Droplet microfluidics enabled cell culture and analysis

Characterized by the features of miniaturization, compartmentalization, and parallelization, droplet microfluidics have emerged as a powerful tool for various biomedical applications, especially cell culture and single cell analysis. However, the currently established methodologies based on droplet microfluidics have not reached its full potential. Although water-oil-water double emulsions produced by microfluidics are shown promising for the cultivation of multicellular spheroids, long term cultivation of spheroids and the characterization in situ remain the main challenges. Single cell analysis has been leveraged to reveal cell heterogeneities by inspections of nuclear/mitochondrial genomics, epigenomics, transcriptomics, proteomics, and metabolomics. However, the whole genome sequences of most human intestinal microbiota remain unknown due to the lack of reliable methods for single cell genomics. Besides, the methodology for single cell full-length mitochondrial DNA (mtDNA) sequencing remains unexplored although some mutations have been implicated in many pathogenetic phenotypes. As to the specific transcription profiling of heterogenous cells at the level of individual cells, it also suffered from obstacles including the lack of simple approaches for high throughput analysis and the requirements of complex microfluidic chip design and operations.This thesis is therefore aimed to address the above-mentioned technical challenges. A platform technology, termed Double Emulsion-Pretreated Microwell Culture, was established to allow for effective initiation, sustained maintenance, and culture of the produced spheroids. To allow in situ characterizations of individual spheroids, double emulsions containing spheroids were first localized in microwells on chips followed by spheroids releasing by removing oil layers of double emulsions. Coupled with a built-in analytical station, drug testing of individual spheroids was made possible, which might reveal spheroids heterogeneities and present possibilities as 3D cell culture model for screening drugs. Secondly, as a complement to metagenomics and cultivation, single bacteria genome sequencing was made possible by technical innovations on cell singularization, genome amplification and barcoding strategies, which was validated by high genome coverage of Escherichia coli and Staphylococcus aureus at the single cell level. Subsequently, we proposed a platform for single cell full-length mtDNA sequencing. Through optimizing polymerase chain reaction (PCR) reagents and lysis buffer, mtDNA amplification from single cells was made feasible. Utilizing barcode templates as primers during PCR, mitochondrial genome was labelled to identify the origin of cells. With the workflow, full-length mtDNA sequences were acquired from single peripheral blood mononuclear cells of healthy male and female subjects by the third-generation sequencing. Lastly, we demonstrated a novel thermo-induced dual-core coalescence method for single cell reverse transcription polymerase chain reaction (RT-PCR) in double emulsions, representing a facile and reliable approach for characterizing targeted transcriptome of heterogeneous biological systems. Taken together, the technical advancements established in this thesis are expected to benefit biomedical applications including the high throughput drug screening for anticancer therapies, the establishment of reference genomes for human intestinal microbiota, the illustration of mtDNA mutation profiles from single cells and the identification of genetic mutations or rare cells with specific expression features.由於具有小型化，可區分性以及並行分析的特點，液滴微流控技術作為一種有力工具已廣泛用於生物醫學研究的各個方面，特別是細胞培養和單細胞分析。然而，目前基於液滴微流控的方法還沒有發揮出其所有潛力。雖然通過微流控技術可以產生水-油-水型雙乳化液滴用於產生細胞聚團，但是還無法實現其長期培養和原位表徵。單細胞分析可以在多個層次（包括核/線粒體基因組學，表觀遺傳組學，轉錄組學，蛋白質組學以及代謝組學）揭示細胞異質性。由於缺少可靠工具用於單細胞核基因組學，目前仍舊無法獲得所有人腸道微生物的全基因組信息。另外，雖然已經證實許多致病表型與線粒體基因組突的相關性，但是現在也沒有單細胞全長線粒體基因組測序工具用於突變分析。由於缺少簡易的高通量分析方法以及目前微流控芯片設計和操作複雜性，單細胞層次的特定轉錄組分析也依然很難做到。因此，本論文旨在解決以上存在的技術挑戰。首先，我們開發了一種技術平臺稱作“雙乳化液滴預處理的微孔培養”，實現了細胞聚集成團，以及長期維護和培養。為了實現單個細胞聚團的原位表徵，首先將雙乳化液滴定位在芯片上的微孔中，然後除去液滴油層將聚團釋放出來。結合自製的分析平臺可以進一步實現對單個細胞聚團的藥物測試，從而揭示聚團之間的異質性以及為用於藥物篩選的三維細胞培養模型提供了可能。通過細胞單一化，基因組擴增以及條形碼策略方面的技術創新，實現了單細菌基因組測序，使其成為宏基因組學和微生物培養的補充。單個大腸桿菌和葡萄球菌的高基因組覆蓋度進一步驗證了該方法的可行性。我們也提出了一個用於單細胞全長線粒體基因組測序的平臺。通過優化聚合酶鏈反應的試劑以及細胞裂解緩衝液，實現了線粒體基因組的全長擴增。為了標記線粒體基因組的細胞來源，我們在聚合酶鏈反應時引入條形碼模板作為擴增引物。根據以上流程和三代測序技術，我們獲得了健康男性和女性外周血單核細胞的全長線粒體基因組序列信息。最後，為了實現單細胞反轉錄聚合酶鏈反應，我們開發了一種基於雙乳化液滴內雙核熱融合的方法。該方法簡單可靠，可以實現對異質性細胞內特定轉錄組的表徵。總結起來，本論文中實現的技術進步將會有益於許多生物醫學應用，例如：抗癌治療中的藥物篩選，人腸道微生物參考基因組的建立，單細胞線粒體基因組突變分析，確定基因突變，以及發現具有特定表達特徵的罕見細胞。Qu, Fuyang.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …)