TWI555847B - Culture and production of adipose - derived stem cells and their stem cell secretions - Google Patents

Culture and production of adipose - derived stem cells and their stem cell secretions Download PDF

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TWI555847B
TWI555847B TW102127874A TW102127874A TWI555847B TW I555847 B TWI555847 B TW I555847B TW 102127874 A TW102127874 A TW 102127874A TW 102127874 A TW102127874 A TW 102127874A TW I555847 B TWI555847 B TW I555847B
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jia-ju Cai
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jia-ju Cai
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脂肪幹細胞及其幹細胞分泌物之培養與量產方法 Culture and mass production method of adipose stem cells and stem cell secretions thereof

本發明係關於一種幹細胞及其幹細胞分泌物之培養與量產方法,特別是關於一種脂肪幹細胞之培養及量產其幹細胞分泌物的方法。 The present invention relates to a method for culturing and mass-producing stem cells and stem cell secretions thereof, and more particularly to a method for culturing a stem cell and producing a stem cell secretion thereof.

基於近年來生技與醫療技術蓬勃發展,幹細胞因其特有的分化、增生及組織修復潛能,使其在生醫產業日趨重要。無論是全球幹細胞相關公司數量、市值的大幅成長,或是美國食品藥物管理局(FDA)接受以幹細胞申請的臨床案件之逐年上升等情況來看,皆顯示出幹細胞治療已成為國際生技產業重點發展項目。 Based on the vigorous development of biotechnology and medical technology in recent years, stem cells have become increasingly important in the biomedical industry due to their unique differentiation, proliferation and tissue repair potential. Whether it is the number of global stem cell-related companies, the rapid growth of market value, or the annual increase in clinical cases of stem cell applications by the US Food and Drug Administration (FDA), it has been shown that stem cell therapy has become the focus of the international biotechnology industry. Development project.

然而,幹細胞的快速分化能力常使其在第3至4次繼代後,逐漸失去其幹細胞特性,以致後續實驗往往無法符合臨床預期。縱使是初代培養的幹細胞,以傳統培養法的幹細胞其特性亦與臨床表現不同。此外,有異於骨髓幹細胞,臍帶血幹細胞及胚胎幹細胞等檢體來源取得較不易,而脂肪為相對容易之檢體取得來源,且檢體取得過程對捐贈者產生的不適感較其他來源輕緩。 However, the ability of stem cells to rapidly differentiate often causes them to lose their stem cell characteristics after the third to fourth passages, so that subsequent experiments often fail to meet clinical expectations. Even though it is a stem cell cultured in the first generation, the characteristics of stem cells in the conventional culture method are different from those in clinical manifestations. In addition, different from bone marrow stem cells, cord blood stem cells and embryonic stem cells, the source of the sample is not easy to obtain, and fat is a relatively easy source of the sample, and the process of obtaining the sample is less responsive to the donor than other sources. .

此外,除了幹細胞本身的醫療應用之外,越來越多針對幹細胞分泌物的研究顯示,幹細胞分泌物的複合組成因子對於再生醫學也有良好表現。歐美均有研究報導,以幹細胞分泌物培養受損之肝臟細胞或其他 哺乳類動物組織,均有細胞增生與組織修復之成果。 In addition, in addition to the medical applications of stem cells themselves, more and more studies on stem cell secretions have shown that the composite component of stem cell secretions also performs well in regenerative medicine. Studies in Europe and the United States have reported that stem cells secrete cultured damaged liver cells or other Mammalian tissue has the result of cell proliferation and tissue repair.

因此,現階段研究之首要目的即在於延緩幹細胞分化、維持其幹細胞特性並同時大量培養幹細胞及其幹細胞分泌物,以利後續研究。如何藉由改良傳統幹細胞培養方法,進而製造生產大量幹細胞與其幹細胞分泌物以符合當下細胞生物學界之需求,已成了亟待解決之一課題。 Therefore, the primary purpose of the current research is to delay stem cell differentiation, maintain its stem cell characteristics and simultaneously culture stem cells and stem cell secretions in large quantities for subsequent research. How to improve the traditional stem cell culture method to produce a large number of stem cells and stem cell secretions to meet the needs of the current cell biology community has become an urgent problem to be solved.

基於上述習知問題及需求,本發明主要以異於傳統細胞培養方式之創新3D培養法延緩細胞分化並維持幹細胞特性,同時搭配定時灌流系統以刺激幹細胞分泌物產生。由於3D立體環境培養法對幹細胞特性的維持與其分泌物的組成分之影響,皆可應用於再生醫學且作為其他醫療領域的發展潛能。因此本發明以立體培養法製造生產大量幹細胞與其幹細胞分泌物符合當下細胞生物學界之需求。 Based on the above-mentioned conventional problems and needs, the present invention mainly delays cell differentiation and maintains stem cell characteristics by an innovative 3D culture method different from the conventional cell culture method, and is accompanied by a timed perfusion system to stimulate stem cell secretion production. Due to the influence of the 3D stereo culture method on the maintenance of stem cell characteristics and the composition of its secretions, it can be applied to regenerative medicine and as a development potential in other medical fields. Therefore, the present invention produces a large number of stem cells and their stem cell secretions by stereo culture to meet the needs of the current cell biology community.

