PROTOCOL

VascuNet™ Pericyte Co-Culture Assay

VascuNet™Pericyte共培养试验

INTRODUCTION 介绍

Vasculogenesis and angiogenesis are the mechanisms responsible for the development of a vascular network. Both endothelial cells and pericytes are key cell types in these processes. While endothelial cells form the lining of the vessels, pericytes are essential to the development of functional vasculature by stabilizing established vessel structures and facilitating local remodeling for network expansion. In addition to conveying structural support, pericytes are also integral in directing endothelial cells via cell-to-cell contact and paracrine signaling. Pericytes have been shown to co-localize with endothelial cells in both normal and abnormal vasculature, and have been implicated in playing a central role in numerous pathologies, including tumorigenesis, neurodegenerative disorders, and diabetic retinopathy.

血管生成和血管生成是血管网络发育的机制。内皮细胞和周细胞都是这些过程中的关键细胞类型。而内皮 细胞是血管的内层，周细胞通过稳定已建立的血管结构和促进网络e的局部重塑，对功能血管的发展至关重要。 xpansion。除了传递结构支持外，周细胞还通过细胞与细胞的接触和旁分泌信号来引导内皮细胞。周细胞已被证明为共室细胞。 在正常和异常血管系统中，内皮细胞与血管内皮细胞结合，并参与多种病理学中的中心作用，包括肿瘤发生、神经退行性疾病。 糖尿病视网膜病变。

The VascuNet Pericyte Co-Culture Assay combines human embryonic stem cell (ESI-017)-derived pericytes (PC-M cells) with primary human umbilical vein endothelial cells (HUVECs) in a co-culture system designed for a 96-well plate format. These unique PC-M cells display several key properties of pericytes, including expression of CD146, pro-angiogenic function, and effective stabilization of endothelial tube networks. The HUVECs and PC-M cell co- culture system supports vasculogenic tube assembly, resulting in the generation of extensive tube networks that persists at least 4 to 6 days in culture. Vasculogenic tube networks formed with the VascuNet Pericyte Co-Culture Assay persist over 4 times longer in culture than those formed by other assay systems, allowing researchers to study the relevant timing of delivery and long-term efficacy of pro- and anti-angiogenic compounds.

人胚胎干细胞(esi-017)衍生周细胞(pc-M细胞)与原代人脐静脉内皮细胞(HUVECs)在共培养系统中的结合。 设计的96井板格式。这些*的pc-M细胞显示了周细胞的几个关键特性，包括CD 146的表达、促血管生成功能以及内皮细胞的有效稳定。 光管网络。HUVECs和pc-M细胞共培养系统支持血管生成管的组装，从而产生了在Cultur至少持续4至6天的广泛的管状网络。 e.用VascuNet Pericyte共培养法形成的致血管网络在培养过程中的持续时间是其他检测系统的4倍以上，这使得研究人员可以研究该方法。 促血管生成化合物和抗血管生成化合物的时间安排和长期疗效。

IMPORTANT TIPS  重要提示

• All work with live cells should be completed in a sterile biological safety cabinet designated for tissue culture. 所有活细胞的工作应在组织培养的无菌生物安全柜内完成。
• Do not allow either PC-M cells or HUVECs to proliferate to more than 90% confluency during expansion. At high confluency, dense cell clusters may form, which can decrease vasculogenic tube assembly in mono- and co-cultures. Be sure to resuspend cells to single-cell suspension before plating for the angiogenic assay.不允许PC-M细胞或HUVECs在扩张过程中增殖到90%以上。在高度汇合的情况下，可形成密集的细胞团簇，从而减少血管生成管的组装。 单一文化和共同文化。在进行血管生成试验之前，一定要将细胞重新悬浮到单细胞悬浮液中.
• Solvents used to dissolve test reagents, such as DMSO. may have inherent pro- or anti-angiogenic properties For this reason, all test reagents should be resuspended in diluents with no greater than 0.1% (v/v) of the solvent.用于溶解试验试剂的溶剂，如dmso，可能具有固有的亲或抗血管生成特性，因此，所有试验试剂应重新悬浮在不超过0的稀释剂中。 .1%(v/v)的溶剂。
• Optimal vascular tube growth and stability is achieved when the HUVECs and PC-M cells are plated at a total of 42,000 cells per well in a ratio of 20:1 for HUVECs:PC-M cells, respectively.当HUVECs与PC-M细胞以42，000细胞/孔的比例分别以20：1的比例镀膜时，可获得宜的血管生长和稳定性。

REQUIRED MATERIALS所需材料

The VascuNet Pericyte Co-Culture Assay kit contains PC-M cells, HUVECs, and all media components for cell expansion and vasculogenic assay. Each kit undergoes extensive quality control to ensure reproducible vasculogenic tube assembly.

