MA Hua?gen, LIU Zhao?de, LIU Hai?qin, TANG Yuan?yu

1. College of TCM,Beijing University of Chinese Medicine,Beijing 102488,China

2. Basic Medical College,Nanjing Medical University,Nanjing 211166,China

3. College of Integrated Traditional Chinese and Western Medicine,Fujian University of TCM, Fuzhou 350122,China

4. College of TCM,Fujian University of TCM,Fuzhou 350122,China

Keywords:Rabbit Brain Microvascular endothelial cells Primary culture Morphologic observation Tube formation test

ABSTRACT Objective: To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells, provide important carriers and tool cells for the research of related cerebrovascular diseases. Methods: The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting, passing through cell sieve, bovine serum albumin density gradient centrifugation, typeⅡ collagenase digestion, finally inoculated and cultured. The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results: Under the inverted microscope, the cells were short fusiform or polygonal, and grew in clusters and adhere to the wall. After the cells were densely fused, they would be in a typical monolayer flat, “pebbled" mosaic arrangement. Tube formation test had the ability to form tubes structure. Conclusion: This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.

1. Introduction

Rabbits have a unique lipid metabolism system, which has a high absorption rate of exogenous cholesterol and a low ability to clear blood lipids. The results show that rabbits have great advantages in simulating various cerebrovascular diseases caused by human hyperlipidemia and atherosclerosis induced brain microvascular endothelial cells (BMECs) damage[1-2].Studies have shown that: the brain tissue is vigorously metabolized, and the demand for blood sugar and blood oxygen is high. The blood flow of patients with long?term hyperlipidemia and atherosclerosis is slow, and the blood oxygen saturation is low, which is easy to cause cerebral ischemia and hypoxia. Lipid peroxidation occurs in BMECs in small branches of cortical arterial vessels.It causes cells degeneration and necrosis, which is also the common pathological basis for the occurrence and development of various cerebrovascular diseases such as vascular dementia[3-5], ischemic stroke[6-7], and cerebral hemorrhage[8-9] and so on[10- 11]. Therefore, the successful isolation and culture of rabbit BMECs can provide a reasonable and practical in vitro cell biological model for the study of the pathogenesis of various cerebrovascular diseases caused by hyperlipidemia and atherosclerosis?induced BMECs damage.However, only Chen Zhi[12] and Xue Qingshan[13] have reported the primary culture method of rabbit BMECs in China, due to the long and tedious cultivation process, susceptibility to microbial contamination and high investment cost. Since then, the exploration and optimization of its primary culture methods in academia have basically stagnated.Therefore, after repeated practice and exploration, the research group expected to establish a more mature and perfect primary culture method of rabbit BMECs, which is reported as follows.

2. Materials and methods

2.1 Laboratory Animals

2 Japanese big?eared white rabbits, 7～10 days old, male or female,purchased from Penghushan Agricultural Development Co., Ltd.,Minhou County, Fuzhou City. The code number of the certificate of animal epidemic prevention conditions: 059111120160016.

2.2 Main Reagents

M199 Medium(1839543, Gibco), Fetal bovine serum(1275860,Gibco), L?glutamine(56?85?9, Leagene), 4?hydroxyethyl piperazine ethyl sulfonic acid(7365?45?9, Leagene), Heparin sodium(120904, Nanjing Xinbai Pharmaceutical), Bovine serum albumin(WXBC44547V, Vetec), Type II collagenase(461699,Beijing Dingguo Changsheng Biotechnology), 0.25% trypsin/0.02%EDTA mixed enzyme digestion solution(20170602, Nanjing KGI Biology), Matrix Matrigel(8302009, Corning).

2.3 Main instruments

Ultra?clean workbench(Suzhou Antai Airtech), Low speed centrifuge(Hunan Xiangyi), Inverted biomicroscope(Leica), CO2 incubator(Thermo).

