TW202200781A - 細胞培養組合物及其用途 - Google Patents
細胞培養組合物及其用途 Download PDFInfo
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Abstract
本發明實施例提供一種細胞培養組合物及其用途，細胞培養組合物包含培養基以及粒線體。包含粒線體的細胞培養組合物可促進細胞生長、改善受損或老化的幹細胞的功能，藉以改善細胞的生長狀況。
Description
本發明係關於細胞培養組合物及其用途，尤其係關於包含粒線體之細胞培養組合物及其用途。
細胞培養基（cell culture medium）是一種模擬動植物體內細胞的生長環境的營養基礎物質，其提供細胞本身不能合成的營養物並維持適宜的pH值及滲透壓，使細胞能在體外存活並增殖。在培養細胞的過程中，為了提高細胞的生長效率與細胞的功能，會添加輔助性的培養添加物。
在貧瘠的培養基中或是嚴苛的培養條件下，細胞成長的速率較慢，生長出來的細胞可能較脆弱、不易存活，甚至無法成長。培養出的細胞若是不夠健康或是成長的速度過慢，對後續的實驗或研究皆會造成不利的影響。
根據本發明一實施例，透過包含粒線體與培養基的細胞培養組合物，可改善細胞的生長狀況。
本發明一實施例提供一種細胞培養組合物，包含培養基以及粒線體。
本發明一實施例提供一種細胞培養組合物在促進細胞生長的用途，其中該細胞培養組合物包含一培養基以及粒線體。
本發明一實施例提供一種細胞培養組合物在改善受損或老化的幹細胞的功能的用途，其中該細胞培養組合物包含一培養基以及粒線體。
根據本發明一實施例，藉由在細胞培養基中添加粒線體，可幫助細胞生長、提高細胞的增生率。對於受損或老化的細胞而言，根據本發明一實施例亦能改善受損或老化的細胞的生長、降低老化細胞比例。除此之外，根據本發明一實施例還能改善受損或老化的細胞的功能。在學術方面，本發明確保細胞的生長效率及穩定性有助於進行後續的研究或實驗，以奠定科學發展、生物學研究的基礎。在產業方面，本發明確保細胞的生長效率及穩定性有助於穩定產率及良率，可降低細胞培養成本並控管產品品質，以達成利益最大化。
於以下實施方式中詳細敘述本發明之詳細特徵及優點，其內容足以使任何熟習相關技藝者了解本發明之技術內容並據以實施，且根據本說明書所揭露的內容、申請專利範圍及圖式，任何熟習相關技藝者可輕易理解本發明相關之目的及優點。以下實施例係進一步詳細說明本發明之觀點，但非以任何觀點限制本發明之範疇。
粒線體是細胞內進行氧化磷酸化反應及合成三磷酸腺苷（ATP）的場所，除了提供細胞正常代謝所需的能量外，還負責調控細胞內氧化壓力處理、訊號傳遞等功能。在細胞的培養過程中，粒線體的功能與細胞的生長有相當大的關聯。粒線體功能較差的細胞在產生能量方面的效率亦較差，造成細胞生長緩慢或細胞生長不健全。因此，發明人藉由以粒線體作為細胞培養組合物中的添加物來改善細胞的生長情況。
根據本發明一實施例，細胞培養組合物包含培養基及粒線體。細胞培養組合物在常溫常壓下可呈液態，可保存於室溫，以保存於4°C為佳。
培養基可包含DMEM、F12、DMEM/F12、MEM、MEM-α、Keratinocyte SFM (1X)、IMEM、RPMI 1640、M-199、Opti-MEM、Ham’s F-10 Nutrient Mixture、Ham’s F-12 Nutrient Mixture或IMDM，但不以此為限。培養基可為液體或固體，可依據所欲培養之細胞選擇適當的培養基並添加適當的營養添加物及水。
粒線體可為取自動物細胞或植物細胞的粒線體，但不以此為限。在本發明部分實施例中，培養動物細胞時添加動物細胞的粒線體至培養基中。提供粒線體的動物細胞可包含脂肪幹細胞、單核球細胞、胚胎幹細胞、間質幹細胞、造血幹細胞、CD34+幹細胞、骨髓幹細胞、骨骼肌細胞、肝臟細胞、腎臟細胞、血小板、纖維母細胞、內皮細胞等具有粒線體的細胞。細胞培養組合物中，每一毫升之細胞培養組合物可包含1微克至100微克的粒線體，但不以此為限。在其他實施例中，每一毫升之細胞培養組合物可包含5微克至80微克的粒線體。在其他實施例中，每一毫升之細胞培養組合物可包含15微克至40微克的粒線體。
根據本發明其他實施例，細胞培養組合物可包含培養基、至少一營養添加物及粒線體。
營養添加物可包含胎牛血清（Fetal Bovine Serum，FBS）、鹽類、胺基酸、馬血清、人類血清、維生素、葡萄糖、生長因子、蛋白質、動物體萃取物、碳水化合物、肌醇、巰乙醇或葉酸，但不以此為限。鹽類包含鈉鹽、鉀鹽、鎂鹽、鈣鹽、氯鹽、碳酸鹽、丙酮酸鹽、硝酸鹽、磷酸鹽或無機鹽，但不以此為限。可依據所欲培養之細胞選擇適當的營養添加物。
