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  An cell-assembly derived physiological 3D model of the metabolic syndrome, based on adipose-derived stromal cells and a gelatin/alginate/fibrinogen matrix

一個基於脂肪衍生的間質細胞以及明膠/海藻酸鈉/纖維蛋白原等基質的細胞組裝所得到的代謝症候群的生理學上的3D模型


HyperLink An cell-assembly derived physiological 3D model of the metabolic syndrome

abstract

One of the major obstacles in drug discovery is the lack of in vitro three-dimensional(3D) models that can capture more complex features of a disease.

Here we established a in vitro physiological model of the metabolic syndrome (MS) using cell-assembly technique (CAT), which can assemble cells into designated places to form complex 3D structures.

摘要

在藥物發掘的主要障礙之一, 是缺乏體外能捕捉疾病的更複雜特徵的三維(3D)模型.

在這裡我們用細胞組裝技術(CAT)建立了一個代謝症候群(MS)的體外生理模型, 它可以將細胞組裝到預指定的位置以形成複雜的三維結構.

Adipose-derived stromal (ADS) cells were assembled with gelatin/alginate/fibrinogen. Fibrin was employed as an effective material to regulate ADS cell differentiation and self-organization along with other methods.

ADS cells differentiated into adipocytes and endothelial cells, meanwhile, the cells were induced to self-organize into an analogous tissue structure.

脂肪衍生的間質細胞是以明膠/海藻酸鈉/纖維蛋白原等組裝的. 與其他方法的配合下, 纖維蛋白作為一種有效的材料, 以調節ADS細胞的分化及自我組織. ADS細胞分化成為脂肪細胞和內皮細胞, 同時這些細胞被誘導使其自我組織成為一個類似的組織結構.

Pancreatic islets were then deposited at designated locations and constituted the adipoinsular axis with adipocytes.  Analysis of the factors involved in energy metabolism showed that this system could capture more pathological features of MS.

Drugs known to have effects on MS showed accordant effects in this system, indicating that the model has potential in MS drug discovery.

Overall, this study demonstrated that cell differentiation and self-organization can be regulated by techniques combined with CAT.  The model presented could result in a better understanding of the pathogenesis of MS and the development of new technologies for drug discovery.

胰島細胞接著被置放在指定地點, 並且與脂肪細胞構成adipoinsular. 分析參與能量代謝的因素表明了這個系統可以獲得更多的MS病理特徵.

已知對MS有作用的藥物, 在此系統中表現出一致的效果, 這指出這個模型具有在MS的藥物的發現上的潛力.

總的來說, 這項研究證明了細胞分化和自我組織, 可以經由結合CAT的技術進行調節. 所提出的模型, 可能會達成對於MS的發病機制更好的理解, 以及用於發現新藥物的技術的發展.

1. Introduction

Researchers have recently developed techniques to fabricate tissues and organs in which both cells and biomaterials have carefully defined architectures.

A three-dimensional (3D) bio-assembly tool is capable of extruding cells and biomaterials into spatially organized 3D constructs [1]. Cell printing equipment can print cells as a stream of drops in 3D positions that mimic their respective positions in organs [2,3].

We have recently developed a 3D cell-assembly technique (CAT) based on Rapid Prototyping (RP) [4,5].  This technique can put different cells and materials into designated places to form complex 3D structures.

1引言

研究人員最近開發了建造組織和器官的技術, 其中細胞和生物材料具有仔細定義的結構.

一種三維(3D)生物組裝的工具, 能夠將細胞和生物材料擠壓在空間上有組織的三維構造[1]. 細胞的印刷設備可以以一個滴狀串流的方式, 將細胞印在三維的位置, 模仿它們在器官裏的對應位置[2,3].

我們最近開發了一種基於快速原型(RP) [4,5]的三維細胞組裝技術(CAT).這種技術可以把不同的細胞和材料放到指定地點, 以形成複雜的三維結構.

The designed architecture facilitates cell growth, organization, and differentiation.

Hepatocytes have been assembled with gelatin and alginate hydrogel to build 3D structures, in which the viabilities and functions of the cells could remain for more than 60 days [6].

Gelatin and alginate have been used as biomaterials in many simple scaffold structures such as sheets, fibers, and micro-capsules.

