CN106854639A - One kind is based on three-dimensional semisolid adult stem cell cultural method - Google Patents

Abstract

A three-dimensional semi-solid culture method for adult stem cells. The medium uses methylcellulose and matrigel as a three-dimensional skeleton. The formula allows cells to grow in a relatively fixed three-dimensional space. Methylcellulose solution is a viscous semi-solid, which is convenient for packaging and taking, and is also conducive to the collection and transfer of cells. The Matrigel is liquid at 4°C, which is convenient for taking and mixing, and is agar-like solid at 37°C, providing a certain support strength for the three-dimensional semi-solid system. The lack of fluidity of single cells in semi-solid media and the formation of colonies can be accurately traced to single cells, so it is a reliable solution to verify the existence of stem cells in adult tissues and organs, and to study the differentiation of stem cells.

Description

A three-dimensional semi-solid-based method for culturing adult stem cells

technical field

The invention belongs to the cell culture technology in the field of biotechnology, and in particular relates to an in vitro three-dimensional semi-solid culture method for adult stem cells with methyl cellulose and matrigel as the skeleton.

technical background

Adult stem cells are a kind of stem cell subsets existing in tissues and organs of adult individuals. It is characterized by a certain self-renewal ability and multi-differentiation potential. Adult stem cells account for a relatively small proportion in tissues and organs, and most of them are in a resting state. They are relatively immature pluripotent cells. They can be activated under specific conditions to start the process of self-renewal and differentiate into mature cells with specific functions in that tissue or organ.

Due to the different sources of adult stem cells, their growth characteristics and nutritional requirements vary. Traditional two-dimensional adherent culture is difficult to meet the growth requirements of adult stem cells from various sources (such as hematopoietic stem cells, tumor stem cells). In addition, cells will grow in clusters during two-dimensional culture, which cannot be distinguished from colonies formed by single-cell clonal growth, and it is difficult to distinguish whether the differentiation of cells comes from a single stem cell or from multiple precursor cells. This shortcoming is a great obstacle for analyzing the multidifferentiation potential of cells. The three-dimensional culture method has one more dimension than the two-dimensional culture. Under the support of the semi-solid matrix, the cells can stretch and grow in the three-dimensional space, which relieves the restriction that some cells grow poorly on the two-dimensional plane. In addition, the relatively fixed three-dimensional matrix can hinder the free flow of cells, so the colonies formed by the clonal growth of single cells are isolated, which is very important for analyzing the differentiation potential of single cells. Since stem cells can grow in the form of monoclonal colonies in vitro, the semi-solid three-dimensional culture method with limited cell fluidity is also an effective way to prove that a single adult cell has stemness.

Methylcellulose is a long-chain polysaccharide compound, which can form a colloidal solution with a certain viscosity after fully swelling in the solution, and is a pseudoplastic fluid with certain fluidity. This three-dimensional skeleton material is not absorbed and degraded by cells, is stable and non-toxic, and can allow cells to grow within a three-dimensional framework. Methylcellulose can be dissolved using the hot water method. Fully stir and swell in hot water at 70°C, and then gradually cool down to form a uniform colloidal solution. The fluidity of methylcellulose with different viscosities (cPs) at the same concentration is also different, and the fluidity of fully swollen methylcellulose at the same concentration is inversely proportional to its viscosity.

Matrigel is the basement membrane matrix isolated from mouse tumors. It is rich in outer matrix proteins such as laminin, type IV collagen, heparan sulfate glycoprotein, etc., and also contains a variety of cytokines and metalloproteinases. The Matrigel is characterized in that it is in a liquid state at 4°C, which is convenient for transfer operation, while at 37°C, Matrigel forms a stable solid gel, providing a three-dimensional skeleton structure for cell culture. In addition, the extracellular protein components in Matrigel can simulate the growth state of cells in vivo, which is closer to the living environment in vivo.

At present, most of the three-dimensional cell culture methods use methylcellulose or matrigel alone. Using methylcellulose alone can only provide a relatively stable three-dimensional environment for cells, but it cannot simulate the nutritional status of cells in tissues in vivo. However, the solid fluidity formed by using matrigel alone is not conducive to the rapid expansion and extension of cells. The semi-solid formed by mixing Matrigel and methylcellulose according to a certain concentration not only provides a three-dimensional skeleton for the growth of cells, but also simulates the growth environment in the body. Methyl cellulose and Matrigel are filled with each other to form a semi-solid with certain fluidity. Compared with the solid medium formed by using methyl cellulose and Matrigel alone, it is more convenient for subsequent operations, making the collection and transfer of colonies more convenient .

