恒流中原发性人类内皮细胞的肺炎球菌感染

Summary

本研究描述了在定义的流动条件下，在剪切应力下在分化人类原发内皮细胞表面产生的对冯·威勒布兰德因子字符串的肺炎球菌依从的微观监测。通过应用微分免疫染色程序，此协议可扩展到特定细胞结构的详细可视化和细菌定量。

Abstract

肺炎链球菌与内皮细胞表面的相互作用通过对美能敏感蛋白（如冯·威勒布兰德因子（VWF）在血液流动中介导。这种糖蛋白改变其分子构象，以响应剪切应力，从而暴露结合位点，用于广泛的宿主-配体相互作用。一般来说，在定义的剪切流下培养原发内皮细胞可以促进特定的细胞分化，并形成一个稳定且紧密相连的内皮层，类似于血管内壁的生理。.因此，对细菌病原体与涉及对肌敏感的蛋白质的宿主血管之间的相互作用的功能分析需要建立泵系统，以模拟已知影响表面的生理流动力。血管细胞。

本研究中使用的微流体装置使具有定义流速的流体能够连续和无脉冲再循环。计算机控制的气压泵系统通过生成连续、单向和控制的介质流，在内皮细胞表面应用定义的剪切应力。通过使用专为微观可视化而设计的特殊通道幻灯片，可以在流中微观监控和量化细胞和细菌附着物的形态变化。与静态细胞培养感染（一般要求在免疫标记和微观分析之前进行样品固定）不同，微流体滑道可实现基于荧光的蛋白质、细菌和细胞成分的检测样品固定后;系列免疫荧光染色;和直接荧光检测。结合荧光细菌和特定的荧光标记抗体，这种感染程序为与血管过程相关的大量科学应用提供了一个高效的多组分可视化系统。

Introduction

肺炎球菌感染的发病机制的特点是与细胞外基质化合物和人类血变成分的多样性相互作用，如质原和VWF^1，2， 3，4，5，6，7，8 。多域糖蛋白VWF作为平衡血变的关键调节器，通过调解血栓细胞招募和纤维蛋白结合在血管^血栓形成位9。功能，活跃的VWF对出血控制和伤口愈合的重要性证明了冯·威尔布兰德的疾病，一种常见的遗传性出血紊^乱10。

球状VWF在人体血液系统中循环，浓度高达14.0微克/mL^11，10。为了应对血管损伤，内皮韦贝尔帕拉德体（WBP）局部释放的VWF^{明显增加11，12。}以前的研究表明，肺炎球菌粘附于人类内皮细胞及其产生的孔隙毒素肺炎溶血蛋白显著刺激发光VWF分^泌13。血流的流体动力诱导机械反应VWF域的结构开放。在10dyn/cm2的流速下，VWF多变到长度高达几百微米的长蛋白串，仍然附着在子内皮10，12。

为了理解在剪切应力下产生的多面化VWF字符串在肺炎球菌与内皮表面相互作用中的功能，建立了基于微流体的细胞培养感染方法。使用一种微流体装置，采用软件控制的气压泵系统。这使得细胞培养基的连续、单向再循环具有定义的流速。因此，系统在内皮细胞表面应用了定义的剪切应力，内皮细胞仍附着在专用通道滑道内。这种方法能够模拟人类血管系统血流中的剪切力，在定义的恒定流动条件下，VWF字符串在分化的内皮细胞上生成。为此，内皮细胞在特定的通道幻灯片中培养（见材料表），这些细胞适用于流中的微小分析。微流体泵系统提供了在可融合的内皮细胞层上形成扩展 VWF 字符串所需的高度定义和控制的剪切应力情况。通过组胺补充刺激人类脐带静脉内皮细胞（HUVEC）的VWF分泌后，通过施加10 dyn/cm²的剪切应力（*）诱导弦形成。剪切应力定义为作用于细胞层的力。它是大约根据康尼什等人计算的。al.¹⁴与方程 1：

其中 = = 在 dyn/cm²中剪切应力 ，= 在 （dyn_s）/cm²中的粘度，h = 通道高度的一半，w = 通道宽度的一半，而 = = 流量（以 mL/min 表示）。