根據本發明其他實施例，細胞培養組合物可包含培養基、粒線體及血小板。
血小板可為動物血液中的血小板，但不以此為限。在細胞培養組合物中，每一毫升的細胞培養組合物可包含1×106
至1×108
個血小板，但不以此為限。在其他實施例中，每一毫升的細胞培養組合物可包含1×108
個血小板。
根據本發明其他實施例，細胞培養組合物可包含培養基、粒線體及補體C3。
在細胞培養組合物中，每一毫升之細胞培養組合物可包含0.1微克至20微克的補體C3，但不以此為限。在其他實施例中，每一毫升之細胞培養組合物可包含10微克的補體C3 。
根據本發明一實施例，細胞培養組合物可促進細胞生長，提高細胞增生率。使用本發明實施例之細胞培養組合物所培養的細胞可包含任意體細胞。體細胞可包含視網膜細胞、肝臟細胞、腎臟細胞、肌腱細胞、皮膚細胞或免疫細胞。免疫細胞可包含自然殺手細胞，自然殺手細胞可具有CD56+、CD3-、CD94+、CD122+、CD127+、KIR+、NKG2A+、NKG2D+、NKp30+、NKp44+、NKp46+及NKp80+之至少一標記。
根據本發明一實施例，細胞培養組合物可改善受損或老化的細胞的老化程度。使用本發明實施例之細胞培養組合物所培養的細胞可包含任意體細胞或任意幹細胞。體細胞可為視網膜細胞、肝臟細胞、腎臟細胞、肌腱細胞、皮膚細胞或免疫細胞。幹細胞可包含間質幹細胞，間質幹細胞可具有CD44+、CD90+、CD105+、CD106+、CD166+、Stro-1+及CD34-之至少一標記。間質幹細胞可為脂肪幹細胞、臍帶幹細胞、胎盤幹細胞、骨髓幹細胞、羊水幹細胞、皮膚幹細胞、周邊血幹細胞、子宮內膜幹細胞、羊膜幹細胞、牙齦幹細胞或牙髓幹細胞。
以下說明如何製備本發明實施例之細胞培養組合物。
本發明一實施例所使用之粒線體取自人類脂肪幹細胞（adipose-derived stem cell，ADSC）。幹細胞培養液包含Keratinocyte SFM (1X)溶液（Gibco）、bovine pituitary extract（BPE，Gibco）、重量百分濃度10%之胎牛血清（HyClone）。首先，在培養皿中將人類脂肪幹細胞培養至細胞數為1.5×108
個細胞，再以杜氏磷酸鹽緩衝液（DPBS）沖洗培養皿中的人類脂肪幹細胞。接著，移除培養皿中的杜氏磷酸鹽緩衝液後，在培養皿中加入細胞剝離用之胰蛋白酶（Trypsin），並在37℃下反應3分鐘後，再加入幹細胞培養液以終止反應。接著，將人類脂肪幹細胞自培養皿中沖洗下來後打散，以600 g離心10分鐘後，移除上清液。接著，將離心後留下的人類脂肪幹細胞及80毫升之IBC-1緩衝液（225 mM甘露醇、75mM蔗糖、0.1 mM EDTA、30 mM Tris-HCl pH 7.4）加入均質器中，並在冰上以均質器對人類脂肪幹細胞進行研磨15次。接著，以1000 g離心研磨後的人類脂肪幹細胞15分鐘，將上清液收集至另一離心管，再以9000 g再次離心收集的上清液10分鐘，移除再次離心後得到的上清液，獲得之沉澱物即為粒線體。在粒線體沉澱物中加入1.5毫升之IBC-2緩衝液（225 mM甘露醇、75mM蔗糖、30 mM Tris-HCl pH 7.4）及蛋白質分解酶抑制劑，並置於4℃下保存。
進行細胞培養時，根據細胞種類選用適當的培養基，接著依期望濃度加入所需之營養添加物以及如上所述之粒線體並混合均勻即可使用。細胞培養組合物以新鮮配製為佳，但不以此為限，亦可事先配製完成細胞培養組合物後保存於4℃，待進行細胞培養時再取用。
以下說明使用本發明實施例之細胞培養組合物進行細胞培養。
〔實驗一，提高細胞增生率〕
在本實驗中，使用實施例之細胞培養組合物作為培養液，來培養人類視網膜色素上皮細胞株（Human retinal pigment epithelium cell，ARPE-19）、肝臟細胞（HepG2）、腎臟上皮細胞（Madin-Darby Canine Kidney cell，MDCK）、人類肌腱細胞（Human tenocyte）及自然殺手細胞（Nature killer cell，NK92MI）。並且，透過阿爾瑪藍檢測試劑來評估包含粒線體的細胞培養組合物對於細胞生長的影響，並以增生率（亦稱為細胞增生率）來表示。
阿爾瑪藍（Alamar blue）係用於檢測細胞活力的檢測試劑。檢測套組內的刃天青（resazurin）是一種氧化還原指示劑，其為無毒、可穿透細胞膜且低螢光性之深藍色染料。當刃天青進入健康的細胞中，會因活細胞體內的還原環境而被還原成粉紅色且具高螢光性的試鹵靈（resorufin）。可藉由量測試鹵靈的光吸收值或螢光值來評估細胞的增生率（cell proliferation）。試鹵靈的光吸收值或螢光值愈高，表示細胞量越多，細胞的增生率愈高。細胞的增生率愈高也表示細胞愈健康、增生能力愈強。因此，本實驗使用阿爾瑪藍作為評估細胞增生率或細胞存活率的指標。
本實驗所使用各實施例之細胞培養組合物的詳細成分如表1至表5所示。
ARPE-19的培養基可為DMEM/F12（Gibco），營養添加物可包含2.5 mM麩醯胺酸、15 mM HEPES、0.5 mM丙酮酸鈉、1200毫克/升（mg/L）碳酸氫鈉及重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為15μg/mL或40 μg/mL。
HepG2的培養基可為DMEM/F12，營養添加物可包含重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為15 μg/mL或40 μg/mL。
MDCK的培養基可為含有Earle’s Balanced Salt的MEM-α（Thermo Fisher Scientific），營養添加物可包含重量百分濃度5%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為15 μg/mL或40 μg/mL。
人類肌腱細胞的培養基可為DMEM/F12，營養添加物可包含重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為1 μg/mL、15 μg/mL、40 μg/mL或100 μg/mL。
自然殺手細胞的培養基可為含有Earle’s Balanced Salt的MEM-α（Thermo Fisher Scientific），營養添加物可包含0.02 mM肌醇、0.1 mM巰乙醇、0.02 mM葉酸、重量百分濃度12.5%胎牛血清及重量百分濃度12.5%馬血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為1 μg/mL、15 μg/mL、40 μg/mL或100 μg/mL。
〔表1〕
| 培養細胞 | ARPE-19 | |
| 組別 | 實施例1-1 | 實施例1-2 |
| 培養基 | DMEM/F12 | |
| 營養添加物 | 2.5 mM麩醯胺酸 15 mM HEPES 0.5 mM丙酮酸鈉 1200 mg/L碳酸氫鈉 10%胎牛血清 | |
| 粒線體 | 15 μg/mL | 40 μg/mL |
〔表2〕
| 培養細胞 | HepG2 | |
| 組別 | 實施例2-1 | 實施例2-2 |
| 培養基 | DMEM/F12 | |
| 營養添加物 | 10%胎牛血清 | |
| 粒線體 | 15 μg/mL | 40 μg/mL |
〔表3〕
| 培養細胞 | MDCK | |
| 組別 | 實施例3-1 | 實施例3-2 |
| 培養基 | MEM-α（含有Earle’s Balanced Salt） | |
| 營養添加物 | 5%胎牛血清 | |
| 粒線體 | 15 μg/mL | 40 μg/mL |
〔表4〕
| 培養 細胞 | 人類肌腱細胞 | ||||
| 組別 | 對照組4 | 實施例 4-1 | 實施例 4-2 | 實施例 4-3 | 實施例 4-4 |
| 培養基 | DMEM/F12 | ||||
| 營養添加物 | 10%胎牛血清 | ||||
| 粒線體 | - | 1 μg/mL | 15 μg/mL | 40 μg/mL | 100 μg/mL |
〔表5〕
| 培養 細胞 | 自然殺手細胞 | ||||
| 組別 | 對照組5 | 實施例 5-1 | 實施例 5-2 | 實施例 5-3 | 實施例 5-4 |
| 培養基 | MEM-α（含有Earle’s Balanced Salt） | ||||
| 營養添加物 | 0.02 mM肌醇 0.1 mM巰乙醇 0.02 mM葉酸 12.5%胎牛血清 12.5%馬血清 | ||||
| 粒線體 | - | 1 μg/mL | 15 μg/mL | 40 μg/mL | 100 μg/mL |
以下說明各細胞的培養流程。首先，取培養代數為4至10代間的各細胞進行實驗。使用不含粒線體的培養液，將各細胞培養至其體積為培養皿的八分滿時，移除培養皿中的培養液並使用磷酸鹽緩衝液（phosphate buffered saline，PBS）潤洗細胞。接著，在培養皿中加入0.25%之胰蛋白酶並使胰蛋白酶在37℃下反應5分鐘，再加入培養液以終止胰蛋白酶的反應。接者，將培養皿中的各細胞及培養液移至離心管中，以每分鐘轉速1000（Revolutions Per Minute，rpm）離心5分鐘後，移除上清液。接著，再加入新的培養液至離心管中，進行細胞計數。接著，將ARPE-19以每孔2×104
