201231087 六、發明說明： 【發明所屬之技術領域】 本發明為提供一種保養品滋養液，尤指一種 富含多種生長因子之保養品原料製程。 【先前技術】 按，一般化妝品中，生物有效成分添加劑分 別來自動物脂肪或植物萃取物兩大類，而植物 • 萃取物多為藥用植物有效部位加工、純化所得 產品。 現今市面所販售的化妝保養品各自含有許多 不同的成份，所宣稱的效果各異，有的具保濕 功能、有的具美白功能、有的具皮膚緊緻功能， 然而這些所販售的化妝保養品來源不易，製作 成本較高。 是以，要如何解決上述習用之問題與缺失， • 即為本發明之發明人與從事此行業之相關廠商 所亟欲研究改善之方向所在者。 201231087 【發明内容】 故，本發明之發明人有鑑於上述缺失，乃搜 集相關資料，經由多方評估及考量，並以從事 於此行業累積之多年經驗，經由不斷試作及修 改，始設計出此種種富含多種生長因子之保養 品原料製程的發明專利者。 本發明之主要目的在於：以培養脂肪幹細胞 (adipose derived stem cells)以得到富含多種 • 生長因子之保養品原料，進而大幅提升修護作 用。 為達上述目的，本發明係一種含有多種生長 因子之保養品原料製程，其主要依下列方式進 行：首先取出健康脂肪；再將一預定容量的溶劑 混入脂肪組織，以對脂肪組織進行清洗；按預 定劑量的試劑加入預定容量之脂肪組織，並進 行離心分離；經分離後產生的脂肪組織則混入 φ 預定劑量的酵素於一離心管，並對該離心管進 行震盪；將產生的沉澱物進行細胞培養以產生 體細胞；及取得該體細胞後，透過培養上述方 式所取得的細胞培養液富含細胞分泌之生長因 子（如：VEGF、HGF、b — FGF、T GF、I GF )’以完成而效保養品原料。 【實施方式】 為達成上述目的及功效，本發明所採用之技 4 201231087 術手段及構造，茲繪圖就本發明較佳實施例詳 加說明其特徵與功能如下，俾利完全了解。 請參閱第一圖及第二圖所示，係為本發明較 佳實施例之流程示意圖及運作流程示意圖，由 圖中可清楚看出本發明主要由下列方式進行： a )取出健康脂肪組織； b )將一預定容量的溶劑混入脂肪組織， 以對脂肪組織進行清洗； • c )將預定劑量的試劑加入預定容量之脂 肪組織，並進行離心分離； d )經分離後產生的脂肪組織則混入預定 劑量的酵素於一離心管，並對該離心 管進行震盪； e )將產生的沉澱物進行細胞培養以產生 體細胞； f )取得該體細胞後，經培養來取得特定 Φ 比例之生長因子。 g )原料產品出貨刖，與活性碳混和後過 濾.。 上述步驟a的健康脂肪組織裝於收集袋並 放置於生物安全操作櫃中，以手術刀割開收集 袋。隨後進行步驟b將一預定容量的溶劑混入 脂肪組織，而此溶劑係為生理食鹽水並以2 0 0 m 1倒入與脂肪組織組織混合，以清洗脂肪組 織。如有大的組織塊，用剪刀剪碎，之後用吸 5 201231087 取器將收集袋底部血水部分吸出，直到血水部 分剩下0 . 5 cm厚度即可。 根據步驟c，c，係將脂肪組織利用吸取器取出 並放置到新的離心管，而每管放置3 〇 ml的 脂肪組織’之後再用PBS(Phosphate-Buffered Saline)補至5 〇ml。而後再進行離心分離6 〇 〇 g / 5分鐘。 完成離心分離後’再此將分離出的脂肪組 • 織以吸取器取出2 5 m 1的脂肪組織，並放置到 新的離心管’每管放置的脂肪組織則需依當時 之脂肪量而平均分配。 完成上述步驟後，則進行步驟d及d ’ ，將 每一管加入已配置完成的1ml劑量的酵素（c〇1 lagenase I )，之後得到稀釋液（最終的濃度為 0 . 0 7 5 %)。此時將離心管斜放於3 7 t： f亙溫 搖震蘯培養箱中反應4 0分鐘，轉速為2 〇 Φ pm’ 而後加入 1 5 ml DMEM(Dulbecco’s Modifi e d E a g 1 e M e d i u m)並混合均勻，再次進行離心 分離1 2 0 0 g/ 5分鐘。 完成離心分離後，則進行步驟e ，利用吸 取器取出游離出之液態脂肪並丟棄於廢〉夜桶。 另以新的吸取器取出沉澱物而放置於新的離心、 管，並加入試劑 DMEM (Du 1 becco’s Mod i f j e(j g agle Medium)至總量5 0 ml並進行離心分離 1 2 0 0 g/ 5分鐘，而後用手術刀片切沉^殿物 201231087 直到無明顯塊狀物。201231087 VI. Description of the Invention: [Technical Field of the Invention] The present invention provides a maintenance product nourishing liquid, and more particularly to a process for preparing a skin care product rich in various growth factors. [Prior Art] According to the general cosmetics, the biological active ingredient additives are mainly derived from animal fats or plant extracts, and the plant extracts are mostly processed and purified products of effective parts of medicinal plants. The cosmetics and skin care products sold in the market today contain many different ingredients, and the claimed effects are different. Some have moisturizing functions, some have whitening functions, some have skin tightening functions, but these are sold cosmetics. The source of skin care products is not easy and the production cost is high. Therefore, how to solve the above problems and problems in the past, is the inventor of the present invention and the relevant manufacturers engaged in this industry are eager to study the direction of improvement. 