PROTOCOL

VascuNet™ Pericyte Co-Culture Assay

VascuNet™Pericyte共培養(yǎng)試驗

INTRODUCTION 介紹

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網(wǎng)絡(luò)發(fā)育的機制。內(nèi)皮細(xì)胞和周細(xì)胞都是這些過程中的關(guān)鍵細(xì)胞類型。而內(nèi)皮 細(xì)胞是血管的內(nèi)層，周細(xì)胞通過穩(wěn)定已建立的血管結(jié)構(gòu)和促進(jìn)網(wǎng)絡(luò)e的局部重塑，對功能血管的發(fā)展至關(guān)重要。 xpansion。除了傳遞結(jié)構(gòu)支持外，周細(xì)胞還通過細(xì)胞與細(xì)胞的接觸和旁分泌信號來引導(dǎo)內(nèi)皮細(xì)胞。周細(xì)胞已被證明為共室細(xì)胞。 在正常和異常血管系統(tǒng)中，內(nèi)皮細(xì)胞與血管內(nèi)皮細(xì)胞結(jié)合，并參與多種病理學(xué)中的中心作用，包括腫瘤發(fā)生、神經(jīng)退行性疾病。 糖尿病視網(wǎng)膜病變。

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.

人胚胎干細(xì)胞(esi-017)衍生周細(xì)胞(pc-M細(xì)胞)與原代人臍靜脈內(nèi)皮細(xì)胞(HUVECs)在共培養(yǎng)系統(tǒng)中的結(jié)合。 設(shè)計的96井板格式。這些*的pc-M細(xì)胞顯示了周細(xì)胞的幾個關(guān)鍵特性，包括CD 146的表達(dá)、促血管生成功能以及內(nèi)皮細(xì)胞的有效穩(wěn)定。 光管網(wǎng)絡(luò)。HUVECs和pc-M細(xì)胞共培養(yǎng)系統(tǒng)支持血管生成管的組裝，從而產(chǎn)生了在Cultur至少持續(xù)4至6天的廣泛的管狀網(wǎng)絡(luò)。 e.用VascuNet Pericyte共培養(yǎng)法形成的致血管網(wǎng)絡(luò)在培養(yǎng)過程中的持續(xù)時間是其他檢測系統(tǒng)的4倍以上，這使得研究人員可以研究該方法。 促血管生成化合物和抗血管生成化合物的時間安排和長期療效。

IMPORTANT TIPS  重要提示

• All work with live cells should be completed in a sterile biological safety cabinet designated for tissue culture. 所有活細(xì)胞的工作應(yīng)在組織培養(yǎng)的無菌生物安全柜內(nèi)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.不允許PC-M細(xì)胞或HUVECs在擴(kuò)張過程中增殖到90%以上。在高度匯合的情況下，可形成密集的細(xì)胞團(tuán)簇，從而減少血管生成管的組裝。 單一文化和共同文化。在進(jìn)行血管生成試驗之前，一定要將細(xì)胞重新懸浮到單細(xì)胞懸浮液中.
• 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.用于溶解試驗試劑的溶劑，如dmso，可能具有固有的親或抗血管生成特性，因此，所有試驗試劑應(yīng)重新懸浮在不超過0的稀釋劑中。 .1%(v/v)的溶劑。
• 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.當(dāng)HUVECs與PC-M細(xì)胞以42，000細(xì)胞/孔的比例分別以20：1的比例鍍膜時，可獲得宜的血管生長和穩(wěn)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-周細(xì)胞共培養(yǎng)檢測試劑盒含有PC-M細(xì)胞、HUVECs和所有用于細(xì)胞擴(kuò)增和血管生成測定的培養(yǎng)基成分。每個套件都進(jìn)行了全面的質(zhì)量控制，以確保RPR。 可排卵的血管生成管組裝。

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.