The special property of gelatin in which it can be gelled at a low temperature allows the CAT extruded mixture to take shape at a low temperature.  The combination of gelatin and alginate is advantageous because of chemical similarity to the extracellular matrix (ECM) [7].

However, the gelling processes of gelatin and alginate are not normal physiological process in living organisms and could not be controlled expediently. Consequently, it is difficult to induce cells in gel differentiation and facilitate self-organization into a functional structure [8].

所設計的結構可供細胞的生長, 組織和分化.

肝細胞是用明膠和海藻酸鹽水凝膠所組合以構建三維結構,在此, 細胞的存活率和功能能維持超過60天以上[6].

明膠和海藻酸鹽已經在許多簡單的支架結構, 例如片材, 纖維, 和微膠囊中被用作生物材料.

明膠可以在低溫下進行凝膠化的特殊屬性, 允許CAT擠出的混合物可在低溫下得到型狀因為與細胞外基質(ECM) 的化學相似性, 明膠和藻酸鹽的組合是有優勢的[7].

然而, 明膠和海藻酸鹽的凝膠化過程是並不是在活的生物體內的正常的生理過程, 而並也不能方便地進行控制. 因此, 很難以誘導細胞凝膠分化並促進其自我組織成一個功能結構[8].

Fibrin is normally used by the body as a temporary scaffold for tissue regeneration and healing [9]. Research shows that fibrin gel combines a number of important properties of an ideal scaffold to grow a variety of cells and tissue constructs [10].

The polymerization, constriction, and degradation of fibrin are controllable with the use of thrombin and aprotinin.  Therefore, we conceived that a composite ECM with fibrin would help regulate cells' differentiation and self-organization into a functional tissue structure.

纖維蛋白通常用被人體作為組織再生和癒合的臨時支架的[9].  研究顯示, 纖維蛋白凝膠結合了很多生長各種細胞和組織結構的理想支架的重要性質[10].

纖維蛋白的聚合, 收縮, 和降解是可以用凝血酶和抑肽酶控制的. 因此, 我們設想, 一個含有纖維蛋白的合成ECM, 將有助於調節細胞的分化和自我組織成一個功能性的組織結構.

Recently, tissue engineering has endeavored to expand into new horizons aside from the formation of tissues [11,12].

In the field of drug discovery, there is an increasing demand for in vitro 3D models that can capture more complex pathological features compared with what traditional two-dimensional (2-D) models have thus achieved [13,14].

Some in vitro 3D models have been used in physiological and pathological research [15,16], but their applications have been limited owing to their poorly controlled structure.   For example, researchers have endeavored to develop drugs that can target the metabolic syndrome (MS) as a whole [17], but to date, there are still no approved drugs that can reliably reduce all metabolic risk factors.

近年來, 除了組織的形成, 組織工程已在努力拓展新視野[11,12].

在藥物發現領域中, 能獲得比傳統的二維(2-D)模型已達到的更複雜的病理特徵的體外三維模型需求不斷增加[13,14].

一些體外3D模型已被用於生理和病理的研究[15,16], 但由於其控制不佳的結構, 使其應用受到限制例如, 研究人員一直在努力開發出藥物, 可以針對整體的代謝徵候群, 但到目前為止, 仍然還沒有能夠可靠地減少所有代謝危險因此子的認可藥物.

One of the major obstacles is the lack of in vitro 3D models that can capture most pathological features of MS [18–20].

MS is a cluster of growing epidemic diseases which present as energy metabolic disorders (obesity, diabetes) and cardiovascular diseases (hyper-tension, atherosclerosis) [21,22].

Adipocytes and b-cells constitute the adipo-insular axis that regulates energy metabolism [23], and endothelial cell dysfunction connects the pathogenesis of energy metabolic disorders with that of cardiovascular diseases [24].  With these mechanisms in mind, we attempt to organize the related cells to establish MS models using CAT.

主要障礙之一, 是缺乏能獲得大部份MS病理特徵的體外三維(3D)模型[18–20].

MS是日益流行的疾病群, 呈現能量代謝失序(肥胖, 糖尿病)和心血管疾病(高血壓, 動脈粥樣硬化)[21,22].