201231087 [Summary content] Therefore, the inventors of the present invention have collected such relevant materials in view of the above-mentioned deficiencies, and have designed and developed such a variety through continuous evaluation and modification through multi-party evaluation and consideration, and with years of experience accumulated in the industry. An inventor of a process for the maintenance of a variety of growth factors. The main object of the present invention is to cultivate adipose derived stem cells to obtain a raw material of a skin care product rich in various growth factors, thereby greatly improving the repairing effect. In order to achieve the above object, the present invention is a process for preparing a skin care product containing a plurality of growth factors, which is mainly carried out in the following manner: first, removing healthy fat; then mixing a predetermined volume of solvent into the adipose tissue to wash the adipose tissue; The predetermined dose of the reagent is added to the adipose tissue of a predetermined volume and centrifuged; the adipose tissue produced after the separation is mixed with a predetermined dose of the enzyme in a centrifuge tube, and the centrifuge tube is oscillated; the resulting precipitate is subjected to the cell. Culture to produce somatic cells; and after obtaining the somatic cells, the cell culture medium obtained by the above-mentioned method is rich in cell-secreted growth factors (eg, VEGF, HGF, b-FGF, TGF, IGF) to complete And the skin care products. [Embodiment] In order to achieve the above object and effect, the technique and structure of the present invention will be described in detail with reference to the preferred embodiments of the present invention. Please refer to the first embodiment and the second figure, which are schematic diagrams of the flow and the operational flow of the preferred embodiment of the present invention. It can be clearly seen from the figure that the present invention is mainly carried out in the following manner: a) taking out healthy fat tissue; b) mixing a predetermined volume of solvent into the adipose tissue to clean the adipose tissue; c) adding a predetermined dose of the reagent to the adipose tissue of a predetermined volume and performing centrifugation; d) mixing the adipose tissue produced after separation The predetermined dose of the enzyme is in a centrifuge tube, and the centrifuge tube is oscillated; e) the resulting precipitate is subjected to cell culture to produce somatic cells; f) the somatic cells are obtained, and cultured to obtain a specific Φ ratio growth factor . g) After the raw materials are shipped, they are mixed with activated carbon and filtered. The healthy fat tissue of the above step a is placed in a collection bag and placed in a biosafety operation cabinet, and the collection bag is cut with a scalpel. Subsequently, step b is carried out to mix a predetermined volume of solvent into the adipose tissue, and the solvent is physiological saline and is poured into the adipose tissue at 200 mm 1 to wash the fat tissue. If there is a large tissue block, cut it with scissors, then use the suction 5 201231087 to absorb the blood from the bottom of the collection bag until the blood water part is 0. 