Vascune-周细胞共培养检测试剂盒含有PC-M细胞、HUVECs和所有用于细胞扩增和血管生成测定的培养基成分。每个套件都进行了全面的质量控制，以确保RPR。 可排卵的血管生成管组装。

VascuNet™ Pericyte Co-Culture Assay Kit Contents and Storage Conditions

Ensure that kit components are stored at the indicated temperatures upon kit arrival. The VascuNet Pericyte Co- Culture Assay components are stable for a minimum of 3 months from date of receipt when stored as directed.

确保组件在组件到达时以的温度存储。VascuNet Pericyte共同培养测试组件从收到时起至少3个月内是稳定的。 按指示存储。

Additional Required Reagents and Materials

附加所需试剂和材料

• Sample test compounds to assay
• 样品检测化合物
• Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV- Free
• Corning Matrigel生长因子减少(GFR)基底膜基质，*LDEV-无
• Phosphate Buffered Saline (PBS)
• 磷酸盐缓冲盐(PBS)
• Accutase® Cell Detachment Solution
• 酸性磷酸酶细胞脱离液
• Trypan blue or alternative cell viability assay
• 台盼蓝或替代细胞活力测定

• 0.22 µm sterile filtration unit, 500 mL 
• 0.22毫升无菌过滤装置，500 mL
• 0.22 µm sterile filtration unit, 150 mL
• 0.22毫升无菌过滤装置，150 mL
• T150 tissue culture flasks
• T 150组织培养瓶
• T75 tissue culture flasks
• T75组织培养瓶
• 96-well tissue culture plate

96孔组织培养板

EXPERIMENTAL OVERVIEW

Experimental Timeline

Assay Set-up

 实验时间线

Row 1: Control Row

H: HUVEC Monoculture Reference (40,000 cells/well)

P: PC-M Monoculture Reference (2,000 cells/well)

C: Co-Cultures (20:1 ratio of HUVEC:PC-M; 40,000 HUVECs and 2,000 PC-M cells/well)

Blue: HUVEC & PC-M Cell Co-Culture (Positive Control)

Yellow: HUVEC & PC-M Cell Co-Culture with 50 µM Suramin Hexasodium Salt (Negative Control)

第1行：控制行

h：HUVEC单一培养参考资料(40，000个细胞/井)

P：PC-M单细胞培养参比(2，000个细胞/井)

C：共培养(20：1的HUVEC：PC-M；40，000 HUVECs和2，000个PC-M细胞/井)

蓝：HUVEC&PC-M细胞共培养(阳性对照)

黄：HUVEC&PC-M细胞与50 M苏拉明六钠盐共培养(阴性对照

Figure 1. Example assay set-up in a 96-well plate. The first row of the VascuNet Pericyte Co-Culture Assay is a control row, containing triplicate samples of monoculture wells and positive and negative control co-culture wells. The remaining 84 wells of the plate may be used for test compounds. Test compound assays may be performed following the same mono- and co-culture set-up as the control row, or simply as co-culture wells.

图1.在一个96井的平板上进行测试。VascunetPericyte共培养试验的行是对照行，包含三份单一培养井的样本以及阳性和阴性的样本。 控制共培养井。该板的其余84口井可用作试验化合物。测试复合测试可以按照与控制行相同的单文化和共培养设置执行。 或者简单地说是共文化水井。

EXPERIMENTAL PROTOCOL

EXPANSION OF HUVECs AND PC-M CELLS

HUVECs和PC-M细胞的扩增

The HUVECs and PC-M cells must be thawed and expanded individually for two days before they can be plated for the experimental set-up in a co-culture format.

