、1248357 九、發明說明： 【發明所屬之技術領域】 本發明係有關生物性真皮、皮膚替代物之製作方法及用途。 【先前技術】 ^皮膚為人體最大的組織結構，並為防禦外界病原入侵的第一道防線，故皮膚一旦遭 受極大損傷時，人體即可能遭受嚴重感染的威脅甚至喪命。利用組織工程以結合皮膚細 胞及天然(或合成)的細胞外基質來製造具生物功能性的生物人工皮膚(artiflcial skin)，不 僅可以解決異體皮膚排斥的問題，克服自體皮膚移植限量的困難，並且可因應需求 同，提供多樣化的生物人工皮膚。 傳統皮膚移植的方式，是將身體某處完整皮膚移植到受傷的皮膚表面，除維持皮膚 的^旎外亦有利於皮膚再生及使傷口癒合的功能。但由於身體的表面皮膚有限，因此對 於k性傷口、燒燙傷面積廣大或在治療上需要重複植皮的病患而言，傳統的自體植皮便 不實用。因此在治療上，人類的屍皮就被廣泛應用作為暫時性的敷料，不過屍皮敷料除 外，亦有供應短缺及保存不易的問題，特別是還有傳染性疾病(例如肝炎或 AIDS)感染的潛在危險。 Λ *為解決前述問題及因應大量皮膚傷口植皮所需，研究人員則致力於人工皮膚的開發 研九。自體上皮細胞膜（cultured epidermal autograft;簡稱CEA)即為其一，它的傣點 票f iff膚於2〜3週内複製1〇3倍，供應大面積燒烫傷患者^皮，以促進i 癒合。但疋自體上皮細胞膜的移植亦面臨一些問題：操作不易、易有水泡生成、皮 口的接受(take)差異大及上皮層與真皮層間的結合力弱容易落。 $=¾層傷日，則需要真皮層填充物以避免傷口過度收縮^成^癒=25 心為解決述問題及目應不同的皮膚傷口的治療，填充真皮層的基質填充物、真皮替代 ϊ 丄皮严物恤n *滅)即陸續被開發*來無細胞ί真g i皮t τ膝®原蛋白凝膠(C0U啊gel)]或天然的基質組織(如人類 便是分別於1995及1996树過美國食品藥物管理 i有生^功1^近林結合麟母細胞及無細胞真妓質所形成 於基礎研究、細^的^= 的齡。另外，生祕人卫歧也可以應用 1248357 於其上以製作皮膚相等物^kin equivalent)，如此製備之皮膚相等物其基本 造相似，應用於大鼠皮膚時，並無排斥反應發生且大鼠接受良好，並"血、其 (vascularization)a^^tffM 〇 ^ Bell 4?6045346 利(4,485,096號)中之真皮替代物為基質，植人穿洞⑦職的的皮膚組 角胎的 接來，，，，，該專利強調已分化完成之表皮與真皮基質 3分化分層的時間外，亦可藉由製備大面積的皮膚相等物作為大面積燒1248357 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method and a method for producing a biological dermis and a skin substitute. [Prior Art] ^The skin is the largest tissue structure of the human body and is the first line of defense against external pathogen invasion. Therefore, once the skin is greatly damaged, the human body may be threatened with serious infection or even die. The use of tissue engineering to combine skin cells and natural (or synthetic) extracellular matrices to produce biofunctional artificial skin (artiflcial skin) can not only solve the problem of allogeneic skin rejection, but also overcome the difficulty of autologous skin grafting. And can provide a variety of bio-artificial skin according to the needs. The traditional way of skin transplantation is to transplant the intact skin of the body to the surface of the injured skin. In addition to maintaining the skin, it also helps the skin to regenerate and heal the wound. However, due to the limited surface skin of the body, traditional autologous skin grafting is not practical for patients with k-sex wounds, extensive burns or repeated skin grafts for treatment. Therefore, in terms of treatment, human corpses are widely used as temporary dressings, except for corpse dressings, which also have shortages and difficulties in storage, especially infectious diseases (such as hepatitis or AIDS). Potentially dangerous. Λ * In order to solve the above problems and to respond to a large number of skin wound skin grafts, the researchers are committed to the development of artificial skin. The cultured epidermal autograft (CEA) is one of its kind, and its f vote f iff skin replicates 1〇3 times in 2~3 weeks, supplying a large area of burn patients with scalp to promote i Heal. However, the transplantation of autologous epithelial cell membranes also faces some problems: the operation is not easy, the formation of blisters is easy, the difference in the acceptance of the skin is large, and the binding force between the epithelial layer and the dermis