TWI303278B

TWI303278B - A method of forming spheroids from the cultured cells

Info

Publication number: TWI303278B
Application number: TW94145899A
Authority: TW
Inventors: Mei Ju Yang; Wannhsin Chen; Peiju Lin; Chunhung Chen; Hsing Wen Sung
Original assignee: Ind Tech Res Inst
Priority date: 2005-12-22
Filing date: 2005-12-22
Publication date: 2008-11-21
Also published as: TW200724684A

Description

1303278 玖、發明說明：【發明所屬之技術領域】本發明有關於一種培養多細胞球體（multicellular spheroids)或胚球體（embryoid bodies)的方法，該多細胞球體或胚球體適於提供細胞以供諸如細胞治療、藥物筛檢等多種醫藥用途使用。詳言之，本發明提供用以形成多細胞1303278 发明, DESCRIPTION OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to a method of culturing multicellular spheroids or embryoid bodies suitable for providing cells for use in, for example, It is used in many medical applications such as cell therapy and drug screening. In particular, the invention provides for the formation of multicellular

已塗覆有纖Coated with fiber

維素和/或纖維素衍生物之細胞培養容器中的步驟。【先前技術】三維（three-dimensional，3-D)細胞培育方式提供研究人員可於類似動物體内環境下研究細胞生長、增生及分化，以及在二維（two-dimensional，2-D)培育環境下可能受到限制或排除而無法完全觀察到的不同程度的細胞與細胞間的反應及細胞與細胞外基質（extracellular matrix， ECM)間的反應（Edlman and Keefer， Experimental Neurology 2005，192: 1-6) ° 目前，3 -D球體培育物系統已被用在腫瘤研究中，其中被培育的細胞會形成多細胞腫瘤球體（multicellular tumor spheroids，MTS)。此外，多細胞球體也被用在諸如腫瘤細胞生物學、治療抗性、細胞與細胞間的反應、侵入、藥物滲透、模建、腫瘤標記物、營養梯度、腫瘤細胞代謝等研究中（Kunz-Schughart W α/，2004; Bates RC e，α/· 6 1303278The steps in the cell culture vessel of the vitamin and/or cellulose derivative. [Prior Art] Three-dimensional (3-D) cell culture methods provide researchers with the ability to study cell growth, proliferation and differentiation in a similar animal environment, as well as in two-dimensional (2-D) incubation. Different degrees of cell-to-cell response and reactions between cells and extracellular matrix (ECM) that may be restricted or excluded from the environment (Edlman and Keefer, Experimental Neurology 2005, 192: 1- 6) ° Currently, the 3-D sphere culture system has been used in tumor research, in which the cells to be cultured form multicellular tumor spheroids (MTS). In addition, multicellular spheroids have also been used in studies such as tumor cell biology, therapeutic resistance, cell-to-cell response, invasion, drug penetration, modeling, tumor markers, nutritional gradients, tumor cell metabolism, etc. (Kunz- Schughart W α/, 2004; Bates RC e,α/· 6 1303278

Crit Rev Oncol Hematol· 2000，36(2-3):61-74.)。球體培育物系統也被用在包括哺乳類細胞生物學（Dontu and Wicha, Journal of Mammary Gland Biology and 、滋養葉母細胞（Thomas K.Experimental Cell Research 2004,297: 41 5— 423)、軟骨細胞（Ursula Anderer and Jeanette Libera，J Bone MinerCrit Rev Oncol Hematol· 2000, 36(2-3): 61-74.). The spheroidal culture system is also used in mammalian cell biology (Dontu and Wicha, Journal of Mammary Gland Biology and trophoblasts (Thomas K. Experimental Cell Research 2004, 297: 41 5-423), chondrocytes (Ursula) Anderer and Jeanette Libera, J Bone Miner

Res 2002;17:1420-1429)、肝細胞（Cervenkova et al，5 Papers 2001,145: 57-60; Kazumori Funatsu, Artificial 25(3):194-20 0)、骨體間質細胞（Braz· J. Med. Bio. Res. 2005，38:1455-1462)、神經幹細胞 /祖（或前趨）細胞（Edlman and Keefer，Experimental Neurology 2005, 192 : 1-6, Reynolds，1992)等研究中。Res 2002;17:1420-1429), hepatocytes (Cervenkova et al, 5 Papers 2001, 145: 57-60; Kazumori Funatsu, Artificial 25(3): 194-20 0), bone stromal cells (Braz· J. Med. Bio. Res. 2005, 38: 1455-1462), neural stem cell/progenitor (or pre-existing) cells (Edlman and Keefer, Experimental Neurology 2005, 192: 1-6, Reynolds, 1992) and the like.

目前最常用來產生3-D培育物的5種方式包括：（i) 器官移植型培育物（organotypic explant cultures)，其係讓所收取到的整個器官或器官切片生長在有培養基的基材上，（2)靜態或旋轉式地微載體培育物（stationary or rotating microcarrier cultures)，其係將解離的細胞凝聚在具有黏性的環狀或圓錐狀之多孔基材上；（3)微質量培育物（micromass cultures)，其係將細胞懸浮在含有適量營養物及分化因子之培養基中；（4)讓自由浮動在轉動容器中的細胞彼此黏附而形成類似組織或器官的結構（又稱為「轉動壁容器（rotating wall vessels)」或「微重力生物反應器 (microgravity bioreactors)」）；及（5)凝膠類技術，其係將細胞包埋在可含有或不含有膠原蛋白骨架或其他可模才疑 ⑧ 7 1303278 ECM之有機纖維或合成纖維之類的基材中，例如瓊脂或 Matrigel® (Edlman and Keefer，Experimental Neurology 2005， 192 : 1-6) ° 在許多細胞研究中，都顯示細胞必須能夠附著在一表面上方能生長，且該表面多設計成可提供細胞良好的附著力。但是，在某些情況下，例如從胚胎幹細胞來形成胚球體的情況下，附著可能會促使細胞產生不希望發生的分化現象。大部分的胚胎幹細胞（ES細胞）在活體外分化時，會經過一個稱為「胚球體形成（embryoid bodies (EBs) formation)」的步驟。在此過程中，ES細胞會先聚集形成含有三胚層（中胚層、内胚層及外胚層）的球體，之後再進一步經由或不經由生長因子的誘發，而分化成具有功能性的體細胞。但是，目前利用幹細胞進行的細胞治療上一項很大的缺點在於無法充分、且大量地提供具均一表現型、已分化之具有功能性的體細胞群，以用於諸如胚胎研究、藥物篩檢及組織移植等廣泛用途中。此領域中具備通常知識者目前採用數種胚球體形成的方式，希望能大量培育出衍生自E S細胞之具有功能性的體細胞群，這幾種方式包括習知的懸滴培育技術（hanging drop technique)、低附著法（low attachment method)、覆液懸浮培育法 (encapsulated liquid suspension culture，ELSC)、液態生物反應器（liquid bioreactor) 、 3 維培育法 (three-dimensional culture)及高黏稠的半固體培育法 (semi-solid culture)等等。 1303278 習知的懸滴培育技術（h a n g i n g d r o p t e c h n i q u e)係將一定數目的細胞聚集在自組織培養盤頂面懸垂而下的一小量體積的液體内，來進行胚球體的形成。低附著法（low attachment method)係由 Thomson 等人提出（參見美國專利第6,602,71 1號），其係以機械性刮抹、酵素解離或攪拌培育物容器等方式，使黏附在培養基材上的靈長類E S聚落保持在懸浮狀態下，來進行培育。覆液懸浮培育法（encapsulated liquid suspension culture，ELS C)則是藉由將胚球體包覆在一基質中，並於高密度、液態懸浮狀態下形成胚球體，其中各別細胞或細胞聚集物係被包覆並置於一可控制的攪拌懸浮生物反應器中或是在覆液懸浮培育中，使細胞不會產生大量凝集並使其增生、分化成欲求的細胞型態（參見WO 03/004626)。半固體培育方式則係將細胞封在不會使細胞產生大量凝集的咼黏稠半固體培養基中，亦即，在此高黏稠半固體培養基中細胞會因培養基的高黏稠性可懸浮生長，亦不會因細胞密度過高而使細胞彼此產生碰撞或凝聚(Steph^ M. D. e/ α/·，Biotechnol. Bioeng. 2002 78(4)，442-453.) 〇The five most commonly used methods for producing 3-D cultures include: (i) organotypic explant cultures, which allow the entire organ or organ slice collected to grow on a substrate with medium. (2) static or rotating microcarrier cultures, which condense dissociated cells on a porous or conical porous substrate; (3) micro-quality cultivation Micromass cultures, which suspend cells in a medium containing appropriate amounts of nutrients and differentiation factors; (4) allowing cells that float freely in a rotating container to adhere to each other to form a structure resembling a tissue or organ (also known as " Rotating wall vessels or microgravity bioreactors; and (5) gel-based techniques for embedding cells in or with collagen backbones or other It is suspected that 8 7 1303278 ECM organic or synthetic fibers, such as agar or Matrigel® (Edlman and Keefer, Experimental Neurology 20 05, 192 : 1-6) ° In many cell studies, cells must be able to adhere to a surface to grow, and the surface is designed to provide good cell adhesion. However, in some cases, such as the formation of blastocysts from embryonic stem cells, attachment may cause undesirable undesirable differentiation of the cells. Most embryonic stem cells (ES cells) undergo a process called "embryoid bodies (EBs) formation" when they are differentiated in vitro. In this process, ES cells first aggregate to form spheres containing three germ layers (mesoderm, endoderm, and ectoderm), and then further differentiate into functional somatic cells with or without induction by growth factors. However, the current major drawback of cell therapy using stem cells is the inability to adequately and extensively provide a homogeneous, differentiated, functional somatic cell population for use in, for example, embryo research, drug screening. And a wide range of applications such as tissue transplantation. Those with ordinary knowledge in this field are currently using several blastocyst formation methods, and it is hoped that a large number of functional somatic cell populations derived from ES cells can be cultivated in large quantities, including conventional hanging drop breeding techniques (hanging drop). Technique), low attachment method, encapsulated liquid suspension culture (ELSC), liquid bioreactor, three-dimensional culture, and highly viscous half Semi-solid culture and the like. 1303278 The conventional technique of hanging drop culture (h a n g i n g d r o p t e c h n i q u e) is to form a globular body by collecting a certain number of cells in a small volume of liquid suspended from the top surface of the tissue culture plate. The low attachment method is proposed by Thomson et al. (see U.S. Patent No. 6,602,71 1), which is attached to the culture substrate by mechanical scraping, enzyme dissociation or agitating the culture vessel. The primate ES colonies on the top remain in suspension for cultivation. The encapsulated liquid suspension culture (ELS C) is formed by coating the blastocysts in a matrix and forming blastocysts in a high-density, liquid suspension state, wherein individual cells or cell aggregates are formed. It is coated and placed in a controlled stirred suspension bioreactor or in suspension culture, so that the cells do not produce a large amount of agglutination and proliferate and differentiate into the desired cell type (see WO 03/004626). . The semi-solid cultivation method is to seal the cells in a viscous semi-solid medium which does not cause a large amount of agglutination of the cells, that is, the cells in the highly viscous semi-solid medium can be suspended and grown due to the high viscosity of the medium, nor Cells may collide or aggregate with each other due to excessive cell density (Steph^ MD e/α/·, Biotechnol. Bioeng. 2002 78(4), 442-453.)