確保組件在組件到達(dá)時以的溫度存儲。VascuNet Pericyte共同培養(yǎng)測試組件從收到時起至少3個月內(nèi)是穩(wěn)定的。 按指示存儲。

Additional Required Reagents and Materials

附加所需試劑和材料

• Sample test compounds to assay
• 樣品檢測化合物
• Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV- Free
• Corning Matrigel生長因子減少(GFR)基底膜基質(zhì)，*LDEV-無
• Phosphate Buffered Saline (PBS)
• 磷酸鹽緩沖鹽(PBS)
• Accutase® Cell Detachment Solution
• 酸性磷酸酶細(xì)胞脫離液
• Trypan blue or alternative cell viability assay
• 臺盼藍(lán)或替代細(xì)胞活力測定

• 0.22 µm sterile filtration unit, 500 mL 
• 0.22毫升無菌過濾裝置，500 mL
• 0.22 µm sterile filtration unit, 150 mL
• 0.22毫升無菌過濾裝置，150 mL
• T150 tissue culture flasks
• T 150組織培養(yǎng)瓶
• T75 tissue culture flasks
• T75組織培養(yǎng)瓶
• 96-well tissue culture plate

96孔組織培養(yǎng)板

EXPERIMENTAL OVERVIEW

Experimental Timeline

Assay Set-up

 實驗時間線

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單一培養(yǎng)參考資料(40，000個細(xì)胞/井)

P：PC-M單細(xì)胞培養(yǎng)參比(2，000個細(xì)胞/井)

C：共培養(yǎng)(20：1的HUVEC：PC-M；40，000 HUVECs和2，000個PC-M細(xì)胞/井)

藍(lán)：HUVEC&PC-M細(xì)胞共培養(yǎng)(陽性對照)

黃：HUVEC&PC-M細(xì)胞與50 M蘇拉明六鈉鹽共培養(yǎng)(陰性對照

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.

圖1.在一個96井的平板上進(jìn)行測試。VascunetPericyte共培養(yǎng)試驗的行是對照行，包含三份單一培養(yǎng)井的樣本以及陽性和陰性的樣本。 控制共培養(yǎng)井。該板的其余84口井可用作試驗化合物。測試復(fù)合測試可以按照與控制行相同的單文化和共培養(yǎng)設(shè)置執(zhí)行。 或者簡單地說是共文化水井。

EXPERIMENTAL PROTOCOL

EXPANSION OF HUVECs AND PC-M CELLS

HUVECs和PC-M細(xì)胞的擴(kuò)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細(xì)胞必須單獨解凍和擴(kuò)張兩天，然后才能以共同培養(yǎng)的形式進(jìn)行實驗設(shè)置。

監(jiān)測細(xì)胞增殖，不允許HUVECs或PC-M細(xì)胞在擴(kuò)張過程中增殖至90%以上。在高匯合度時，可形成密集的細(xì)胞團(tuán)簇。 單培養(yǎng)和共培養(yǎng)時血管生成管的組裝。

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細(xì)胞擴(kuò)增用VascuNet生長培養(yǎng)基的制備

將以下VascuNet生長介質(zhì)從-20℃儲存中移除，并允許下列試劑在2至8℃解凍：

rhVEGF，rhEGF，rhIGF-1，rhFGF堿性，抗壞血酸，硫酸肝素，半琥珀酸氫皮質(zhì)酮，F(xiàn)BS，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

通過結(jié)合下面列出的所有試劑，準(zhǔn)備VascuNet生長培養(yǎng)基。

VascuNet Basal培養(yǎng)基475 mL

rhVEGF 0.5 mL

rhEGF 0.5 mL

rhIGF-1 0.5 mL

rhFGF堿性0.5 mL

抗壞血酸0.5mL

硫酸肝素0.5 mL氫曲松半琥珀酸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.濾器用0.22m孔徑、低蛋白質(zhì)結(jié)合的過濾裝置消毒VascuNet培養(yǎng)基，制成無菌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.將75 mL的VascuNet培養(yǎng)基轉(zhuǎn)移到無菌的100 mL瓶中，置于37°C水浴中30分鐘取暖。
3. Store the remaining VascuNet Growth Medium at 2 to 8°C for up to 2 weeks.將剩余的VascuNet培養(yǎng)基保存在2至8°C處，多2周。

