TWI779450B - 去細胞的細胞外基質及其製備方法與用途 - Google Patents
去細胞的細胞外基質及其製備方法與用途 Download PDFInfo
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- TWI779450B TWI779450B TW110100186A TW110100186A TWI779450B TW I779450 B TWI779450 B TW I779450B TW 110100186 A TW110100186 A TW 110100186A TW 110100186 A TW110100186 A TW 110100186A TW I779450 B TWI779450 B TW I779450B
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Abstract
本發明提供一種去細胞的細胞外基質及其製備方法與用途。本發明去細胞的細胞外基質衍生自一個三維細胞球體，該去細胞的細胞外基質具有一為三維的球狀結構。本發明去細胞的細胞外基質可用於製備一促進組織再生及修復的生醫材料支架。
Description
本發明是有關於一種去細胞的細胞外基質及其製備方法與用途。
臨床與研究中常使用於再生醫學與組織修復的生醫材料支架(scaffold)，其成分主要可區分為人工高分子以及天然高分子。合成高分子材料以聚乙醇酸、聚乳酸、聚乳酸-甘醇酸以及聚己內脂最為廣泛運用，具有生物相容性、生物降解性以及可精確控制等優點，缺點則是無法建構出類似於細胞外基質(extracellular matrix, ECM)的微結構與成分，故用於促進細胞生長與組織再生的潛能有限。
儘管天然高分子，例如從動物組織中萃取而得的膠原蛋白及透明質酸皆為ECM的成份，但天然ECM的結構與組成皆相當複雜，僅用少數種類的ECM分子並不能完美模擬體內環境，因而無法提供適當的區位(niche)來有效支持細胞的各項功能或促進組織再生。如何建構一類天然組織之基質支架，是再生醫學領域尚待解決的問題。
為了克服高分子材料無法完整建立類似組織內部的基質微環境，先前技術曾以生物組織甚至器官為原料，將其進行去細胞處理後，作為一具有生物活性之支架。儘管去細胞組織已被實際應用於臨床上，其仍有諸多缺點與安全風險需要面對，如免疫排斥、疾病傳染、批次間品質差異、大小與尺寸受限等。此外，受限於來源組織的特性，該去細胞組織無法被用於多種目標組織的修復。
為了解決上述生物組織衍生之去細胞支架所面對的問題，先前技術曾使用二維體外培養細胞所產生的基質作為支架。然而二維培養方式耗時漫長(數週至數月)，製作出來的去細胞的細胞外基質(decellularized extracellular matrix, dECM)卻相當薄且脆弱，不利於後續的操作與使用。此外，二維培養所形成的dECM不僅尺度受限於培養容器的大小，其微結構與三維dECM有相當顯著的差異。
為了解決上述問題，本領域的技術人員亟需研發出新穎且有效組織再生及修復的去細胞的細胞外基質、其製備方法與生醫材料支架以造福有此需求的廣大族群。
有鑑於此，本發明之目的為提供一種去細胞的細胞外基質(decellularized extracellular matrix)，係衍生自一個三維細胞球體，該去細胞的細胞外基質具有一三維的球狀結構。
本發明之另一目的為提供一種用於製備一去細胞的細胞外基質(decellularized extracellular matrix)的方法，包含：(a)對複數個細胞進行體外培養以構築一個三維細胞球體；以及(b)使用一鹼性之非離子介面活性劑及一去氧核醣核酸酶對該三維細胞球體進行一去細胞處理，得到該去細胞的細胞外基質，其中該去細胞的細胞外基質具有一為三維的球狀結構。
在本發明的一實施例中，去細胞的細胞外基質包含至少一生物活性成分。
在本發明的一實施例中，該至少一生物活性成分為一生長因子、一細胞激素或一外泌體。
在本發明的一實施例中，去細胞的細胞外基質進一步包含一第一型膠原蛋白、一纖維接合素(fibronectin)及一層黏連蛋白(laminin)。
在本發明的一實施例中，該生長因子是一血管內皮生長因子(vascular endothelial growth factor, VEGF)。
在本發明的一實施例中，去細胞的細胞外基質藉由使用一鹼性之非離子介面活性劑及一去氧核醣核酸酶進行一去細胞處理。
在本發明的一實施例中，複數個細胞會貼附於該去細胞的細胞外基質的表面進行增生。
在本發明的一實施例中，去細胞的細胞外基質係藉由至少一醣類巨分子而被誘發大分子擁擠現象(macromolecular crowding, MMC)，俾以促進細胞增生。
在本發明的一實施例中，該三維細胞球體為一由複數個細胞所形成的三維細胞球體。
本發明之另一目的為提供一種如前所述的去細胞的細胞外基質用於製備一促進組織再生及修復的生醫材料支架的用途。
綜上所述，本發明去細胞的細胞外基質的功效在於：由於基質為三維培養的細胞所製造，故其成分與微結構皆相當類似於生物組織中的基質，實驗結果顯示，細胞可以有效貼附於三維的去細胞的細胞外基質表面，並進行增生。由於三維的去細胞的細胞外基質內尚有諸多細胞所分泌的生物活性成分，如生長因子、細胞激素與外泌體等，故具有調節後續貼附細胞行為之功能。藉由改變構築三維細胞球體之細胞種類或控制細胞行為，其後續留存於三維的去細胞的細胞外基質內部的生物活性分子組成也不盡相同，因此具有相當廣泛的應用性，例如使用幹細胞來建立三維球體，其留存下來的生物活性分子即能有助於血管新生與組織再生的發生。藉由醣類巨分子所誘發之大分子擁擠現象(macromolecular crowding, MMC)，三維細胞球體中的細胞外基質(ECM)與生長因子分泌總量可顯著上升，進而使後續衍生之去細胞的細胞外基質內的ECM分子與生長因子含量有所增加，進一步提昇去細胞的細胞外基質的生物活性。此外，每個三維的去細胞的細胞外基質可視為一小型支架，其大小與去細胞前之三維細胞球體相同，因此可依據需求製備不同直徑之三維的去細胞的細胞外基質。將細胞培養於三維的去細胞的細胞外基質後，除了能將其作為細胞傳輸之載體(例如以注射方式將含有細胞之三維的去細胞的細胞外基質移植至欲治療的部位)以外，複數個三維的去細胞的細胞外基質能夠藉由多種方式組裝成為一大型結構。由於三維的去細胞的細胞外基質相當容易大量製造，因此其聚合而成的大型支架在形狀與大小上皆不受限制，能夠高度客製化。再者，由於細胞是先培養於個別的三維的去細胞的細胞外基質上，故聚集後細胞可均勻分布於整個大型支架結構中，解決傳統組織工程所遇到的細胞分布不均問題。
以下將進一步說明本發明的實施方式，下述所列舉的實施例係用以闡明本發明，並非用以限定本發明之範圍，任何熟習此技藝者，在不脫離本發明之精神和範圍內，當可做些許更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
定義
本文中所使用數值為近似值，所有實驗數據皆表示在20%的範圍內，較佳為在10%的範圍內，最佳為在5%的範圍內。
以下實驗數據以平均值
標準偏差(mean
SD)表示。所有
p值使用單因子變異數分析(one-way ANOVA)及杜凱氏校正(Tukey correction)來計算。統計分析是使用GraphPad Prism軟體(8.4.3版，San Diego, CA, USA)進行。
p＜ 0.05表示有統計學顯著差異。
以下實施例所用的人類臍帶血(human umbilical cord blood)間葉幹細胞(mesenchymal stem cells, MSCs)及人類臍帶靜脈內皮細胞(human umbilical cord vein endothelial cells, HUVECs)購自台灣新竹食品工業發展研究所生物資源保存及研究中心(Bioresource Collection and Research Center, BCRC)。MSCs在CMV啟動子(pDsRed-N1載體)的控制下表現紅色螢光蛋白(RFP)，從而易於追蹤細胞(參見C.J. Hung, C.L. Yao, F.C. Cheng, et al., Establishment of immortalized mesenchymal stromal cells with red fluorescence protein expression for
in vivotransplantation and tracing in the rat model with traumatic brain injury, Cytotherapy 12(4) (2010) 455-465)。MSCs維持在補充有20%胎牛血清(fetal bovine serum, FBS)(GE Healthcare Bio-Sciences, Pittsburgh, PA, USA)、4 ng/mL鹼性纖維母細胞生長因子(basic fibroblast growth factor)(PeproTech, Rocky Hill, NJ, USA)、30 mg/mL潮黴素B (hygromycin B)(Thermo Fisher Scientific)及200 mg/mL遺傳黴素(geneticin)(Thermo Fisher Scientific)的最低必需培養基
(minimum essential medium
)(Thermo Fisher Scientific, Waltham, MA, USA)中。HUVECs在內皮細胞生長培養基(endothelial cell growth medium, EGM)-2 (Lonza Walkersville, MA, USA)中培養。將細胞在加濕的培養箱中於37°C、5% CO
2下培育。
實施例 1. 去細胞的細胞外基質 (decellularized extracellular matrix, dECM) 的製備、構形及生物活性成分分析
在本實施例用到的免疫螢光染色的操作流程如下。用4%多聚甲醛(paraformaldehyde)(Sigma-Aldrich)將三維細胞球體或衍生的dECM支架固定20分鐘。在室溫下用0.5% Triton X-100透化(permeabilization)並用5%正常山羊血清(normal goat serum)(Vector Laboratories, Burlingame, CA, USA)封阻2小時後，將樣品與抗血管內皮生長因子(vascular endothelial growth factor, VEGF)、纖維接合素(fibronectin)、層黏連蛋白(laminin)及膠原蛋白(均來自台灣新竹Genetex)的一級抗體(配於5%牛血清白蛋白(bovine serum albumin)中)於4°C培育過夜。用PBS洗滌3次(每次5分鐘)後，使用綴合有Alexa Fluor 488或Alexa Fluor 633的二級抗體(Thermo Fisher Scientific)在4°C下過夜。最後，將測試樣品用PBS洗滌，用4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole, DAPI)(Thermo Fisher Scientific)複染，並用組織清除溶液(tissue-clearing solution)(FocusClear溶液，CelExplorer，新竹，台灣)固定，並用共軛焦顯微鏡(LSM 780, Carl Zeiss, Oberkochen，德國)觀察。
在本實施例用到的DNA定量的操作流程如下。為了確定DNA含量，使用200 μL的含0.1 mg/mL蛋白酶K (proteinase K)(Sigma-Aldrich)的消化緩衝液(digestion buffer)(10 mM Tris-HCl及1 mM乙二胺四乙酸(EDTA))在50°C過夜以裂解25個細胞球體或dECM支架。根據製造商的操作指引，使用Quant-iT PicoGreen dsDNA定量套組(Thermo Fisher Scientific)定量分析裂解液(lysate)中的DNA量。簡言之，將Quant-iT PicoGreen試劑添加到裂解液中，反應5分鐘，然後使用微盤讀取儀(SpectraMax iD3, Molecular Devices, Sunnyvale, CA, USA)在激發波長480 nm及發射波長520 nm下進行螢光測量。相對於使用
DNA (Thermo Fisher Scientific)產生的標準曲線測定絕對DNA含量。
在本實施例用到的酵素結合免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)的操作流程如下。在本實施例中使用一種ELISA套組(RayBiotech, Peachtree Corners, GA, USA; Cat. No. ELH-VEGF-CL)，用於確定裂解液中VEGF的濃度。簡言之，使用200 µL的裂解緩衝液(由製造商提供)裂解25個細胞球體或dECM支架。在4,500 rpm離心10分鐘後，使用二金雞鈉酸測定法(bicinchoninic acid assay)(G-Biosciences, St.Louis, MO, USA)對上清液的總蛋白質水平進行定量。之後，將所有測試樣品標準化為在100 µL的緩衝液中20 µg的總蛋白質，並根據製造商的操作手冊對VEGF含量進行定量。
首先，使用基於甲基纖維素(methylcellulose, MC)水凝膠的方法製造三維細胞球體。簡言之，將在0.5x磷酸鹽緩衝生理鹽水(phosphate buffered saline, PBS)(Sigma-Aldrich)中製備的50 µL的12% (w/v) MC (Sigma-Aldrich, St. Louis, MO, USA)溶液裝入96孔盤的每個孔中，並加熱至37°C歷時30分鐘。藉由使用TrypLE試劑(Thermo Fisher Scientific)對融合細胞(confluent cells)進行輕度胰蛋白酶消化來製備MSC懸浮液。然後使用培養基將細胞稀釋至所需的細胞密度。或者，對培養基補充各種擁擠試劑(包括Ficoll 70 (37.5 mg/mL; Cytiva, Marlborough, MA)、Ficoll 400 (25 mg/mL; Cytiva)、Ficoll混合物(37.5 mg/mL Ficoll 70及25 mg/mL Ficoll 400)，或葡聚糖硫酸(dextran sulfate)(DxS, MW: 500kDa; 10 µg/mL; Sigma-Aldrich; D8906))被用來懸浮MSC，以誘導大分子擁擠現象(macromolecular crowding,MMC)的排除體積效應(excluded volume effect)。最後，將150μL的MSC懸浮液轉移到裝有MC的96孔盤的每個孔中，並培育24小時以進行球體組裝(spheroid assembly)。
培育24小時後，在每個孔中觀察到三維細胞球體。共軛焦影像表明，MSC在形成的三維細胞球體內沉積多種ECM蛋白，包括第一型膠原蛋白(collagen I)、纖維接合素(fibronectin)及層黏連蛋白(laminin)(參見圖1A)，證明它們有潛力用作細胞衍生的dECM支架的來源。
在處理細胞球體進行去細胞之前，本發明試圖藉由延長培育期來最大化細胞球體內MSCs分泌的生物活性成分的含量。在此，選擇血管內皮生長因子(vascular endothelial growth factor,VEGF)作為標靶因子(target factor)，因為VEGF誘導的血管生成(VEGF-induced angiogenesis)產生新生的脈管系統(vasculature)，這對於隨後的移植物整合(graft integration)及組織再生至關重要。藉由免疫螢光顯微鏡(參見圖1B)檢測球體中的VEGF，並藉由ELISA進行定量(參見圖1C)。由圖1B及1C可見，細胞球體組裝後，VEGF含量增加並在2天內達到高峰(與第1天相比，升高了5.4倍，p<0.001)，而長時間的培育導致VEGF水平顯著下降。因此，將在接種後48小時形成的三維MSC球體用於後續實驗。
接著，製備衍生自三維細胞球體的去細胞的細胞外基質(decellularized extracellular matrix,dECM)。收集三維MSC細胞球體並進行處理以除去細胞及核內含物。藉由使用鹼性清潔劑處理(alkaline detergent treatment)或凍融循環法(freeze-thaw cycling method)處理的三維細胞球體中的細胞。在此，採用凍融循環法(比較組)及Triton X-100處理這兩種不同的方法。兩種方法均顯示出去除核及細胞內含物的高效率，分別由DAPI及RFP的螢光強度顯著降低所表明(參見圖2A)。對於多個凍融循環，將收集的細胞球體懸浮在離心管中的PBS中，將其冷卻至-80℃歷時30分鐘並在37℃水浴中融化。此程序重複三次。對於基於清潔劑(detergent)的方法，將細胞球體浸入含有0.5%(v/v)Triton X-100(Sigma-Aldrich)及20mM氫氧化銨(ammonium hydroxide)的去細胞溶液中，並在室溫下培育1小時。洗滌3次PBS後，兩個程序均在37℃下與1kU/mL DNase I(Sigma-Aldrich)培育30分鐘，並在進一步實驗前用PBS沖洗兩次，得到去細胞的細胞外基質。