5 cm thick. According to steps c, c, the adipose tissue was removed by a pipette and placed in a new centrifuge tube, and 3 〇 ml of adipose tissue was placed in each tube', and then supplemented with PBS (Phosphate-Buffered Saline) to 5 〇ml. Then centrifuge for 6 〇 〇 g / 5 minutes. After the centrifugation is completed, 'the fat group to be separated will be removed. Take 2 5 m 1 of adipose tissue with a pipette and place it in a new centrifuge tube. The fat tissue placed in each tube is averaged according to the amount of fat at that time. distribution. After the above steps are completed, steps d and d ' are performed, and each tube is added to the configured 1 ml dose of enzyme (c〇1 lagenase I ), followed by a dilution (final concentration of 0.77 %). . At this point, the centrifuge tube was placed in a 3 7 t: f 亘 warm shaking shaker incubator for 40 minutes at a speed of 2 〇 Φ pm' and then added to 15 ml DMEM (Dulbecco's Modifi ed E ag 1 e M edium) Mix well and centrifuge again for 1 2 0 0 g / 5 minutes. After the centrifugation is completed, step e is performed, and the free liquid fat is taken out by the aspirator and discarded in the waste>night barrel. The precipitate was taken out with a new aspirator and placed in a new centrifuge, tube, and reagent DMEM (Du 1 becco's Mod ifje (jg agle Medium) was added to a total of 50 ml and centrifuged 1 2 0 0 g / 5 Minutes, then cut with a surgical blade ^ Temple 201231087 until no obvious mass.
完成對沉澱物切割後，則進行步驟f ，首 先加入1 〇 ml/2 0% FBS DMEM的體細胞分 離培養液，取出體細胞後放置於容器中，並於 容器正面標示日期、細胞編號、代數（P 〇 )，將 容器放入3 7 °C細胞培養箱。隔日，再用吸取 器吸出原有2 0 % F B S D Μ E Μ體細胞分離培養 液，以P B S清洗體細胞，並加入新鮮1 〇 % F B S • DMEM體細胞分離培養液。4 8小時之後，在顯 微鏡下檢查細胞有無異狀，如無異狀，直接添 加培養液。如有異狀，立即報告品保人員處理。 原則上，如果顯微鏡下觀察確定是微生物感 染，則蓋緊蓋子，視同感染性廢棄物滅菌銷毁。 第3 — 7天若細胞生長密度已達9 0 % ，進行 細胞繼代與細胞計數，若細胞數達2 * 1 0 6， 將細胞全數種入Τ1 5 0 容器中（medium内含 • 1 %FBS DMEM);若細胞數未達2 * 1 0 6，則將 細胞全數種入T75容器中。收集 ADSCmediu m，單一細胞來源置入同一容器内。檢測並記錄 生長因子含量並於_8 0°C保存。 產品原料出貨前，以P i s t e m a 1 :活性碳4 的比例混合並攪拌一小時。之後，用濾紙過濾 掉活性碳，無菌包裝出貨。 而步驟f中的生長因子係包括VEGF 、 HGF、b — FGF、TGF、 IGF。而在 201231087 體細胞内以特定比例混入生長因子，其比 為：VEGF26%-30%、HGF19 -23%、b — FGF3%— 5%、TGF %— 5%、IGF23%-27%。 藉由此種以特定比例將生長因子混入體 胞以得到富含多種生長因子之保養品，進而 幅提升修護作用。 惟，以上所述僅為本發明之較佳實施例 藝已，非因此即拘限本發明之專利範圍，故舉 運用本發明說明書及圖式内容所為之簡易修 及等效結構變化，均應同理包含於本發明之 利範圍内，合予陳明。 綜上所述，本發明之一種含有多種生長 子之保養品製程於使用時，為確實能達到其 效及目的，故本發明誠為一實用性優異之 明，為符合發明專利之申請要件，爰依法提 參 申請，盼 審委早曰賜准本發明，以保障發 人之辛苦發明，倘若 鈞局審委有任何稽疑 請不吝來函指示，發明人定當竭力配合，實 公便。 例 % 3 細 大 而 凡 飾 專 因 功 發 出 明 感 8 201231087 意 示 程 流 之 例 施 實 佳 較 明 1 發 明本 說為。 單係圖 簡 式圖 圖一 t第 示 程 流 作 il 之 例 施 實 佳 較 明 發 本。 