Rennard等人則提出一種類似前述3-D球體培育物率、統的三維（three-dimensional，3-D)細胞培育方式（參見美國專利公告第2005/0054 1 00號），其係將胚球體封在—3 維架具材料及細胞培養基中，使胚球體在該3維架具材料及細胞培養基中生長。此類3維架具材料可選自白蛋白、 9 1303278 膠原蛋白、明膠、透明質酸、澱粉、藻酸、果膠、或纖維素衍生物、酪蛋白、葡聚醣、多醣或纖維蛋成的物質群組中。雖然以上揭示多種用來培育E S細胞的系統，少含有一下述缺點，包括費時、人力需求高、系統無法放大量產、經濟效益不佳、形成球體的比例太化率太低等等。因此，亟需發展出一種簡單、容易使用的方法造大量可形成球體的細胞，以於短時間内為醫療應充分的細胞來源，同時此方法還需具備經濟效益，要高度技術背景即可進行操作。【發明内容】本發明人發現以習知的懸滴培育技術或是低培養盤（P e t r i d i s h)來培育細胞時，並無法有效地形球體。因此，本發明一態樣即為提供一種用以形成體或胚球體的方法，該方法使用上相當簡單、具經且細胞球體生成率超過9 0 %，效果遠遠超越上述任的細胞培育技術。本發明另一態樣則在於提供使用法所培育出來之一成熟細胞的細胞球體或一未分胞的胚球體。在一較佳實施例中，本發明提供一種用以形成的方法，包括將人類胚胎幹細胞（HES細胞）培育在纖維素白所組但其至複雜、低、分，來製用提供且不需附著性成細胞細胞球濟效益一習知上述方化幹細胚球體一已塗 10 1303278 覆有纖維素和/或纖維素衍生物之培育容器中。在一較佳實施例中，係以甲基纖維素（methylcellulose，MC)作為該培育容器表面的一種塗層物質；但是，亦可使用其他的纖維素衍生物作為該培育容器表面的塗層物質，較佳係選用叛曱基纖維素（carboxymethylcellulose，CMC)、經曱基纖維素（hydroxymethylcellulose，HMC)、經乙基纖維素 (hydroxyethylcellulose， HEC)、經丙基纖維素 (hydroxypropylcellulose, HPC)及羥丙曱基纖維素 (hydroxypropylmethylcellulose, HPMC) 〇本發明另一實施態樣為提供一種可分化成多種細胞糸的胚球體。在一較佳實施例中’依據本發明方法而形成的胚球體可繼續分化成為肝細胞或衍生自胚胎三胚層中任一層的細胞。在另一較佳實施例中，提供一種用以形成肝細胞球體的方法，包括將諸如肝腫瘤細胞（C3 A細胞）之類具肝臟功能的肝臟細胞培育在一已塗覆有纖維素和/或纖維素衍生物之培育容器中。在另一較佳實施例中，提供一種依據本發明方法而形成的肝細胞球體。在參閱以下詳細說明及實施例後，將可更了解上述本發明的目的及其優點。須知，上述的一般性描述及以下的詳細說明，均為闡述本發明而為的例示說明，非用以限制本發明之範疇。 11 1303278 【實施方式】所揭示實施例及名詞僅係作為說明之用，本發明範疇，且本發明亦涵蓋未於此述及、但知技術人士在閱讀過本文後所能思及並用以的其他實例。名詞定義為方便敘述起見，在此將全文提及的專業用語做統一說明，除非另有定義，否則所述及學名詞均採此領域具備通常知識者所能理解g 若無特別於上下文中清楚記述其他意義泪容及後附申請專利範圍中所使用之如「一（“a’ 與「該（the)」等特定用語均包含其複數型態本文中所使用之名詞「多潛能型幹細胞 s t e m c e 11)」係指一種在適當條件下，可不斷可分化形成所有類型之成體細胞的細胞，特另J 生自三胚層（包括中胚層、内胚層及外胚層）中有類型的細胞。文中所用之名詞「胚球體（或稱EBs)」係育期間由ES細胞凝集所形成的一種可幫助後的細胞三維空間結構。當ES細胞培育在懸浮下，ES細胞會形成小型細胞凝集物，且其外層内臟内皮層（visceral endoderm)。由於其大力及基因表現模式類似著床後的胚胎早期，因非用以限制為此領域習實施本發明名詞及特定之技術與科 '一般定義。 =，說明書内 or “an”）」 (pluripotent 自我複製並是，那些衍任一層之所指一種在培續細胞分化生長的環境圍包覆著一小、分化能此這些細胞 12 1303278 凝集物又被稱為胚球體，且多作為可研究發育初期之細胞分化及基因表現的模型系統使用。若讓ES細胞生長並凝集，其會形成一般稱為「簡單胚球體（simple embryoid bodies)」之小型細胞球體。此簡單胚球體具有一大型内臟及壁内皮細胞（large visceral and parietal endoderm cells) 所形成的外層；若繼續成長則會成為囊性胚球體（cystic embryoid bodies)，其具有一充滿液體之腔室及一層由類似外皮細胞組成的内層。因此，文中所用之名詞「球體（s p h e r 〇 i d s)」係指在細胞培育期間，由細胞凝集物所形成的球型、3 -D細胞結構。在一較佳實施例中，可由本發明方法形成一肝細胞球體或肝細胞之3 - D凝集物。文中所用之名詞「培育容器」包括任何一種適合用來裝載一液態細胞培育物的容器。可使用習知的各種細胞培育容器，包括三角燒瓶（Erlenmeyer flask)、長頸培養瓶 (baffled flask)、培養im、培養盤、燒杯、試管等。除非另行定義，文中所使用之所有專業與科學用語與化學及生物領域公開文獻中所使用的相對應用語之意義相同。需知於實施例或他處内容中所顯示之成分用量、反應條件等數字或是本申請說明書内容所使用之數字均為大約數值。因此，除非文中特別注明，本說明書之上述數字參數均為近似值，其可根據欲求得之本發明結果而加以變 1303278 化。並且，這些參數並非用來限定與本發明申請專利範均等之原理，而是應用正常操作技術下所得到之較佳據。雖然上述用來指出本發明之最大範圍之數據範圍參數均為近似值，但上述特定較佳實施例中所記述之數值已盡可能地精準。然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。於本申請書中所提到之所有參考文獻均全體納入考，以揭露並敘述該文獻所記載之相關方法及/或材料此外，文中所討論之文獻僅揭露本發明申請日前之習知術。並且無任何文獻顯示本發明内容曾為習知技術所露。本發明内容所得到之實際數據會因個別的實施條件與本發明揭露於說明書内容中之數據有所不同。本發明係關於用來產生大量衍生自E S細胞之具有能的細胞或具有功能的成熟細胞的方法，該些細胞係可用在諸如細胞治療、藥物篩檢等各種臨床應用上，該方包含於半固體凝膠培養容器製備出大量適於進一步分的胚球體（EBs)或成熟細胞組成之細胞球體。參照第1圖，其示出本發明形成細胞球體或胚球體方法所需步驟。用於培育細胞的容器係先行塗覆了一層維素和/或纖維素衍生物，以在容器底部表面形成一低著性的半固體凝膠薄層。之後，再將細胞接種在該半固凝膠表面上，並保持懸浮以避免培育中的細胞凝聚，以成更均一、大尺寸且具備活性的胚球體或細胞球體。圍數及據參〇技揭而功應法化之纖附體形 14 1303278 因此，在本發明一實施態樣中，係提供一種用以形成細胞球體的方法，包含將細胞培育在一已塗覆有纖維素和 /或纖維素衍生物之培育容器中。該塗覆有纖維素和/或纖維素衍生物之培育容器的製備方式如下：於室溫（〜20。(：）下，在一培養盤中央傾倒入少量纖維素溶液，之後將該培養盤略微傾斜，使該纖維素溶液能夠均勻分散開以覆蓋整個培養盤底面並形成一層透明薄膜。之後，將該已塗覆有纖維素溶液的培養盤在高於室溫（如，3 7 °C )的溫度下靜置一段時間（例如，1小時），使培養盤底面上該透明薄膜硬化成為一不透明的凝膠層，在此又稱「纖維素凝膠」。之後，即可在後續的實驗中以此種塗覆有纖維素凝膠的培養盤來形成胚球體或細胞球體。在一較佳實施例中，係以甲基纖維素（methylcellulose， MC)作為該培育容器表面的一種塗層物質；但是，亦可使用其他的纖維素衍生物作為該培育容器表面的塗層物質，較佳係選用竣甲基纖維素（carboxymethylcellulose, CMC)、經甲基纖維素（hydroxymethylcellulose，HMC)、經乙基纖維素（hydroxyethylcellulose，HEC)、經丙基纖維素 (hydroxypropylcellulose， HPC)及經丙甲基纖維素 (hydroxypropylmethylcellulose，HPMC) ° 可用於本發明之適當的培育容器包括三角燒瓶、長頸培養瓶、培養孤、培養盤、燒杯、試管等。在一較佳實施 15 1303278 例中，係使用一細胞培養盤（F a 1 c ο n e ® Β e c t ο η D i c k i η s ο η Labware，Franklin Lakes，NJ，USA) ο 在一較佳實施例中，提供一種用以形成人類胚胎幹細胞胚球體的方法，其中形成囊胚球體的比例超過90%，效果遠優於所揭示之懸滴技術和/或低附著性培養盤等細胞培育技術的成果。本發明涉及培育人類胚胎幹細胞（HES 細胞）的方法已揭示於同一申請人於2005年9月23曰向美國專利商標局提申之序列編號為：1 1 /2 3 3，0 5 5，標題為「人類胚胎幹細胞及其之培育方法」、目前仍在審查中的另一申請案中，其全部内容在此併入作為參考。簡言之，將HES 細胞培育在無滋養層的培育系統中，其包含一覆蓋有細胞外基質（extracellular medium，ECM)的基材，該 ECM 係分離自已經過γ -射線照射或C -型絲裂黴素（m i t 〇 m y c i n C)處理過而使其停止生長的滋養層細胞中；及一種已經由該滋養層細胞調節過的調節培養基（con(|iti〇ned medium)。該調節培養基的主要成分包括氨基酸、維生素、碳水化合物、無機離子、生長因子及某些其他生物活性物質。發明人發現組合使用依據所揭示方法製備而成的ECM及調節培養基’可明顯提升未分化之HES細胞的生長情形。本發明用以形成HES細胞胚球體的方法，包含將培育於滋養層的 HES細胞或依據上述美國申請案u/23 3,〇55所揭示方法培育而成之HES細胞，培育在一塗覆有纖維素和/或纖維素衍生物之培育容器中。但是，此方法同樣也適用於培育其他任何可形成球體的細胞，這類可形成球體的細胞可源自 16 1303278 哺乳動物、鼠和/或人類，其包含（但不限於）心臟細胞、造血細胞、内皮細胞、神經細胞、神經膠原細胞、腎臟細胞、肝臟、、，田胞或血管祖細胞（vascular pr〇genit〇r cells)。在一較佳實施例中’可依據本發明所揭示方法形成肝臟細胞球體 (實施例2)。您方法所製備而成之細胞的用途本發明人揭示藉由將細胞培育在一半固態凝膠的培養基質上’可促使細胞形成諸如胚球體之類的球體狀結構’並因而形成肝細胞球體。依據本發明方法所獲得的諸如胚球體之類的細胞球體可作為一種細胞來源，用以提供組織移植時所需的體細胞。在一較佳實施例中，依據本發明方法而形成的胚球體可在諸如bEGF之類的生長因子存在下，被進一步誘導分化成為肝譜系細胞（實施例i . 3 · 2)。因此，本發明提供一種可產生大量適於後續醫療用途之細胞的方法。可形成球體的細胞（例如，HES細胞），及衍生自該球體的細胞（例如’肝細胞）可用來治療各種疾病，包括（但不限於）癌症、白也病、自體免疫疾病、器官衰竭及組織複製等，以及培養成熟具組織功能性的細胞以應用於藥物篩選與人工肝臟輔助器等醫療用途。實施例以下將藉由實施例來闡述本發明，以幫助此領域習知技藝人士來實施本發明，需知所揭示之實施例，僅係供闡述 ⑧ 17 1303278 本發明之用，本發明範疇並不侷限於所揭示的該等實例。實施例1 形成胚球體（EBs)及分析其表現特徵 1·1 培育hes細胞 -1—· 1 · 1—-製備調節培養基用以維持HES細胞培育的調節培養基係依據以下方法進行製備。簡言之’將初代培養的小鼠胚胎纖維母細胞 (proimary mouse embryonic fibroblasts (PMEF)培育在添加有10%胎牛血清（FBS)的DMEM培養基中（購自Gibco Invitrogene)。將細胞培育在37°C、5% c〇2及飽和溼度下，直到其生長至將近全滿（confluence)為止，之後再加入1〇 gg/ml的C-型絲裂黴素使纖維母細胞停止增生。隨後，將已停止增生的纖維母細胞再次重新培育於添加有2 〇 %胎牛血清、ImM β-氫硫基乙醇（購自Gibco Invitrogene)、1〇/〇非必要氨基酸（購自 Gibco Invitrogene)、1%穀胺酸（講自 Gibco Invitrogene)及1 %之胰島素-輸鐵蛋白-硒g型添加物（ITS G添加物，購自Gibco Invitrogene)的DMEM培養基中（參見 Richards, M. ei a/.， Nat. Biotechnol.， 20(9):93 3-93 6, 2002)。也可省略不加入FBS以血清替代物取代的無血清培養基。收集培養過停止生長的纖維母細胞至少1天的培養基，供後續實驗立即使用或將其儲存在_80 °C以於日後實驗中使用。所收集的培養基稱為「調節培養基（conditioned medium)」。依據所培育細胞之需求，該培養基中還可包含其他在此並未述及的成分。Rennard et al. proposed a three-dimensional (3-D) cell culture method similar to the aforementioned 3-D sphere culture rate (see US Patent Publication No. 2005/0054 00), which is a globule body. The glomerulus is grown in the 3-dimensional scaffold material and the cell culture medium in a 3-dimensional racking material and a cell culture medium. Such 3-dimensional scaffolding materials may be selected from the group consisting of albumin, 9 1303278 collagen, gelatin, hyaluronic acid, starch, alginic acid, pectin, or cellulose derivatives, casein, dextran, polysaccharides or fibrous eggs. In the substance group. Although a variety of systems for culturing E S cells have been disclosed above, they have the following disadvantages, including time consuming, high labor requirements, inability to scale up the system, economical efficiency, and the proportion of spheres that are too low. Therefore, there is an urgent need to develop a simple and easy-to-use method for making a large number of cells capable of forming spheres, so as to provide a sufficient source of cells for medical treatment in a short period of time, and at the same time, this method needs to have economic benefits and can be carried out with a high technical background. operating. SUMMARY OF THE INVENTION The present inventors have found that when a cell is cultivated by a conventional hanging drop culture technique or a low culture disk (P e t r i d i s h), the sphere cannot be effectively formed. Therefore, an aspect of the present invention provides a method for forming a body or a globule, which is relatively simple to use, has a cell spheroid formation rate of more than 90%, and the effect far exceeds the above cell culture technique. . Another aspect of the present invention is to provide a cell sphere or an undivided blastocyst of a mature cell cultivated by the method of use. In a preferred embodiment, the present invention provides a method for forming comprising culturing human embryonic stem cells (HES cells) in a group of cellulose whites but providing them to complex, low, and fractional, and providing and not required Adhesive cell-forming cell globular benefits. It is known that the above-described phytochemical dry fine blastomeres have been coated with 10 1303278 in a culture vessel covered with cellulose and/or cellulose derivatives. In a preferred embodiment, methylcellulose (MC) is used as a coating material on the surface of the incubation vessel; however, other cellulose derivatives may also be used as the coating material on the surface of the incubation vessel. Preferably, carboxymethylcellulose (CMC), hydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC) and hydroxy group are selected. Hydroxypropylmethylcellulose (HPMC) Another aspect of the present invention provides a blastocyst that can differentiate into a variety of cell rafts. In a preferred embodiment, the blastocyst formed by the method of the present invention can continue to differentiate into hepatocytes or cells derived from any of the three layers of the embryonic germ layer. In another preferred embodiment, a method for forming a hepatocyte spheroid is provided, comprising cultivating liver functioning liver cells such as liver tumor cells (C3 A cells) coated with cellulose and/or Or in a culture vessel for the cellulose derivative. In another preferred embodiment, a hepatocyte spheroid formed in accordance with the methods of the present invention is provided. The objects and advantages of the present invention described above will become more apparent from the detailed description and appended claims. It is to be understood that the following general description and the following detailed description are illustrative of the invention 11 1303278 [Embodiment] The disclosed embodiments and the terms are used for illustration only, the scope of the present invention, and the present invention also covers those that are not described herein, but can be considered and used by those skilled in the art after reading this document. Other examples. The nouns are defined as convenient for narration. The terminology mentioned in the full text is used to make a unified description. Unless otherwise defined, the terms and terms are understood by those who have the usual knowledge in this field. Clearly describe other meanings of tears and the scope of the patent application. The specific terms such as "a" and "the" are used in their plurals. The term "pluripotent stem cells" is used herein. Stem 11) means a cell that, under appropriate conditions, is continually differentiated into all types of adult cells, and is derived from cells of the three germ layers (including mesoderm, endoderm and ectoderm). The term "blastosome (or EBs)" as used herein refers to a three-dimensional structure of cells that can be formed by the agglutination of ES cells during the process of breeding. When ES cells are cultured in suspension, ES cells form small cell aggregates and their outer visceral endoderm. Since its power and gene expression patterns are similar to those of post-implantation embryos, it is not intended to limit the practice of the invention and the specific technical and scientific definitions. =, in the specification or "an")" (pluripotent self-replication is that one of the layers referred to in any layer of the cell is surrounded by a small, differentiated energy cell 12 1303278 agglomerate It is called blastoderm and is used as a model system for studying cell differentiation and gene expression in the early stages of development. If ES cells are grown and aggregated, they form a small size called "simple embryoid bodies". Cell spheroid. This simple blastocyst has an outer layer formed by a large visceral and parietal endoderm cells; if it continues to grow, it becomes a cystic blastocyst (cystic embryoid body) with a liquid filled The chamber and a layer consisting of an outer layer resembling a sheath cell. Therefore, the term "spher 〇ids" as used herein refers to a spherical, 3-D cell structure formed by cell aggregates during cell culture. In a preferred embodiment, a hepatocyte spheroid or a 3-D agglutination of hepatocytes can be formed by the method of the present invention. The term "incubation container" includes any container suitable for loading a liquid cell culture. Various cell culture vessels, including Erlenmeyer flasks, baffled flasks, cultures, can be used. Im, culture trays, beakers, test tubes, etc. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as the relative terms used in the open literature on chemical and biological fields. Need to know about the examples or elsewhere The amounts used in the ingredients, the reaction conditions, and the like, or the numbers used in the specification of the present application are all approximate values. Therefore, unless otherwise stated herein, the above numerical parameters in the specification are approximate, and may be obtained according to the desired The results of the present invention are varied and are not intended to limit the principles of the application of the present invention, but rather to the preferred data obtained under normal operating techniques. The data range parameters of the ranges are approximate, but in the particular preferred embodiment described above The values stated are as accurate as possible. However, each measurement experiment has its standard deviation, so any data value must have some error. All references mentioned in this application are included in the test to expose and describe the document. Related Methods and/or Materials Illustrated In addition, the documents discussed herein disclose only the prior art of the present application, and no document has been shown to be disclosed in the prior art. The data may differ from the data disclosed in the present specification by the individual implementation conditions. The present invention relates to a method for producing a large number of competent cells or functional mature cells derived from ES cells, which can be used in various clinical applications such as cell therapy, drug screening, etc. The solid gel culture vessel produces a plurality of cell spheres composed of blastocysts (EBs) or mature cells suitable for further division. Referring to Figure 1, there is shown the steps required in the method of forming a cell sphere or blastocyst of the present invention. The container for culturing the cells is first coated with a layer of vitamins and/or cellulose derivatives to form a thin layer of semi-solid gel on the bottom surface of the container. Thereafter, the cells are seeded on the surface of the semi-solid gel and kept suspended to avoid cell aggregation in the culture to form more uniform, large-sized and active blastocysts or cell spheres. The invention relates to a method for forming a cell sphere, comprising cultivating a cell in a coated state, in accordance with an embodiment of the present invention. In a culture vessel with cellulose and/or cellulose derivatives. The culture vessel coated with cellulose and/or cellulose derivative is prepared in the following manner: at room temperature (~20. (:), a small amount of cellulose solution is poured into the center of a culture tray, and then the culture tray is poured Slightly tilted to allow the cellulose solution to be evenly dispersed to cover the entire bottom surface of the culture tray and form a transparent film. Thereafter, the culture plate coated with the cellulose solution is above room temperature (eg, 3 7 ° C) The temperature is allowed to stand for a period of time (for example, 1 hour), and the transparent film on the bottom surface of the culture plate is hardened into an opaque gel layer, which is also referred to herein as "cellulose gel". In this experiment, a cellulose gel-coated culture plate is used to form a globule or a cell sphere. In a preferred embodiment, methylcellulose (MC) is used as a coating on the surface of the culture vessel. Layer material; however, other cellulose derivatives may also be used as the coating material on the surface of the cultivation container, preferably carboxymethylcellulose (CMC), methylcellulose (hyd) Roxymethylcellulose, HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC) and hydroxypropylmethylcellulose (HPMC) ° suitable for use in the present invention, including a triangle Flask, long neck culture flask, culture solitary, culture plate, beaker, test tube, etc. In a preferred embodiment 15 1303278, a cell culture plate is used (F a 1 c ο ne ® Β ect ο η D icki η s ο η Labware, Franklin Lakes, NJ, USA) ο In a preferred embodiment, a method for forming human embryonic stem cell blastomers is provided, wherein the proportion of blastocyst spheres formed exceeds 90%, and the effect is much better than disclosed The results of cell culture techniques such as hanging drop techniques and/or low adhesion culture disks. The present invention relates to methods for cultivating human embryonic stem cells (HES cells) which have been disclosed to the United States Patent and Trademark Office on September 23, 2005. The serial number of the reference is: 1 1 /2 3 3,0 5 5, entitled "Human Embryonic Stem Cells and Their Cultivation Methods", currently under review In another application, the entire contents of which is hereby incorporated by reference, in its entirety, the HES cells are incubated in a trophoblast-free cultivating system comprising a substrate covered with an extracellular medium (ECM) The ECM is isolated from trophoblast cells that have been subjected to gamma-irradiation or C-type mitomycin C treatment to stop growth; and an adjustment that has been regulated by the trophoblast cells Medium (con(|iti〇ned medium). The main components of the medium are amino acids, vitamins, carbohydrates, inorganic ions, growth factors and certain other biologically active substances. The inventors have found that the combined use of ECM and regulatory medium prepared according to the disclosed methods can significantly enhance the growth of undifferentiated HES cells. The method for forming a blastoderm of a HES cell of the present invention comprises cultivating a HES cell cultured in a trophoblast or a HES cell cultured according to the method disclosed in the above-mentioned U.S. Patent Application Serial No. 23/55, In a culture vessel of cellulose and/or cellulose derivatives. However, this method is equally applicable to the cultivation of any other spheroid-forming cells. Such spheroid-forming cells may be derived from 16 1303278 mammals, mice and/or humans, including but not limited to cardiac cells, hematopoietic cells. , endothelial cells, nerve cells, neurocollagen cells, kidney cells, liver, , vascular pr〇genit〇r cells. In a preferred embodiment, hepatic cell spheres can be formed in accordance with the methods disclosed herein (Example 2). Use of cells prepared by your method The inventors have revealed that by culturing cells on a culture substrate of a semi-solid gel, the cells can be caused to form a spherical structure such as blastoderm and thus form hepatocyte spheres. Cell spheres such as blastomers obtained by the method of the present invention can be used as a source of cells to provide somatic cells required for tissue transplantation. In a preferred embodiment, blastocysts formed in accordance with the methods of the present invention are further induced to differentiate into hepatic lineage cells in the presence of a growth factor such as bEGF (Example i.3.2). Accordingly, the present invention provides a method of producing a large number of cells suitable for subsequent medical use. Cells that form spheroids (eg, HES cells), and cells derived from the spheroids (eg, 'hepatocytes') can be used to treat a variety of diseases including, but not limited to, cancer, leukocytosis, autoimmune disease, organ failure And tissue replication, etc., as well as the cultivation of mature tissue-functional cells for medical applications such as drug screening and artificial liver assist devices. The present invention will be described by the following examples in order to assist those skilled in the <Desc/Clms Page number> It is not limited to the examples disclosed. Example 1 Formation of blastocysts (EBs) and analysis of their performance characteristics 1·1 Cultivation of hes cells -1 - 1 · 1 - Preparation of regulatory medium The regulatory medium for maintaining HES cell culture was prepared according to the following method. Briefly, 'Proimary mouse embryonic fibroblasts (PMEF) were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) (purchased from Gibco Invitrogene). Cells were grown at 37 At °C, 5% c〇2 and saturated humidity, until it grows to near confluence, then add 1〇gg/ml of C-type mitomycin to stop the fibroblasts from proliferating. The proliferating fibroblasts were re-incubated with 2% fetal calf serum, 1 mM β-thioethyl alcohol (purchased from Gibco Invitrogene), 1 〇/〇 non-essential amino acids (purchased from Gibco Invitrogene), 1 % glutamic acid (from Gibco Invitrogene) and 1% insulin-transferrin-selen g-type supplement (ITS G supplement, purchased from Gibco Invitrogene) in DMEM medium (see Richards, M. ei a/. , Nat. Biotechnol., 20(9): 93 3-93 6, 2002). It is also possible to omit a serum-free medium that is replaced with a serum substitute without adding FBS. A culture medium in which the growth-sustained fibroblasts are cultured for at least 1 day is collected. For subsequent experiments or use it immediately In the presence of _80 ° C for use in future experiments. The collected medium is called "adjustment-yl culture (conditioned medium)." According to the needs of the cultivated cells, the culture medium may also contain other components mentioned here are not.

