TW201418465A - 人類間質幹細胞之培養基 - Google Patents

人類間質幹細胞之培養基 Download PDF

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TW201418465A
TW201418465A TW102141117A TW102141117A TW201418465A TW 201418465 A TW201418465 A TW 201418465A TW 102141117 A TW102141117 A TW 102141117A TW 102141117 A TW102141117 A TW 102141117A TW 201418465 A TW201418465 A TW 201418465A
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Cervantes Jose Maria Diez
Rodriguez Rodrigo Gajardo
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Grifols Sa
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Abstract

本發明係關於一種人類間質幹細胞(hMSC)之新穎培養基。更特定言之，本發明係關於一種可在其中生長hMSC株(包括彼等在通常用於此類型細胞之培養基中不生長者)之培養基。

Description

人類間質幹細胞之培養基
本發明係關於一種人類間質幹細胞(hMSC)之新穎培養基。更特定言之，本發明係關於一種可在其中生長hMSC株(包括彼等因而其已適應特定培養基而不在通常用於此類型細胞之培養基中生長者)之培養基。
hMSC為基本上表徵係其有分化為尤其是諸如骨骼、軟骨、肌腱、肌肉及脂肪組織之各種間質組織之能力的多潛能細胞。可自成人之各種組織分離得hMSC，包括彼等會存在於骨髓、脂肪組織及臍帶血中者。
hMSC之抗增生、免疫調節及抗炎作用已使注意力集中於將該等細胞作為針對因免疫系統引起之疾病(包括移植物抗宿主疾病)、實質器官移植後排斥及自體免疫疾病之有潛力治療劑。
已知hMSC之活體外培養的要務是保持其多潛能能力，為此需要在標準培養基中添加外源因子，諸如(例如)鹼性纖維母細胞生長因子(bFGF)、表皮生長因子(EGF)及轉變生長因子(TGFβ-1)。然而，大多數hMSC依賴於特定培養基而亦無法在通常用於間質幹細胞之培養之培養基(諸如，經驗證可用於間質幹細胞(MSCQ)之補充牛胎血清之DMEM/F12培養基)上培養。
為了克服該等問題，本發明係關於一種可於其上生長不在其他 培養基調配物中生長之hMSC的hMSC之新穎培養基。根據本發明之培養基包含：
a)通常用於間質幹細胞之培養之基礎培養基
b)人類白血球/血小板層溶解物(膚色血球層；LBC)
c)胰島素
d)亞硒酸鈉
e)乙醇胺
f)鹼性纖維母細胞生長因子(bFGF)。
如本文件中所用，術語「培養基」係關於一種用於細胞之培養、生長或增生之營養溶液。術語「細胞培養」係指將細胞維持、培養或生長於人工活體外環境中。
用於本文件中之術語「基礎培養基(basal media/basal medium)」係指當在補充可含或可不含血清之不同補充劑時其中能夠生長人類間質細胞之任何培養基。基礎培養基提供標準無機鹽(諸如鋅、鐵、鎂、鈣及鉀鹽)、以及維他命、葡萄糖、緩衝系統及必需胺基酸。
較佳地，通常用於在根據本發明培養基中培養間質細胞之基礎培養基為DMEM/F12+丙酮酸鹽、為等份數的杜貝卡氏改良依格培養基(Dubelcco's Modified Eagle Medium)(DMEM)(其為由Harry Eagle開發之最低必須培養基之改質物)、與由Ham開發用於生長CHO(中國倉鼠卵巢)細胞之Ham's F12培養基、Ham’s F10、Ham’s F12、MCDB 131(為由Knedler與Ham開發作為用於人類細胞之生長之含減少之血清補充劑之培養基之培養基)之混合物之DMEM/F12培養基、或RPMI 1640，其名稱衍生自Roswell Park Memorial Institute(洛斯維帕克紀念研究所)之首字母，在該研究所其由Moore及其同事研發用於正常及腫瘤性淋巴細胞之培養，然而，當在經適當補充時，其可生長其他不同 類型細胞。該等培養基之調配及組成廣為人知及可從任一家製造商或供應商(諸如(例如)Gibco(Life Technologies))獲得。更佳地，該培養基為DMEM/F12+丙酮酸鹽。
根據本發明之人類間質細胞培養基之組分之一為為白血球/血小板層溶解物。該白血球/血小板層(亦稱為「膚色血球層」)為在全血之離心或沉降後於血漿及紅血球之間所觀察到層。其基本上包含白血球及血小板。根據本發明之培養基中該白血球/血小板層溶解物(膚色血球層)之存在量介於5及20%(v/v)之間，更佳係介於7及15%(v/v)之間，且最佳為10%(v/v)。
根據本發明培養基之另一組分為胰島素。已知胰島素對細胞生長之有益效果(Cartwright,T.y Shah，G.P Culture media.Basic cell culture，第2版，Davis,J.M.編輯2002年Oxford University Press，美國紐約)。根據本發明之培養基中胰島素之存在量較佳介於2及20mg/L之間，更佳係介於5及15mg/L之間且最佳為10mg/L。
根據本發明培養基之又一組分為硒(呈亞硒酸鈉形式)。硒為通常藉由血清提供至培養基且可能為血清所需的微量元素。該培養基中亞硒酸鈉之存在量介於0.005及1.730mg/L之間。
此外，根據本發明之人類間質細胞培養基包含乙醇胺。已知乙醇胺促進細胞之建構。根據本發明之培養基中乙醇胺之存在量介於1及8mg/L之間，較佳係介於2及5mg/L之間。
最後，根據本發明之間質細胞培養基亦包含bFGF。已知bFGF被推薦用於培養間質細胞(Sato,J.等人Specific cell types and their requirements.Basic cell culture，第2版，Davis,J.M.編輯2002年Oxford University Press，美國紐約)。根據本發明之培養基中bFGF之存在量介於5及25ng/mL之間。
令人意外地，藉由根據本發明之培養基中該等組分之組合，可培養在通常用於此類型細胞之培養基中不生長之人類間質細胞株，包括彼等適應一般可自商業上購得之其他培養基者，諸如(例如)由Lonza或PromoCell供應之人類間質細胞。
根據本發明之培養基可視需要包含抗生素。可使用通常用於培養此類型細胞之任何抗生素。較佳地，該抗生素為健他黴素-兩性黴素之混合物。熟習此項技藝者可輕易地確定該培養基中必需之抗生素的用量。
就間質幹細胞之生長而言，根據本發明之培養基必須補充例如胎牛血清(FBS)或例如如美國專利案8,252,590 B2中所述獲得之衍生自人類血漿之細胞培養補充劑(CCS)。若使用FBS，介於10及20%(v/v)之間的量被認為係足夠。另一方面，若使用CCS來補充根據本發明之培養基，則介於10及40%(v/v)之間的量可被視為係足夠。
根據本發明之人類間質幹細胞培養基可呈乾燥形式或呈液體形式。就以乾燥形式使用而言，可將其組分溶解於用於培養間質細胞之適宜液體中。熟習此項技藝者已知其中可溶解根據本發明培養基之該等組分之液體之類型，諸如(例如)無菌蒸餾水。
下文參考許多實施例來更詳細地論述本發明。然而，該等實例並非意圖限制本發明之範疇。
實例1.
製得根據本發明之培養基，其包含作為基礎培養基之DMEM/F12+丙酮酸鹽、10%(v/v)人類白血球/血小板層溶解物(膚色血球層)、10mg/L胰島素、0.0067mg/L亞硒酸鈉、2mg/L乙醇胺及20ng/mL bFGF。將該培養基稱為LV2。培養基LV2係單獨使用或補充有經驗證可用於間質幹細胞(MSCQ)之10%(v/v)胎牛血清(FBS)或衍生自 人類血漿之15%(v/v)細胞培養補充劑(CCS)。培養購自Lonza(巴塞爾，瑞士)或PromoCell(海德爾堡，德國)之hMSC。使用製造商所推薦的培養基作為正對照組。只有一種市售基礎培養基是用為負對照組。結果顯示於表1中。
由表1中可見，當在僅使用DMEM/12基礎培養基作為培養基(包括該基礎培養基補充FBS或CCS之情況)時，Lonza hMSC無法生長。當在根據本發明之LV2培養基係單獨使用時，其亦不生長。然而，其可生長於在補充FBS或CCS之情況下的根據本發明之培養基、及製造商所推薦的培養基中。
實例2.
為了確定將根據本發明培養基之所有組分組合之效應，依實例1中所述製得LV2培養基及除該培養基外亦製得缺乏根據本發明培養基之至少一種組分之各種不同培養基。所製得之培養基及所獲得之結果顯示於表2中。
由表2可見，Lonza及PromoCell hMSC僅能生長於製造商所推薦的培養基及補充CCS之根據本發明之培養基中。當在缺少根據本發明培養基之至少一種組分時，該培養基中無法生長hMSC。
雖然已相對於較佳實施例之實例來論述本發明，但該等實例不應視作是限制本發明，本發明係由對隨後申請專利範圍之最寬泛解讀來界定。