此外，如第一型膠原蛋白及層黏連蛋白的螢光影像所顯示的，沉積的ECM蛋白及其總體框架(gross framework)得以保留(參見圖2A)。然後，本實施例進一步量化殘留DNA (參見圖2B)或VEGF (參見圖2C)的量。結果顯示，各組中殘餘VEGF的水平是可比較的(參見圖2C)。然而，使用DNase I處理的凍融循環僅去除了72.9%的宿主DNA，而基於Triton X-100的萃取方法則去除了98.1%的總雙股DNA (dsDNA)(參見圖2B)。由於應達到至少90%的宿主DNA去除率以防止dECM支架的免疫排斥，因此在以下實驗中使用鹼性Triton X-100。
實施例 2. 去細胞的細胞外基質 (decellularized extracellular matrix, dECM) 的尺寸優化
由於較大的球體尺寸難以確保高效去除細胞核物質，這對於製備支架非常重要，因此本實施例藉由調節細胞接種密度進一步優化球體的直徑。將具有各種細胞接種密度(每個球體5,000、7,000、10,000及12,000個細胞)製備的三維MSC細胞球體進行去細胞處理，然後進行DNA定量。如螢光影像(參見圖3A)及PicoGreen測定結果(參見圖3B)所示，隨著細胞接種密度的增加，生長的細胞球體的直徑及其DNA含量也隨之增加。去細胞後，具有5,000及7,000個細胞的細胞球體的DNA去除率(分別為97.8%及94.7%)比具有10,000及12,000個細胞的細胞球體的DNA去除率(分別為85.7%及84.6%)更高(參見圖3B)。因此，每個球體以7,000個細胞製造的三維MSC細胞球體經去細胞處理而得到的細胞外基質用於後續實驗。
實施例 3. 補充大分子對於增強 三維 MSC 的細胞外基質 (extracellular matrix, ECM) 沉 積的效用評估
在本實施例用到的西方墨點分析(Western blotting analysis)的操作流程如下。藉由與200 µL的含蛋白酶抑制劑雞尾酒錠(protease inhibitor cocktail tablets)(Sigma-Aldrich)的RIPA緩衝液(20 mM Tris-HCl、1 mM EGTA、150 mM NaCl及1% Triton X-100)一起反應來裂解測試樣品(25個細胞球體或dECM支架)。將裂解液在95°C下變性10分鐘，經由十二烷基硫酸鈉(sodium dodecyl sulfate, SDS)-聚丙烯醯胺凝膠電泳(polyacrylamide gel electrophoresis)在8%丙烯醯胺凝膠(Bio-Rad Laboratories, Hercules, CA, USA)中以80 V的解析電壓進行解析，並轉移到聚偏氟乙烯膜(polyvinylidene difluoride membrane)上。用5%脫脂奶封阻1小時並與抗纖維接合素或層黏連蛋白的一級抗體培育過夜後，使用Amersham ECL Select西方墨點檢測試劑(Cytiva)及綴合辣根過氧化酶(horseradish peroxidase, HRP)的二級抗體(GeneTex)檢測膜。
基於碳水化合物的大分子Ficolls及DxS被證明分別經由大分子擁擠現象(macromolecular crowding, MMC)或共沉澱來放大二維培養中的ECM沉積。為了研究這些大分子是否會對三維中的ECM積累產生可比的影響，將MSCs暴露於Ficolls或DxS，同時在MC水凝膠表面組裝成三維球體構形。本實施例利用為二維培養建立的大分子濃度。以100 µg/mL補充DxS，並以25 mg/mL的Ficoll 400及37.5 mg/mL的Ficoll 70的混合物形式添加Ficolls。除此之外，本實施例還探索每種Ficoll子類別在確定濃度下的影響。從形態上而言，具有MMC的三維MSC細胞球體類似於在對照條件下獲得的組別(參見圖4A)，直徑約為270 µm (參見表1)，但Ficoll混合物組的直徑明顯較小。下面表1顯示有無添加擁擠試劑(crowding agents)製造的三維MSC細胞球體的平均大小(n = 5個批次中的30個球體)。*表示與對照組比較，
p＜ 0.05。
表1
| 組別 | 對照組 | Ficoll 70 | Ficoll 400 | Ficoll混合物 | DxS |
| 直徑 (µm) | 264.4 ± 17.1 | 264.9 ± 11.8 | 277.3 ± 9.9 | 204.4 ± 5.6* | 270.2 ± 8.2 |
接著，收集這些球體，進行去細胞，並分別藉由免疫染色及西方墨點法分析其層黏連蛋白分佈及ECM蛋白含量。層黏連蛋白染色的結果表明，大分子的添加促進具有明確定義的層黏連蛋白纖維的體內類原纖維或網狀超結構的形成(
vivo-like fibrillar or reticular ultrastructural organization)(參見圖4B)。相反地，對照組球體顯示層黏連蛋白瀰漫性染色，細胞周圍有細纖維(thin fibrils)(參見圖4B)。此外，補充Ficoll 70及Ficoll 400導致三維dECM支架的層黏連蛋白水平顯著增加(與對照組相比增加了5.3倍及6.1倍；
p＜0.05)，並且用Ficoll 400處理導致最大量的纖維接合素(相對於對照組增強2.6倍；
p＜0.001；參見圖4C)。藉由使用ELISA分析dECM支架中殘留的VEGF的量，本實施例發現所有MMC處理組均表現出升高的VEGF水平，特別是Ficoll 400組(與對照組相比增加4.2倍;
