In 2009, a research paper on single-cell mRNA sequencing was published on Nature Methods. Since then, the development of single-cell sequencing industry has been fully kicked off.
single-cell sequencing is an emerging technology for extraction and amplification of genomes or transcription groups and high-throughput sequencing analysis at the level of single cells. As a major technology in the field of life sciences in the past decade, its application prospects are widely optimistic. At the same time, more and more companies around the world emerge and join this track, and the industry competition is becoming increasingly fierce. Compared with
globally, the domestic single-cell sequencing industry started a little later, and its technology, products and market share are dominated by overseas companies. In order to avoid being "bottled" in the future, it is necessary for domestic companies to independently develop single-cell sequencing technologies and products from the source.
As an enterprise driven by innovative technology, Mozhuo Bio has accumulated innovation from the source based on droplet microfluidic control technology, independently developing end-to-end high-throughput single-cell sequencing complete solutions, and promoting the development of the domestic single-cell sequencing industry from technology to products.
Recently, Shenghui invited Dr. Zheng Wenshan, Mozhuo Bio CTO, to interpret the single-cell sequencing industry based on multiple dimensions such as source innovation, products, technology, and industries.
▲Photo | Mozhuo Biology CTO Dr. Zheng Wenshan (Source: Interviewee)
Origin: occupying the source of innovation highland
From becoming a famous teacher to industrialization practice
After graduating from the undergraduate degree in Chemistry of Peking University, Zheng Wenshan entered Harvard University pursued a doctorate degree. His undergraduate research direction is nanomaterial synthesis. "Although the topic I was studying at that time did not belong to biotechnology, it was essentially methodological development. Up to now, this has always been my research direction - methodological development in different fields," he said.
At Harvard University, Zheng Wenshan was a member of the United States' Third Academy and a foreign academician of the Chinese Academy of Engineering Professor David Weitz. Professor David Weitz is one of the founders of droplet microfluidic control and the inventor of applying droplet microfluidic control to high-throughput single-cell sequencing technology.
Zheng Wenshan The research direction during his Ph.D. mainly revolves around the field of droplet microfluidics. For example, uses droplet microfluidics for high-throughput single-cell sequencing , includes the eukaryotic transcriptome and microbial genome. At present, he has published more than ten papers in high-level international journals such as Science, Nature Communications and other high-level international journals.
Talking about the original intention of founding Mozhuo Biotechnology, Zheng Wenshan admitted that during his studies at Harvard University, he and two other founders, Dr. Pei Hao and Dr. Liu Han, hoped to have some big and positive impact: apply the knowledge learned to product development to help and benefit more people. "The entrepreneurial atmosphere at Harvard University is very strong, and the Boston where Harvard University is located is a holy place for biotechnology entrepreneurship around the world. We have been influenced and influenced by this during our studies," he added.
In 2018, they co-founded Mozhuo Bio on the Harvard University campus in Boston, USA.
▲Picture | From left to right, Chief Operating Officer Liu Han, CEO Pei Hao, and Chief Technology Officer Zheng Wenshan (Source: Company Official Website)
chose to return to China to land, "First of all, we see that there is still a certain gap between the field of biotechnology and foreign countries in China. We hope to use practical actions to contribute to the promotion of the domestic biotechnology industry; secondly, the overall domestic environment strongly supports entrepreneurship and innovation, including national policies and local policies, which are very favorable; thirdly, domestic peripheral supporting facilities, whether it is hardware facilities or scientific research teams, have been very sound and perfect, and the development prospects in China are broader." Zheng Wenshan said.
With this enthusiasm, they returned to China in 2020 and brought Mozhuo Bio to Jiaxing, Zhejiang.
▲Photo | Mozhuo Bio (Source: Company Official Website)
Currently, Zheng Wenshan serves as the chief technology officer of Mozhuo Bio, focusing on innovative technology research and development. Professor David Weitz is the company's chief scientist. "Drop microfluidic control is the core technology of Mozhuo Biologics, which is also the main research topic I followed Professor David Weitz during my PhD, namely the application and technological development of droplet microfluidic control technology in the field of life sciences." Zheng Wenshan said.
In Zheng Wenshan's view, Mozhuo Biotechnology is a truly user-oriented and product-centered biotechnology company. "Our team has a highly consistent internal thinking, implementing the product-centered corporate philosophy. By deeply exploring the needs of users, all departments work together to develop high-quality and high-performing innovative products that can effectively meet user needs, serve life sciences, and benefit human health." He said, "'Serving human health with life sciences' is not only the company's vision, but also one of our own wishes."
