200827017 九、發明說明： 【發明所屬之技術領域】 本發明係相關於一種製備細胞培養用的人類血浆及分 離臍帶原始細胞（umbilical cord progenitor cell)的方 法0 【先前技術】 既有培養細胞的方法，最常應用胎牛血清作為建立初 代細胞培養之用。隨著細胞治療法臨床應用的發展，將欲 _作為移植的細胞安全地使用於病人身上，以胎牛血清所培 養的細胞其安全性是亟待考慮的。 幹細胞為一群具高度潛力的臨床應用細胞群，因此許 多研究團隊投入臨床試驗之研究。造血幹細胞 (hemat〇P〇ietic stem ceUs; HS〇係常用於移植，而間 葉原始細胞（mesenchymal pr〇genit〇r cell)(Mpc)則在目 刖處於床試驗階段。大部分臨床試驗使用的幹細胞皆在 3有月°牛血清的培養液中培養。然而使用胎牛血清卻可能 f有專播病毋或病原性蛋白顆粒的風險，因而造 成使用上的限制。 “、：牛低人類、、田胞的培養受到前述病毒或病原性蛋白顆 粒感木的風險’如能以人類血清/血敷培養人類細胞，將 可：巾田提间人類細胞的利用、應用方式及安全性。然而， 目月厂/方、如何製備人類血清/血漿以取代現有胎牛血清培 養方法仍不清禁。田 α此’目前急待提出一種製備細胞培養 用的人類血清/血將 α /血濃’以期更安全的使用細胞治療法及發 5 200827017 展再生醫學。 另-方面，目前應用於臨床的幹細胞多為成體幹細 胞，如骨髓幹細胞，而胎盤與臍帶係為產婦生產後的醫療 廢棄物’其内含有許多間葉原始細胞。因&，如能有效大 量獲取腾帶中的間葉原始細胞’亦可幫助再生醫學的發 展。 【發明内容】 有鑑於既有的細胞培養用之血清種類的不足，及獲製 細胞培養用的人類血清/血漿的製造方法之闕如，本發明 係欲提出一種可製造細胞培養用之人類血毅為其主要目 的。本發明主要藉由醫療廢棄物：臍帶及胎盤中所保留的 臍帶血，或一般成體血液，加以分離處理，以獲製細胞培 養用之人類血漿。 另一方面，該臍帶及胎盤中含有多數未分化的幹細 胞，如能有效加以分離及保存，將對再生醫學的進展有極 大的幫助。因此，本發明的另一目的係為提供一種自臍帶 及胎盤中分離原始細胞的方法。 為達成本發明的主要目的，本發明係相關於一種製備 細胞培養用的人類血漿的方法，其係包含： 提供人類血液以獲取血漿， 將所獲取的血漿放入透析管以約丨〇mM氣化鈣進行透 析二次，及 獲取細胞培養用的人類血漿。 較佳的是，經透析後的血漿進一步經3500g離心後並 6 200827017 以0 · 2微孔濾紙過濾，以獲取細胞培養用的人類血漿。 本發明另一方面相關於一種自臍帶分離原始細胞的方 法，其係包含： 提供臍帶； - 自臍帶中分離臍帶結缔組織並將臍帶結締組織細碎 化； 加入分解酵素以形成混合物進行分解； 過濾混合物以獲取固體物及液體物；及 _ 分別培養固體物及液體物以獲取臍帶原始細胞。 較佳的是，收集的臍帶先經過酒精清洗後，移除臍靜 脈與臍動脈以進行臍帶結締組織細碎化。 較佳的是，前述分解酵素為胰蛋白酶及膠原蛋白酶。 較佳的是，胰蛋白酶的最終濃度約為〇 〇5%(v/v)，膠 原蛋白酶的最終濃度約為3〇〇U/ml。 較佳的是，於分別培養固體物及液體物時，係分別於 各培養皿内加入α-ΜΕΜ培養液、約2〇% ("Ο臍帶血血漿 及約1% (v/v)抗生素進行培養。 本發明另一方面係相關於一種經前述方法所獲取的細 胞培養用的人類血漿。 藉由本發明之方法，可將乂 供人體細胞… ，可將人類血清/血漿應用於臨床200827017 IX. Description of the Invention: [Technical Field] The present invention relates to a method for preparing human plasma for cell culture and for isolating umbilical cord progenitor cells. [Prior Art] Method for culturing cells The most commonly used fetal bovine serum is used to establish primary cell culture. With the development of clinical applications of cell therapy, the cells to be used as transplanted cells are safely used on patients, and the safety of cells cultured with fetal bovine serum is urgently considered. Stem cells are a group of highly potential clinical application cell populations, so many research teams have invested in clinical trials. Hematopoietic stem cells (hemat〇P〇ietic stem ceUs; HS 〇 is commonly used for transplantation, while mesenchymal pr〇genit〇r cell (Mpc) is in the bed test stage. Most clinical trials use Stem cells are cultured in a medium containing 3 months of bovine serum. However, the use of fetal bovine serum may have a risk of spreading diseased or pathogenic protein particles, thus causing restrictions on use. The culture of the cells is subject to the risk of the aforementioned virus or pathogenic protein particles. If human cells can be cultured in human serum/blood, the use, application and safety of human cells in the field can be improved. However,目月厂/方, how to prepare human serum/plasma to replace the existing fetal bovine serum culture method is still not clear. Tian α this 'currently urgent to propose a preparation of human serum / blood for cell culture will be α / blood thick' in the hope Safer use of cell therapy and regenerative medicine. In addition, the stem cells currently used in clinical practice are mostly adult stem cells, such as bone marrow stem cells, and placenta and umbilical cord. The medical waste after maternal production contains many mesenchymal primordial cells. It can also help the development of regenerative medicine by effectively obtaining large quantities of mesenchymal primordial cells in the sputum. In the case of the deficiency of the serum type for the cell culture and the method for producing the human serum/plasma for cell culture, the present invention is intended to provide a human blood test for cell culture. The invention mainly uses medical waste: umbilical cord blood retained in the umbilical cord and the placenta, or general adult blood, to be separated and processed to obtain human plasma for cell culture. On the other hand, the umbilical cord and the placenta contain most of the Differentiated stem cells, if effectively isolated and preserved, will greatly contribute to the advancement of regenerative medicine. Therefore, another object of the present invention is to provide a method for separating primitive cells from the umbilical cord and the placenta. The main object of the present invention relates to a method for preparing human plasma for cell culture, which comprises: providing Blood is taken to obtain plasma, and the obtained plasma is placed in a dialysis tube to be dialyzed twice with about mM mM calcium carbonate, and human plasma for cell culture is obtained. Preferably, the dialyzed plasma is further centrifuged at 3500 g. Subsequently, 6 200827017 is filtered with 0. 2 microporous filter paper to obtain human plasma for cell culture. Another aspect of the invention relates to a method for isolating primitive cells from a umbilical cord, comprising: providing an umbilical cord; - separating from the umbilical cord The umbilical cord connective tissue and the umbilical cord connective tissue are finely divided; the decomposing enzyme is added to form a mixture for decomposition; the mixture is filtered to obtain solid matter and liquid matter; and _ the solid matter and the liquid matter are respectively cultured to obtain umbilical cord original cells. The collected umbilical cord is first washed with alcohol, and the umbilical vein and the umbilical artery are removed to perform sarcomal connective tissue shredding. Preferably, the aforementioned enzymes are trypsin and collagenase. Preferably, the final concentration of trypsin is about 〇 5% (v/v) and the final concentration of collagen is about 3 〇〇 U/ml. Preferably, when the solid matter and the liquid material are separately cultured, the α-ΜΕΜ culture solution is added to each culture dish, and about 2% ("Οum blood plasma and about 1% (v/v) antibiotic. The present invention relates to a human plasma for cell culture obtained by the aforementioned method. By the method of the present invention, sputum can be supplied to human cells, and human serum/plasma can be applied to clinical practice.