1303278 1.1.2 製備細胞外基質（ECM) 依據以下步驟製備可維持HES細胞生長之細胞外基質。簡言之，將初代培養小鼠胚胎纖維母細胞（PMEF)或人類包皮纖維母細胞（human forskin fibroblast，HFF，購自台灣動物技術研究所）培育在添加有 1 0%胎牛血清的 DMEM培養基中。當細胞生長達到將近 90%全滿 (confluence)時，加入1〇 gg/ml的C-型絲裂黴素使纖維母細胞停止生長。以胰蛋白酶將細胞取下、計算其數目，並再次重新培育在培養盤中，使其達到幾近全滿狀態至少2 天，之後以0_ 05 N的氫氧化鈉或0.1 %之三硝基甲苯（購自 Sigma)處理約1-15分鐘，並以大量磷酸緩衝溶液（PBS)(pH 7.4，購自 Gibco Invitrogene)移除細胞胞器及細胞核。所製備而成之PMEF或HFF的細胞外基質可直接用於後續實驗或可將其保存在4°C的PBS溶液中至少9個月。 1.1.3 培育HES細臉將包括 HES-3 (ES 細胞株’講自 ESI cell international) 及TW1 (工業技術研究院與李茂盛婦產科）在内的heS細胞接種在塗覆有依據實施例1 · 1.2所製備出來的E c Μ之培養盤上，並加入依據實施例1 .1 . 1所製備出來的調節培養基進行細胞培育。每隔1到2天便更換一次培養基，在3 7 °C、5 % C Ο2及飽和渔度的環境下培育6至8天。每隔6 至8天即以中性蛋白酶（dispase)(5單位/亳升，bd)將其繼代培育，並以毛細管玻璃拉成的玻璃刀將其機械性地切割成數小塊。 19 1303278 1.2. 形成胚球體（EBs) 1 · 2 · 1 盤備曱基纖維素溶液在50C下’將已秤重的甲基纖維素（methylcellulose， MC)粉末（Μ 7027，細胞培育級，sigma，St. Louis, USA)溶解在添加有各種濃度（〇· 25倍至1倍）PBS溶液的熱水中，以製備出各種濃度（9〜12%，重量/體積比）的MC溶液。以滲透壓儀（Model 3300，Advanced Instruments，Inc.，Norwood, MA，USA)來測量所製備而成的MC溶液之滲透壓。 ί·2·2-1備塗覆有MC-凝勝的細胞培育容器將實施例1.2.1所製備而成之MC溶液（其會在低於37 C的度下產生/破膠）塗覆在細胞培養盤（Faic〇ne⑧，Bect〇n1303278 1.1.2 Preparation of extracellular matrix (ECM) The extracellular matrix that maintains the growth of HES cells was prepared according to the following procedure. Briefly, primary cultured mouse embryonic fibroblasts (PMEF) or human forskin fibroblasts (HFF, purchased from Taiwan Institute of Animal Technology) were cultured in DMEM medium supplemented with 10% fetal bovine serum. in. When the cell growth reached nearly 90% confluence, the addition of 1 〇 gg/ml of C-type mitomycin stopped the growth of the fibroblasts. The cells were removed by trypsin, the number was counted, and re-cultured in the culture plate to reach a nearly full state for at least 2 days, followed by 0 0 05 N sodium hydroxide or 0.1% trinitrotoluene. (purchased from Sigma) was treated for about 1-15 minutes and cell organelles and nuclei were removed in a large amount of phosphate buffered saline (PBS) (pH 7.4, purchased from Gibco Invitrogene). The extracellular matrix of the prepared PMEF or HFF can be used directly in subsequent experiments or it can be stored in a 4 ° C PBS solution for at least 9 months. 1.1.3 cultivating HES fine face will include HES-3 (ES cell line 'speaking from ESI cell international) and TW1 (Industrial Technology Research Institute and Li Maosheng Obstetrics and Gynecology) inoculated with HES cells coated according to Example 1 · 1.2 prepared E c 培养 on the culture plate, and added to the conditioning medium prepared according to Example 1.1.1 for cell culture. The medium was changed every 1 to 2 days and incubated for 6 to 8 days at 37 ° C, 5 % C Ο 2 and saturated fish. It was subcultured every 7 to 8 days with a neutral dispase (5 units/liter, bd) and mechanically cut into small pieces with a glass knife drawn from capillary glass. 19 1303278 1.2. Formation of blastocysts (EBs) 1 · 2 · 1 Preparation of thiol-based cellulose solution at 50C 'When weighed methylcellulose (MC) powder (Μ 7027, cell culture grade, sigma) , St. Louis, USA) was dissolved in hot water to which various concentrations (〇·25 times to 1×) of PBS solution were added to prepare various concentrations (9 to 12%, weight/volume ratio) of the MC solution. The osmotic pressure of the prepared MC solution was measured by an osmometer (Model 3300, Advanced Instruments, Inc., Norwood, MA, USA). ί·2·2-1 is coated with MC-stabilized cell culture container. The MC solution prepared in Example 1.2.1 (which will produce/break at less than 37 C) is coated. In cell culture tray (Faic〇ne8, Bect〇n