2.4 Primary culture of BMECs

Two 7～10 day old rabbits were selected. After anesthesia and neck amputation, they were disinfected with iodophor for 3 minutes.The skull was cut off and taken out the whole brain under sterile conditions. Remove the pia mater and large blood vessels on the surface of brain stem, cerebellum and cortex, and separate the cerebral cortex on sterilized filter paper. After the cerebral cortex was rinsed 2～3 times with pre?cooled PBS, the cerebral cortex was cut with ophthalmic scissors and passed through a 75 μm cell mesh. The screen was rinsed with PBS solution, filtrate under the screen was collected, and centrifuged at a short time and low speed. The precipitation at the bottom was mixed with 20% bovine serum albumin density gradient centrifugation was performed. The supernatant was discarded, the precipitation was collected at the bottom of the centrifuge tube, 10 mL 0.1% type Ⅱ collagenase was added, and digested by shaking in water bath at 37℃ for 15～20 min. After centrifugal, the precipitation was mixed with 3 mL M199 complete medium (containing 20% fetal bovine serum, 4 mmol/L L?glutamine, 20 mmol/L HEPES, 100 mg/L heparin sodium,and 1000 IU/L insulin) and inoculated into an imported Nunc petri dish with a diameter of 6 cm. Culture in 37℃, 5% CO2constant temperature incubator. After 24 h, the medium was replaced in full,and half of the medium was changed every other day.

2.5 Subculture of BMECs

When the primary cells grew close to the fusion state, the old medium was discarded, and residual liquid was washed with PBS preheated at 37℃, then 4 mL 0.25% trypsin / 0.02% EDTA mixed enzyme solution preheated at 37℃ was added for digestion for 2～3 min. During this period, the bottom of the dish was slightly shaken to promote cell ablution. When it was observed under the microscope that most of the cells contracted and became rounded and began to peel off, add 4 ml of 37℃ preheat complete medium termination of digestion, the cells suspension out into a centrifuge tube and centrifugal supernatant on abandoned, add 6 ml fresh medium, the gentle blowing after blending, to extend the scale of 1∶2, incubate in a 37℃, 5% CO2incubator.

2.6 Morphological observation of BMECs

The morphology, adherence and fusion degree of the primary and first generation cells were observed and recorded under an inverted microscope.

2.7 Tube formation test

The 24-well culture plate was coated with 300 μL Matrigel gel per well, and then lay at 37℃ 45 min. The first?generation rabbit BMECs in the logarithmic growth phase were digested with 0.25% trypsin /0.02% EDTA mixed enzyme solution to prepare single?cell suspension,and the supernatant was centrifuged. The cell pellet was mixed with the culture medium containing 10% fetal bovine serum by pipetting and inoculated in a 24-well culture plate (500 μL per well, 2～3×104cells).2 h later, the formation of tubules was observed under 40× microscope,and then observed at intervals of 1 h.

3 Results

3.1 Isolation of cerebral microvascular segments in rabbits

The rabbit cerebral cortex excised by craniotomy was cut into pieces and sieved, and the cerebral microvascular segments obtained before and after digestion were "short stick" and "bead?like" of different lengths, and were evenly distributed on the bottom of the petri dish (Figure 1).

3.2 Morphological observation of primary BMECs

After 2 days of primary culture, a small number of short fusiform or polygonal endothelial cells crawled out from around the vascular segment and migrated outwards (Fig2. A, B). After 3 days, the cells formed "island?like" and "cluster?like" colonies that expanded to the periphery (Fig2. C). 4～5 days later, the cells entered the logarithmic growth phase, and the colonies fused into patches (Fig2. D, E). After 6 days, the cells covered the bottom of the dish, showing a typical monolayer, cobblestone?like, mosaic arrangement, and local contact inhibition occurred (Figure 2. F).

3.3 Observation of passaged BMECs and angiogenesis experiments

The morphology of the first generation rabbit BMECs is basically the same as that of the primary rabbit BMECs (Fig3. A). Rabbit BMECs inoculated on Matrigel began to adhere to the wall after 2 h, and gradually extended. After 4 hours, the cells can be connected into a network, forming a temporary stable lumen?like structure(Fig3. B). 24 h later, the structure disintegrated, and the cells clustered into clusters, scattered in the distribution (Figure 3. C).

