201200593 六、發明說明： 1 【發明所屬之技術領域】 本發明關於一種細胞培養盤，尤指具有特定排列方 式之複數個生醫材料之多槽體之細胞培養盤，其可用於 觀察細胞行為及篩選生醫材料。 、 、 【先前技術】 生醫材料泛指可用以取代活體部分組織而可直接 和活體接觸並執行該組織之功能者，此類物質多屬反應 鈍性物質，不易和身體有免疫反應的產生，目前較常^ 使用的生醫材料’大致可分為：高分子（p〇lymer )、複 合材料（composite)、金屬（metal)、合金（aU〇y)或其 組合。而臨床方面的生醫材料又可再分為二大類：合^ 材料和天然材料，其可應用於各種植入物或輔助材^， 例如：人工心血管、縫線等，因此對於各種不同生醫材 料於人體細胞、組織中的研究隨著科技進步而顯得越益 重要。 ’ 1 目前雖已有許多研究著眼於生醫材料對生物體外 之細胞行為的影響’例如：Young等人研究發現乙稀乙 烯醇共聚物（poly ( vinyl alcohol-co-ethylene )，EVAL ) 可以誘導神經幹細胞分化[Young et el.，说26 (2005) 4291-4299] ; Chen等人研究發現聚乳酸_甘醇 酸（PLGA)和聚偏二氟乙烯（PVDF)等生醫材料所培 養出的牙胚細胞具有不同的形態[Chen et al.，Part A 83 (2007) 241-248 ] 〇 而Daniel G.等人在2005年及2009年提出，利用 高傳輸量的方法（high throughput method)在玻璃板上 201200593 製造出一個高分子微陣列，並將人類胚胎幹細胞培養於 此微陣列後，觀察細胞生長。[Daniel G. et al·， Biomaterials 26 ( 2005 ) 4892-4897, and Daniel G et al.5 Comb Chen High Troughput Screen. 12 (2009)554-561]; 雖此方式可以快速掃描出細胞與不同摻混比例之生醫 材料間的行為’但是此技術在一般實驗室並不容易操 作，無法將不同種之生醫材料依特定方式排列並篩選 之。201200593 VI. Description of the Invention: 1 Technical Field of the Invention The present invention relates to a cell culture tray, and more particularly to a multi-tank cell culture tray having a plurality of biomedical materials in a specific arrangement, which can be used for observing cell behavior and Screen biomedical materials. [Previous technique] Biomedical materials generally refer to those that can be used to replace part of living tissue and can directly contact the living body and perform the function of the tissue. Such substances are mostly reactive blunt substances and are not easily immune to the body. The biomedical materials currently used more generally can be roughly classified into: a polymer (p〇lymer), a composite (composite), a metal (metal), an alloy (aU〇y), or a combination thereof. The clinical aspects of biomedical materials can be further divided into two categories: materials and natural materials, which can be applied to various implants or auxiliary materials, such as artificial cardiovascular, suture, etc., so for different kinds of students The research of medical materials in human cells and tissues has become more and more important with the advancement of science and technology. '1 Although many studies have focused on the effects of biomedical materials on the behavior of cells outside the body', for example, Young et al. found that poly(vinyl alcohol-co-ethylene) (EVAL) can induce Neural stem cell differentiation [Young et el., 26 (2005) 4291-4299]; Chen et al. found that polylactic acid-glycolic acid (PLGA) and polyvinylidene fluoride (PVDF) and other biomedical materials were cultured. Tooth germ cells have different morphologies [Chen et al., Part A 83 (2007) 241-248] and Daniel G. et al. proposed in 2005 and 2009 using the high throughput method. On the glass plate 201200593, a polymer microarray was fabricated, and human embryonic stem cells were cultured in the microarray to observe cell growth. [Daniel G. et al., Biomaterials 26 (2005) 4892-4897, and Daniel G et al. 5 Comb Chen High Troughput Screen. 12 (2009) 554-561]; although this method can quickly scan out cells and different blends Behavior between mixed proportions of biomedical materials' However, this technique is not easy to operate in general laboratories, and it is not possible to arrange and screen different types of biomedical materials in a specific manner.
綜上所述，發展一套可快速篩選具不同特性生醫材 料之細胞培養盤，並可同時觀察不同特性生醫材料對細 胞生長的影響之方法，實為一亟待開發的課題。 【發明内容】 有鑑於先前技術之缺失，本發明之主要目的為提供 -細胞培養盤，其係為-快速_觀f材料對細胞影響 胞培養盤’其具有不同特性之生醫材料於其複數個 槽體=前述不同特性之生醫材料係以特殊規則排列。 培養盤觀察細胞行為的方法，發明細胞 種不同特性之生醫材料，並同時二 於各 =行為，—不-===: 培養=:二的方:在==用本發明細胞 =料對細胞行為的影響後，再經由:== 適合的生醫材料進行後續研究或細胞培養。察方法&出 含·· 二發莫明提供-細胞培養盤，其包 3权處理之細胞培養盤’其具有複數個槽體；複 201200593 材料’其係置放於前述複數個槽體中.其中前 ;生:4醫，包含:親疏水性材料二 且前材料、掺混材料、水膠材料或其組合； 盤：=個生醫材料係依下列方式排列於細胞培養 帶電性材料係依正負、負正或其組== ，剛述生物可降解性材料係依其生物可降解性之強弱 ，序排W述摻混材料係依掺⑧比例之高低依序排 列，且前述複數個生醫材料係可以行或列之方式排列於 前述細胞培養盤上。 、 較佳地，前述細胞培養盤係用以觀察細胞行為。 較佳地，前述細胞培養盤係用以筛選生醫材料。 較佳地，前述親疏水性材料包含：聚偏二氟乙烯 (poly ( vinylidene difluoride )，下稱 PVDF)或聚乙烯 (poly (ethylene),下稱PE)、乙烯乙烯醇共聚物_44 ((vinyl alcohol-co-ethylene) -44，下稱 EVAL-44)、乙 稀乙稀醇共聚物-38(( vinyl alcohol-co-ethylene)-38，下 稱EVAL-38 )、乙烯乙烯醇共聚物_32 ( ( vinyl alcohol-co-ethylene) -32，下稱 EVAL-32)、乙烯乙烯醇 共聚物-27 (( vinyl alcohol-co-ethylene ) -27,下稱 EVAL-27)、聚乙烯醇（poly ( vinyl alcohol),下稱 PVA) 或其組合。 較佳地’前述帶電性材料包含：聚丙稀胺（p〇ly (allylamine)，下稱 PAA)、聚離胺酸（p〇iy( D-lysine)， 下稱PDL)、聚丙烯醯胺（Poly( acrlacid)，下稱PAAc)、 玻尿酸（Hyaluronic acid，下稱HA)或其組合。 