動物的污染 胞的方法， 的利用。 200827017 【實施方式】 本發明係相關於一種製備細胞培養用的人類血漿及自 臍帶中分離原始細胞的方法。在本發明中係利用獲取人類 血液後以透析方式處理所獲取的人類血漿，以製備適合細 胞培養用的人類血漿，作為體外幹細胞初代培養之培養液 添加劑（medium supplement)。由於一般血清的製備是利 用未加抗凝劑的血液經過血液凝固做用所製備而成的；本 發明所製備的血漿，由於一開始所取得的臍帶血已添加抗 凝劑，因此透過本發明所揭露之較佳透析方式，例如以1〇mM each，進行透析以去除纖微素原（fibrinogen)m製備而 成，以供培養人類細胞之用。 利用本發明提供之方法所製備的細胞培養用人類血 漿，培養臨床試驗用的細胞，可避免因動物來源的血清因 可月b受到污^源感染所導致的風險，而能有效確保臨床使 用或臨床試驗的安全。 另外，本發明另相關於自臍帶中分離原始細胞的方 法。由於原始細胞在經過適當的外在刺激後，可分化成不 同的母細胞，因此，藉由自臍帶分離原始細胞，而能提供 更多樣化的幹細胞臨床應用。 本叙明長：供製備細胞培養用的人類血漿的主要步驟包 含，先提供人類血液以獲取血漿，將所獲取的血漿放入透 析管以約10mM氯化鈣進行透析一次後再以生理食鹽水透 析一次，最後，獲取細胞培養用的人類血槳。 本發明提供自臍帶分離原始細胞的方法，主要包含先 8 200827017 提供並收集臃册％ 茱脚▼後，將所收集的膪帶加 化的臍帶内加入分 4鲆化，於細碎 刀胖言以形成混合物 過濾混合物以嬅阑麯‘ 進仃刀解，之後經 物以獲取固體物及液體物 及液體物以獲取膪;A 更刀別培養固體物 紋取朌π原始細胞。在本發明較佳具啤者·#如 中，該分解酵素可為胰蛋白酶或膠原蛋白酶。、虹“ ’ 列名詞對發明所屬技術領域中具有通常知識者而 ^可以充分理解；下収義係為避免在本發明的具體實 施例中任何意義不明確的解釋。 在本發明中所使用的專有名詞『人類也液』在此包含 人體血液或臍帶血；較佳係為臍帶血。 在本發明中所使用的專有名詞『生長培養液』在此意 指在α -MEM培養液中含有约20%臍帶血血漿及1%抗生素。 本發明其他的特徵及優點將可明顯見於下列較佳具體 事實及申請專利範圍。 實施例 實施例一：細胞培養用的臍帶血血漿之製備 將已添加抗凝劑的臍帶血經血球分離後所得之新鮮臍 帶血血漿以4°C保存，血球分離技術係為所屬領域中具有 通常知識者，可依申請前之先前技術進行。取約2〇毫升 的臍帶血血漿放入透析管（3500 MWC0)，以1公升l〇mM CaCh 於室溫下進行透析，透析平衡時間在4°c下隔夜透析。而 後再以500毫升生理食鹽水於室溫下進行透析’透析平衡 時間約4小時（或於4°c下隔夜透析）° 可在透析管内觀察到血液凝塊產生。而後將透析管内 9 200827017 的内容物移至50毫升的離心管，以35〇〇G離心15分鐘， 以去除血液凝塊，所得上清液即為可供細胞培養用的臍帶 血血漿。而後以〇. 2微孔濾紙過濾上清液，收集並以4〇c 儲存經過濾後的臍帶血血漿。 * 實施例二：臍帶原始細胞（umbilical cord progenitor cel)之分離 m 將含有切碎後臍帶結締組織的各管培養於37〇c的二氧 化石反培養箱一小時，之後以無菌篩網過濾前述混合物以分 離固體物及液體物，並將過濾後的液體收集至50毫升的 試管中。經過濾後的固體部分以10毫升（ml)含2〇%濟帶 血血漿培養液培養在培養皿上，並額外加入約1毫升臍帶 血血漿（cord blood plasma)，此部份稱為組織分離法培 將所收集的臍帶以75%酒精清洗消毒，而後以pBS清 洗。將臍帶剪成4至6片段，且使各片段放置在培養皿上。 以鋒利的剪刀插入臍帶並剪開靜脈，並以鑷子將臍帶展 開。以鑷子將二條臍動脈分離並小心剝離，以避免任何血 液濺落在臍帶結締組織上，同時將臍靜脈移除。將臍帶绪 締組織放置在少量α-ΜΕΜ上以保持濕潤。而後以外科手術 韵刀將臍帶結締組織剪成細碎狀。將切碎後的臍帶結缔組 織分至經標記的離心管内，使每管約有J 〇毫升1 )的量， 並加入4毫升（ml )約〇· 25%的胰蛋白爾及1毫升（ml )約 300U/ml的膠原蛋白酶溶液，使胰蛋白酶的最終濃度約為 〇·〇5%(ν/ν)，膠原蛋白酶的最終濃度約為3〇〇u/ml，並使 每管的體積成為20毫升的混合物。 200827017 養（diffusion culture) 〇 過;慮後的液體經4 0 0 G離心1 〇分鐘後，去除至剩5毫 升的上清液後，重新使細胞呈懸浮狀態的懸浮液。再加入 10 *升含20%臍帶血血漿培養液混合，將含有細胞的15 *升生長培養液種於1 〇公分培養皿中，並將各培養皿培 養於3 7 C的二氧化碳培養箱内。待隔天，於顯微鏡下確認 培養孤是否遭受污染，如無污染，則丟棄舊培養液並以5 耄升的1XPBS清洗兩次。以巴斯德微量滴管（pasteur pipettes)進行前述操作。復加6毫升的含2〇%臍帶血血漿 培養液於培養皿中，並使培養孤重新放置在二氧化碳培養 箱中培養。每週更換培養液兩次，每次更換時先以顯微鏡 觀察’再以5毫升PBS清洗一次後，加入6毫升的新鮮培 養液。The use of animal methods of pollution. [Embodiment] The present invention relates to a method for preparing human plasma for cell culture and separating original cells from the umbilical cord. In the present invention, the obtained human plasma is treated by dialysis using human blood to prepare human plasma suitable for cell culture, and is used as a medium supplement for primary culture of in vitro stem cells. Since the preparation of the general serum is prepared by blood coagulation using blood without an