CN108715836B - Separation and bionic culture method of pericytes in tumor tissue - Google Patents

Abstract

The invention discloses a method for separating pericytes from tumor cells and performing biomimetic culture, and relates to the field of tumor biology. The method breaks through the previous method of only using two non-specific antibodies to mark the pericytes; the first time 10 antibody markers (5 positive markers, 5 negative markers) were isolated, purified and characterized for pericytes. And through morphological identification under a microscope, the pericytes can obviously form microtubules in the bionic matrix culture medium, which accords with the morphological characteristics of the pericytes, and the growth of the pericytes in vivo is reproduced, thereby laying a foundation for further functional research.

Description

Separation and bionic culture method of pericytes in tumor tissue

Technical Field

The invention relates to the field of tumor biology, in particular to a method for separating pericytes from tumor cells and performing biomimetic culture.

Background

Pericytes are structural cells that are widely distributed throughout the microvascular wall and, together with endothelial cells, form the barrier to the microvascular and interstitial space (1). The pericytes talk with the microvascular endothelial cells through cell connection or paracrine signals, and play an important role in regulating and controlling microcirculation blood flow, microvascular permeability, angiogenesis and the like. Furthermore, in tumor tissues, pericytes are functional cells that have been shown to mediate endothelial maturation and stability and reduce vascular leakage. Microvascular lesions of various diseases are accompanied by abnormal structures and functions of pericytes, and the regulation of the pericytes becomes a research hotspot. Based on their morphology, pericytes are often confused with vascular smooth muscle cells (vsmcs), fibroblasts, macrophages, and even epithelial cells. Although the same cell markers as vSMC are currently commonly used, there is no specific antibody marker that clearly identifies pericytes. The cell markers commonly used are largely unsuccessful in distinguishing pericytes from other vascular cells. Therefore, the methods of pericyte identification as a compromise are usually defined or described using a combination of criteria for location, morphology and gene/protein expression pattern. Several recent reviews provide a list of pericyte molecular markers. However, from a practical point of view, not all markers are useful (2). It is also important that no single complete pericyte specific marker is known and that all markers currently used are in their expression dynamics and may be down-regulated together with developmental status, pathological response, in vitro culture etc. (3). A negative marker of endothelial cells and a combination of two or more pericyte markers is therefore necessary. Table 1 provides a list of pericyte markers currently validated and frequently used. Since pericytes are similar in morphology to fibroblasts and therefore difficult to identify unambiguously, labeled antibodies currently used for human pericytes (table 1). However, none of these antibodies is specific for pericytes, and they are expressed in other cells (e.g., endothelial cells, mesenchymal stem cells, fibroblasts, etc.) (4-6), so that pericyte identification is a technical problem. In addition, pericytes are easily differentiated into fibroblasts in the culture process, and the problem of finding a suitable environment is also a problem to be solved urgently.

TABLE 1 murine pericyte markers

Reference documents:

1.Armulik A,GenovéG,Betsholtz C.Pericytes:developmental,physiological,and pathological perspectives,problems,and promises.[J].Developmental Cell,2011,21(2):193.

2.Ozerdem,U.,Grako,K.A.,Dahlin-Huppe,K.,et al.NG2proteoglycan isexpressed exclusively by mural cells duringvascularmorphogenesis.Dev.Dyn.2001,222,218–227.

3.Winkler F,Kozin S V,Tong R T,et al.Kinetics of vascularnormalization by VEGFR2 blockade governs brain tumor response to radiation:Role of oxygenation,angiopoietin-1,and matrix metalloproteinases[J].CancerCell,2004,6(6):553-63.

4.Nehls V,Drenckhahn D.The versatility of microvascular pericytes:from mesenchyme to smooth muscle？[J].Histochemistry,1993,99(1):1-12.

5.Geraldes P,Hiraokayamamoto J,Matsumoto M,et al.Activation of PKC-|[delta]|and SHP-1 by hyperglycemia causes vascular cell apoptosis anddiabetic retinopathy[J].Nature Medicine,2009,15(11):1298-1306.

