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  • br Cell lines and culture

    2020-07-06


    2.1. Cell lines and culture
    The human lung cancer cell line PC9 was used as the primary lung cancer model. To mimic microenvironments for in situ lung cancers and BM of lung cancers, human bronchial epithelial cells (16HBE), human pulmonary microvascular endothelial cells (hPMEC), human lung fibroblasts (HFL1), and human mononuclear cells (THP-1) were introduced into the upstream unit as previous reported [35]. Human astrocytes (HA-1800) and human brain microvascular endothelial cells (hBMVEC) were cultured to mimic the cerebral BBB structure.
    All cell lines were purchased from the Chinese Academy of Medical Sciences (Beijing, China). The 16HBE, HFL1, THP-1 and PC9 cells were cultured in Roswell Park Memorial Institute medium-1640 (RPMI1640), Ham’s F12 medium (F12K), Dulbecco’s modified Eagle’s medium (DMEM), and DMEM/F12, respectively. The media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin (all of these reagents were from Gibco, Invitrogen, Inc, USA). The hPMEC, HA-1800, and hBMVEC cells were purchased from Sciencell (Sciencell, USA) and cultured in the corresponding culture medium recom-mended by the manufacturer. All cell lines were maintained in a humidified Oxaliplatin with 5% CO2 at 37 LC.
    To investigate the mechanism of BM in lung cancer, brain meta-static subpopulations (PC9-BrM1, PC9-BrM2, and PC9-BrM3) were created by injecting tumor cells into the left-ventricle of immunod-eficient mice and isolating the metastatic cells from harvested brain metastases as previously described [36]. The highly brain metastatic lung cancer cell line PC9-BrM3 was established by repeating injection-isolation-expansion cycling three additional times and cultured in DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 LC in a humidified atmosphere with 5% CO2.
    2.2. Establishment and evaluation of the microfluidic system
    The multi-organ-on-a-chip system consisted of an upstream ‘‘lung organ” and a downstream ‘‘brain organ”. The upstream
    pathological biomimetic microfluidic model of lung cancer was built as previous reported [35], while the downstream metastasis target organ ‘‘brain” was characterized by its specific functional
    BBB structure (Fig. 1A). The downstream unit was comprised of
    two 300 lm wide vascular chambers (outer side), one of which was connected to the upstream as a pathway for the transport of metastatic tumor cells, while the other one was not connected
    and functioned as a control. The two vascular chambers were sep-arated by a circular brain parenchyma chamber with a radius of 600 lm (center side). To study the migration and extravasation of tumor cells, the chamber was built with micro-gaps 50 lm in length and 20 lm in width, which mimicked the filters used in the Transwell apparatus. All of the chambers were 100 lm in depth, which is the typical depth of microvascular networks
    Fig. 1. Construction of a multi-organ microfluidic chip to study lung cancer-derived BM. (A) Schematic illustration of the pathological process of lung cancer BM and the multi-organ microfluidic chip, which were reproduced corresponding to each part of a chip-upstream ‘‘lung” and downstream ‘‘brain”. The upstream ‘‘lung” adopts the sandwich structure for multicell co-culture (bronchial epithelial cells, fibroblasts, immune cells, pulmonary vascular endothelial cells, and tumor cells), while two vacuum channels on both sides simulate the respiratory rhythm to reconstitute the tumorigenesis and intravasation of lung cancer in situ. The downstream ‘‘brain” is comprised of a brain parenchyma chamber circled by two vascular channels, one of which is connected to the upstream as a pathway for the transport of metastatic tumor cells, while the other one without further connection serves as the control. The micro-gaps connect the brain parenchyma chamber with the vascular channels to allow co-culturing of the cells and extravasation of tumor cells. (B) General configuration of the biomimetic multi-organ microfluidic chip. The microfluidic chip was fabricated with two PDMS layers and a thin microporous. (C) The image of an actual biomimetic multi-organ microfluidic chip as viewed from above, scale bar,1 cm.
    observed in vivo. Each chamber had its own inlet and outlet ports, which allowed independent perfusion and sampling. To mimic blood circulation, a syringe pump (Pump 11 Elite Series Pumps, USA) was utilized to drive the flow of cell culture medium to go through the microvascular channel.
    Two PDMS (polydimethylsiloxane) layers and one thin microp-orous membrane were used to fabricate the multi-organ microflu-idic chip (Fig. 1B and C). The PDMS layers and the microporous membrane were produced as previously described [35].
    2.2.3. Establishment of the dynamic BBB
    The operation of the upstream biomimetic microfluidic model of lung cancer was generated as previously described [35]. The equal proportions mixture media of their corresponding culture medium was applied while different cell types are grown together in the same channel on the chip. To mimic the formation of natural ECM in the brain, collagen I and fibronectin (each 100 lg/ml) were injected into the vascular chambers, which were then placed in an inverted position for 2 h in a 37 LC incubator supplied with 5% CO2. The chambers were then filled with serum free media and incu-bated at 37 LC for 30 min to equilibrate the chambers with the media contents. Next, hBMVECs were harvested and loaded at a density of 1 107 cells/ml into the vascular channels via the inlets. The loaded cells were further incubated in the 37 LC incubator for 4 h to allow their attachment to the bottom surface of the chan-nels. Then freshly isolated hBMVECs were reloaded and allowed to attach to the side of the micro-gap at a vertical position for another 4 h. After the attachment of hBMVECs to both surfaces, human astrocytes HA-1800 were harvested and injected into the circular brain parenchyma chamber at a concentration of 106/ml to build the co-culture microenvironment. The HA-1800 cells were allowed to attach for 4 h in the 37 LC incubator to form the bionic