本發明之一目的為提供一種脂肪幹細胞培養及量產其幹細胞分泌物的方法,利用創新研發之3D培養技術大量培育脂肪幹細胞且量產並收集其幹細胞分泌物以利後期研究發展出個人化幹細胞產品。 An object of the present invention is to provide a method for culturing and mass producing stem cell secretions of adipose stem cells, and using the 3D culture technology developed by the invention to mass culture adipose stem cells, mass-produce and collect stem cell secretions for later research and development of individualized stem cells. product.

上述脂肪幹細胞之培養方法之步驟包括:(A)收集脂肪細胞樣本;(B)分離該脂肪細胞樣本中的脂肪幹細胞;(C)將該脂肪幹細胞以乾式細胞種植法分布於三維載體上,並置於一生物反應器中,以定時定量培養液灌流與細胞定時斷食法進行培養;(E)收集由該脂肪幹細胞分泌之幹細胞分泌物;其中該幹細胞分泌物至少包括血管內皮生長因子(Vascular endothelial growth factor,VEGF)、肝細胞生長因子(Hepatocyte growth factor,HGF)、介白素-6(Interleukin-6,IL-6)及第一型膠原蛋白(collagen type I)。 The steps of the method for culturing the above-mentioned adipose stem cells include: (A) collecting a sample of the fat cell; (B) isolating the adipose stem cell in the sample of the fat cell; (C) distributing the adipose stem cell to the three-dimensional vector by dry cell implantation, and juxtaposing In a bioreactor, the culture medium is perfused with a timed quantitative culture solution and a cell timed fasting method; (E) the stem cell secretion secreted by the adipose stem cell is collected; wherein the stem cell secretion comprises at least a vascular endothelial growth factor (Vascular endothelial) Growth factor, VEGF), hepatocyte growth factor (HGF), interleukin-6 (IL-6), and collagen type I.

在上述方法中,較佳地,步驟(C)更包括以定時定量培養液灌流與細胞定時斷食法進行培養;每個生物反應器裝載之三維載體之數目係介於100~1500個;培養獲取之脂肪幹細胞總數量為1×108~1×109個/培養瓶。幹細胞分泌物之血管內皮生長因子之濃度可介於593.5~2090pg/ml、肝細胞生長因子之濃度可介於656.4~1780pg/ml、介白素-6之濃度可介於151.9~523pg/ml及第一型膠原蛋白之濃度可介於23.41~42ng/ml,且脂肪幹細胞於三維載體上之貼覆率較佳地可達到90%。 In the above method, preferably, the step (C) further comprises culturing with a timed quantitative culture medium perfusion and a cell timing fasting method; the number of three-dimensional vectors loaded in each bioreactor is between 100 and 1500; The total number of obtained adipose stem cells was 1 × 10 8 ~ 1 × 10 9 / culture flask. The concentration of vascular endothelial growth factor in stem cell secretion may range from 593.5 to 2090 pg/ml, the concentration of hepatocyte growth factor may range from 656.4 to 1780 pg/ml, and the concentration of interleukin-6 may range from 151.9 to 523 pg/ml. The concentration of the first type collagen may be between 23.41 and 42 ng/ml, and the adhesion rate of the adipose stem cells on the three-dimensional carrier is preferably 90%.

本發明相較於傳統細胞培養法有下列優勢:藉由三維培養脂肪幹細胞之方法,可省去繁複操作步驟與人力、減少設備空間、量產幹細胞分泌物以及節省細胞培養液之經濟成本。此外,乾式細胞植入技術可改善以往3D培養法細胞貼覆率較差之瓶頸、並以定時定量培養液灌流與細胞定時斷食法,改變幹細胞分泌物之組成及生產量,且在經濟效益方面可節約8至10倍的培養耗材用量與支出。藉由本發明揭露內容,可快速大量培養脂肪幹細胞及大量生產其幹細胞分泌物,以提供疾病治療、細胞修復及再生醫學等領域之研究應用。獨創之細胞培養系統亦可做為其他種類細胞培養之研究開發應用。 Compared with the traditional cell culture method, the present invention has the following advantages: by three-dimensional cultivation of adipose stem cells, the complicated operation steps and manpower, the reduction of equipment space, the mass production of stem cell secretions, and the economic cost of saving the cell culture fluid can be eliminated. In addition, dry cell implantation technology can improve the bottleneck of poor cell attach rate in previous 3D culture methods, and change the composition and production of stem cell secretions by timing quantitative culture perfusion and cell timing fasting, and in terms of economic benefits. It can save 8 to 10 times the consumption and consumption of culture consumables. Through the disclosure of the present invention, adipose stem cells can be rapidly cultured in large quantities and their stem cell secretions can be produced in large quantities to provide research applications in the fields of disease treatment, cell repair and regenerative medicine. The original cell culture system can also be used as a research and development application for other types of cell culture.