Claims (14)

  1. 一種人類間質細胞之培養基，其包含：a)用於間質幹細胞之基礎培養基；b)人類白血球/血小板層溶解物(膚色血球層(buffy coat))；c)胰島素；d)亞硒酸鈉；e)乙醇胺；及f)鹼性纖維母細胞生長因子(bFGF)。
  2. 如請求項1之人類間質細胞培養基，其中該間質幹細胞基礎培養基為DMEM/F12+丙酮酸鹽、Ham’s F10、Ham’s F12、MCDB 131或RPMI 1640。
  3. 如請求項1或2之人類間質細胞培養基，其中該培養基中人類白血球/血小板層溶解物(膚色血球層)之存在量為5至20%(v/v)。
  4. 如請求項3之人類間質細胞培養基，其中該培養基中人類白血球/血小板層溶解物(膚色血球層)之存在量為7至15%(v/v)。
  5. 如請求項1或2之人類間質細胞培養基，其中胰島素之存在量為2至20mg/L。
  6. 如請求項5之人類間質細胞培養基，其中胰島素之存在量為5至15mg/L。
  7. 如請求項1或2之人類間質細胞培養基，其中亞硒酸鈉之存在量為0.005至1.730mg/L。
  8. 如請求項1或2之人類間質細胞培養基，其中乙醇胺之存在量為1至8mg/L。
  9. 如請求項8之人類間質細胞培養基，其中乙醇胺之存在量為2至5mg/L。
  10. 如請求項1或2之人類間質細胞培養基，其中bFGF之存在量為5至25ng/mL。
  11. 如請求項1或2之人類間質細胞培養基，其進一步包含抗生素。
  12. 如請求項11之人類間質細胞培養基，其中該抗生素為健他黴素/兩性黴素之混合物。
  13. 如請求項1或2之人類間質細胞培養基，其中該培養基係補充10%至20%(v/v)含量之胎牛血清(FBS)。
  14. 如請求項1或2之人類間質細胞培養基，其中該培養基係補充10%至40%(v/v)含量之衍生自人類血漿之細胞培養補充劑(CCS)。
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