Monitor cell proliferation, and do not allow either the HUVECs or PC-M cells to proliferate to more than 90% confluency during expansion. At high confluency dense cell clusters may form, which can decrease vasculogenic tube assembly in mono- and co-cultures.

HUVECs和PC-M细胞必须单独解冻和扩张两天，然后才能以共同培养的形式进行实验设置。

监测细胞增殖，不允许HUVECs或PC-M细胞在扩张过程中增殖至90%以上。在高汇合度时，可形成密集的细胞团簇。 单培养和共培养时血管生成管的组装。

DAY -4

Prepare VascuNet Growth Medium for HUVECs and PC-M Cell Expansion

1. Remove the following VascuNet Growth Medium supplements from -20°C storage and allow the following reagents to thaw at 2 to 8°C:

rhVEGF, rhEGF, rhIGF-1, rhFGF basic, Ascorbic Acid, Heparin Sulfate, Hydrocortosone Hemisuccinate, FBS, L-Glutamine

HUVECs和PC-M细胞扩增用VascuNet生长培养基的制备

将以下VascuNet生长介质从-20℃储存中移除，并允许下列试剂在2至8℃解冻：

rhVEGF，rhEGF，rhIGF-1，rhFGF碱性，抗坏血酸，硫酸肝素，半琥珀酸氢皮质酮，FBS，L-谷氨酰胺

1. Prepare VascuNet Growth Medium by combining all the reagents listed below.

VascuNet Basal Medium 475 mL

rhVEGF 0.5 mL

rhEGF 0.5 mL

rhIGF-1 0.5 mL

rhFGF basic 0.5 mL

Ascorbic Acid 0.5 mL

Heparin Sulfate 0.5 mL Hydrocotrisone Hemisuccinate 0.5 mL FBS 25 mL

• Glutamine 25 mL

通过结合下面列出的所有试剂，准备VascuNet生长培养基。

VascuNet Basal培养基475 mL

rhVEGF 0.5 mL

rhEGF 0.5 mL

rhIGF-1 0.5 mL

rhFGF碱性0.5 mL

抗坏血酸0.5mL

硫酸肝素0.5 mL氢曲松半琥珀酸0.5 mL FBS 25 mL

L-谷氨酰胺25 mL

1. Filter sterilize the VascuNet Growth Medium using a 0.22 µm pore size, low protein-binding filter unit into a sterile 500 mL bottle.滤器用0.22m孔径、低蛋白质结合的过滤装置消毒VascuNet培养基，制成无菌500 mL瓶。
2. Transfer 75 mL of the VascuNet Growth Medium to a sterile 100 mL bottle and place in a 37°C water bath for 30 minutes to warm.将75 mL的VascuNet培养基转移到无菌的100 mL瓶中，置于37°C水浴中30分钟取暖。
3. Store the remaining VascuNet Growth Medium at 2 to 8°C for up to 2 weeks.将剩余的VascuNet培养基保存在2至8°C处，多2周。

Thaw and Plate HUVECs for Expansion 用于膨胀的解冻板HUVECs

1. Thaw the vial of HUVECs briefly in a 37°C water bath.将HUVECs瓶在37°C水浴中短暂解冻。
2. Transfer the entire volume of the vial into 4 mL of pre-warmed VascuNet Growth Medium in a 15 mL conical tube.将整个体积的小瓶转移到4毫升的预热VascuNet生长培养基中，放入15 mL的锥形管中。

1. Centrifuge the cell suspension for 5 minutes at 200 x g. 200×g离心细胞悬液5分钟。

1. Aspirate the supernatant and gently resuspend the cell pellet in 10 mL of VascuNet Growth Medium.

吸出上清液，在10 mL的VascuNet培养基中轻轻悬浮细胞颗粒。

1. Count the total number of viable cells using Trypan blue.使用台盼蓝计算活细胞总数。
2. Add 10 mL of VascuNet Growth Medium to each of two T150 flasks.在两个T 150瓶中各加入10 mL VascuNet生长培养基。
3. Divide the HUVEC suspension evenly between the two T150 flasks, such that each flask contains approximately 3.75 to 4.5 × 105 HUVECs at a density of 2.5 to 3.0 × 103 HUVECs/cm2.将HUVEC悬浮液均匀地分成两个T 150瓶，使每个瓶在2.5~3.0×103HUVECs/cm2的密度范围内含有约3.75~4.5×105个HUVECs。
4. Add a sufficient volume of the VascuNet Growth Medium to each flask to bring the total volume to 30 mL per T150 flask.在每个瓶中加入足够量的VascuNet生长培养基，使总体积达到每T 150瓶30 mL。