101~104‧‧‧步驟 101~104‧‧‧Steps

201~208‧‧‧步驟 201~208‧‧‧Steps

301~310‧‧‧步驟 301~310‧‧‧Steps

第一圖顯示本發明脂肪幹細胞之培養方法之步驟流程圖;第二圖為脂肪幹細胞之純化步驟流程圖;第三圖為脂肪幹細胞之培養以及幹細胞分泌物之生產步驟流程圖;第四圖顯示經純化培養後之脂肪幹細胞存活率; 第五圖顯示以乾式細胞種植法將細胞液均勻分布於培養瓶中的三維載體後的細胞貼覆率;第六圖顯示本發明之方法與習知培養液中的葡萄糖含量比較結果;第七A~七D圖顯示各種幹細胞分泌物之濃度比較。 The first figure shows a flow chart of the steps of the method for culturing the adipose stem cells of the present invention; the second figure is a flow chart of the purification steps of the adipose stem cells; the third figure is a flow chart of the steps of the cultivation of the adipose stem cells and the production steps of the stem cell secretions; Adipose stem cell survival rate after purification and culture; The fifth graph shows the cell coverage rate after the three-dimensional vector in which the cell liquid is uniformly distributed in the culture flask by the dry cell seeding method; the sixth graph shows the comparison result of the glucose content in the method of the present invention and the conventional culture solution; The A~7D plot shows the concentration comparison of various stem cell secretions.

參閱第一圖,其顯示本發明脂肪幹細胞培養及量產其幹細胞分泌物的方法之步驟流程圖。本發明主要提供一種脂肪幹細胞及其幹細胞分泌物之培養與量產方法,步驟包括:收集脂肪細胞樣本(步驟101);分離該脂肪細胞樣本中的脂肪幹細胞(步驟102);將該脂肪幹細胞以乾式細胞種植法分布於三維載體上,並置於一生物反應器中進行培養(步驟103);收集該培養液中由該脂肪幹細胞分泌之幹細胞分泌物(步驟104)。其中該幹細胞分泌物至少包括血管內皮生長因子(Vascular endothelial growth factor,VEGF)、肝細胞生長因子(Hepatocyte growth factor,HGF)、介白素-6(Interleukin-6,IL-6)及第一型膠原蛋白(collagen type I)。 Referring to the first panel, there is shown a flow chart of the steps of the method for culturing and mass producing stem cell secretions of the adipose stem cells of the present invention. The present invention mainly provides a method for culturing and mass-producing adipose stem cells and stem cell secretions thereof, the steps comprising: collecting a fat cell sample (step 101); isolating the adipose stem cells in the fat cell sample (step 102); The dry cell seeding method is distributed on a three-dimensional carrier and placed in a bioreactor for cultivation (step 103); the stem cell secretion secreted by the adipose stem cells in the culture solution is collected (step 104). The stem cell secretion includes at least vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-6 (IL-6) and first type. Collagen type I.

在上述方法中,較佳地,幹細胞分泌物之血管內皮生長因子之濃度介於593.5~2090pg/ml,肝細胞生長因子之濃度介於656.4~1780pg/ml、介白素-6之濃度介於151.9~523pg/ml及第一型膠原蛋白之濃度介於23.41~42ng/ml,培養獲取之脂肪幹細胞總數量為1×108~1×109個/培養瓶,且脂肪幹細胞於三維載體上之貼覆率較佳地可達到90%,每個生物反應器裝載之三維載體之數目係介於100~1500。上述內容僅是先行簡述各步 驟,具體實施方式將詳述如后。 In the above method, preferably, the concentration of the vascular endothelial growth factor of the stem cell secretion is between 593.5 and 2090 pg/ml, the concentration of the hepatocyte growth factor is between 656.4 and 1780 pg/ml, and the concentration of the interleukin-6 is between The concentration of 151.9~523pg/ml and type I collagen is between 23.41~42ng/ml, and the total number of adipose stem cells obtained by culture is 1×10 8 ~1×10 9 /culture flask, and the adipose stem cells are on the three-dimensional carrier. The coverage rate is preferably 90%, and the number of three-dimensional carriers loaded in each bioreactor is between 100 and 1500. The above content is only a brief description of each step, and the specific embodiment will be described in detail later.

脂肪幹細胞之分離及純化Isolation and purification of adipose stem cells

參閱第二圖,其顯示脂肪幹細胞之純化步驟流程圖。如圖所示,步驟201~208分別為:清洗脂肪,待脂肪分層後去掉下方血水廢液(步驟201),將脂肪移至離心管中離心(步驟202),將脂肪塊倒入盤中剪碎後移至離心管中(步驟203),加入膠原蛋白酶後放入培養箱,並慢速轉動以混合均勻(步驟204),將混合液過濾至離心管內離心(步驟205),離心後取出細胞沉澱物清洗,然後再次離心(步驟206),將細胞沉澱物以dPBS分散,然後再過濾離心(步驟207),取離心之上清液並送檢驗部門進行無菌檢驗、黴漿菌及內毒素含量測試(步驟208),離心後所得之脂肪幹細胞再於傳統培養瓶中培養。由於步驟201~208之脂肪檢體收集及幹細胞分離與習知技術相同,在此不針對細節部份進行說明。 Referring to the second figure, it shows a flow chart of the purification steps of adipose stem cells. As shown in the figure, steps 201-208 are respectively: cleaning the fat, removing the lower blood water waste liquid after the fat layering (step 201), transferring the fat to the centrifuge tube for centrifugation (step 202), and pouring the fat block into the tray. After cutting, move to the centrifuge tube (step 203), add collagenase, put it into the incubator, and rotate slowly to mix well (step 204), filter the mixture into a centrifuge tube for centrifugation (step 205), after centrifugation The cell pellet is removed for washing, and then centrifuged again (step 206), the cell pellet is dispersed in dPBS, and then filtered and centrifuged (step 207), and the supernatant is centrifuged and sent to the inspection department for sterility test, mold fungus and internal Toxin content test (step 208), the adipose stem cells obtained after centrifugation are cultured in a conventional culture flask. Since the fat sample collection and stem cell separation in steps 201 to 208 are the same as those in the prior art, the details are not described here.

