201040258 六、發明說明： 【發明所屬之技術領域】 • 本發明係有關於用來分離、體外擴增及收獲造血幹細 胞的方法與系統。 【先前技術】 幹細胞因可做為多種疾病、異常或失能情況之可再生 0 的組織或細胞來源，因而受到相當大的關注。幹細胞主 要來自於胚胎幹細胞與成人幹細胞。胚胎幹細胞是源自 胚胎，至於成人幹細胞則通常以極少量的方式存在於每 一種組織中’且已知在包括腦、骨髓、週邊血液、血管、 骨骼肌、皮膚、臍帶、脂肪組織及肝臟等多種組織中都 可發現成人幹細胞的蹤影。以胚胎幹細胞來治療疾病常 有倫理道德上的爭議，相反的，成人幹細胞則沒有這層 顧慮。以成人幹細胞進行治療的另一項優點在於可將源 ❹ 自患者自己的幹細胞在體外擴增後，再重新送回患者自 己體内，因此，也不會有免疫反應方面的困擾，更無需 使用免疫抑制藥物。 造血幹細胞是最常被用於臨床治療的成人幹細胞之 一，但是’因為這類型幹細胞在成人體内的數量極為稀 少，且不易利用細胞培育擴增其數量，因此有許多的研 究專注於尋找可分離及體外增生成人幹細胞的方法，希 望藉此獲得足夠數目的成人幹細胞，以用於後續的臨床 治療。日本專利H08-69中揭示一種以Fic〇ii_Hypaque方 法自臍帶血中分離單核細胞的操作，但是這種方法採用 3 201040258 =放因此極容易受到細菌或黴菌的汗染。此 時門就1擴增之前，其用來分離單核細胞的操作 時間就長達3小時， 2_-237136中揭干不經濟。日本公開專利 „ $晏不了 一種極費力的方法，此方法是先利 ’—利用磁珠，來分離出CD34+造血細胞。同樣 4 >、進行體外擴增之前，單是分離CD34+造血細胞的 由門就長達3〜5 *時之久。另一日本公開專利Hi〇_8495〇 Ο 揭不了帛透析法，其是將血液細胞通過塗佈有親水 性共聚物的領布，其中CD34、血細胞會被留置在不 織布上/、可惜此方法的產率太低，以致於沒有實際的 產業價值。 基於以上，此領域中亟需一種用以分離及增生成人幹 細胞’特別疋，人類造血幹細胞，的改良方法，以便將 其應用到包括骨髓移植在内的各種醫學治療上。 【發明内容】 在此廣義地揭示及實施用來分離、體外擴增及收獲造 血幹細胞的方法與系統。所揭示的方法與系統，非常省 時、谷易使用且谷許以批次式地或連續式地方式來分 離、體外擴增及收獲造血幹細胞。 在一方面，本發明提供用以分離、體外擴增及收獲造 血幹細胞的系統。 在一實施方式中’本發明系統為一種批次式系統，此 系統包括：一過濾腔室，其中包含一孔徑在約2μπι至約 ΙΟΟμιη間的濾膜；一第一入口，用來將一造血幹細胞來 4 201040258 源引入至該過濾腔室中；一第二入口，用來將一清洗溶 液引入至該過濾腔室中；及一第一出口，用來將該清洗 . 溶液從該過濾腔室中排出。在一實例中，此系統更包括 一儲存腔室，用來儲存已穿透(permeate)通過該過濾腔室 ' 的該造血幹細胞來源。在另一實例中，此造血幹細胞是 被滯留在該過濾腔室中的該濾膜上，之後，將該濾膜從 該過濾腔室中取出，並放在一幹細胞培養基中進行體外 擴增。 Ο 在另一實施方式中，本發明系統為一種連續式系統， 此系統包括：一過濾腔室，其中包含一孔徑在約2μιη至 約ΙΟΟμηι間的濾膜；一第一入口，用來將一造血幹細胞 來源引入至該過濾腔室中；一第二入口，用來將一清洗 溶液引入至該過濾腔室中；一第三入口，用來將一幹細 胞培養基引入至該過濾腔室中；一泵，用來使該幹細胞 培養基得以在該系統内循環；一第一出口，用來將該清 洗溶液從該過遽腔室中排出；及一第二出口，用來收集 〇造崎細胞。在—實财，此系統更包括-儲存腔室， 用來儲存e^it(penneate)iii^該n腔冑❺該造金幹 、田胞來源。在另—實例中，可由該第二出口中所收集的 幹細胞培養基巾連續缝穫造*幹細胞。 在第一方面，本發明提供透過本發明系統來分離、體 外擴增及收獲造金幹細胞的方法。 在一實施方式中’本發明方法是有關以批次方式來分 體外擴增及收獲造血幹細胞的方法，此方法 含以下步騍： 5 201040258 (a) 提供一批次式系統來分離、體外擴增及收獲造血 幹細胞’此系統包含： 一過濾腔室’其中包含一孔徑在約2pm至約 ΙΟΟμιη間的濾膜； 一第一入口，用來將一造血幹細胞來源引入 至該過濾、腔室中； 一第二入口，用來將一清洗溶液引入至該過 濾腔室中；及 一第一出口’用來將該清洗溶液從該過濾腔 室中排出； (b) 從該第一入口將該造血幹細胞來源引入至該過 濾腔室内； (c) 從該第二入口將該清洗溶液引入至該過濾腔室 中；及 (d) 將該濾膜培育在一幹細胞培養基中，以擴增滯留 在該濾膜上的造血幹細胞。 在一實例中，本方法更包含：（cl)在步驟（c)之後，將該 清洗溶液從該過遽腔室中排出。 在一第二實施方式中，本發明方法是有關以連續方式 來为離、體外擴增及收獲造血幹細胞的方法，此方法依 序包含以下步驟： (a)提供一連續式系統來分離、體外擴增及收獲造 血幹細胞，此系統包含： 6 201040258 一過濾腔室，其中包含一孔徑在約2μιη 至約ΙΟΟμιη間的濾膜； 一第一入口 ’用來將一造金幹細胞來源 引入至該過濾腔室中； 一第一入口’用來將一清洗溶液引入至 該過濾腔室中； 一第三入口 ’用來將一幹細胞培養基引 入至該過濾腔室中； 一幫浦，用來使該幹細胞培養基可在該 系統中循環； 一第一出口’用來將該清洗溶液從該過濾 腔室中排出；及 一第二出口 ’用來收集該造血幹細胞； (b) 從該第一入口將該造血幹細胞來源引入至該 過濾腔室内； (c) 從該第二入口將該清洗溶液引入至該過濾腔 室中； (d) 從該第三入口將該幹細胞培養基引入至該過 濾腔室中； (e) 啟動該幫浦，使該幹細胞培養基得以在該系統 中循環； (f) 自該第二出口所收集的該幹細胞培養基中收 獲該造血幹細胞。 7 201040258 在一實例中，上述方法更包含以下步驟：（bi)在步 驟(b)之後，將已穿透(permeate)通過該過濾腔室的該造 血幹細胞來源儲存在-儲存腔室# ;⑷）在步驟⑷之 後，從該第一出口將該清洗溶液排出該過濾腔室外·及 ⑷）在步驟⑷之後，湘從該第二出口採取少量該幹細 胞培養基來進行該造血幹細胞的採樣。201040258 VI. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to methods and systems for isolating, in vitro expanding and harvesting hematopoietic stem cells. [Prior Art] Stem cells have received considerable attention because of their ability to be a regenerable tissue or cell source for a variety of diseases, abnormalities, or disability. Stem cells are mainly derived from embryonic stem cells and adult stem cells. Embryonic stem cells are derived from embryos, while adult stem cells are usually present in every tissue in a very small amount 'and are known to include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, umbilical cord, adipose tissue, liver, etc. Adult stem cells can be found in a variety of tissues. It is often ethical and controversial to treat diseases with embryonic stem cells. Conversely, adult stem cells do not have this concern. Another advantage of treatment with adult stem cells is that the patient's own stem cells can be re-introduced back into the patient's body after being expanded in vitro, so there is no immune response or use. Immunosuppressive drugs. Hematopoietic stem cells are one of the most commonly used adult stem cells for clinical treatment, but 'because this type of stem cells is extremely rare in adults, and it is not easy to use cell culture to amplify its number, so many studies focus on finding A method of isolating and in vitro proliferating adult stem cells, in which it is desirable to obtain a sufficient number of adult stem cells for subsequent clinical treatment. Japanese Patent No. H08-69 discloses an operation for separating monocytes from cord blood by the Fic〇ii_Hypaque method, but this method uses 3 201040258 = it is extremely susceptible to bacterial or mold staining. At this time, before the amplification of the gate, the operation time for separating monocytes was as long as 3 hours, and it was uneconomical to uncover it in 2_-237136. Japanese public patents „ 晏 can not be a very laborious method, this method is the first profit - using magnetic beads to separate CD34 + hematopoietic cells. The same 4 > The door is as long as 3 to 5 *. Another Japanese patent, Hi〇_8495〇Ο, does not reveal the dialysis method, which is to pass blood cells through a collar cloth coated with a hydrophilic copolymer, in which CD34, blood cells Will be left on the non-woven fabric / Unfortunately, the yield of this method is too low, so that there is no actual industrial value. Based on the above, there is a need in the field to separate and proliferate adult stem cells 'special sputum, human hematopoietic stem cells, Improved methods for its application to various medical treatments including bone marrow transplantation. SUMMARY OF THE INVENTION Methods and systems for isolating, in vitro expanding and harvesting hematopoietic stem cells are broadly disclosed and implemented herein. With the system, it is very time-saving, easy to use, and it is possible to separate, in vitro expand and harvest hematopoietic stem cells in batch or continuous manner. The present invention provides a system for isolating, in vitro expanding and harvesting hematopoietic stem cells. In one embodiment, the system of the present invention is a batch system comprising: a filtration chamber containing a pore size of about 2 μm a filter between about ΙΟΟμιη; a first inlet for introducing a hematopoietic stem cell 4 201040258 source into the filtration chamber; a second inlet for introducing a cleaning solution into the filtration chamber; And a first outlet for discharging the cleaning solution from the filtration chamber. In an example, the system further includes a storage chamber for storing permeate through the filtration chamber The hematopoietic stem cell source. In another example, the hematopoietic stem cell is retained on the filter membrane in the filtration chamber, after which the filter membrane is removed from the filtration chamber and placed in a stem cell culture medium. In vitro expansion is carried out. Ο In another embodiment, the system of the present invention is a continuous system comprising: a filtration chamber comprising a pore size of from about 2 μm to about ΙΟΟμηι a filter; a first inlet for introducing a source of hematopoietic stem cells into the filtration chamber; a second inlet for introducing a cleaning solution into the filtration chamber; and a third inlet for Introducing a stem cell culture medium into the filtration chamber; a pump for circulating the stem cell culture medium; and a first outlet for discharging the cleaning solution from the buffer chamber; a second outlet for collecting the sputum cells. In the real money, the system further includes a storage chamber for storing e^it(penneate) iii^ the n cavity, the gold stalk, the field cell In another example, the stem cells can be continuously sewn from the stem cell culture tissue collected in the second outlet. In a first aspect, the invention provides for isolation, in vitro expansion and harvesting of gold stem cells by the system of the invention. Methods. In one embodiment, the method of the present invention relates to a method for in vitro expansion and harvesting of hematopoietic stem cells in a batch manner, the method comprising the following steps: 5 201040258 (a) providing a batch system for separation and expansion in vitro Increasing and harvesting hematopoietic stem cells 'This system comprises: a filtration chamber containing a membrane having a pore size between about 2 pm and about ΙΟΟμιη; a first inlet for introducing a source of hematopoietic stem cells into the filtration, chamber a second inlet for introducing a cleaning solution into the filtration chamber; and a first outlet 'for discharging the cleaning solution from the filtration chamber; (b) from the first inlet a hematopoietic stem cell source is introduced into the filtration chamber; (c) introducing the cleaning solution into the filtration chamber from the second inlet; and (d) cultivating the filter in a stem cell culture medium to amplify retention Hematopoietic stem cells on the filter. In one example, the method further comprises: (cl) discharging the cleaning solution from the buffer chamber after step (c). In a second embodiment, the method of the present invention relates to a method for ex vivo, in vitro expansion and harvesting of hematopoietic stem cells in a continuous manner, the method comprising the steps of: (a) providing a continuous system for isolation, in vitro Amplifying and harvesting hematopoietic stem cells, the system comprising: 6 201040258 a filtration chamber comprising a membrane having a pore size between about 2 μm and about ;μιη; a first inlet' for introducing a source of gold stem cells to the filtration a first inlet 'for introducing a cleaning solution into the filtration chamber; a third inlet' for introducing a stem cell culture medium into the filtration chamber; a pump for making Stem cell culture medium can be circulated in the system; a first outlet 'used to drain the cleaning solution from the filtration chamber; and a second outlet' to collect the hematopoietic stem cells; (b) from the first inlet The hematopoietic stem cell source is introduced into the filtration chamber; (c) introducing the cleaning solution into the filtration chamber from the second inlet; (d) the stem cell culture from the third inlet Group is introduced into the filtering chamber; (e) to start the pump, so that the stem cell culture medium is circulated in the system; the stem cell culture medium (f) from the second outlet of the collected harvested the hematopoietic stem cells. 