Thaw and Plate HUVECs for Expansion 用于膨脹的解凍板HUVECs

1. Thaw the vial of HUVECs briefly in a 37°C water bath.將HUVECs瓶在37°C水浴中短暫解凍。
2. Transfer the entire volume of the vial into 4 mL of pre-warmed VascuNet Growth Medium in a 15 mL conical tube.將整個體積的小瓶轉(zhuǎn)移到4毫升的預(yù)熱VascuNet生長培養(yǎng)基中，放入15 mL的錐形管中。

1. Centrifuge the cell suspension for 5 minutes at 200 x g. 200×g離心細(xì)胞懸液5分鐘。

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培養(yǎng)基中輕輕懸浮細(xì)胞顆粒。

1. Count the total number of viable cells using Trypan blue.使用臺盼藍(lán)計算活細(xì)胞總數(shù)。
2. Add 10 mL of VascuNet Growth Medium to each of two T150 flasks.在兩個T 150瓶中各加入10 mL VascuNet生長培養(yǎng)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.將HUVEC懸浮液均勻地分成兩個T 150瓶，使每個瓶在2.5~3.0×103HUVECs/cm2的密度范圍內(nèi)含有約3.75~4.5×105個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.在每個瓶中加入足夠量的VascuNet生長培養(yǎng)基，使總體積達(dá)到每T 150瓶30 mL。

1. Incubate the cells overnight at 37°C with 5% CO2 humidified atmosphere.細(xì)胞在37°C和5%CO2加濕氣氛中過夜

DAY -2

Thaw and Plate PC-M Cells for Expansion 用于膨脹的解凍板PC-M電池

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.將45 mL的VascuNet培養(yǎng)基轉(zhuǎn)移到無菌的50 mL錐形管中，在37°C水浴中加熱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.將PC-M細(xì)胞在37°C水浴中解凍。將小瓶的全部內(nèi)容轉(zhuǎn)移到4mL預(yù)加熱的VascuNet生長培養(yǎng)基中，放入15 mL的錐形管中。
3. Centrifuge the cell suspension for 5 minutes at 200 x g.200×g離心細(xì)胞懸液5分鐘。
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培養(yǎng)基中輕輕懸浮細(xì)胞顆粒。

1. Count the total number of viable cells using Trypan blue.使用臺盼藍(lán)計算活細(xì)胞總數(shù)。
2. Add 10 mL of VascuNet Growth Medium to each of two T75 flasks.將10毫升VascuneT培養(yǎng)基加入到兩個T75燒瓶中。
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.將PC-M細(xì)胞懸浮液均勻地劃分到兩個T75瓶之間，使每瓶容量約為2.5×105個PC-M細(xì)胞，密度為1.0×104pC-M細(xì)胞/cm2。

Note: The PC-M cells are plated at a higher density than the HUVECs.注：PC-M細(xì)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.在每個瓶中加入足夠量的VascuNet生長培養(yǎng)基，使總體積達(dá)到每T75瓶15 mL。
2. Incubate the cells overnight at 37°C with 5% CO2 humidified atmosphere.細(xì)胞在37°C和5%CO2加濕氣氛中過夜。

Exchange Medium in HUVEC Culture HUVEC培養(yǎng)中的交換培養(yǎng)基

1. Warm 60 mL of VascuNet Growth Medium at 37°C.37°C溫60 mL VascuNet培養(yǎng)基。
2. Observe HUVEC cultures under microscope to assess cell proliferation, confluency, and overall health.顯微鏡下觀察HUVEC培養(yǎng)，觀察細(xì)胞增殖、融合及整體健康狀況。
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培養(yǎng)瓶中抽吸培養(yǎng)基，每瓶更換30毫升新鮮VascuneT培養(yǎng)基。
4. Incubate cells at 37°C with 5% CO2 humidified atmosphere.