1. Incubate the cells overnight at 37°C with 5% CO2 humidified atmosphere.细胞在37°C和5%CO2加湿气氛中过夜

DAY -2

Thaw and Plate PC-M Cells for Expansion 用于膨胀的解冻板PC-M电池

1. Transfer 45 mL of VascuNet Growth Medium to a sterile 50 mL conical tube and warm in a 37°C water bath for 30 minutes.将45 mL的VascuNet培养基转移到无菌的50 mL锥形管中，在37°C水浴中加热30 min。
2. Thaw the vial of PC-M cells briefly in a 37°C water bath. Transfer the entire contents of the vial into 4 mL of pre-warmed VascuNet Growth Medium in a 15 mL conical tube.将PC-M细胞在37°C水浴中解冻。将小瓶的全部内容转移到4mL预加热的VascuNet生长培养基中，放入15 mL的锥形管中。
3. Centrifuge the cell suspension for 5 minutes at 200 x g.200×g离心细胞悬液5分钟。
4. Aspirate the supernatant and gently resuspend the cell pellet in 10 mL of VascuNet Growth Medium.吸出上清液，在10 mL的VascuNet培养基中轻轻悬浮细胞颗粒。

1. Count the total number of viable cells using Trypan blue.使用台盼蓝计算活细胞总数。
2. Add 10 mL of VascuNet Growth Medium to each of two T75 flasks.将10毫升VascuneT培养基加入到两个T75烧瓶中。
3. Divide the PC-M cell suspension evenly between the two T75 flasks, such that each flask contains approximately 2.5 × 105 PC-M cells at a density of 1.0 × 104 PC-M cells/cm2.将PC-M细胞悬浮液均匀地划分到两个T75瓶之间，使每瓶容量约为2.5×105个PC-M细胞，密度为1.0×104pC-M细胞/cm2。

Note: The PC-M cells are plated at a higher density than the HUVECs.注：PC-M细胞的密度高于HUVECs.

1. Add a sufficient volume of the VascuNet Growth Medium to each flask to bring the total volume to 15 mL per T75 flask.在每个瓶中加入足够量的VascuNet生长培养基，使总体积达到每T75瓶15 mL。
2. Incubate the cells overnight at 37°C with 5% CO2 humidified atmosphere.细胞在37°C和5%CO2加湿气氛中过夜。

Exchange Medium in HUVEC Culture HUVEC培养中的交换培养基

1. Warm 60 mL of VascuNet Growth Medium at 37°C.37°C温60 mL VascuNet培养基。
2. Observe HUVEC cultures under microscope to assess cell proliferation, confluency, and overall health.显微镜下观察HUVEC培养，观察细胞增殖、融合及整体健康状况。
3. Aspirate the medium in both of the T150 culture flasks, and replace with 30 mL of fresh VascuNet Growth Medium per flask.在T150培养瓶中抽吸培养基，每瓶更换30毫升新鲜VascuneT培养基。
4. Incubate cells at 37°C with 5% CO2 humidified atmosphere.

37°C条件下，5%CO2加湿培养细胞

ANGIOGENESIS ASSAY 血管生成试验

Once the HUVECs and PC-M cells have been expanded, the HUVEC and PC-M cells are plated in co-cultures and monoculture wells of a 96-well plate for the angiogenesis assay. Experimental and control assay wells are established to determine the effects of test compounds on angiogenesis.一旦HUVECs和PC-M细胞被扩增，HUVEC和PC-M细胞被镀在96孔板的共培养和单培养井中进行血管生成实验。试验与控制驴 我们建立了测试井，以确定试验化合物对血管生成的影响。

DAY 0

Prepare 96-well Assay Plate制备96井试井板

1. Thaw sufficient amounts of Growth Factor Reduced (GFR) Matrigel aliquot(s) on ice at 2 to 8°C.在冰上解冻足够数量的生长因子减少(GFR)Matrigel ali“(S)在2到8°C。

Note: Thaw a sufficient amount of GFR Matrigel to coat the total amount of wells used for the experiment, including the control row (see Fig. 1). Each well used in the assay will require 50 µL of Matrigel solution.注：融化足够数量的GFR Matrigel，以覆盖总数量的井用于实验，包括控制排(见图1)。在分析中使用的每一口井都需要50升的MAT。 Rigel溶液

1. Place a 96-well tissue culture plate and P100 pipet tips at -20°C for 1 hour to chill.放置96孔组织培养板和P 100管尖，温度-20℃，冷藏1小时。
2. Transfer the chilled 96-well plate, chilled pipet tips, and thawed GFR Matrigel bottle to an ice bucket in the tissue culture hood.将冰镇的96井板、冰镇管尖和融化的GFR Matrigel瓶转移到组织培养罩中的冰桶中。

Note: The GFR Matrigel will solidify rapidly when warmed above 2 to 8°C. To ensure even coating and distribution of the matrix, keep all of the reagents, tips, and plate on ice.注：GFR Matrigel在温度高于2°~8°C时会迅速凝固，以保证基体的均匀涂层和分布，使所有试剂、针尖和平板保持在冰上。

1. Add 50 µL of GFR Matrigel to each well of the 96-well plate, changing tips often for even plating.在96井板的每口井中加入50升GFR Matrigel，经常改变镀液的。
2. Incubate the coated 96-well plate at room temperature for 1 hour, then transfer to a 37°C incubator with 5% CO2 humidified atmosphere for 1 to 2 hours prior to plating cells.将包覆的96孔板在室温下孵育1h，再在37℃、5%CO2加湿气氛下孵育1~2小时。

Prepare VascuNet Assay Medium制备VascuNet检测介质

1. Thaw the L-Glutamine (for assay medium) at 2 to 8°C. Allow the VascuNet Basal Assay Medium to warm to room temperature.将L-谷氨酰胺(用于检测介质)在2到8°C解冻。允许VascuNet Basal分析介质加热到室温。
2. Prepare the VascuNet Assay Medium by combining the reagents below.通过组合下面的试剂，准备VascuNet测试介质。

VascuNet Basal Assay Medium 95 mL  VascuNet Basal试验培养基95 mL

L-Glutamine 5 mL   L-谷氨酰胺5mL

1. Filter sterilize the VascuNet Assay Medium using a 0.22 µm pore size, low protein-binding filter and a sterile 100 mL bottle. 滤池用0.22m孔径、低蛋白质结合过滤器和无菌100 mL瓶对VascuNet分析培养基进行消毒。
2. Transfer 50 mL of VascuNet Assay Medium to a 50 mL conical tube. Warm this aliquot in a 37°C water bath for 30 minutes. The required amount of medium may vary depending on the number of test component wells.将50 mL VascuNet试剂盒转入50 mL锥形管。在37℃的水浴中加热30分钟。所需介质的数量可能会根据测试组件的数量而有所不同。
3. Store the remaining VascuNet Assay Medium at 2 to 8°C for up to 2 weeks.

将剩余的VascuNet检测介质保存在2至8°C处，多2周。

Harvest HUVECs  收获HUVECs

1. Aspirate the culture medium from each of the T150 flasks, and add 10 mL of PBS per flask to wash. Aspirate the PBS wash.分别从T 150瓶中抽吸培养基，每瓶加入10 mL PBS进行洗涤。吸入PBS洗涤液。
2. Add 5 mL of Accutase Cell Detachment Solution to each flask and incubate at room temperature for 5 minutes, or until cells appear rounded.在每瓶中加入5毫升酸性磷酸酶细胞脱落液，在室温下孵育5分钟，或直至细胞呈圆形。
3. Add 5 mL of VascuNet Assay Medium to each flask. Firmly tap the side of the flask to release the cells from the culture surface.在每个瓶中加入5毫升VascuNet分析培养基。牢固地敲击瓶的侧面，将细胞从培养表面释放出来。
4. Collect the HUVECs from each flask and transfer the cells within each flask to a 15 mL conical tube. Pipet gently to dissociate any remaining cell clumps.从每个烧瓶收集HUVECs，并将每个烧瓶内的细胞转移到15毫升锥形管中。轻轻吸管以分离的任何剩余的细胞团。
5. Centrifuge the cells for 5 minutes at 250 x g.250×g离心5分钟。
6. Aspirate the supernatant from each tube and resuspend the cell pellets to a single cell suspension in 5 mL of VascuNet Assay Medium per tube.每管抽取上清液，再将细胞颗粒悬浮到单个细胞悬液中，每管5 mL VascuNet检测培养基。