anticoagulant; the plasma prepared by the present invention, since the cord blood obtained at the beginning has been added with an anticoagulant, is thus passed through the present invention. The preferred dialysis methods disclosed are, for example, dialysis at 1 mM each to remove fibrinogen m for culture of human cells. The cell culture for human cells prepared by the method provided by the present invention can be used for culturing cells for clinical trials, thereby avoiding the risk of the animal-derived serum being contaminated by the contaminated source, thereby effectively ensuring clinical use or The safety of clinical trials. Additionally, the invention is further related to a method of separating protocells from an umbilical cord. Since the original cells can differentiate into different mother cells after appropriate external stimulation, it is possible to provide a more diverse clinical application of stem cells by isolating the original cells from the umbilical cord. The main steps of preparing human plasma for cell culture include first providing human blood to obtain plasma, and placing the obtained plasma into a dialysis tube for dialysis with about 10 mM calcium chloride, followed by physiological saline. Dialysis is performed once, and finally, a human blood paddle for cell culture is obtained. The invention provides a method for separating original cells from an umbilical cord, which mainly comprises the first 8 200827017, and collects and collects the 茱 茱 ▼ ▼ ▼ ▼ ▼ , , , , , , , , , , , 脐 脐 脐 脐 脐 脐 脐 脐 脐 脐The mixture is filtered to form a mixture to distort the solution, and then the material is obtained to obtain solids and liquids and liquids to obtain hydrazine; A further cultures the solid matter to extract 朌 π original cells. In the preferred embodiment of the present invention, the degrading enzyme may be trypsin or collagenase. And the "column nouns" can be fully understood by those of ordinary skill in the art to which the invention pertains; the lower connotation is to avoid any ambiguous interpretation in the specific embodiments of the present invention. The term "human fluid" includes human blood or cord blood; preferably cord blood. The term "growth culture fluid" as used in the present invention means herein in α-MEM culture medium. Containing about 20% cord blood plasma and 1% antibiotic. Other features and advantages of the present invention will be apparent from the following preferred specific facts and the scope of the patent application. EXAMPLES Example 1: Preparation of cord blood plasma for cell culture The cord blood of the umbilical cord blood to which the anticoagulant is added is stored at 4 ° C, and the blood cell separation technique is generally known in the art, and can be carried out according to the prior art before the application. About 2 ml is taken. The cord blood plasma was placed in a dialysis tube (3500 MWC0), dialyzed at room temperature with 1 