TWI715874B - Serum-free culture medium and stem cell isolation and expansion method using the same in vitro - Google Patents
Serum-free culture medium and stem cell isolation and expansion method using the same in vitro Download PDFInfo
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
Description
本發明係關於一種細胞分離和放大培養之技術，特別是一種體外幹細胞分離和放大培養方法以及此方法中使用的無血清培養液。 The present invention relates to a cell separation and scale-up culture technique, in particular a method for in vitro stem cell separation and scale-up culture and the serum-free culture medium used in the method.
幹細胞是一種原始未分化的細胞，具有分化成其他具功能性細胞與自我新生新幹細胞的能力。常見的幹細胞分為胚胎幹細胞、成體幹細胞與腫瘤幹細胞。其中，成體幹細胞的多分化能性對於再生醫學以及幹細胞療法有很大的應用潛力。成體幹細胞例如有脂肪間質幹細胞，其具有取得容易、易於放大培養、分化能力強、可調控免疫之優點，因而目前在醫學用途方面的幹細胞培養多使用脂肪間質幹細胞。 Stem cells are primitive undifferentiated cells that have the ability to differentiate into other functional cells and self-new stem cells. Common stem cells are divided into embryonic stem cells, adult stem cells and cancer stem cells. Among them, the pluripotency of adult stem cells has great application potential for regenerative medicine and stem cell therapy. Adult stem cells include, for example, adipose mesenchymal stem cells, which have the advantages of being easy to obtain, easy to scale up and culture, strong differentiation ability, and adjustable immunity. Therefore, adipose mesenchymal stem cells are mostly used in stem cell culture for medical purposes.
目前已知的體外放大幹細胞之方法，主要係於培養基中添加各種不同物質，如細胞激素、血清及重組蛋白等營養物所組成之培養液來促使幹細胞增生。然而，血清的成分過於複雜且容易有異種間病毒及傳染病的交叉感染，因此含有血清之培養液雖然在學術研究上被廣泛使用，但對於預計製作成臨床用製劑的幹細胞來說是不適合的培養液。 The currently known methods for amplifying stem cells in vitro are mainly based on adding a variety of different substances to the culture medium, such as cytokine, serum, and recombinant protein and other nutrients to promote the proliferation of stem cells. However, the composition of serum is too complicated and prone to cross-infection of heterogeneous viruses and infectious diseases. Therefore, although the culture medium containing serum is widely used in academic research, it is not suitable for stem cells that are expected to be made into clinical preparations. Culture medium.
此外，大部分幹細胞之培養時間需長達7天甚至2週以上，並無法在短期間內獲取臨床上有效之幹細胞族群。由於幹細胞分離不易且 極為脆弱，因此如何有效提升幹細胞之回收率與產率，實為體外放大幹細胞培養需要克服的課題。 In addition, most stem cells need to be cultured for as long as 7 days or even more than 2 weeks, and clinically effective stem cell populations cannot be obtained in a short period of time. Because the separation of stem cells is not easy and It is extremely fragile, so how to effectively improve the recovery and yield of stem cells is actually a problem that needs to be overcome in vitro to scale up stem cell culture.
鑒於以上的問題，本發明揭露一種無血清培養液以及使用此無血清培養液的幹細胞分離及放大培養之方法，有助於解決含血清培養液不適用於培養臨床用途的幹細胞以及細胞放大速率較慢的問題。 In view of the above problems, the present invention discloses a serum-free culture medium and a method for separating and enlarging stem cells using the serum-free culture fluid, which helps to solve the problem that the serum-containing culture fluid is not suitable for culturing stem cells for clinical purposes and the cell amplification rate is relatively low. Slow problem.
本發明提供一種無血清培養液，包含培養基、副甲狀腺素(PTH)、纖維母細胞生長因子-2(FGF-2)、表皮生長因子(EGF)、人血清白蛋白(Human serum albumin)、茴香黴素(Anisomycin)、運鐵蛋白(Transferrin)以及胰島素(Insulin)。 The present invention provides a serum-free culture medium, comprising culture medium, parathyroid hormone (PTH), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), human serum albumin (Human serum albumin), fennel Anisomycin, Transferrin and Insulin.