高效能脂肪幹細胞培養及其幹細胞分泌物之收集High-performance adipose stem cell culture and collection of stem cell secretions

完成上述脂肪幹細胞之分離及純化步驟後,先進行第一次放大培養,其存活率如第四圖所示。接著進行後續之高效能脂肪幹細胞培養及其幹細胞分泌物之收集,其分別為步驟301~310,如第三圖所示。 After the separation and purification steps of the above adipose stem cells are completed, the first amplification culture is performed first, and the survival rate is as shown in the fourth figure. Subsequent high-performance adipose-derived stem cell culture and collection of stem cell secretions are performed, which are steps 301-310, respectively, as shown in the third figure.

將培養盤中之細胞懸浮於容器中(步驟301)。 The cells in the culture dish are suspended in a container (step 301).

以乾式細胞種植法將細胞液均勻分布於培養瓶中的三維載體上(步驟302)。三維載體數量較佳為100~1,500個,其材質可以是玻璃纖維、PET或其他具有孔隙、孔洞,以及適用於脂肪細胞貼覆培養、兼具生物相容性材質。習知的濕式細胞種植法是將三維載體、培養液和細胞置於離心管中,讓細胞自行貼附於載體上,唯此法需靜置擺放且需定時進行搖晃, 步驟較繁瑣且所需時間較長。而在此採用的乾式細胞種植法和習知濕式細胞種植法不同之處在於,乾式細胞種植法是將含有特定數量的細胞液直接分布在乾燥的三維載體上,節省了上述靜置擺放、搖晃等步驟及所需時間,且藉此種改良之乾式種植法大幅提高了細胞於載體上的貼覆率。 The cell fluid is evenly distributed on the three-dimensional vector in the culture flask by dry cell seeding (step 302). The number of three-dimensional carriers is preferably from 100 to 1,500, and the material may be glass fiber, PET or other pores, pores, and a biocompatible material suitable for fat cell coating culture. The conventional wet cell cultivation method is to place a three-dimensional carrier, a culture solution and a cell in a centrifuge tube, and let the cells attach themselves to the carrier, but the method needs to be placed statically and shakes regularly. The steps are cumbersome and take longer. The dry cell seeding method used here differs from the conventional wet cell seeding method in that the dry cell seeding method directly distributes a specific amount of cell liquid on a dry three-dimensional carrier, thereby saving the above-mentioned static placement. The steps such as shaking, and the time required, and the improved dry planting method greatly improves the cell coverage on the carrier.

參閱第五圖,在經過約200分鐘後,以乾式細胞種植法所得之細胞貼覆率可達到約90%,確實地改善習知脂肪幹細胞培養技術貼覆率不高之缺點。乾式細胞種植法之步驟係如下所示:前置作業 Referring to the fifth figure, after about 200 minutes, the cell attachment rate obtained by the dry cell implantation method can reach about 90%, and the disadvantage of the conventional fat stem cell culture technique is not improved. The steps of the dry cell cultivation method are as follows: front work

a.以細胞培養技術於傳統培養瓶將脂肪幹細胞放大培養。 a. Amplify culture of adipose stem cells in a conventional culture flask by cell culture technique.

b.於操作前準備100~1500個三維載體。 b. Prepare 100~1500 three-dimensional carriers before operation.

操作步驟Steps

1.移除培養盤中的舊培養液。2.以dPBS(杜式磷酸緩衝液)清洗細胞2次。3.加入適量胰蛋白酶-EDTA溶液,使所有細胞懸浮。4.以適量培養液將細胞懸浮液中和後離心300-500g/3-10分鐘。5.去除上清液,保留管底細胞,以培養液將細胞沖散懸浮至每毫升約1千萬至1千5百萬顆細胞。6.將細胞懸浮液均勻分布於生物反應器專用瓶中的三維載體上。此步驟較佳為使用上述之乾式細胞種植法,可獲得較好的細胞貼覆率。7.將生物反應器專用瓶置於37℃,5% CO2培養箱中靜置60-120分鐘(步驟303)。 1. Remove the old culture medium from the culture tray. 2. Wash the cells twice with dPBS (DuPont phosphate buffer). 3. Add appropriate amount of trypsin-EDTA solution to suspend all cells. 4. Neutralize the cell suspension with an appropriate amount of the culture solution and centrifuge 300-500 g / 3-10 minutes. 5. Remove the supernatant, retain the bottom cells, and pulsate the cells to about 10 to 15 million cells per ml. 6. The cell suspension is evenly distributed on a three-dimensional carrier in a dedicated bottle for the bioreactor. Preferably, this step uses a dry cell seeding method as described above to obtain a better cell coverage rate. 7. Place the bioreactor-specific bottle in a 37 ° C, 5% CO 2 incubator for 60-120 minutes (step 303).