layer is weak. $=3⁄4 layer of injury day, you need dermal layer filling to avoid excessive contraction of the wound ^ ^ ^ = 25 heart to solve the problem and the treatment of different skin wounds, filling the dermal layer of matrix filler, dermal replacement ϊ Suede strict mercenary n * extinction) is gradually developed * to have no cells ί 真皮皮 t τ knee ® proprotein gel (C0U ah gel)] or natural matrix tissue (such as humans are in 1995 and 1996 respectively The tree has passed the US food and drug management, and there is a result of the birth of the mother and the cell-free true enamel, which are formed in the basic research, and the age of ^=. In addition, the secret person Weiqi can also apply 1248357 to The skin equivalents thus prepared are basically similar, and when applied to rat skin, no rejection occurs and the rats receive good, and "blood, vascularization a^^tffM 〇^ Bell 4?6045346 The dermis substitute in Li (4,485,096) is the substrate, and the skin group of the 7th position of the implanted hole is picked up, and, the patent emphasizes that the differentiation has been completed. The time between the epidermis and the dermal matrix 3 can be differentiated and stratified. Large areas of skin as a large-area burn equivalents
Bernard氏等人在他們美國專利第5,639,654號中揭露結合^ ^ 母細胞及培養㈣絲魏，賊齡錄aif腿de)於歸上 以製，，如此製備之皮射代物其真皮替代物與表皮。 細胞为化为層7〇全’、與十類皮膚結構類似。另Elsenberg氏在其美國專利第6〇3976〇垆 中則寿型或第^^(typelll；)膠原蛋自或其混合之膠原蛋白為基“ ; 雉母細胞及角質細胞之培養，以製作複合皮膚替代物(eGmpGsitesWns制 : $試者皆可射其皮㈣代物且傷°癒合情況良好，並皮膚#代物$有收 另f國Organogenesis公司所出產已通過FDA認可上 Ap^af^Gra趾in)則是利用牛第-型(type D膠原蛋白與人類纖維母細胞共同層形 hvmgskmequivalent)。此皮膚替代物無感染性病原及引發免疫反應之抗原存在\ / 與人類皮膚相似，並已被廣泛應用於慢性皮膚潰瘍傷口之治療。 由於一般皮膚替代物絕大部分是由萃取之膠原蛋白進行基質製作後， 代物之製備，其結構不似天然之基質骨架(scaffold)。由於天然之基質骨架提供完 層内膠原纖維立體架構，利於細胞的攀附、生長及胞外間質的生成。、為^ 之 膚替代物，仍需尋找更適合且來源豐富並具天然立體結構之基質成再、=匕fig 技術，以發展便利有效之皮膚替代物。 胃表作 【發明内容】 本發明係關於生物性真皮、皮射代物之製作方法，其侧 的緒皮所開發出新穎且具天然立體結構的真皮基質，植人人類纖維 胞。^維=胞可在豬真皮基質表騎著及伸展，並在豬皮基質内職著其膠原纖維 束’形成a有人類纖維母細胞的生物性真皮替代物。另人類皮膚肖質胞丨在物:陡 真皮替代物的乳突狀基質上附著並分化成多層的上皮層結構，胃=成=二 物0 本發明另提供本發明所製得之生物性真皮、皮膚替代物之方法。 【實施方式】 '1248357 本發明之目的為提供製備生物性真皮、皮膚替代物之方法。該方法包括取裁成適當 ^小之無細胞豬真皮基質，種上人類纖維母細胞，待細胞附著伸展後以形成生物性真皮 替代物。將人類皮膚角質細胞種植於前述製得之生物性真皮替代物上，加入使角質細胞 生長及分化之培養液，待角質細胞分層後即可製得生物性皮膚替代物。較佳地，所使用 之角質細胞生長及分化之培養液為自行開發之階段性培養液。 本發明之另一目的係提供生物性真皮、皮膚替代物，其係利用清潔、無污染及無疾 病的豬皮所開發出新穎且具天然立體結構的真皮基質，植入人類纖維母細胞及皮膚角質 細胞。其中該皮膚替代物之纖維母細胞可在豬真皮基質表面附著及伸展，並在豬皮基質 内攀附其膠原纖維束，形成含有人類纖維母細胞的生物性真皮替代物，且皮膚角質細胞 可在$物性真皮替代物的乳突狀基質表面上附著並分化成多層的上皮層結構。根據本發 明所製得之生物性真皮、皮膚替代物其結構完整緻密，在結構及性質上與人類皮膚更為 接近，而增加其在醫療、基礎研究及體外檢測系統應用之便利性及範疇。 依本發明所製得之生物性真皮、皮膚替代物，具有下列優點4丨)所使用豬真皮基質 無特定感染性病源污染，且在去細胞過程中已去除會引起免疫反應之細胞及組'成；（2) 所使用豬真皮基質具天然立體結構，此使得真皮纖維母細胞易於附著、伸展及分化，除 有助於生物性皮膚替代物上皮層之形成外，所得生物性真皮、皮膚替代物更 皮 膚結構及性質。 本發明另一目的為所使用之豬皮基質具天然的真皮層，除具有天然基質成分(膠原 蛋白，胞外間質)外並擁有原始的膠原纖維束三度空間結構。三度空間的立體社構士利 ^深層傷口的填充，避免傷口過度收縮，基質内膠原蛋白可協助細胞的附著、移、生 長及分化’加速傷口的復癒。豬皮基質在去除皮膚細胞後，可以除去免疫及感染的問 ，可以直接應用於臨床治療。台灣豬皮的來源豐沛，加上製程簡便，於是▲夠大量降低 ^質的成本，故本發明係利用自行開發之豬真皮基質作為生物性真皮、皮膚替代物之基 貝0 依士發明所製得之生物性真皮、皮膚替代物可作為皮膚移植或植入材料、 研九、藥物穿透性測試系統或化妝品抗紫外線檢測系統。 土 ^發明提供下列實施例進一步說明本發明，該等實施例之目的係為說明本發明並 作為本發明之限制。 卜 實施例1 :豬真皮基質之製作 新鮮豬皮經過清洗、除去皮下脂肪後再以脫毛劑徹底清除豬毛及毛根。以 =皮劇至0.016英吋厚。剷好的豬皮先以含抗生素盤尼西林(6〇〇〇u/m 浓 (Phosphate-buffered saline，簡稱PBS，ΡΗ7·0)浸泡，再以無菌水清洗數次後 ' 二$ 述去細胞步驟。 