liter of l mM CaCh, and the dialysis equilibrium time was dialysis overnight at 4 ° C. Then 500 mM Physiological saline was dialyzed at room temperature for 'dialysis equilibration time of about 4 hours (or overnight dialysis at 4 ° C) ° Blood clot production was observed in the dialysis tube. The contents of 9 200827017 in the dialysis tube were then moved to 50 The milliliter centrifuge tube was centrifuged at 35 〇〇G for 15 minutes to remove blood clots, and the resulting supernatant was used for umbilical cord blood plasma for cell culture. The supernatant was then filtered with 微. 2 microporous filter paper and collected. The filtered cord blood plasma was stored at 4 〇c. * Example 2: Separation of umbilical cord progenitor cel m The tubes containing the chopped umbilical cord connective tissue were cultured at 37 ° C for oxidation. The stone was incubator for one hour, after which the mixture was filtered through a sterile mesh to separate solids and liquids, and the filtered liquid was collected into a 50 ml test tube. The filtered solid portion was contained in 10 ml (ml). 2〇% of the blood plasma culture medium was cultured on the culture dish, and an additional 1 ml of cord blood plasma was added. This part was called tissue separation method and the collected umbilical cord was cleared with 75% alcohol. Disinfect and then wash with pBS. Cut the umbilical cord into 4 to 6 fragments, and place each fragment on the culture dish. Insert the umbilical cord with sharp scissors and cut the vein, and spread the umbilical cord with forceps. Two umbilical arteries with forceps Separate and carefully peel off to avoid any blood splashing on the umbilical cord connective tissue while removing the umbilical vein. Place the umbilical cord tissue on a small amount of α-ΜΕΜ to keep it moist. Then use the surgical rhythm to cut the umbilical cord connective tissue. It is divided into finely divided. The chopped umbilical cord connective tissue is divided into labeled centrifuge tubes, so that each tube has about 1 ml of J 〇 ml, and 4 ml (ml) of about 25% of the trypsin is added. And 1 ml (ml) of about 300 U / ml of collagenase solution, the final concentration of trypsin is about 〇 · 〇 5% (ν / ν), the final concentration of collagenase is about 3 〇〇 u / ml, and The volume of each tube became a 20 ml mixture. 200827017 Diffusion culture ; After the centrifuged liquid was centrifuged at 400 G for 1 〇 minutes, the supernatant was removed to 5 ml, and the suspension was resuspended. Further, 10 * liter of a 20% cord blood plasma culture solution was mixed, and 15 * liter of the growth medium containing the cells was seeded in a 1 〇 centimeter culture dish, and each culture dish was cultured in a 3 7 C carbon dioxide incubator. On the next day, it was confirmed under the microscope whether the cultured orphan was contaminated. If there was no pollution, the old culture solution was discarded and washed twice with 5 liters of 1X PBS. The foregoing operation was carried out with Pasteur pipettes. 