較佳地，前述生物可降解性材料包含：聚甘醇酸 (Poly( glycolic acid)，下稱 PGA)、聚乳酸（p〇ly( lactic 201200593 acid ),下稱PLLA )、聚乳酸-甘醇酸（p〇iy (DL-lactide-co-glycolide),下稱 PLGA)、聚[(穴）-3-經基丁酸](poly[(穴）-3-hydroxybutyric acid],下稱 PHB)或其組合。 較佳地，前述摻混材料包含：聚己酸内酯（P〇ly (caprolactone)，下稱PCL)、甲殼素或其組合。 較佳地，前述水膠材料包含：聚乙烯醇（p〇ly( vinyl alcohol ),下稱PVA )、聚甲基丙晞酸乙g旨（p〇ly (2-hydroxyethyl methacrylate)，下稱 pHEMA)、聚甲 基丙缔酸丙醋（poly ( 2-hydroxypropyl methacrylate ), 下稱pHPMA)、甲殼素（Chitosan)或其組合。 較佳地，前述細胞培養盤進一步包含一組織培養用 聚苯乙烯（Tissue-culture polystyrene，下稱 TCPS )、聚 甲基丙浠酸甲g旨（polymethylmethacrylate, 下稱 PMMA)或其組合。 較佳地，前述且前述生醫材料之排列方式係為··第 一列自左至右係為：聚偏二氟乙烯（PVDF)或聚乙烯 (PE )、乙烯乙烯醇共聚物-44 ( EVAL-44 )、乙烯乙烯 醇共聚物-38 ( EVAL-38 )、乙烯乙烯醇共聚物-32 (EVAL-32)、乙烯乙烯醇共聚物-27(EVAL-27)及聚 乙烯醇（PVA);第二列自左至右係為：聚丙烯胺（PAA)、 聚離胺酸（PDL)、聚丙烯醯胺（PAAc)'玻尿酸（HA)、 組織培養用聚苯乙烯（TCPS )、聚甲基丙烯酸丙酯 (ΡΗΡΜα);第三列自左至右係為：聚甘醇酸（PGA)、 聚乳酸（PLLA)、聚乳酸-甘醇酸（PLGA)、聚[(/〇 -3-說基丁酸](ΡΗΒ)、聚曱基丙烯酸甲酯（ΡΜΜΑ)、聚甲 基内烯酸丙酯（ρΗΡΜΑ );第四列自左至右係為：1〇〇 死的聚己酸内酯（PCL)、80%的聚己酸内酯（PCL )和 201200593 20%的甲殼素、60%的聚己酸内酯（PCL)和40%的甲 殼素、40%的曱殼素和60%的聚己酸内酯（PCL)、20 %的聚己酸内酯（PCL)和80%的甲殼素、100%的曱 殼素。 本發明之又一·目的為提供一種利用本發明細胞培 養盤觀察細胞行為之方法，其包含：（a)取一本發明細 胞培養盤；（b)將細胞培養於前述細胞培養盤；（c)觀 察前述步驟（b )之細胞行為。 本發明之再一目的為提供一種利用本發明細胞培 養盤篩選生醫材料之方法，其包含：（a)取一本發明細 胞培養盤；（b)將細胞培養於前述細胞培養盤；（c)觀 察前述步驟（b)之細胞行為；（d)篩選前述生醫材料。 較佳地，前述細胞係包含原生細胞、原核細胞、植 物細胞、動物細胞、真菌細胞或其組合。 較佳地，前述細胞係為神經幹細胞、人類間葉幹細 胞、人類牙胚細胞或其組合。 較佳地，前述步驟（c)之觀察係透過免疫染色法 (Immunostaining method)、穿透光光學顯微鏡、掃描 式電子顯微鏡、穿透式電子顯微鏡或其組合。 綜上所述，本發明之細胞培養盤具有不同特性之生 醫材料於其複數個槽體中，且前述不同特性之生醫材料 係以特殊規則排列，並可利用此細胞培養盤進行細胞行 為觀察及篩選生醫材料。 【實施方式】 本發明透過將各種不同特性之生醫材料分成各種 群組，如：親疏水性、表面帶電性、生物可降解性、摻 混材料、水膠，而後再依特殊規則排列之，分別置放於 201200593 未經處理之細胞培養盤上，得本發明細胞培養盤。將前 述生醫材料置放於未經處理之細胞培養盤上之方法 用，何習知之方法’包括：塗佈（coating)、電聚嫁接 (plasma graft)、放置薄臈等方法。 本發明之細胞培養盤之槽體個數可為一般市面上 可購得之細胞培養盤槽體個數，即為96、48、24 12 或1等槽體個數，亦可視需要開發任何槽體個數之細胞 培養盤。 性材^發細胞培養盤上之生醫材料係包含：親疏水 、生物可降解性材料、摻混材料、 合；前述生醫材料係以下列規則排列： 材料係依其親疏水性之強弱依序排列 =係依正負、貞正或其組合之方式排列；生物 其生物可降解性之強弱依序排列;掺混材料 係二:;二之尚低依序排列;且前述複數個生醫材料 '、仃或列之方式排列於前述細胞培養盤上。 _ 要符及排列方式可視情況調整，只 鍵和水㈣料之親疏水性係指分子透過氫 明暫鍵結，則該分子之親水性越強，而本發 或含，但不限於：聚偏二氟乙稀（卿） 乙烯乙嫌iT E)、6烯6埽醇共聚物_44(EVAL-44)、 -32 (EVA3聚物(Μ。38)、乙烯乙烯醇共聚物 聚乙浠醇醇共聚物·27(Ε·27)、 前述乙埽乙_共聚物（EVAL)後方編號意為其 201200593 結構（如化學式（i)所千、士 , 整體結構的莫耳百分比（—％)，當此 (EVAL)疏水性越高；）表不乙婦乙埽醇共聚物 結構式（I):In summary, the development of a method for rapidly screening cell culture plates with different characteristics of biomedical materials and simultaneously observing the effects of different characteristics of biomedical materials on cell growth is a subject to be developed. SUMMARY OF THE INVENTION In view of the deficiencies of the prior art, the main object of the present invention is to provide a cell culture tray which is a fast-acting material that affects cells in a cell culture tray, which has different characteristics of biomedical materials in its plural The tanks = the different characteristics of the above-mentioned biomedical materials are arranged by special rules. A method of observing cell behavior in a culture dish, inventing a biomedical material with different characteristics of the cell species, and simultaneously at the same time = each behavior, - not -===: culture =: two squares: at == using the cell of the invention = material pair After the effects of cell behavior, follow-up studies or cell cultures are performed via: == appropriate biomedical materials. The method of <exhibition····································································· Among them; raw: 4 doctors, including: pro-hydrophobic materials and pre-materials, blending materials, hydro-adhesive materials or combinations thereof; disc: = a biomedical material is arranged in the following manner in cell culture charged materials Positive or negative, negative or its group ==, just described the biodegradable material is based on the strength of its biodegradability, the order of the blended materials is arranged according to the ratio of the ratio of 8, and the above multiple The medical material can be arranged in rows or columns on the aforementioned cell culture tray. Preferably, the aforementioned cell culture tray is used to observe cell behavior. Preferably, the aforementioned cell culture tray is used to screen biomedical materials. Preferably, the aforementioned hydrophilic and hydrophobic material comprises: poly(vinylidene difluoride, hereinafter referred to as PVDF) or polyethylene (poly (ethylene), hereinafter referred to as PE), ethylene vinyl