Note: It is essential to thoroughly resuspend the cells to single-cell suspension before plating for the angiogenic assay.注：在进行血管生成试验前，必须将细胞*重新悬浮到单细胞悬浮液中。

1. Combine single cell suspensions (10 mL total volume) and pipet to mix.将单细胞悬液(总体积10 mL)与吸管混合。
2. Count the total number of viable cells in the combined cell suspension using Trypan blue.用台盼蓝计数组合细胞悬液中的活细胞总数。
3. Dilute the HUVECs in VascuNet Assay Medium to a concentration of 5 x 105 viable cells/mL.在VascuNet检测培养基中稀释HUVECs至5x105个活细胞/mL。

Harvest PC-M Cells 收获PC-M细胞

1. Aspirate the culture medium from each of the T75 flasks, and add 5 mL of PBS per flask to wash. Aspirate the PBS wash.分别从T75瓶中抽吸培养基，每瓶加入5 mL PBS进行洗涤。吸入PBS洗涤液。
2. Add 2.5 mL of Accutase Cell Detachment Solution to each flask and incubate at room temperature for 5 minutes, or until cells appear rounded.每瓶加入2.5mL的酸性磷酸酶细胞脱落液，在室温下孵育5分钟，直至细胞呈圆形。
3. Add 2.5 mL of VascuNet Assay Medium to each flask. Firmly tap the side of the flask to release the cells from the culture surface.在每个瓶中加入2.5mL的VascuNet分析培养基。牢固地敲击瓶的侧面，将细胞从培养表面释放出来。
4. Collect the PC-M cells from each flask and transfer to a single 15 mL conical tube. Pipet gently to dissociate any remaining cell clumps.从每个烧瓶中收集PC-M细胞，转移到一个15 mL的锥形管中。轻轻地将任何剩余的细胞团分离。
5. Centrifuge cells for 5 minutes at 250 x g.250×g离心细胞5 min。
6. Aspirate the supernatant and resuspend the cell pellet to a single cell suspension in 10 mL of VascuNet Assay Medium.取上清液，在10 mL VascuNet检测培养基中，将细胞颗粒再悬浮于单个细胞悬液中。

Note: It is essential to thoroughly resuspend the cells to single-cell suspension before plating for the angiogenic assay.注：在进行血管生成试验前，必须将细胞*重新悬浮到单细胞悬浮液中。

1. Measure the total number of viable cells in the sample by performing a cell count using Trypan blue.通过使用台盼蓝执行细胞计数来测量样本中可行的细胞总数。
2. Dilute the PC-M cells in VascuNet Assay Medium to a concentration of 1 x 105 viable cells/mL.

在VascuNet检测培养基中稀释PC-M细胞，浓度为1x105个活细胞/mL。

Plate HUVECs and PC-M Cells平板HUVECs和PC-M细胞

Refer to sample plate set-up diagram (Fig. 1). In addition to the experimental wells, one row of the 96-well plate will be utilized for control wells. Determine the total number of cells needed, in addition to the control row, based on the number of test samples.参考样板设置图(图1).除试验井外，96口井板中的一行井将用于控制井。确定所需单元格的总数，在 到控制行，根据测试样本的数量。