人1交丹進仃下 •首先將前述處理後之豬皮，先以0.25%胰蛋白酶(trypsin)溶液及0.04%中性|合 (dispase)溶液於37。(：作用，直到上皮層剝離。再以0.1〇/〇TritonX-100於4°c作用脾畫In U.S. Patent No. 5,639,654, the disclosure of U.S. Patent No. 5,639,654, the disclosure of which is incorporated herein by reference to U.S. Patent No. 5,639,654, the disclosure of which is incorporated herein by reference. . The cells are turned into layers, which are similar to the ten types of skin structures. In addition, Elsenberg's US patent No. 6〇3976〇垆 is a life-type or a ^^(typellll;) collagen egg from its mixed collagen-based "culture of hernia cells and keratinocytes to make a composite Skin substitutes (eGmpGsitesWns system: $ testers can shoot their skin (four) substitutes and wounds healed well, and skin #代物$有收其他f country Organogenesis company has produced FDA approved on Ap^af^Gra toe in ) is the use of bovine-type (type D collagen and human fibroblasts in the same layer hvmgskmequivalent). This skin substitute has no infectious pathogens and antigens that trigger immune responses \ / similar to human skin, and has been widely It is used in the treatment of chronic skin ulcer wounds. Since most of the skin substitutes are made from the extracted collagen, the preparation of the substitutes does not resemble the natural matrix skeleton (scaffold). The stereoscopic structure of the collagen fibers in the layer is beneficial to the cell's climbing, growth and extracellular matrix formation. For the skin substitute, it is still necessary to find a more suitable and abundant source and natural. The matrix of the body structure is re-, 匕fig technology to develop a convenient and effective skin substitute. [Summary of the invention] The present invention relates to a method for producing a biological dermis and a skin-eating substitute, and the side of the dermatology is developed. A novel and natural steric structure of the dermal matrix, implanted human fiber cells. ^ Wei = cells can ride and stretch on the porcine dermal matrix, and in the pigskin matrix, working with its collagen fiber bundles to form a human fiber matrix a biological dermal substitute for cells. Another human skin cytoplasmic cytoplasm adheres to and differentiates into a multi-layered epithelial structure on a papillary matrix of steep dermal substitutes, stomach = Cheng = two objects 0 The invention provides a method for preparing a biological dermis and a skin substitute. [1210357] The object of the present invention is to provide a method for preparing a biological dermis and a skin substitute. The method comprises the step of cutting into a suitable small cell-free method. Pig dermal matrix, seeded with human fibroblasts, to form a biological dermal substitute after cell attachment and extension. Plant human keratinocytes in the above-mentioned biological dermis replacement On the object, a culture solution for growing and differentiating keratinocytes is added, and a dermal cell substitute is stratified to obtain a biological skin substitute. Preferably, the culture medium for keratinocyte growth and differentiation used is a self-developed stage. Sexually cultured liquid. Another object of the present invention is to provide a biological dermis, skin substitute which utilizes a clean, non-polluting and disease-free pig skin to develop a novel dermal matrix with a natural three-dimensional structure, implanted into human fibers. Mother cells and skin keratinocytes, wherein the fibroblasts of the skin substitute can adhere and stretch on the surface of the porcine dermal matrix, and adhere to the collagen fiber bundle in the porcine skin matrix to form a biological dermal substitute containing human fibroblasts. And the skin keratinocytes can adhere to and differentiate into a multi-layered epithelial structure on the surface of the papillary matrix of the physical dermal substitute. The biological dermis and skin substitutes prepared according to the present invention are structurally complete and compact, and are closer in structure and properties to human skin, thereby increasing their convenience and scope in medical, basic research and in vitro detection systems. The biological dermis and skin substitute prepared by the invention have the following advantages: 4) The porcine dermal matrix used has no specific infectious pathogen contamination, and the cells and groups which cause an immune reaction have been removed during the decellularization process. (2) The porcine dermal matrix used has a natural three-dimensional structure, which makes the dermal fibroblasts easy to adhere, stretch and differentiate, in addition to contributing to the formation of the biodermal skin substitute epithelial layer, the resulting biological dermis, skin replacement The skin is more skin structure and nature. Another object of the invention is that the pigskin substrate used has a natural dermis layer which, in addition to having a natural matrix component (collagen, extracellular matrix), possesses a three-dimensional structure of the original collagen fiber bundle. The three-dimensional space of the three-dimensional community Shili deep filling of the wound, to avoid excessive contraction of the wound, collagen in the matrix can help the cell adhesion, migration, growth and differentiation 'accelerated wound healing. After removing the skin cells, the pigskin matrix can remove the immune and infection problems and can be directly applied to clinical treatment. The source of Taiwan pig skin is abundant, and the process is simple, so ▲ is enough to reduce the cost of quality. Therefore, the present invention utilizes a self-developed pig dermal matrix as a substitute for bio-dermis and skin substitutes. Bio-derived dermis and skin substitutes can be used as skin grafting or implant materials, research ninth, drug penetration test system or cosmetic anti-ultraviolet detection system. The invention is further described in the following examples, which are intended to illustrate the invention and are to be construed as limiting. Example 1: Preparation of Porcine Dermal Substrates Fresh pigskins were washed and removed from subcutaneous fat, and then the hairs and roots were thoroughly removed with a depilatory agent. Take the = leather to 0.016 inches thick. The shovel pig skin is first soaked with antibiotic penicillin (Phosphate-buffered saline (PBS, ΡΗ7·0), and then washed several times with sterile water. Human 1 crosses into the armpits • First, the previously treated pig skin is firstly treated with a 0.25% trypsin solution and a 0.04% neutral dispase solution at 37. (: action until the epithelial layer is peeled off. Spleen painting at 4 ° C with 0.1 〇 / 〇 Triton X-100
