Rat brain microvascular endothelial cell primary culture experiment

Summary

Primary culture of rat brain microvascular endothelial cells can be used for: (1) research on blood-brain barrier; (2) pathophysiological and molecular biological research on cerebrovascular diseases; (3) screening of new drugs; (4) physiological, biochemical and pharmacological research on brain microvascular endothelial cells.

Operation method

enzymatic digestion

Principle

Using cerebral cortex as material, enzyme digestion and gradient centrifugation to isolate cerebral microvascular segments and primary culture, we have successfully figured out the method of isolation and primary culture of rat cerebral microvascular endothelial cells, and obtained cerebral microvascular endothelial cells with high purity.

Materials and Instruments

SD rat
Fibronectin Type IV collagen Gelatin Sodium heparin Fibroblast growth factor Penicillin Streptomycin NaHCO3 Bovine serum albumin Rhamnosus Gum Dextran DNAase I D-Hanks' liquid Type II collagenase
Cryo-centrifuge Centrifuge tubes Pipette guns

Move

I. Preparation of experimental materials
1. Laboratory animals
For each experiment, 10 SD rats of 2-3 weeks of age, both male and female, weighing 40-60 g were used.

2. Reagents
Fibronectin, type IV collagen, rat tail gum, dextran (molecular weight 100-200 kDa) and DNase I were purchased from Sigma; D-Hanks' liquid and 10xPBS were prepared according to the formula; type II collagenase was purchased from Worthington; gelatin, bovine serum albumin (BSA) and HEPES were purchased from A&P. (BSA) and HEPES were purchased from Amresco; Percoll was purchased from Amersham Biosciences; DMEM (high sugar) was purchased from GIBCO/BRL; sodium heparin and NaHCO3 were purchased from Shanghai Chemical Reagent Company of China Pharmaceutical Group; penicillin and streptomycin were purchased from Shanghai Sangong Biological Engineering Co. Fetal bovine serum (FBS) was purchased from PAA Laboratories; basic fibroblast growth factor (bFGF), collagenase/dispase were purchased from Roche.
3. Instruments
Cryogenic centrifuge (Centrifuge 5810 R, purchased from Eppendorf; Himac CR 22F, purchased from Hitachi).
4. Reagent preparation
(1) 1 mg/ml rat tail gum (prepared with 0.2% acetic acid), 1% gelatin (prepared with D-Hanks' solution), 1% type II collagenase (prepared with DMEM, diluted to a concentration of 0.1% when used), 1% collagenase/dispersase (prepared with DMEM, diluted to a concentration of 0.1% when used), 15% dextran (prepared with PBS), 20% BSA (prepared with DMEM (prepared with DMEM, adjust the pH to 7.4 and then filtered with 0.45 um filter membrane for sterilization, which is more difficult to filter), DNase I (prepared with cold PBS to 2 U/ul), the above reagents were filtered and sterilized by 0.22 μm filter membrane and then stored at -20℃ in separate packages.

(2) 50% Percoll (prepared 12 ml, 6 ml Percoll stock solution, 0.67 ml 10xPBS, 0.4 ml FBS, 4.93 ml PBS were required); DMEM culture solution (containing 4000 mg/L D-glucose, 4 mmol/L L-glutamine, added 3.7 g/L NaHCO3, 20 mmol/L HEPES, 100 mg/L sodium heparin, 100 U/ml penicillin, and 100 ug/ml streptomycin), pH adjusted to 7.4, filtered to remove bacteria and stored in portions at 4 ℃, with 20% FBS and 1 ng/ml bFGF added at the time of use.
Pretreatment of Petri dishes
1. Coat with 3 different substrates, add 1 ml of 1% gelatin to 35 mm plastic petri dishes one day before culture, place them in the incubator at 37 ℃ overnight, and rinse them with D-Hanks' solution twice before inoculation. 2.

2. 4 h before inoculation, add 6~10 ^ug/cm2 of rat-tail gum, fumigate with ammonia for 5~10 min in a closed vessel, put it at room temperature for 1~2 h to dry, and then rinse with D-Hanks' solution for 2 times.

3. 50 ul 0.1% fibronectin, 50 ul 1 mg/ml type IV collagen and 400 ul double-distilled water were mixed, added to each petri dish and coated uniformly and then sucked out, which could be coated with 10 petri dishes, and then placed in the incubator at 37 ℃ for 1~2 h to dry, and then rinsed with D-Hanks' solution for 2 times.
Isolation of cerebral microvascular segments and primary culture of cerebral microvascular endothelial cells
1. The rat was dislocated from the cervical vertebrae and sterilized in 75% ethanol for 3~5 min, then the severed head was placed in a glass petri dish, and the whole brain was taken out and placed in a glass petri dish with cold D-Hanks' liquid for dissecting and removing the cerebellum and the mesencephalon (including the hippocampus).

2. Subsequently, the cerebral hemispheres were slowly rolled on dry filter paper to remove the soft meninges and large meningeal blood vessels, and then placed in a new glass Petri dish containing cold D-Hanks' solution, and the white matter of the cerebrum, the residual large blood vessels and the soft meninges were removed with fine dissecting forceps, while the cerebral cortex was retained.

3. After rinsing with D-Hanks' solution for 3 times, 1 ml of DMEM culture medium was added, which was cut into 1 mm³ size with iris scissors, and 0.1% type II collagenase (containing 30 U/ml DNase I, 1 ml/cerebrum) was added and mixed well, and then digested in 37 ℃ water bath for 1.5 hours.

4. Centrifuge (1 000 rpm, 8 min, room temperature), remove the supernatant, add 20% BSA suspension mixing and centrifugation (1000 g, 20 min, 4 ℃) or add 15% dextran suspension mixing and centrifugation (4 000 rpm, 20 min, 4 ℃), to remove the upper and middle layers of neural tissue and large blood vessels, and retain the bottom precipitate.

5. Add 2 ml 0.1% collagenase/dispersase (containing 20 U/ml DNase I), suspend and mix well, then digest for 1 h at 37 ℃ in a water bath, centrifuge (1 000 rpm, 8 min, room temperature), remove the supernatant, and then add 2 ml of DMEM culture medium to suspend and spread on 12 ml of 50% Percoll (25 000 g, 60 min, 4 ℃), which had formed a continuous gradient after centrifugation. ).

6. Centrifuge (1 000 g, 10 min, 4 ℃), near the bottom of the erythrocyte layer above the white-yellow layer that is the purified microvascular segments, sucked out and rinsed twice with DMEM (centrifugation 1 000 rpm, 5 min, room temperature), to remove the supernatant.

7. Add DMEM complete culture solution (containing 20% FBS, 100 μg/ml sodium heparin) after suspension and inoculation in 35 mm disposable plastic petri dishes coated with substrate (1.5 ml/petri dish, can be inoculated with 1 petri dish/brain), placed in 37 ℃, 5% CO2 incubator static incubation, 12~24 h after fluid exchange, and add 1 ng/ml bFGF, followed by fluid change every other day.Identification method
Morphology, immunohistochemical detection of factor VIII-related antigen.
V. Results
1. Cell morphology observation
At the time of inoculation, the brain microvascular segments composed of round endothelial cells were seen to be in the shape of single branch or branch, and scattered single cells and tissue fragments could be seen (see Figure 1-1); after 12~24 h of culture, the cultured cells could be seen to grow out from around the walled microvascular segments, and the cells were in the form of a short shuttle, with a regional monolayer growth, and the residual part of the microvascular segments was decreasing after the fluid change, with less content of fibroblasts, pericytes and other heterocytes; with the prolongation of culture time, the endothelial cells continued to multiply, and a "swirling" distribution could be seen. With the prolongation of culture time, the endothelial cells proliferated continuously, and the distribution of "vortex" could be seen, and the cells reached fusion in about 5-7 days, and the short shuttle-shaped endothelial cells accounted for more than 90% of the cells, but a small number of pericytes and other heterogeneous cells could be seen growing on the surface of the endothelial cell monolayer or in the interstitial space of the cell clones (results are not shown). The cell growth process is shown in Figure 1, and the cells were inoculated in fibronectin/type IV collagen-coated culture dishes.


2. Growth of cells coated with different matrices
Gelatin and rat tail gum coated culture dish microvessel segment adhering to the wall for a longer time, 6 h can see a small number of microvessel segment adhering to the wall but no endothelial cell growth, 12 ~ 24 h can see some endothelial cell growth, 24 h after the cerebral microvessel segment completely adhering to the wall and more endothelial cell growth, there is no obvious difference between the two (see Fig. 2-1-4); Fibronectin / type IV collagen coated culture dish culture after 6 h that is, the microvessel segment adhering to the wall and some endothelial cell growth, the growth process of cells is not visible. The microvessel segments were adherent to the wall and some endothelial cells grew out at 6 h, and they were basically completely adherent to the wall and more endothelial cells grew out at 12~24 h (see Fig. 2-5,6).