培養液倒入生物反應器專用瓶中後置於培養箱中培養(步驟304)。在此使用之培養液成分包括培養液(DMEM、DMEM/F12、F12、StemPro...等任何一種)、胎牛血清(FBS)、抗生素(gentamicin、penicillin...等),與習知培養皿法相似,在此不再贅述。惟在進行細胞植入以及細胞數 目放大時使用含有動物來源的添加物,例如FBS,須為CTS等級或是狂牛症非疫區之來源。 The culture solution is poured into a special bottle for the bioreactor and then placed in an incubator for cultivation (step 304). The culture solution used herein includes a culture solution (DMEM, DMEM/F12, F12, StemPro, etc.), fetal bovine serum (FBS), antibiotics (gentamicin, penicillin, etc.), and conventional culture. The dish method is similar and will not be repeated here. Only in cell implantation and cell count Additions containing animal sources, such as FBS, are required to be a source of CTS or a source of FF-PFA.

串接生物反應器專用瓶與含培養液的血清瓶(步驟305)。 The bioreactor-specific bottle and the serum bottle containing the culture solution are connected in series (step 305).

再將串接管線裝於蠕動幫浦上(步驟306)。 The series line is then mounted on the peristaltic pump (step 306).

定時定量灌流細胞培養液(步驟307)。在此步驟可配合給予特定次數及特定時間長度的斷食,以刺激幹細胞調整其幹細胞分泌物之組成比例及產量。 The cell culture fluid is periodically perfused (step 307). In this step, a specific number of times and a certain length of time can be used to stimulate the stem cells to adjust the composition ratio and yield of the stem cell secretions.

培養數十小時後即可收集含高濃度生長因子之幹細胞分泌物(步驟308)。進行幹細胞分泌物收集時,不可使用動物血清,以無動物來源(xeno-free)、無抗生素的培養液或是基礎培養液進行,以避免因為成品中含有非人類成分而造成使用者過敏。以下為脂肪幹細胞於生物反應器中培養並收集幹細胞分泌物之詳細步驟,以使該上述步驟更為明確: Stem cell secretions containing high concentrations of growth factors can be collected after tens of hours of incubation (step 308). When collecting stem cell secretions, animal serum should not be used, and it should be carried out in xeno-free, antibiotic-free medium or basal medium to avoid allergies caused by non-human ingredients in the finished product. The following is a detailed step of culturing adipose stem cells in a bioreactor and collecting stem cell secretions to make the above steps clearer:

1.將脂肪幹細胞培養於三維載體上,於生物反應器中進行培養,培養液為含10%胎牛血清之DMEM/F12。 1. The adipose stem cells are cultured on a three-dimensional vector and cultured in a bioreactor, and the culture solution is DMEM/F12 containing 10% fetal bovine serum.

2.培養時將生物反應器專用瓶裝置於生物反應器上。 2. The bioreactor special bottle is placed on the bioreactor during the cultivation.

3.培養期間每2-3日以緩衝液清洗並更換培養液。緩衝液可為dPBS或PBS。 3. Wash and replace the culture solution with buffer every 2-3 days during the culture. The buffer can be dPBS or PBS.

4.培養7~10日後即可開始收集脂肪幹細胞分泌物。 4. After 7 to 10 days of culture, the secretion of adipose stem cells can be collected.

5.去除舊培養液後以約1-2公升緩衝液清洗含有三維載體之生物反應器專用瓶。緩衝液可為dPBS或PBS。 5. After removing the old culture solution, wash the bioreactor-specific bottle containing the three-dimensional carrier with about 1-2 liters of buffer. The buffer can be dPBS or PBS.

6.加入培養液DMEM/F12。使用的培養液可為DMEM、DMEM/F12、F12、StemPro...等任何一種,並非僅限於在此所述之 DMEM/F12。 6. Add the culture medium DMEM/F12. The culture solution used may be any one of DMEM, DMEM/F12, F12, StemPro, etc., and is not limited to the ones described herein. DMEM/F12.

7.以灌流管線串連血清瓶與生物反應器專用瓶,並搭載蠕動幫浦以調控流速及流量,藉此達成定時定量及細胞定時斷食之目的。 7. The serum bottle and the bioreactor special bottle are connected in series with the perfusion line, and the peristaltic pump is arranged to regulate the flow rate and the flow rate, thereby achieving the purpose of regular quantitative and cell timing fasting.

8.培養48~72小時後收集細胞培養液中之脂肪幹細胞分泌物。 8. After 48 to 72 hours of culture, the secretion of adipose stem cells in the cell culture medium was collected.

細胞可繼續進行培養與收集幹細胞分泌物(步驟309)。 The cells can continue to culture and collect stem cell secretions (step 309).

收集之幹細胞分泌物以恆溫恆濕保存(步驟310)。 The collected stem cell secretions are stored at a constant temperature and humidity (step 310).

就習知利用培養皿之培養方法而言,10-cm培養皿所能培養的人類脂肪幹細胞約為1×106個,所能獲取的培養液體積約為10~15ml;T-175培養角瓶所能培養的人類脂肪幹細胞約為2.5×106個,所能獲取的培養液體積約為40~100ml,而本方法之脂肪幹細胞總數量較佳可達到1×109個/培養瓶,而一次所能獲取之細胞培養液最多為12L。 As far as the culture method of the culture dish is used, the human fat stem cells which can be cultured in a 10-cm culture dish are about 1×10 6 , and the volume of the culture medium which can be obtained is about 10-15 ml; the T-175 culture angle The human fat stem cells that can be cultured in the bottle are about 2.5×10 6 , and the volume of the culture medium that can be obtained is about 40-100 ml, and the total number of the fat stem cells in the method is preferably 1×10 9 /culture flask. The cell culture fluid that can be obtained at one time is up to 12L.