皮層内細胞及其殘骸移除。去掉細胞的豬真皮基質保存在15%甘油中，再經強产 3^0照射4小時進行滅菌，完成的豬皮基質可冷;東保存㈣。。，藉由組g切^ J 木务、與伊紅染色；簡稱H&E staining)觀察豬真皮基質内膠原纖維結構及細胞殘留产巴^穌 1248357 結果顯示，去除細胞之豬真皮基質内沒有細胞的殘留並且仍保有豬真皮的原始架構(圖 ΙΑ、1B)。另外，如圖1C所示豬真皮基質膠原纖維束呈絲狀或束狀疏鬆纏繞，顯示去 細胞的處理過程並未破壞基質真皮層的纖維結構。 實施例2 :人類皮膚細胞之分離及培養 自醫院取得的人類皮膚組織，利用含珍他黴素(gentamicin)(10ug/ml)的培養液清洗 3-5次，再利用2.4U/ml中性蛋白酶(Dispase)將皮膚上皮層與真皮層分離，兩皮層再分別 以0.25%胰蛋白酶及0.1%膠原蛋白酶(collagenase)作用，分離出單一的人類皮膚上皮層 角質細胞及人類真皮層纖維母細胞。分離出的皮膚細胞分別培養於適合細胞生長及分化 的細胞培養液中，並培養於37°C，5% C02培養箱内，每2-3天更換一次培養液。結果 顯示以中性蛋白酶及胰蛋白酶分離培養的人類皮膚角質細胞，在前三代培養階段，細胞 聚落形成率平均在20%以上，顯示前三代的人類皮膚角質細胞具有良好的增殖能力，如 圖2A所示人類皮膚角質細胞的細胞形態及聚落的形成。另外，以膠原蛋白酶分離皮膚 真皮層的人類推纖維母細胞亦有良好的生長速率及型態，如圖2B所示。 實施例3 :生物性皮膚替代物之製作 1·生物性真皮替代物之製作 無細胞豬皮基質於實驗前先用緩衝液(Dulbecco’s Phosphate-buffered saline，簡稱 DPBS)清洗數遍’裁成適當大小後放入培養盤孔槽中，於每一孔槽中加入含%牛胚胎 血清(fetal bovine serum)之 Dulbecco’s Modified Eagle Medium (簡稱 DMEM)的人類纖維 母細胞培養液浸泡待用。繼代培養中的人類纖維母細胞經由005%胰蛋白酶作用將細胞 自培養瓶中取下，計算細胞數目後，調整細胞濃度為1X105cell/cm2再植入豬皮基質網狀 真皮面(reticular dermis)。經過培養7天後，以掃瞄式電子顯微鏡(scanning electlOI1 microscope;簡稱SEM)及組織切片染色觀察人類纖維母細胞在基質内附著、增殖及遷移 的情形。結果發現已去細胞的豬真皮基質為架構，有利於人類纖維母細胞的附著、生長 及生物功能的表現。由SEM照片可以看到人類纖維母細胞不僅附著在基質表面，並藉 由偽足攀附在膠原纖維上，如圖3A所示。另外由H&E組織切片染色的結果可知，人 類纖維母細胞會從豬基質表面遷移延伸至基質内部，並攀附在膠原纖維上，細長的人類 $維母細胞會隨著天數的增加逐漸伸展，並開始分化進行基質的再建(rem〇deli 圖 3B所不，箭頭所指者即為人類皮膚纖維母細胞。 2·生物性皮唐替代物之製作The cells in the cortex and their debris are removed. The porcine dermal matrix from which the cells were removed was stored in 15% glycerol, and then sterilized by vigorously producing 3^0 for 4 hours, and the finished pigskin substrate was cold; the east was preserved (4). . By group g cutting, J wood, and eosin staining; referred to as H&E staining) to observe the collagen fiber structure and cell residue production in the porcine dermal matrix. The results showed that there were no cells in the porcine dermal matrix of the removed cells. Residues and still retain the original structure of pig dermis (Figure ΙΑ, 1B). Further, as shown in Fig. 1C, the porcine dermal matrix collagen fiber bundle was loosely entangled in a filamentous or bundle shape, indicating that the decellularized treatment did not destroy the fibrous structure of the stromal dermis layer. Example 2: Isolation and culture of human skin cells Human skin tissue obtained from a hospital was washed 3-5 times with a culture solution containing gentamicin (10 ug/ml), and then neutralized with 2.4 U/ml. Protease (Dispase) separates the skin epithelial layer from the dermis layer, and the two cortexes are separated by 0.25% trypsin and 0.1% collagenase to separate single human epithelial keratinocytes and human dermal fibroblasts. The isolated skin cells were separately cultured in a cell culture medium suitable for cell growth and differentiation, and cultured at 37 ° C in a 5% CO 2 incubator, and the culture medium