為圖 係意 圖 二 第After the cleavage of the precipitate is completed, step f is performed, firstly, a somatic cell separation culture solution of 1 〇ml/2 0% FBS DMEM is added, the somatic cells are taken out, placed in a container, and the date, cell number, and algebra are marked on the front of the container. (P 〇), place the container in a 3 7 °C cell culture incubator. On the next day, the original 20% F B S D Μ E Μ somatic cell isolation medium was aspirated by aspirator, and the somatic cells were washed with P B S, and fresh 1 〇 % F B S • DMEM somatic cells were separated and cultured. After 4 hours, check the cells for any abnormalities under the microscope. If there is no abnormality, add the culture solution directly. If there is any abnormality, report it to the quality assurance personnel immediately. In principle, if microscopic infection is determined under the microscope, the lid is closed and sterilized as if it were infected with infectious waste. On the 3rd to 7th day, if the cell growth density has reached 90%, the cell is subcultured and counted. If the number of cells reaches 2 * 1 0 6, the cells are all seeded into the Τ1 0 0 container (medium inclusion • 1 %) FBS DMEM); if the number of cells is less than 2 * 1 0 6, the cells are all seeded into the T75 container. ADSCmediu m was collected and a single cell source was placed in the same container. The growth factor content was measured and recorded and stored at _80 °C. The raw materials of the product were mixed and stirred for one hour in the ratio of P i s t e m a 1 : activated carbon 4 before shipment. After that, the activated carbon is filtered off with filter paper and shipped aseptically. The growth factors in step f include VEGF, HGF, b-FGF, TGF, IGF. In 201231087, growth factors were mixed in a specific ratio in the somatic cells, and the ratio was: VEGF26%-30%, HGF19-23%, b-FGF3%-5%, TGF%-5%, IGF23%-27%. By mixing the growth factors into the body cells in a specific ratio to obtain a skin care product rich in various growth factors, the dressing effect is enhanced. However, the above description is only the preferred embodiment of the present invention, and thus the scope of the present invention is not limited thereto, so that the simple repair and equivalent structural changes of the present specification and drawings should be applied. The same is included in the scope of the present invention and is combined with Chen Ming. In summary, the process of the skin care product containing a plurality of growers of the present invention can achieve its effectiveness and purpose when used. Therefore, the present invention is excellent in practicality and is in accordance with the application requirements of the invention patent.提Apply for the application according to law, and hope that the review committee will grant the invention as soon as possible to protect the hard work of the person. If there is any doubt in the arbitral tribunal, please do not hesitate to give instructions, the inventor will try his best to cooperate and be fair. Example % 3 is large and the special decoration is made by the special function. 8 201231087 The example of the flow is shown in the example of the implementation. A simple diagram of a single diagram Figure 1 shows the example of a flow of il. For the picture
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