Product: Independently designing the first product from the source,
reaches the international mainstream level
This year is the first year of the commercialization of Mozhuo Bio's first set of products.
As a product-centric biotechnology company, MobiNova® high-throughput single-cell transcriptome solution, was launched in April this year.
▲Picture | MobiZhuo BiomobioMobiNova® high-throughput single-cell transcriptome solution (Source: Company Promotional Information)
MobiNova® high-throughput single-cell transcriptome solution is a complete solution covering instruments, reagents and bioinformatics analysis software. It consists of MobiNova®-100 high-throughput single-cell sequencing library construction system, MobiCube® high-throughput single-cell transcriptome kit and MobiVision® bioinformatics analysis software, covering the needs of high-throughput single-cell transcriptome experiments throughout the process.
It is understood that the cell population quality of MobiNova® high-throughput single-cell transcriptome solution is comparable to that of international mainstream products. The cell capture rate is 70%, and rare cell types can be captured, with a multicellular rate of 5% per 10,000 cells.
has achieved high-level performance indicators. In the view of Zheng Wenshan , it is mainly due to the technical accumulation of Mozhuo team. "Using droplet microfluidic technology to conduct high-throughput single-cell sequencing is actually the first of our team in David Weitz, the founder of Mozhuo. From the invention of microfluidic technology to the subsequent expansion and application research and development, we have had deep technical precipitation and accumulation of experience over the years." He said, "In actual product development, since the entire product system is relatively complex, various ports such as instruments, microfluidic chips, reagents, and biotechnology need to cooperate highly with each other to achieve precise control of each port. This requires us to carry out independent and starting from scratch design, research and development and industrialization.”
Specifically, "we optimize the design and instrument control of microfluidic chips, and stably realize cell isolation and co-wrap of microspheres, reagents, and cells during droplet generation, thereby achieving a high cell capture rate. At the same time, the high-speed generation of droplets ensures that the multicellularity rate per 10,000 cells is controlled within 5%. "Zheng Wenshan introduces .
In addition, the stability of data in different batches and the repeatability of experiments have always been the pain points of the industry and one of the most difficult challenges to solve, because this is a consideration of comprehensive performance. It requires both the instruments to be precise enough and the reagents are stable enough, and even the convenience of the experimental process has extremely high requirements. Looking at the entire scientific research instrument and in vitro diagnostic industries, this is the biggest gap between domestic instruments and imported instruments.
MobiNova® The stability of different batch data of single-cell transcriptome solutions and the repeatability of experiments are also at the international leading level. In this regard, Zheng Wenshan said, "As mentioned just now, the entire system's instruments, microspheres, reagents, biotechnology software, microfluidic chips, etc. need to be closely coordinated between each port, from the core components to the smallest process. We all start from the source and carry out independent research and development and production, so we can achieve accurate control of the entire process. Therefore, the experimental data obtained by our products have high stability, and the stability in batches and inter-batches are very high. "
" In short, we have complete independent intellectual property rights, and the entire system can be developed and improved internally. Therefore, we can achieve high performance parameter indicators by constantly polishing the products, and be as good as the quality of international mainstream products. "Zheng Wenshan concluded.
technology: From substitution to surpassing
3 three major technological innovations occupy the international leading
Create an excellent product naturally cannot be separated from the support of advanced technology.
As for the moment, high There are two main technical routes for flux single-cell sequencing, one is the micropore method and the other is the droplet method. The two technical routes have their own advantages. As the core technology of Mozhuo Bio - droplet microfluidic control, its advantage is to turn each droplet into a micro "reactor", which can greatly increase the flux and reduce costs. Zheng Wenshan said, "During my doctoral studies, I hoped to truly productize the droplet microfluidic control technology, and using droplet microfluidic control for high-throughput biological experiments is a very good application direction, which can allow more users to use this advanced technology and provide support for life science research. "
▲Picture | Mozhuo Bio Reagent R&D Laboratory (Source: Company Official Website)
In order to ensure the superior performance and stability of the product, Mozhuo Bio has further innovated on the basis of the existing droplet microfluidic control technology. independently developed the "zoning" design of cells and reagents, high-speed runners wrapped by microspheres, and Lightcut™ photoexcitation separation.
It is understood that the "zoning" design of cells and reagents is designed to design an independent runner for sample cells, so the original 2 The increase of one aqueous flow channel and one oil-phase flow channel to three aqueous flow channels and one oil-phase flow channel can minimize interference to cells.