alcohol copolymer _44 ((vinyl) Alcohol-co-ethylene) -44, hereinafter referred to as EVAL-44), vinyl alcohol-co-ethylene-38 (hereinafter referred to as EVAL-38), ethylene vinyl alcohol copolymer_ 32 ((vinyl alcohol-co-ethylene) -32, hereinafter referred to as EVAL-32), ethylene vinyl-co-ethylene -27 (hereinafter referred to as EVAL-27), polyvinyl alcohol ( Poly (vinyl alcohol), hereinafter referred to as PVA) or a combination thereof. Preferably, the aforementioned chargeable material comprises: polyallylamine (plyly (allylamine), hereinafter referred to as PAA), polyphosphoric acid (p〇iy (D-lysine), hereinafter referred to as PDL), and polypropylene decylamine ( Poly (acrlacid), hereinafter referred to as PAAc), hyaluronic acid (hereinafter referred to as HA) or a combination thereof. Preferably, the aforementioned biodegradable material comprises: poly(glycolic acid) (hereinafter referred to as PGA), polylactic acid (p〇ly (lactic 201200593 acid), hereinafter referred to as PLLA), polylactic acid-glycol Acid (p〇iy (DL-lactide-co-glycolide), hereinafter referred to as PLGA), poly[(hole)-3-butyric acid] (poly[(hole)-3-hydroxybutyric acid], hereinafter referred to as PHB) Or a combination thereof. Preferably, the aforementioned blending material comprises: poly(caprolactone) (hereinafter referred to as PCL), chitin or a combination thereof. Preferably, the water-repellent material comprises: polyvinyl alcohol (p〇ly (vinyl alcohol), hereinafter referred to as PVA), and poly(2-hydroxyethyl methacrylate), hereinafter referred to as pHEMA. ), poly(2-hydroxypropyl methacrylate, hereinafter referred to as pHPMA), chitosan or a combination thereof. Preferably, the cell culture tray further comprises a tissue-culture polystyrene (hereinafter referred to as TCPS), polymethylmethacrylate (hereinafter referred to as PMMA) or a combination thereof. Preferably, the foregoing and the arrangement of the aforementioned biomedical materials are: · The first column from left to right is: polyvinylidene fluoride (PVDF) or polyethylene (PE), ethylene vinyl alcohol copolymer - 44 ( EVAL-44), ethylene vinyl alcohol copolymer-38 (EVAL-38), ethylene vinyl alcohol copolymer-32 (EVAL-32), ethylene vinyl alcohol copolymer-27 (EVAL-27) and polyvinyl alcohol (PVA) The second column from left to right is: polyacrylamide (PAA), polylysine (PDL), polyacrylamide (PAAc) 'hyaluronic acid (HA), polystyrene for tissue culture (TCPS), poly Propyl methacrylate (ΡΗΡΜα); the third column from left to right is: polyglycolic acid (PGA), polylactic acid (PLLA), polylactic acid-glycolic acid (PLGA), poly[(/〇-3 - said butylbutyric acid] (ΡΗΒ), polymethyl methacrylate (ΡΜΜΑ), polymethyl methacrylate (ρΗΡΜΑ); the fourth column from left to right is: 1 聚 dead polyhexanoic acid Lactone (PCL), 80% polycaprolactone (PCL) and 201200593 20% chitin, 60% polycaprolactone (PCL) and 40% chitin, 40% chitin and 60% polycaprolactone (PCL), 20% polycaprolactone (PCL) And 80% chitin, 100% chitin. Another object of the present invention is to provide a method for observing cell behavior using the cell culture tray of the present invention, comprising: (a) taking a cell culture tray of the invention; (b) culturing the cells in the aforementioned cell culture tray; (c) observing the cell behavior of the aforementioned step (b). A further object of the present invention is to provide a method for screening biomedical materials using the cell culture tray of the present invention, comprising: (a) taking a cell culture tray of the invention; (b) culturing the cells in the aforementioned cell culture tray; (c) observing the cell behavior of the aforementioned step (b); (d) screening the aforementioned biomedical material. Preferably, the foregoing The cell line comprises a primary cell, a prokaryotic cell, a plant cell, an animal cell, a fungal cell or a combination thereof. Preferably, the aforementioned cell line is a neural stem cell, a human mesenchymal stem cell, a human tooth germ cell or a combination thereof. Preferably, the foregoing The observation of the step (c) is by an immunostaining method, a penetrating light optical microscope, a scanning electron microscope, a transmission electron microscope or a combination thereof. The cell culture tray of the invention has different characteristics of the biomedical material in a plurality of tanks, and