本發明提供一種體外幹細胞放大培養之方法，包含將幹細胞置入上述無血清培養液中進行初代培養或繼代培養。 The present invention provides a method for scale-up and culture of stem cells in vitro, which comprises placing the stem cells in the aforementioned serum-free culture medium for primary culture or secondary culture.
根據本發明所揭露的無血清培養液以及體外幹細胞放大培養之方法，適合培養幹細胞之無血清培養液能有效促進幹細胞放大培養之效率以及存活率，且經繼代培養後，幹細胞仍保有需要的表面抗原特性和分化能力以作為臨床醫療使用。 According to the serum-free culture medium and the method of in vitro stem cell amplification culture disclosed in the present invention, the serum-free culture medium suitable for stem cell culture can effectively promote the efficiency and survival rate of stem cell amplification culture, and after subculture, the stem cells still retain the needed Surface antigen properties and differentiation ability for clinical medical use.
以上之關於本揭露內容之說明及以下之實施方式之說明係用以示範與解釋本發明之精神與原理，並且提供本發明之專利申請範圍更進一步之解釋。 The above description of the content of the disclosure and the description of the following embodiments are used to demonstrate and explain the spirit and principle of the present invention, and to provide a further explanation of the patent application scope of the present invention.
以下在實施方式中詳細敘述本發明之詳細特徵以及優點，其內容足以使任何熟習相關技藝者瞭解本發明之技術內容並據以實施，且根據本說明書所揭露之內容、申請專利範圍及圖式，任何熟習相關技藝者可輕易地理解本發明相關之目的及優點。以下之實施例進一步詳細說明本發明之觀點，但非以任何觀點限制本發明之範疇。 The detailed features and advantages of the present invention are described in detail in the following embodiments, and the content is sufficient to enable anyone familiar with the relevant art to understand the technical content of the present invention and implement it accordingly, and according to the content disclosed in this specification, the scope of patent application and the drawings Anyone who is familiar with the relevant art can easily understand the related purpose and advantages of the present invention. The following examples further illustrate the viewpoint of the present invention in detail, but do not limit the scope of the present invention by any viewpoint.
首先說明本發明一實施例的無血清培養液，包含有培養基、副甲狀腺素、纖維母細胞生長因子-2、表皮生長因子、人血清白蛋白、茴香黴素、運鐵蛋白以及胰島素。無血清培養液可用於幹細胞從脂肪組織中的分離、初代培養以及繼代培養，有效率地擴增細胞數量，並且在數代後的幹細胞仍保有間質幹細胞的表面抗原特性(CD34(-)、CD45(-)、CD90(+)、CD105(+)、CD73(+))及分化成脂肪、軟骨和硬骨之能力。幹細胞例如為脂肪間質幹細胞(Adipose-Derived Stem Cell，ADSC)。 First, the serum-free culture medium of an embodiment of the present invention will be described, which contains culture medium, parathyroid hormone, fibroblast growth factor-2, epidermal growth factor, human serum albumin, anisomycin, transferrin, and insulin. Serum-free culture medium can be used for the separation, primary culture and secondary culture of stem cells from adipose tissue, efficiently expanding the number of cells, and the stem cells after several generations still retain the surface antigen characteristics of mesenchymal stem cells (CD34(-) , CD45(-), CD90(+), CD105(+), CD73(+)) and the ability to differentiate into fat, cartilage and bone. The stem cells are, for example, Adipose-Derived Stem Cell (ADSC).
培養基例如為MCDB-131培養基。MCDB-131培養基的配方為本領域所通曉，故在此不加以詳述。在其他實施例中，培養基可以是MCDB-135或MCDB-138培養基。 The medium is, for example, MCDB-131 medium. The formula of MCDB-131 medium is well known in the art, so it will not be detailed here. In other embodiments, the medium may be MCDB-135 or MCDB-138 medium.
副甲狀腺素的濃度為0.10微克/公升至0.20微克/公升，較佳地為0.12微克/公升至0.16微克/公升。更佳地，副甲狀腺素的濃度為0.14微克/公升至0.15微克/公升。 The concentration of parathyroid hormone is 0.10 microgram/liter to 0.20 microgram/liter, preferably 0.12 microgram/liter to 0.16 microgram/liter. More preferably, the concentration of parathyroid hormone is 0.14 microgram/liter to 0.15 microgram/liter.
纖維母細胞生長因子-2的濃度為4微克/公升至12微克/公升，較佳地為6微克/公升至10微克/公升。更佳地，纖維母細胞生長因子-2的濃度為8微克/公升至10微克/公升。 The concentration of fibroblast growth factor-2 is 4 micrograms/liter to 12 micrograms/liter, preferably 6 micrograms/liter to 10 micrograms/liter. More preferably, the concentration of fibroblast growth factor-2 is 8 micrograms/liter to 10 micrograms/liter.
表皮生長因子的濃度為2.5微克/公升(μg/L)至7.5微克/公 升，較佳地為3.5微克/公升至6微克/公升。更佳地，表皮生長因子的濃度為5微克/公升至6微克/公升。 The concentration of epidermal growth factor is 2.5 micrograms/liter (μg/L) to 7.5 micrograms/liter Liter, preferably 3.5 micrograms/liter to 6 micrograms/liter. More preferably, the concentration of epidermal growth factor is 5 micrograms/liter to 6 micrograms/liter.
人血清白蛋白的濃度為0.25公克/公升至0.75公克/公升，較佳地為0.3公克/公升至0.6公克/公升。更佳地，人血清白蛋白的濃度為0.4公克/公升至0.5公克/公升。 The concentration of human serum albumin is 0.25 g/liter to 0.75 g/liter, preferably 0.3 g/liter to 0.6 g/liter. More preferably, the concentration of human serum albumin is 0.4 g/liter to 0.5 g/liter.
茴香黴素的濃度為75微克/公升至125微克/公升，較佳地為90微克/公升至115微克/公升。更佳地，茴香黴素的濃度為100微克/公升至110微克/公升。 The concentration of anisomycin is 75 micrograms/liter to 125 micrograms/liter, preferably 90 micrograms/liter to 115 micrograms/liter. More preferably, the concentration of anisomycin is 100 micrograms/liter to 110 micrograms/liter.
運鐵蛋白的濃度為3.75毫克/公升(mg/L)至5.75毫克/公升，較佳地為4.5毫克/公升至6毫克/公升。更佳地，運鐵蛋白的濃度為5.5至6毫克/公升。 The concentration of transferrin is 3.75 milligrams/liter (mg/L) to 5.75 mg/liter, preferably 4.5 mg/liter to 6 mg/liter. More preferably, the concentration of transferrin is 5.5 to 6 mg/liter.
胰島素的濃度為8毫克/公升至12毫克/公升，較佳地為8.5毫克/公升至10.5毫克/公升。在一實施例中，胰島素的濃度為10毫克/公升。 The concentration of insulin is 8 mg/liter to 12 mg/liter, preferably 8.5 mg/liter to 10.5 mg/liter. In one embodiment, the concentration of insulin is 10 mg/L.
在一實施例中，無血清培養液更包含硒(Selenium)。較佳地，硒的濃度為3微克/公升至7微克/公升。在一實施例中，硒的濃度為5微克/公升。 In one embodiment, the serum-free culture medium further contains Selenium. Preferably, the concentration of selenium is 3 micrograms/liter to 7 micrograms/liter. In one embodiment, the concentration of selenium is 5 micrograms/liter.
以下說明本發明一實施例的體外幹細胞放大培養之方法。 The following describes a method for in vitro stem cell amplification culture according to an embodiment of the present invention.
從一捐贈者體內抽取適量的含有幹細胞之人體組織。將人體組織用酵素作用30min~1小時，之後以離心機分離幹細胞與其他組織成分，以取得純化的幹細胞。在一實施例中，係從捐贈者體內抽取脂肪組織，並從脂肪組織中純化出脂肪間質幹細胞作為P0代細胞。 Extract an appropriate amount of human tissue containing stem cells from a donor. The human tissues are treated with enzymes for 30 minutes to 1 hour, and then the stem cells and other tissue components are separated by a centrifuge to obtain purified stem cells. In one embodiment, adipose tissue is extracted from the donor's body, and adipose-derived mesenchymal stem cells are purified from the adipose tissue as P0 generation cells.