脂肪細胞和B細胞構成調節能量代謝的adipo-insular[23],並且, 內皮細胞功能障礙連接了心血管疾病的能量代謝失序的發病機理. 有了這些機制為思考基礎, 我們試圖使用CAT組織這些相關的細胞來建立MS模型.

In this work, we report on the feasibility of constructing an in-vitro multicellular 3D model of the energy metabolic system for MS using CAT.

Adipose-derived stromal (ADS) cells [25,26] and gelatin/algi-nate/fibrinogen were assembled in a well-designated 3D structure, and their differentiation into adipocytes and endothelial cells was controlled based on their respective positions within the structure; pancreatic islets were then deposited at the designated micro-holes.

We also tested the factors involved in the energy metabolism and endothelial dysfunction of the multicellular model, whether chronic exposure to high glucose, a major inducement of MS, could lead to similar pathological changes in the multicellular model to MS, and whether drugs known to have effects on MS manifest accordant effects in the multicellular system.

在這項工作中, 我們報告了用CAT構建一個用於MS能量代謝系統的體外多細胞3D模型的可行性.

脂肪衍生的間質(ADS)細胞[25,26]和明膠/藻酸鹽/纖維蛋白原被組裝在一個良好定位的三維結構,而它們分化為脂肪細胞和內皮細胞是基於其各自在結構中的位置的所控制. 然後胰島被沉積放在指定的微孔.

我們也測試了這個多細胞模型與能量代謝和內皮功能障礙有關的因素, 包括, 長期暴露於高血糖, MS的主要誘因, 是否在這個MS的多細胞模型可能導致類似的病理變化, 以及已知對MS有作用的藥物, 是在這個多細胞系統呈現一致的效果.

2. Methods

2.1. Cell culture

ADS cells were isolated from rat subcutaneous adipose tissues [26].   The epididymal adipose tissues from Sprague-Dawley rats (100–150 g, Beijing university Medical Center of Laboratory Animals) were excised, washed and finely minced in PBS.

Tissues were digested with 0.075% Type II collagenase (sigma) at 37℃ for 30 min.

Neutralized cells were centrifuged to separate mature adipocytes and stromal-vascular fraction. Floating adipocytes were removed and pelleted stromal cells were filtered through a 100 mm cell strainer before plating.

ADS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) at 37℃ in an atmosphere of 5% CO2.

Cells were grown to subconfluence and passaged by standard methods of trypsinization.Pancreatic Islets were isolated from the rat pancreas [27].

2 .方法

2.1. 細胞培養

ADS的細胞是從老鼠皮下脂肪組織中分離得到[26].SD大鼠的附睾脂肪組織(100-150, 北京大學實驗動物醫學中心)被切取, 洗滌並剁碎於PBS.

組織37℃0.075II型膠原酶(Sigma)消化30分鐘.去活化的細胞離心分離成熟的脂肪細胞和間質血管部分.去除浮動脂肪細胞, 顆粒間質細胞以100mm的細胞過濾器在培養前過濾.

ADS細胞培養在含有10%胎牛血清(FCS)Dulbecco的改良的Eagle培養基(DMEM),37 ℃, 5CO2的氣體環境中.

細胞生長至次飽和, 並且以胰蛋白酶消化的標準方法進行傳代.胰島細胞從大鼠胰腺分離出來[27].

Pancreas of SD rats was infused with 0.2% type V collagenase(Sigma), quickly excised, minced, and incubated at 37℃ for 30 min. Neutralized cells was washed in Hank's solution and centrifuged. Islets were separated using ficoll gradient centrifugation (ficoll 400 DL, Sigma).

Islets were cultured in DMEM containing 100 mL/L FCS, 200 kU/L penicillin, 100 mg/L streptomycin, and 2 mmol/L L-glutamine(Gibco) at 37℃ in an atmosphere of 5% CO2. Medium was changed every second day.

SD大鼠胰腺注入0.2 %的V型膠原酶( Sigma公司), 迅速切除剁碎, 並在37℃孵育30分鐘. 去活化的細胞離心分離成熟的脂肪細胞和間質血管部分. 去活化的細胞以Hank溶液洗滌, 以及離心. 胰島用ficoll梯度離心(聚蔗糖400DL, Sigma公司)分離出來.

