類器官研究的現狀和發展趨勢

類器官研究的現狀和發展趨勢——諾貝爾生理學或醫學獎獲得者專訪

圖1 熒光顯微鏡下的類器官（圖片源自網路）

類器官（Organoids）是一種在體外環境下培育而成的具備三維結構的微器官（圖1），擁有類似真實器官的複雜結構，並能部分模擬來源組織或器官的生理功能。藉助類器官，研究人員可深入觀察人體組織的變化，更好地理解發育過程，並可用於再生醫學以及藥物的療效篩選。因此，類器官研究具有廣闊的發展前景。

圖2「Organoid Modeling of the Tumor Immune Microenvironment」文章封面

中國生物技術發展中心針對2018年12月美國斯坦福大學Calvin J. Kuo教授團隊發表在國際頂尖雜誌Cell的文章「Organoid Modeling of the Tumor Immune Microenvironment」（圖2），特邀諾貝爾生理學或醫學獎獲得者Thomas C. Südhof教授、著名環境毒理學及神經類器官專家Ellen Fritsche教授就類器官研究的現狀、瓶頸、應用價值以及未來的發展方向展開評述。下面將為讀者呈現訪談內容。

1. 問： 什麼是類器官？類器官的主要類型和他們各自的關鍵特徵是什麼？

Question：What is organoid?What are the main organoid types and their key characteristics?

Thomas C. Südhof答：目前有很多該領域的綜述和專著可供參考，以下是我推薦的一些綜述。(編者按：綜述列表見英文回答)

Thomas C. Südhof』s answer：There are innumerable review articles and textbooks on organoids that I would suggest you consult.

Here are some reviews:

[1] Little MH, Hale LJ, Howden SE, Kumar SV. Generating Kidney from Stem Cells. Annu Rev Physiol. 2019 Feb 10;81:335-357

[2] Rowe RG, Daley GQ. Induced pluripotent stem cells in disease modeling and drug discovery. Nat Rev Genet. 2019 Feb 8.

[3] Sontheimer-Phelps A, Hassell BA, Ingber DE. Modeling cancer in microfluidic human organs-on-chips. Nat Rev Cancer. 2019 Feb;19(2):65-81.

[4] Mittal R, Woo FW, Castro CS, Cohen MA, Karanxha J, Mittal J, Chhibber T, Jhaveri VM. Organ-on-chip models: Implications in drug discovery and clinical applications. J Cell Physiol. 2019 Jun;234(6):8352-8380.

[5] Amin ND, Pa?ca SP. Building Models of Brain Disorders with Three-Dimensional Organoids. Neuron. 2018 Oct 24;100(2):389-405.

Ellen Fritsche答：類器官是一種在體外培育而成的具有來源器官顯微解剖特徵的多細胞三維結構。迄今為止，不同組織、疾病模型及模擬發育的類器官已問世。類器官的工具細胞主要為組織特異性多能幹細胞。類器官的主要特徵包括基於細胞類別的自我組織及空間限制的定向分化，與體內發育過程相似。他們（類器官）含有多種器官特異性細胞，這些細胞的空間組織、排列與來源器官類似。另外，他們（類器官）具有一些來源器官特有的功能。迄今，來源於多種器官的類器官業已面世，包括腦、腸道、胃、舌、甲狀腺、胸腺、睾丸、肝臟、胰腺、皮膚、肺、腎、心臟及視網膜。除了來源於健康組織的類器官，大量疾病模型（包括腫瘤模型）的類器官也不斷湧現。最後，類器官為科研人員進行發育生物學研究提供了絕佳模型。

Ellen Fritsche』s answer：An organoid is a three-dimensional (3D), multicellular structure with microanatomical features of the organ of origin produced in vitro. So far, organoids of different tissues, disease models, as well as organoids resembling development have been created. Cellular basis for organoids are mainly pluripotent or tissue-specific stem cells. Key features of organoids include their self-organization through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo. They contain multiple, organ-specific cell types which are spatially organized in a manner similar to the organ. In addition, they recapitulate some specific organ functions. Organoids from multiple organs have so far been created. These include brain, intestine, stomach, tongue, thyroid, thymus, testis, liver, pancreas, skin, lung, kidney, heart and retina. In addition to the healthy organoids, a plethora of disease models including tumor models, have been developed. Last, organoids offer researchers an exceptional model to study developmental biology.