公式 1 的结果取决于所使用的不同幻灯片的不同高度和宽度（参见材料表）。在本研究中，使用了 0.4 μm 的 Luer 通道滑动，导致室滑动系数为 131.6（参见公式 2）。

37°C时介质的粘度为0.0072 dyn_s/cm2，剪切应力为10 dyn/cm2。这导致流量为 10.5 mL/min（参见公式 3）。

本文详细介绍了使用单向层流系统对宿主血管中细菌感染机制进行调查和可视化的微流体细胞培养过程的适应和进展。内皮层上的VWF字符串的生成也可以通过使用其他泵系统，能够应用连续和稳定的剪切^应力15刺激。

在原发内皮细胞培养到流动汇合和刺激VWF弦形成后，在恒定的微观控制下，将表达红荧光蛋白（RFP）16的肺炎球菌添加到内皮细胞层中。使用VWF特异性荧光标记抗体，对内皮细胞表面的细菌附着在VWF串上进行微观可视化和实时监测长达三个小时。用这种方法，VWF作为促进细菌附着到血管内皮的粘附辅助因子的作用被^确定8。

除了蛋白质分泌和构象变化的微观可视化外，该方法还可用于实时监测细菌感染过程的单步骤，并量化不同时间点的附着细菌数量。感染。特定的软件控制的泵系统还提供在定义的恒定流动条件下培养内皮细胞长达数天的可能性，并实现定义的脉冲介质流孵育。此外，该方法可以使用不同的单元格类型应用。调整染色方案还能够检测和可视化内化为真核细胞的细菌。

本手稿描述了这种高级实验协议，可用作一种定义、可靠和可重复的方法，用于对病理生理学过程进行高效和通用的表征。

Protocol

微流体细胞培养用商业性原发性人类脐带静脉内皮细胞（HUVEC）进行。公司在未经捐赠者知情同意后对细胞进行隔离。这项研究得到了联邦巴登-符腾堡州医生委员会伦理委员会的批准，参考号为219-04。 注：有关协议供应，请参阅材料表。 1. 原内皮细胞的预培养 在37°C下从三个不同的捐赠者轻轻解冻含有1 x 105初级H…

Representative Results

在恒定的单向流动中培养初级HUVEC，形成一个融合和紧密包装的细胞层，促进细胞WPB的生成，填充了对美氯介素VWF13，14。该协议描述了使用基于气压泵的无脉冲再循环系统的感染分析，需要模拟人类血流中的剪切应力情况。 该系统支持定义的软件控制流量条件设置。图1中的流方案说明了主要工作流程，从主?…

Discussion

模拟细菌与对美能敏感宿主蛋白（如 VWF）的相互作用，需要一个可渗透的细胞培养系统，该系统能够生成定义、单向和连续流动的液体，从而产生可靠的剪切应力.已经描述了几个微流体泵系统。Bergmann等人的综合综述总结了不同二维和三维细胞培养模型的关键^方面17。