Contents of the invention

The invention provides a three-dimensional semi-solid in vitro culture method of adult stem cells. It uses a semi-solid solution mixed with methylcellulose solution and matrigel as a three-dimensional framework. The methylcellulose solution is a viscous semi-solid, which is convenient for packaging and taking, and is also beneficial for the collection and transfer of cells. The Matrigel is liquid at 4°C, which is convenient for taking and mixing, and is agar-like solid at 37°C, providing a certain support strength for the three-dimensional semi-solid system. Further, the cell culture plate used for culture is an ultra-low adhesion culture plate, in which the adherent cells will not proliferate in large quantities and consume nutrients, and at the same time, the plate does not adsorb Matrigel, which is convenient for cell handling. This formula allows cells to grow in a relatively fixed three-dimensional space. The lack of fluidity of single cells in semi-solid media and the formation of colonies can be accurately traced to single cells, so it is a reliable solution to verify the existence of stem cells in adult tissues and organs, and to study the differentiation of stem cells. Furthermore, the present invention can not only cultivate and identify adult stem cells derived from normal tissues, but also be used as a method for cultivating and identifying tumor stem cells.

The implementation method of this technology includes the following steps:

1. Slowly add methylcellulose (1500cPs) into sterile water at 70°C, and stir continuously with a magnetic stirrer to fully swell. When the water temperature drops to close to room temperature, add penicillin and streptomycin (final concentration: penicillin 100U/ml, streptomycin 0.1mg/ml) and an equal volume of pre-cooled 2 times liquid medium to form a normal concentration liquid medium Dissolved 3.3% (w/v) methylcellulose solution. After continuous stirring at 4°C for 24 hours, it became a viscous semi-solid solution for later use.

2. Adult tissues or organs are digested with collagenase for 30 minutes (use a syringe with a needle to beat repeatedly during the period), use liquid complete medium (containing 10% fetal bovine serum) to stop digestion, centrifuge at 2000rpm for 5 minutes, resuspend and filter A 200-mesh cell sieve was used to obtain a fully digested single-cell suspension.

3. Add liquid complete medium (containing 8.4% fetal bovine serum), 3.3% methylcellulose and Matrigel in a 5mL centrifuge tube, the ratio of the three is 6:3:1, to obtain 3.3% methylcellulose to make formazan The final concentration of base cellulose is 0.99%, the final concentration of matrigel is 10% (w/w), and the final concentration of fetal bovine serum is 5%. Add cell suspension to the above system, shake well and evenly, add to 24-well ultra-low adhesion cell culture plate, and culture at 37°C in an incubator containing 5% carbon dioxide.

4. The culture medium does not need to be changed. Subsequent operations were carried out after the colonies grown by single-cell clones were sufficiently enriched. After 2 weeks of culture, Matrigel condensed and separated from the methylcellulose solution. Colonies were concentrated in pyknotic Matrigel. Use a Pasteur pipette to aspirate the condensed matrigel from the methylcellulose solution, transfer it to a plate, and use ophthalmic forceps and needles to isolate individual clonal colonies under a microscope for immunofluorescence experiments, RNA or protein extraction, or their Place in trypsin to digest into a single cell suspension and continue to subculture.

Figure 1 is a graph of colony growth after 2 weeks of culture.

Figure 2 is a graph showing the change of colony growth over time.

Figure 3 is a statistical diagram of colony diameter and number.

Figure 4 is an analysis diagram of self-renewal ability.

Fig. 5 is a graph showing changes in genes of cystic colonies and cells before culture.

Figure 6 is a graph of gene analysis of a single colony.

Figure 7 is a diagram of immunofluorescence analysis of a single colony.

Fig. 8 is a diagram of colony formation of MCF-7 cells.

specific implementation plan

The content of the present invention will be further described below in conjunction with the embodiments. This embodiment is only used to illustrate the present invention, not to limit the applicable scope of the present invention. The embodiments produced by applying the present invention without creative efforts by those skilled in the art all belong to the protection scope of the present invention.

Example 1: Culture of adult stem cells derived from mouse pancreas

Main reagents: methylcellulose (1500cPs), recombinant human epidermal growth factor (EGF), Noggin, and Nicotinamide were purchased from Sigma Company, Matrigel and Cell recovery solution were purchased from BD Company, DMEM/F12 medium, fetal bovine Serum, penicillin, collagenase type II, and B27 were purchased from Invitrogen.