37°C條件下，5%CO2加濕培養(yǎng)細(xì)胞

ANGIOGENESIS ASSAY 血管生成試驗

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細(xì)胞被擴(kuò)增，HUVEC和PC-M細(xì)胞被鍍在96孔板的共培養(yǎng)和單培養(yǎng)井中進(jìn)行血管生成實驗。試驗與控制驢 我們建立了測試井，以確定試驗化合物對血管生成的影響。

DAY 0

Prepare 96-well Assay Plate制備96井試井板

1. Thaw sufficient amounts of Growth Factor Reduced (GFR) Matrigel aliquot(s) on ice at 2 to 8°C.在冰上解凍足夠數(shù)量的生長因子減少(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：融化足夠數(shù)量的GFR Matrigel，以覆蓋總數(shù)量的井用于實驗，包括控制排(見圖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孔組織培養(yǎng)板和P 100管尖，溫度-20℃，冷藏1小時。
2. Transfer the chilled 96-well plate, chilled pipet tips, and thawed GFR Matrigel bottle to an ice bucket in the tissue culture hood.將冰鎮(zhèn)的96井板、冰鎮(zhèn)管尖和融化的GFR Matrigel瓶轉(zhuǎn)移到組織培養(yǎng)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在溫度高于2°~8°C時會迅速凝固，以保證基體的均勻涂層和分布，使所有試劑、針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，經(jīng)常改變鍍液的。
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.將包覆的96孔板在室溫下孵育1h，再在37℃、5%CO2加濕氣氛下孵育1~2小時。

Prepare VascuNet Assay Medium制備VascuNet檢測介質(zhì)

1. Thaw the L-Glutamine (for assay medium) at 2 to 8°C. Allow the VascuNet Basal Assay Medium to warm to room temperature.將L-谷氨酰胺(用于檢測介質(zhì))在2到8°C解凍。允許VascuNet Basal分析介質(zhì)加熱到室溫。
2. Prepare the VascuNet Assay Medium by combining the reagents below.通過組合下面的試劑，準(zhǔn)備VascuNet測試介質(zhì)。

VascuNet Basal Assay Medium 95 mL  VascuNet Basal試驗培養(yǎng)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. 濾池用0.22m孔徑、低蛋白質(zhì)結(jié)合過濾器和無菌100 mL瓶對VascuNet分析培養(yǎng)基進(jìn)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.將50 mL VascuNet試劑盒轉(zhuǎn)入50 mL錐形管。在37℃的水浴中加熱30分鐘。所需介質(zhì)的數(shù)量可能會根據(jù)測試組件的數(shù)量而有所不同。
3. Store the remaining VascuNet Assay Medium at 2 to 8°C for up to 2 weeks.

將剩余的VascuNet檢測介質(zhì)保存在2至8°C處，多2周。

Harvest HUVECs  收獲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.分別從T 150瓶中抽吸培養(yǎng)基，每瓶加入10 mL PBS進(jìn)行洗滌。吸入PBS洗滌液。
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毫升酸性磷酸酶細(xì)胞脫落液，在室溫下孵育5分鐘，或直至細(xì)胞呈圓形。
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.在每個瓶中加入5毫升VascuNet分析培養(yǎng)基。牢固地敲擊瓶的側(cè)面，將細(xì)胞從培養(yǎng)表面釋放出來。
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.從每個燒瓶收集HUVECs，并將每個燒瓶內(nèi)的細(xì)胞轉(zhuǎn)移到15毫升錐形管中。輕輕吸管以分離的任何剩余的細(xì)胞團(tuán)。
5. Centrifuge the cells for 5 minutes at 250 x g.250×g離心5分鐘。
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，再將細(xì)胞顆粒懸浮到單個細(xì)胞懸液中，每管5 mL VascuNet檢測培養(yǎng)基。

Note: It is essential to thoroughly resuspend the cells to single-cell suspension before plating for the angiogenic assay.注：在進(jìn)行血管生成試驗前，必須將細(xì)胞*重新懸浮到單細(xì)胞懸浮液中。

1. Combine single cell suspensions (10 mL total volume) and pipet to mix.將單細(xì)胞懸液(總體積10 mL)與吸管混合。
2. Count the total number of viable cells in the combined cell suspension using Trypan blue.用臺盼藍(lán)計數(shù)組合細(xì)胞懸液中的活細(xì)胞總數(shù)。
3. Dilute the HUVECs in VascuNet Assay Medium to a concentration of 5 x 105 viable cells/mL.在VascuNet檢測培養(yǎng)基中稀釋HUVECs至5x105個活細(xì)胞/mL。