6.Gerhardt H,Semb H.Pericytes:gatekeepers in tumour cell metastasis？[J].Journal of Molecular Medicine-jmm,2008,86(2):135-144.

disclosure of Invention

In order to overcome the defects that the conventional pericytes are not labeled by specific antibodies and are difficult to identify, the invention aims to provide a method for identifying and separating the pericytes in the tumor tissue by the antibody markers. The method is a method for simultaneously marking, identifying, separating and purifying pericytes based on multiple antibodies. In addition, if pericytes are cultured in a common culture bottle, the pericytes lose the growth forms under the normal environment and are differentiated into other cells (such as fibroblasts), and in order to simulate the growth environment of the pericytes in the culture process, the invention provides a coating bionic culture.

To study how pericytes promote vascularization of endothelial cells in tumor tissue, we first isolated pericytes with high purity from tumor tissue.

The purpose of the invention is realized by the following technical scheme:

a method for separating pericytes from tumor tissues and performing biomimetic culture, wherein all separation operations are performed in a super clean bench, and comprises the following steps:

(1) taking a fresh tumor tissue sample, washing the tumor tissue sample by using normal saline until the color of the tumor tissue sample is free from blood, and removing coagulated blood;

(2) cutting the tissue into pieces;

(3) adding an enzymolysis solution into the sheared tissue, then crushing the tissue, adding DMEM after enzymolysis is finished to complete culture and terminate the reaction, wherein the enzymolysis solution comprises a DMEM culture medium, 1-2.5% (g/100m L) of collagenase Type I, 1-2.5% of collagenase Type III, 1-2.5% of collagenase Type IV and 0.5-1.5% of DNase, the set temperature is 37 ℃, and the enzymolysis time is 0.5-2 hours;

(4) centrifuging after enzymolysis, sucking upper layer adipose tissue, adding physiological saline into residual liquid for resuspending, standing, and primarily filtering with a cell filter screen; the aperture of the cell filter screen is 150-200 mu m; preferably 200 μm;

(5) centrifuging, discarding the supernatant, and adding physiological saline for resuspension; cell screen wetted with normal saline, filtering the cells; the aperture of the cell screen is 70-100 mu m; preferably 100 μm;

(6) filtering the filtered cells by using a cell screen; removing the filtrate, and removing the unfiltered cells on the upper layer of the screen; the aperture of the cell screen is 40 μm;

(7) centrifuging and removing the supernatant; adding PBS buffer solution to resuspend the cells and counting the cells;

(8) the pericytes and the endothelial cells are marked and identified by flow sorting, 10 antibodies (positive markers comprise CD13, CD140b, CD146, NG2 and alpha SMA; negative markers comprise CD31, CD34, CD11b, CD140a and CD45) are respectively selected as positive and negative identifications of the pericytes, 3 antibodies are taken as identification antibodies of the endothelial cells (positive markers comprise CD31 and CD 34; negative markers comprise CD45), and each reaction system 5 × 10⁵(ii) individual cells; adding the antibody and the DAPI dye in a dark place, then, uniformly mixing by vortex, and carrying out a dark reaction at 4 ℃ for 30-60 minutes (preferably 60 minutes); flicking the tube wall every 15 minutes to promote complete reaction; adding PBS buffer solution to wash the incubated sample twice, and centrifuging; removing supernatant, adding PBS buffer solution, and performing flow sorting;

(9) taking preparation of 12m L culture coating solution as an example, adding 90-120 mu L30 mu g/m L collagen solution and 20-40 mu L fibronectin into 11.84-11.89 m L0.1.1% gelatin solution, slightly shaking to pave the culture plate, placing the culture plate with the coating in a 37 ℃ incubator for culturing for 30-60 min, then placing the culture plate into a 4 ℃ incubator for solidification for more than 16 hours, immediately sucking out excessive solution before inoculating cells, and simply washing with a small amount of culture medium to neutralize acidity;

(10) adding the pericytes after flow sorting into a pericyte culture medium PM, placing the pericytes into a culture plate with a coating, and culturing at 37 ℃; note: the culture medium is not changed;

(11) after the culture, the cells were determined to be pericytes by observation.