7 201040258 In an example, the above method further comprises the step of: (bi) storing, after step (b), the source of hematopoietic stem cells that have been permeated through the filtration chamber in a storage chamber #; (4) After the step (4), the cleaning solution is discharged from the first outlet to the outside of the filter chamber, and (4). After the step (4), a small amount of the stem cell culture medium is taken from the second outlet to perform sampling of the hematopoietic stem cells.
以下將詳述本發明一或多實施方式之細節，並可由以 下的發明詳細說明和中請專利來了解本發明的優點及其 他特徵。並且，可以藉由單獨地或組合地使用記載於隨 附申請專利範圍中之手段而理解本發明之目的與優點。 此外，上述有關本發明的一般性敘述及以下詳細說 明，都只是例示，並非用以限定本發明之範疇。 【實施方式】 定義 在本文中，「幹細胞（stem ceii)」意指一種全能或多能 細胞，其可自我複製並形成特化的組織細胞或器官。幹 細胞可自我分裂產生兩個子代幹細胞，或是一個子代幹 細胞和一個前驅細胞（progenitor cell)，然後進一步辦生 成為一組織之成熟、具完整形態的細胞。在本文中j幹 細胞（stem cell)」意指多能性細胞，此「多能性細胞 (multipotent cell)」意指多能性幹細胞，其可成長為哺乳 動物體内特定組織或器官中之二或多種不同形態之細 胞。在本文中，「造血幹細胞」意指可成長為成熟:球細 8 201040258 胞的多能性細胞’其包括，但不限於，紅血球細胞、白 血球細胞、巨核細胞、血小板及τ_型和㈣淋巴細胞。 ‘ 本文中用來指出本發明之最大範圍之數據範圍及參 •數均為近似值’特定較佳實施财所記述之數據值已盡 可能地精準ϋ而各個測量實驗均有其標準偏差，因此 任何數據值必有部分誤差。 此外’若無特別於上下文中清楚記述其他意義，則說 ‘明^内容及後附申請專利範圍中所使用之如「一（“a” 〇r an )」與「該（the)」等特定用語均包含其複數形態。 习除非另行定義，文中所使用之所有專業與科學用語與 =头技藝者所熟悉之意義相同。此外，任何與所記載内 谷相似或均等之方法及材料皆可應用於本發明方法中。 =中所述之較佳實施方法與材料僅做示範 之用。於本申 請書中所提到之所有參考文獻均全體納入參考， =揭露並敘述該文獻所記載之相關方法及/或材 ❹；〜二此外，文中所討論之文獻僅揭露本發明申請 I :之習知技術。並且無任何文獻顯示本發明内 容曾為習知技術所揭露。本發明内容所得到之實際數 據會因個別的實施條件而與本發明揭露於說明書内容中 之數據有所不同。 以下將詳細說明有關用來分離、體外擴增及收獲造血 幹細胞的方法及系統。 用 分離、體也於w m ^.......... 201040258 依據本發明一實施方式，一種用來分離、體外擴增及 收獲造血幹細胞的批次式系統包含：一過滤腔室，其中 包含一孔徑在約2μιη至約ΙΟΟμιη間的濾膜；一第一入 口，用來將一造jk幹細胞來源引入至該過滤腔室中；一 • 第二入口，用來將一清洗溶液引入至該過濾腔室中；及 一第一出口，用來將該清洗溶液從該過濾腔室中排出。 參照第1圖，圖中緣示出依據本發明一實施方式之批 次式系統100。此系統100包括一過濾、腔室1 〇 1、一第一 〇 入口 104、一第二入口 105、一第一出口 106和一集液容 器107。此過濾腔室101中設有一濾膜，且此濾膜之孔 徑在約2μιη至約ΙΟΟμιη間，例如，約2μιη至約25μιη 間、約3μιη至約20μιη間、或是約3μιη至約15μιη間。 操作此系統時’從第一入口 1〇4引入一造幹細胞來源 102使通過過濾腔室ιοί ;然後，從第二入口 ι〇5將一清 洗溶液103饋送入過濾腔室ιοί ;接著，用過的清洗溶 液103可從第一出口 1〇6離開過濾腔室101而流到集液 〇 容器ι〇7中。造血幹細胞會被過濾腔室ιοί中的濾膜擋 下而滯留在濾膜上，因此，可從過濾腔室101中將整個 濾膜拆卸下來，放在培養容器中，加入適量的幹細胞培 養基，進行體外擴增。 適於用在本發明此實施方式中的造血幹細胞來源包 括，但不限於，臍帶血、骨髓液或週邊血液。在一實例 中，此造血幹細胞來源是臍帶血，此臍帶血是向接受剖 腹產手術或自然產之婦女請求其同意後，取得濟帶，然 後以針筒自臍帶中收集血液並儲存在内含抗凝血劑 201040258 (如’檸檬酸或肝素）之血袋中。在另一實例中，此造血 幹細胞來源是骨髓液，此同樣是先取得捐贈者同意，然 後依標準操作流程自捐贈者骨髓中抽取出骨髓液。待造 血幹細胞來源102滲透通過過濾腔室丨〇1後，接著就從 • 第二入口 1〇5將清洗溶液1〇3引入至系統100中。此清 洗步驟的目地在於透過移除其中所含的紅血球細胞來進 一步純化造血幹細胞來源1〇2，因為一般認為紅血球細 胞可能會抑制造血幹細胞。可用於本發明實施方式的適 〇 當清洗溶液103包括，但不限於，無血清的細胞培養基、 含血清的細胞培養基、生理食鹽水、緩衝溶液、内含 EDTA的生理食鹽水、内含EDTA的緩衝溶液、低血小 板含；!：的血漿（platelet-poor plasma)及其之組合。可以血 液做為來源物，依照一般習知的方法來製造上述之低血 小板含量的血漿。在一實例中，將臍帶血以適當速度離 心’接著以濾膜（孔徑為〇.22μπι)過濾離心後所得的上清 液，藉以移除其中的紅血球細胞，並形成低血小板含量 Q 的血漿。或者，也可以細胞培養基、生理食鹽水、緩衝 溶液做為本實施方式所需的清洗溶液103。當以細胞培 養基做為清洗溶液103時，其中可包含或不包含血清； 而當以生理食鹽水或緩衝溶液做為本實施方式所需的清 洗溶液103時，則其中可包含或不包含乙二胺四醋酸 (ethylene diamine tetraacetic acid, EDTA)。在一實例中， 是以不含血清的細胞培養基做為清洗溶液103,例如購 自 StemCell Technologies (USA)的 StemSpan SFEM 培養 基，此StemSpan SFEM培養基中可包含或不包含生長因 201040258 子或其他類型的細胞素（cytokines)。在另一實施方式 中，則是組合使用不含血清的細胞培養基與上述低血小 板含量的血漿兩者，來做為清洗溶液103。此外，可藉 助於一外加的幫浦，例如螺動式幫浦（peristaltic pump)， • 來將造血幹細胞來源102或是清洗溶液103引入至系統 100中。而且，無論是造血幹細胞來源102或是清洗溶 液103，其流速都在約1〜1〇毫升/分鐘間。在一實例中， 上述造血幹細胞來源102或是清洗溶液103中任一溶液 〇 的流速約為1毫升/分鐘。 過濾腔室101的腔室主體中設有一孔徑在約2μιη至 約ΙΟΟμιη間之濾膜。舉例來說，此濾膜的孔徑可在約2μιη 至約25μιη間、約3μπι至約20μιη間、或是約3μιη至約 15μηι間。目前有多種技術可用來測量濾膜的孔徑，例如 掃描式電子顯微鏡或是孔洞分析儀（liquid extrusion porosimetry) ’或是其他任何適當的技術。在所揭示的實 例中，濾膜孔徑是利用孔洞分析儀進行估算。此領域中 〇 熟悉技藝人士應能了解，濾膜孔徑大小的估算數值一般 會隨著所採用的特定技術不同而有所差異。對細胞來 說，適合做為濾膜的材料本身必須具備良好的生物相容 性、良好的模造性（moldability)、良好的無菌性、以及低 毒性。此濾膜一般是由人工合成的聚合物製成，適當的 人工合成聚合物包括，但不限於，聚乙烯、聚丙烯、聚 苯乙烯、丙烯樹脂、尼龍、聚酯、聚碳酸酯、聚丙烯醯 胺和聚尿烷；或是由天然的聚合物製成，適當的天然聚 合物包括’但不限於，壤脂（agarose)、纖維素、纖維素 12 201040258 乙酸酯、幾丁質、曱殼素（chitosan)、和藻酸酯（alginate> ; 或是由無機材料製成，適當的無機材料包括，但不限於， 氫氧基構灰石（hydroxyapatite)、玻璃、氧化銘和氧化鈦； 或是由金屬製成，例如不鏽鋼、鈦及銘。此遽膜較佳是 " 由聚尿烷為底的聚合物或是聚對苯二曱酸二乙酯為底的 聚合物（即，不織布）材料製成。還可透過接枝反應在這 些聚合物主鏈或侧鏈上嫁接上其他分子，進一步修飾這 些聚合物。適合嫁接在聚合物主鏈或側鏈上的分子，包 〇 括，但不限於，氨基酸、胜肽、葡萄糖胺聚醣 (glycosaminoglycans)、和糖蛋白。在一實例中，利用習 知的電漿放電法（如，揭示在JP 2005-323534中的方法， 其内容在此併入做為參考），在上述聚尿烧為底的聚合物 表面上嫁接上諸如碳酸基之類的官能基團。在另一實例 中，將表面嫁接有碳酸基之聚尿烷為底的聚合物做為濾 膜，直接用來分離及擴增造血幹細胞。相反的，若是使 用聚對苯二曱酸二乙酯為底的聚合物做為濾膜材料，則 Q 可在濾膜上額外鍍覆上一層以至少一種選自下列之單體 所聚合而成的聚合物層，適合的單體是選自以下：曱丙 稀酸經乙醋（hydroxyethyl methacrylate，HEMA)、甲丙婦 酸二曱基氨乙醋（dimethylaminoethyl methacrylate, DM)、曱丙稀酸正-丁酉旨（n-butyl methacrylate，BMA)、 N，N-二曱基丙稀醯胺（N，N-dimethylacrylamide, DMA)、N-丙浠酿嗎淋(N_acryloylmorpholine，AMO)和 N-乙稀0比p各烧_ (N_vinylpyrrolidone，VP)。在一實例中，此 濾膜是由聚對苯二曱酸二乙酯為底的聚合物（亦即，不織 13 201040258 布）製成，且其上鍍覆有一層由BMA和DMA兩種單體 聚合而成之聚合物層。 在造血幹細胞來源102通過過濾腔室101後，其中的 造血幹細胞會被過濾腔室101中的濾膜擋下而留在濾膜 * 上。接著，從過濾腔室101中將其上留有造血幹細胞的 濾膜取出，並將整個濾膜浸泡在内含適當幹細胞培養基 的細胞培養盤中，進行體外直接培養。在一實例中，此 幹細胞培養基為StemSpan SFEM培養基，此培養基中可 〇 更添加一種細胞素混合物以及低密度脂蛋白（LDL)。此細 胞素混合物中包括有重組人類幹細胞因子、重組人類血 小板生成素（recombinant human thrombopoietin)及重組 人類Flt-3配體（Flt-3 ligand)。每一種細胞素的濃度在開 始進行細胞培育時是介於約5 ng/ml至500 ng/ml間，且 較佳是在約10 ng/ml至100 ng/ml間。至於LDL，其使 用情形則是非必要的，當選擇添加LDL時，LDL的濃度 在開始進行細胞培育時，一般是介於約0.1 mg/ml至20 ❹ mg/ml間，且較佳是在約5 mg/ml間。在一實例中，以 一種不含血清的細胞培養基作為清洗溶液103，並使通 過過濾腔室101，將此過濾腔室101中留有造血幹細胞 於其上之濾膜取出，直接進行體外細胞擴增約10天，相 較於控制組（S卩，從造血幹細胞來源中直接分離出來的造 血幹細胞，其直接進行體外細胞擴增後所得的幹細胞數 目），可使造血幹細胞的數目擴增至少約63%。在另一實 例中，使用低血小板含量的血清做為清洗溶液1 〇3，並 201040258 使通過過濾腔室101，相較於控制組，可使造血幹細胞 的數目擴增至少約280%。 本實驗之批次式用以分離造血幹細胞的系統在使用 上極為方便，且省時，從造血幹細胞來源通過過濾腔室 . 到分離出幹細胞可進行體外擴增止，所需花費的時間相 當短，僅約10分鐘至約60分鐘。在一較佳實施方式中， 操作時間約為18分鐘。 J來分離、體外擴增及收藉造血幹細臉的連锖式率怂热 Ο 方法 依據本發明另一實施方式，一種用來分離、體外擴增 及收獲造血幹細胞的連績式系統包含：一過濾腔室，其 中包含一孔控在約2μηι至約1 ΟΟμιη間的濾膜；一第一入 口，用來將一造血幹細胞來源引入至該過濾腔室中；一 第二入口’用來將一清洗溶液引入至該過濾腔室中；一 第三入口’用來將一幹細胞培養基引入至該過濾腔室 中；一幫浦’用來循環該幹細胞培養基於整個系統中； 〇 一第一出口，用來將該清洗溶液從該過濾腔室中排出； 及一第二出口，用來收集該造血幹細胞。 參照第2圖’圖中繪示出依據本發明一實施方式之連 續式系統200。此系統200包括一過濾腔室201、一第一 入口 204、一第一入口 205、一第一出口 206、一集液容 •器207、一第二出口 208、一造血幹細胞收集容器209、 -一弟二入口 211和一幫浦213。此系統更包括一非必要 的儲存腔室210,其連接至該過濾腔室201。此過濾腔室 201中設有一遽膜’且此遽膜之孔徑在約2μηι至約1 〇〇pm 15 201040258 Ο Ο 間。操作此系統時，從第一入口 204引入一造血幹細胞 來源202使通過過濾腔室2〇1。非必要地，可再次收集 通過過濾腔室201之造血幹細胞來源2〇2，並將其儲存 在一非必要的儲存腔室210中。接著，從第二入口 2〇5 將一清洗溶液203饋送入過濾腔室2〇1 ;用過的清洗溶 液203則可從第一出口 206離開過濾腔室201而流到集 液谷器207中。接著，從第三入口 211將幹細胞培養基 212饋送至過濾腔室2〇1中並透過幫浦213之助，使此 幹細，培養基212可在系統細中循環。每隔數天，例 如’母隔2天’即简鮮的培養基212來置換原先在系 統200中循環的幹細胞培養基212，並將此用過的幹細 2。養基212由第一出口 2〇6排出，流到集液容器 二同時過濾腔室201中的幹細胞仍維持在培育狀態。 ’也可從第二出口 2G8中採取少量的培養基 2^刀析其中所含的幹細胞之數目，以決定系統雇 二。育的，血幹細胞之數目是否已到達足夠採收的程 ^㈣旦所採樣的培養基212中的幹細胞的數目到達可 度’即可從第二出口細將過濾腔室2〇1中的 J ; ^12流出，並集中在幹細胞收集容器209中，之 ΐ的中所收集的培養基212中分離出欲 所收二二/或者’也可每隔數天即從容器209中 吓叹杲的培養基212中磕嬙i丄八站，i 胞，然後再將數±/、 、，，77離出欲求的造血幹細 續庫用所得的幹細胞集中在—起，供後 可在系統内適當位置處設置2—向間或3-(未不出）’以便在系統中形成適當的迴路。舉例來 16 201040258 説’當從第三入口 211 3丨λ 8± , >δ ,± ^丨入幹細胞培養基212至系統中 岈了將。又在通往第一入 51 9Π7 ^ Εδ ζυ4、第二入口 205、集液容 斋207、容|§ 209和非必|的蚀— ? ^ aa 要的儲存腔室210等路徑上的 2-向閥或3-向閥均關閉 212 〇 使仵有在幫浦213、培養基 212和過濾腔室201 =去π丄、 一者間形成一封閉迴路。類似的， 虽培月期間需要進;f干;^ ★举 ^ , ^ Ρ3 π ± 采樣時，則可打開通往容器209路 徑上的閥，同時關閉通往第一 ^入口 204、第二入口 205、The details of one or more embodiments of the invention are set forth in the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Further, the objects and advantages of the present invention can be understood by using the means described in the appended claims. In addition, the above general description of the present invention and the following detailed description are not intended to limit the scope of the invention. [Embodiment] Definitions As used herein, "stem ceii" means a totipotent or pluripotent cell that self-replicates and forms specialized tissue cells or organs. Stem cells can self-divide to produce two progeny stem cells, or one progeny stem cell and one progenitor cell, and then further develop into a mature, fully morphological cell of a tissue. In the present context, "stem cell" means a pluripotent cell, and "multipotent cell" means a pluripotent stem cell which can grow into a specific tissue or organ in a mammal. Or a variety of different forms of cells. As used herein, "hematopoietic stem cells" means pluripotent cells that can grow into maturity: globular cells, including, but not limited to, red blood cells, white blood cells, megakaryocytes, platelets, and τ-type and (four) lymphocytes. cell. 'The data range and the number of parameters used to indicate the maximum range of the invention are approximations'. The data values described in the specific preferred implementation have been as accurate as possible, and each measurement experiment has its standard deviation, so any The data value must have some error. In addition, if there is no particular meaning in the context, the specific meanings such as "one ("a" 〇r an )" and "the" are used in the scope of the patent application. The terms include their plural forms. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to the artisan. In addition, any methods and materials similar or equivalent to those described are applicable to the methods of the present invention. The preferred embodiment and materials described in the specification are for illustrative purposes only. All references mentioned in this application are hereby incorporated by reference in their entirety in their entireties in the entire disclosure of the disclosure of the disclosure of the disclosure of the disclosure of Known technology. And there is no literature showing that the contents of the present invention have been disclosed in the prior art. The actual data obtained in the context of the present invention may differ from the data disclosed herein in the context of the specification due to individual implementation conditions. Methods and systems for isolating, in vitro expanding and harvesting hematopoietic stem cells will be described in detail below. According to an embodiment of the present invention, a batch system for separating, in vitro expanding and harvesting hematopoietic stem cells comprises: a filtration chamber, The membrane comprises a membrane having a pore size between about 2 μm and about ΙΟΟμιη; a first inlet for introducing a source of stem cells into the filtration chamber; and a second inlet for introducing a cleaning solution to a filter chamber; and a first outlet for discharging the cleaning solution from the filter chamber. Referring to Fig. 1, a marginal system 100 in accordance with an embodiment of the present invention is shown. The system 100 includes a filter, a chamber 1 〇 1, a first inlet 104, a second inlet 105, a first outlet 106, and a header container 107. A filter membrane is disposed in the filtration chamber 101, and the membrane has a pore diameter of from about 2 μm to about ,μηη, for example, from about 2 μm to about 25 μm, from about 3 μm to about 20 μm, or from about 3 μm to about 15 μm. When operating the system, 'a stem cell source 102 is introduced from the first inlet 1〇4 to pass through the filtering chamber ιοί; then, a cleaning solution 103 is fed from the second inlet ι〇5 into the filtering chamber ιοί; The cleaning solution 103 can exit the filtration chamber 101 from the first outlet 1〇6 and flow into the collection container 〇7. The hematopoietic stem cells are blocked by the filter membrane in the filtration chamber ιοί and remain on the filter membrane. Therefore, the entire filter membrane can be removed from the filtration chamber 101, placed in a culture vessel, and an appropriate amount of stem cell culture medium is added. In vitro amplification. Sources of hematopoietic stem cells suitable for use in this embodiment of the invention include, but are not limited to, cord blood, bone marrow fluid, or peripheral blood. In one example, the source of the hematopoietic stem cell is cord blood, which is obtained from a woman who has undergone caesarean section or natural production, obtains a meticulous zone, and then collects blood from the umbilical cord with a syringe and stores the antibiotic. In the blood bag of coagulant 201040258 (such as 'citric acid or heparin). In another example, the source of the hematopoietic stem cells is bone marrow fluid, again with the consent of the donor, and then the bone marrow fluid is withdrawn from the donor's bone marrow according to standard protocols. After the blood stem cell source 102 has permeated through the filtration chamber 丨〇1, the cleaning solution 1〇3 is introduced into the system 100 from the second inlet 1〇5. The purpose of this washing step is to further purify the hematopoietic stem cell source 1 2 by removing the red blood cells contained therein, since it is generally believed that red blood cells may inhibit hematopoietic stem cells. Suitable cleaning solution 103 for use in embodiments of the present invention includes, but is not limited to, serum-free cell culture medium, serum-containing cell culture medium, physiological saline solution, buffer solution, physiological saline containing EDTA, and EDTA-containing. Buffer solution, low platelet content; platelet-poor plasma and combinations thereof. Blood can be used as a source, and the above-mentioned low blood platelet content plasma can be produced according to a conventional method. In one example, cord blood is centrifuged at an appropriate rate' followed by filtration of the supernatant obtained after centrifugation with a filter (pore size: 22.22 μm) to remove red blood cells therein and form plasma with low platelet Q content. Alternatively, the cell culture medium, physiological saline, or buffer solution may be used as the cleaning solution 103 required for the present embodiment. When the cell culture medium is used as the cleaning solution 103, serum may or may not be contained therein; and when the physiological saline solution or the buffer solution is used as the cleaning solution 103 required for the present embodiment, the second or the second may or may not be included. Ethylene diamine tetraacetic acid (EDTA). In one example, serum-free cell culture medium is used as a cleaning solution 103, such as StemSpan SFEM medium from StemCell Technologies (USA), which may or may not contain growth factor 201040258 or other types. Cytokines. In another embodiment, both the serum-free cell culture medium and the above-mentioned low blood platelet content plasma are used in combination as the cleaning solution 103. In addition, hematopoietic stem cell source 102 or cleaning solution 103 can be introduced into system 100 by means of an additional pump, such as a peristaltic pump. Moreover, whether it is the hematopoietic stem cell source 102 or the cleaning solution 103, the flow rate is between about 1 and 1 ml/min. In one example, the flow rate of any of the hematopoietic stem cell source 102 or the cleaning solution 103 is about 1 ml/min. A filter membrane having a pore diameter of between about 2 μm and about ΙΟΟ μη is provided in the chamber body of the filtration chamber 101. For example, the filter membrane may have a pore size of from about 2 μm to about 25 μm, from about 3 μm to about 20 μm, or from about 3 μm to about 15 μm. A variety of techniques are currently available for measuring the pore size of a filter, such as a scanning electron microscope or liquid extrusion porosimetry, or any other suitable technique. In the disclosed example, the membrane pore size is estimated using a pore analyzer. In this field 熟悉 Those skilled in the art should be able to understand that the estimated values for the pore size of the membrane will generally vary depending on the particular technology used. For cells, materials suitable for use as filters must have good biocompatibility, good moldability, good sterility, and low toxicity. The filter membrane is generally made of a synthetic polymer, and suitable synthetic polymers include, but are not limited to, polyethylene, polypropylene, polystyrene, acrylic, nylon, polyester, polycarbonate, polypropylene. Indoleamine and polyurethane; or made of natural polymers, suitable natural polymers include, but are not limited to, agarose, cellulose, cellulose 12 201040258 acetate, chitin, strontium Chitosan, and alginate (alginate); or made of inorganic materials, suitable inorganic materials include, but are not limited to, hydroxyapatite, glass, oxidized and titanium oxide; Or made of metal, such as stainless steel, titanium and Ming. The ruthenium film is preferably a polyurethane-based polymer or a polyethylene terephthalate-based polymer (ie, Non-woven fabrics are made of materials. These polymers can be further modified by grafting reactions on these polymer backbones or side chains to further modify these polymers. Suitable for grafting on the polymer backbone or side chains, including , but not limited to, ammonia Acids, peptides, glycosaminoglycans, and glycoproteins. In one example, a conventional plasma discharge method is utilized (e.g., the method disclosed in JP 2005-323534, the contents of which are incorporated herein For reference, a functional group such as a carbonate group is grafted onto the surface of the above-mentioned polyurethane-based polymer. In another example, a polymer having a surface-grafted polyurethane-based polymer is used. It is a filter that is used directly to separate and expand hematopoietic stem cells. Conversely, if a polymer based on polyethylene terephthalate is used as the filter material, Q can be additionally plated on the filter. a polymer layer polymerized by at least one monomer selected from the group consisting of hydroxyethyl methacrylate (HEMA) and dimethyl acetoacetate Dimethylaminoethyl methacrylate (DM), n-butyl methacrylate (BMA), N,N-dimethylacrylamide (DMA), N-propyl N_acryloylmorpholine (AMO) and N-diethyl 0-p Burning (N_vinylpyrrolidone, VP). In one example, the filter is made of a polyethylene terephthalate-based polymer (ie, non-woven 13 201040258 cloth) and is plated