1. For each set of triplicate HUVEC monoculture samples, combine 1.32 x 105 HUVECs with a sufficient volume of VascuNet Assay Medium to bring the total volume to 495 µL per triplicate set.对于每一组三重的HUVEC单培养样品，将1.32×105 HUVEC与足够体积的Vascuneta试验培养基相结合，使总体积为每三套495 L。
2. Dispense 150 µL of the HUVEC cell suspension into each well of the assay, for a total of 4.0 x 104 HUVECs per monoculture well.将150 L的HUVEC细胞悬液分装于每口培养井中，平均每孔培养4.0x104个HUVECs。
3. For each set of triplicate PC-M monoculture samples, combine 6.6 x 103 PC-M cells with a sufficient volume of VascuNet Assay Medium to bring the total volume to 495 µL per triplicate set.
4. Dispense 150 µL of the PC-M cell suspension into each well of the assay, for a total of 0.2 x 104 PC-M cells per monoculture well.
5. For each set of triplicate co-culture samples, combine 1.32 x 105 HUVECs with 6.6 x 103 PC-M cells. Add a sufficient volume of VascuNet Assay Medium to bring the total volume to 495 µL per triplicate set.对于每组三份PC-M单细胞培养样品，将6.6×103个PC-M细胞与足够体积的VascuNet检测培养基结合，使总体积达到每三份样品495 L。

Note: This will make a 20:1 ratio of HUVEC:PC-M cells.注：这将使HUVEC：PC-M细胞的比例达到20：1.

1. Dispense 150 µL of the combined cell suspension into each well of the assay, for a total of 4.2 x 104 cells per co-culture well.将150 L的PC-M细胞悬液注入每孔，每孔培养一次，共培养0.2x104个PC-M细胞。
2. Incubate the plate at 37°C with 5% CO2 humidified atmosphere undisturbed for 4 to 6 hours to allow cells to adhere and initial tube networks to form.每组三份共培养样品，将1.32x105个HUVECs与6.6×103个PC-M细胞结合.加入足够量的VascuNet检测介质，使总体积达到每三份495升 部分，段

Addition of the Test Compounds添加试验化合物

Following the 4 to 6 hour incubation period, visualy inspect the wells to confirm attachment and tube formation in both HUVEC monocultures and co-culture conditions (see Fig. 2). After confirmation of initial tube formation,

the cells are ready to be treated with the test reagents. For best results, this is done 4 to 6 hours after plating, but must be completed within 24 hours after cells are plated.在4到6小时的潜伏期后，目视检查水井，以确定在HUVEC单细胞培养和共培养条件下的附着和管状形成(见图2)。确认后 初的管状，这些细胞已准备好用试验试剂处理。为了取得良好的效果，这是在电镀后4至6小时，但必须在24小时内完成电池被镀。

Figure 2. VascuNet HUVEC and PC-M Cell Co-Culture at 4 hours. Co-Culture of HUVEC and PC-M cells seeded at a 20:1 ratio begin formation of tube networks as early as 4 hours.

图2.VascuNet HUVEC和PC-M细胞共培养4小时.人脐静脉内皮细胞(HUVEC)与PC-M细胞按20：1比例共培养，4小时开始形成管状网络。

Positive Control Wells 正控井

Positive control wells contain cells in co-culture at a 20:1 (HUVEC:PC-M cell) ratio in VascuNet Assay Medium. No additional compounds are added to these wells.阳性对照井在VascuNet检测培养基中含有20：1(HUVEC：PC-M细胞)比例的细胞。在这些井中不添加任何额外的化合物。

Negative Control Wells负压井

The negative control samples are run in triplicate co-culture conditions with 50 µM Suramin Hexasodium Salt.阴性对照样品一式三份，共培养条件为50 m苏拉明六钠盐。

1. Thaw the vial of Suramin Hexasodium Salt for 1 hour at room temperature.在室温下解冻苏拉明六钠盐1小时。Add 25 µL of 1 mM Suramin Hexasodium Salt to 475 µL of VascuNet Assay Medium, for a final Suramin Hexasodium Salt concentration of 50 µM. Warm the 50 µM Suramin Hexasodium Salt solution to 37°C before use.在475升VascuNet分析介质中加入25升1毫米苏拉明六钠盐，使终的六钠浓度为50 M，然后将50 M苏拉明六钠盐溶液加热至37°C。 e使用。
2. Aspirate the VascuNet Assay Medium from each well of cells and replace with 150 µL per well of the 50 µM Suramin Hexasodium Salt solution.从每口细胞中抽取VascuNet检测介质，用50 M苏拉明六钠盐溶液中的每口150毫升代替。