2. 問：可否談談類器官在生物醫學領域的主要應用？

Question：What are the main applications of the organoids in the field of biomedicine?

Thomas C. Südhof答：類器官的價值在於其具有在體外培養環境下構建人類器官疾病模型的潛力。這非常適用於像心臟這樣的組織，在人類早期腦發育的研究上也逐漸變得可行。但類器官在再生醫學上的應用依然前路漫漫。

Thomas C. Südhof』s answer：The attraction of organoids is that they potentially allow disease modeling of human organs in a dish. This works best for tissues such as heart, and is becoming feasible for early human brain development. The use of organoids in regenerative medicine is still far in the future.

Ellen Fritsche答：作為一項重大的技術突破，類器官目前已被公認為生物研究的重要工具，並具有重要的臨床應用價值。類器官允許在一個模擬內源性細胞組織和器官結構的環境中進行組織生物學、發育、再生、疾病建模 (包括癌症研究)、器官移植技術改良、藥物發現/療效評估以及毒理學的研究。

Ellen Fritsche』sanswer：Starting as a major technological breakthrough,organoids are now well-established as an essential tool in biological research and also have important implications for clinical use. Organoids allow research on tissue biology, development, regeneration, disease modeling (including cancer research), improvements in organ transplantation, drug discovery/response as well as toxicological studies in an environment that mimics endogenous cell organization and organ structures.

3. 問：在腫瘤生物學及新葯開發領域，類器官相對於細胞系、動物模型的主要優勢在哪裡？

Question：What are the main advantages of using organoids instead of cell lines, or animal models in the field of tumor biology and new drug development?

Thomas C. Südhof答：相對於細胞系而言，類器官構建了一個具備三維結構的器官樣組織，儘管並不完全（模擬人類器官）。相較於動物模型，類器官的優勢體現在其實現了應用人源性組織進行實驗研究。

Thomas C. Südhof』s answer：The advantage over cell lines is that organoids model a three dimensional organ, although not completely. The advantage over animal models is that organoids enable studies of human material.

Ellen Fritsche答：傳統的二維 (2D) 腫瘤細胞系培養和動物人源性腫瘤異種移植物 (PDTXs) 長期以來一直被用作腫瘤模型, 並為癌症研究做出了巨大貢獻。然而，各種缺點阻礙了這些模型的臨床應用，這主要是由於與腫瘤治療相關的藥物開發是成功率最低的。二維細胞培養體系不具備免疫細胞、微環境、間質成分和器官特異性的功能。其他限制包括腫瘤細胞系經多次傳代後缺乏來源腫瘤的遺傳異質性，原因是細胞在培養皿二維生長的環境下會發生優勢克隆選擇，但這並不符合生理。此外，PDTX 模型還經歷了小鼠特異性的腫瘤演化。在資源方面，這些模型也是極度的費錢費時。類器官可以克服其中的一些限制。類器官的基因修飾可實現在接近生理環境的情況下進行疾病建模。比如，將腫瘤性突變導入健康幹細胞可以產生遺傳控制的腫瘤類器官。此外，類器官可以從患者來源的健康組織和腫瘤組織中迅速培育，從而使患者特異性藥物檢測和個性化治療方案的開發成為可能。在這種患者特異性的腫瘤類器官中，可觀察到組織穩態(histostasis)，如3D培養保留了與來源患者腫瘤相一致的組織病理學特徵，為未來個性化腫瘤治療的發展提供了希望。與 PDTX 不同，類器官維護便利，具有整合免疫細胞的可能性，易進行基因改造 (遺傳性腫瘤建模)，支持匹配對照的研究，並可用於高通量藥物篩選和生物庫的建設。