Dickinson Labware，Franklin Lakes，NJ，USA)中。首先，在室溫（〜20 °C )下，將約450 μΐ的MC溶液倒入培養盤中央’之後將該培養盤略微傾斜，使該M C溶液能夠均勻分月欠開並覆蓋整個培養盤底面以形成一層透明薄膜。之後，將該已塗覆有M C溶液的培養盤靜置在3 7 t (或5 〇 )的溫度下1小時，使培養盤底面上該透明薄膜硬化成為一不透明的/邊膠層’在此又稱「纖維素凝膠（或稱M c -凝膠）」。之後’即可在後續的實驗中以此種塗覆有M c _凝膠的培養盤來形成胚球體或細胞球體。 1.2.3 形成胚跋艚將實施例1 · 1 · 3中的HES細胞團塊移轉到依據實施例 1 · 2.2所製備完成之含有M C -凝膠塗層的培養盤上，並將細胞培養在添加有2 0 %胎牛血清、1 m μ β -氫硫基乙醇（購自 ⑧ 20Dickinson Labware, Franklin Lakes, NJ, USA). First, at a room temperature (~20 °C), about 450 μM of the MC solution is poured into the center of the culture tray, and then the culture tray is slightly tilted, so that the MC solution can be evenly distributed and covers the entire bottom of the culture tray. To form a transparent film. Thereafter, the culture plate coated with the MC solution is allowed to stand at a temperature of 3 7 t (or 5 Torr) for 1 hour to harden the transparent film on the bottom surface of the culture plate to become an opaque/edge layer. Also known as "cellulose gel (or M c - gel)". The blastocyst or cell sphere can then be formed in such a subsequent experiment with such a culture plate coated with a Mc-gel. 1.2.3 Formation of embryos The HES cell pellets of Example 1 · 1 · 3 were transferred to a MC-gel coated disc prepared according to Example 1 2.2, and the cells were cultured. Added 20% fetal calf serum, 1 m μ β-thiol ethanol (purchased from 8 20