The fresh tumor tissue sample in the step (1) is a sample which is stored in physiological saline containing 1% of double antibody (v/v) of cyan chain and 0.2% of heparin (v/v) and is stored in an ice box within 4 hours;

preferably, the enzymolysis solution in the step (3) comprises a DMEM culture medium, 1% (g/100m L) of collagenase Type I, 1% of collagenase Type III, 1% of collagenase Type IV and 1% of DNase, and the enzymolysis time is 1-2 hours;

the centrifugation condition in the step (4) is 500g, and the centrifugation is carried out for 5 minutes at 20 ℃;

the centrifugation condition in the step (5) is 500g, and the centrifugation is carried out for 5 minutes at 20 ℃;

the centrifugation condition in the step (7) is 350g, and the centrifugation is carried out for 5 minutes at 20 ℃;

the centrifugation condition in the step (8) is 1200rpm centrifugation for 4 min;

the 0.1% gelatin solution described in step (9) was prepared by adding Milli-Q water to 0.4g of pig gelatin to 400m L, sterilizing for 30min, cooling to room temperature and storing at 4 ℃.

Preferably, in the step (9), for example, to prepare 12m L culture coating solution, 90 μ L30 μ g/m L collagen solution and 30 μ L fibronectin are added into 11.88m L0.1.1% gelatin solution, and gently shaken to flatten;

the method successfully separates out high-purity pericytes from the tumor tissues and identifies the pericytes. And the growth of the pericytes in the body is reproduced by a bionic culture mode.

Compared with the prior art, the invention has the following advantages and effects:

the pericytes are difficult to identify because of no specific antibody markers. In addition, the content of the compound in the tissues is low (0.5-3%), and if the compound cannot be separated and purified, the compound is difficult to survive in the culture process. The method breaks through the previous method of only using two non-specific antibodies to mark the pericytes; the first time 10 antibody markers (5 positive markers, 5 negative markers) were isolated, purified and characterized for pericytes. And the shape identification (figure 1) under a microscope shows that the pericytes can form microtubules in the bionic matrix culture medium, which accords with the shape characteristics of the pericytes, and lays a foundation for further functional research.

Drawings

FIG. 1 is a microscopic observation of pericytes in tumor tissue.

FIG. 2 shows the flow analysis results of the separation and purification of tumor tissues, wherein ① NG2, ② CD31, ③ CD45, ④ CD140b, ⑤ CD146 and ⑥ alpha SMA.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental procedures, in which specific conditions are not specified, in the following examples are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Example 1

The invention relates to a method for separating, purifying and culturing pericytes in tumor tissues by using a novel method, wherein all separation operations are carried out in a super clean bench, and the method sequentially comprises the following steps:

1. fresh tumor tissue samples were stored in saline containing 1% diabody (v/v, penicillin and streptomycin) and 0.2% heparin (v/v), stored in ice boxes and subjected to enzymatic separation within 4 hours.

2. The tissue was placed in a 100mm petri dish, washed with physiological saline until there was no blood color, and coagulated blood was removed with forceps.

3. The tissue was minced with sterilized scissors. Note: the more broken the shears are, the better; cells can be destroyed if a homogenizer and a blade cannot be used; and (4) operating on ice.

4. The enzymolysis liquid contains DMEM culture medium, 1% collagenase Type I, 1% collagenase Type III, 1% collagenase Type IV (Worthington) and 1% DNA enzyme (Roche, 100 ×), and DMEM is added after enzymolysis is finished to complete culture and stop the reaction.

5. Adding 8m L enzymolysis solution slowly into the cut tissue with a sterilized pipette, and washing the residual tissue residue with 2m L. transferring the tissue added with the enzymolysis solution into a gentlem MACS C Tubes (Miltenyl), wherein the mixed solution of the enzymolysis solution should not exceed 10m L in the C Tubes, if the mixed solution exceeds, the tissue should be divided into two Tubes, and the tissue is smashed in a gentlem ACS separator (Miltenyl) by selecting h _ knob _01.01C Tubes for 30 seconds.

6. Placing the smashed tissue in a molecular hybridization chamber (UVP, HB-1000) and setting the temperature at 37 ℃; the enzymolysis time is about 1 to 2 hours, and the whole tissue (including fibrous tissue, fat tissue and the like) is evenly rotated and enzymolyzed. Note: every 15 minutes, the tissue was disrupted by placing it in a GentleMeS Disociator. If the turbidity is not achieved, the time for mixing is prolonged.

7. Eppendorf centrifuge 500g, 5 minutes, 20 degrees C centrifugation, pipet to remove the upper adipose tissue, the remaining 5m L liquid plus physiological saline heavy suspension, static 5 minutes, with 200 u m cell filter screen for primary filter.