the above-mentioned different characteristics of the biomedical materials are arranged by special rules, and the cell culture tray can be used for cell behavior observation and screening of biomedical materials. [Embodiment] The present invention divides biomedical materials of various characteristics into various groups, such as: hydrophobicity, surface chargeability, biodegradability, blending materials, water gel, and then arranged according to special rules. The cell culture plates of the present invention were obtained by placing them on an untreated cell culture plate of 201200593, respectively. A method of placing the above-mentioned biomedical material on an untreated cell culture tray, and a conventional method includes: coating, plasma grafting, and placing a thin crucible. The number of tanks of the cell culture tray of the present invention may be the number of cell culture trays available on the market, that is, the number of tanks of 96, 48, 24 12 or 1, etc., and any tank may be developed as needed. A number of cell culture plates. The biomedical materials on the cell culture tray include: pro-hydrophobic, biodegradable materials, blending materials, and combinations; the aforementioned biomedical materials are arranged according to the following rules: The materials are in accordance with the strength of their hydrophilicity Arrangement = is arranged according to positive and negative, 贞 positive or a combination thereof; biological biodegradability is arranged in order; blending material is two:; second is still low in order; and the above plurality of biomedical materials' Arranged on the aforementioned cell culture tray in the form of sputum or column. _ The arrangement and arrangement can be adjusted according to the situation. Only the hydrophilicity of the bond and the water (four) material refers to the temporary bond of the molecule through the hydrogen, so the hydrophilicity of the molecule is stronger, and the present or the inclusion is not limited to: Difluoroethylene (Q) Ethylene B. iT E), 6-ene 6-sterol copolymer _44 (EVAL-44), -32 (EVA3 polymer (Μ.38), ethylene vinyl alcohol copolymer polyacetate The alcohol copolymer · 27 (Ε · 27), the aforementioned ethylene glycol copolymer (EVAL) is numbered after its 201200593 structure (such as the chemical formula (i) thousand, the total structure of the molar percentage (-%), When this (EVAL) is more hydrophobic;) Table Ethyl Acetate Copolymer Structure (I):
本發明帶電性材料帶 性，當㈣於細胞❷養赫分子表面帶電 (I) 其帶電性排列’其 列，例如：「正正負負」」或n將正1或負電連續排 負交錯」排列，例如:「i負負2正正「」等;亦可為「正 其他各種組合，例如：「正、正」、負正負正」；或是 為示例性表示，而;^以=1性材料之排列方式僅 性材料包含，但不限於:聚丙稀胺 i酸上==叫聚罐胺(叫玻 本發明生物可降解性材料之生物可降解性係指生 醫材料在有足_濕度、氧氣含量與適當環i條= 可被微生物或生物體所代謝分解產生水、二氧化碳或f 炫的特性，當前述_分解之時間越短，顺述生物可 降解性越強；本發明生物可降解性材料包含，但不限 於.聚甘醇酸（PGA)、聚乳酸（pLLA)、聚乳酸-甘醇 酸（PLGA)、聚[(/〇 -3-經基丁酸](PHB)或其組合。 201200593 本發明摻混材料係指將二種以上不同材料依照不 同比例相互混合後而得；本發明摻混材料包含，但不限 於：聚己酸内酯（PCL)、甲殼素或其組合。 本發明水膠材料係指高分子鍊段經由化學或物理 交聯後所形成之可吸水性膠體；本發明水膠材料包含， 但不限於：聚乙烯醇（PVA )、聚甲基丙烯酸乙酯 (pHEMA )、聚甲基丙烯酸丙酯（pHPMA )、甲殼素 (Chitosan )。 本發明細胞培養盤可進一步包含一組織培養用聚 苯乙烯（TCPS)，該生醫材料係為電漿改質過之聚苯乙 烯，於較佳實施態樣中係作為對照組使用。當所使用細 胞培養盤之材料為組織培養用聚苯乙烯（TCPS)時， 即不需再塗佈任何生醫材料而直接培養細胞即可；此 外，亦可進一步包含聚曱基丙烯酸甲酯（PMMA)，其 係用於骨科手術時之骨水泥。 本發明之細胞培養盤100之較佳實施態樣之一可參 考第一圖（此係以24個槽體之細胞培養盤為例），該細 胞培養盤100包含：複數個槽體；複數個生醫材料，其 係置放於前述複數個槽體中；其中前述複數個生醫材料 係包含：親疏水性材料10、帶電性材料20、生物可降 解性材料3 0、推混材料4 0、水膠材料5 0及組織培養用 聚苯乙烯（TCPS)及聚曱基丙烯酸甲酯（PMMA)。其 中前述各種生醫材料排列依下列方式： 第一列係為前述親疏水性材料10，依其親水性自 弱至強、由左至右依序排列為：聚偏二氟乙烯（PVDF) 或聚乙烯（PE )、乙烯乙烯醇共聚物-44 ( EVAL-44 )、 乙烯乙烯醇共聚物_38 ( EVAL-38)、乙烯乙烯醇共聚物 -32 ( EVAL-32 )、乙烯乙烯醇共聚物-27 ( EVAL-27 )及 11 201200593 聚乙埽醇（p VA ); 第二列係為前述帶電性材料2〇，依其帶φ 電、正電、負電、負電」的順序、由左以「正 =材料排列為:聚丙烯胺(ΡΑΑ)、聚離胺二’ 汆丙烯醯胺（PAAc)、玻尿酸（HA) ; ^ 〇L)> 第三列係為前述生物可降解性材料3〇， 解性自強至弱、由左至右依序排列為依聚生^降 (PGA)、聚乳酸（PLLA)、聚乳酸·甘_ ，酸 聚[(及）-3-經基丁酸](PHB ); 、 第四列係為聚己酸内酯（PCL)和曱殼素、言_ 料（摻混後即為本發明所述之摻混材料4〇)'，、=種材 例之高低依序、由左至右排列為：1〇〇%的/二2比 (PCL )、_的聚己酸内酯（PCL )和2〇%的H酉旨 60%的聚己酸内酯（PCL)和40%的甲殼素、4〇f、、 殼素和60%的聚己酸内醋（PCL)、2〇%的聚〇内: (PCL)和80%的甲殼素、1〇〇%的甲殼素； ·曰 最右行係為前述不同種類之水膠材料5〇， 下依序為：聚乙烯醇（PVA)、聚曱基丙烯酸^ = (pHEMA )、^^曱基丙稀酸丙酉旨（pHpjyfA )、田± (Chitosan) ； T 喊素 而組織培養用聚苯乙烯（TCPS)排列於破尿 (HA)之右方，聚甲基丙烯酸曱酯（PMMa)排列二 聚[(/〇 -3-經基丁酸](PHB)之右方。 '; 本發明之不同生醫材料各有多種特性，而非僅具 前述分類之特性，不同類之生醫材料亦可具有其他^ 特性，此僅為示例性分類而非用以侷限本發明之實施= 樣；舉例而言，本發明細胞培養盤1〇〇中，聚乙西= (PVA)屬於親疏水性材料1〇中的疏水性材料，亦g 12 201200593 於水膠材料50之一；而甲殼素（Chitosan)在本發明細 胞培養盤100中作為摻混材料40之一’亦屬於水膠材 料50之一。因此，適用於本發明之生醫材料當其具有 多種特性時，其排列方式並不僅限於第一圖之實施態 樣，而可視情況而調整，亦即，在一細胞培養盤中，可 有二個以上的槽體置放相同的生醫材料。 