細胞分化程度比較Comparison of cell differentiation

參閱第六圖,其顯示本發明之方法與習知培養液中的葡萄糖含量比較結果。由於過去研究發現幹細胞對葡萄糖利用率與細胞分化程度有關,細胞分化程度越大時,對葡萄糖的利用率越高,透過分析傳統法與本發明之三維培養法的培養液中葡萄糖含量可發現,三維培養法的培養液葡萄糖含量顯著高於傳統法,顯示脂肪幹細胞在三維立體環境下培養較能維持幹細胞之特性。 Referring to the sixth graph, there is shown a comparison of the method of the present invention with the glucose content in a conventional culture solution. Since past studies have found that stem cells are related to the degree of cell differentiation and the degree of cell differentiation, the greater the degree of cell differentiation, the higher the utilization of glucose, and the glucose content in the culture solution of the three-dimensional culture method of the present invention and the present invention can be found. The glucose content in the culture medium of the three-dimensional culture method was significantly higher than that of the traditional method, indicating that the adipose stem cells cultured in a three-dimensional environment can maintain the characteristics of stem cells.

幹細胞分泌物濃度比較Comparison of stem cell secretion concentrations

經由上述步驟收集得到的幹細胞分泌物,其中包括了多種幹細胞分泌物,其中至少包含有血管內皮生長因子(Vascular endothelial growth factor,VEGF)、肝細胞生長因子(Hepatocyte growth factor,HGF)、介白素-6(Interleukin-6,IL-6)及第一型膠原蛋白(collagen type I)。除上述幾種因子外,培養所得之幹細胞尚可能含有其他多種細胞激素(cytokine)與細胞外基質(ECM);例如:生長因子(Growth Factor)、細胞介素(Interlukin)及細胞骨架(cytoskeleton)等等,在此僅以血管內皮生長因子、肝細胞生長因子、介白素-6及第一型膠原蛋白之檢測為例,並非僅限於此。 Stem cell secretions collected through the above steps, including a variety of stem cell secretions, including at least vascular endothelial growth factor (Vascular endothelial growth factor) Factor, VEGF), Hepatocyte growth factor (HGF), Interleukin-6 (IL-6), and collagen type I. In addition to the above factors, the stem cells cultured may contain other cytokines and extracellular matrices (ECM); for example, growth factor, interlukin, and cytoskeleton. Etc. Here, the detection of vascular endothelial growth factor, hepatocyte growth factor, interleukin-6 and type I collagen is exemplified, and is not limited thereto.

將本發明方法與傳統培養盤之結果作比較,以上述因子的濃度作為比較基準。請參閱第七A~七D圖,並同時參閱表1。創新法即為本發明揭露之培養方法,在培養時培養瓶內含1000片三維載體,黑、白、灰色bar分別代表以500mL、2700mL及6000mL培養液灌流48~50小時後收取,並以ELISA kit分別偵測VEGF、HGF、LI-6及Collagen type I濃度。傳統法為使用T75培養瓶,以34mL DMEM/F12培養液培養48~50小時後收取,並以ELISA kit分別偵測VEGF、HGF、LI-6及Collagen type I濃度。 The method of the present invention was compared with the results of a conventional culture dish, and the concentration of the above factors was used as a comparison. Please refer to the seventh A~7D diagram and refer to Table 1. The innovative method is the culture method disclosed in the present invention. In the culture, the culture bottle contains 1000 three-dimensional carriers, and the black, white and gray bars respectively represent 500 mL, 2700 mL and 6000 mL culture solution perfusion for 48-50 hours, and are collected by ELISA. The kit detects VEGF, HGF, LI-6 and Collagen type I concentrations, respectively. The traditional method was to use a T75 flask and cultured in 34 mL of DMEM/F12 medium for 48-50 hours, and the concentrations of VEGF, HGF, LI-6 and Collagen type I were detected by ELISA kit.