1. Preparation of adult mouse pancreas single cell suspension: 4-8 week-old adult C57BL/6 mice were sacrificed by cervical dislocation and soaked in alcohol for 3 minutes. An incision was made on the left side of the abdomen, and the pancreas connected to the spleen was taken out, and the head of the pancreas was cut close to the duodenum. The pancreas was washed three times with PBS containing 0.1% BSA, placed in a pre-cooled plate, and shredded for 2 minutes. Place in 0.2% type II collagenase digestion solution (containing 30U DNaseI), digest in a 15mL centrifuge tube with a 37°C water bath for 20-25 minutes, during which every 7-10 minutes use a syringe with a needle to blow 10 times. After digestion, a cloudy tissue suspension was obtained. An equal volume of DMEM/F12 medium containing 10% fetal bovine serum was used to terminate the digestion, and centrifuged at 2000 rpm for 5 minutes. After discarding the supernatant, resuspend in DMEM/F12 medium containing 8.4% fetal bovine serum, and pass through a 200-mesh cell sieve to obtain a single cell suspension of adult mouse pancreas. Cell counts were performed under a microscope by trypan blue exclusion.

2. Medium preparation and plating: DMEM/F12 medium containing 8.4% fetal bovine serum (containing penicillin 100U/ml, streptomycin 0.1mg/ml), 3.3% methylcellulose, Matrigel according to 6:3 : 1 ratio, and then add recombinant human epidermal growth factor (EGF), Nicotinamide (Nicotinamide), Noggin protein and 100x B27, so that the final concentration of EGF is 20ng/mL, the final concentration of Nicotinamide is 10mmol/L, and the final concentration of Noggin is 100ng/L mL, B27 final concentration 1x, methylcellulose final concentration 0.99% (w/v), matrigel final concentration 10% (w/v). According to the calculation of inoculating 10,000 cells per well of a 24-well plate, the cell suspension and the above components were fully shaken and mixed, and then stood still until the bubbles disappeared. Use a 1 ml syringe to draw up the fully dispersed semi-solid cell suspension, and add it to an ultra-low adhesion 24-well cell culture plate at a volume of 500 μl per well. Add 1mL sterile water to the left and right rows of culture wells to prevent medium evaporation in the middle row. Place them in an incubator at 37°C with 5% carbon dioxide for 2 weeks.

3. Colony generation rate statistics: After 2 weeks of culture, the cells formed a hollow sac-like colony that aggregated and grew (circular section under the light microscope, as shown in Figure 1). This colony is a clonal colony from the proliferation of a single cell. The colony growth relationship is shown in Figure 2. Select 4 culture wells, count the number of colonies with different diameters under a light microscope of 400 times, and calculate the colony formation rate according to the number of inoculated cells, as shown in Table 1. Based on the seeding of 10,000 cells per well, the colony formation rate per well was 1.18%±0.06%. The proportion of colonies with different diameters is shown in Figure 3.

Table 1: Colony formation and formation rate of adult stem cells derived from mouse pancreas (10,000 cells per well)

4. Verification of self-renewal ability: self-renewal (self-renwal) is one of the signs of cell stemness. Stem cells can activate the process of cell cycle under certain conditions and have stable proliferation ability, that is, they can be continuously passaged in vitro. The experimental scheme is as follows: the single cells cultured for 2 weeks expanded and formed cystic colonies, which aggregated into clusters along with the pyknotic Matrigel. Use ophthalmic tweezers and needles to isolate a single colony under a microscope, place several isolated colonies in trypsin digestion solution, and digest at 37°C for 10 minutes until obvious cell gaps appear on the surface of cystic colonies, and then use an equal amount to completely culture basal (containing 10% fetal bovine serum) to stop the digestion, use a pipette to pipette into a single cell suspension and count with a hemocytometer. Repeat step 2 to plate the cell suspension again, and count the number of colonies in step 4 after 2 weeks. Repeat the self-renewal experiment 4 times to obtain the value-added curve (see Figure 4). It can be seen from the figure that the single-cell clonal colony obtained by this culture method has the ability of self-renewal.