4 Discussion

Rabbit BMECs have important vector value in the study of many cerebrovascular diseases such as hyperlipidemia, atherosclerotic vascular dementia, ischemic stroke and cerebral hemorrhage.However, there are few reports about the primary culture methods.Therefore, the research group successfully established a simple,repeatable and low experimental cost primary culture method of rabbit BMECs through repeated and extensive practice and explorati on.

In the primary culture of rabbit BMECs, the selection of appropriate rabbit age is the key to ensure the inoculation density of cerebral microvascular segment and the proliferation activity of BMECs.After several experiments, the research group found that compared with the rabbits aged 8～12 weeks selected by Chen Zhi[12], rabbits aged 7～10 days were easier to separate microvascular segments from brain tissues, and the cultured BMECs were better at dividing and proliferating, making them good samples. Obtaining relatively pure cerebral microvascular segments is the premise to ensure the purity of BMECs. Taking advantage of the fact that the diameter of cerebral microvessels is mostly between 6～80 μm[14], the research group selected a 75 μm mesh to filter out larger and longer blood vessel segments, so as to reduce the fibroblasts in the adventitia and the media of the large and medium vessels. smooth muscle cell contamination, And through 20% bovine serum albumin density gradient centrifugation to further remove the myelin sheath around the blood vessels, and finally obtain relatively pure cerebral microvascular segments.

In addition, moderate digestion of microvessel segments with chemoenzymes to loosen their peripheral tissues can promote the crawl of endothelial cells. But its digestion effect is affected by many factors such as enzyme type, potency, dosage, acting time and temperature. Xue Qingshan[13] advocated using 0.05% collagenase to digest the blood vessel segment for 2～4 h. The research group also tried this scheme in the early stage, but it was found that the vascular segments were prone to disintegrate after digestion in 0.05% collagenase for more than 2 h, and the cell viability was significantly reduced, and the experiment took a long time.Therefore, the concentration of collagenase can be appropriately increased and the digestion time can be greatly shortened, which can not only improve the experimental efficiency, but also prevent the excessive digestion of vascular segments. After many practical explorations, 10 ml 0.1% typeⅡ collagenase was finally used for digestion in water bath at 37℃ for 15～20 min, and digestion was terminated when most of the vascular segments showed "bead?like"changes under the microscope.

Finally, the identification of BMECs is also one of the key indicators to evaluate the success of primary culture. Previous studies have mostly used factor Ⅷ immunocytochemical staining to identify BMECs[15-17]. However, due to the large difference between the rabbit genome and the human genome, and the large biological companies at home and abroad have not developed anti?rabbit factorⅧ antibody. Therefore, the research group took advantage of the important biological feature that “vascular endothelial cells can form a lumen?like structure when cultured on matrigel, fibronectin and other matrices”[18], and used angiogenesis experiments to identify the cultured cells. The positive result that the cells could form a “l(fā)umen?like structure” on Matrigel, combined with its morphological observation, fully proved that the cultured cells were rabbit BMECs. In addition, by detecting the membrane protein CD31, the tight junction protein ZO?1, the vascular endothelial cadherin VE?cadherin, the von Willebrand factor vWF and other molecular indicators specifically expressed by endothelial cells, it is also possible to determine whether the cultured cells are vascular endothelial cells [19-20], the research group will further verify in follow?up experiments.

In conclusion, this study has carried out various practices and explorations on the selection of rabbit age, the acquisition of microvascular segments, the use of digestive enzymes, and the identification of target cells. A simple, reproducible, low cost, high purity and good activity primary rabbit BMECs culture method was successfully established. This also laid an important cell biology experimental foundation for the next stage of the research group to study the effect of mesenchymal stem cells and their exosomes on the high?fat?induced BMECs injury model.

Conflict of Interest Statement: All authors declare no conflict of interest.

Author Contribution: The experimental design was Tang Yuanyu,Ma Huagen, Liu Zhaode, and Liu Haiqin, the experimental implementation was Ma Huagen, Liu Zhaode, Liu Haiqin, and Tang Yuanyu, the experimental evaluation was Tang Yuanyu, and the data collection was Ma Huagen, Liu Zhaode, Liu Haiqin, and Tang Yuanyu.