在相同細胞來源、培養液條件、溫度和二氧化碳濃度的培養狀況下,以T75培養瓶所獲取培養液中所測得VEGF濃度為246.78±58.64pg/ml,而本發明培養方法為1987.18±103.25pg/ml。以T75培養瓶所獲取培養液中所測得HGF濃度為299.31±86.3pg/ml,而本發明培養方法為1613.25±166.71pg/ml。以T75培養瓶所獲取培養液中所測得IL-6濃度為76.21±22.65pg/ml,而本發明培養方法為514.12±9.29pg/ml。以T75培養瓶所獲取培養液中所測得collagen type I濃度為17.75±6.96ng/ml,而本發明培養方法為39.45±2.55ng/ml。兩種培養方式之VEGF、HGF、IL-6及collagen type I濃度以Student’st test進行雙尾統計分析,結果顯示皆具有顯著差異, 故使用本發明方法所測得之VEGF、HGF、IL-6及collagen type I濃度顯著較高。 Under the same cell source, culture condition, temperature and carbon dioxide concentration, the concentration of VEGF measured in the culture solution obtained from the T75 flask was 246.78±58.64 pg/ml, and the culture method of the present invention was 1987.18±103.25 pg. /ml. The concentration of HGF measured in the culture solution obtained from the T75 flask was 299.31±86.3 pg/ml, and the culture method of the present invention was 1613.25±166.71 pg/ml. The concentration of IL-6 measured in the culture solution obtained from the T75 flask was 76.21 ± 22.65 pg / ml, and the culture method of the present invention was 514.12 ± 9.29 pg / ml. The concentration of collagen type I measured in the culture solution obtained from the T75 flask was 17.75 ± 6.96 ng / ml, and the culture method of the present invention was 39.45 ± 2.55 ng / ml. The two-tailed statistical analysis of VEGF, HGF, IL-6 and collagen type I concentrations in Student's test showed significant differences. Therefore, the concentrations of VEGF, HGF, IL-6 and collagen type I measured by the method of the present invention are significantly higher.

綜上所述,本發明揭露之培養方法相較於傳統細胞培養法有下列優勢:短期大量培養脂肪幹細胞、省去繁複操作步驟與人力、減少設備空間、量產幹細胞分泌物以及節省細胞培養液之經濟成本。此外,具有乾式細胞植入技術可改善習知培養法細胞貼覆率較差之瓶頸、以及藉由定時定量培養液灌流與細胞定時斷食法,以改變幹細胞分泌物之組成及生產量。 In summary, the culture method disclosed by the present invention has the following advantages over the conventional cell culture method: short-term mass culture of adipose stem cells, elimination of complicated operation steps and manpower, reduction of equipment space, mass production of stem cell secretions, and saving of cell culture fluid. The economic cost. In addition, dry cell implantation technology can improve the bottleneck of poor cell attachment rate in conventional culture methods, and change the composition and production of stem cell secretions by timed quantitative culture perfusion and cell timing fasting.

以T175細胞培養瓶為例,可培養的面積為175cm2,每次操作僅能操作單一細胞培養瓶。本計畫使用之三維載體的有效培養面積約為25cm2,而每個生物反應器最多可裝載1,500個載體總共僅需500ml培養液,相當於同時培養214個T175細胞培養瓶,此需約4,000-6,500ml。經濟效益方面可節約8至10倍的培養耗材用量與支出。以一個培養批次(一整個細胞培養箱)為例,一個細胞培養箱可同時培養144個T25細胞培養瓶或是2個生物反應器。若分別以T25細胞培養瓶及本發明之3D高效能系統進行培養,與利用T25細胞培養瓶的傳統生產方式相比,3D高效能系統的每個培養批次可減 少工時至少24小時,同時達成約27倍的產量。 Taking the T175 cell culture flask as an example, the culturable area is 175 cm 2 , and only a single cell culture flask can be operated per operation. The effective culture area of the three-dimensional vector used in this project is about 25 cm 2 , and each bioreactor can carry up to 1,500 carriers in total, only 500 ml of the culture solution, which is equivalent to simultaneously culturing 214 T175 cell culture flasks, which requires about 4,000. -6,500ml. Economic benefits can save 8 to 10 times the amount of cultivation supplies and expenditure. Take a culture batch (one whole cell incubator) as an example. A cell culture incubator can simultaneously culture 144 T25 cell culture flasks or 2 bioreactors. If the T25 cell culture flask and the 3D high-performance system of the present invention are separately cultured, the culture batch of the 3D high-performance system can reduce the working time by at least 24 hours compared with the conventional production method using the T25 cell culture flask. Achieved about 27 times the output.

幹細胞分泌物之檢測Detection of stem cell secretions

為驗證所生產之幹細胞分泌物,不會對細胞產生不正常增生等影響,將收取之培養液,進行人類纖維母細胞(human foreskin fibroblast,HSF)培養。 In order to verify the secretion of the stem cells produced, the cells are not affected by abnormal proliferation, and the culture solution to be collected is cultured for human foreskin fibroblast (HSF).

培養3天後進行細胞增殖倍率分析,以20%或是100%幹細胞分泌物培養之細胞,細胞增殖倍率分別為1.7±0.3和1.3±0.2。 After 3 days of culture, cell proliferation magnification analysis was performed, and cells cultured at 20% or 100% of stem cell secretions had cell proliferation rates of 1.7 ± 0.3 and 1.3 ± 0.2, respectively.

以5%或10%FBS培養3天的HSF細胞增殖倍率分別為4.4±0.2和6.3±0.4,均比幹細胞分泌物顯著較高。經由上述測試得以證明,本發明之培養方法所生產之幹細胞分泌物沒有人體使用上的安全疑慮。 The proliferation rates of HSF cells cultured for 3 days at 5% or 10% FBS were 4.4 ± 0.2 and 6.3 ± 0.4, respectively, which were significantly higher than stem cell secretions. It was confirmed by the above test that the stem cell secretion produced by the culture method of the present invention has no safety concerns in human use.