除了腫瘤學，類器官也為新葯開發提供了絕佳模型。新葯開發的失敗率很高，這在一定程度上是由於動物葯代動力學和藥效學的差異或動物疾病模型並不能完全模擬人體病理過程。具有人體特異生理和病理特徵的類器官有助於克服這些問題。基於特定疾病，甚至特定個體，以高通量方式培育的類器官預計將發展成為精確治療的強大工具。未來可藉助生物庫進行篩選，不僅是為了鑒定新葯，還可揭示哪些患者可以從某些 (現有) 藥物的治療中受益。此外，對潛在藥物的重點檢測可為製藥業提供新的指引。另外，類器官未來可能用於毒理學檢測，以作為動物試驗的有力補充(如果不是部分取代的話)。

Ellen Fritsche』s answer：Traditional two-dimensional (2D) tumor cell line cultures and patient-derived tumorxenografts (PDTXs) in animals have long been employed as tumor models and have made tremendous contribution to cancer research. However, a varietyof drawbacks hamper these models for clinical useas success rates for tumor therapeutics are lowest in the field of drug development. 2D cell line cultures do not contain immune cells, microenvironment, stromal compartments, and organ-specific functions. Other limitations include the lack of genetic heterogeneity of original tumors after many passages for cancer cell lines because clonal selection in the dish happens for superiority in 2D growth, which is not physiologic. Moreover, PDTX models experience mouse-specific tumor evolution. On the resource side, such models arehighly money- and time-consuming.Organoids can overcome some of these constraints. Genetic modification of organoids allows disease modeling in a setting that approaches the physiological environment. Here, insertions of tumor mutations into healthy stem cells allow generation of genetically-controlledtumoroids. Additionally, organoids can be grown with high efficiency from patient-derived healthy and tumour tissues, potentially enabling patient-specific drug testing and the development of individualized treatment regimens. In such patient-specific tumoroids, histostasis is observed, i.e. conservation of histopathological traits between 3D cultures and the matched patient tumor, promising advances in personalized tumor therapies in the future. In contrast to PDTX, organoids are of easier maintenance, bear the possibility to integrate immune cells, are amenable to genetic modification (genetic cancer modeling), allow study of matched controls, can be used for high throughput drug screening and biobanking.

Besides oncology, organoids are promising models for drug development. Attrition rates in new drug development are high. This is partly reasoned indifferences between animal pharmacokinetics and –dynamics or in animal disease models that do not correctly resemble human pathology. Organoids with human-relevant physiology and pathology are thought to help overcoming these issues. Organoid cultures based on a specific disease and even on a specific individual used in a high-throughput manner are expected to develop into powerful tools for precision therapy. Future screens may be performed using biobanks with the aim of not only identifying new drugs but also revealing which patients may benefit from treatment with certain (existing) drugs. In addition, focused tests of potential drugs should identify new leads for the pharmaceutical industry.Furthermore, organoidsmay be used in the future for toxicology testing to complement, if not in part replace, animal testing.

4. 問：當前類器官的局限是什麼？為了滿足腫瘤生物學、幹細胞生物學、移植、新葯開發領域的研究需要，類器官需要在哪些方面進一步改進？

Question：What are the limitations of organoids and what aspects of organoids can be further improved to meet the demand for research in tumor biology, stem cell biology, transplantation and drug development?

Thomas C. Südhof答:類器官領域的研究仍在起步階段。即使對於如心臟和肝臟這樣的組織，類器官也很不成熟，僅能部分模擬人體器官。對於腦組織則更甚。許多基本的（腦組織）生理功能，如細胞生理、生化功能仍有待突破。這將花費數年的時間。

Thomas C. Südhof』s answer：The field of organoid research is still in the beginning. Even for tissues like heart and liver, organoids are very immature and only partly model the human organ. This is worse for brain. Much fundamental biology, such as cell biology and biochemistry, is needed to advance the field. This will take many years.