6 ml of a 2% umbilical cord blood plasma culture medium was added to the culture dish, and the culture was re-placed in a carbon dioxide incubator for cultivation. The culture medium was changed twice a week, and each time it was replaced, it was observed under a microscope. After washing once again with 5 ml of PBS, 6 ml of fresh culture solution was added.
另外，前述以組織分離法培養的固體部分經約3 — 4天 後，將固體部分及培養液移除，並以前述方式清洗及更新 培養液。臍帶原始細胞可自濾液及固體部分獲得。 經前述方法所獲取的臍帶原始細胞胞如第一圖所示， 當經培養後期外觀形成如纖維母細胞狀。同時，請分別來 閱弟一及二圖所示，進一步以流式細胞儀分析所獲得之細 胞表面抗原，發現所獲得的細胞表面帶有CD29、CD44、 CD73、CD90、CD105、CD166 及 HLA-ABC 抗原標記，而未帶 有 CD14、CD34、CD45 及 HLA-DR 表面抗原。 實施例三：臍帶原始細胞的繼代培養 約10-15天後，當細胞群落（c〇i〇nies)長成，細胞已 200827017 了進竹繼代培養。首先，預備一 1 5毫升離心管及ο 2 5 %胰 蛋白酶-EDTA(tΓypsin_EDTA)。將培養皿自培養箱移出， 清除培養液後，以5毫升的PBS清洗一次。每個培養皿加 入3毫升的胰蛋白酶_5；])7^後，放入37X培養箱5分鐘。 ^ 將培養皿所分離下的細胞移至15毫升的離心管中，並以 .400G離心5分鐘以收集細胞。移除離心後的上清液，並以 1 - 2毫升的生長培養液重新懸浮細胞。取1 〇微升（# 1)的 細胞懸浮液與10微升1)的錐蟲藍混合，而後以血球計 #數器進行計數（稀釋因子2 )。 經鈾述分離的細胞以每個培養皿含有3 —5X 105個細胞 的濃度進行種植（seed)，並於每個培養皿中加入含2〇%臍 帶血血漿培養液至6毫升後放入3rc的二氧化碳培養箱培 養。這些細胞稱為初代細胞，即p 1世代。 每3-4天更新培養液。首先需先以顯微鏡觀察細胞， 如生長至90%的生長密度，則再進一步將細胞進行繼代培 養。自培養箱取出培養皿，去除培養液後以5毫升pBS清 !洗兩次。每個培養皿加入3毫升的胰蛋白酶—edta後，將 培養皿放入37°C培養箱培養5分鐘，之後將培養皿所分離 下的細胞移至15毫升的離心管中，並以4〇〇G離心5分鐘 以收集細胞。移除部分上清液並留下2毫升上清液以重新 懸洋細胞。取1 0微升（#丨）的細胞懸浮液與丨〇微升的錐 蟲藍混合’以血球計數器計算細胞數（稀釋因子2)。將前 述細胞懸浮液以每個培養皿含有3-5 χ 1〇5個細胞，平均 地種入各培養皿中，並於每個培養皿中加入含2〇%臍帶血 12 200827017 血製培養液至6毫升後使細胞培養於咖二氧化碳培養 箱。每3-4天更換培養液。每次更換前先以顯微鏡觀察細 胞’當細胞成長至佈滿9G%左右的培養皿面積時，細胞則 進行FACS分析並冷凍保存。 ' 根據本發明可作之不同修正及變化對於熟悉該項技術 者而5均顯然不會偏離本發明的範圍與精神。雖然本發明 已敘述特定的較佳具體事實，必須瞭解的是本發明不應被 不當地限制於該等特定具體事實上。事實上，在實施本發 明之已述模式方面，對於熟習該項技術者而言顯而易知之 不同修正亦被涵蓋於下列申請專利範圍之内。 【圖式簡單說明】 第一圖係為間葉原始細胞培養狀態的照片圖。 第二圖係為間葉原始細胞帶有CD44、CD73及CD105 抗原標記的流式細胞儀結果圖。 第三圖係為間葉原始細胞帶有CD29及HLA-ABC抗原 才示e而未帶有CD34抗原標記的流式細胞儀結果圖。 【主要元件符號說明】 益 13Further, after the solid portion cultured by the tissue separation method described above is removed for about 3-4 days, the solid portion and the culture solution are removed, and the culture solution is washed and renewed as described above. Umbilical cord primitive cells are obtained from the filtrate and solid fraction. The umbilical cord primitive cell obtained by the foregoing method is as shown in the first figure, and forms a fibroblast-like appearance when it is cultured. At the same time, please read the cell surface antigens obtained by flow cytometry, and find that the obtained cell surface carries CD29, CD44, CD73, CD90, CD105, CD166 and HLA- as shown in the first and second pictures. The ABC antigen is labeled without the CD14, CD34, CD45 and HLA-DR surface antigens. Example 3: Subculture of umbilical cord primitive cells After about 10-15 days, when the cell population (c〇i〇nies) grows, the cells have been subcultured in 200827017. First, prepare a 15 ml centrifuge tube and ο 2 5 trypsin-EDTA (tΓypsin_EDTA). The culture dish was removed from the incubator, and after the culture solution was