CN101812428A - Method for culturing tissue blocks of mammary epithelial cells - Google Patents

Abstract

The invention discloses a tissue block planting method of mammary gland epithelial cells, which is characterized in that it comprises the following steps: 1) pretreatment: covering the culture dish with serum, and standing still; 2) planting: planting mammary gland in the culture dish Tissue block, mammary gland epithelial cells and fibroblasts are collected; 3) purification: fibroblasts are removed to obtain mammary gland epithelial cells. The above method omits the cumbersome steps of preparing collagen in the traditional method, obtains a higher rate of tissue block adhesion, and finally obtains mammary gland epithelial cells with good shape and a large number.

Description

Method for planting tissue blocks of breast epithelial cells

technical field

The invention relates to a tissue block planting method of mammary gland epithelial cells.

Background technique

The mammary gland is one of the few organs in mammals that can repeatedly undergo the process of growth, functional differentiation and degeneration. Since foreign genes can be expressed in the mammary gland and then flow out with milk, mammary epithelial cells become important target cells in transgenic research. The in vitro culture of mammary epithelial cells is of great significance for studying the growth and development of mammary glands and elucidating the molecular regulation mechanism of lactation. It also provides an effective model for transgenic research.

To culture mammary gland epithelial cells in vitro, a certain amount of primary mammary gland epithelial cells must be obtained first. Currently the most widely used method is the tissue block planting method. The tissue block planting method needs to plant the tissue block into a culture dish, and cells will gradually migrate out 4-8 days after attachment. If planted directly, most of the tissue pieces will float after adding culture medium, which is not conducive to cell migration. The traditional method is to lay a layer of collagen on the bottom of the dish in advance to make the tissue block adhere to the bottom of the dish and promote the adhesion of the tissue block.

At present, homemade rat tail collagen is generally used in China. The preparation process is very cumbersome, and it needs to go through many steps such as soaking in ethanol, pickling, 4 overnight and autoclaving. For laboratories that have long been engaged in the study of lactation mechanism and transgenics, it will be very beneficial if they can find a simple, efficient and easy-to-obtain substance to replace collagen.

Contents of the invention

In order to solve the above technical problems, the present invention provides a method for planting mammary gland epithelial cell tissue blocks.

The present invention provides a method for planting tissue blocks of breast epithelial cells, comprising the steps of:

1) Pretreatment: Cover the culture dish with serum and let stand. The above-mentioned serum is preferably fetal bovine serum (FBS), and the static condition is: room temperature, 30min.

2) Planting: plant mammary gland tissue blocks, preferably Xinong Saanen sheep mammary gland acinar tissue blocks, in the culture dish, and collect mammary gland epithelial cells and fibroblasts. The specific steps of planting are:

a. Clean the breast tissue and cut it into pieces;

b. Inoculate the tissue block in the culture dish and place it in the incubator for 20-30 minutes;

c. Add the culture medium to the culture dish, add the culture medium after 1-2 hours, and culture it in a 37°C, 5% CO ₂ , saturated humidity incubator for two days, replace the culture medium on the third day, and then The culture medium is changed every two to three days to obtain mammary gland epithelial cells and fibroblasts. The above-mentioned culture solution is a D-MEM/F-12 medium containing the following components: 10% by volume of FBS, 5 mg/L bovine insulin, 5 mg/L hydrocortisone, 5 mg/L epidermal growth factor and 100,000 IU /L penicillin and streptomycin double antibody.

3) Purification: removing fibroblasts to obtain mammary gland epithelial cells. The specific steps of purification are:

a. Add digestive fluid to the mixture of epithelial cells and fibroblasts, digest at 37°C, stop digestion when the fibroblasts are detached from the bottle wall, suck out the digestive juice, then add appropriate amount of digestive juice, continue digestion at 37°C for 5-6 minutes After adding the culture medium, the pipette blows the suspended cells, and the cells are collected after centrifugation;

b. Add the culture solution to the cells collected in step a, transfer to the culture dish, after the fibroblasts adhere to the wall, suck out the culture solution, replace the cells in the culture dish to a new culture dish, and then Adding the culture solution, and doing this 3-4 times, mammary gland epithelial cells can be obtained.

As preferably, the above-mentioned digestive juice is an aqueous solution containing the following concentration components: trypsin 0.5g/L, NaCl 8g/L, KCl 0.4g/L, glucose 1g/L, NaHCO30.58g/L, EDTA 0.2g/L, phenol Red 0.002g/L and penicillin and streptomycin double antibody 100,000 IU/L.

The beneficial effect of the present invention is that: it provides an optimal plan for planting breast tissue blocks, saves the cumbersome steps of preparing collagen in the traditional method, uses serum planting, obtains a higher rate of tissue block adhesion, and finally obtains a breast tissue block with good shape and quantity. More mammary epithelial cells.

Description of drawings

Figure 1. Primary cells in digestion.

Figure 2 is the purified mammary epithelial cells.

Figure 3 is the immunofluorescence identification of mammary epithelial cytokeratin.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

(1) Primary culture of breast epithelial cells

Surgical method The healthy purebred Xinong Saanen sheep mammary acinar tissue from the Saanen sheep breeding farm of Northwest Agriculture and Forestry University. Wash the breast tissue repeatedly with 3 times double antibody D-Hank's solution until the tissue turns white and the washing solution is clear. Separation and trimming in ultra-clean bench, peel off adipose tissue and connective tissue, and cut white granular acinar tissue into about 1mm3 size. Pretreat the culture dish with fetal bovine serum (FBS), then inoculate tissue pieces in the dish at intervals of about 0.5 cm, and place them in an incubator at 37°C, 5% CO ₂ , and saturated humidity for about 20 to 30 minutes to make the tissue pieces microscopic. Dry up, then gently add about 1mL of culture solution to the culture dish, put it in a _CO2 incubator for 1 to 2 hours and then add 1mL of culture solution, keep it in the incubator for two days, and replace the culture solution on the third day. Thereafter, the culture medium was changed every two to three days. Fibroblasts can be seen migrating out around the tissue block in 3-4 days, and mixed growth of mammary epithelial cells and fibroblasts can be seen around the 8th day.

The preparation method of the above culture solution is: (1) dissolving bovine insulin (Sigma, dry powder) in 0.01M HCl solution to obtain solution a; (2) dissolving hydrocortisone (Wolsen, dry powder) in absolute ethanol , to obtain solution b; (3) dissolve the epidermal growth factor in triple-distilled water to obtain solution c; (4) dissolve the penicillin-streptomycin double antibody in triple-distilled water to obtain solution d; (5) take a certain amount of FBS , solutions a, b, c, and d are dissolved in D-MEM/F-12 medium, so that the volume percentage of FBS in the above-mentioned D-MEM/F-12 medium is 10%, bovine insulin is 5mg/L, hydrocortisone Pine is 5mg/L, epidermal growth factor is 5mg/L and penicillin and streptomycin double antibody is 100,000 IU/L.

Screening for optimal pretreatment conditions:

According to the pretreatment conditions of the culture dish, different serum additions and storage time were set respectively, and then the tissue blocks were planted according to the conventional method. After 3 days, the adhesion rate of the tissue blocks in each plan was compared, and the mammary epithelial cells obtained by each treatment were observed after 10 days. shape and growth status.

The final pretreatment protocol was as follows: add 1mL serum (Tianjin Haoyang) to a 60mm dish (Corning, USA), shake gently to spread evenly, cover the bottom of the entire culture dish, then suck it out, and place it at room temperature for 30 minutes.