3. Immunohistochemical identification
Immunohistochemical detection of factor VIII-associated antigen showed brownish-yellow staining in the cytoplasm and perinuclear area of cultured brain microvascular endothelial cells, and the nuclei were vacuolated; control staining was negative.

Caveat

1. Maintain all tissue cells in sterile conditions from the time of sampling. Cell counting may be performed in a sterile environment.

2. In the ultra-clean table, tissue cells, culture solutions, etc. should not be exposed for too long to avoid evaporation of solutions.

3. where steps are operated outside the ultra-clean bench, each vessel needs to be covered with a lid or rubber stopper to prevent bacteria from falling in.

4. Wash your hands before operation, and wipe your hands with 75% alcohol or 0.2% Neosporin after entering the ultra-clean bench. The mouth of the reagent bottle should also be wiped.

5. Light the alcohol lamp, operate near the flame, heat-resistant items should always be burned on the flame. Metal instruments should not be cauterized for too long to avoid annealing and should be cooled before clamping the tissue. Appliances that have absorbed nutrient solution should not be cauterized again to avoid charring and forming a carbon film.

6. the operation should be precise and agile, but not too fast to prevent air flow, increasing the chance of contamination.

7. the working part of the sterilized utensils should not be touched by hand, and the arrangement of supplies on the workbench should be well laid out.

8. bottles should be kept in a 45° slanting position as far as possible after opening.

9. Pipettes, etc., for sucking up solutions should not be mixed.

Common Problems

I. Experimental discussion

We used two enzyme digestion, gradient centrifugation to obtain brain microvascular segments, explored different culture conditions, and successfully carried out primary culture of purer rat brain microvascular endothelial cells, the purity of endothelial cells can reach more than 90%, and the cell yield is higher, 10 animals can be cultured 8~10 35 mm disposable plastic Petri dishes, and the culture area is about 80~100 ^cm2, which is significantly improved compared with some domestic methods [3~5]. ^Compared with some domestic methods ^[3~5], the cell yield was significantly improved.

Because neonatal rats are prone to mix more heterogeneous cells and have smaller brains, and because lactating rats are preferred to rats older than 1 month ^[9], we used 2~3- 12 - Clove Garden Cytotechnology Discussion Board eCollection rat cerebral microvascular endothelial cell cultures of week-old lactating SD rats as culture materials. The key to primary culture of cerebral microvascular endothelial cells is to first isolate and obtain cerebral microvascular segments with high purity and good viability. Careful removal of the soft meninges, large blood vessels and cerebral white matter to collect the cerebral cortex during dissection can reduce the growth of fibroblasts and smooth muscle cells, which is important for improving the purity of endothelial cells. Since collagenase has less damage to endothelial cells, we used 0.1% type II collagenase to digest the dispersed tissue of sheared cerebral cortex. Compared with the tissue homogenization method ^[3,10], the enzyme digestion method avoids the damage to endothelial cells caused by tissue homogenization, which is conducive to the enhancement of cell viability. After collagenase digestion, 20% BSA or 15% dextran centrifugation can be used to separate the cerebral microvascular segments from the neural tissues and large blood vessels, and obtain the underlying cerebral microvascular segments, which has been widely used in the separation of cerebral microvascular segments ^{[5-9,11,12], and} we have found that the number of cerebral microvascular segments obtained by 20% BSA is higher than that obtained by 15% dextran and the cerebral microvascular segments are in a better condition. easier to grow adherently to the wall. Since pericytes are the most common stray cells in primary culture of brain microvascular endothelial cells, it will obviously inhibit the growth of endothelial cells ^[7], so the number of pericytes should be minimized in primary culture, we used two enzyme digestion method ^[7], controlled the time of two enzyme digestion, dispersed the pericytes at the periphery of the endothelial cells with 0.1% collagenase/digestive enzyme, and finally used a certain concentration of continuous gradient of Percoll separation to remove pericytes and erythrocytes to obtain purer brain microvascular segments. The addition of an appropriate amount of DNAase to the enzymatic digestion process can disperse the clumping of the microvascular segments caused by the DNA released during the digestion process, which is conducive to increasing the yield of microvascular segments. For Percoll centrifugation, we tried three concentrations (33%, 45% and 50%) and found that the higher the concentration, the farther the brain microvascular segments were from the erythrocytes and pericytes near the bottom of the centrifuge tube, and the better the separation effect was, therefore, the best result was obtained by using 50% Percoll, and it was best to use a low-temperature centrifuge with a horizontal rotary head to improve the centrifugation effect. Other methods of endothelial cell purification include mechanical scraping, clonal culture, FACS classification ^[13], Thy1.1 immunoreactive killing ^[7,14], use of plasma-derived fetal bovine serum ^{[8], and} magnetic bead incorporation ^{[10], etc}. However, we have found that the mechanical scraping method and the clonal culture method are not suitable for the purification of rat cerebral microvascular endothelial cells because the primary endothelial cells are not good and easily damaged by heterocytes after transmission. However, we found that mechanical scraping method and clonal culture method are not suitable for purification of rat brain microvascular endothelial cells, because the primary endothelial cells are in poor condition after transmigration and are easily inhibited by heterocytes [7], and the latter methods are more expensive and not recommended; plasma source of fetal bovine serum does not contain PDGF, and it does not promote heterocyte growth, but it is more expensive, so it can be chosen for primary culture.

