PROTOCOL

VascuNet™ Pericyte Co-Culture Assay

VascuNet™Pericyte共培養試驗

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絡發育的機制。內皮細胞和周細胞都是這些過程中的關鍵細胞類型。而內皮 細胞是血管的內層，周細胞通過穩定已建立的血管結構和促進網絡e的局部重塑，對功能血管的發展至關重要。 xpansion。除了傳遞結構支持外，周細胞還通過細胞與細胞的接觸和旁分泌信號來引導內皮細胞。周細胞已被證明為共室細胞。 在正常和異常血管系統中，內皮細胞與血管內皮細胞結合，并參與多種病理學中的中心作用，包括腫瘤發生、神經退行性疾病。 糖尿病視網膜病變。

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.

人胚胎干細胞(esi-017)衍生周細胞(pc-M細胞)與原代人臍靜脈內皮細胞(HUVECs)在共培養系統中的結合。 設計的96井板格式。這些*的pc-M細胞顯示了周細胞的幾個關鍵特性，包括CD 146的表達、促血管生成功能以及內皮細胞的有效穩定。 光管網絡。HUVECs和pc-M細胞共培養系統支持血管生成管的組裝，從而產生了在Cultur至少持續4至6天的廣泛的管狀網絡。 e.用VascuNet Pericyte共培養法形成的致血管網絡在培養過程中的持續時間是其他檢測系統的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. 所有活細胞的工作應在組織培養的無菌生物安全柜內完成。
• 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細胞或HUVECs在擴張過程中增殖到90%以上。在高度匯合的情況下，可形成密集的細胞團簇，從而減少血管生成管的組裝。 單一文化和共同文化。在進行血管生成試驗之前，一定要將細胞重新懸浮到單細胞懸浮液中.
• 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HUVECs與PC-M細胞以42，000細胞/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-周細胞共培養檢測試劑盒含有PC-M細胞、HUVECs和所有用于細胞擴增和血管生成測定的培養基成分。每個套件都進行了全面的質量控制，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時以的溫度存儲。VascuNet Pericyte共同培養測試組件從收到時起至少3個月內是穩定的。 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，*LDEV-無
• Phosphate Buffered Saline (PBS)
• 磷酸鹽緩沖鹽(PBS)
• Accutase® Cell Detachment Solution
• 酸性磷酸酶細胞脫離液
• Trypan blue or alternative cell viability assay
• 臺盼藍或替代細胞活力測定

• 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組織培養瓶
• T75 tissue culture flasks
• T75組織培養瓶
• 96-well tissue culture plate

96孔組織培養板

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單一培養參考資料(40，000個細胞/井)

P：PC-M單細胞培養參比(2，000個細胞/井)

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

藍：HUVEC&PC-M細胞共培養(陽性對照)

黃：HUVEC&PC-M細胞與50 M蘇拉明六鈉鹽共培養(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井的平板上進行測試。VascunetPericyte共培養試驗的行是對照行，包含三份單一培養井的樣本以及陽性和陰性的樣本。 控制共培養井。該板的其余84口井可用作試驗化合物。測試復合測試可以按照與控制行相同的單文化和共培養設置執行。 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細胞的擴增

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細胞必須單獨解凍和擴張兩天，然后才能以共同培養的形式進行實驗設置。

監測細胞增殖，不允許HUVECs或PC-M細胞在擴張過程中增殖至90%以上。在高匯合度時，可形成密集的細胞團簇。 單培養和共培養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細胞擴增用VascuNet生長培養基的制備

將以下VascuNet生長介質從-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

通過結合下面列出的所有試劑，準備VascuNet生長培養基。

VascuNet Basal培養基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、低蛋白質結合的過濾裝置消毒VascuNet培養基，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培養基轉移到無菌的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移到4毫升的預熱VascuNet生長培養基中，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培養基中輕輕懸浮細胞顆粒。

1. Count the total number of viable cells using Trypan blue.使用臺盼藍計算活細胞總數。
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生長培養基，使總體積達到每T 150瓶30 mL。

1. Incubate the cells overnight at 37°C with 5% CO2 humidified atmosphere.細胞在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培養基轉移到無菌的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細胞在37°C水浴中解凍。將小瓶的全部內容轉移到4mL預加熱的VascuNet生長培養基中，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培養基中輕輕懸浮細胞顆粒。

1. Count the total number of viable cells using Trypan blue.使用臺盼藍計算活細胞總數。
2. Add 10 mL of VascuNet Growth Medium to each of two T75 flasks.將10毫升VascuneT培養基加入到兩個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細胞，密度為1.0×104pC-M細胞/cm2。

Note: The PC-M cells are plated at a higher density than the HUVECs.注：PC-M細胞的密度高于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生長培養基，使總體積達到每T75瓶15 mL。
2. Incubate the cells overnight at 37°C with 5% CO2 humidified atmosphere.細胞在37°C和5%CO2加濕氣氛中過夜。

Exchange Medium in HUVEC Culture HUVEC培養中的交換培養基

1. Warm 60 mL of VascuNet Growth Medium at 37°C.37°C溫60 mL VascuNet培養基。
2. Observe HUVEC cultures under microscope to assess cell proliferation, confluency, and overall health.顯微鏡下觀察HUVEC培養，觀察細胞增殖、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培養瓶中抽吸培養基，每瓶更換30毫升新鮮VascuneT培養基。
4. Incubate cells at 37°C with 5% CO2 humidified atmosphere.