13032781303278

Gibco Invitrogene)、1% 非必要氨基酸（購自 Gibco Invitrogene)、1% 榖胺酸（購自 Gibcolnvitrogene)及 1%之騰島素-輸鐵蛋白-石西G型添加物（ITS G添加物，購自Gibco Invitrogene)的DMEM培養基中，並保持懸浮狀態，使形成胚球體，且每隔2至3天即更換一次培養基。細胞在懸浮培養11天後，可觀察到在塗覆有M C -凝膠之培育容器中，形成HES細胞之囊性胚球體的比例為 95%，至於以習知懸滴技術或低附著性培養盤培養之HES 細胞其出現嚢性胚球體的比例則分別為8 5 %及6 0 %。此量化數據總結於下表1中，至於形成在實施例1.2.2之塗覆有M C -凝膠之培育容器表面的胚球體的照片則顯示於第2 圖中。表1 比較本發明方法與習知方法所形成之囊性胚球體比例培育方法懸滴法低附著性培養盤塗覆有M C -凝膠之培養盤第11天時的囊性胚球體比例 (%) 85 60 95 13 實施例1·2·3之胚球體的表現性分析 1-3.1 實施例1 · 2.3之胚球體的公彳h -牿徵為可表現出 21 1303278 三胚層之每一層的某因成蛋白質此實施例證實依據本發明方法所形成之胚球體具有分化能力（亦即’具有多能性）。以實施例1 · 2 · 3所述方法形成之胚球體的多能性，可藉由偵測源自該三胚層之每一層的基因（第3圖）或蛋白質（第4圖）之表現來獲得證實。以反轉錄酶鏈反應（RT-PCR)來分析第11天之胚球體的總RNA 中源自内胚層（例如，α-胎蛋白（oc-FP)及GATA4之基因）、中胚層（例如，心肌動蛋白、烯醇酶及腎素之基因）及外胚層（例如角蛋白及神經絲重鏈蛋白（NFH)之基因）之基因，結果示於第3圖中。以培育在小鼠ECM或人類ECM上（分別為第3圖之第3、4行）之HES細胞所形成的胚球體，其胚球體中諸如a-FP、GATA4、心肌動蛋白、烯醇酶及神經絲重鏈蛋白（NFH)的基因表現量與培育在含有PMEF滋養層之HES細胞（第3圖之第2行，正控制組）所形成的胚球體的基因表現量相當，表示以本發明方法所形成的胚球體具備多能性，且在適當誘導下可分化成所有三胚層中任一層之細胞。其分化能力亦可再經下述之實施例1 . 3.2來證實’弟4圖疋以免疫^&(immunohistochemistry)法分析，顯示出第30天之胚球體中源自内胚層之α_ρρ、中胚層之心肌動蛋白及外胚層之巢蛋白（nestin)等基因的蛋白質表現情形。 ――2——將實施.1 ·2 · 3 驻球體分化成旰譜系的細胞胚球體可被分化成多種譜系的細胞。此實施例闡示以本發明方法开> 成的胚球體，特別是，實施例丨· 2 · 3之胚球體， 1303278 可於適當誘導下分化成肝譜系的細胞。簡言之，細胞經本發明方式懸浮培育1 〇天後，收集所形成的胚球體，並將其培育在塗覆有0.1 %板素的24-孔培養盤中，在第14天後，加入10 ng/ml的鹼性纖維母細胞生長因子（bFGF)至培養基中。在第1 7天時，收集細胞樣本並以RT-PCR分析是否有肝細胞特有的基因表現。Gibco Invitrogene), 1% non-essential amino acids (purchased from Gibco Invitrogene), 1% proline (purchased from Gibcolnvitrogene) and 1% of Tengdasu-transferrin-ishixi G-type additive (ITS G additive, The DMEM medium was purchased from Gibco Invitrogene and kept in suspension to form blastocysts, and the medium was changed every 2 to 3 days. After the cells were cultured for 11 days in suspension, it was observed that the proportion of cystic blastomers forming HES cells in the MC-gel-coated culture vessel was 95%, as in the conventional hanging drop technique or low adhesion culture. The proportion of sputum blastomers in the cultured HES cells was 85 % and 60%, respectively. The quantified data is summarized in Table 1 below, and a photograph of the blastocyst formed on the surface of the incubation container coated with the M C-gel of Example 1.2.2 is shown in Fig. 2. Table 1 Comparison of the ratio of cystic blastocysts formed by the method of the present invention and the conventional method. The ratio of cystic blastocysts on the 11th day of the suspension plate coated with the MC-gel of the low adhesion culture disc (%) 85 60 95 13 Expression analysis of blastocysts of Example 1·2·3 1-3.1 Example 1 · The 彳牿牿牿胚 2.3 2.3 2.3 2.3 2.3 胚 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 This embodiment demonstrates that the globules formed by the method of the present invention have the ability to differentiate (i.e., 'have pluripotency'). The pluripotency of the blastocyst formed by the method described in Example 1 2 can be detected by detecting the gene (Fig. 3) or protein (Fig. 4) derived from each of the three germ layers. Obtained confirmation. Reverse transcriptase chain reaction (RT-PCR) was used to analyze the total RNA derived from endoderm (for example, α-fetoprotein (oc-FP) and GATA4 genes) and mesoderm (for example, Genes for cardiac actin, enolase and renin) and ectoderm (for example, genes for keratin and neurofilament heavy chain protein (NFH)) are shown in Figure 3. A blastocyst formed by HES cells cultured on mouse ECM or human ECM (Fig. 3, lines 3 and 4, respectively), such as a-FP, GATA4, cardiac actin, enolase in blastocysts And the gene expression of the neurofilament heavy chain protein (NFH) is equivalent to the gene expression of the blastocyst formed by the HES cells (Fig. 3, line 2, positive control group) containing the PMEF trophoblast, indicating The blastocyst formed by the method of the invention is pluripotent and can be differentiated into cells of any of the three germ layers under appropriate induction. The differentiation ability can also be confirmed by the following Example 1.3.2 to confirm that 'Dia 4' is analyzed by immunological method and shows the α_ρρ derived from endoderm in the glomerulus on the 30th day. Protein expression of genes such as cardiac actin and ectoderm nestin in the germ layer. ——2—The cells that will perform the .1·2·3 spheroid differentiation into the 旰 lineage blastoderm can be differentiated into cells of multiple lineages. This example illustrates the blastocysts formed by the method of the present invention, in particular, the blastocysts of the Examples 22.3, 3303278 can be differentiated into cells of the liver lineage under appropriate induction. Briefly, cells were suspended for 1 day in the manner of the present invention, and the formed blastocysts were collected and cultured in a 24-well plate coated with 0.1% platin. After the 14th day, 10 was added. Ng/ml of basic fibroblast growth factor (bFGF) to the medium. At day 17, cell samples were collected and analyzed by RT-PCR for the presence of hepatocyte-specific gene expression.