Experimental Wells 实验井

Experimental wells should be tested in co-culture wells in triplicates. Triplicate monoculture samples can also be run for comparison.实验威尔斯应在共培养威尔斯中试验三次。三份的单一栽培样品也可用于比较。

1. Prepare additional pro- or anti-angiogenic test compounds by diluting with VascuNet Assay Medium to desired concentrations. Warm diluted test compound solutions to 37°C before use.用VascuNet分析培养基稀释至所需浓度，制备额外的亲或抗血管生成试验化合物。使用前将温热稀释的复合溶液稀释至37°C。
2. Aspirate VascuNet Assay Medium from each well to be assayed and replace with 150 µL per well of VascuNet Assay Medium containing the appropriate test compound in solution.从每口井中抽吸VascuNet检测介质，用含适当试液的VascuNet分析介质每井150 L代替。

Day 1 to Day 4+ 第1天到第4天

1. Observe and image the cells every 4 to 24 hours to monitor vasculogenic tube assembly and stability.

Note: Vascular tube formations in co-cultures containing both HUVECs and PC-M cells will be stable for at least 4 days in culture without the need to replace media or add exogenous factors.每4至24小时观察和成像细胞，以监测血管生成管的组装和稳定性。

注：在含有HUVECs和PC-M细胞的共培养中，血管管状细胞在培养中至少稳定4天，不需要更换培养基或添加外源因子。

EXPECTED RESULTS预期结果

The following data describes the expected results for co-culture and monoculture conditions at the recommended cell seeding density of 42,000 cells per well containing a 20:1 ratio of HUVECs to PC-M cells (40,000 HUVECs and 2,000 PC-M cells).以下数据描述了在建议的细胞播种密度为每井42，000个细胞时，共培养和单一培养条件下的预期结果，其中含有20：1的HUVECs与pc-m的比例。 细胞(40，000 HUVECs和2，000个PC-M细胞).

• HUVEC monocultures will begin to form tube networks as early as 4 hours. A complete tube network can be observed at 24 hours. HUVEC tubes lacking PC-M cell support should destabilize by Day 2 of the angiogenesis assay and do not re-assemble.HUVEC单细胞早可在4小时内形成管状网络。24小时可观察到完整的管状网络。缺乏pc-M细胞支持的HUVEC管应在第2天发生不稳定。 血管生成实验和不重新组装。

• PC-M cell monocultures do not form complete tube networks. Minimal branching may be observed.PC-M细胞不形成完整的管状网络.可以观察到小分支。

• Co-cultures form tube networks within four days and are maintained for up to 6 days.共培养在4天内形成管状网络，并维持6天

• The addition of 50 µM Suramin Hexasodium Salt (negative control) to both monocultures and co- cultures will reduce tube formation by > 90% within 4 to 24 hours after treatment. The presence of Suramin Hexasodium Salt will prevent tube re-assembly for at least 4 days.在单一培养和共培养中加入50μm苏拉明六钠盐(阴性对照)，可在处理后4~24小时内使试管形成减少90%以上。苏拉米的存在 N六钠盐可以防止管重新组装至少4天。

Image Timeline

Figure 3. Stability of tube structures over time. HUVEC monocultures (rows 1 and 2) seeded at 120,000 cells/cm2 and stained with Vybrant DiO (green), show tube formation on Day 1 post seeding. By day 2, degredation of the vessels is already observed. In contrast, tube structures with multiple branching points are visible from Day 1 and are still stable at Day 6 in the HUVEC and PC-M cell co-cultured wells plated at a 20:1 ratio (rows 3 and 4). PC-M cells are stained red with Vybrant Dil.

Images were taken at 4X magnification.

图3.随着时间的推移，管状结构的稳定性。HUVEC单眼(第1行和第2行)在12万个细胞/cm2下播种，用VybrantDio(绿色)染色，在播种后第1天出现管状形成。白天 2、已观察到船舶的高度。相比之下，具有多个分支点的管状结构从第1天就可以看到，在HUVEC和pc-M细胞共培养中，在第6天仍保持稳定。 红色水井按20：1比例镀制(第3和第4行)。PC-M细胞用Vybrant Dil染红。

图像以4X放大倍数拍摄。

APPENDIX

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