37°C條件下，5%CO2加濕培養細胞

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細胞被擴增，HUVEC和PC-M細胞被鍍在96孔板的共培養和單培養井中進行血管生成實驗。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：融化足夠數量的GFR Matrigel，以覆蓋總數量的井用于實驗，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.將冰鎮的96井板、冰鎮管尖和融化的GFR Matrigel瓶轉移到組織培養罩中的冰桶中。

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

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)在2到8°C解凍。允許VascuNet Basal分析介質加熱到室溫。
2. Prepare the VascuNet Assay Medium by combining the reagents below.通過組合下面的試劑，準備VascuNet測試介質。

VascuNet Basal Assay Medium 95 mL  VascuNet Basal試驗培養基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、低蛋白質結合過濾器和無菌100 mL瓶對VascuNet分析培養基進行消毒。
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試劑盒轉入50 mL錐形管。在37℃的水浴中加熱30分鐘。所需介質的數量可能會根據測試組件的數量而有所不同。
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瓶中抽吸培養基，每瓶加入10 mL PBS進行洗滌。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毫升酸性磷酸酶細胞脫落液，在室溫下孵育5分鐘，或直至細胞呈圓形。
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分析培養基。牢固地敲擊瓶的側面，將細胞從培養表面釋放出來。
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，并將每個燒瓶內的細胞轉移到15毫升錐形管中。輕輕吸管以分離的任何剩余的細胞團。
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，再將細胞顆粒懸浮到單個細胞懸液中，每管5 mL VascuNet檢測培養基。

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

1. Combine single cell suspensions (10 mL total volume) and pipet to mix.將單細胞懸液(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.用臺盼藍計數組合細胞懸液中的活細胞總數。
3. Dilute the HUVECs in VascuNet Assay Medium to a concentration of 5 x 105 viable cells/mL.在VascuNet檢測培養基中稀釋HUVECs至5x105個活細胞/mL。

Harvest PC-M Cells 收獲PC-M細胞

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瓶中抽吸培養基，每瓶加入5 mL PBS進行洗滌。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的酸性磷酸酶細胞脫落液，在室溫下孵育5分鐘，直至細胞呈圓形。
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分析培養基。牢固地敲擊瓶的側面，將細胞從培養表面釋放出來。
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細胞，轉移到一個15 mL的錐形管中。輕輕地將任何剩余的細胞團分離。
5. Centrifuge cells for 5 minutes at 250 x g.250×g離心細胞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檢測培養基中，將細胞顆粒再懸浮于單個細胞懸液中。

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

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

在VascuNet檢測培養基中稀釋PC-M細胞，濃度為1x105個活細胞/mL。

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

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，在 到控制行，根據測試樣本的數量。

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單培養樣品，將1.32×105 HUVEC與足夠體積的Vascuneta試驗培養基相結合，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細胞懸液分裝于每口培養井中，平均每孔培養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單細胞培養樣品，將6.6×103個PC-M細胞與足夠體積的VascuNet檢測培養基結合，使總體積達到每三份樣品495 L。

Note: This will make a 20:1 ratio of HUVEC:PC-M cells.注：這將使HUVEC：PC-M細胞的比例達到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細胞懸液注入每孔，每孔培養一次，共培養0.2x104個PC-M細胞。
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.每組三份共培養樣品，將1.32x105個HUVECs與6.6×103個PC-M細胞結合.加入足夠量的VascuNet檢測介質，使總體積達到每三份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單細胞培養和共培養條件下的附著和管狀形成(見圖2)。確認后 初的管狀，這些細胞已準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細胞共培養4小時.人臍靜脈內皮細胞(HUVEC)與PC-M細胞按20：1比例共培養，4小時開始形成管狀網絡。

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檢測培養基中含有20：1(HUVEC：PC-M細胞)比例的細胞。在這些井中不添加任何額外的化合物。

Negative Control Wells負壓井

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.從每口細胞中抽取VascuNet檢測介質，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.實驗威爾斯應在共培養威爾斯中試驗三次。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。使用前將溫熱稀釋的復合溶液稀釋至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，用含適當試液的VascuNet分析介質每井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小時觀察和成像細胞，以監測血管生成管的組裝和穩定性。

注：在含有HUVECs和PC-M細胞的共培養中，血管管狀細胞在培養中至少穩定4天，不需要更換培養基或添加外源因子。

EXPECTED RESULTS預期結果

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).以下數據描述了在建議的細胞播種密度為每井42，000個細胞時，共培養和單一培養條件下的預期結果，其中含有20：1的HUVECs與pc-m的比例。 細胞(40，000 HUVECs和2，000個PC-M細胞).

• 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單細胞早可在4小時內形成管狀網絡。24小時可觀察到完整的管狀網絡。缺乏pc-M細胞支持的HUVEC管應在第2天發生不穩定。 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細胞不形成完整的管狀網絡.可以觀察到小分支。

• Co-cultures form tube networks within four days and are maintained for up to 6 days.共培養在4天內形成管狀網絡，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.在單一培養和共培養中加入50μm蘇拉明六鈉鹽(陰性對照)，可在處理后4~24小時內使試管形成減少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，管狀結構的穩定性。HUVEC單眼(第1行和第2行)在12萬個細胞/cm2下播種，用VybrantDio(綠色)染色，在播種后第1天出現管狀形成。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構從第1天就可以看到，在HUVEC和pc-M細胞共培養中，在第6天仍保持穩定。 紅色水井按20：1比例鍍制(第3和第4行)。PC-M細胞用Vybrant Dil染紅。

圖像以4X放大倍數拍攝。

APPENDIX

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