Ellen Fritsche答：類器官是融合了各種器官特異性細胞類型、組織形態和功能的組織模型。但類器官僅為有限度的模擬，困擾這項技術應用的一個重要限制是它的體積。當類器官體積增加時，缺氧和缺乏可溶性因子所致的組織壞死是亟需解決的問題。解決這個問題的一個可能方案是激活血管生成途徑, 從而使類器官血管化。這已經在hiPSC衍生的肝臟類器官上成功實現。類器官領域的另一個挑戰在於一個完整的有機體中所自然存在的器官「對話」。類器官研究可滿足生物工程的要求，通過培育包含不同類型hiPSC衍生類器官（呈現多個器官系統的結構和功能）的器官晶元設備，用以在更類似於體內的環境中篩選藥物。另外，通過在類器官中添加免疫細胞，還可模擬具有免疫系統的組織間「對話」。另外，在藥理和毒理學研究中，物質的肝臟代謝至關重要，這可通過以器官晶元的形式包含肝臟代謝來實現。

Ellen Fritsche』s answer：Organoids are organ models recapitulating an assortment of organ-specific cell types, tissue morphogenesis and functions. Yet, there are limitations in their mimicry. One important limitation plaguing the application of this technologyis their size. When the organoids』 volume increases, the issue of tissue necrosis caused by the lack of diffusion of oxygen and soluble factors needs to be addressed. One solution for this problemmight be the activation ofangiogenic pathways that will lead to vascularisedorganoids.This was already succeeded with hiPSC-derived liver organoids. One more challenge of the organoid field lies in organ crosstalk, which is naturally present in an intact organism. Here, organoid research meets bioengineering by producing organ-on-a-chip devices containing different types of hiPSC-derived organoids representing the structure and function of multiple organ systems for screening the effects of drugs in more in vivo-like settings. The crosstalk of tissues with the immune system can be modelled by adding immune cells to the organoids. For pharmacological and toxicological applications, liver metabolism of compounds is crucial. Including such metabolism via an organ-on-a-chip approach can solve this issue.

5. 問：當前類器官研究的發展方向如何？

Question：What are the current trends for organoidsresearch?

Thomas C. Südhof答:（當前的現狀是）所有人都在盲目追求應用，卻忽略了一個堅實的科學基礎。我認為未來會有數以百計的公司在那裡販賣希望，但他們大多數將以失敗告終。因為相關生理學研究成果並不足以支撐這些應用項目。類器官最有前景的應用領域應是用於肝臟、心臟和腫瘤的藥物篩選。

Thomas C. Südhof』s answer：Everybody rushes towards applications, without a solid scientific basis. I think hundreds of companies will be founded that will sell hope, but will mostly fail because the biology isn"t there to support applications. Most promising are drug screens in tissue organoids such as liver or heart and in cancer.

Ellen Fritsche答：目前類器官研究的趨勢包括建立用於高通量篩選的類器官庫和平台，建立其他疾病模型，以及建立用於整個生物體建模的器官晶元和微流體晶元。在此特別要強調的是培養基的限制亟待解決。對微流體晶元來講，需要一種晶元上所有類器官均適用的通用培養基。此外，根據器官系統的不同，需要開發與生理過程相關的來自類器官的高通量數據輸出裝置。在臨床方面，為了開發最佳個體化治療方案，使用源自患者特異性hiPSC類器官的個體化醫療研究亟需開展。在毒理學領域，類器官目前已被用來替代動物進行毒性測試。

Ellen Fritsche』s answer：Current trends for organoid research include generation of organoid banks and platforms for high-throughput screening approaches, generation of additional disease models, and set up of organ-on-a-chip and microfluidics devices for whole organism modeling. Here, especially medium constrictions have to be solved. For microfluidics a common medium for all organoids on the chip is needed. Moreover, depending on the organ system, physiologically relevant high-throughput readouts from organoids need to be developed. On the clinical side, research on personalized medicine using organoids derived from patient-specific hiPSC is warranted for optimal individual treatment regimes. In the toxicology field, organoids as substrates for toxicity testing replacing animals is currently exploited.