thereon. There is a polymer layer formed by polymerizing two monomers, BMA and DMA. After the hematopoietic stem cell source 102 passes through the filtration chamber 101, the hematopoietic stem cells therein are blocked by the filter membrane in the filtration chamber 101 and left in the filter. On the membrane*. Next, the filter membrane on which the hematopoietic stem cells are left is taken out from the filtration chamber 101, and the entire filter membrane is immersed in a cell culture tray containing an appropriate stem cell culture medium, and directly cultured in vitro. In one example, the stem cell culture medium is StemSpan SFEM medium, and a cytokine mixture and low density lipoprotein (LDL) are added to the medium. This mixture of cytokines includes recombinant human stem cell factor, recombinant human thrombopoietin, and recombinant human Flt-3 ligand. The concentration of each cytokine is between about 5 ng/ml and 500 ng/ml, and preferably between about 10 ng/ml and 100 ng/ml, at the start of cell culture. As for LDL, its use is not necessary. When LDL is added, the concentration of LDL is generally between about 0.1 mg/ml and 20 ❹ mg/ml when starting cell culture, and preferably about 5 mg/ml. In one example, a serum-free cell culture medium is used as the cleaning solution 103, and the filtration chamber 101 is passed through the filter chamber 101, and the filter membrane on which the hematopoietic stem cells are left is taken out, and the cell expansion is performed directly in vitro. Increasing the number of hematopoietic stem cells by at least about 10 days compared to the control group (ie, hematopoietic stem cells directly isolated from hematopoietic stem cell sources, which directly obtain the number of stem cells obtained after in vitro cell expansion) 63%. In another example, a low platelet content serum is used as the wash solution 1 〇 3, and 201040258 is passed through the filtration chamber 101 to amplify the number of hematopoietic stem cells by at least about 280% compared to the control group. The batch-based system for isolating hematopoietic stem cells in this experiment is extremely convenient to use and time-saving, from the source of hematopoietic stem cells through the filtration chamber. The isolation of stem cells can be expanded in vitro, which takes a relatively short time. , only about 10 minutes to about 60 minutes. In a preferred embodiment, the operating time is approximately 18 minutes. J. Separation, in vitro expansion, and collection of hematopoietic dry face 锖 怂 Ο Ο method According to another embodiment of the present invention, a continuous performance system for isolating, expanding and harvesting hematopoietic stem cells in vitro comprises: a filtration chamber comprising a membrane having a pore size between about 2 μm and about 1 μm; a first inlet for introducing a source of hematopoietic stem cells into the filtration chamber; a second inlet for a cleaning solution is introduced into the filtration chamber; a third inlet ' is used to introduce a stem cell culture medium into the filtration chamber; a pump' is used to circulate the stem cell culture medium throughout the system; For discharging the cleaning solution from the filtration chamber; and a second outlet for collecting the hematopoietic stem cells. Referring to Figure 2, there is shown a continuous system 200 in accordance with an embodiment of the present invention. The system 200 includes a filtration chamber 201, a first inlet 204, a first inlet 205, a first outlet 206, a collection reservoir 207, a second outlet 208, a hematopoietic stem cell collection container 209, One brother two entrance 211 and one pump 213. The system further includes a non-essential storage chamber 210 coupled to the filtration chamber 201. The filter chamber 201 is provided with a diaphragm ' and the aperture of the diaphragm is between about 2 μm to about 1 〇〇 pm 15 201040258 Ο 。. When operating the system, a hematopoietic stem cell source 202 is introduced from the first inlet 204 to pass through the filtration chamber 2〇1. Optionally, the hematopoietic stem cell source 2 2 passing through the filtration chamber 201 can be collected again and stored in a non-essential storage chamber 210. Next, a cleaning solution 203 is fed from the second inlet 2〇5 into the filtration chamber 2〇1; the used cleaning solution 203 can exit the filtration chamber 201 from the first outlet 206 and flow into the liquid collection tank 207. . Next, the stem cell culture medium 212 is fed from the third inlet 211 into the filtration chamber 2〇1 and passed through the help of the pump 213 to make it dry, and the medium 212 can be circulated in the system. Every few days, for example, 'mother 2 days', a fresh medium 212, replaces the stem cell medium 212 that was originally circulated in the system 200, and the used dry fine 2 is used. The nutrient 212 is discharged from the first outlet 2〇6 and flows to the liquid collection container. At the same time, the stem cells in the filtration chamber 201 are maintained in a state of cultivation. 'A small amount of medium can also be taken from the second outlet 2G8 to determine the number of stem cells contained therein to determine the system. Whether the number of blood stem cells has reached a sufficient harvesting process ^ (4) Once the number of stem cells in the sampled medium 212 is reached, the J in the filtration chamber 2〇1 can be finely filtered from the second outlet; ^12 flows out and is concentrated in the stem cell collection container 209, and the medium 212 collected in the crucible is separated from the medium 212 which is to be collected or/or may be scared from the container 209 every few days. In the middle of the 磕嫱i丄 eight stations, i cells, and then the number of ± /,,,, 77 from the desired hematopoietic stem cell continued to use the stem cells in the collection, for later can be set at the appropriate location in the system 2—Interval or 3-(not shown) to form the appropriate loop in the system. For example, 16 201040258 says 'When the third inlet 211 3 丨 λ 8± , > δ , ± ^ into the stem cell medium 212 into the system will be smashed. Also on the path leading to the first entry 51 9Π7 ^ Εδ ζυ4, the second inlet 205, the collection liquid 207, the capacitance § 209 and the non- must | ^ ^ aa storage chamber 210, etc. The valve or 3-way valve is closed 212 〇 so that a closed loop is formed between the pump 213, the medium 212, and the filter chamber 201 = π 丄. Similarly, although it is required during the training period; f dry; ^ ★ ^, ^ Ρ 3 π ± when sampling, the valve leading to the path of the container 209 can be opened, and the first entrance 204 and the second entrance are closed. 205,
Ο 207和非必要的儲存腔室21〇等路徑上的2_向 使得只有在幫浦213、培養基心過濾腔 至201和谷器2〇9之間形成一封閉迴路。 類^的，可用在本實施方式中之適當的造讀細胞來 "、1，但不限於’臍帶血、骨髓液或週邊血液。在一 實例中，此造血幹細胞來源是臍帶▲，此臍帶i是向接 ^剖腹產手術或自然產之婦女請求其同意後，取得臍 帶，然後以針筒自臍帶中收集錢並儲存在内含抗凝金 劑（如’檸檬酸或肝素）之血袋中。在另—實例中，此造 血幹細胞來源是週邊血液。在又一實例中，此造血幹細 胞來源疋骨髓液’此同樣是先取得捐贈者同意，然後依 標準操作流程自捐贈者骨射抽取出㈣液。此造血幹 細胞來源202可經由第一入口 2〇4滲透通過過滤腔室 201 ’並可被再次收集並存放在非必要的儲存腔室210 中。接著，從第二入口 205將清洗溶液203引入至系統 200中。此清洗步驟的目地在於透過移除其中所含的紅 血球細胞來進一步純化造血幹細胞來源202，因為一般 I忍為紅血球細胞可能會抑制造血幹細胞。可用於本發明 17 201040258 實施方式的適當清洗溶液21〇3包括，但不限於，無也清 的細胞培養基、含血清的細胞培養基、生理食鹽水、緩 衝溶液、内含EDTA的生理食鹽水、内含EDTA的緩衝 ’ 溶液、低血小板含量的血漿（platelet-poor plasma)及其之 * 組合。可以血液做為來源物，依照一般習知的方法來製 造上述之低血小板含量的血漿。在一實例中，將臍帶血 以適當速度離心’接著以濾膜（孔徑為〇22μπι)過濾離心 後所得的上清液’藉以移除其中的紅血球細胞，並形成 〇 低血小板含量的血漿。或者，也可以細胞培養基、生理 食鹽水、緩衝溶液做為本實施方式所需的清洗溶液 203。當以細胞培養基做為清洗溶液203時，其中可包含 或不包含血清；而當以生理食鹽水或緩衝溶液做為本實 施方式所需的清洗溶液203時，則其中可包含或不包含 乙二胺四醋酸(EDTA)。在一實例中，是以不含血清的細 胞培養基做為清洗溶液203，例如講自StemCell Technologies (USA)的 StemSpan SFEM 培養基，此 q StemSpan SFEM培養基中可包含或不包含生長因子或其 他類型的細胞素（cytokines)。在另一實施方式中，則是 組合使用不含血清的細胞培養基與上述低血小板含量的 血·紫·兩者，來做為清洗溶液203。此外，可藉助於一外 加的幫浦，例如螺動式幫浦（peristaltic pump)，來將造也 幹細胞來源202或是清洗溶液203引入至系統200中。 而且，無論是造血幹細胞來源202或是清洗溶液203， 其流速都在約1〜10毫升/分鐘間。 18 201040258 過濾腔室201的腔室主體中設有一孔徑在約2Pm至 約ΙΟΟμπι間之濾膜。舉例來說，此濾膜的孔徑可在約2叫 至約25μπι間、約3μιη至約20μιη間、或是約3μιη至約 • 15μπι間。目前有多種技術可用來測量濾膜的孔徑’例如 • 掃描式電子顯微鏡或是孔洞分析儀（Hquid extrusion porosimetry)，或是其他任何適當的技術。在所揭示的實 例中，濾膜孔徑是利用孔洞分析儀進行估算。此領域中 熟悉技藝人士應能了解，濾膜孔徑大小的估算數值一般 0 會隨著所採用的特定技術不同而有所差異。對細胞來 說，適合做為濾膜的材料本身必須具備良好的生物相容 性、良好的模造性（moldability)、良好的無菌性、以及低 毒性。此濾膜一般是由人工合成的聚合物製成，適當的 人工合成聚合物包括，但不限於，聚乙烯、聚丙烯、聚 苯乙烯、丙烯樹脂、尼龍、聚酯、聚碳酸酯、聚丙烯醯 胺和聚尿烷；或是由天然的聚合物製成，適當的天然聚 合物包括，但不限於，瓊脂（agarose)、纖維素、纖維素 Q 乙酸酯、幾丁質、曱殼素（chitosan)、和藻酸酯（alginate); 或是由無機材料製成，適當的無機材料包括，但不限於， 氫氧基鱗灰石（hydroxyapatite)、玻璃、氧化鋁和氧化鈦； 或是由金屬製成，例如不鏽鋼、鈦及鋁。此濾膜教佳是 由聚尿燒為底的聚合物或是聚對苯二曱酸二乙醋為底的 聚合物（即，不織布）材料製成。還可透過接枝反應在這 些聚合物主鏈或侧鏈上嫁接上其他分子，進一步修飾這 些聚合物。適合嫁接在聚合物主鏈或侧鏈上的分子，包 括，但不限於’氨基酸、胜肽、葡萄糖胺聚醣 201040258 (glycosaminoglycans)、和糖蛋白。在一實例中，利用習 知的電漿放電法（如，揭示在JP 2005-323534中的方法， 其内容在此併入做為參考），在上述聚尿烷為底的聚合物 表面上嫁接上諸如碳酸基之類的官能基團。在另一實例 ' 中，將表面嫁接有碳酸基之聚尿烷為底的聚合物做為濾 膜，直接用來分離及擴增造血幹細胞。相反的，若是使 用聚對苯二甲酸二乙酯為底的聚合物做為濾膜材料，則 可在濾膜上額外鍍覆上一層以至少一種選自下列之單體 〇 所聚合而成的聚合物層，適合的單體是選自以下：曱丙 稀酸經乙醋（hydroxyethyl methacrylate，HEMA)、甲丙稀 酸二曱基氨乙酯（dimethylaminoethyl methacrylate， DM)、曱丙烯酸正-丁醋（n_butyl methacrylate, BMA)、 N，N-二曱基丙烯酿胺（N，N-dimethylacrylamide， DMA)、N-丙烯醢嗎琳(N-acryloylmorpholine，AMO)和 N-乙稀0比洛烧酮（N-vinylpyrrolidone，VP)。在一實例中，此 濾膜是由聚對苯二曱酸二乙酯為底的聚合物（亦即，不織 ◎ 布）製成，且其上鍍覆有一層由BMA和DMA兩種單體 聚合而成之聚合物層。 在造血幹細胞來源202通過過濾腔室201後，其中的 造血幹細胞會被過濾腔室201中的濾膜擋下而留在濾膜 上。