p＜0.005，參見圖4D)。此外，藉由比較三維dECM支架與相應的三維MSC細胞球體中的殘留ECM蛋白或VEGF的量，Ficoll 400處理組在保留這些生物活性成分方面顯示出顯著增強的潛力(
p＜ 0.05，參見圖4C及4D)。基於這些分析結果，在三維球體製備過程中，將Ficoll 400用作擁擠試劑來誘導MMC。
有關MMC，一般認為效果最好的是葡聚糖硫酸(dextran sulfate, DxS)或Ficoll混合物(或又稱Ficoll雞尾酒；由Ficoll 70與Ficoll 400所組成)(參見Chen C, Loe F, Blocki A, Peng Y, Raghunath M. Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv Drug Deliv Rev 2011;63:277-90; Gaspar D, Fuller KP, Zeugolis DI. Polydispersity and negative charge are key modulators of extracellular matrix deposition under macromolecular crowding conditions. Acta Biomaterialia 2019;88:197-210; Kumar P, Satyam A, Fan X, Collin E, Rochev Y, Rodriguez BJ, Gorelov A, Dillon S, Joshi L, Raghunath M, Pandit A, Zeugolis DI. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies. Sci. Rep. 2015;5)。但根據圖4C及4D的實驗結果，DxS與Ficoll混合物在三維環境中增強細胞外基質(ECM)沈積的效率皆不如單獨使用Ficoll 400。
在先前技術中，DxS在濃度為10 µg/mL的情形下，經過6天的培養後，可以使纖維接合素的沈積量增加為對照組的2倍(參見Assunção M, Wong CW, Richardson JJ, Tsang R, Beyer S, Raghunath M, Blocki A. Macromolecular dextran sulfate facilitates extracellular matrix deposition by electrostatic interaction independent from a macromolecular crowding effect. Mater Sci Eng C Mater Biol Appl 2020;106:110280的圖3A)。而在本實施例的圖4C及4D的結果中，在三維培養的情形下加入同樣濃度的DxS，僅需2天就可以使纖維接合素的沈積量最多上升至1.85倍。
此外，在先前技術中，MMC會降低MSC的移動能力(參見Zeiger AS, Loe FC, Li R, Raghunath M, Van Vliet KJ. Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior. PLoS ONE 2012;7的圖3D)。然而，根據本實施例的圖4E的實驗結果，當MSC聚集成三維細胞球體後，其移動能力並不會受是否引發MMC影響。圖4E是將三維細胞球體加入至培養皿內的觀察情形，各組間的細胞球貼附與細胞移動情形並無太顯著的差異。
實施例 4. 三維間葉幹細胞 (mesenchymal stem cell, MSC) 細胞球體衍生的去細胞的細胞外基質 (decellularized extracellular matrix, dECM) 所展現 的生物活性的效用評估
在本實施例用到的細胞增殖分析及Matrigel管形成測定的操作流程如下。將懸浮在補充有10%FBS的杜貝可氏改良的最低必需培養基(minimum essential medium, DMEM)中的HUVECs接種到6孔盤的每個孔中，密度為2
10
5細胞/孔，並培育4小時以實現細胞貼附。隨後，將50個三維dECM支架轉移到孔中進行24小時培養。未處理的HUVECs用作對照組。細胞在位相差顯微鏡(Carl Zeiss)下照相。此外，根據製造商的操作流程，使用細胞計數套組-8 (cell counting kit-8)(Dojindo Molecular Technology, Kumamoto，日本)對HUVECs的增殖進行定量。
此外，藉由將具有10% FBS的DMEM中的10,000 HUVECs添加至96孔盤中預塗覆以40 µL的生長因子減少的Matrigel (growth factor-reduced (GFR) Matrigel)(Corning, Corning, NY, USA)的一孔中。培育4小時後，每個孔中的細胞接受10個三維dECM支架。對形成的管狀結構照相並監控3天。使用ImageJ軟體定量管的總長度。
由於在整個去細胞過程中都保留由MSCs分泌的VEGF，因此本實施例接下來研究這些裝載VEGF的dECM支架是否可以影響內皮細胞的行為。首先，將HUVECs暴露於dECM支架，然後將未經處理的HUVECs用作對照組。結果表明，HUVECs響應三維dECM支架而顯示出增加的細胞增殖(參見圖5A及5B)。再者，用MMC製備的支架比不使用MMC製備的支架表現出顯著更高的增殖潛能(相對於未處理的對照組是1.29倍)(與未處理的對照組相比是1.16倍；