Harvest PC-M Cells 收獲PC-M細(xì)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.分別從T75瓶中抽吸培養(yǎng)基，每瓶加入5 mL PBS進(jìn)行洗滌。吸入PBS洗滌液。
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的酸性磷酸酶細(xì)胞脫落液，在室溫下孵育5分鐘，直至細(xì)胞呈圓形。
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.在每個瓶中加入2.5mL的VascuNet分析培養(yǎng)基。牢固地敲擊瓶的側(cè)面，將細(xì)胞從培養(yǎng)表面釋放出來。
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.從每個燒瓶中收集PC-M細(xì)胞，轉(zhuǎn)移到一個15 mL的錐形管中。輕輕地將任何剩余的細(xì)胞團(tuán)分離。
5. Centrifuge cells for 5 minutes at 250 x g.250×g離心細(xì)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檢測培養(yǎng)基中，將細(xì)胞顆粒再懸浮于單個細(xì)胞懸液中。

Note: It is essential to thoroughly resuspend the cells to single-cell suspension before plating for the angiogenic assay.注：在進(jìn)行血管生成試驗前，必須將細(xì)胞*重新懸浮到單細(xì)胞懸浮液中。

1. Measure the total number of viable cells in the sample by performing a cell count using Trypan blue.通過使用臺盼藍(lán)執(zhí)行細(xì)胞計數(shù)來測量樣本中可行的細(xì)胞總數(shù)。
2. Dilute the PC-M cells in VascuNet Assay Medium to a concentration of 1 x 105 viable cells/mL.

在VascuNet檢測培養(yǎng)基中稀釋PC-M細(xì)胞，濃度為1x105個活細(xì)胞/mL。

Plate HUVECs and PC-M Cells平板HUVECs和PC-M細(xì)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.參考樣板設(shè)置圖(圖1).除試驗井外，96口井板中的一行井將用于控制井。確定所需單元格的總數(shù)，在 到控制行，根據(jù)測試樣本的數(shù)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.對于每一組三重的HUVEC單培養(yǎng)樣品，將1.32×105 HUVEC與足夠體積的Vascuneta試驗培養(yǎng)基相結(jié)合，使總體積為每三套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.將150 L的HUVEC細(xì)胞懸液分裝于每口培養(yǎng)井中，平均每孔培養(yǎng)4.0x104個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.對于每組三份PC-M單細(xì)胞培養(yǎng)樣品，將6.6×103個PC-M細(xì)胞與足夠體積的VascuNet檢測培養(yǎng)基結(jié)合，使總體積達(dá)到每三份樣品495 L。

Note: This will make a 20:1 ratio of HUVEC:PC-M cells.注：這將使HUVEC：PC-M細(xì)胞的比例達(dá)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.將150 L的PC-M細(xì)胞懸液注入每孔，每孔培養(yǎng)一次，共培養(yǎng)0.2x104個PC-M細(xì)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.每組三份共培養(yǎng)樣品，將1.32x105個HUVECs與6.6×103個PC-M細(xì)胞結(jié)合.加入足夠量的VascuNet檢測介質(zhì)，使總體積達(dá)到每三份495升 部分，段

Addition of the Test Compounds添加試驗化合物

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小時的潛伏期后，目視檢查水井，以確定在HUVEC單細(xì)胞培養(yǎng)和共培養(yǎng)條件下的附著和管狀形成(見圖2)。確認(rèn)后 初的管狀，這些細(xì)胞已準(zhǔn)備好用試驗試劑處理。為了取得良好的效果，這是在電鍍后4至6小時，但必須在24小時內(nèi)完成電池被鍍。

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.