為了進行體外擴增，從第三入口 211引入一適當的 幹細胞培養基到過濾腔室201中，並利用幫浦213使培 養基212可在整個系統中循環流動。此幫浦213較佳是 一種蠕動式幫浦，可幫助培養基212在整個系統中循環 流動，特別是在過濾腔室201中循環流動。此幫浦213 201040258 可設置在系統中任何適當的位置上，前提是只要能形成 一封閉迴路，使培養基得以在其中循環流動即可。在一 種設置方式中，將通往第一入口 204、第二入口 205、非 必要的儲存腔室210、集液容器207和第二出口 208和 ' 等路徑上的2-向閥或3-向閥關閉，使得只有在幫浦213、 培養基212與過濾腔室201間形成一封閉迴路。在另一 設置方式中，將通往第一入口 204、第二入口 205、集液 容器207、非必要的儲存腔室210等路徑上的2-向閥或 〇 3·向閥關閉’使得只有在幫浦213、培養基212、過濾腔 室201與容器209間形成一封閉迴路。在一實例中，此 幹細胞培養基為StemSpanSFEM培養基，此培養基中可 更添加一種細胞素混合物以及低密度脂蛋白（LDL)。此 StemSpan SFEM培養基是直接購自 StemCell Technologies (USA)公司，其中可包含或不包含造血幹細 胞生長因子或其他類型的細胞素混合物。此細胞素混合 物中包括有重組人類幹細胞因子、重組人類血小板生成 0 素（recombinant human thrombopoietin)及重組人類 Flt-3 配體（Flt-3 ligand)。每一種細胞素的濃度在開始進行細胞 培育時是介於約5 ng/ml至500 ng/ml間，且較佳是在約 10 ng/ml至100 ng/ml間。至於LDL，其使用情形則是 非必要的，當選擇添加LDL時，LDL的濃度在開始進行 細胞培育時，一般是介於約0.1 mg/ml至20 mg/ml間， 且較佳是在約5 mg/ml間。每隔兩天，即可以新鮮的培 養基212來置換過濾腔室201中的培養基，並採取適量 之已用過的培養基212來分析其中所含造血幹細胞數 21 201040258 目。-旦採樣的培養基212中的幹細胞數目達到一預定 數值，即可彳之第二出口 208將過濾腔室2〇1中的培養基 212流出，並收集在容器2〇9中，接著，再從容器2〇9 ’中所收集的培養基212中分離出造血幹細胞。或者，也 •可連續地每隔數天即從容器209中所收集的培養基212 中分離出造企幹細胞，然後再將不同天所收集到的造血 幹細胞集中起來，供後續應用。在一實 增!天後，從培養基212中所收集到二 ❹目比控制組（即，將從臍帶血中直接分離出來的造血幹細 胞進行體外擴增1天後的幹細胞數目）高出約丨倍；若是 體外擴增10天後，從培養基212中所收集到的造血幹細 胞的數目可比控制組尚出約14〜15倍之多。 此外，利用本實施方式之連續式用以分離、體外擴增 及收獲造血幹細胞的系統，所分離出來的造血幹細胞， 仍然保有形成細胞群落（colony)的能力。在一實例中，由 所分離並擴增之造血幹細胞形成的細胞群落數目，與控 ❹制組相去不遠，約為150個細胞群落（數據未顯示）。 類似的，本實施方式之用以分離、體外擴增及收獲造 血幹細胞的連續式系統，在使用上極為方便，且省時’ 從造血幹細胞來源通過過濾腔室到分離出幹細 體外擴增止，所需花費的時間相當短，僅約1〇分鐘至約 60分鐘。在一較佳實施方式中，操作時間約為18分鐘。 、下文中，將經由實施例詳細敘述本發明。然而，本發 明並不限於實驗例，而且在後附的申請專利範圍之範疇 内可以實施各種的實施例。 可 22 201040258 實施例 實施例1 製備表面改質之PU濾膜 1.1製備Ρϋ-GMA濾膜 依照先前技藝JP 2005-323534中所公開的電漿放 • 電法來製備PU-GMA濾膜，其揭示内容在此併入做 為參考。簡言之，將取自白血球移除濾器（Imguard IH-RC，Terumo Corporation)中的聚尿烧濾膜用於本實 施例中。在反應容器中，透過氩氣電漿之助，於聚尿 Ο 烷（PU)膜表面上嫁接上曱丙烯酸環氧丙酯（glycidyl methacrylate，GMA)基團。在反應容器中，在一复氣 流（壓力約為26.6 Pa)上施加高頻電力（約200 瓦）（Adtec Co.，AX-300)約30秒，可產生氩氣電漿， 接著在容器中執行GMA與PU濾膜間的嫁接聚合反 應約5分鐘，壓力約0.65Pa。接著，以水與甲醇（1 : 1混合）所形成的混合液來清洗此嫁接有GMA的PU 濾膜，接著再用超純水清洗約30分鐘。PU濾膜上所 〇 嫁接的GMA比例是由下式來決定： 所引入的 GMA 比例（％) = [(X/y)-1] X 100 其中，GMA比例定義為電漿放電後之PU-GMA濾膜乾 重（X克）（真空乾燥或是8(TC下乾燥24小時）除以PU 濾膜乾重（Y克）（真空乾燥或是80°C下乾燥24小時）。 在此實例中，所引入之GMA比例約為0.61%。 1.2製備PU-COOH濾膜The 2_ direction on the path of Ο 207 and the unnecessary storage chamber 21 使得 is such that only a closed loop is formed between the pump 213, the culture medium filter chamber 201 and the valley 2〇9. For the appropriate reading cells in the present embodiment, ", 1, but not limited to, cord blood, bone marrow fluid or peripheral blood. In one example, the source of the hematopoietic stem cell is the umbilical cord ▲, which is obtained from the cesarean section or the natural product of the woman, obtains the umbilical cord, and then collects the money from the umbilical cord with a syringe and stores the anti-inclusion. A blood bag (such as 'citric acid or heparin') in a blood bag. In another example, the source of the hematopoietic stem cells is peripheral blood. In yet another example, the hematopoietic stem cell is derived from sputum bone marrow solution. This is also the first to obtain the donor's consent, and then extract the (iv) fluid from the donor's bone by standard procedures. This hematopoietic stem cell source 202 can be infiltrated through the filtration chamber 201' via the first inlet 2' and can be collected again and stored in the non-essential storage chamber 210. Next, the cleaning solution 203 is introduced into the system 200 from the second inlet 205. The purpose of this washing step is to further purify the hematopoietic stem cell source 202 by removing the red blood cell cells contained therein, since it is generally accepted that red blood cells may inhibit hematopoietic stem cells. Suitable cleaning solutions 21〇3 which can be used in the embodiment of the present invention 17 201040258 include, but are not limited to, cell culture medium without serum, serum-containing cell culture medium, physiological saline solution, buffer solution, physiological saline containing EDTA, and EDTA-containing buffer 'solution, platelet-poor plasma, and combinations thereof*. Blood can be used as a source, and the above-mentioned low platelet-containing plasma can be produced according to a conventional method. In one example, cord blood is centrifuged at an appropriate rate' followed by filtration of the supernatant obtained after centrifugation with a filter (pore size 〇22 μm) to remove red blood cells therein and form plasma with low platelet content. Alternatively, the cell culture medium, physiological saline, or buffer solution may be used as the cleaning solution 203 required for the present embodiment. When the cell culture medium is used as the cleaning solution 203, serum may or may not be contained therein; and when the physiological saline solution or the buffer solution is used as the cleaning solution 203 required for the present embodiment, the second or the second may or may not be included. Amine tetraacetic acid (EDTA). In one example, serum-free cell culture medium is used as the cleaning solution 203, such as StemSpan SFEM medium from StemCell Technologies (USA), which may or may not contain growth factors or other types of cells. Cytokines. In another embodiment, a serum-free cell culture medium and the above-mentioned low platelet-containing blood and purple are used in combination as the cleaning solution 203. Alternatively, the stem cell source 202 or the cleaning solution 203 can be introduced into the system 200 by means of an additional pump, such as a peristaltic pump. Moreover, whether it is the hematopoietic stem cell source 202 or the cleaning solution 203, the flow rate is between about 1 and 10 ml/min. 18 201040258 A filter membrane having a pore diameter of between about 2 Pm and about ΙΟΟμπι is provided in the chamber body of the filtration chamber 201. For example, the pore size of the filter may range from about 2 to about 25 μm, from about 3 μm to about 20 μm, or from about 3 μm to about 15 μm. A variety of techniques are currently available to measure the pore size of a filter, such as a scanning electron microscope or a Hquid extrusion porosimetry, or any other suitable technique. In the disclosed example, the membrane pore size is estimated using a pore analyzer. Those skilled in the art should be able to understand that the estimated value of the pore size of the membrane will generally vary depending on the particular technology employed. For cells, materials suitable for use as filters must have good biocompatibility, good moldability, good sterility, and low toxicity. The filter membrane is generally made of a synthetic polymer, and suitable synthetic polymers include, but are not limited to, polyethylene, polypropylene, polystyrene, acrylic, nylon, polyester, polycarbonate, polypropylene. Indoleamine and polyurethane; or made of natural polymers, including, but not limited to, agarose, cellulose, cellulose Q acetate, chitin, chitin (chitosan), and alginate; or made of an inorganic material, including, but not limited to, hydroxyapatite, glass, alumina, and titania; Made of metal, such as stainless steel, titanium and aluminum. The filter is made of a polymer that is based by polyurea or a polymer of polyethylene terephthalate (ie, non-woven). These polymers can be further modified by grafting other molecules onto the polymer backbone or side chains by grafting. Molecules suitable for grafting onto the polymer backbone or side chains include, but are not limited to, 'amino acids, peptides, glycosaminoglycans 201040258 (glycosaminoglycans), and glycoproteins. In one example, a conventional plasma discharge method (e.g., the method disclosed in JP 2005-323534, the contents of which is incorporated herein by reference) A functional group such as a carbonic acid group is added. In another example, a polymer-based polyurethane-based polymer grafted on the surface is used as a filter to directly separate and expand hematopoietic stem cells. Conversely, if a polyethylene terephthalate-based polymer is used as the filter material, the filter may be additionally plated with a layer of at least one monomer selected from the group consisting of the following monomers. The polymer layer, suitable monomers are selected from the group consisting of: hydroxyethyl methacrylate (HEMA), dimethylaminoethyl methacrylate (DM), ruthenium acrylate butyl vinegar (n_butyl methacrylate, BMA), N,N-dimethylacrylamide (DMA), N-acryloylmorpholine (AMO) and N-ethyl ketone (N-vinylpyrrolidone, VP). In one example, the filter is made of a polyethylene terephthalate-based polymer (ie, a non-woven fabric), and is plated with a layer of BMA and DMA. A polymer layer formed by polymerization. After the hematopoietic stem cell source 202 has passed through the filtration chamber 201, the hematopoietic stem cells therein are blocked by the filter membrane in the filtration chamber 201 and remain on the filter membrane. For in vitro expansion, a suitable stem cell medium is introduced from the third inlet 211 into the filtration chamber 201, and the pump 213 is used to allow the culture substrate 212 to circulate throughout the system. The pump 213 is preferably a peristaltic pump that assists in the circulation of the medium 212 throughout