個細胞的密度於實施例1-1及1-2之細胞培養組合物中培養24或48小時。將HepG2以每孔5×104
個細胞的密度於實施例2-1及2-2之細胞培養組合物中培養24小時。將MDCK以每孔5×104
個細胞的密度於實施例3-1及3-2之細胞培養組合物中培養24小時。將人類肌腱細胞以每孔2×104
個細胞的密度於實施例4-1至4-4之細胞培養組合物中培養24或48小時。將自然殺手細胞以每孔2×104
個細胞的密度於實施例5-1至5-4之細胞培養組合物中培養24或48小時。培養完成後，使用磷酸鹽緩衝液清洗各細胞，並將培養液更換為含有阿爾瑪藍的培養液，培養3小時。培養完成後，以OD530/595的波長量測螢光，計算各細胞的增生率。
ARPE-19的實驗結果請參考表6及圖1A及圖1B，圖1A及圖1B顯示使用實施例之細胞培養組合物培養ARPE-19的細胞增生率，圖1A之培養時間為24小時，圖1B之培養時間為48小時。HepG2的實驗結果請參考表7及圖2，圖2顯示使用實施例之細胞培養組合物培養HepG2的細胞增生率，培養時間為24小時。MDCK的實驗結果請參考表8及圖3，圖3顯示使用實施例之細胞培養組合物培養MDCK的細胞增生率，培養時間為24小時。人類肌腱細胞的實驗結果請參考表9及圖4，圖4顯示使用實施例之細胞培養組合物培養人類肌腱細胞的細胞增生率，培養時間為24小時及48小時。自然殺手細胞的實驗結果請參考表10及圖5，圖5顯示使用實施例之細胞培養組合物培養自然殺手細胞的細胞增生率，培養時間為24小時及48小時。初始對照組為細胞剛開始培養時（第0小時）的細胞數，將其訂為1。增生率為細胞培養後之細胞數相對於初始對照組的倍數。對照組為未添加粒線體的組別。圖1A至圖3中，#表示相對初始對照組具有顯著差異（P＜0.05），##表示相對初始對照組具有高顯著差異（P＜0.01），###表示相對初始對照組具有非常顯著差異（P＜0.001）。圖4及圖5中，#表示相對於對照組具有顯著差異（P＜0.05）。
〔表6〕
| 培養細胞：ARPE-19 | |||
| 粒線體 （μg/mL） | 24小時 增生率（倍） | 48小時 增生率（倍） | |
| 實施例1-1 | 15 | 1.18±0.16 | 1.25±0.05 |
| 實施例1-2 | 40 | 1.26±0.16 | 1.39±0.07 |
〔表7〕
| 培養細胞：HepG2 | ||
| 粒線體 （μg/mL） | 24小時 增生率（倍） | |
| 實施例2-1 | 15 | 1.16±0.15 |
| 實施例2-2 | 40 | 1.28±0.09 |
〔表8〕
| 培養細胞：MDCK | ||
| 粒線體 （μg/mL） | 24小時 增生率（倍） | |
| 實施例3-1 | 15 | 1.05±0.05 |
| 實施例3-2 | 40 | 1.1±0.05 |
〔表9〕
| 培養細胞：人類肌腱細胞 | |||
| 粒線體 （μg/mL） | 24小時 增生率（倍） | 48小時 增生率（倍） | |
| 對照組4 | - | 1.09±0.21 | 1.42±0.02 |
| 實施例4-1 | 1 | 1.26±0.17 | 1.46±0.09 |
| 實施例4-2 | 15 | 1.45±0.22 | 1.63±0.12 |
| 實施例4-3 | 40 | 1.46±0.18 | 2.12±0.10 |
| 實施例4-4 | 100 | 1.58±0.28 | 2.23±0.07 |
〔表10〕
| 培養細胞：自然殺手細胞 | |||
| 粒線體 （μg/mL） | 24小時 增生率（倍） | 48小時 增生率（倍） | |
| 對照組5 | - | 1.19±0.08 | 1.57±0.13 |
| 實施例5-1 | 1 | 1.22±0.08 | 1.63±0.09 |
| 實施例5-2 | 15 | 1.32±0.09 | 1.70±0.13 |
| 實施例5-3 | 40 | 1.33±0.08 | 1.81±0.10 |
| 實施例5-4 | 100 | 1.37±0.10 | 1.83±0.14 |
上述實驗結果顯示，使用含有粒線體的細胞培養組合物來培養細胞，可使細胞具有提高的增生率，且增生率隨著粒線體的濃度增加而提升，表示含有粒線體的細胞培養組合物確實有助於細胞的生長。並且，增生率的提高隨培養時間增長而更加明顯，表示含有粒線體的細胞培養組合物可穩定幫助細胞生長。
〔實驗二，改善受損或老化的幹細胞的老化程度〕