胰島以含100 mL/L FCS, 200 KU/L青黴素, 100 mg/L的鏈黴素和2毫摩爾/L L-谷氨酰胺(Gibco)DMEM培養液, 37 ℃, 5CO2的氣體環境中培養. 培養基每隔一天更換.

2.2. 3-D multicellular system construct and operation

2.2. 3-D多細胞系統結構和操作

The ADS Cells were trypsinized off the culture dishes upon subconfluence, washed and quantified. Then cells were mixed with gelatin (Tianjin green-island Company, 96 kDa, type B), alginate (SIGMA, 75–100 kDa, guluronic acid 39%) and fibrinogen(SIGMA)gel (gelatin: alginate: fibrinogen, 2:1:1) at a density of 3x10(7) cells/mL.

After mixing, 1 mL of the mixture was loaded into a sterilized syringe (1mL, 0.45 x16 RW LB).

We used a software package (Microsoft, AT6400) to design the complex 3-D structure, which consisted of square grids and orderly channels about 400 um in diameter (fig. 1a), this structure has been used in our previous work and has been proved effective.

ADS細胞在次飽和時用胰蛋白酶從培養皿切出來, 洗滌及定量.然後細胞以3×107次方個細胞/ml的密度, 與明膠(天津綠色島公司, 96 kDa, B), 藻酸鹽(SIGMA公司, 75-100 kDa 的古洛糖醛酸的39) , 以及纖維蛋白原(SIGMA公司)的凝膠(明膠:藻酸鹽:纖維蛋白原, 2:1:1) 混合.

混合後, 1毫升混合物裝入已滅菌的注射器(1毫升之, 0.45 x16RW LB).

我們使用了一套裝軟體(微軟, AT6400)來設計複雜的3-D結構,其中有方形網格, 直徑約400微米的有規則的通道(1A), 這種結構已經被應用在我們以前的工作, 且已證明是有效的.

Following the designed structure, a refit nozzle controlled by computer was used to deposit the mixture on a glass chip at a temperature of 10℃(Gelatin at gel state).

The program was run 8 times consecutively at the same position to generation of a 10 x 10 x 2 mm (3) 3-D configuration with the square pattern.

When the process was finished, the 3-D structure was cross linked with 10% CaCL2 for 1 min(crosslink the alginate), washed with DMEM three times. Then the 3-D structure was further stabilized by 50mU/ml Thrombin (polymerize fibrinogen) in a culture medium containing DMEM, 10% FCS, 1 mmol/L insulin, EGF and 50 U/mL aprotinin (Sigma), placed in a CO2 incubator at 37℃.

根據所設計的結構, 一個由電腦控制的改裝噴嘴被用於在10℃(明膠凝膠態)的溫度下, 將混合物沉積於玻璃晶片上.

程式在相同的位置連續執行8, 以產生有方形圖案的 10 ×10 ×2 毫米立方的3-D結構.

當過程完成後, 3-D結構用10 %的氯化鈣交聯1分鐘(使藻酸鹽交聯), DMEM洗滌3. 然後將3-D結構用50mU/ml凝血酶(聚合纖維蛋白原), 在個含有DMEM, 10FCS, 1 mmol/L 的胰島素, EGF50 U/ml的抑肽酶(Sigma公司), 放在37℃下的 CO2培養箱, 的培養基中進一步被穩定.

After 3 days of culture, the medium was switched to another containing 10% FCS, 1 m M insulin, 1 mM dexamethasone, 0.5 mmol/L isobutyl-methylxanthine (IBMX; Sigma), and 50 u/mL aprotinin for 3 d. At the 6th day, the pancreatic islets were aspirated and deposited at designated position of the 3-D structure. The medium was changed every other day.

For 2-D control experiment, the cell culture plates were soaked with solution (gelatin: alginate: fibrinogen, 2:1:1,0.5%) for 60 min, and then the solution was dumped and the plate was processed with 5% CaCL2 for 1 min.

ADS cells were plated at 5x10(5) cells/well plates and induced to adipocytes by IBMX or to endothelial cells by EGF.