8. Eppendorf centrifuge 500g, 5 minutes, 20 degrees C centrifugal, discard the supernatant to about 2m L, add physiological saline to about 50m L heavy suspension, with physiological saline wet cell screen (100 m), filter cells.

9. The filtered cells were then filtered through a cell screen (40 μm). The filtrate was removed and the unfiltered cells (pericytes) in the upper layer of the screen were removed.

10. 350g, centrifuged at 20 ℃ for 5 minutes, the supernatant removed, 5m L PBS added to resuspend the cells, and the cells counted.

11. We used flow sorting to mark and identify pericytes and endothelial cells, we selected 10 antibodies as positive and negative identification of pericytes, 3 antibodies as identification of endothelial cells, 5 × 10 per reaction system⁵Individual cells, 50 μ L, 5 μ L antibody per singly stained cell.

1) Blank group;

2) pericyte PE anti-human CD31 (cat No. 303106, Biolegend);

3) pericyte PE anti-human CD11b (cat No. 101208, Biolegend);

4) pericyte PE anti-human CD140a (cat No. 303506, Biolegend);

5) pericyte PE anti-human CD45 (cat No. 368510, Biolegend);

6) pericyte Brilliant Violet 510^TManti-human CD34 (cat # 343528, Biolegend);

7) pericyte alpha SMA-Cy3 (cat # C6198, Sigma).

8) Pericyte APC anti-human CD140b (PDGFR β, cat No. 323608, Biolegend);

9) pericyte APC anti-human CD146 (cat # 556002, BD);

10) pericyte APC anti-human CD13 (cat No. 301706, Biolegend);

11) pericyte FITC-NG2 (cat #: AB5324A, Millipore);

12) pericyte mixed staining (positive markers include: CD13, CD140b, CD146, NG2, alpha SMA; negative markers include: CD31, endothelial cell antibody; CD34, endothelial cell antibody; CD11b, lymphocyte antibody; CD140a, fibroblast antibody; CD45, lymphocyte antibody);

13) endothelial cell co-staining (positive markers include: CD31, CD 34; negative markers include: CD45)

Adding the antibody and the DAPI dye in a dark place, then vortexing and mixing uniformly, reacting for 60 minutes in a dark place at 4 ℃, flicking the tube wall every 15 minutes to promote complete reaction, washing the incubated sample twice by adding 1m L PBS, centrifuging for × 4 minutes at 1200rpm, discarding the supernatant, and adding the PBS for flow sorting.

12. The pericytes of the structure analyzed by a flow analyzer (model BD FACSVerse, three laser 8 colors) account for about 3% of the whole tumor tissue; endothelial cells account for around 7%.

13. Preparation of pericyte coating:

(1) 0.1% (w/V) gelatin solution is prepared by adding 0.4G of pig gelatin (product number V900863-100G, SigmaVETEC) into Milli-Q water to 400m L, sterilizing for 30min, cooling to room temperature, and storing at 4 deg.C.

(2) Culture coating solution taking preparation of 12m L coating as an example, 90 μ L30 μ g/m L collagen solution (cat # C3867-1V L, Sigma) and 30 μ L fibronectin (cat # FC010, Millipore) were added to 11.88m L0.1.1% gelatin solution, gently shaken to plate 6 well plates with 1m L per well, and 24 well plates with 0.5m L per well, the coated plates were placed in a 37 ℃ incubator to culture for 30-60 min and then frozen in a 4 ℃ freezer for 16 hours or more, excess solution was immediately aspirated before cell inoculation, and the acidity was neutralized by simple rinsing with a small amount of medium.

14. Pericytes after flow sorting were added with pericyte medium PM (cat 1201, seiressel science cell). The cells were placed in coated 6-well plates and cultured at 37 ℃ for 7 days. Note: no change of medium was required during the culture.

15. After 7 days of culture, the cells were confirmed to be pericytes by microscopic observation (see FIG. 1), and biomimetic culture of the pericytes was achieved.

