture medium used for CSC enrichment and culture, the present invention establishes a tumor cell reprogramming method with wider application range, shorter experimental period, lower cost and higher success rate. method. The CSC obtained by the present invention can be stably passaged to more than 10 passages, and has good experimental repeatability and consistency, and provides a highly applicable operation method for the related research on CSC and vascular microenvironment.

Contents of the invention

The purpose of the invention is to provide a method for promoting tumor cell reprogramming.

In order to achieve the above object, the technical scheme adopted by the present invention is: adding one or more serum albumins to the cell culture medium.

The serum albumin is human serum albumin or bovine serum albumin.

The method is to add 1-55g/L of bovine serum albumin or human serum albumin to the cell culture medium.

The method for promoting tumor cell reprogramming is applicable to most malignant tumor cells, including most tumor cells derived from mesenchymal cells. According to the source and metastasis characteristics of CSC, if you want to simulate the area of primary tumor CSC in the environment of tumor vascular bleeding, adding 1g-8g/L serum albumin can significantly improve the reprogramming efficiency of tumor cells; if you want to simulate the plasma microenvironment In CSC, adding 35-55g/L serum albumin can maximize the reprogramming efficiency of tumor cells, and under this colloid osmotic pressure, non-CSC tumor cells that cannot tolerate the environment can be removed, and finally obtained Tumor-initiating cells with enhanced metastatic ability and resistance to anoikis.

The specific operation method of the method for promoting tumor cell reprogramming described in the present invention is: after simply removing the serum from the tumor cells, digest them into single cells, and then inoculate them in serum-free cell culture medium, adding the appropriate amount according to the experimental requirements and different cell types. concentration of serum albumin, and then cultivated under suitable culture conditions (37°C, 5%-10% CO2) for 48-72h, the suspended agglomerated or spherical CSCs can be seen in the culture medium.

The positive effect of obtaining CSC by the present invention is:

(1) Compared with the traditional serum-free suspension method, the efficiency of CSCs obtained by promoting tumor cell reprogramming is higher, which is reflected in the shorter time used, the greater proportion of CSCs obtained, and the higher success rate of CSCs; (2) The CSCs obtained by promoting the reprogramming of tumor cells are stable in nature and can be stably passaged in the state of suspension culture, which can maintain the physiological activity and cell activity of tumor stem cells to the greatest extent. (3) By adjusting different concentrations of serum albumin, the changes in the tumor vascular microenvironment in which tumor cells are transferred from the primary tumor to the circulatory system can be simulated to a great extent, and the tumor metastasis model and circulating tumor cells in the plasma microenvironment The study provides a more suitable tumor cell model.

Description of drawings

Figures 1a-1c are microscopic cell pictures of CSCs obtained from osteosarcoma cell lines and colon cancer cell lines using the method provided by the present invention, wherein Figure 1a is MNNG osteosarcoma stem cells, Figure 1b is MG63 osteosarcoma stem cells, and Figure 1c For Sw620 colon cancer stem cells.

Fig. 2a, Fig. 2b, Fig. 2c are respectively under the condition of adding bovine serum albumin of 0g/L (that is, no addition group), 2.5g/L and 50g/L in serum-free medium by using the method provided by the present invention , The reprogramming status of osteosarcoma stem cells cultured to the third day.

Fig. 3 is to utilize the method provided by the present invention to contrast under the condition of adding bovine serum albumin of 0g/L (that is, no addition group) and 2.5g/L respectively in the serum-free medium, osteosarcoma cells are cultivated to the first day , reprogramming on day 3 and day 5. It can be seen from the figure that after the addition of serum albumin, the reprogramming time of tumor cells is advanced, and the number of CSCs finally obtained is more than that of traditional serum-free medium.

Figures 4a to 4c are the results of self-renewal of CSCs obtained by the method provided by the present invention and stable passage in suspension culture. Figure 4a, Figure 4b, and Figure 4c are images under the microscope of the second, seventeenth, and thirty-fourth passages of MNNG osteosarcoma stem cells, respectively.

Figures 5a to 5b are the marker genes of the stem cells detected by immunofluorescent staining of CSCs obtained by the method provided by the present invention, wherein Figure 5a shows the expression of CD133 molecular cells of MNNG non-CSC (bulk) and CSC (Sphere) by immunofluorescence Situation; Figure 5b shows the cellular immunofluorescence expression of C-kit and TRA-1-81 of MNNG non-CSC (bulk) and CSC (Sphere).

specific embodiment

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention.

Example 1

This example is a preparation example of serum-free medium and serum albumin.

Prepare bovine serum albumin stock solution, dissolve bovine serum albumin powder with double distilled water to a final concentration of 100g/L, filter through a 0.2 μM filter and place in a 4-degree refrigerator for later use.

Serum-free culture medium takes another disclosed invention of our laboratory as an example, the publication number is CN101984051A, the publication date is March 9, 2011, and the title of the invention is "serum-free cell culture medium suitable for enrichment and cultivation of tumor stem cells" , the medium is based on DMEM/F12, and the final concentration is 0.52mg/L of β-mercaptoethanol, 3.6mg/L of transferrin, 0.0017mg/L of sodium selenite, 1.6 mg/L insulin and 0.48mg/L aminoethanol.

Bovine serum albumin stock solution was added to the above-mentioned serum-free medium to a final concentration of 2.5 g/L, 5 g/L and 50 g/L. Specifically, the serum albumin concentration will depend on the source and characteristics of the tumor cells to be reprogrammed. In order to fit the real environment in the body, the serum albumin concentration fluctuates within the range of 1g/L-55g/L.