RT-PCR 簡言之，以 Rneasy 迷你套組（Rneasy Mini Kit) (QIAGENE，日本東京市）來抽取細胞中全部的RNA。用於 PCR反應中的核酸引子對及其放大產物的序列和長度如下表所示：基因_引子對序列及放大產物之長度_ /3-肌動蛋白 5，_TGGCACCACACCTTCTACAATGAGC-3,及 3,-GCACAGCTTCTCCTTAATGTCACGC-5,; 387 bpRT-PCR In short, all the RNA in the cells was extracted with the Rneasy Mini Kit (QIAGENE, Tokyo, Japan). The sequence and length of the nucleic acid primer pair and its amplified product used in the PCR reaction are shown in the following table: Gene_Introduction sequence and length of amplified product _ /3-actin 5, _TGGCACCACACCTTCTACAATGAGC-3, and 3,-GCACAGCTTCTCCTTAATGTCACGC -5,; 387 bp

Oct-4 5’-GACMCAATGAGAACCTTCAGGAGA-3,及 3，-TTCTGGCGCCGGTTACAGAACCA-5，； 217 bpOct-4 5'-GACMCAATGAGAACCTTCAGGAGA-3, and 3,-TTCTGGCGCCGGTTACAGAACCA-5,; 217 bp

α-FP 5，-AGAACCTGTCACAAGCTGTG-3,及 3，-GACAGCAAGCTGAGGATGTC-5，； 675 bp ALB 5’-CCTTTGGCACAATGAAGTGGGTAACC-3,及 3’-CAGCAGTCAGCCATTTCACCATAGG-5，； 350 bp G6P 5，-CAGGACTGGTTCATCTTGGT-3,及 3，-CAGACATTCAGCTGCACAGC-5，； 421 bp 23 1303278 TAT 5，-CTAGAAGCTAAGGACGTCAT-3,及 3，-GAGGAAGCTCAGAGTGTTGT-5，; 642 bp CYP3A4 5，-CAAGACCCCTTTGTGGAAAA-3,及 3，-TGCAGTTTCTGCTGGACATC-5，； 398 bpα-FP 5,-AGAACCTGTCACAAGCTGTG-3, and 3,-GACAGCAAGCTGAGGATGTC-5,; 675 bp ALB 5'-CCTTTGGCACAATGAAGTGGGTAACC-3, and 3'-CAGCAGTCAGCCATTTCACCATAGG-5,; 350 bp G6P 5,-CAGGACTGGTTCATCTTGGT-3, and 3 , -CAGACATTCAGCTGCACAGC-5,; 421 bp 23 1303278 TAT 5,-CTAGAAGCTAAGGACGTCAT-3, and 3,-GAGGAAGCTCAGAGTGTTGT-5,; 642 bp CYP3A4 5,-CAAGACCCCTTTGTGGAAAA-3, and 3,-TGCAGTTTCTGCTGGACATC-5,; 398 bp

依據下歹1J反應條件，以QIAGENE —步驟之RT-PCR套組來執行RT-PCR反應：在50°C下進行反轉錄30分鐘；在 9 5 °C下啟始PCR反應及進行變性反應 15分鐘30秒；在 52-60°C下進行黏合反應30秒（詳言之，對α-胎蛋白（a-FP) 的反應條件是56°C、對白蛋白的反應條件是68°C、對葡萄糖-6-磷酸酶（G6P)及酪氨酸氨基轉移酶（TAT)的反應條件是 62°C、對Oct-4及/3 肌動蛋白的反應條件是60°C );在72 t下進行延長反應1分鐘，總計進行25個循環；在72 °C 下進行最後的延長反應7分鐘；其中係以α-FP係作為内胚層分化以及是否出現初期胚胎肝臟細胞的標記；白蛋白（ALB) 與葡萄糖-6-磷酸酶（G6P)是作為後期或新生周圍階段之肝臟發育的標記；且酪氨酸氨基轉移酶（TAT)是作為新生周圍或新生後階段肝臟分化的酵素標記。Oct-4及/3 -肌動蛋白則是作為一未分化之ES標記及一内源性控制標記。 RT-PCR的結果確認内胚層專一性基因，即，a-FP，其表現程度會隨著肝臟的發育而逐漸減少。相反的，相較於未分化的HES細胞來說，其他包括白蛋白、G6P及TAT在内的三種肝細胞標記基因的表現，則隨著生長因子誘發肝細胞 24 1303278 分化而逐步增加。 f施例2 形成肝細胞球體將人類肝臟腫瘤細胞株，即，C3A細胞，培育在添加有10% FBS的DMEM培養基中。將細胞維持在37〇c、5% C 〇2及飽和溼度的環境下培育直到其接近9 0%全滿狀態為止，之後，以胰蛋白酶將細胞收取下來，計算數目後，再重新培育在習知的細胞培養盤、低附著性培養盤（Falc〇n) 或實施例1.2.2之塗覆有MC-凝膠的培養盤上。第6A圖顯示細胞培養在習知的細胞培養盤上附著生長 4天後的型態，第6B圖顯示細胞培養在習知的低附著性培養盤上4 天後，大部分的細胞係附著在培養盤底部生長。相反的，大部分培養在實施例1.2.2之塗覆有MC-凝膠之培養盤上的細胞，則會形成肝細胞球體（第6C圖），且其均會表現出諸如〇^?、八1^、06卩、及0丫？3八4等專一性的肝細胞基因（第 7圖，第3行）。 >則里肝臟；酵素、活i生依據製造商提供的指示說明，以五氧試鹵靈（pentoxyresorufin，PR0D)試驗（購自 M〇lecuUrRT-PCR reaction was performed according to the QIAGENE-step RT-PCR kit according to the conditions of the lower 1J reaction: reverse transcription was carried out at 50 ° C for 30 minutes; PCR reaction was initiated at 15 ° C and denaturation reaction was carried out 15 30 seconds in minutes; bonding reaction at 52-60 ° C for 30 seconds (in detail, the reaction conditions for α-fetoprotein (a-FP) are 56 ° C, the reaction conditions for albumin are 68 ° C, The reaction conditions of glucose-6-phosphatase (G6P) and tyrosine aminotransferase (TAT) are 62 ° C, the reaction conditions for Oct-4 and /3 actin are 60 ° C); at 72 t The extension reaction was carried out for 1 minute for a total of 25 cycles; the final extension reaction was carried out at 72 ° C for 7 minutes; among them, the α-FP line was used as a marker for endoderm differentiation and whether early embryonic liver cells appeared; albumin (ALB) Glucose-6-phosphatase (G6P) is a marker for liver development in the late or neonatal peripheral phase; and tyrosine aminotransferase (TAT) is an enzyme marker for liver differentiation in the neonatal or neonatal stages. Oct-4 and /3 -actin are used as an undifferentiated ES marker and an endogenous control marker. The results of RT-PCR confirmed that the endoderm-specific gene, i.e., a-FP, showed a degree of expression that gradually decreased with the development of the liver. Conversely, the performance of three other hepatocyte marker genes, including albumin, G6P, and TAT, increased progressively with growth factor-induced hepatocyte 24 1303278 differentiation compared to undifferentiated HES cells. f Example 2 Formation of hepatocyte spheroids Human liver tumor cell lines, i.e., C3A cells, were cultured in DMEM medium supplemented with 10% FBS. The cells were maintained under the conditions of 37 ° C, 5% C 〇 2 and saturated humidity until they were close to 90% full state. Thereafter, the cells were collected by trypsin, and the number was counted, and then re-cultivated. Known cell culture plates, low adhesion culture plates (Falc〇n) or MC-gel coated plates of Example 1.2.2. Fig. 6A shows the morphology of cell culture after attachment growth for 4 days on a conventional cell culture plate, and Fig. 6B shows that after 4 days of cell culture on a conventional low adhesion plate, most of the cell lines are attached. The bottom of the culture plate grows. Conversely, most of the cells cultured on the MC-gel coated plates of Example 1.2.2 will form hepatocyte spheres (Fig. 6C), and both will exhibit such as 〇^?, Eight 1^, 06卩, and 0丫? 3 8 4 and other specific hepatocyte genes (Fig. 7, line 3). >The liver; enzymes, live isheng according to the instructions provided by the manufacturer, with pentoxyresorufin (PR0D) test (purchased from M〇lecuUr

Probe)來分析C3A細胞中專一性肝酵素，即cYp3A4，之活性。簡言之，在37 °C下，將生長在不同培養盤之C3 a 細胞以CYP的螢光PROD受質（其在經過CYp催化後會發出榮光）加以染色30分鐘。因此，可測量螢光強度的變化來表示細胞是否有表現肝臟酵素（即，CYP_45〇)，榮光強度 25 1303278 係以流式細胞分析儀（flow cytometer)進行測量，激發光波長設定在5 7 1 nm，發射光波長則設定在5 8 5 nm，結果示於第8圖中。相較於附著生長的細胞（第8 A圖）來說，懸浮生長的C 3 A細胞（第8 B圖）可表現出較高的肝臟酵素活性。產業利用性Probe) to analyze the activity of specific liver enzyme, cYp3A4, in C3A cells. Briefly, C3 a cells grown in different culture dishes were stained with CYP fluorescent PROD receptor (which glory after CYp catalysis) at 37 °C for 30 minutes. Therefore, the change in fluorescence intensity can be measured to indicate whether the cell has a liver enzyme (ie, CYP_45〇), and the glory intensity 25 1303278 is measured by a flow cytometer with the excitation wavelength set at 5 7 1 The wavelength of the emitted light is set at 585 nm, and the results are shown in Fig. 8. Suspension-grown C3 A cells (Fig. 8B) can exhibit higher liver enzyme activity than adherently grown cells (Fig. 8A). Industrial utilization