利用本發明細胞培養盤1〇〇觀察細胞行為之方 法，其包含下列步驟：（a)取本發明細胞培養盤1〇〇 ; (b)將細胞培養於前述細胞培養盤100; (c)觀察前The charged material of the present invention is band-like, when (4) the surface of the cell ❷ 赫 分子 分子 ( ( ( ( ( ( I I I I I I I I I , , , , , I I 表面 表面 表面 表面 表面 表面 表面 表面 表面 表面 表面 表面 表面 表面 ❷ ❷ ❷ ❷ ❷ For example: "i negative and negative 2", etc.; can also be "positive various combinations, such as: "positive, positive", negative positive and negative positive"; or for exemplary representation, and ^^ to = The arrangement of materials is only included in the material, but not limited to: polyacrylamide i acid == called poly-tank (the biodegradability of the biodegradable material of the invention) refers to the biomedical material in the presence of _ humidity , oxygen content and appropriate ring i = can be metabolized by microorganisms or organisms to produce water, carbon dioxide or fluffy characteristics, when the aforementioned _ decomposition time is shorter, the biodegradability is stronger; the bioreactor of the present invention Degradable materials include, but are not limited to, polyglycolic acid (PGA), polylactic acid (pLLA), polylactic acid-glycolic acid (PLGA), poly[(/〇-3-butyric acid) (PHB) or The combination of the present invention 201200593 The mixed material of the present invention means that two or more different materials are mixed with each other according to different ratios. The blending material of the present invention comprises, but is not limited to, polycaprolactone (PCL), chitin or a combination thereof. The water gel material of the present invention refers to a polymer segment formed by chemical or physical crosslinking. Water-absorbent colloid; the hydrocolloid material of the present invention comprises, but is not limited to, polyvinyl alcohol (PVA), polyethyl methacrylate (pHEMA), polypropyl methacrylate (pHPMA), chitin (Chitosan). The cell culture tray may further comprise a polystyrene (TCPS) for tissue culture, which is a plasma-modified polystyrene, and is used as a control group in a preferred embodiment. When the material of the culture tray is polystyrene (TCPS) for tissue culture, the cells may be directly cultured without applying any biomedical material; in addition, polymethyl methacrylate (PMMA) may be further included. It is used for bone cement during orthopedic surgery. One of the preferred embodiments of the cell culture tray 100 of the present invention can be referred to the first figure (this is an example of a cell culture tray of 24 tanks), which is a cell culture tray. 100 contains: a plurality of slots a plurality of biomedical materials placed in the plurality of troughs; wherein the plurality of biomedical materials comprise: a hydrophilic and hydrophobic material 10, a charged material 20, a biodegradable material 30, and a push-mixed material 4 0, hydrocolloid material 50 and polystyrene (TCPS) for tissue culture and polymethyl methacrylate (PMMA). The above various biomedical materials are arranged in the following manner: The first column is the aforementioned hydrophilic and hydrophobic material 10, According to its hydrophilicity, it is weak to strong, and is arranged from left to right in order: polyvinylidene fluoride (PVDF) or polyethylene (PE), ethylene vinyl alcohol copolymer-44 (EVAL-44), ethylene vinyl alcohol copolymerization. _38 (EVAL-38), ethylene vinyl alcohol copolymer-32 (EVAL-32), ethylene vinyl alcohol copolymer-27 (EVAL-27) and 11 201200593 polyacetone (p VA ); second column For the above-mentioned charging material 2〇, in the order of φ electric, positive, negative, negative, in the order of left, “positive = material arrangement: polyacrylamide (ΡΑΑ), polyamine amine ' 汆 醯 醯 醯(PAAc), hyaluronic acid (HA); ^ 〇L)> The third column is the aforementioned biodegradable material, From strong to weak, from left to right, sequentially arranged as PGA, polylactic acid (PLLA), polylactic acid·Gan_, acid poly[(and)-3-butyric acid] (PHB) The fourth column is polycaprolactone (PCL) and chitin, which is the blending material of the present invention after mixing, and = the height of the seed material Order, from left to right: 1〇〇% / 2 2 ratio (PCL), _ polycaprolactone (PCL) and 2% H 酉 60% polycaprolactone (PCL) ) and 40% of chitin, 4〇f, crustin and 60% polycaprolactone (PCL), 2〇% of polypeptone: (PCL) and 80% of