取適量的幹細胞置入培養皿，並以PBS沖洗數次後，注入培養液進行初代培養。初代培養的時間約為數十小時至數天。在一實施例中，係採用本發明揭露的無血清培養液進行初代培養(P1代)，並且初代培養的時間約為4天。 Put an appropriate amount of stem cells into a petri dish, wash them with PBS several times, and then inject the culture medium for primary culture. The initial culture time is about tens of hours to several days. In one embodiment, the serum-free culture medium disclosed in the present invention is used for primary culture (P1 generation), and the time for primary culture is about 4 days.
初代培養完成後，移除培養液，並以酵素(例如膠原蛋白酶或胰蛋白酶)使P1代細胞脫離培養皿。取適量的幹細胞置入新的培養皿，並同樣以本發明揭露的無血清培養液進行繼代培養。在一實施例中，繼代培養的時間約為5天。 After the primary culture is completed, the culture medium is removed, and the P1 generation cells are removed from the culture dish with enzymes (such as collagenase or trypsin). An appropriate amount of stem cells are placed in a new culture dish, and the serum-free culture medium disclosed in the present invention is also used for subculture. In one embodiment, the time for subculture is about 5 days.
以下提供本發明之具體實施例，以說明本發明所揭露之無血清培養液在幹細胞放大培養上的功效。 The following provides specific examples of the present invention to illustrate the efficacy of the serum-free culture medium disclosed in the present invention on the scale-up and culture of stem cells.
〔實施例一〕 [Example 1]
提供一種無血清培養液，包含有25毫升的MCDB-131培養基、0.15微克/公升的副甲狀腺素、4微克/公升的纖維母細胞生長因子-2、5微克/公升的表皮生長因子、0.5公克/公升的人血清白蛋白、100微克/公升的茴香黴素、5.5毫克/公升的運鐵蛋白、10毫克/公升的胰島素以及10微克/公升的硒。將脂肪間質幹細胞置入填充有無血清培養液的培養皿中，以進行初代培養與數次繼代培養，進而依序得到P1代至P5代脂肪間質幹細胞。培養皿為直徑10公分的圓形培養皿。此外，進行初代培養與繼代培養所使用之脂肪間質幹細胞的細胞密度(即在每一代培養中置入無血清培養液的細胞密度)為400細胞/平方公分(cell/cm2)至1500細胞/平方公分。 Provide a serum-free culture medium containing 25 ml of MCDB-131 medium, 0.15 micrograms/liter of parathyroid hormone, 4 micrograms/liter of fibroblast growth factor-2, 5 micrograms/liter of epidermal growth factor, 0.5 grams /L of human serum albumin, 100 micrograms/liter of anisomycin, 5.5 mg/liter of transferrin, 10 mg/liter of insulin, and 10 micrograms/liter of selenium. The adipose-derived mesenchymal stem cells are placed in a culture dish filled with serum-free culture solution for primary culture and several subcultures, and then the adipose-derived mesenchymal stem cells from generation P1 to generation P5 are obtained sequentially. The petri dish is a round petri dish with a diameter of 10 cm. In addition, the cell density of adipose-derived mesenchymal stem cells used for primary culture and secondary culture (that is, the cell density of serum-free culture medium in each culture) is 400 cells/cm 2 (cell/cm 2 ) to 1500 Cells/cm².
〔比較例〕 [Comparative example]
提供習知的MCDB-131培養基。將脂肪間質幹細胞置入 MCDB-131培養基進行初代培養與數次繼代培養，依序得到P1代至P5代脂肪間質幹細胞。進行初代培養與繼代培養所使用之脂肪間質幹細胞的細胞密度為2000細胞/平方公分。 Provide conventional MCDB-131 medium. Place adipose mesenchymal stem cells into MCDB-131 medium was subjected to primary culture and several subcultures to obtain adipose-derived mesenchymal stem cells from P1 to P5 in sequence. The cell density of adipose-derived mesenchymal stem cells used for primary culture and secondary culture is 2000 cells/cm².
脂肪間質幹細胞的放大(增殖)狀況如下表1所示。根據表1，於實施例一中，P1代至P5代脂肪間質幹細胞的細胞數量倍增所需要的時間皆小於30小時，並且細胞倍增所需時間皆小於比較例，代表實施例一的無血清培養液能有效加快脂肪間質幹細胞之增殖。 The amplification (proliferation) status of adipose-derived mesenchymal stem cells is shown in Table 1 below. According to Table 1, in Example 1, the time required for cell number doubling of P1 to P5 adipose-derived mesenchymal stem cells is less than 30 hours, and the time required for cell doubling is less than that of the comparative example, which represents the serum-free example of Example 1. The culture fluid can effectively accelerate the proliferation of adipose-derived mesenchymal stem cells.