5. Analysis of stemness-related genes of pancreatic stem cells: The pancreas goes through several stages of development: "specialization-pancreatic progenitor cells-pancreatic endocrine precursor cells-endocrine cells". Each stage expresses multiple specific transcription factors. Among them, Pdx1 is a specific transcription factor of pancreatic progenitor cells, and the progenitor cells expressing Pdx1 will differentiate into all the constituent cells of the pancreas during development. In addition, Ngn3 is a specific transcription factor of pancreatic endocrine precursor cells. During development, endocrine precursor cells expressing Ngn3 finally differentiate into various mature endocrine cells under the action of different specialized signals. Therefore, Pdx1 and Ngn3 are important markers of pancreatic adult stem cells and differentiation into endocrine precursor cells, respectively. In addition, transcription factors such as Onecut1 and Pax4 can also serve as auxiliary markers of pancreatic adult stem cells. In particular, Ptfla is a pancreatic exocrine cell marker. Ck7, Sox9, and CD133 are markers of pancreatic ductal cells, and ductal cells expressing the latter two have been reported to have characteristics of pancreatic adult stem cells. The gene analysis method is as follows: after two weeks of culture, suck out the condensed matrigel that wraps the colony, place it in Cell revcovery solution for 2 hours on ice at 4°C to dissolve the matrigel around the colony, and release the single cells that have not grown into a colony. Centrifuge at 300g for 5 minutes to discard the supernatant, and add Trizol to dissolve the lower colony precipitate. RNA was extracted according to the Trizol method, and qPCR was performed after reverse transcription of cDNA. The results of gene analysis are shown in Figure 5. In addition, the colony cultured for 2 weeks was isolated under the microscope and the RNA of a single colony was extracted using an RNA extraction kit as an object, and the expression of pancreas-related genes was also analyzed by qPCR after reverse-transcribing the cDNA (see Figure 6). As shown in Figure 5 and Figure 6, compared with freshly extracted primary cells, the cultured colonies were enriched in cells expressing genes related to pancreatic progenitor cells and pancreatic endocrine precursor cells, and duct-related genes were also significantly increased, but Mature endocrine and exocrine cells were not enriched. It shows that the clonal colony obtained by this culture method exhibits the characteristics of pancreatic progenitor cells and differentiates into pancreatic endocrine precursor cells to a certain extent. At the same time, the high expression of ductal genes is also consistent with the reported that pancreatic adult stem cells are derived from ductal cells.

6. Immunofluorescence analysis: Colonies cultured for 2 weeks were isolated separately and then fixed in 4% paraformaldehyde overnight at 4°C. Then block overnight at 4°C in Blocking buffer containing 5% donkey serum and 1% triton. Incubate overnight at 4°C with 1 antibody, wash with PBST three times, and incubate with fluorescent secondary antibody at 37°C for 1 hour. If DAPI nuclear staining is used, add 50 μg/mL DAPI and incubate for 5 minutes, wash with PBST 3 times, and use a fluorescence microscope to observe the protein expression. As shown in Figure 7, laser confocal photography shows that there are Pdx1 and Ngn3 positive cells in cystic colonies, which proves that there are differentiations similar to pancreatic progenitor cells and pancreatic endocrine precursor cells in the colonies grown from single cell clones, indicating that the generation of this The initial cells of the colony are a subpopulation of cells with certain stem cell characteristics, which can self-proliferate during culture and have the ability to differentiate into endocrine precursor cells.

Example 2: Three-dimensional semi-solid culture of breast cancer stem cells

Main reagents: MCF-7 cells (purchased from ATCC). Other reagents are the same as in Example 1.

1. Preparation of single cell suspension: Adhesively cultivated to 80% confluent MCF-7 breast cancer cell line in a culture flask, after trypsin hydrolysis and digestion, centrifuge at 1200rpm for 5 minutes, use DMEM/F12 medium (containing 10% fetus bovine serum) to obtain a single cell suspension.

2. Medium preparation and plating: take 16mL DMEM/F12 medium, add 0.8mL fetal bovine serum, 0.3mL 20μg/mL bFGF (final concentration 200ng/mL), 0.03mL 20μg/mL EGF (final concentration 20ng/mL) mL), 0.4mL 100X B27 (final concentration 1X), 7mL 3.3% methylcellulose (final concentration 0.99%), 3mL Matrigel (final concentration 10%), and an appropriate amount of cell suspension, mixed well and inoculated into 24 wells board.

3. Clonally grown tumor stem cell spheres can be obtained by culturing in a 37° C., 5% carbon dioxide incubator for 2 weeks, as shown in FIG. 8 .

4. Calculation of colony formation rate: Count the number of spherical monoclonal colonies formed in each well, and calculate the colony formation rate based on the initial plated cells (3000). In this example, the colony formation rate of MCF-7 was about 5%.

Claims (5)

1. A method for culturing adult stem cells based on a three-dimensional semi-solid, characterized in that, using methylcellulose and Matrigel as a three-dimensional framework, in an ultra-low adhesion cell culture plate, and placed in a carbon dioxide constant temperature cell incubator for cultivation . 2. according to the described in vitro three-dimensional semi-solid culture method of claim 1, it is characterized in that, described methylcellulose concentration is 0.5% to 2%, preferred 0.99% (w/v), viscosity is 15cPs to 4000cPs, preferred 1500cPs . 3. The in vitro three-dimensional semi-solid culture method according to claim 1, characterized in that the Matrigel concentration is 5% to 20%, preferably 10% (w/w). 4. The in vitro three-dimensional semi-solid culture method according to claim 1, wherein the culture medium used is a DMEM/F12 medium containing fetal bovine serum. 5. The three-dimensional semi-solid culture method according to claim 1, characterized in that the carbon dioxide concentration is 5%, and the culture temperature is 37°C.