Primary culture of brain microvascular endothelial cells requires a matrix coated with gelatin, rhamnogel, and fibronectin to facilitate the adherence of brain microvascular segments to the wall and the growth of endothelial cells. We tried different matrices and found that they had different effects on the walling of brain microvascular segments and the growth of endothelial cells. Fibronectin/type IV collagen was better than sage gelatin, while sage gelatin was better than gelatin, and the inoculation density requirement of the former was lower than that of the latter two, which needed to reach a certain density in order to be favorable for the walling of brain microvascular segments. In addition, we found that endothelial cells were less likely to grow on slides and more likely to grow on plastic petri dishes, which is basically consistent with the literature ^[8,14]. The growth of endothelial cells requires the addition of endothelial growth factor to promote the proliferation of endothelial cells and inhibit the growth of heterogeneous cells. bFGF can effectively promote the growth of endothelial cells, while the addition of 100 ug/ml heparin synergizes with the effect of bFGF and inhibits the growth of smooth muscle cells ^[9]. At the same time, in order to ensure the nutrition of endothelial cells and to remove the residual debris of cerebral microvascular segments, we used 20% FBS serum concentration and changed the fluid every other day.

The endothelial cells in our cultured brain microvessels were short spindle-shaped, with regional monolayer growth. With the prolongation of the culture time and the proliferation of endothelial cells, a "swirling" distribution could be seen, and after about 5-7 days, the regions could be gradually fused with each other, and the morphology and growth characteristics were similar to those reported in most of the literatures ^[6-9,11,12]. The morphology and growth characteristics were similar to those reported in most of the literature [6-9,11,12]. Based on the short spindle-shaped morphology of the endothelial cells and the positive expression of factor VIII-associated antigen, it was possible to identify the cells we cultured as cerebral microvascular endothelial cells ^[3,5,8].

The establishment of our rat brain microvascular endothelial cell culture model in vitro is a better tool for the study of the physiology, biochemistry and pharmacology of the brain endothelium, and at the same time can be co-cultured with astrocytes for the construction of the blood-brain barrier model ^[1]. It is believed that with the continuous improvement of technical methods, the model of cerebral microvascular endothelial cells cultured in vitro will be gradually close to their in vitro characteristics and be widely used in related research ^[1].

References

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[3] Wang JM et al. Journal of Anatomy, 1998, 21:495

[4] Xu YG et al. Journal of Microcirculation Technology, 1997, 2: 63

[5] Qian ZY et al. Journal of Cell Biology, 1999, 21: 42

[6] Kis B et al. Neuroreport, 2001, 12: 4139

[7] Szabo CA et al. Neurobiology (Bp), 1997, 5:1

[8] Abbott NJ et al. J Cell Sci, 1992, 103:23

[9] Gordon EL et al. In Vitro Cell Dev Biol, 1991, 27A:312

[10] Diglio CA et al. Lab Invest, 1982, 46:554

[11] Rupnick MA et al. In Vitro Cell Dev Biol, 1988, 24:435

[12] Ichikawa N et al. J Pharmacol Toxicol Methods, 1996, 36:45

[13] Scott PA et al. J Cell Sci, 1993, 105:269

[14] Domotor E et al. Neurochem Int, 1998, 33:473

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