(2) Purification of mammary gland epithelial cells:

According to the different sensitivities of epithelial cells and fibroblasts to trypsin digestion, in mixed-growing primary or passage cell cultures, first add digestion solution (1×ATV digestion solution: trypsin 0.5g/L, NaCl 8g/L , KCl 0.4g/L, glucose 1g/L, NaHCO ₃ 0.58g/L, EDTA 0.2g/L, phenol red 0.002g/L and penicillin and streptomycin double-antibody, of which penicillin and streptomycin are 100,000 IU/L L.), digest at 37°C, observe under an inverted microscope, stop the digestion when the fibroblasts are detached from the bottle wall, and suck out the digestive juice, most of the discarded fibroblasts at this time. Then add an appropriate amount of digestive fluid, and continue to digest at 37°C for 5-6 minutes. After adding the above-mentioned culture fluid, blow the suspended cells with a pipette. Most of the cells collected after centrifugation are epithelial cells. After adding an appropriate amount of culture medium to the collected epithelial cells to resuspend the cells, transfer them to a new culture dish. Fibroblasts adhere to the wall quickly, and can adhere to the wall 30-40 minutes after digestion. Therefore, after 40 minutes, the cell culture solution is sucked out and replaced into a new culture dish, so that the purified mammary gland epithelium can be obtained after 3-4 passages. Cells, see Figure 2.

(3) Immunofluorescence identification of keratin in purified mammary epithelial cells

The immunofluorescence identification of keratin was performed on the purified mammary epithelial cells, as shown in Figure 3. For the specific method, refer to the instructions of the immunofluorescence kit of Wuhan Boster Bioengineering Co., Ltd. After the operation was completed, the nuclei were stained with Hoechst33342.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims (8)

1. a culturing tissue blocks of mammary epithelial cells method is characterized in that, comprises the steps:

1) pre-treatment:, leave standstill with serum confluent culture ware;

2) plantation: plantation mammary tissue piece in described culture dish, collect mammary epithelial cell and inoblast;

3) purifying: remove inoblast, obtain mammary epithelial cell.

2. tissue block kind method for planting according to claim 1 is characterized in that the described serum of step 1) is foetal calf serum.

3. tissue block kind method for planting according to claim 1 is characterized in that the described condition of leaving standstill of step 1) is: room temperature, 30min.

4. tissue block kind method for planting according to claim 1 is characterized in that step 2) described mammary tissue piece be Xi Nongsa can sheep mammar gland the acinar tissue piece.

5. tissue block kind method for planting according to claim 1 is characterized in that step 2) step of described plantation is:

A. clean mammary tissue, be cut to piece;

B. in described culture dish, inoculate tissue block, and place incubator, 20～30 minutes;

C. in culture dish, add nutrient solution, add nutrient solution after 1～2 hour again, at 37 ℃, 5%CO ₂, leave standstill in the saturated humidity incubator and cultivated two days, changed nutrient solution, changed a nutrient solution, and promptly got mammary epithelial cell and inoblast in later per two to three days in the 3rd day.

6. tissue block kind method for planting according to claim 5, it is characterized in that described nutrient solution is the D-MEM/F-12 substratum that contains following concentration composition: the mycillin of the FBS of volume percent 10%, 5mg/L Sigma I8405,5mg/L hydrocortisone, 5mg/L Urogastron and 100,000 IU/L is two anti-.

7. tissue block kind method for planting according to claim 1 is characterized in that the step of the described purifying of step 3) is:

A. in epithelial cell and inoblast mixture, add Digestive system, 37 ℃ of digestion, when treating that inoblast breaks away from the bottle wall, stop digestion, with the Digestive system sucking-off, add an amount of Digestive system again, 37 ℃ are continued digestion 5～6min, add suction pipe piping and druming suspension cell behind the described nutrient solution, centrifugal back collecting cell;

B. in the cell that step a collects, add described nutrient solution, be transferred in the culture dish, after inoblast is adherent, with described nutrient solution sucking-off, cell in the culture dish is changed to a new culture dish, add described nutrient solution again, so, can obtain mammary epithelial cell through 3～4 times.

8. tissue block kind method for planting according to claim 7, it is characterized in that, described Digestive system is the aqueous solution that contains the composition of following concentration: trypsinase 0.5g/L, NaCl 8g/L, KCl 0.4g/L, glucose 1g/L, NaHCO3 0.58g/L, EDTA 0.2g/L, two anti-100,000 IU/L of phenol red 0.002g/L and mycillin.

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