p＜0.01；參見圖5B)。接下來，進一步進行Matrigel管形成測定以驗證保留的VEGF的促血管生成潛力(pro-angiogenic potential)。藉由用dECM支架進行處理，HUVECs形成的新生管狀結構可以被穩定並因此持續更長的時間，而未處理的對照組觀察到生長的管狀網絡迅速消退(參見圖5C)。管長度的分析證實所獲得的成像數據(參見圖5D)。另外，在接受帶有MMC的dECM支架的組中，觀察到的管長增加1.6倍，這表明這些支架的促血管生成潛能要優於沒有MMC的支架(
p＜0.005，參見圖5D)。總體而言，這些結果表明，保留在dECM支架中的可溶性分子(如VEGF)具有生物活性，可用於調節細胞功能及行為。
實施例 5. 三維間葉幹細胞 (mesenchymal stem cell, MSC) 細胞球體衍生的去細胞的細胞外基質 (decellularized extracellular matrix, dECM) 用作 細胞化 (cellularization) 的支架
在本實施例用到的再細胞化(recellularization)的操作流程如下。為了用細胞重新填充支架，將懸浮在100 µL培養基中的1×10
6HUVECs添加到含有100個三維dECM支架的管中。在37°C下培育1小時後，將支架用PBS沖洗以除去未附著的細胞，然後在具有超低附著表面(ultralow-attachment surface)(Corning)的盤中進行培養。培養後，使用抗CD31的一級抗體(Agilent Technologies, Santa Clara, CA, USA)處理載有細胞的支架進行免疫染色。
為了評估三維MSC細胞球體衍生的dECM作為生物支架並誘導血管生成的潛力，將HUVECs重新填充三維MSC細胞球體衍生的dECM。對於細胞接種，將三維dECM支架添加到含有HUVEC懸浮液的試管中，並在37°C下培育60分鐘，然後轉移到超低附著盤(ultralow-attachment plate)中。如共軛焦影像所示，HUVECs能夠在1小時後貼附到dECM支架上(參見圖6A)。隨著時間增長，細胞在dECM支架上增殖(參見圖6A)。此外，藉由觀察構建體(construct)的最寬橫截面(赤道板(equatorial plane))來驗證HUVECs向支架的遷移及向內生長(參見圖6B)，證明三維MSC球體衍生的dECM可以用作支架系統，並且從而成為一個裝有細胞的構建體(cell-laden construct)。此外，來自連續光學切片(serial optical section)的重建的三維共軛焦影像證明多個三維細胞接種dECM支架的細胞驅動宏組裝(cell-driven macroassembly)(參見圖6C)，表明它們有潛力用作工程化大型構建體(engineering large construct)的基礎。培養7天後，在構建體中觀察到由HUVECs形成的毛細血管樣血管前結構(capillary-like prevascular structure)(參見圖6D)。
實施例 6. 三維間葉幹細胞 (mesenchymal stem cell, MSC) 細胞球體衍生的去細胞的細胞外基質 (decellularized extracellular matrix, dECM) 誘導體內血管生成 的效用評估
在本實施例用到的體內生物活性評估的操作流程如下。動物實驗是根據2000年中華民國實驗動物學會(Chinese-Taipei Society of Laboratory Animal Sciences)出版的實驗動物管理與使用指南(Guidebook for the Care and Use of Laboratory Animals)(第三版)進行的。實驗方案由台灣新竹國立清華大學實驗動物照護及使用委員會審查並批准。六週大的雌性裸鼠(BALB/cAnN.Cg-
Foxn1 nu/CrlNarl)從台灣南港國家實驗動物中心購買。為了進行植入，將400個三維dECM支架與20 µL的GFR Matrigel充分混合，並在37°C下培育30分鐘，然後皮下植入裸鼠的背側。1週後，藉由吸入CO
2使動物犧牲，取回植入物，固定在10%磷酸鹽緩衝的福馬林(phosphate-buffered formalin)(Sigma-Aldrich)中，並包埋在石蠟(paraffin)中製備7 µm厚的切片。組織切片用蘇木精及伊紅(hematoxylin and eosin)染色，或使用抗溫韋伯氏凝血因子(von Willebrand factor, vWF)或
-平滑肌肌動蛋白(
-smooth muscle actin,
SMA)(均來自Agilent Technologies)的抗體進行免疫染色。
將本發明的三維dECM支架與GFR Matrigel混合，並經由局部注射皮下植入裸鼠，以評估其體內生物活性。將接受普通GFR Matrigel的動物用作對照組。兩種植入物均貼附在皮膚內部。在第7天，空的Matrigel表現為半透明的外觀，沒有明顯的血管生長到植入物中(參見圖7A)。攜帶Matrigels的三維dECM支架表現出不透明的整體外觀，有少許淺血管(superficial vessels)生長到植入物的邊緣(參見圖7A)。對收獲的Matrigel進行處理以進行組織學分析。蘇木精及伊紅染色的結果表明，植入的空Matrigel內部總體上沒有細胞和脈管系統(vasculature)(參見圖7B)，而在含有三維dECM支架的凝膠中可以發現顯著的細胞浸潤和血管形成(參見圖7B)。浸潤細胞的vWF (內皮細胞)及
SMA (血管週細胞(perivascular cells))染色進一步證實此點(參見圖7C)，證明本發明三維dECM支架在體內吸引細胞並誘導血管形成(vascularization)的生物活性潛力。
在另一實施例中，可以結合二種或多種細胞種類來建構三維細胞球體。參見圖8，其顯示不同種細胞(包括足細胞(podocyte)、間葉幹細胞(mesenchymal stem cell, MSC)及人類臍帶靜脈內皮細胞(human umbilical cord vein endothelial cell, HUVEC))皆可建構三維細胞球體。