6. 問：可否預測一下接下來5年內類器官研究領域的發展？

Question：How the organoids research field will be look like in 5 years?

Thomas C. Südhof答:我的預測是在接下來的5年內好的實驗室將學會如何促進類器官的成熟，並明確該方法的局限。我認為，儘管類器官為幹細胞研究提供了巨大的機會，如促進新發現和療法的出現；但這將耗費10年或更長的時間來發展。到那時候，也只有到那時候，走嚮應用才能真正成為可能。在那之前，大量初創公司將會燒掉數以億計的資金，他們中的少部分將會走向成功，並找到增加營收的新途徑。

Thomas C. Südhof』s answer：My prediction is that in 5 years, good science labs will have learned how to mature organoids and the limitations of the approach will have been defined. I think organoids are a tremendous opportunity in stem cell approaches that will enable novel discoveries and therapies, but that this will take at least 10 years to develop. Then and only then will it be possible to rationally move towards applications. Until then, lots of start-ups will have spent hundreds of millions of dollars, and a few of them will have been successful in generating some future avenue of revenue.

Ellen Fritsche答：在5年內，類器官的遺傳操作與類器官庫相結合將給生物醫學研究帶來翻天覆地的變化。購買來源於具有不同遺傳背景患者的疾病特異性類器官將成為可能。器官晶元平台將具有特定標準，由合同研究組織（CRO）以與目前動物試驗類似的方式提供。類器官將極大地促進藥物療效試驗和安全性測試的開展，因此也將進入藥物開發和化學安全性評估研究的監管領域。

Ellen Fritsche』s answer：In 5 years, genetic manipulation of organoids in combination with organoid banking will have revolutionized biomedical research. It will be possible to purchase disease-specific organoids from broad ranges of patients with distinct genetic backgrounds. Organ-on-a-chip platforms will be standard and offered by CROs in a similar manner than currently animal testing. Organoids will have tremendously facilitated drug efficacy and safety testing and thus will have entered also into the regulatory areas of research for drug development as well as chemical safety assessment.

附：

1、Thomas C. Südhof教授簡介

斯坦福大學醫學院教授、霍華德-休斯醫學研究所 (HHMI) 研究員、美國科學院院士、美國醫學科學院院士、英國皇家學會外籍院士、2013年諾貝爾生理學或醫學獎獲得者。1955年生於德國哥廷根，1982年獲得哥廷根大學醫學博士學位。Südhof教授的研究主要聚焦於突觸前神經遞質釋放的分子機制，為該領域的頂級科學家。他發現了囊泡內神經遞質釋放過程中的多種關鍵蛋白，並闡明了神經遞質釋放的具體分子機制。鑒於在囊泡轉運領域的開創性工作，他先後榮獲拉斯克基礎醫學獎及諾貝爾生理學或醫學獎等重要醫學獎項。

2. Ellen Fritsche教授簡介

德國IUF-萊布尼茨環境醫學研究所（IUF-Leibniz Research Institute for Environmental Medicine）環境毒理學教授，球模型和風險評估專家組組長。1998年獲雷根斯堡大學和杜塞爾多夫大學醫學博士學位，曾先後在美國國立環境衛生研究所（NIEHS)和IUF-萊布尼茨環境醫學研究所完成博士後研究工作，2009-2012年任亞琛工業大學皮膚毒理學教授。目前為Neurotoxicology雜誌副主編、歐洲化學理事會（cefic）顧問、歐盟地平線2020計劃專家組成員、歐洲替代動物試驗研究中心（CERST-NRW）項目牽頭人、替代法信託大會（ACT）成員和OECD發育神經毒性專家組委員。歷任歐洲替代動物試驗協會（EUSAAT）副主席、主席。

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