chitin, 1〇〇% Chitin; · The rightmost line of the 曰 is the different types of water-based glue material 5 〇, the following order: polyvinyl alcohol (PVA), poly-mercapto acrylate ^ = (pHEMA), ^ ^ 曱 丙 acrylic acid p 酉 (pHpjyfA ), 田± (Chitosan); T 素 而 and tissue culture polystyrene (TCPS) arranged on the right side of the broken urine (HA), polymethyl methacrylate (PMMa) arranged dimerization [ (/〇-3-Pyridinic acid] (PHB) to the right. The different biomedical materials of the present invention each have a plurality of characteristics, and not only the characteristics of the foregoing classification, and different types of biomedical materials may have other characteristics, which are merely exemplary classifications and are not intended to limit the present invention. For example, in the cell culture tray of the present invention, polyethyl oxime = (PVA) is a hydrophobic material in the hydrophobic material, and is also one of the water gel materials 50; Chitosan as one of the blending materials 40 in the cell culture tray 100 of the present invention is also one of the water gel materials 50. Therefore, when the biomedical materials suitable for use in the present invention have various characteristics, the arrangement thereof is not limited to the embodiment of the first figure, but may be adjusted as appropriate, that is, in a cell culture tray, there may be two More than one tank is placed with the same biomedical material. A method for observing cell behavior using the cell culture tray of the present invention, comprising the steps of: (a) taking the cell culture tray of the present invention; (b) culturing the cells in the cell culture tray 100; (c) observing before
述步驟（b)之細胞行為。前述「細胞行為」（cell behavior ) 係指細胞懸浮（cell suspension )、細胞貼附（cell attachment )、細胞爬行（cell migration )、細胞分化（cell differentiation )、細胞生長（cell growth )、細胞侵潤（cell invasion )、細胞增生（cell proliferation )、細胞 >周亡（cell apoptosis)、細胞壞死（cell necrosis)、細胞聚集（cell aggregation)等一般習知之細胞行為。 利用本發明細胞培養盤1 〇〇篩選生醫材料之方 法，其包含下列步驟：（a)取本發明細胞培養盤1〇〇 ; (b)將細胞培養於前述細胞培養盤1〇〇;(c)觀察前 述步驟⑴之細胞行為；（d)篩選細胞培養盤之生醫 材料。刖述「選」之方法係為透過依照觀察不同特性 之生醫材料所培養出之特定細胞行為，賴最為恰當的 生醫材料做後續研究’例如依照細胞之爬行、貼附、懸 浮等等細齡為，^料為所欲 選擇該生醫材料做後續培養。 甚f述觀察細^為或筛選生醫材料之方法中，細胞 Γΐ跑，包含原生細胞、原核細胞、 植物細胞、動物細胞、真菌細胞或其組合，舉例如：體 13The cell behavior of step (b). The aforementioned "cell behavior" refers to cell suspension, cell attachment, cell migration, cell differentiation, cell growth, cell infiltration. Cellular behavior such as cell invasion, cell proliferation, cell > cell apoptosis, cell necrosis, cell aggregation, and the like. The method for screening biomedical materials by using the cell culture tray 1 of the present invention comprises the following steps: (a) taking the cell culture tray of the present invention; (b) culturing the cells in the aforementioned cell culture tray; c) observing the cell behavior of the aforementioned step (1); (d) screening the biomedical material of the cell culture plate. The method of "selecting" is to conduct follow-up studies on the most appropriate biomedical materials through the specific cell behaviors of biomedical materials that observe different characteristics, such as cell crawling, attachment, suspension, etc. The age is, and the material is selected for the subsequent cultivation of the biomedical material. In the method of observing or screening biomedical materials, the cells are run, including primary cells, prokaryotic cells, plant cells, animal cells, fungal cells, or a combination thereof, for example, a body 13
I 201200593 細胞（soma cell)、癌細胞（cancer cell)、幹細胞（stem cell)、生殖細胞（germ cell)等，於較佳實施態樣中， 前述細胞係為神經幹細胞。 前述觀察細胞行為或篩選生醫材料之方法中，其步 驟（c)之「觀察」可透過任何習知之細胞行為觀察方 法，例如：細胞一般形態學觀察法，其係利用普通顯微 鏡或電子顯微鏡觀察細胞或組織樣本；免疫化學技術觀 察法，其係透過螢光分子或酵素作為抗體進行細胞或組 織樣本的標記’再使用螢光顯微鏡或普通顯微鏡觀察被 標記之細胞或組織樣本；活細胞直接觀察法；培養細胞 生物學性狀檢測，其係自細胞初代培養至繼代培養皆詳 細紀錄細胞形態、增殖或特定細胞之行為；前述「觀察」 亦可為前述各種細胞行為觀察方法之組合；其中細胞一 般形態學觀察法和免疫化學技述觀察法如：免疫染色法 (Immunostaining method)、穿透光光學顯微鏡、掃描 式電子顯微鏡、穿透式電子顯微鏡或其組合；於本發明 較佳實施態樣中，前述觀察係透過免疫螢光染色法 (Immunofluorescence labeling method) ° 將細胞培養於本發明細胞培養盤100上，藉由同時 將細胞培養於各種不同特性之生醫材料上，同時觀察各 種生長環境下的細胞行為，一般使用者在初始接觸某種 細胞時，可快速的了解其與各種不同特性之生醫材料的 關係。不僅如此，可藉由觀察前述細胞與各種生醫材料 之間的行為，篩選出最適合研究特定細胞或研究特定細 胞行為之生醫材料，並進行後續研究或測試。 