微流体技术是一种非常年轻的技术，始于20世纪90年代初，在微米甚至纳米^尺?…

Materials

1 mL Luer-syringe	Fisher Scientific	10303002	with 1 mL volume for gelatin injection using the luer-connection of the slides
2 mL Luer-syringe	Sarstedt	9077136	For pieptting/injecting fluids into the luer connections of the channel chamber slides
Accutase	eBioscience now thermo fisher	00-4555-56	protease mix used for gentle detachment of endothelial cells
AlexaFluor350-conjugated Phalloidin	Abcam	ab176751	no concentration available from the manufacturer, stock solution is sufficient for 300 tets, company recommends to use 100 µl of a 1:1000 dilution, blue fluorescence (DAPI-filter settings)
AlexaFluor488-conjugated goat-derived anti-mouse antibody	Thermo Fisher Sientific	A11001	stock concentration: 2 mg/mL for immunostaining of human VWF in microfluidic slide after PFA fixation, green fluorescence
AlexaFluor568-conjugated goat-derived anti-rabbit-antibody	Thermo Fisher Scientific	A-11011	stock concentration: 2 mg/mL for immunostaining of pneumococci in microfluidic slide after pFA fixation, red fluorescence
Bacto Todd-Hewitt-Broth	Becton Dickinson GmbH	BD 249210	complex bacterial culture medium
Bovine Serum Albumin (BSA)	Sigma Aldrich	A2153-25G	solubilized, for preparation of blocking buffer
Cell culture flasks with filter	TPP	90026	subcultivation of HUVEC in non-coated cell culture flasks of 25 cm2 surface
Centrifuge Allegra X-12R	Beckman Coulter Life Sciences	392304	spinning down of bacteria (volumes of > 2mL)
Centrifuge Allegra X-30	Beckman Coulter Life Sciences	B06314	spinning down of eukaryotic cells
Centrifuge Z 216 MK	Hermle	305.00 V05 – Z 216 M	spinning down of bacteria (volumes of less than 2 mL)
Chloramphenicol	Carl Roth GmbH + Co. KG, Karlsruhe	3886.2	used in a concentration of 0.2 /mL for cultivation of pneumococci transformed with genetic construct carrying red fluorescent protein and chloramphenicol resistance cassette
Clamp for perfusion tubing	ibidi	10821	for holding the liquid in the tube bevor connecting the slide to the pump system
CO2-Incubator	Fisher Scientific	MIDI 40	incubator size is perfectly adapted to teh size of the fluidic unit with connected channel slide and was used for flow cultivation at 37°C and 5% CO2
CO2-Incubator	Sanyo	MCO-18 AIC	for incubation of bacteria and cells in a defined atmosphere at 5% CO2 and 37°C
Colombia blood agar plates	Becton Dickinson GmbH	PA-254005.06	agar-based complex culture medium for S. pneumoniae supplemented with 7% sheep blood
Computer	Dell	Latitude 3440	Comuter with pressure pump software
Confocal Laser Scanning Microscope (CLSM)	Leica	DMi8	An inverse microscope with a stage covered by a heatable chamber and with a fluorescence unit equipped with fluorescence filter, Xenon-light source (SP8, DMi8) and DFC 365 FX Kamera (1392 x 1040, 1.4 Megapixel)
Di Potassium hydrogen phosphate (KH2PO4)	Carl Roth GmbH + Co. KG, Karlsruhe	3904.1	used for PBS buffer
Drying material	Merck	101969	orange silica beads for drying used in a glass bottle with a tubing adaptor
ECGM supplement Mix	Promocell	C-39215	supplement mix for ECGM -medium, required for precultivation of endothelial cells: 0.02 mL/mL Fetal calf serum, 0.004 mL/ mL endothelial cell growth supplement, 0.1 ng / mL epidermal growth factor, 1 ng / mL basic fibroblast growth factor, 90 µg / mL heparin, 1 µg / mL Hydrocortisone
ECGMS	Promocell	C-22010	ECGM supplemented with 5 % [w/w] FCS and 1 mM MgSO4 to increase cell adhesion
Endothelial Cell growth medium (ECGM, ready to use)	Promocell	C-22010	culture medium of HUVECs, already supplemented with all components of the supplement mix
Fetal Calf Serum (FCS)	biochrome now Merck	S 0415	supplement for cell culture, used for infection analyses
FITC-conjugated goat anti-human VWF antibody	Abcam	ab8822	stock concentration: 10 mg/mL, for immunodetection of globular and multimerized VWF in flow
Fluidic Unit	ibidi	10903	fluidic unit for flow cultivation