在本實驗中，使用醣化終產物（advanced glycation end product，AGE）誘導幹細胞損傷或老化，再使用實施例之細胞培養組合物作為培養液來培養受損或老化的幹細胞。並且，透過SA-β-gal套組來評估包含粒線體的細胞培養組合物對於受損或老化的幹細胞的影響，並以老化細胞比例（%）來表示老化程度。本實驗使用之幹細胞包含脂肪幹細胞（Adipose-derived stem cell，ADSC）及羊膜幹細胞（Amniotic membrane stem cell，AMSC）。
在老化的細胞中，老化相關-β-半乳糖苷酶（Senescence-associated beta-galactosidase，SA-β-gal）會被過度表達，因此SA-β-gal可作為細胞衰老的標記之一。本實驗使用SA-β-gal套組（Senescence β-Galactosidase Staining Kit #9860，Cell Signaling technology）來評估幹細胞的老化狀態。
本實驗所使用之細胞培養組合物的詳細成分如表11及表12所示。
ADSC的培養基可為Keratinocyte SFM (1X)（Catalog number: 17005042，Life Technologies），營養添加物可包含重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為1 μg/mL、15 μg/mL或40 μg/mL。
AMSC的培養基可為DMEM/F12（Gibco），營養添加物可包含重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為1 μg/mL、15 μg/mL或40 μg/mL。
［表11］
| 培養 細胞 | ADSC | |||
| 組別 | 對照組6 | 實施例6-1 | 實施例6-2 | 實施例6-3 |
| 培養基 | Keratinocyte SFM (1X) | |||
| 營養 添加物 | 10%胎牛血清 | |||
| 粒線體 | - | 1 μg/mL | 15 μg/mL | 40 μg/mL |
［表12］
| 培養 細胞 | AMSC | |||
| 組別 | 對照組7 | 實施例7-1 | 實施例7-2 | 實施例7-3 |
| 培養基 | DMEM/F12 | |||
| 營養 添加物 | 10%胎牛血清 | |||
| 粒線體 | - | 1 μg/mL | 15 μg/mL | 40 μg/mL |
以下說明幹細胞的培養流程。首先，取培養代數為第10代的幹細胞進行實驗。使用不含粒線體的培養液，將幹細胞培養至其體積為培養皿的八分滿時，移除培養皿中的培養液並使用磷酸鹽緩衝液（phosphate buffered saline，PBS）潤洗幹細胞。接著，在培養皿中加入0.25%之胰蛋白酶並使胰蛋白酶在37℃下反應5分鐘，再加入培養液以終止胰蛋白酶的反應。接者，將培養皿中的各細胞及培養液移至離心管中，以每分鐘轉速1000（Revolutions Per Minute，rpm）離心5分鐘後，移除上清液。接著，再加入新的培養液至離心管中，進行細胞計數。接著，將幹細胞以每孔1.5×104
個細胞的密度培養24小時。接著，於孔盤中加入AGE，使幹細胞在含有濃度為400 μg/mL的AGE的培養液中培養4小時。接著，將含有AGE的細胞培養液移除，加入實施例之細胞培養組合物，使幹細胞在含有不同粒線體濃度的細胞培養組合物中培養24小時。細胞培養組合物中粒線體的濃度為0、1 μg/mL、15 μg/mL或40 μg/mL。培養完成後，使用磷酸鹽緩衝液清洗各細胞並使用SA-β-gal套組進行細胞老化的評估。
ADSC的實驗結果請參考表13及圖6，圖6顯示使用實施例之細胞培養組合物培養受損或老化的ADSC的老化細胞比例。AMSC的實驗結果請參考表14及圖7，圖7顯示使用實施例之細胞培養組合物培養受損或老化的AMSC的老化細胞比例。對照組為添加AGE且不包含粒線體的培養組合物。控制組為未添加AGE且未添加粒線體的組別。老化細胞比例為在單位面積中老化細胞的數量佔所有細胞的數量的百分比，以代表老化程度。圖6至圖7中，縱軸為老化細胞比例（%），#表示相對對照組具有顯著差異（P＜0.05），##表示相對對照組具有高顯著差異（P＜0.01），###表示相對對照組具有非常顯著差異（P＜0.001）。
〔表13〕
| 培養細胞：ADSC | |||
| AGE （μg/mL） | 粒線體 （μg/mL） | 老化細胞比例 （%） | |
| 控制組6 | - | - | 11.8±2.32 |
| 對照組6 | 400 | - | 30.8±6.7 |
| 實施例6-1 | 1 | 29±5.9 | |
| 實施例6-2 | 15 | 19.33±6.25 | |
| 實施例6-3 | 40 | 18±5.55 |