was changed every 2-3 days. The results showed that the human skin keratinocytes isolated and cultured by neutral protease and trypsin had an average cell colony formation rate of more than 20% in the first three generations of culture, indicating that the first three generations of human skin keratinocytes have good proliferative capacity, as shown in Figure 2A. The cell morphology and colony formation of human skin keratinocytes are shown. In addition, human push fibroblasts which separate collagen dermis layers with collagenase also have good growth rates and patterns, as shown in Fig. 2B. Example 3: Preparation of biological skin substitutes 1. Preparation of biological dermal substitutes The cell-free pig skin matrix was washed several times with buffer (Dulbecco's Phosphate-buffered saline, DPBS) before being cut into appropriate sizes. Thereafter, the cells were placed in a culture well, and a human fibroblast culture solution containing Dulbecco's Modified Eagle Medium (abbreviated as DMEM) containing % fetal bovine serum was added to each well for use. The human fibroblasts in the subculture were removed from the culture flask via 005% trypsin. After calculating the cell number, the cell concentration was adjusted to 1×105 cells/cm2 and then implanted into the reticular dermis. . After 7 days of culture, the adhesion, proliferation and migration of human fibroblasts in the matrix were observed by scanning electron microscopy (SEM) and tissue section staining. It was found that the decellularized porcine dermal matrix was a framework that facilitated the attachment, growth and biological function of human fibroblasts. It can be seen from the SEM photograph that the human fibroblasts adhere not only to the surface of the substrate but also to the collagen fibers by pseudopods, as shown in Fig. 3A. In addition, the results of H&E tissue section staining show that human fibroblasts migrate from the surface of the porcine matrix to the inside of the matrix and adhere to the collagen fibers, and the elongated human virgin cells will gradually expand as the number of days increases. And began to differentiate for the reconstruction of the matrix (rem〇deli Figure 3B does not, the arrow refers to the human skin fibroblasts. 2) the production of biological Pi Tan substitutes
人類皮膚角質細胞利用0·05%胰蛋白酶將細胞自培養皿中取下，所得细胎於 後，調整細胞濃度為3X1Q6ceU/em2，再種植於本發明之生物性真皮替S 面，此時所用細胞培養液為促進皮膚角質細胞生長的第一 ρ白叙不3血π之角質細胞培養液，其主要為以MCDB_153培養液為主培養液。 1248357 化合物所配製成之水i液，較;i者t氣 荖、fi7八染色觀察t類皮膚脱細胞在生物性活真皮替代物上附 ΐ菩ΐ人類皮膚角f細胞在豬基質基底層不規則的凹&面上 2貝者生長，並沿著表面’形成一多層化的上皮層（圖 β( ^ · ”、、貝曰（Stl=Um comeum)、顆粒層(stratum granulosum)及棘狀細胞 f (Saturn spmosum)形成，而且表皮底層有排列規則組織化並呈矮小柱狀的基底 g (basal cell layer)，形成與人類皮膚組織結構(圖4C)十分相近的皮膚替代物。- 由於皮膚角質細胞生長分化，須有技術性的培養條件及方式，才能 备 f上f層二此實驗結果顯示，採用目前培養條件有利於角質細胞生長及分化，並可以ί 成全皮功能的生物性皮膚替代物。故依本發明方法所製得生物性真 膚 枓、皮膚基礎研九、樂物牙透性測試系統或化妝品抗紫外線檢測系統之用。 實施例4 :生物性活皮替代物動物測試 2 8週大之裸鼠(BALB/c-nu)以ketamine麻醉之後，以銳剪進行全皮層開創約15对 =λ·ί· ’植入物^真皮替代物及生物性活皮替代物，以縫線固定，覆蓋 上^ucidm，以3 M Tegaderm將整個傷口包住，最後再以3M Coban™繃帶纏繞。2l 天後解開傷口，進行組織切片觀察，如圖5所示，未植入基質之傷口沒有角質声覆芸， fit佳’植人活真皮及活皮替代物之傷口癒合程度、角質層皮膚層數均較只^入^基 【圖式簡單說明】 圖1A為正常豬皮膚之組織切片H&E染色圖(2〇〇χ): 圖1B為無細胞豬真皮基質之組織切片圖(2〇οχ) 圖1C為豬皮基質電子顯微鏡圖(250X)。 