removed, it was washed once with 5 ml of PBS. Each dish was added with 3 ml of trypsin_5;]) 7^, and placed in a 37X incubator for 5 minutes. ^ The cells separated from the culture dish were transferred to a 15 ml centrifuge tube and centrifuged at .400 G for 5 minutes to collect the cells. The supernatant after centrifugation was removed and the cells were resuspended in 1-2 ml of growth medium. A cell suspension of 1 〇 microliter (# 1) was mixed with 10 μl of 1) trypan blue, and then counted by a hemocytometer (dilution factor 2). The cells separated by uranium were seeded at a concentration of 3 - 5×105 cells per dish, and 2% umbilical cord blood plasma culture solution was added to each dish to 6 ml and then placed in 3rc. The carbon dioxide incubator is cultured. These cells are called primary cells, ie the p 1 generation. The culture solution was updated every 3-4 days. First, the cells should be observed under a microscope. If they grow to a growth density of 90%, the cells are further cultured. The culture dish was taken out from the incubator, and the culture solution was removed, and washed with 5 ml of pBS. After adding 3 ml of trypsin-edta to each culture dish, the culture dish was placed in a 37 ° C incubator for 5 minutes, after which the cells separated from the culture dish were transferred to a 15 ml centrifuge tube and 4 〇. The cells were centrifuged for 5 minutes to collect cells. Part of the supernatant was removed and 2 ml of the supernatant was left to resuspend the cells. A cell suspension of 10 μl (#丨) was mixed with 丨〇 microliter of trypan blue, and the number of cells (dilution factor 2) was calculated by a hemocytometer. The above cell suspension is contained in each culture dish containing 3-5 χ 1〇5 cells, and is evenly implanted into each culture dish, and 2%% cord blood is added to each culture dish 12 200827017 blood culture liquid After 6 ml, the cells were cultured in a coffee carbon dioxide incubator. The medium was changed every 3-4 days. The cells were observed under a microscope before each change. When the cells grew to a petri dish area of about 9 G%, the cells were subjected to FACS analysis and cryopreserved. The various modifications and variations of the present invention are apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred specific embodiments, it should be understood that the invention should not be In fact, the various modifications that are obvious to those skilled in the art are also covered by the following claims. [Simple description of the diagram] The first figure is a photographic picture of the culture state of mesenchymal cells. The second panel is a flow cytometry result plot of the mesenchymal blast cells with CD44, CD73 and CD105 antigen markers. The third panel is a flow cytometry result plot of mesenchymal cells with CD29 and HLA-ABC antigens showing e without CD34 antigen labeling. [Main component symbol description] Benefit 13