實施例一：製作本發明細胞培養盤 取一未經塗佈之細胞培養盤（市面上可購得之24 201200593I 201200593 A soma cell, a cancer cell, a stem cell, a germ cell, etc. In a preferred embodiment, the cell line is a neural stem cell. In the foregoing method for observing cell behavior or screening biomedical materials, the "observation" of step (c) can be observed by any conventional cell behavior observation method, for example, general morphological observation of cells, which is observed by ordinary microscope or electron microscope. Cell or tissue sample; immunochemical technique observation method for labeling cells or tissue samples by using fluorescent molecules or enzymes as antibodies 'reviewing labeled cells or tissue samples using a fluorescent microscope or ordinary microscope; direct observation of living cells Method; the detection of cell biological traits, the cell morphology, proliferation or specific cell behavior is recorded in detail from primary cell culture to subculture; the aforementioned "observation" can also be a combination of various cell behavior observation methods described above; General morphological observation method and immunochemical technique observation method such as immunostaining method, penetrating light optical microscope, scanning electron microscope, transmission electron microscope or a combination thereof; in the preferred embodiment of the present invention In the above observation, the immunofluorescence staining method (Imm) Unofluorescence labeling method) ° The cells are cultured on the cell culture plate 100 of the present invention, and the cells are cultured on various biomedical materials of different characteristics, and the cell behavior in various growth environments is observed at the same time. When planting cells, you can quickly understand the relationship between them and various biomedical materials with different characteristics. Moreover, by observing the behavior between the aforementioned cells and various biomedical materials, the biomedical materials that are most suitable for studying specific cells or studying the behavior of specific cells can be screened and subjected to subsequent research or testing. Example 1: Preparation of cell culture plate of the present invention An uncoated cell culture plate (commercially available 24 201200593
I 個槽體的細胞培養盤’ 24-well tissue culture plate)，其 材料係為組織培養用聚苯乙烯（TCPS)(廠牌：COSTAR 3524)，並取以下生醫材料，且依照下列順序塗佈至前 述細胞培養盤之各槽體内。前述塗佈生醫材料之方式係 以一般習知方式達成。 本實施例之細胞培養盤排列方式如前述第一圖所 示。 實施例二··利用本發明細胞培養盤觀察細胞行為的方法 • 取前述實施例一之細胞培養盤，將神經幹細胞分別 選擇培養於聚乙烯醇（pVA)、聚偏二氟乙烯（pVDF)、 聚離胺酸（PDL)和甲殼素之生醫材料中，其培養過程 皆為放入無菌培養箱（37°C，含有5%C02)，培養基為 DMEM ( Dulbecco's Modified Eagle Medium ) /F12 (廠 商：GIBICO型號：10565 )並含有N2添加劑（N2 supplement)(廠商：GIBICO，型號：17502-048)，其 pH 值為7.4，培養天數各如下表一： 表一、幹細胞培養於各生醫材料之天數及結果圖 生醫材料 培養天數 結果 PVA 二天 第一* A圖 PVDF 三天 第二B圖 PDL 三天 第二C圖 曱殼素 五天 第二D圖 將前述四組之神經幹細胞中的星狀細胞 (Astrocytes)進行免疫螢光染色，其所使用之一級抗體 為··抗神經膠質酸性蛋白（anti-Glial Fibrillary Acidic Protein，下稱 GFAP)(廠牌：Millipore，型號：AB5804)’ 15 201200593 所使用之二級抗體為：螢光黃結合親合性純化二級抗體' (Fluorescein conjugated affinity purified secondary antibody，廠牌：chemicon，型號：API82F)，其染色結 果顯示各如上表所載各圖所示。 觀察前述細胞免疫螢光染色結果，其當細胞培養於 親水性高的聚乙烯醇（PVA)時，結果如第二A圖所示， 觀察此時神經幹細胞會懸浮於此親水基材上；而當細胞 培養於疏水性高之聚偏二氟乙烯（PVDF)上時，結果 如第二B圖所示，神經幹細胞會貼附於此基材上，並開 始有突觸向外生長。 當將神經幹細胞培養於帶正電性的聚離胺酸 ( )上時，結果如第二C圖所不，神經幹細胞會貼 附於基材上並開始分化。 當神經幹細胞培養於生物可降解性強的Chit〇san上 時’其結果如第二D圖所示’神經幹細胞會在基材上分 化成星狀細胞，並開始向外爬出神經幹細胞球。 當觀察細胞行為後，可透過以上細胞行為篩選最為 適备^生醫材料。例如：當欲研究神經幹細胞中星狀細 ^ f、生長狀況時，則取疏水性較高的聚偏二氟乙稀 *做生醫材料以培養細胞，進行後續研究；而 虽欲研九神經幹細胞分化狀況時，則取 ^醫材料以進行培養；當欲研究神經幹細胞之_^ 為時，取聚離㈣（PDL)做生醫材料以培養細胞 欲研究神經幹細胞之懸浮行為時，則以不塗佈任何生醫 胞接觸之生醫材料為細胞培養盤之材料-組織培 蚕用t本乙烯（TCPS))做細胞培養。 ，利用本發明細胞培養盤培養細胞，可同 時觀察不同特性之生醫材料對於細胞行為的影塑，並可 201200593 進一步篩選特定的生W材料做後續研究，著實縮短 究人員在甫進人特L研究領域時，了解細胞 必須花費的時間。 所屬領域之技術人S當可了解，在錢背本發明精 神下’依據本案實施態樣所能進行的各種變化。因此， 顯見所列之實施態樣並非用以限制本發明，而β八 所附申請專利範_定義下’涵蓋於本發=斑 嘴中所做的修改。 【圖式簡單說明】 第一圖係本發明之細胞培養盤較佳實施態樣之一。 r m,匕？係為將神經胚胎幹細胞培養“聚乙烯醇 (P VA )材料一天後之免疫螢光染色圖。 第二B圖係為將神經胚胎幹細胞培養於 烯（^VDF)材料三天後之免疫螢光染色圖。 第二C圖係為將神經胚胎幹細胞培養於聚離 (PDL)材料三天後之免疫螢光染色圖。 第二D圖係為將神經胚胎幹細胞培養於甲殼素 五天後之免疫螢光染色圖。 ^ 【主要元件符號說明】 10 親疏水性材料 20 帶電性材料 30生物可降解性材料 40 摻溫材料 50 水勝材料 100細胞培養盤 17I-well cell culture plate '24well tissue culture plate>, the material of which is polystyrene (TCPS) for tissue culture (label: COSTAR 3524), and the following biomedical materials are taken and coated in the following order The cloth is placed in each of the tanks of the aforementioned cell culture tray. The foregoing method of coating biomedical materials is accomplished in a conventional manner. The arrangement of the cell culture dishes of this embodiment is as shown in the first figure above. Example 2············· In the biomedical materials of poly-lysine (PDL) and chitin, the culture process is carried out in a sterile incubator (37 ° C, containing 5% CO 2 ), and the medium is DMEM ( Dulbecco's Modified Eagle Medium ) / F12 (manufacturer) :GIBICO model: 10565) and contains N2 additive (manufacturer: GIBICO, model: 17502-048), its pH value is 7.4, the number of culture days are as follows: Table 1, stem cells cultured in various biomedical materials Days and Results Figure Biomedical Materials Culture Days Results PVA Two Days First* A Map PVDF Three Days Second B Map PDL Three Days Second C Maps Shellfish Five Days Second D Maps The Stars of the Four Groups of Neural Stem Cells Astrocytes were immunofluorescently stained, and the primary antibody used was anti-Glial Fibrillary Acidic Protein (GFAP) (label: Millipore, model: AB5804) 15 201200593 The secondary antibody used is: Fluorescein conjugated affinity purified secondary antibody (label: chemicon, model: API82F), and the staining results are shown in the above table. The figure shows. Observing the results of the above-mentioned cellular immunofluorescence staining, when the cells were cultured in a highly hydrophilic polyvinyl alcohol (PVA), the results were as shown in FIG. 2A, and it was observed that the neural stem cells were suspended on the hydrophilic substrate at this time; When the cells were cultured on highly hydrophobic polyvinylidene fluoride (PVDF), as shown in Figure B, the neural stem cells adhered to the substrate and began to have synaptic outgrowth. When the neural stem cells are cultured on the positively charged polylysine ( ), the results are as shown in the second panel C, and the neural stem cells are attached to the substrate and start to differentiate. When the neural stem cells are cultured on the biodegradable Chit〇san, the results are shown in Figure 2D. The neural stem cells are differentiated into stellate cells on the substrate and begin to climb out of the neural stem cell sphere. After observing the behavior of the cells, it is possible to screen the biomedical materials through the above cell behavior. For example, when studying the star-shaped fineness and growth of neural stem cells, take the highly hydrophobic polyvinylidene fluoride* as a biomedical material to culture the cells for subsequent research; When the stem cells are differentiated, the medical material is taken for culture; when the neural stem cells are to be studied, the poly (4) (PDL) is used as the biomedical material to culture the cells to study the suspension behavior of the neural stem cells, The biomedical material that is not coated with any biomedical cells is the cell culture plate material - tissue culture silkworm is t cell ethylene (TCPS) for cell culture. By using the cell culture tray of the present invention to culture cells, the biochemical characteristics of different characteristics of the biomedical materials can be observed at the same time, and 201200593 can be further screened for specific raw W materials for subsequent research, and the research personnel are shortened. When studying the field, understand the time that cells must spend. Those skilled in the art can understand various changes that can be made in accordance with the embodiments of the present invention. Therefore, it is apparent that the embodiments described are not intended to limit the invention, and that the modifications made in the present invention are covered by the invention. BRIEF DESCRIPTION OF THE DRAWINGS The first drawing is one of the preferred embodiments of the cell culture tray of the present invention. r m, 匕? This is an immunofluorescence staining of "PVA" material after one day of culture of neural embryonic stem cells. The second B is an immunofluorescence after culture of neural embryonic stem cells for three days after the olefin (^VDF) material. The second C map is an immunofluorescence staining map of neural embryonic stem cells cultured on a poly(PDL) material for three days. The second graph is the immunization of neural embryonic stem cells cultured for five days after chitin. Fluorescent staining image. ^ [Main component symbol description] 10 Hydrophilic material 20 Charged material 30 Biodegradable material 40 Temperature-doped material 50 Water wins material 100 cell culture plate 17