圖2A為人類皮膚纖維母細胞照片（2〇〇χ): 圖2B人類皮膚角質細胞(200X)。 圖3A為本發明之生物性真皮替代物電子顯微鏡圖(ιοοοχ); 圖3B為本發明之生物性真皮替代物組織切片h&e染色圖(200X)。 圖4A為本發明之生物性皮膚替代物電子顯微鏡圖(LQoox) 圖4B為本發明之生物性皮膚替代物組織切片H&E染色圖(2⑻X) 1248357The human skin keratinocytes were removed from the culture dish by using 0.05% trypsin, and the resulting fine fetus was adjusted to a cell concentration of 3×1Q6ceU/em2, which was then planted in the biological dermis of the present invention. The cell culture fluid is a keratinocyte culture medium which promotes the growth of keratinocytes of the skin, and is mainly a culture medium of MCDB_153 culture medium. 1248357 compound prepared by the water i liquid, compared with i; t gas 荖, fi7 eight staining observation t type skin decellularization in biological live dermal substitute attached to ΐ ΐ ΐ human skin angle f cells in the porcine matrix basal layer Irregular concave & surface 2 shells grow and form a multi-layered epithelial layer along the surface (Fig. β(^ · 、, 曰 曰 (Stl=Um comeum), stratum granulosum) And the spine cell f (Saturn spmosum) is formed, and the bottom layer of the epidermis has a basal cell layer which is regularly organized and has a short columnar shape, forming a skin substitute which is very similar to the human skin tissue structure (Fig. 4C). - Due to the growth and differentiation of skin keratinocytes, technical conditions and methods must be used to prepare the f layer. The results of this experiment show that the current culture conditions are beneficial to the growth and differentiation of keratinocytes, and can be used as a whole skin function. A skin substitute. Therefore, it can be used in the method of the present invention to prepare a biological skin lotion, a skin foundation research, a music tooth permeability test system or a cosmetic anti-ultraviolet detection system. Example 4: Biological living skin substitute move Substance test 2 Eight-week-old nude mice (BALB/c-nu) were anesthetized with ketamine, and the whole cortex was created with sharp scissors for about 15 pairs = λ·ί· 'implants ^ dermal substitutes and biological live skin substitutes The object was fixed with suture, covered with ^ucidm, wrapped around the entire wound with 3 M Tegaderm, and finally wrapped with 3M CobanTM bandage. After 1 day, the wound was unwound and examined by tissue section, as shown in Figure 5, There is no horny sound in the wound implanted in the matrix. The degree of wound healing and the number of skin layers in the stratum corneum are better than those of the skin and the skin substitute. The figure is simple. Figure 1A is normal. H&E staining of pig skin tissue (2〇〇χ): Figure 1B is a histogram of acellular porcine dermal matrix (2〇οχ) Figure 1C is an electron micrograph of a pig skin matrix (250X). Human skin fibroblast photograph (2〇〇χ): Figure 2B Human skin keratinocytes (200X). Figure 3A is an electron micrograph of a biological dermal substitute of the present invention (ιοοο); Figure 3B is a biological dermis of the present invention Substitute tissue section h&e staining map (200X). Figure 4A is a biological skin substitute of the present invention. Was electron micrograph (LQoox) in FIG. 4B biological skin substitutes of the present invention, the tissue sections were H & E staining pattern (2⑻X) 1248357