培養3天後, 將培養基換為另一個含有10FCS, 1mM 胰島素, 1mMdexamethasone, 0.5 mmol/L的異丁基甲基黃嘌呤( IBMX, Sigma公司), 50u/ml的抑肽酶, 3, 在第6, 胰島被吸出並存放於3-D結構的指定位置. 培養基每隔一天更換.

2 -D控制實驗, 細胞培養板用溶液浸泡(明膠:藻酸鹽:纖維蛋白原, 2:1:1, 0.5 ) 60分鐘, 然後將溶液倒出, 並用5%的CaCl2 處理板子1分鐘.

ADS的細胞以 5×105次方個細胞/孔培養, 並以IBMX誘導為脂肪細胞或以表皮生長因子誘導為內皮細胞。

2.3. Structural analyses by scanning electron microscopy

At the 6th day of culture, the multicellular 3-D structure were washed in phosphate buffer (pH 7.4) and fixed with 3% glutaraldehyde for 2 h. Then the samples were post-fixed with 0.5% OsO4 and rinsed with PB again.

The samples were dried in vacuum freeze dryer for 12 h. After dehydrated, samples were sputter coated with gold–palladium. All micrographs were obtained in a scanning electron microscope (Hitachi S450, JAP).

2.3. 以掃描電子顯微鏡的結構分析

在培養的第6, 此多細胞的3-D結構用磷酸鹽緩衝液(pH 7.4)洗滌, 以及用3%戊二醛固定2小時. 然後用0.5%的OsO 4將樣品進行後固定, 以及用PB再漂洗.

將樣品在真空冷凍乾燥機中乾燥12小時. 在脫水後, 樣品用金-鈀濺鍍. 所有顯微照片是在一掃描型電子顯微鏡裏(日立S450, JAP)獲得的.

2.4. Immunostaining

For immunofluorescence analyses, the assembled multicellular 3-D structure was fixed with 4% glutaraldehyde for 20 min at 20℃, and washed 3 times with PBS.

The structure was incubated with 50 mg/mL propidium iodide (PI, sigma USA) for 20 min (nuclear staining), then the structure were incubate with primary anti-bodies : rabbit anti-rat:CD31 (1:20 in PBS); rabbit anti-rat CD34; rabbit anti-ratinsulin (1:50) (all from Santa Cruz, USA) for 30 min respectively.

2.4. 免疫染色法

對於免疫熒光分析, 這個組裝的多細胞3-D結構在20℃下用4%戊二醛固定20分鐘, 然後用PBS清洗3.

50毫克/毫升的碘化丙啶(PI, 美國Sigma公司)孵育此結構20分鐘(核染色), 然後分別以兔抗大鼠:CD31(PBS1:20); 兔抗大鼠CD34; 兔抗大鼠胰島素(1:50) (全部來自Santa Cruz, USA) 原發性抗體孵育此結構30分鐘.

And then the structure was incubated with a secondary antibody (FITC-conjugated anti rabbit IgG, Santa Cruz, 1:20 in PBS) for 30 min. Finally, the samples were washed with PBS and observed by fluorescence microscope (OLYMPUS BX51, JAP) or confocal microscopy (Leica TCS SP2, Germany).

Image acquisition and analysis were performed using the Applied DP-Controller system (OLYMPUS, JAP) and Image-pro Plus 5.0 (Media Cybernetics, USA).

然後將該結構物用二級抗體(FITC標記的抗兔IgG, Santa Cruz, 1:20 in PBS) 孵育此30分鐘. 最後, 樣品用用PBS洗滌, 以及用熒光顯微鏡(Olympus BX51, JAP)或共聚焦顯微鏡(Leica公司TCS SP2, 德國)觀察.

圖像採集和分析是用 Applied DP-Controller 系統(OLYMPUS, JAP) 以及 Image-pro Plus 5.0 (Media Cybernetics, USA)進行的.

2.5. Dynamic insulin secretion experiment

We measured Insulin secretion kinetics by perfusion experiments.