the system, particularly in the filtration chamber 201. This pump 213 201040258 can be placed in any suitable position in the system, provided that a closed loop is formed to allow the medium to circulate therein. In one arrangement, there will be a 2-way valve or 3-way to the first inlet 204, the second inlet 205, the optional storage chamber 210, the sump container 207 and the second outlet 208, and the 'equal path. The valve is closed so that only a closed loop is formed between the pump 213, the medium 212 and the filtration chamber 201. In another arrangement, the 2-way valve or the 〇3. valve is closed to the first inlet 204, the second inlet 205, the sump container 207, the non-essential storage chamber 210, etc. A closed loop is formed between the pump 213, the medium 212, the filtration chamber 201 and the container 209. In one example, the stem cell culture medium is StemSpan SFEM medium, and a cytokine mixture and low density lipoprotein (LDL) may be added to the medium. This StemSpan SFEM medium is purchased directly from StemCell Technologies (USA) and may or may not contain hematopoietic stem cell growth factor or other types of cytokine mixtures. The cytokine mixture includes recombinant human stem cell factor, recombinant human thrombopoietin, and recombinant human Flt-3 ligand. The concentration of each cytokine is between about 5 ng/ml and 500 ng/ml at the start of cell culture, and preferably between about 10 ng/ml and 100 ng/ml. As for LDL, its use is not necessary. When LDL is added, the concentration of LDL is generally between about 0.1 mg/ml and 20 mg/ml when starting cell culture, and preferably at about 5 Between mg/ml. The culture medium in the filtration chamber 201 can be replaced every two days by fresh culture medium 212, and an appropriate amount of used medium 212 is used to analyze the number of hematopoietic stem cells contained therein. Once the number of stem cells in the sampled medium 212 reaches a predetermined value, the second outlet 208 can be used to flow out the medium 212 in the filtration chamber 2〇1 and collect it in the container 2〇9, and then from the container. Hematopoietic stem cells are isolated from medium 212 collected in 2〇9'. Alternatively, it is also possible to continuously separate the stem cells from the culture medium 212 collected in the container 209 every few days, and then collect the hematopoietic stem cells collected on different days for subsequent application. After a day of increase, the number of stem cells in the diptera control group (i.e., the number of stem cells expanded in vitro from the cord blood for one day in vitro) collected from the medium 212 was higher than that of the control group (i.e., the number of stem cells expanded one day after in vitro expansion from cord blood). The number of hematopoietic stem cells collected from the medium 212 after about 10 days of in vitro expansion may be about 14 to 15 times greater than that of the control group. Further, with the continuous system of the present embodiment for separating, in vitro expanding and harvesting hematopoietic stem cells, the hematopoietic stem cells isolated still retain the ability to form a colony. In one example, the number of cell populations formed by the isolated and expanded hematopoietic stem cells is not far from the control group, which is about 150 cell populations (data not shown). Similarly, the continuous system for isolating, in vitro expanding and harvesting hematopoietic stem cells of the present embodiment is extremely convenient to use, and saves time from the source of hematopoietic stem cells through the filtration chamber to the separation of dry and fine in vitro expansion. The time it takes is quite short, from about 1 minute to about 60 minutes. In a preferred embodiment, the operating time is approximately 18 minutes. Hereinafter, the present invention will be described in detail through examples. However, the present invention is not limited to the experimental examples, and various embodiments can be implemented within the scope of the appended claims. 22 201040258 EXAMPLES Example 1 Preparation of Surface Modified PU Filters 1.1 Preparation of Ρϋ-GMA Filters PU-GMA filters were prepared according to the plasma discharge method described in the prior art JP 2005-323534, which reveals The content is incorporated herein by reference. Briefly, a polyurea filter membrane from a white blood cell removal filter (Imguard IH-RC, Terumo Corporation) was used in this example. In the reaction vessel, a glycidyl methacrylate (GMA) group was grafted onto the surface of the polyurethane (PU) membrane by means of argon plasma. In the reaction vessel, high frequency power (about 200 watts) (Adtec Co., AX-300) is applied to a complex gas stream (pressure of about 26.6 Pa) for about 30 seconds to produce argon plasma, which is then placed in a vessel. Graft polymerization between GMA and PU membranes was carried out for about 5 minutes with a pressure of about 0.65 Pa. Next, the grafted GMA-coated PU membrane was washed with a mixture of water and methanol (1:1 mixed), followed by washing with ultrapure water for about 30 minutes. The proportion of GMA grafted on the PU filter is determined by the following formula: The ratio of the introduced GMA (%) = [(X/y)-1] X 100 where the GMA ratio is defined as the PU after the plasma discharge. GMA filter dry weight (X grams) (vacuum drying or 8 (24 hours drying under TC) divided by PU filter dry weight (Y grams) (vacuum drying or drying at 80 ° C for 24 hours). The ratio of GMA introduced is about 0.61%. 1.2 Preparation of PU-COOH filter
將上述依照實施例1.1所述方法製成PU-GMA濾 膜減成多個直徑約25mm的圓形。將3層PU-GMA 23 201040258 濾膜浸泡在内含甘胺酸的 NaOH溶液中（20ml， 0·1Μ ;甘胺酸在NaOH溶液中的濃度約為0.4M)，並 在80°C下培育約24小時，以使PU濾膜表面上產生 ^ COOH基團。待反應完成之後，在25°C以純水清洗濾 膜約10分鐘。重複此清洗步驟2次，最後將濾膜浸 泡在純水中，並置於4°C下冷藏直到要用為止。以下 將依此方式製備而成的濾膜稱為「PU-COOH濾膜」。 〇 實施例2 用以分離、體外擴增及收獲造血幹細胞的批 次式系統 在取得懷孕生產婦女之書面同意後，於其分娩後 取得新生兒臍帶，然後依照標準流程抽出臍帶血，儲 存在血袋中（CPDA-1 Termo Co)，此血袋中含有諸如 檸檬酸鹽之類的抗凝血劑與葡萄糖。接著，將3層依 照上述實施例1.2所揭示方法所製成的PU-COOH濾 膜，架設在過濾、器支架上（濾膜直徑約25 mm，Millipore 〇 Co)，形成一過濾裝置。接著，以約1毫升/分鐘之流速 將6毫升的臍帶血通入至此過濾裝置内，接著，以約1 毫升/分鐘之流速饋入清洗溶液，此清洗過程持續進行約 6分鐘。在此實施例中所使用的清洗溶液為血漿A (plasma A)或StemSpan SFEM培養基。jk衆A是以如下 方式製備：以1，800 rpm的速度將臍帶血離心，然後以 0.22 μιη 孔徑的拋棄式濾器（Millex GS，Millipore Co) 過濾離心後的上清液，以便移除其中所有的紅血球， 如此可獲得低血小板含量的ik裂（platelet-poor 24 201040258 plasma)，又稱「血聚A」。至於StemSpan SFEM培養基， 則可直接自供應商處購得（#09650, StemCell Technologies Co) ° 清洗後，從過濾裝置中將其中的PU-COOH濾膜拆 • 下，直接放入培養基中於37°C下、5% C02環境中培育 10天。在此實施方式中，用來擴增所分離之造血幹細胞 的培養基乃是添加有細胞素雞尾酒StemSpan CC110 (#02697，StemCell Technologies)以及濃度為 5 毫克/毫 0 升之低密度脂蛋白（LDL)的StemSpan SFEM培養基，以 下稱此用來擴增造血幹細胞的培養基為「HSC培養基 A」。從將臍帶血通過PU-COOH濾膜到將分離出來的造 血幹細胞放入培養盤中進行體外擴增，這整個操作過程 所需時間只有約18分鐘左右，非常快。 經過體外擴增10天後，所擴增出來的造血幹細胞的 數目可以流式細胞儀（Beckman-Coulter Co.，EPICStm XL)進行估算。依據國際血液治療與移植工程協會 0 (International Society of Hematotherapy and Graft Engineering (ISHAGE), Keeney M. et al., Cytometry (1998) 34, 61 -70)所頒佈準則（或稱ISHAGE準則）中規 範的標準流程來分析CD34+的細胞數目。依據樣品數準 備相同數量的樣品試管，在每一樣品試管中加入2 0微升 的抗-CD34抗體，攪拌後注入20微升可測試細胞存活 力的染料（Beckman-Coulter Co” 7-AAD Viability Dye)，並在室溫下避光培育約15分鐘。接著，在每一 樣品試管中加入500微升的溶解液（OptiLyse C)，混合 25 201040258 ❹ Ο 均勻後在室溫下培育約10分鐘。繼續在每一樣品試管中 加入500微升的磷酸緩衝液（PBS)、攪拌均勻後在室溫 下培育約10分鐘。最後，以流式細胞儀來估算細胞數 目。首先，以直接自臍帶血中收集而得的CD34+造如件 細胞數目當作參考數值，並以此數值當作基準點來此較 以「血漿Α」或StemSpan SFEM培養基做為清洗溶浪時’ 其對分離造血幹細胞的效果以及後續體外擴增CD34 & 血幹細胞的效果。分析體外擴增後自培養基中所收振& CD34+造血幹細胞的數目。CD34+造血幹細胞之體外擴 增比（Hx wVo expansion ratio, EVER)可依以下數學 # 來計算： NVn xiooThe PU-GMA filter film prepared as described above in Example 1.1 was reduced to a plurality of circles having a diameter of about 25 mm. Soak 3 layers of PU-GMA 23 201040258 filter in NaOH solution containing glycine (20ml, 0·1Μ; concentration of glycine in NaOH solution is about 0.4M), and incubate at 80 °C About 24 hours to produce a COOH group on the surface of the PU filter. After the reaction was completed, the filter was washed with pure water at 25 ° C for about 10 minutes. This washing step was repeated twice, and finally the filter was immersed in pure water and stored at 4 ° C until it was used. The filter prepared in this manner is hereinafter referred to as "PU-COOH filter". 