圖2.VascuNet HUVEC和PC-M細(xì)胞共培養(yǎng)4小時.人臍靜脈內(nèi)皮細(xì)胞(HUVEC)與PC-M細(xì)胞按20：1比例共培養(yǎng)，4小時開始形成管狀網(wǎng)絡(luò)。

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.陽性對照井在VascuNet檢測培養(yǎng)基中含有20：1(HUVEC：PC-M細(xì)胞)比例的細(xì)胞。在這些井中不添加任何額外的化合物。

Negative Control Wells負(fù)壓井

The negative control samples are run in triplicate co-culture conditions with 50 µM Suramin Hexasodium Salt.陰性對照樣品一式三份，共培養(yǎng)條件為50 m蘇拉明六鈉鹽。

1. Thaw the vial of Suramin Hexasodium Salt for 1 hour at room temperature.在室溫下解凍蘇拉明六鈉鹽1小時。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分析介質(zhì)中加入25升1毫米蘇拉明六鈉鹽，使終的六鈉濃度為50 M，然后將50 M蘇拉明六鈉鹽溶液加熱至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.從每口細(xì)胞中抽取VascuNet檢測介質(zhì)，用50 M蘇拉明六鈉鹽溶液中的每口150毫升代替。

Experimental Wells 實驗井

Experimental wells should be tested in co-culture wells in triplicates. Triplicate monoculture samples can also be run for comparison.實驗威爾斯應(yīng)在共培養(yǎng)威爾斯中試驗三次。三份的單一栽培樣品也可用于比較。

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分析培養(yǎng)基稀釋至所需濃度，制備額外的親或抗血管生成試驗化合物。使用前將溫?zé)嵯♂尩膹?fù)合溶液稀釋至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.從每口井中抽吸VascuNet檢測介質(zhì)，用含適當(dāng)試液的VascuNet分析介質(zhì)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小時觀察和成像細(xì)胞，以監(jiān)測血管生成管的組裝和穩(wěn)定性。

注：在含有HUVECs和PC-M細(xì)胞的共培養(yǎng)中，血管管狀細(xì)胞在培養(yǎng)中至少穩(wěn)定4天，不需要更換培養(yǎng)基或添加外源因子。

EXPECTED RESULTS預(yù)期結(jié)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).以下數(shù)據(jù)描述了在建議的細(xì)胞播種密度為每井42，000個細(xì)胞時，共培養(yǎng)和單一培養(yǎng)條件下的預(yù)期結(jié)果，其中含有20：1的HUVECs與pc-m的比例。 細(xì)胞(40，000 HUVECs和2，000個PC-M細(xì)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單細(xì)胞早可在4小時內(nèi)形成管狀網(wǎng)絡(luò)。24小時可觀察到完整的管狀網(wǎng)絡(luò)。缺乏pc-M細(xì)胞支持的HUVEC管應(yīng)在第2天發(fā)生不穩(wěn)定。 血管生成實驗和不重新組裝。

• PC-M cell monocultures do not form complete tube networks. Minimal branching may be observed.PC-M細(xì)胞不形成完整的管狀網(wǎng)絡(luò).可以觀察到小分支。

• Co-cultures form tube networks within four days and are maintained for up to 6 days.共培養(yǎng)在4天內(nèi)形成管狀網(wǎng)絡(luò)，并維持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.在單一培養(yǎng)和共培養(yǎng)中加入50μm蘇拉明六鈉鹽(陰性對照)，可在處理后4~24小時內(nèi)使試管形成減少90%以上。蘇拉米的存在 N六鈉鹽可以防止管重新組裝至少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.

圖3.隨著時間的推移，管狀結(jié)構(gòu)的穩(wěn)定性。HUVEC單眼(第1行和第2行)在12萬個細(xì)胞/cm2下播種，用VybrantDio(綠色)染色，在播種后第1天出現(xiàn)管狀形成。白天 2、已觀察到船舶的高度。相比之下，具有多個分支點的管狀結(jié)構(gòu)從第1天就可以看到，在HUVEC和pc-M細(xì)胞共培養(yǎng)中，在第6天仍保持穩(wěn)定。 紅色水井按20：1比例鍍制(第3和第4行)。PC-M細(xì)胞用Vybrant Dil染紅。

圖像以4X放大倍數(shù)拍攝。

APPENDIX

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