After the islets were deposited in the structure, the structures were precultured in normal glucose (5 mM ) or high glucose (15 mM ) medium. After 5 days, islets in or not in the 3-D structures (20 islets/per) were introduced to a 1-mL perfusion chamber and exposed to flowing perfusate (DMEM, 0.2 mL/min) with a basal glucose concentration (2mM) for 1 h for islet cell starvation.

Basal insulin secretion was estimated at the same medium and then switched to a high glucose content (15 mM) perfusate and collected every 2 min for 20 min. Then the perfusate was returned to the basal solution and collected every 5 min for 15 min.

The insulin content of collected samples was measured by using a rat insulin ELISA kit according to the manufacturers instructions (RB, USA) by a microplate reader (Bio-Rad 550).

For drug experiment, rosiglitazone (3 mg/mL) and nateglinide (5 mg/mL) were added to the culture medium at the 11th day respectively.

2.5. 動態胰島素分泌實驗

我們用灌注實驗測量胰島素分泌的動力學.

在胰島被沉積在結構之後, 這些個結構被預培養在正常的葡萄糖(5mM)或高葡萄糖(15mM)的培養基中. 5天之後, 在或不在 3-D結構中的胰島(20胰島/), 被引到一個1毫升灌注腔, 並且暴露於具有基底葡萄糖濃度(2mM)的灌流液(DMEM, 0.2毫升/分鐘)1小時使胰島細胞挨餓.

基礎胰島素分泌在相同的培養基中作評估, 然後切換到一高葡萄糖含量(15 mM)的灌注液每2分鐘收集一次, 20分鐘. 然後灌注液返回到基礎溶液, 並且每5分鐘收集一次, 15分鐘.

所收集的樣品的胰島素含量, 使用一大鼠胰島素ELISA試劑盒, 根據製造商的說明書, microplate reader (Bio-Rad 550)測定.

對於藥物試驗, rosiglitazone (3 mg/mL) 以及 nateglinide (5 mg/mL)在第11, 分別地加入到培養基中.