16. The results of flow analysis of tumor tissue by separation and purification are shown in FIG. 2, and it is found that the pericytes account for 3% of the total cells and have a purity of 80%.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (8)

1. A method for separating pericytes and performing biomimetic culture in tumor tissues is characterized by comprising the following steps: all separation operations are carried out in a clean bench, comprising the following steps:

(1) taking a fresh tumor tissue sample, washing the tumor tissue sample by using normal saline until the color of the tumor tissue sample is free from blood, and removing coagulated blood; the fresh tumor tissue sample is stored in physiological saline containing 1% of double antibody and 0.2% of heparin, and the sample is stored in an ice box within 4 hours;

(2) cutting the tissue into pieces;

(3) adding the enzymolysis liquid into the sheared tissue, then smashing the tissue, adding DMEM after enzymolysis is finished, and terminating the reaction; the enzymolysis solution comprises a DMEM culture medium, 1% of collagenase Type I, 1% of collagenase Type III, 1% of collagenase Type IV and 1% of DNA enzyme; setting the temperature to 37 ℃; the enzymolysis time is 0.5-2 hours;

(4) centrifuging after enzymolysis, sucking upper layer adipose tissue, adding physiological saline into residual liquid for resuspending, standing, and primarily filtering with a cell filter screen; the aperture of the cell filter screen is 150-200 mu m;

(5) centrifuging, discarding the supernatant, and adding physiological saline for resuspension; cell screen wetted with normal saline, filtering the cells; the aperture of the cell screen is 70-100 mu m;

(6) filtering the filtered cells by using a cell screen; removing the filtrate, and removing the unfiltered cells on the upper layer of the screen; the aperture of the cell screen is 40 μm;

(7) centrifuging and removing the supernatant; adding PBS buffer solution to resuspend the cells and counting the cells;

(8) labeling and identifying pericytes and endothelial cells by flow sorting, selecting 10 antibodies as positive and negative identification antibodies of pericytes respectively, 3 antibodies as identification antibodies of endothelial cells, and each reaction system 5 × 10⁵(ii) individual cells; adding the antibody and the DAPI dye in a dark place, then, uniformly mixing by vortex, and carrying out a dark reaction at 4 ℃ for 30-60 minutes; flicking the tube wall every 15 minutes to promote complete reaction; adding PBS buffer solution to wash the incubated sample twice, and centrifuging; removing supernatant, adding PBS buffer solution, and performing flow sorting;

the 10 antibodies were antibodies identifying the positive markers CD13, CD140b, CD146, NG2 and alpha SMA, and the negative markers CD31, CD34, CD11b, CD140a and CD45, respectively;

the 3 antibodies are antibodies for identifying positive markers CD31 and CD34 and a negative marker CD45 respectively;

(9) adding 90-120 mu L30 mu g/m L collagen solution and 20-40 mu L fibronectin into 11.84-11.89 m L0.1.1% gelatin solution to prepare 12m L culture coating solution, slightly shaking to pave the culture plate, culturing the coated culture plate in a 37 ℃ incubator for 30-60 min, then placing the culture plate into a 4 ℃ incubator to coagulate for more than 16 hours, immediately sucking out excessive solution before inoculating cells, and simply washing with a small amount of culture medium to neutralize acidity;

(10) adding the pericytes after flow sorting into a pericyte culture medium PM, placing the pericytes into a culture plate with a coating, and culturing at 37 ℃; the culture medium is not changed;

(11) after the culture, the cells were determined to be pericytes by observation.

2. The method of claim 1, wherein the method comprises:

and (4) carrying out enzymolysis for 1-2 hours.

3. The method of claim 1, wherein the method comprises: the aperture of the cell strainer in the step (4) is 200 μm.

4. The method of claim 1, wherein the method comprises: the aperture of the cell screen in the step (5) is 100 μm.

5. The method of claim 1, wherein the method comprises:

the centrifugation condition in the step (4) is 500g, and the centrifugation is carried out for 5 minutes at 20 ℃;

the centrifugation in step (5) was carried out at 500g for 5 minutes at 20 ℃.

6. The method of claim 1, wherein the method comprises:

the centrifugation condition in the step (7) is 350g, and the centrifugation is carried out for 5 minutes at 20 ℃;

the centrifugation in the step (8) is carried out for 4min at 1200 rpm.

7. The method of claim 1, wherein the method comprises:

in step (9), 90. mu. L30 collagen solution at 30. mu.g/m L and 30. mu. L fibronectin were added to 11.88m L0.1.1% gelatin solution, and gently shaken to spread the plates.

8. The method of claim 1, wherein the method comprises:

the 0.1% gelatin solution described in step (9) was prepared by adding Milli-Q water to 0.4g of pig gelatin to 400m L, sterilizing for 30min, cooling to room temperature and storing at 4 ℃.

Images