〔表14〕
| 培養細胞：AMSC | |||
| AGE （μg/mL） | 粒線體 （μg/mL） | 老化細胞比例 （%） | |
| 控制組7 | - | - | 11.3±1.75 |
| 對照組7 | 400 | - | 62.67±12.03 |
| 實施例7-1 | 1 | 53.17±11.57 | |
| 實施例7-2 | 15 | 27.33±8.5 | |
| 實施例7-3 | 40 | 23.67±7.92 |
上述實驗結果顯示，使用含有粒線體的細胞培養組合物來培養受損或老化的幹細胞，可降低老化細胞比例，且老化細胞比例隨著粒線體的濃度增加而降低，表示含有粒線體的細胞培養組合物確實有助於改善受損或老化的幹細胞的生長，同時有助於改善受損或老化的幹細胞的功能。
〔實驗三，細胞培養組合物包含血小板〕
在本實驗中，進一步在細胞培養組合物中添加血小板，以包含血小板的細胞培養組合物作為培養液來培養纖維母細胞CCD-996SK。並且，透過阿爾瑪藍檢測試劑來評估包含粒線體的細胞培養組合物對於細胞生長的影響，並以增生率（亦稱為細胞增生率）來表示。
以下說明本實驗所使用之血小板的製備流程。首先，將血液抽取至含有抗凝血劑的採血管或離心管中，採血管為例如CPT管（BD Vacutainer® CPTTM
Cell Preparation Tube，REF362761）。接著，將血液離心使血液分層，例如以1500 g離心10分鐘使血液分層為紅血球層、膠體層、白細胞層（buffy coat）及血漿層，將白細胞層收集至新的離心管中，其中白細胞層包含單核球細胞及血小板。接著，將白細胞層與HEP buffer（140 mM NaCl、2.7mM KCl、3.8 mM HEPES、5 mM EGTA，pH 7.4）以1：1的比例均勻混合，再加入Prostaglandin E1使其濃度為1 μM以防止血小板活化。接著，將白細胞層以100 g離心15至20分鐘使白血球及殘餘的紅血球沉澱，將含有血小板的上清液收集至新的離心管中。接著，將含有血小板的上清液以800 g離心15至20分鐘使血小板沉澱。移除上清液，使用wash buffer（10 mM檸檬酸鈉、150 mM NaCl、1 mM EDTA、1%(w/v) dextrose）將所沉澱的血小板潤洗兩次。接著，使用Tyrode’s buffer（134 mM NaCl、12 mM NaHCO3
、2.9 mM KCl、0.34 mM Na2
HPO4
、1 mM MgCl2
、10 mM HEPES，pH 7.4）進行回溶，以血球計數器進行小小板計數。製備完成的血小板可保存於20°C至24°C，待培養細胞時再加入細胞培養組合物中，但以配製後立即使用為佳。
本實驗所使用之細胞培養組合物的詳細成分如表15及表16所示。
CCD-996SK的培養基可為含有2 mM麩醯胺酸的DMEM（Gibco；10566016），營養添加物可包含重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為1 μg/mL、15 μg/mL、40 μg/mL，血小板可為1×106
至1×108
個/mL或者可為1×106
個、1×107
個或1×108
個/mL。
〔表15〕
| 培養細胞 | CCD-996SK | ||
| 組別 | 對照組8-1 | 對照組8-2 | 對照組8-3 |
| 培養基 | DMEM（含有2 mM麩醯胺酸） | ||
| 營養添加物 | 10%胎牛血清 | ||
| 血小板 | 1×106 個 | 1×107 個 | 1×108 個 |
| 粒線體 | - | - | - |
〔表16〕
| 培養 細胞 | CCD-996SK | ||||
| 組別 | 控制組8 | 實施例8-1 | 實施例8-2 | 實施例8-3 | 實施例8-4 |
| 培養基 | DMEM（含有2 mM麩醯胺酸） | ||||
| 營養 添加物 | 10%胎牛血清 | ||||
| 血小板 | - | - | - | - | 1×106 個 |
| 粒線體 | - | 1 μg/mL | 15 μg/mL | 40 μg/mL | 40 μg/mL |
本實驗之細胞培養流程請參考實驗一，進行細胞計數時，將CCD-996SK以每孔1.5×104
個細胞的密度培養於控制組、對照組及實施例8-1至8-4之細胞培養組合物中培養24小時。培養完成後，使用磷酸鹽緩衝液清洗各細胞，並將培養液更換為含有阿爾瑪藍的培養液，培養3小時。培養完成後，以OD530/595的波長量測螢光，計算各細胞的增生率。