2.6. Measurement of glucose consumption, FFA release, and adipogenesis

2.7. Measurement of adipocytokine by ELISA

2.8. DNA assays

2.9. Measurement of endothelin-1(ET-1) and nitric oxide (NO)

2.10. Statistical analysis

3. Results

3.1. Assembly of the 3D multicellular system

3.2. Dynamic insulin secretion

3.3. Glucose consumption, FFA release, and adipogenesis

3.4. Adipocytokine

3.5. Endothelial dysfunction

4. Discussion

5. Conclusion

Acknowledgements

Appendix

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台中 伴手禮 文化節 禮物 母親節 端午節 高美濕地 佳節倍思親,好禮表孝心, 父母的健康,就是子女的孝心. 佳節好禮, WWW.chromnet.net 穩達F3靈芝多醣體在使用中可持續保有可視圖文的肥皂 (Soaps with Sustained Visible Characters and Graphics while applying) 一.特點, 二.可客製化設計三.訂購方式特點: (專利申請中:101150476, 102105651)  1.含有金玉良言及圖案 2.一直洗,一直洗,仍可看得到圖文 3.可多層組成一組詩詞短句本項發明的目的在於提供各種內含有圖案或文字的肥皂, 此圖案或文字具有深度方向的厚度, 以使得在肥皂的在使用過程中, 仍可持續保有可以被看到的圖案或文字. The purpose of this invention is to provide soaps with characters and graphics included. The included characters and graphics have their depths in the thickness direction to sustain their visibilities while applying the soaps. 現有的各種肥皂所內含的圖案或文字, 主要都以表面淺層凹版或凸版方式製作, 在短暫的使用後, 肥皂表面的圖案或文字即被消耗而看不見了, 因而未能在其使用過程中持續保有可以被看到的圖案或文字. 本項發明的目的在於提供各種內含有圖案或文字的肥皂, 此圖案或文字具有深度方向的厚度, 以使得在肥皂的在使用過程中, 仍可持續保有可以被看到的圖案或文字.本系統發明的重點在於其應用方式, 組構型態及製作, 所用零組件能達到所須功能即可. 利用各種常見或特定的肥皂的材料及色料, 製作含有圖案或文字的肥皂,此圖案或文字具有深度方向的厚度. 可客製化設計: 我們可以依據您們的指定,設計所須要的"深度圖文耐用皂", 包括: 皂體尺寸,圖文內容,皂體顏色,圖文顏色,香精成份,單一皂體包裝材質及印刷內容,多皂體禮盒構造材質及印刷內容等... "深度圖文耐用皂" 是可以重疊多層,形成一個含有多層文字圖案的耐用皂,其特點是可在一個多層文字圖案耐用皂裏表達出一篇簡短的古詩,現代詩,對聯或短句等,是我們的專利主要訴求之一.  另我們也可有涵蓋到固體芳香劑及馬桶小便斗除臭劑的應用上,使得芳香劑及馬桶小便斗除臭劑上可以有不同顏色的文字或線條圖案,如"早安"或"請對準","花朵"或"蒼蠅"(圖案),深度都是與固體芳香劑及馬桶小便斗除臭劑的厚度一樣,在用完以前就可以一直被看到,這也是既實用有創新的應用. 客製化的設計, 可以增進客戶的在創新服務層面的評價, 使得產品及服務內容更具有吸引力. 訂購方式: 請至各子項目查看各現有產品訂價,確定所需品項,香精種類及數量之後,請直接以EMAIL連繫,經我們確定可交件日期後,即可付款訂購. 常見香精有:香茅,艾草,薰衣草,樟樹,茶樹,玫瑰麝香,甜菊,月桂,... 運費及運送方式:一次購滿500元免運費,500元以下酌收50-80元運費.以郵局包裹方式運送.  匯款資訊:台中市清水區農會 活期存款帳號名稱:林坤益 (+) 太陽系分析基礎開發中心, 帳號: 45000100033625 通匯代碼: 9544505 NOTE:1.香精種類很多,部份香精對特定體質具有刺激性或過敏性,一般建議先少量短期使用,以確保適用. NOTE:2.不同成份的香精成本差距相當大(5-20倍),在您確認訂購時,會適度酌收高單價香精的成本費用. NOTE:3.不加香精也是種很好的選項,完全不用顧慮刺激性或過敏性問題,但使用時會有甘油皂基的原本不香的味道.能夠體認習慣了也是很好的選項. NOTE4:客製化部份, 煩均以email連繫及確認需求及可交件日期,     良言一句三冬暖(星雲--遠見雜誌)  語言是思想和觀念的表象, 用來聯繫人心的般若風光. 若能時時慎之於口, 與他人共享, 陽光,花朵.淨水般的話語, 人生必然豐美... (http://www.gvm.com.tw/Boardcontent_4574.html) 多年以前,曾經在一篇文章裡,讀到這麼一句話:「語言,要像陽光、花朵、淨水。」