Gelatin (porcine)	Sigma Aldrich	G-1890-100g	for precoating of microslide channel surface
histamine dihydrochloride	Sigma Aldrich	H-7250-10MG	for induction of VWF secretion from endothelial Weibel Palade Bodies
Human Umbilical Vein Endothelial Cell (HUVEC)	Promocell	C-12203 Lot-Nr. 396Z042	primary endothelial cells from pooled donor, stored crypcoserved in liquid nitrogen
Human VWF-specific antibody derived from mouse (monoclonal)	Santa Cruz	sc73268	stock concentration: 200 µg/mL for immunostaining of VWF in microfluidic slide after PFA fixation
Injection Port	ibidi	10820	for injection of histamin or bacteria into the reservoir tubing during the flow circulation
Light microscope	Zeiss	Axiovert 35M	inverse light microscope for control of eukaryotic cell detachement and cell counting using a 40 x water objective allowing 400 x magnification
Luer-slides I0.4 (ibiTreat472microslides)	ibidi	80176	physically modified slides for fludic cultivation (μ–Slide I0.4Luer with a channel hight of 0.4 mm, a channel volume of 100 μl, a growth area of 2.5 cm and a coating area of 25.4 cm2) suitable for all kinds of flow assay, the physical treatment generates a hydrophilic and adhesive surface.
Magnesium sulfate (MgSO4, unhydrated)	Sigma Aldrich	M7506-500G	For preparation of ECGMS medium
Microfluidic Pump	ibidi	10905	air pressure pump
Neubauer cell counting chamber	Karl Hecht GmbH&Co KG	40442002	microscopic counting chamber for HUVECs
Paraformaldehyde 16% (PFA)	Electron Microscopy Sciences	15710-S	for cross linking of samples
Perfusion Set	ibidi	10964	Perfusion Set Yellow/Green has a tubing diameter of 1.6 mm, a tube length of 50 cm, a total working volume of 13.6 mL, a dead tube volume of 2.8 mL and a reservoir size of 10 mL. combined with the µ-slide L0.4Luer, at 37°C and a viscosity of 0.0072 dyn x s/cm2 a flow rate range of 3.8mL/min up to 33.9 mL/min and shear stress between 3.5 dyn/cm2 and 31.2 dyn/cm2 can be reached. with 50 cm lenght for microfluidic
Phosphate-buffered saline (PBS)			the solution was prepared using the following chemicals: 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4 , pH 7.4
Plastic cuvettes	Sarstedt	67,741	(2 x optic) for OD measurement at 600 nm
Pneumococcus-specific antiserum	Pineda		raised in rabbit using heat-inactivated Streptococcus pneumoniae NCTC10319 and D39, IgG-purified using proteinA-sepharose column.
Polystyrene or Styrofoam plate			this is a precaution step to avoid cold stress on the cells seeded in the channel slides. Any type of styrofoam such as packaging box-material can be used. The plate might by 0.5 cm thick and should have a size of 20 cm2.
Potassium chloride (KCl)	Carl Roth GmbH + Co. KG, Karlsruhe	6781.1	used for PBS buffer
Pump Control Software (PumpControl v1.5.4)	ibidi	v1.5.4	Computer software for controlling the pressure pump, setting the flow conditions and start/end the flow
reaction tubes 1.5/ 2.0mL	Sarstedt	72.706/ 72.695.500	required for antibody dilutions
reaction tubes with 50 mL volume	Sarstedt	6,25,48,004
RFP-expressing pneumococci	National Collection of Type Cultures, Public Health England	10,319	Streptococcus pneumoniae serotype 47 expressing RFP fused to ahistone-like protein integrated into the genome
serological pipets 5, 10 mL	Sarstedt	86.1253.025/ 86.1254.025	for pipeting larger volumes
Sodium Carbonate (Na2CO3, water free)	Sigma Aldrich	451614-25G	for preparation of 100 mM Sodium Carbonate buffer
Sodium dihydrogen phosphate (NaH2PO4)	Carl Roth GmbH + Co. KG, Karlsruhe	P030.2	used for PBS buffer
Spectral Photometer Libra S22	Biochrom	80-2115-20	measurement of optical density (OD) of bacterial suspension at 600 nm
Sucrose	Sigma Aldrich	S0389-500G	for preparation of blocking buffer
Triton X-100	Sigma Aldrich	T9284-500ML	Used in 0.1% end concentration diluted in dH20 for eukaryotic cell permeabilization after PFA fixation
Yeast extract	oxoid	LP0021	bacteria are cultivated in THB supplement with 1% [w/w] yeast extract = complete bacterial cultivation medium THY