依據上述內容,本發明可快速大量培養脂肪幹細胞及量產其幹細胞分泌物,以提供疾病治療、細胞修復及再生醫學等領域之研究應用。獨創之細胞培養系統亦可做為其他種類細胞培養之研究開發應用。 According to the above, the present invention can rapidly and mass culture adipose stem cells and mass produce their stem cell secretions, and provide research applications in the fields of disease treatment, cell repair and regenerative medicine. The original cell culture system can also be used as a research and development application for other types of cell culture.

由以上實施例可知,本發明所提供之脂肪幹細胞之培養方法及其幹細胞分泌物確具產業上之利用價值,惟以上之敘述僅為本發明之較佳實施例說明,凡精於此項技藝者當可依據上述之說明而作其它種種之改良,惟這些改變仍屬於本發明之精神及以下所界定之專利範圍中。 It can be seen from the above examples that the method for culturing the adipose stem cells and the stem cell secretion thereof provided by the present invention have industrial utilization value, but the above description is only a description of the preferred embodiment of the present invention, and the skill is fine. Other modifications may be made by the above description, but such modifications are still within the spirit of the invention and the scope of the invention as defined below.

101~104‧‧‧步驟 101~104‧‧‧Steps

Claims (7)

一種脂肪幹細胞培養及量產其幹細胞分泌物的方法,其步驟至少包括:(A)收集脂肪細胞樣本;(B)分離該脂肪細胞樣本中的脂肪幹細胞;(C)將含有預定數量的脂肪幹細胞的細胞液直接分布於乾燥的三維載體上,並置於一生物反應器之培養液中進行培養;(D)收集由該脂肪幹細胞分泌之幹細胞分泌物;其中該幹細胞分泌物至少包括血管內皮生長因子(Vascular endothelial growth factor,VEGF)、肝細胞生長因子(Hepatocyte growth factor,HGF)、介白素-6(Interleukin-6,IL-6)及第一型膠原蛋白(collagen type I)。 A method for culturing and mass producing a stem cell secretion thereof, the method comprising the steps of: (A) collecting a fat cell sample; (B) isolating the adipose stem cell in the fat cell sample; (C) containing a predetermined number of adipose stem cells The cytosol is directly distributed on a dry three-dimensional carrier and cultured in a culture solution of a bioreactor; (D) collecting stem cell secretion secreted by the adipose stem cell; wherein the stem cell secretion comprises at least vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), Hepatocyte growth factor (HGF), Interleukin-6 (IL-6), and collagen type I (collagen type I). 如申請專利範圍第1項所述之方法,其中步驟(C)所述的培養更包括以定時定量培養液灌流與細胞定時斷食法進行培養。 The method of claim 1, wherein the culturing according to step (C) further comprises culturing with a timed quantitative broth perfusion and a cell timed fasting method. 如申請專利範圍第1項所述之方法,其中該幹細胞分泌物之血管內皮生長因子之濃度介於593.5~2090pg/ml,肝細胞生長因子之濃度介於656.4~1780pg/ml、介白素-6之濃度介於151.9~523pg/ml及第一型膠原蛋白之濃度介於23.41~42ng/ml。 The method of claim 1, wherein the concentration of the vascular endothelial growth factor of the stem cell secretion is between 593.5 and 2090 pg/ml, and the concentration of the hepatocyte growth factor is between 656.4 and 1780 pg/ml, and the interleukin- The concentration of 6 is between 151.9 and 523 pg/ml and the concentration of the first type collagen is between 23.41 and 42 ng/ml. 如申請專利範圍第1項所述之方法,其中培養獲取之該脂肪幹細胞總數量為1×108~1×109個/培養瓶。 The method according to claim 1, wherein the total number of the adipose stem cells obtained by the culture is 1×10 8 to 1×10 9 /culture flask. 如申請專利範圍第1項所述之方法,其中該脂肪幹細胞於三維載體上之貼覆率可達到90%。 The method of claim 1, wherein the fat stem cell has a coverage of 90% on the three-dimensional carrier. 如申請專利範圍第1項所述之方法,其中該每個生物反應器裝載之三維載體之數目係介於100~1500個。 The method of claim 1, wherein the number of three-dimensional carriers loaded in each bioreactor is between 100 and 1500. 一種幹細胞分泌物,係以如申請專利範圍第1項所述之方法所取得,其中該幹細胞分泌物至少包括血管內皮生長因子(Vascular endothelial growth factor,VEGF)、肝細胞生長因子(Hepatocyte growth factor,HGF)、介白素-6(Interleukin-6,IL-6)及第一型膠原蛋白(collagen type I),其中該幹細胞分泌物之血管內皮生長因子之濃度介於593.5~2090pg/ml,肝細胞生長因子之濃度介於656.4~1780pg/ml、介白素-6之濃度介於151.9~523pg/ml及第一型膠原蛋白之濃度介於23.41~42ng/ml。 A stem cell secretion obtained by the method of claim 1, wherein the stem cell secretion comprises at least Vascular endothelial growth factor (VEGF) and Hepatocyte growth factor (Hepatocyte growth factor, HGF), Interleukin-6 (IL-6) and collagen type I (collagen type I), wherein the concentration of vascular endothelial growth factor of the stem cell secretion is between 593.5 and 2090 pg/ml, and the liver The concentration of cell growth factor is between 656.4 and 1780 pg/ml, the concentration of interleukin-6 is between 151.9 and 523 pg/ml, and the concentration of type I collagen is between 23.41 and 42 ng/ml.
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