當時深深感到十分受用,於是謹記心田,時刻反省,隨著年歲的增長,益發覺得其中意味深長。 http://www.sfes.tc.edu.tw/index.asp(上楓國小好話實踐) http://www.sfes.tc.edu.tw/index.asp(上楓國小唐詩河洛語線上教學)       節氣指二十四時節和氣候,是中國古代訂立的一種用來指導農事的補充曆法,... http://zh.wikipedia.org/wiki/节气二十四節氣(農委會)http://www.coa.gov.tw/view.php?catid=284 今日星象、行星動向、節氣 http://web2.nmns.edu.tw/constellation/home.php AEEA 天文教育資訊網 http://aeea.nmns.edu.tw/ 太陽系的前世今生, 影像提供:NASA/ESA,陳輝樺(NMNS) http://aeea.nmns.edu.tw/index1.html   大地藏無盡, 勤勞資有生, 念哉斯意厚,努力事春耕. 一.特點,二.可客製化設計,三.訂購方式   大地藏無盡  念哉斯意厚 勤勞資有生 努力事春耕298元/組(320g) 誠歡迎有天文學,民俗學及相關的教育及創新構想的原創者及推廣者,與我們密切合作 !    氨基酸分子模型系列(19種常見氨基酸) 由丙氨酸的基礎結構可研衍生出各種常見氨基酸分子結構一.特點,二.可客製化設計,三.訂購方式   分子球 89元/個(80g)    丙安酸(Alanine)  丙氨酸 369元/個(330g) 麩氨酸(圓柱) 469元/個(430g) 誠歡迎有科學教育創新構想的原創者及推廣者,與我們密切合作 ! RainbowDash 及UNLIGHT 是最經點的動漫符號之一網路搜尋即有豐富的資訊. Ranbow Dash 動畫My Little Pony:Friendship is Magic 之主角群之一 Cutie mark的圖案為打著彩虹閃電的雲在故事中代表的精神為"忠誠" UNLIGHT FACEBOOK網頁遊戲以故事為中心的對戰型卡牌養成遊戲玩家人數突破30萬人   好香皂結合好男人及撿肥皂的梗,並將其實體化梗的詳細資料有請 網路搜尋   誠歡迎有文化及新構想的原創者及推廣者,與我們密切合作. 先民從大陸移居台灣,渡過黑水溝,也把馬祖信仰帶來台灣。媽祖信仰起源於西元10世紀的宋朝,幾乎每個台灣人都耳熟能詳福建莆田湄洲林默娘傳說,為了指引出海的父兄返航而犧牲自己,後來成為漁民信仰的「航海女神」。 一.特點,二.可客製化設計,三.訂購方式   保庇 89元/個(80g)     恭賀新喜_1 79元/個(30g)      金鎖片 79元/個(80g) 元寶錢幣 79元/個(80g)   風調雨順 99元/個(80g) 國泰民安_2 99元/個(80g) 富貴吉祥_1 99元/個(80g) 年年有餘_1 79元/個(80g) 年年有餘_3 99元/個(80g) 誠歡迎有宗教學,民俗學及相關的教育及創新構想的原創者及推廣者,與我們密切合作 ! 牛牽到北京 還是牛,.. 一.特點,二.可客製化設計,三.訂購方式    牛牽到北京   牛牽到北京還是牛 188元/組(160g) 還是牛 台灣諺語_動物篇 http://163.21.2.41/t128/chinesepassport/animals.htm 動物之台灣諺語 www.taiwan123.com.tw/local/a_index.htm 誠歡迎有趣味文學相關的教育及創新構想的原創者及推廣者,與我們密切合作 ! 沒辦法再更傳統了特出的文化產品, 如沐春風粿模經典(雕:餅印.粿印.糕印.糖印) 粿模_財子壽中草藥種苗很專業的中草藥種苗來源, 值得珍藏綬草沒辦法再更先進了用最先進的技術, 提供最優化的生活營養保健美肌保養居家潔身居家潔身深度圖文耐用手工皂唯一可以讓我們的日常生活充滿文學品味的手工皂, 具有無可替代的專利特點發現金玉良言系列良言一句三冬暖媽祖(風調雨順, 國泰民安, 保庇每一個手工皂都有一句媽祖的提示良言, 直到用完前都還能看得見節氣(驚執:大地藏無盡, 勤勞資有生, 念哉斯意厚, 努力事春耕清明節母親節端午節畢業紀念四書, 五經經典文學系列, 詩, 詞, 曲勵志追思永懷親恩祝賀五世其昌情意與關懷 LOVE, WISH, 平安文具禮頻贈品不只是高CP值,還特別具有創新及創意科教禮贈用品結合科學, 教育, 文創, 既有趣又有豐富的科學人文內涵天文科學星座觀測, 觀測工具發現生命的起源及意義考古人類學探索, 學習, 驚奇, 有趣 ... 追思(城鄉差距與世代正義) 記憶伴隨著歲月, 從鄉村到城市, 一切是否留下寶貴的智慧, 或是只是無限的追思 ! 小時候, 母親曾說:"如果我們這邊有一條較直的大路直接到清水鎮上就好了" 這事實上也是我們鄉下人共同的期望與夢想. 記得每年農曆10月13前後, 母親難得年回甲南娘家一次 , 我們總是既期待又害羞. 回甲南娘家其實不遠, 走路只要約50-60分鐘 , 不過幾十年來一直也沒有公車... 有時後總是在想, 這人類的社會到底是怎麼回事 ? 人口總是往都市集中, 留在鄉下卻是一些無法被提起的記憶 ? 因此, 容我們有一點能力與機會, 來訴說我們的所見所知與所感 , 也希望這一切是留下來的不只是無限的追思.