綜上所述，本發明去細胞的細胞外基質(dECM)確實可以達成以下功效：由於基質為三維培養的細胞所製造，故其成分與微結構皆相當類似於生物組織中的基質，實驗結果顯示，細胞可以有效貼附於三維的去細胞的細胞外基質表面，並進行增生。由於三維的去細胞的細胞外基質內尚有諸多細胞所分泌的生物活性成分，如生長因子、細胞激素與外泌體等，故具有調節後續貼附細胞行為之功能。藉由改變構築三維細胞球體之細胞種類或控制細胞行為，其後續留存於三維的去細胞的細胞外基質內部的生物活性分子組成也不盡相同，因此具有相當廣泛的應用性，例如使用幹細胞來建立三維球體，其留存下來的生物活性分子即能有助於血管新生與組織再生的發生。藉由醣類巨分子所誘發之大分子擁擠現象(MMC)，三維細胞球體中的細胞外基質(ECM)與生長因子分泌總量可顯著上升，進而使後續衍生之去細胞的細胞外基質內的ECM分子與生長因子含量有所增加，進一步提昇去細胞的細胞外基質的生物活性。此外，每個三維的去細胞的細胞外基質可視為一小型支架，其大小與去細胞前之三維細胞球體相同，因此可依據需求製備不同直徑之三維的去細胞的細胞外基質。將細胞培養於三維的去細胞的細胞外基質後，除了能將其作為細胞傳輸之載體(例如以注射方式將含有細胞之三維的去細胞的細胞外基質移植至欲治療的部位)以外，複數個三維的去細胞的細胞外基質能夠藉由多種方式組裝成為一大型結構。由於三維的去細胞的細胞外基質相當容易大量製造，因此其聚合而成的大型支架在形狀與大小上皆不受限制，能夠高度客製化。再者，由於細胞是先培養於個別的三維的去細胞的細胞外基質上，故聚集後細胞可均勻分布於整個大型支架結構中，解決傳統組織工程所遇到的細胞分布不均問題。
以上所述僅為舉例性，而非為限制性者。任何未脫離本發明之精神與範疇，而對其進行之等效修改或變更，均應包含於後附之申請專利範圍中。
無
圖1A顯示三維細胞球體的共軛焦螢光影像，其中DAPI表示4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole)，一種能夠與DNA強力結合的螢光染料；RFP表示紅色螢光蛋白；比例尺為100
m。
圖1B顯示三維細胞球體內血管內皮生長因子(VEGF)的代表性共軛焦影像，比例尺為100
m。
圖1C顯示利用酵素結合免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)(n = 4)測定的對應定量結果，其中****表示
p＜ 0.001；ns表示不具統計學顯著差異。
圖2A顯示去細胞前後三維MSC細胞球體的代表性螢光影像，比例尺為100
m。
圖2B顯示藉由PicoGreen分析法測定的三維去細胞的細胞外基質(decellularized extracellular matrix, dECM)支架內的殘餘DNA含量，其中****表示
p＜ 0.001。
圖2C顯示藉由PicoGreen分析法測定的三維去細胞的細胞外基質(decellularized extracellular matrix, dECM)支架內的殘餘VEGF含量，其中****表示
p＜ 0.001，***表示
p＜ 0.005，ns表示不具統計學顯著差異。
圖3A顯示去細胞前後利用各種不同細胞數量製造的三維MSC細胞球體的代表性DAPI染色影像，比例尺為100
m。
圖3B顯示去細胞前後利用各種不同細胞數量製造的三維MSC細胞球體的對應DNA含量(n = 6)，其中*表示
p＜ 0.05，**表示
p＜ 0.01，ns表示不具統計學顯著差異。
圖4A顯示組裝過程中補充各種擁擠試劑(Ficoll 70、Ficoll 400、Ficoll混合物(mix)或葡聚糖硫酸(dextran sulfate, DxS))的三維MSC細胞球體的位相差顯微照片，比例尺為200
m。
圖4B顯示三維dECM支架的層黏連蛋白染色的螢光影像，下圖為上面白色框放大的區域，上圖比例尺為100
m，下圖比例尺為20
m。
圖4C顯示層黏連蛋白、纖維接合素(均藉由西方墨點法測定)的水平及相應的殘留比率，DxS表示葡聚糖硫酸(dextran sulfate)。
圖4D顯示擁擠試劑處理的三維MSC細胞球體及衍生的dECM支架的VEGF (藉由ELISA測定)(n = 4)，將纖維接合素及層黏連蛋白的量(相對於
-肌動蛋白)相對於未處理對照組的量標準化；殘留含量的百分比定義為三維dECM支架中目標蛋白質的量與三維MSC細胞球體中目標蛋白質的量之比，*表示
p＜ 0.05，**表示
p＜ 0.01，***表示
p＜ 0.005，****表示
p＜ 0.001，ns表示不具統計學顯著差異，control表示對照組。
圖4E顯示組裝過程中補充各種擁擠試劑(Ficoll 70、Ficoll 400、Ficoll混合物(mix)或葡聚糖硫酸(dextran sulfate, DxS))的三維MSC細胞球體的位相差顯微照片。
圖5A顯示用製備的三維dECM支架處理的人類臍帶靜脈內皮細胞(human umbilical cord vein endothelial cells, HUVECs)的代表性位相差影像，比例尺為400
m，MMC+表示以大分子擁擠現象(macromolecular crowding, MMC)製備的支架，MMC-表示未以MMC製備的支架。
圖5B顯示相應的細胞增殖定量分析(n = 6)，**表示
p＜ 0.01，***表示
p＜ 0.005，****表示
p＜ 0.001，MMC+表示以MMC製備的支架，MMC-表示未以MMC製備的支架。
圖5C顯示由接受Matrigel上三維dECM支架的HUVECs形成的管狀結構，比例尺為400
m，MMC+表示以MMC製備的支架，MMC-表示未以MMC製備的支架。