脂肪間質幹細胞的成長速率如下表2所示。根據表2，在相同的培養時間下，實施例一的P1至P3代脂肪間質幹細胞成長速率會高於比較例的成長速率，例如在培養P1代細胞時，實施例一的細胞增加倍數為107.0，代表P1代細胞培養完成後，取得的存活細胞數量是在培養P1代細胞之初始階段置入無血清培養液的細胞數量的107倍。 The growth rate of adipose-derived mesenchymal stem cells is shown in Table 2 below. According to Table 2, under the same culture time, the growth rate of the P1 to P3 generation adipose mesenchymal stem cells in Example 1 will be higher than that of the comparative example. For example, when the P1 generation cells are cultured, the cell growth rate of Example 1 is 107.0, representing that the number of viable cells obtained after the completion of the P1 generation cell culture is 107 times the number of cells placed in the serum-free culture medium at the initial stage of the P1 generation cell culture.
每代脂肪間質幹細胞的表面抗原分析結果如下表3所示。以流式細胞儀分析表面抗原，能得知實施例一中的脂肪間質幹細胞符合CD34(-)、CD45(-)、CD73(+)、CD90(+)、CD105(+)的表面抗原特性，因此可應用於誘導脂肪分化以及誘導硬骨/軟骨細胞分化。 The surface antigen analysis results of each generation of adipose-derived mesenchymal stem cells are shown in Table 3 below. Using flow cytometry to analyze the surface antigen, it can be known that the adipose-derived mesenchymal stem cells in Example 1 meet the surface antigen characteristics of CD34(-), CD45(-), CD73(+), CD90(+), CD105(+) Therefore, it can be used to induce adipose differentiation and induce differentiation of bone/chondrocytes.
實施例一中，脂肪間質幹細胞的存活率如下表4所示。於進行P2代培養時，在填充有無血清培養液的5個培養皿中分別置入相同細胞密度(400cell/cm2)的脂肪間質幹細胞。根據表4，在P2代細胞培養完成後，每個培養皿中的細胞存活率(培養皿中存活細胞數量/培養皿中所有細胞數量)皆大於90%。 In Example 1, the survival rate of adipose mesenchymal stem cells is shown in Table 4 below. During the P2 generation culture, adipose-derived mesenchymal stem cells of the same cell density (400 cells/cm 2 ) were placed in 5 petri dishes filled with serum-free culture medium. According to Table 4, after the completion of the P2 generation cell culture, the survival rate of cells in each culture dish (the number of surviving cells in the culture dish/the number of all cells in the culture dish) is greater than 90%.
綜上所述，本發明所揭露的無血清培養液以及體外幹細胞放大培養之方法中，適合培養幹細胞之無血清培養液能有效促進幹細胞放大培養之效率以及存活率，且經繼代培養後，幹細胞仍保有需要的表面抗原特性以作為臨床醫療使用。 In summary, in the serum-free culture medium and the method for in vitro stem cell amplification culture disclosed in the present invention, the serum-free culture medium suitable for stem cell culture can effectively promote the efficiency and survival rate of stem cell amplification culture, and after subculture, Stem cells still retain the required surface antigen properties for clinical medical use.
此外，採用本發明所揭露的無血清培養液時，只需要加入少量的幹細胞便能成功培養，例如最少只需要400細胞/平方公分的細胞密度就能成功培養。 In addition, when using the serum-free culture medium disclosed in the present invention, only a small amount of stem cells need to be added to successfully culture, for example, a cell density of at least 400 cells/cm² is required to successfully culture.
雖然本發明以前述之實施例揭露如上，然而這些實施例並非用以限定本發明。在不脫離本發明之精神和範圍內，所為之更動與潤飾，均屬本發明之專利保護範圍。關於本發明所界定之保護範圍請參考所附之申請專利範圍。 Although the present invention is disclosed in the foregoing embodiments, these embodiments are not intended to limit the present invention. All changes and modifications made without departing from the spirit and scope of the present invention fall within the scope of patent protection of the present invention. For the scope of protection defined by the present invention, please refer to the attached patent scope.
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