摘要 Abstract

This review highlights our major developments in the fields of organoids

and organ-on-chip to address issues in fundamental and biomedical

research by modeling development and cancer.

這篇文章強調我們的類器官領域和器官晶片的發展，通過建模開發去解決基礎

醫學和生物醫學中研究癌症的問題

We illustrate how contemporary miniaturized technologies (microfluidics,

3D scaffolding, 3D imaging) combined with RNAi-based organoids HTS

(High-Throughput Screening), would help forming and analyzing

consistent, efficient and reproducible organoids.

我們將說明如何將現在的技術像是(微流體，3D 腳手架，3D 成像）去結合類器

官(高通量篩選)，將有助於形成和分析，高效和可重複性的類器官。

we illustrate the potential of engineered organ-on-chip devices for

creating novel human organ and disease models, with particular focus on

“prostate-on-a-chip”.

此外我們還說明了設計器官晶片對於創造特別類器官的潛在性，特別要關注

“前列腺上的晶片”
介紹 INTRODUCTION
A major limitation in the development of new disease treatments is the

lack of good models to identify potential drug targets screen toxicity and

predict clinical drug efficacy in humans

發展中有一個主要限制條件新疾病治療方法缺乏了好的模型去確定藥物靶標，

對人類做毒性篩選和預測臨床藥物的效果。

藥物靶標
是指體內具有藥效功能並能被藥物作用的生物大分子，如某些蛋白質和核酸等

生物大分子。那些編碼靶標蛋白的基因也被稱為靶標基因。事先確定靶向特定

疾病有關的靶標分子是現代新藥開發的基礎。
Although the physiological relevance of human organoids as models to

mimic cancer and to increase the predictive values of in vitro drug

evaluation is now accepted,

雖然生理相關的類器官現在被做為模擬癌症的模型和增加藥物評價的預測價

值，

the practical use of organoids in HTS is still limited by the low yield of

mature and viable organoids.

但是實際上類器官對於 HTS(高通量篩選)還是有純熟度限制

One key-aspect is the use of microtechnologies，

一個關鍵方面是使用微技術，

microfluidic-based encapsulation for engineering organoids from single

cells and HT functional genomics to exhaustively characterize cell

properties

以微流體為主的封裝對於工程的類器官從單細胞高通量和實用的基因詳盡地表

徵細胞特性
Deciphering key signaling cascades, which control organoid generation is

a pre-requisite to generate robust and standardized organoids that would

be amenable to HTS applications.

要解讀這關鍵傳訊級聯反應，產生健康和標準化的類器官是控制類器官先決條

件，它將會是高通量篩選應用。

Another aspect is the development of organ-on-chip combining 3D-

shaped models and microfluidics to better access the exocrine function of

glandular organs (e.g., prostate, breast) to identify new cancer “liquid-

biopsy” biomarkers for prognostics and diagnostic.

另一方面器官晶片發展結合了 3D 模型和微流體，使得腺體器官能獲得更好的

外分泌功能像是(前列腺和乳腺)來識別新“癌症液態活檢” 用於預後和診斷的

生物標誌。
The potential of micro- and nanotechnologies in cancer research has long

been reported as offering precise control over cell cluster size and

geometry but this approach lacks control over dynamic, spatial and

temporal cell responses

長期以來微奈米技術對癌症的研究提供精確控制細胞簇大小和幾何，但這個方

法缺乏控制動態，空間和時間細胞反應

To overcome this limitation, more sophisticated approaches combining

3D cell culture and microfluidics emerged in 2010.

為了克服這個限制，更複雜的方法結合 3D 細胞培養和微流體出現(2010 年)。

Microfluidics has indeed revolutionized the way in which cells can be

studied and manipulated.

而微流體確實改變了細胞的研究和操作方式。

On small amounts of cells and samples, it can combine cell culture with

the sampling and the potential for direct analysis of metabolites.

再少許的細胞樣本上，細胞的培養可以取樣和直接分析代謝物的可能性。
However, current culture systems for HT applications in 3D rely on

multiwell plates where cells embedded in extracellular matrix scaffolds

develop into heterogeneous and overlapping organoids

然而，當前的培養系統對於高通量 HT 應用 3D 多孔版

在細胞嵌入的多孔板上

細胞外的模型支架開發成了不均勻且

This approach limits experiment reproducibility, analysis and gene

delivery.

這種方法限制了實驗的可重複性，分析和基因傳遞。

Altogether, there is a need for new 3D culture formats to produce

homogeneous

and easy-to-handle organoids

總而言之，需要新的 3D 培養的規格來產生均勻的且易處理的類器官
Building on our previous microfluidic device in which fully-differentiated

organoids

grow within Matrigel microbeads under highly controlled conditions , we

designed an automatic and robust platform allowing HT production of

homogeneous organoids and

demonstrated the feasibility of organoid-based genetic screen.

基於我們以前的微流體設備高度控制的條件下其中完全分化的類器官增長在基

底膜微珠下，我們設計了一個自動且強大的平台允許高通量的生長均質的類器

官，證明了類器官為主的遺傳篩選是可行的。

In the field of “organ on chip”, most standard models have been

developed from the original idea of Huh et al.