CCD-996SK的實驗結果請參考表17及圖8至圖10。圖8顯示使用僅含血小板之對照組之細胞培養組合物培養CCD-996SK的細胞增生率，培養時間為24小時及48小時。圖9顯示使用僅含粒線體之實施例之細胞培養組合物培養CCD-996SK的細胞增生率，培養時間為24小時及48小時。圖10顯示使用含粒線體及血小板之實施例之細胞培養組合物培養CCD-996SK的細胞增生率，培養時間為24小時及48小時。初始對照組為細胞剛開始培養時（第0小時）的細胞數，將其訂為1。增生率為細胞培養後之細胞數相對於初始對照組的倍數。對照組為未添加粒線體的組別。控制組為未添加粒線體且未添加血小板的組別。圖8至圖10中，#表示相較於控制組具有顯著差異（P＜0.05），##表示相較於控制組具有高顯著差異（P＜0.01），###表示相較於控制組具有非常顯著差異（P＜0.001）。
〔表17〕
| 培養細胞：CCD-996SK | ||||
| 粒線體 （μg/mL） | 血小板 （個） | 24小時 增生率（倍） | 48小時 增生率（倍） | |
| 控制組8 | - | - | 1.16±0.08 | 1.77±0.06 |
| 對照組8-1 | - | 1×106 | 1.20±0.06 | 1.85±0.10 |
| 對照組8-2 | - | 1×107 | 1.23±0.13 | 1.96±0.07 |
| 對照組8-3 | - | 1×108 | 1.30±0.04 | 2.08±0.10 |
| 實施例8-1 | 1 | - | 1.17±0.06 | 1.77±0.07 |
| 實施例8-2 | 15 | - | 1.28±0.08 | 1.90±0.12 |
| 實施例8-3 | 40 | - | 1.31±0.07 | 2.20±0.16 |
| 實施例8-4 | 40 | 1×106 | 1.42±0.05 | 2.13±0.06 |
根據上述實驗結果，由對照組可知，在細胞培養基中添加血小板可提高細胞增生率，且增生率隨著血小板的數量增加而提升。由實施例8-1至8-3可知，使用含有粒線體的細胞培養組合物來培養細胞，可使細胞具有提高的增生率，且增生率隨著粒線體的濃度增加而提升，表示含有粒線體的細胞培養組合物確實有助於細胞的生長。由對照組8-1、實施例8-3及實施例8-4可知，使用含有粒線體及血小板的細胞培養組合物來培養細胞，可進一步提升細胞的增生率。實施例8-3及實施例8-4證實在粒線體濃度皆為40 μg/mL的情況下，添加1×106
個/mL之血小板可使細胞增生率進一步提升。實施例8-4及對照組8-1證實在血小板數量皆為1×106
的情況下，添加40 μg/mL之粒線體可使細胞增生率超過血小板數量為1×108
（對照組8-3）始能達到的效果。因此，相較於僅添加血小板或僅添加粒線體的細胞培養組合物，同時含有粒線體及血小板的細胞培養組合物在提升細胞增生率上可具有加乘的效果。並且，增生率的提高隨培養時間增長而更加明顯，表示含有粒線體的細胞培養組合物可穩定幫助細胞生長。於此，特別說明，實施例8-4之48小時增生率略低於實施例8-3之48小時增生率，原因在於細胞已生長至飽和，細胞生長至飽和時細胞總數應略為相等，故細胞生增生率的差異不明顯。
〔實驗四，細胞培養組合物包含補體C3〕
在本實驗中，進一步在細胞培養組合物中添加補體C3（Sigma-Aldrich，204885），以包含補體C3的細胞培養組合物作為培養液來培養ARPE-19。並且，透過阿爾瑪藍檢測試劑來評估包含粒線體的細胞培養組合物對於細胞生長的影響，並以增生率（亦稱為細胞增生率）來表示。
本實驗所使用之細胞培養組合物的詳細成分如表18所示。
ARPE-19的培養基可為DMEM/F12（Gibco），營養添加物可包含2.5 mM麩醯胺酸、15 mM HEPES、0.5 mM丙酮酸鈉、1200毫克/升（mg/L）碳酸氫鈉及重量百分濃度10%胎牛血清，粒線體濃度可為1微克/毫升（μg/mL）至100 μg/mL，可為5 μg/mL至80 μg/mL，可為15 μg/mL至40 μg/mL，或者可為15 μg/mL。補體C3濃度可為0.1 μg/mL至20 μg/mL，或者可為10 μg/mL。
〔表18〕
| 培養細胞 | ARPE-19 | |
| 組別 | 控制組9 | 實施例9-1 |
| 培養基 | DMEM/F12 | |
| 營養添加物 | 2.5 mM麩醯胺酸 15 mM HEPES 0.5 mM丙酮酸鈉 1200 mg/L碳酸氫鈉 10%胎牛血清 | |
| 補體C3 | - | 10 μg/mL |
| 粒線體 | - | 15 μg/mL |
本實驗之細胞培養流程請參考實驗一，進行細胞計數時，將ARPE-19以每孔2×104