〇Example 2 A batch system for isolating, in vitro expansion and harvesting hematopoietic stem cells obtains the newborn umbilical cord after delivery after obtaining the written consent of the pregnant woman, and then withdraws the cord blood according to the standard procedure and stores it in blood. In the bag (CPDA-1 Termo Co), this blood bag contains an anticoagulant such as citrate and glucose. Next, three layers of the PU-COOH filter film prepared according to the method disclosed in the above Example 1.2 were placed on a filter holder (filter membrane diameter of about 25 mm, Millipore® Co) to form a filter device. Next, 6 ml of cord blood was introduced into the filter device at a flow rate of about 1 ml/min, and then the washing solution was fed at a flow rate of about 1 ml/min. This washing process was continued for about 6 minutes. The cleaning solution used in this example was plasma A (Stage A) or StemSpan SFEM medium. Jk A was prepared by centrifuging cord blood at 1,800 rpm, and then filtering the centrifuged supernatant with a 0.22 μη pore size disposable filter (Millex GS, Millipore Co) to remove all of them. Red blood cells, so that low platelet content of IK (platelet-poor 24 201040258 plasma), also known as "blood aggregation A". For StemSpan SFEM medium, it can be purchased directly from the supplier (#09650, StemCell Technologies Co) ° After cleaning, remove the PU-COOH filter from the filter unit and place it directly in the medium at 37°. Incubate for 10 days in C and 5% C02 environment. In this embodiment, the medium used to amplify the isolated hematopoietic stem cells is a cytokine cocktail StemSpan CC110 (#02697, StemCell Technologies) and a low density lipoprotein (LDL) at a concentration of 5 mg/ml. The StemSpan SFEM medium, hereinafter referred to as the medium for amplifying hematopoietic stem cells, is "HSC Medium A". From the passage of cord blood through the PU-COOH filter to the in vitro expansion of the isolated hematopoietic stem cells into a culture dish, the entire operation takes only about 18 minutes, which is very fast. After 10 days of in vitro expansion, the number of expanded hematopoietic stem cells can be estimated by flow cytometry (Beckman-Coulter Co., EPICStm XL). According to the guidelines promulgated by the International Society of Hematotherapy and Graft Engineering (ISHAGE), Keeney M. et al., Cytometry (1998) 34, 61-70 (or ISHAGE guidelines) The standard procedure was used to analyze the number of cells in CD34+. Prepare the same number of sample tubes according to the number of samples, add 20 μl of anti-CD34 antibody to each sample tube, and inject 20 μl of dye that can test cell viability (Beckman-Coulter Co) 7-AAD Viability Dye), and incubated for about 15 minutes at room temperature in the dark. Next, add 500 μl of the solution (OptiLyse C) to each sample tube, mix 25 201040258 ❹ 均匀 and mix for about 10 minutes at room temperature. Continue to add 500 μl of phosphate buffer (PBS) to each sample tube, stir well and incubate for about 10 minutes at room temperature. Finally, estimate the number of cells by flow cytometry. First, directly from the umbilical cord. The number of CD34+ cells collected in the blood is used as a reference value, and this value is used as a reference point. When compared with "plasma sputum" or StemSpan SFEM medium as a cleaning solution, it is used to separate hematopoietic stem cells. Effect and subsequent in vitro expansion of CD34 & blood stem cells. The number of & CD34+ hematopoietic stem cells harvested from the culture medium after in vitro expansion was analyzed. The in vitro expansion ratio (Hx wVo expansion ratio, EVER) of CD34+ hematopoietic stem cells can be calculated according to the following mathematical #: NVn xioo
EVER 其中，Ν2代表體外擴增後自培養基中所收獲之CD34 造血幹，細胞的數目；川代表直接自臍帶血中收集而得的 CD34+造血幹細胞數目。結果顯示於表1中’其中使用 「血聚A」及StemSpan SFEM培養基做為清洗溶浪’ < 使CD34+造血幹細胞的體外擴增比分別增加約2.5得及 0.63 倍。 表i :橡用血漿A或StemSpanSFEM培養基做為清洗漆1 液時之CD34+造血幹細胞的體外擴增比 _ 血漿A__StemSpan SFEM培養基 EVER (%) 253 63.6 26 201040258 實施例3 以實施例2之批次式系統來分離、體外擴增 及收獲造血幹細胞 除了使用1毫升的臍帶血（而非6毫升的臍帶血） 且直到細胞培育開始後的第5天才更換培養基（即， ' HSC培養基A)之外，大致依照上述實施例2所述的 方法來分離、體外擴增及收獲造血幹細胞。在本實驗中 是以血漿A做為清洗溶液。結果示於表2中，且依據本 實施方式所分離出來的造血幹細胞之體外擴增比約為 0 2.7〜6.2倍（相較於控制組而言）。 從將造血幹細胞來源通過過濾腔室到分離出幹細胞 可進行體外擴增為止，整個操作過程所需花費的時間相 當短，少於約15分鐘。 表2:使用血漿A做為清洗溶液且不更換培養基之CD34+ 造金幹細胞的體外擴增比 不更換培養基 更換培養基 ❹ EVER (%) 271 623 實施例4用以分離、體外擴增及收獲造血幹細胞之連續 式系統 依照實施例2所述的方法收集臍帶血，並依照第 2圖所揭示配置架設出一連續式系統來分離、體外擴 增及收獲造血幹細胞。以約2毫升/分鐘的流速將20克 的臍帶血送入架設有6層聚尿烷（PU)濾膜（丨mugard 27 201040258 IH_RC，Terumo Co·)的過濾腔室201中。每一層PU濾 膜的平均孔徑在約5μιη至12μπι間，此平均孔徑是以 毛細管流孔洞分析儀（capillary flow porometer) (Porous Materials Inc.)所估算出來的數值。可將滲透通過過遽腔 ， 室201的臍帶血再次收集存放在儲存腔室206中。接 著，以約2毫升/分鐘的流速將20克的血漿A送入過濾 腔室201中，接著繼續以60克的StemSpan SFEM培養 基來清洗過濾腔室201。然後，在過濾腔室201中通入 0 HSC培養基A (其中添加有雞尾酒細胞素（stemSpan CC110, #02697’ StemCell Technologies)和 5 毫克/ 毫升之LDL)，並透過蠕動式幫浦以0.5毫升/分鐘的速 度在系統内循環。分別在第1、5天將20克的新鮮HSC 培養基A引入至系統中，並由出口 206將用過的HSC 培養基A引出系統外而集中在集液容器2〇7中，而同一 時間被留在過濾腔室201之濾膜上的造血幹細胞的體外 擴增則繼續進行並未中斷。分別在第1、6及1〇天從集 〇 液容器207中採取少量的培養基，依據實施例2所揭示 方法分析其中所含的造血幹細胞數目。結果示於表3中。 從表3的數據可知，本實施例之連續式系統可提供 較佳的幹細胞體外擴增比，相較於控制組（即，直接從濟 帶iL中分離出造企幹細胞後進行體外擴增）至少高出 6.8倍。此外，從將臍帶血通過過濾腔室到引入HSC培 養基A至過濾腔室中進行體外擴增為止，整個操作過^ 所需花費的時間相當短’僅約30分鐘’再次顯示相較於 習知用以分離和或擴增幹細胞的技術來說，本實施例用 28 201040258 以分離、體外擴增及收獲造血幹細胞的連續式系統極其 容易使用的本質。 表3 :細胞培育期間，在不同天時所量測到的CD34+造 血幹細胞的體外擴增比 _第1天 第6天 第10天 總計 EVR (%) 179 375 130 684 0 實施例5以實施例4之連續式系統來分離、體外擴增及 收獲造血幹細胞 除了在細胞培育開始後的第2、4、6及8天後才 更換培養基（即，HSC培養基A)之外，大致依照上述 實施例4所述的方法來分離、體外擴增及收獲造血幹 細胞。同樣的，從將臍帶血通過過濾腔室到引入HSC培 養基A至過濾腔室中進行體外擴增為止，整個操作過程 q 所需花費的時間相當短，僅約30分鐘。結果示於表4 中，且從表4的數據可知，依據本實施例之方法所分離 且擴增出來的幹細胞體外擴增比，是控制組（即，直接從 臍帶血中分離出造血幹細胞後進行體外擴增）的46 倍。 表4 :細胞培育期間，在不同天時所量測到的CD34+造 血幹細胞的體外擴增比 29 201040258 第2天 第4天 第6天 第8天 第10天 總計 EVR (%) 547 168 1,104 1,281 1,468 4,567 實施例6以不織布做為濾膜並以實施例4之連績式系統 來分離、體外擴增及收獲造血幹細胞 除 了使用不織布（Asahi Medical Co.，SepaCell R， R-500B2(3)-1)來取代過濾腔室中的PU濾膜且在細胞 〇 培育開始後的第2、4、6及8天後才更換培養基（即， HSC培養基A)之外，大致依照上述實施例4所述的 方法來分離、體外擴增及收獲造血幹細胞。 結果示於表5中，且從表5的數據可知，依據本實 施例之方法所分離且擴增出來的幹細胞體外擴增比，是 控制組（即，直接從臍帶血中分離出造血幹細胞後進行 體外擴增）的3.7倍。 q 表5 :細胞培育期間，在不同天時所量測到的CD34+造 血幹細胞的體外擴增比 _第2天 第4天 第6天 第8天 第10天 總計 EVR (%) 1.1% 2.4% 136% 167% 65% 371% 實施例7以實施例4之連續式系統來自週邊血液中分 離、體外擴增及收獲造血幹細胞EVER where Ν2 represents the number of CD34 hematopoietic stem cells harvested from the culture medium after in vitro expansion, and represents the number of CD34+ hematopoietic stem cells collected directly from cord blood. The results are shown in Table 1 in which "Gym A" and StemSpan SFEM medium were used as a cleaning solution" < In vitro amplification ratio of CD34+ hematopoietic stem cells was increased by about 2.5 and 0.63 times, respectively. Table i: In vitro expansion ratio of CD34+ hematopoietic stem cells when using PET A or StemSpanSFEM medium as a wash lacquer 1 _ Plasma A__StemSpan SFEM medium EVER (%) 253 63.6 26 201040258 Example 3 Batch of Example 2 System to separate, in vitro expand and harvest hematopoietic stem cells except 1 ml of cord blood (rather than 6 ml of cord blood) and until the medium is changed (ie, 'HSC medium A) on the 5th day after the start of cell culture, Hematopoietic stem cells are isolated, expanded in vitro, and harvested in substantial accordance with the methods described in Example 2 above. In this experiment, plasma A was used as a cleaning solution. The results are shown in Table 2, and the in vitro expansion ratio of the hematopoietic stem cells isolated according to the present embodiment was about 0 2.7 to 6.2 times (compared to the control group). From the passage of the hematopoietic stem cell source through the filtration chamber to the isolation of stem cells for in vitro expansion, the time required for the entire procedure is relatively short, less than about 15 minutes. Table 2: In vitro expansion of CD34+ gold-producing stem cells using plasma A as a washing solution without changing medium. Change medium without changing medium EVER EVER (%) 271 623 Example 4 for isolation, in vitro expansion and harvesting of hematopoietic stem cells The continuous system collects cord blood according to the method described in Example 2, and constructs a continuous system to separate, in vitro expand and harvest hematopoietic stem cells according to the configuration disclosed in FIG. 20 g of cord blood was sent to a filtration chamber 201 equipped with a 6-layer polyurethane (PU) filter (丨mugard 27 201040258 IH_RC, Terumo Co.) at a flow rate of about 2 ml/min. The average pore diameter of each of the PU membranes is between about 5 μm and 12 μm, and the average pore diameter is a value estimated by a capillary flow porometer (Porous Materials Inc.). The umbilical cord blood of the chamber 201 can be collected and stored in the storage chamber 206 by permeating through the sputum cavity. Next, 20 grams of plasma A was fed into the filtration chamber 201 at a flow rate of about 2 ml/min, and then the filtration chamber 201 was washed with a 60 g StemSpan SFEM medium. Then, in the filtration chamber 201, 0 HSC medium A (with cocktail cytokines (stemSpan CC110, #02697' StemCell Technologies) and 5 mg/ml LDL) was introduced, and the peristaltic pump was passed through 0.5 ml/ The speed of minutes circulates within the system. 20 grams of fresh HSC medium A was introduced into the system on days 1 and 5, respectively, and the used HSC medium A was taken out of the system by outlet 206 and concentrated in the collection container 2〇7, while being retained at the same time. In vitro expansion of hematopoietic stem cells on the filter membrane of the filtration chamber 201 continues without interruption. A small amount of medium was taken from the sputum container 207 on days 1, 6, and 1, respectively, and the number of hematopoietic stem cells contained therein was analyzed according to the method disclosed in Example 2. The results are shown in Table 3. As can be seen from the data in Table 3, the continuous system of the present example can provide a better in vitro expansion ratio of stem cells compared to the control group (i.e., in vitro expansion after isolation of stem cells from the iL zone). At least 6.8 times higher. In addition, from the passage of the cord blood through the filtration chamber to the introduction of HSC medium A into the filtration chamber for in vitro expansion, the time required for the entire operation is relatively short 'only about 30 minutes' again showing compared to the conventional For techniques for isolating and or expanding stem cells, this example uses 28 201040258 to isolate, in vitro expand and harvest the essence of a continuous system of hematopoietic stem cells that is extremely easy to use. Table 3: In vitro expansion ratio of CD34+ hematopoietic stem cells measured at different days during cell culture_ Day 1 Day 6 Day 10 Total EVR (%) 179 375 130 684 0 Example 5 by way of example 4 continuous system for separation, in vitro expansion, and harvesting of hematopoietic stem cells except for the medium (i.e., HSC medium A) after the second, fourth, sixth, and eighth days after the start of cell culture, substantially in accordance with the above examples 4 described method for isolating, in vitro expanding and harvesting hematopoietic stem cells. Similarly, the entire procedure q takes a relatively short period of time, from about 30 minutes, from the passage of the cord blood through the filtration chamber to the introduction of the HSC medium A into the filtration chamber for in vitro expansion. The results are shown in Table 4, and it can be seen from the data in Table 4 that the in vitro expansion ratio of stem cells isolated and amplified according to the method of the present example is the control group (i.e., after hematopoietic stem cells are directly isolated from cord blood). 46 times the in vitro amplification). Table 4: In vitro expansion ratio of CD34+ hematopoietic stem cells measured at different days during cell culture 29 201040258 Day 2 Day 4 Day 6 Day 8 Day 10 Total EVR (%) 547 168 1,104 1,281 1,468 4,567 Example 6 Separation, in vitro expansion and harvesting of hematopoietic stem cells using a non-woven fabric as a filter and the continuous performance system of Example 4 except for the use of non-woven fabric (Asahi Medical Co., SepaCell R, R-500B2(3)- 1) In place of the PU filter in the filtration chamber and the medium (ie, HSC medium A) is replaced after the 2nd, 4th, 6th and 8th days after the start of cell culture, substantially in accordance with Example 4 above. The method described is for isolating, in vitro expanding and harvesting hematopoietic stem cells. The results are shown in Table 5, and it can be seen from the data in Table 5 that the in vitro expansion ratio of stem cells isolated and amplified according to the method of the present example is the control group (i.e., after hematopoietic stem cells are directly isolated from cord blood). 3.7 times the in vitro expansion). q Table 5: In vitro expansion ratio of CD34+ hematopoietic stem cells measured at different days during cell culture_ Day 2 Day 4 Day 6 Day 8 Day 10 Total EVR (%) 1.1% 2.4% 136% 167% 65% 371% Example 7 was isolated from peripheral blood, expanded in vitro, and harvested hematopoietic stem cells using the continuous system of Example 4.
除了使用週邊血液來取代臍帶血且在細胞培育 開始後的第2、4、6及8天後才更換培養基（即，HSC 30 201040258 培養基A)之外，大致依照上述實施例4所述的方法 來分離、體外擴增及收獲造血幹細胞。可預期依據本實 施例之方法所分離且擴增出來的幹細胞體外擴增比，會 較控制組P卩，直接從週邊血液中分離出造血幹細胞後 進行體外擴增）高。 實施例8以實施例4之連續式系統來自骨髄液中分離、 體外擴增及收獲造血幹細胞 〇 除了使用骨髓液來取代臍帶血且在細胞培育開 始後的第2、4、6及8天後才更換培養基（即，HSC 培養基A)之外，大致依照上述實施例4所述的方法 來分離、體外擴增及收獲造血幹細胞。可預期依據本實 施例之方法所分離且擴增出來的幹細胞體外擴增比，會 較控制組（即，直接從週邊血液中分離出造血幹細胞後 進行體外擴增）高。 〇 比較例 以習知的Ficoll-Paque方法（參見Fotino M.，ei a/., Ann. Clin· Lab. Sci·，（1971) 1:131-133)自臍帶企 中分離出單核細胞。簡言之，以内含2 mmol/L EDTA 及0.5%胎牛血清（BSA)之磷酸緩衝液（以下稱此溶液 為「PBS A」）來稀釋臍帶血，稀釋比例為臍帶血：PBS A = 1 : 4。接著將35毫升經過稀釋的臍帶血加到15 毫升的Ficoll-Paque溶液（Pharmacia)表面上，然後離 心（400 X g，40分鐘、20°C )。離心後，小心地分離出 31 201040258 其中的單核細胞。將PBS A加到單核細胞溶液中直到 總體積為50毫升為止。接著，攪拌溶液並再次以3〇〇 x g的速度離心約10分鐘。 移除離心後的上清液，留下底部約〇 5毫升的溶 液。依據上述ISHAGE準則所規範的方法，分析所製 備出來的造血幹細胞溶液，其中含有5,181個細胞/ 微升的紅血球細胞、11，935個細胞/微升的白血球細 胞、171，812個細胞/微升的血小板、和大約185個細 〇 胞/微升的cr)34+造血幹細胞》整個細胞分離過程所 需化費的時間長達3小時。 將依照上述方法分離出來的約1，〇〇〇個CD34+造 血幹細胞種植在有24孔的培養盤中，每一培養孔中 含有約1毫升的HSC培養基A。經過1〇天培育後，以 上述實施例2所述方式來分析培養基中造血幹細胞的體 外擴增比（EVER)，其約為143%。 综合以上’以習知Ficoll-Paque方法來分離造血幹 ® 細胞’其所需花費的時間較本發明實施例2、3、4、 5及6所需的時間來得長；且以Ficoll_Paque方法所 分離出來的造血幹細胞之體外擴增比（EVER)也比依照 本發明實施例2、3、4、5及ό方法分離出來的造血 幹細胞之體外擴增比來的低。 其他實施方式 如上所述’本發明之較佳實施例已經參照附圖而 詳細地敘述。然而，應瞭解當中所表示為本發明之較 32 201040258 佳，鉍例者，其中詳細的敘述以及特定的實驗例僅為 說月之目的而已’對本發明所屬技術領域中之技術人 士而s，由以上詳細地說明，在本發明之範疇與精神 内為各式變化與修改是顯而易見的。 【圖式簡單說明】 处為讓本發明之上述和其他目的、特徵、優點與實施 例能更明顯易懂，所附圖式之說明如下： 第1圖為依據本發明一實施方式之批次式培育系統 之示意圖；及 第2圖為根據本發明一實施方式之連續式培育系統 之示意圖。 【主要元件符號說明】 100 批次式系統 101、201過濾腔室 103、203清洗溶液 105、205 第二入口 107、207集液容器 209造血幹細胞收集容器 211第三入口 213幫浦 200連續式系統 102、202造血幹細胞來源 104、204 第一入口 106、206 第一出口 208第二出口 210儲存腔室（非必要） 212幹細胞培養基 33The method substantially according to the above embodiment 4 is used except that the peripheral blood is used to replace the cord blood and the medium is replaced after the 2nd, 4th, 6th and 8th days after the start of the cell culture (ie, HSC 30 201040258 medium A). To isolate, in vitro expand and harvest hematopoietic stem cells. It is expected that the in vitro expansion ratio of stem cells isolated and amplified according to the method of the present embodiment is higher than that of the control group P卩, which directly separates hematopoietic stem cells from peripheral blood and is expanded in vitro. Example 8 was isolated from the bone sputum by the continuous system of Example 4, in vitro expansion and harvesting of hematopoietic stem cells, except that bone marrow fluid was used to replace cord blood and after days 2, 4, 6 and 8 after the start of cell culture. In addition to the medium (i.e., HSC medium A), hematopoietic stem cells were isolated, expanded in vitro, and harvested in substantial accordance with the methods described in Example 4 above. It is expected that the in vitro expansion ratio of stem cells isolated and amplified according to the method of the present embodiment will be higher than that of the control group (i.e., in vitro expansion of hematopoietic stem cells directly from peripheral blood). 〇 Comparative Example Mononuclear cells were isolated from the umbilical cord using the conventional Ficoll-Paque method (see Fotino M., ei a/., Ann. Clin. Lab. Sci., (1971) 1: 131-133). Briefly, cord blood was diluted with phosphate buffer (hereinafter referred to as "PBS A") containing 2 mmol/L EDTA and 0.5% fetal bovine serum (BSA) in a dilution ratio of cord blood: PBS A = 1 : 4. Next, 35 ml of the diluted cord blood was added to the surface of a 15 ml Ficoll-Paque solution (Pharmacia), followed by centrifugation (400 X g, 40 minutes, 20 ° C). After centrifugation, the mononuclear cells of 31 201040258 were carefully isolated. PBS A was added to the monocyte solution until the total volume was 50 ml. Next, the solution was stirred and centrifuged again at a rate of 3 Torr x g for about 10 minutes. The supernatant after centrifugation was removed, leaving a solution of about 5 ml at the bottom. The prepared hematopoietic stem cell solution containing 5,181 cells/μl of red blood cells, 11,935 cells/μl of white blood cells, and 171,812 cells/μl was analyzed according to the method specified in the above ISHAGE guidelines. Platelets, and approximately 185 fine cells/microliters of cr) 34+ hematopoietic stem cells require up to 3 hours for the entire cell separation process. About one CD34+ hematopoietic stem cells isolated according to the above method were planted in a 24-well culture dish containing about 1 ml of HSC medium A in each well. After 1 day of incubation, the in vitro expansion ratio (EVER) of hematopoietic stem cells in the medium was analyzed in the manner described in Example 2 above, which was about 143%. The time taken to separate the hematopoietic stem cells by the conventional Ficoll-Paque method is longer than the time required for the inventive examples 2, 3, 4, 5 and 6; and separated by the Ficoll_Paque method. The in vitro expansion ratio (EVER) of the resulting hematopoietic stem cells was also lower than the in vitro expansion ratio of hematopoietic stem cells isolated according to Examples 2, 3, 4, 5 and the sputum method of the present invention. Other Embodiments As described above, the preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings. However, it should be understood that the present invention is described as a preferred embodiment of the present invention, and the detailed description and the specific experimental examples are merely for the purpose of the month, and are intended to be used by those skilled in the art to which the present invention pertains. It will be apparent that various changes and modifications can be made within the scope and spirit of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. A schematic diagram of a cultivation system; and Figure 2 is a schematic illustration of a continuous cultivation system in accordance with an embodiment of the present invention. [Main component symbol description] 100 batch system 101, 201 filter chamber 103, 203 cleaning solution 105, 205 second inlet 107, 207 liquid collection container 209 hematopoietic stem cell collection container 211 third inlet 213 pump 200 continuous system 102, 202 Hematopoietic stem cell source 104, 204 First inlet 106, 206 First outlet 208 Second outlet 210 Storage chamber (not necessary) 212 Stem cell medium 33