在器官晶片的領域，
ORGANOIDS-BASED GENETIC SCREENING

基於類器官的基因篩選
3D transfection
Direct 3D transfection on already formed organoids remains challenging.

直接 3D 轉染在已經形成的類器官上仍然充滿挑戰性。
To address this issue,

要解決這個問題，

we developed an innovative approach for transgene expression in 3D

organoids by combining single-cell encapsulation in microbeads and

electroporation (Fig. 4).

我們開發了一個創新的方法，3D 類器官的轉基因表示通過塑膠微珠和電穿孔裡

結合單細胞封裝

如圖.4

We showed that 3D direct transfection preserves the natural self-

organization of organoids along with their spatial architecture and

polarity,

我們發現 3D 直接轉染保留了類器官的自然自組織及其空間結構和極性

which demonstrated that electroporation reached up to 80% transfection

efficiency.

這證明電穿孔達到高達 80％的轉染效率。
For the more refractory breast organoids (23% transfection efficiency vs.

80% for prostatic cells), after modulating the microbead size and

composition, we achieved 57% transfection efficiency.

對於更難治療的乳腺類器官(23%的轉染效率對上 80%前列腺細胞)，在調節微

珠尺寸和組成後，我們實現了 57％的轉染效率。

We further validated the role of p63 and pTEN as key-genes in acinar

development in breast and prostate tissues (Fig. 4),

我們進一步的驗證腫瘤蛋白 p63 和磷酸酯酶與張力蛋白同源物 pTEN 作為乳腺

癌和前列腺組織中腺泡發育的關鍵基因如圖 4

hence demonstrating the feasibility of organoid-based genetic screening

on directly transfected organoids and not on dissociated cells from

organoids, as usually performed.

於是證明了通常進行的組織為主的器官基因篩選是直接轉染的類器官而不是從

類器官解離細胞。

Such microcapsules can be sorted by LP-(Large particles) FACS.

這樣微膠囊可以分類 LP-(Large particles) FACS.

We anticipate that the combination of microencapsulation and LP-FACS

will open new avenues in 3D organoids RNAi-based screening

approaches using microfluidics.

我們預計結合微膠囊和 LP-FACS 將開啟使用微流體的 3D 類器官核糖核酸干擾

的篩選方法的新途徑

RNAi-based screening approach 核糖核酸干擾篩選方

法
The feasibility of large scale HCS using prostatic 3D organoids was

demonstrated by combining 3D culture, lens-free imaging, and

holographic reconstruction of 3D images.

通過 3D 的培養、無透鏡成像，大規模 HCS 人絨毛膜生長激素運用到前列腺 3D

類器官證明是可行的。

We provide here preliminary results on the consequences of RNAi-

mediated down-regulation of each kinase and phosphatase in the human

genome on the dynamic proportion of fully differentiated acini versus

proliferating unorganized tumor-like spheroids.

我們在這裡提供初步結果在核糖核酸干擾後過下調激酶和磷酸酶，在人類基因

組中，完全分化的腺泡與增殖的無組織腫瘤球狀體的動態比例。

To allow a direct, parallel and abel-free read-out of organoid fate (acini vs.

tumor-like spheroids),
prostate on a chip 前列腺晶片
In the aim to closely mimic the structural and physiological function of

prostatic human organs, we created a functional in vitro model that

reproduce acinar and ductal structures of exocrine organs, while

providing controlled growth conditions and access to their secretions.

為了模仿人體前列腺結構和生理功能，我們創造了一個模型，複製了有腺泡和

導管結構的外分泌器官，提供了受控制的生長條件來獲取他們的分泌物。

PE-membrane based scaffolds 聚電解質膜基於支架

Biomimetic strategies based on 3D self-assembling polyelectrolytes (PE)

produced using the layer-by-layer (LbL) technique have been introduced

to provide 3D scaffolds (e.g.

circular microtubes ) to mimic the natural ductal structures that connect

acini in exocrine glands (Fig. 5).

基於 3D 層層自組裝技術所產生的聚電解質的模仿策略，已經被介紹提供到 3D

支架上(例如: 圓形微管)來去模仿連接外分泌腺體腺泡的天然導管結構
More recently, a first patented prototype (Fig. 5) has been created by

additive manufacturing technologies (3D printing) and populated with

prostate epithelial cells.

最近，第一個（3D 打印）專利原型已經創建用來填充前列腺上皮細胞

The device has been characterized structurally and functionally tested by

analyzing the cell secretome by proteomics.

該設備已經通過分析進行功能測試應用在蛋白質組學分析細胞分泌蛋白

Functional signature of cancer cells 癌細胞的功能特徵

With the aim at providing the building-blocks of a “liquid biopsy”

concept based on secretome fingerprints, we present preliminary data

obtained by collecting secretions from control and cancer cell models

within the scaffold and analysis by ELISA, protein arrays and Mass

Spectrometry.
為了提供液體活檢的建築模塊上，我們提出了通過收集支架內對照和癌細胞模

型的分泌物並通過酶聯法 ELISA，蛋白質晶片和質譜法獲得的初步數據。