圖5D顯示相應的管的總長度(n = 5)，**表示
p＜ 0.01，***表示
p＜ 0.005，ns表示不具統計學顯著差異。
圖6A顯示最大強度投影共軛焦影像，顯示在三維dECM支架上生長1小時或2天的CD31陽性HUVECs，比例尺為50
m。
圖6B顯示球狀赤道板(spheroid equatorial plane)的代表性光學切片，白色箭頭表示細胞生長到三維支架內部，比例尺為50
m。
圖6C顯示圖6A中來自組裝的三維dECM球體的三維渲染(3D rendering)，比例尺為50
m。
圖6D顯示培育7天的裝有HUVEC的構建體的三維渲染及相應的最大強度投影影像，黃色箭頭表示由HUVECs在構建體內形成的毛細血管樣血管前結構(capillary-like prevascular structure)，比例尺為50
m。
圖7A顯示皮下植入一週後取回的測試樣品的照片，虛線區域表示植入了帶有dECM支架的Matrigel，白色框在右圖中放大，箭頭表示新生的血管。
圖7B顯示皮下植入一週後取回的測試樣品的蘇木精及伊紅染色影像，i、ii、iv、v及vii中的比例尺為500
m，iii、vi及viii中的比例尺為100
m。
圖7C顯示圖7B的viii中溫韋伯氏凝血因子(von Willebrand factor, vWF)及平滑肌肌動蛋白(smooth muscle actin, SMA)的免疫螢光影像，Nuclei表示核，比例尺為50
m。
圖8顯示不同種細胞(包括足細胞(podocyte)、間葉幹細胞(mesenchymal stem cell, MSC)及人類臍帶靜脈內皮細胞(human umbilical cord vein endothelial cell, HUVEC))皆可建構三維細胞球體。
Claims (6)
- 一種去細胞的細胞外基質(decellularized extracellular matrix)，係衍生自一個三維細胞球體，該去細胞的細胞外基質具有一三維的球狀結構，該去細胞的細胞外基質藉由使用一0.5%(v/v)Triton X-100進行一去細胞處理，該去細胞的細胞外基質包含一血管內皮生長因子(vascular endothelial growth factor,VEGF)，該三維細胞球體為一由複數個人類臍帶血(human umbilical cord blood)間葉幹細胞(mesenchymal stem cells,MSCs)或人類臍帶靜脈內皮細胞(human umbilical cord vein endothelial cells,HUVECs)所形成的三維細胞球體，該去細胞的細胞外基質藉由至少一醣類巨分子而被誘發大分子擁擠現象(macromolecular crowding,MMC)，俾以增加細胞外基質及生長因子含量，其中該至少一醣類巨分子為Ficoll 70或Ficoll 400。
- 如請求項1的去細胞的細胞外基質，進一步包含一第一型膠原蛋白、一纖維接合素(fibronectin)及一層黏連蛋白(laminin)。
- 如請求項1的去細胞的細胞外基質，其中複數個細胞會貼附於該去細胞的細胞外基質的表面進行增生。
- 一種用於製備一去細胞的細胞外基質(decellularized extracellular matrix)的方法，包含：(a)對複數個人類臍帶血(human umbilical cord blood)間葉幹細胞(mesenchymal stem cells,MSCs)或人類臍帶靜脈內皮細胞(human umbilical cord vein endothelial cells,HUVECs)進行體外培養以構築一個三維細胞球體；以及(b)使用一0.5%(v/v)Triton X-100對該三維細胞球體進行一去細胞處理，得到該去細胞的細胞外基質，其中該去細胞的細胞外基質具有一為三維的球狀結構，該去細胞的細胞外基質包含一血管內皮生長因子(vascular endothelial growth factor,VEGF)，該去細胞的細胞外基質藉由至少一醣類巨分子而被誘發大分子擁擠現象(macromolecular crowding,MMC)，俾以增加細胞外基質及生長因子含量，其中該至少一醣類巨分子為Ficoll 70或Ficoll 400。
- 如請求項4的方法，其中該去細胞的細胞外基質進一步包含一第一型膠原蛋白、一纖維接合素(fibronectin)及一層黏連蛋白(laminin)。
- 一種如請求項1至3中任一項的去細胞的細胞外基質用於製備一促進組織再生及修復的生醫材料支架的用途。
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| 期刊 Ylostalo et al., "Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions", J Vis Exp., Vol. 121, No. 55126, Jove, 2017 Mar 18, p. 1-13. * |
| 期刊 Yu et al., "Autologous decellularized extracellular matrix protects against H2O2-induced senescence and aging in adipose-derived stem cells and stimulates proliferation in vitro", Bioscience Reports, Vol. 39, 1 portland Press, May 2019, p. 1-10.; * |
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