切片職染色聊Χ)。其中a為角質層;b為上皮層;C 圖圖圖圖 未植入無細胞豬基質之組織切片H&E染色圖(200X) β為植入無細胞豬基質之組織切片H&E染色圖(200X) 植入生物性真皮替代物之組織切片Η&Ε染色圖(200Χ) 為植入生物性皮膚替代物之組織切片Η&Ε染色圖(200Χ) 【主要元件符號說明】 、申請專利範圍： •申請專利範圍 1. 種生物性真皮替代物之製作方法，包括下列步驟： (1) 無細胞豬皮基質放入培養盤孔槽中，於每一孔槽中加入添加血清之DMEM的人 類皮膚纖維母細胞培養液浸泡待用； (2) 將人類皮膚纖維母細胞植入於(1)之無細胞豬皮基質網狀真皮面，待纖維母細胞 在基質内及表面附著及伸展即得。 2. 如申請專利範圍第i項所述之方法，其中之人類皮膚纖維母細胞可為初代培養細胞、 細胞株或基因操控之細胞而得。 3. —種生物性皮膚替代物之製作方法，包括下列步驟： (1) 生物性真皮替代物之製作 (i)無細胞豬皮基質放入培養盤孔槽中，於每一孔槽中加入添加血清之DMem的 人類皮膚纖維母細胞培養液浸泡待用； (η)將人類皮膚纖維母細胞植入於①之無細胞豬皮基質網狀真皮面，待纖維母細 胞在基質内及表面附著及伸展即得； 、 (2) 生物性皮膚替代物之製作 細胞分裂生長 ⑼繼以糊第-階段肖f細胞培養液加人添加血清之纖維母細胞 第二階段角質細胞培養液，提供角質細胞多層分化； "彳于 ⑽再利用第二階段㈣細胞培養液再添加娜子及▲清馳成之第三階段 ①利用步驟⑴所得之真錢代物為基質，將人類皮膚角質細胞種植於真皮替代 物ίϋΐίί層面表面，並以第—階段不含血清之角f細胞培養液提供角質 之 角 質細胞培養液，提供角質細胞分化形成角質層即得Slice job dyeing chat). Where a is the stratum corneum; b is the epithelial layer; C is a non-cellular porcine matrix tissue section H&E staining map (200X) β is the tissue section of the acellular porcine matrix H&E staining (200X) Tissue section 植入 & Ε staining map (200Χ) implanted with biological dermal substitutes. Tissue section Η & Ε staining map for implanted biological skin substitutes (200Χ) [Main component symbol description], patent application scope : • Patent Application 1. A method for making a biological dermal substitute, comprising the following steps: (1) Acellular pig skin matrix is placed in a culture dish, and serum-added DMEM is added to each well. Soaking the skin fibroblast culture medium for use; (2) implanting human skin fibroblasts into the reticular dermis of the cell-free pigskin matrix of (1), and attaching and stretching the fibroblasts in the matrix and on the surface . 2. The method of claim i, wherein the human skin fibroblasts are derived from primary cultured cells, cell lines or genetically manipulated cells. 3. A method for preparing a biological skin substitute, comprising the following steps: (1) preparation of a biological dermal substitute (i) a cell-free pig skin matrix is placed in a culture tray hole, and is added to each well. Human dermal fibroblast culture medium supplemented with serum DMem is used for immersion; (η) Human skin fibroblasts are implanted into the reticular dermal surface of cell-free pig skin matrix, and the fibroblasts are attached to the matrix and surface. And stretching to obtain;, (2) biological skin substitute production cell division growth (9) followed by paste phase-stage xiao f cell culture fluid plus human serum-added fibroblast second stage keratinocyte culture medium to provide keratinocytes Multi-layer differentiation; "彳(10) Reuse the second stage (4) Cell culture solution and then add Nazi and ▲ Qingchicheng to the third stage 1 Use the real money substitute obtained in step (1) as the substrate to plant human skin keratinocytes in the dermis Substitute ίϋΐίί surface, and provide keratinocyte keratinocyte culture medium with the first stage serum-free angle f cell culture medium to provide keratinocyte differentiation to form the stratum corneum.