個細胞的密度於實施例之細胞培養組合物中培養24或48小時。孔盤中粒線體的濃度為15 μg/mL。孔盤中補體C3的濃度為10 μg/mL。培養完成後，使用磷酸鹽緩衝液清洗各細胞，並將培養液更換為含有阿爾瑪藍的培養液，培養3小時。培養完成後，以OD530/595的波長量測螢光，計算各細胞的增生率。
ARPE-19的實驗結果請參考表19及圖11，圖11顯示使用含粒線體及補體C3之實施例之細胞培養組合物培養ARPE-19的細胞增生率，培養時間為24小時及48小時。控制組為未添加粒線體且未添加補體C3的組別，將其定為1。增生率為細胞培養後之細胞數相對於控制組的倍數。
〔表19〕
| 培養細胞：ARPE-19 | ||||
| 粒線體 （μg/mL） | 補體C3 （μg/mL） | 24小時 增生率（倍） | 48小時增生率（倍） | |
| 控制組9 | - | - | 1 | 1 |
| 實施例9-1 | 15 | 10 | 1.22 | 1.28 |
上述實驗結果顯示，使用含有粒線體及補體C3的細胞培養組合物來培養細胞，可進一步提升細胞的增生率。並且，增生率的提高隨培養時間增長而更加明顯，表示含有粒線體的細胞培養組合物可穩定幫助細胞生長。
根據本發明一實施例，藉由在細胞培養基中添加粒線體，可幫助細胞生長、提高細胞的增生率。對於受損或老化的細胞而言，根據本發明一實施例亦能改善受損或老化的細胞的生長、降低老化細胞比例。除此之外，根據本發明一實施例還能改善受損或老化的細胞的功能。在學術方面，本發明確保細胞的生長效率及穩定性有助於進行後續的研究或實驗，以奠定科學發展、生物學研究的基礎。在產業方面，本發明確保細胞的生長效率及穩定性有助於穩定產率及良率，可降低細胞培養成本並控管產品品質，以達成利益最大化。
雖然本發明以前述之實施例揭露如上，然其並非用以限定本發明。在不脫離本發明之精神和範圍內，所為之更動與潤飾，均屬本發明之專利保護範圍。關於本發明所界定之保護範圍請參考所附之申請專利範圍。
無。
圖1A及圖1B顯示使用實施例之細胞培養組合物培養ARPE-19的細胞增生率，圖1A之培養時間為24小時，圖1B之培養時間為48小時。
圖2顯示使用實施例之細胞培養組合物培養HepG2的細胞增生率，培養時間為24小時。
圖3顯示使用實施例之細胞培養組合物培養MDCK的細胞增生率，培養時間為24小時。
圖4顯示使用實施例之細胞培養組合物培養人類肌腱細胞的細胞增生率，培養時間為24小時及48小時。
圖5顯示使用實施例之細胞培養組合物培養自然殺手細胞的細胞增生率，培養時間為24小時及48小時。
圖6顯示使用實施例之細胞培養組合物培養受損或老化的ADSC的老化細胞比例。
圖7顯示使用實施例之細胞培養組合物培養受損或老化的AMSC的老化細胞比例。
圖8顯示使用僅含血小板之對照組之細胞培養組合物培養CCD-996SK的細胞增生率，培養時間為24小時及48小時。
圖9顯示使用僅含粒線體之實施例之細胞培養組合物培養CCD-996SK的細胞增生率，培養時間為24小時及48小時。
圖10顯示使用含粒線體及血小板之實施例之細胞培養組合物培養CCD-996SK的細胞增生率，培養時間為24小時及48小時。
圖11顯示使用含粒線體及補體C3之實施例之細胞培養組合物培養ARPE-19的細胞增生率，培養時間為24小時及48小時。
Claims (12)
- 一種細胞培養組合物，包含：一培養基；以及粒線體。
- 如請求項1所述之細胞培養組合物，其中每一毫升的該細胞培養組合物包含1微克至100微克的粒線體。
- 如請求項1所述之細胞培養組合物，更包含血小板，其中每一毫升的該細胞培養組合物包含1×106 至1×108 個血小板。
- 如請求項1所述之細胞培養組合物，更包含補體C3，其中每一毫升的該細胞培養組合物包含0.1微克至20微克的補體C3。
- 一種細胞培養組合物在促進細胞生長的用途，其中該細胞培養組合物包含一培養基以及粒線體。
- 如請求項5所述之用途，其中每一毫升的該細胞培養組合物包含1微克至100微克的粒線體。
- 如請求項5所述之用途，其中該細胞包含視網膜細胞、肝臟細胞、腎臟細胞、肌腱細胞、皮膚細胞或免疫細胞。
- 如請求項7所述之用途，其中該免疫細胞為具有CD56+及CD3-標記的自然殺手細胞。
- 一種細胞培養組合物在改善受損或老化的幹細胞的功能的用途，其中該細胞培養組合物包含一培養基以及粒線體。
- 如請求項9所述之用途，其中每一毫升的該細胞培養組合物包含1微克至100微克的粒線體。
- 如請求項9所述之用途，其中使用該細胞培養組合物培養的幹細胞為間質幹細胞。
- 如請求項9所述之用途，其中使用該細胞培養組合物培養的幹細胞具有CD90+及CD34-標記。
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