本發明優點在於可提供一種形成細胞球體或胚球體之方法，其係簡單、容易使用且符合經濟效益，同時形成細胞球體或囊胚球體的比例超過9 0 %，成效遠優於習知的懸滴培養技術或低附著性培養技術。本發明另一優點是提供一種可形成適於分化成其他種類細胞（例如，肝細胞及軟骨細胞）之胚球體的方法。本發明再一優點係以經濟、有效的方式，提供可用於諸如移植、藥物篩檢等臨床應用之具有功能性之細胞（包括細胞株、初代培養組織細胞，幹細胞之衍生細胞）的細胞球體。The invention has the advantages that a method for forming a cell sphere or a globule body can be provided, which is simple, easy to use and economical, and at the same time, the proportion of forming a cell sphere or a blastocyst sphere is more than 90%, and the effect is far superior to the conventional suspension. Drop culture technique or low adhesion culture technique. Another advantage of the present invention is to provide a method of forming blastocysts suitable for differentiation into other types of cells (e.g., hepatocytes and chondrocytes). Still another advantage of the present invention is to provide a cell sphere of functional cells (including cell strains, primary cultured tissue cells, stem cell-derived cells) useful for clinical applications such as transplantation, drug screening, and the like in an economical and effective manner.

本說明書内的較佳實施例僅作說明本發明之較佳實施例之用，而非用以限制本發明範圍。習知技藝者需明白，本發明尚涵蓋多種其他較佳實施例，且說明書與實施例僅作示範之用，其實際精神與範圍當由後附申請專利範圍所界定。 26 1303278 【圖式簡單說明】第1圖顯示依據本發明用來形成細胞球意圖；第2圖顯示依據本發明一較佳實施例而 MC-凝膠之培育容器表面之HES細胞所形成片；第3圖顯示衍生自生長在有PMEF滋養層小鼠ECM(第3列）或人類ECM(第4列）的無之HES細胞於本發明方式所形成之胚球體，中三個胚層之基因表現情形；基因表現係利行偵測’其中以不含RNA的反應產物作為 (第5列）；第4圖顯示衍生自生長在有PmeF滋養, 所形成之胚球體（第3 0天），此胚球體其三胚的免疫染色分析結果；其中以正常抗體 antibody isotype)作為一種負控制組; 第5圖顯示以RT-PCR分析由依據本發明所培育之E S細胞所衍生出來的肝譜系細胞果；第6A圖為生長在細胞培養盤上貼覆生長細胞（C3A細胞）所形成之細胞型態照片；弟6A圖為生長在低附著性培養盤上4天體之方法的示培育在塗覆有的胚球體的照 (第2列）或在滋養層系統上來自該胚球體用 RT-PCR^ 一種負控制組罾之HES細胞層蛋白質表現同型（Normal 一較佳實施例的基因表現結 4天之肝腫瘤之肝腫瘤細胞 27 1303278 (C3A細胞）所形成办成之細胞球體的照片；第6C圖為生長在在本發明塗覆有MC-凝膠之培養盤上之肝腫瘤細胞（C3A細胞）匕）所形成之細胞球體的照片；第7圖顯示培養於έ 、’、、胞培養盤（1 a n e 1)、低附著性培養盤（lane 2)或依據本發較佳實施例（1 a n e 3 )之C 3 A細胞中屬於肝細胞專一性土因的表現情形，包括a-FP、白蛋白、G6P 及 CYP 3A4 ; 第8A圖顯示以五氧試兩式自莖（PROD)試驗分析培養在一般細胞培養盤表面上之細胞的 J旰酵素（即，細胞色素p-450)活性；第8B圖顯示以五氧試鹵図靈（PROD)試驗分析培養在明塗覆有M C -凝膠之細皰拉 ' 匕&養盤表面上之細胞的肝 (即，細胞色素P-450)活性。听I 【元件代表符號簡單說明】 28The preferred embodiments of the present invention are intended to be illustrative only and not to limit the scope of the invention. It is to be understood by those skilled in the art that the present invention is intended to be limited by the scope of the appended claims. 26 1303278 [Simplified illustration of the drawings] Figure 1 shows the intention of forming a cell ball according to the present invention; Figure 2 shows a sheet formed by HES cells on the surface of an MC-gel culture container according to a preferred embodiment of the present invention; Figure 3 shows the gene expression of three germ layers derived from the blastocysts formed in the blastoderm formed by the present invention in the absence of HES cells grown in PMEF trophoblastic mouse ECM (column 3) or human ECM (column 4). Situation; gene expression is detected by 'in which RNA-free reaction product is used (column 5); Figure 4 shows that blastocysts (day 30) derived from growth in PmeF nourishment, this embryo The result of immunostaining analysis of the three embryos of the sphere; wherein the normal antibody antibody isotype) is used as a negative control group; FIG. 5 shows the analysis of the liver lineage cell fruit derived from the ES cells cultured according to the present invention by RT-PCR; Figure 6A is a photograph of a cell type formed by adhering growth cells (C3A cells) grown on a cell culture plate; Figure 6A shows a method of growing on a low-adhesion plate for 4 days. Blastosphere According to the (column 2) or on the trophoblast system, the HES cell layer protein from the glomerulus was RT-PCR^ a negative control group, and the gene expression was normal (the gene of the preferred embodiment showed a liver tumor of 4 days). Photograph of cell spheres formed by liver tumor cells 27 1303278 (C3A cells); Figure 6C shows liver tumor cells (C3A cells) grown on the MC-gel coated plates of the present invention Photograph of the formed cell sphere; Figure 7 shows culture in έ, ',, cell culture plate (1 ane 1), low adhesion culture plate (lane 2) or according to the preferred embodiment of the present invention (1 ane 3) The expression of hepatocyte-specific soil factors in C 3 A cells, including a-FP, albumin, G6P and CYP 3A4; Figure 8A shows the analysis of cultured cells in general cells by the five-oxygen test from the stem (PROD) test. The activity of the J旰 enzyme (ie, cytochrome p-450) of the cells on the surface of the plate was incubated; Figure 8B shows the analysis of the blister coated with MC-gel by the pentoxide test (PROD) test. Pulling ''' and the liver of the cells on the surface of the plate (ie, cells Prime P-450) activity. Listen I [Simple description of component symbol] 28

Claims

1303278 Π~Γ^Ί Letter picking, patent application scope: 1 · A method for forming human multicellular spheres, comprising: coating a cell culture vessel with cellulose and/or cellulose derivatives; and forming human multicellular cells Most of the human cells of the sphere are cultured in the cell culture vessel, and the human cells are maintained in suspension growth; wherein more than 90% of the human cells can form human multicellular spheres. 2. The method of claim 1, wherein the human cells comprise at least one cell selected from the group consisting of a human pluripotent stem cell, a stem cell derived cell, or Specific tissue cells. 3. The method of claim 2, wherein the specific tissue cell is a hepatocyte. 4. The method of claim 1, wherein the cellulose is selected from the group consisting of methylcellulose (MC), carboxymethylcellulose (CMC), and methylcellulose (hydroxymethylcellulose, HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC) and hydroxypropylmethylcellulose (HPMC). 29 1303278 5. A method of forming a human blastocyst comprising: coating a cell culture vessel with a cellulose and/or a cellulose derivative; and incubating undifferentiated human embryonic stem cells in the cell culture vessel and maintaining the Some undifferentiated human embryonic stem cells are in suspension growth; wherein more than 90% of these undifferentiated human cells can form human blastoderm. 6. The method of claim 5, wherein the cellulose is selected from the group consisting of methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and In the group consisting of hydroxypropionyl cellulose. 7. The method of claim 6, wherein the blastocyst is differentiated into mature tissue cells upon induction. 8. The method of claim 7, wherein the mature tissue cells comprise cells having hepatocyte function. 9. A method of forming a human hepatocyte spheroid comprising: coating a cell culture vessel with cellulose and/or a cellulose derivative; and cultivating human liver cells in the cell culture vessel and maintaining the 30 1303278 liver The cells are in suspension growth; wherein more than 90% of the hepatocytes can form human hepatocyte spheres. The method of claim 9, wherein the cellulose is selected from the group consisting of mercapto cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose. And a group consisting of hydroxypropylmethylcellulose. 31