Example 2

This example is a culture example when the cultures are human osteosarcoma cell lines MNNG, MG63 and colon cancer cell line Sw620.

Using the culture medium provided in Example 1 of the present invention, the human osteosarcoma cell line MNNG was cultivated when the serum albumin concentration was 2.5 g/L, and the human osteosarcoma cell line Mg63 and The colon cancer cell line Sw620 was cultured for five days, and Fig. 1a to Fig. 1c are the obtained CSCs after reprogramming. From Figures 1a to 1c, it can be seen that the application of the medium provided by this patent makes the tumor cells that should have adhered to the wall shrink and aggregate to form a balloon-like shape that can be detached from the culture dish, indicating that we have obtained reprogramming with stronger stemness After the CSC.

Example 3

This example is a comparison example of adding different amounts of serum albumin at the same culture time when the culture is the human osteosarcoma cell line MNNG.

Using the medium provided in Example 1 of the present invention, the human osteosarcoma cell line MNNG was cultured under the conditions of serum albumin concentrations of 2.5 g/L and 50 g/L and no serum albumin was added, and the cell reprogramming situation after three days of culture was compared . As shown in Figure 2a, when cultured for three days without adding serum albumin, MNNG cells still showed an adherent morphology, and only a small number of cells began to aggregate; as shown in Figure 2b, after adding 2.5g/L serum albumin In the case of three days of culture, the MNNG cell population has obvious aggregation and a small amount of balloon formation; as shown in Figure 2c, in the case of adding 50g/L serum albumin for three days, almost all the cells Both aggregated to form more dry CSC balloons. It shows that the reprogramming efficiency of osteosarcoma cells increases with the increase of serum albumin concentration.

Example 4

This example is a comparative example of adding and not adding serum albumin at multiple times of the same culture time when the culture is the human osteosarcoma cell line MNNG.

Using the medium provided in Example 1 of the present invention, the concentration of serum albumin was 2.5g/L and no serum albumin was added to cultivate the human osteosarcoma cell line MNNG, and the first day, the third day and the second day after culture were compared respectively. Cell reprogramming at five days. It can be seen from Figure 3 that the MNNG cells added with 2.5g/L concentration of serum albumin did not show a fully extended morphology in the form of adherent growth on the first day of culture, but on the third day of culture, the MNNG cell population Obvious aggregation and balloons can appear, until the fifth day of culture, obvious CSC balloons can be seen in the culture dish. Compared with the MNNG group without serum albumin, there were still a large number of adherent cells on the fifth day. It can be seen that, compared with the control group without serum albumin, adding serum albumin can significantly improve the reprogramming speed and efficiency of tumor cells.

Example 5

This example is an example of continuous culture when the culture is the human osteosarcoma cell line MNNG.

The MNNG tumor stem cells obtained by using the medium provided in Example 1 of the present invention at a serum albumin concentration of 2.5 g/L were digested into single cells and continued to be cultured under this condition. As shown in Figure 4, in this case, CSC cells can grow in a suspension state in ordinary culture dishes without low adsorption or anti-adherence treatment, and can be stably passaged and have a strong self-renewal ability.

The MNNG tumor stem cells and MNNG adherent cells obtained by using the method provided in Example 2 of the present invention were subjected to immunofluorescent staining to detect the expression of their stem cell marker genes. As shown in Figure 5a and Figure 5b, compared with adherent cells, the CSCs obtained by the reprogramming of the present invention express stemness marker genes such as C-Kit, CD133, TRA-1-81, and have stronger cellular immunofluorescence signals, indicating that The MNNG tumor stem cells obtained through the medium provided by this patent do have stronger stemness.

Claims (5)

1. A kind of 1. method for promoting tumour cell reprogramming, it is characterised in that：Tumour cell is carried out using serum free medium In vitro culture, adds one or more seralbumins that concentration is 1~55g/L, the serum-free in serum free medium Culture medium adds β-sulfydryl second that ultimate density is 0.52~0.88mg/L wherein using DMEM/F12 as basic culture medium Alcohol, the transferrins of 3.6~5.8mg/L, the sodium selenite of 0.0017~0.002mg/L, 1.6~12.8mg/L insulin with And the ethylaminoethanol of 0.48~1.1mg/L.
2. A kind of 2. method for promoting tumour cell reprogramming according to claim 1, it is characterised in that：The white egg of serum White is human serum albumins or bovine serum albumin(BSA).
3. A kind of 3. method for promoting tumour cell reprogramming according to claim 1, it is characterised in that：Tumour cell refers to Tumor cell line or shredded by the tissue block of tumor tissues or digest formed it is unicellular after be seeded in the serum free medium Culture.
4. 4. the method reprogrammed according to a kind of any promotion tumour cell of claims 1 to 3, it is characterised in that：It is described Tumour cell include one kind in human osteosarcoma cell or colon cancer cell.
5. A kind of 5. method for promoting tumour cell reprogramming according to claim 1, it is characterised in that：It is thin for Tumor Stem The source of born of the same parents and transfer characteristic, the region to simulate primary tumor tumor stem cell under tumor vessel oozing of blood environment, addition Seralbumin concentration is 1g-8g/L；To the tumor stem cell in simulating blood plasma microenvironment, the seralbumin concentration of addition For 35-55g/L.