"The difficulty in this design is that more flow channels will produce more interactions, from the original pair of three interactions of three channels to the pair of three interactions of four channels. The overall complexity is several times more, and the difficulty of control is greatly increased. Moreover, higher requirements are put forward for microfluidic flow channel design, droplet generation, chip surface modification, etc. "Zheng Wenshan introduced, "The field of microfluidic control is the expertise of Mozhuo Biological Team. We have carried out technical research and properly solved this problem. At present, the generation of high-stability droplets can be achieved on existing systems."
For high-speed flow channels that wrap microspheres, "the high speed here refers to the relatively high frequency of droplet generation, which means that more droplets can be generated per unit time. "Zheng Wenshan pointed out. To achieve high speed, he summarized the following 3 aspects:
First, structural design. The structural design of the runner must be able to generate droplets very stably.
Second, the surface is hydrophobic. "The surface of the runner is hydrophobic. For example, we are currently using 'water-in-oil' droplets, which puts high requirements on the surface modification of the chip. "He explained.
Third, system control. For example, implement precise regulation of the pressure parameters of the system, etc.
" Ultimately, with the coordination and cooperation of these three aspects, our products can generate hundreds to thousands of droplets per second. "Zheng Wenshan said, "This speed can greatly shorten the on-board time, improve the experimental efficiency, and greatly reduce the possibility of mRNA degradation during the experiment, and obtain more stable and superior experimental results. "
" After the droplet is generated, we expect the reaction inside the droplet to be a 'homogeneous reaction', that is, the primer and the mRNA molecule that needs to be captured are both in the aqueous phase, so we need to release the primers attached to the microspheres. "He pointed out.
According to reports, the traditional method is not to release, but the interface reaction will reduce the reaction efficiency; the other method is to dissolve the microspheres, but the complex components in the microspheres will enter the system, which may cause serious interference to the mRNA reverse transcription efficiency and other aspects in the system.
Both of the above methods have limitations. In contrast, "our new method is to add a new light-sensitive linking bond between the microspheres and primers, and after excitation of specific light, the primers will be released controllable. This is the Lightcut™ photoexcitation separation technology. "Zheng Wenshan explained. The three independent innovative technologies, namely,
cell and reagent "zoning" design, microsphere wrapping high-speed runners and Lightcut™ photoexcitation separation, have given Mozhuo Bio's products further superior performance and stability. At present, these three technologies have been applied in MobiNova® In the high-throughput single-cell transcriptome solution.
Breakthrough: Innovative invention of microbial
High-throughput single-cell genomics technology
In addition to being used in the field of single-cell sequencing of animals and plants, Mozhuo Bio has also made innovative breakthroughs in the field of microbial single-cells.
In June this year, Zheng Wenshan, as the tiered first author, published a entitled "High-throughput," single-Microbe genomics with strain resolution, applied to a human gut Microbiome's new research paper has developed microbiome high-throughput single-cell genomics technology - Microbe-seq, which is a major breakthrough in the research methodology of microbial communities. Just last year, the National Natural Science Foundation of China also clearly proposed in its project guidelines to encourage the development of microbial single-cell research.
▲Picture | Related papers (Source: Science)
h Compared with the current conventional microbial metagenomic sequencing technology, high-throughput single-cell sequencing technology has overwhelming advantages. According to Zheng Wenshan, a sample of a microbial community often contains various microorganisms, and its composition is very complex, with hundreds or even thousands of different species. The traditional metagenomic method is to extract all nucleic acids from all microorganisms and then sequence them. The sequence of each nucleic acid cannot be specially processed on the experimental end, and can only be analyzed with the help of the biotechnology end, which has certain limitations.
In comparison, single-cell sequencing can label the nucleic acid marker in each bacteria with a special encoding, and after the sequencing is completed, its sequence can be re-correlated. In this way, the problem of "what sequences come from which microbial cell" has been solved on the experimental end, greatly improving the accuracy and reliability of the results.
Therefore, this high-throughput single-cell sequencing technology for microorganisms can allow scientific researchers to truly study microbial flora genomics, explain the correlation between each sequence measured clearly, and the results will be more credible. Therefore, we can also expand the research on the characteristics of some complex microbial communities.
compared with single-cell sequencing of animals and plants focuses on transcriptome . At this stage, single-cell sequencing of microorganisms focuses on genome. In this regard, Zheng Wenshan said, "For omics of microorganisms, the current research is still relatively early. Even if you want to study the transcriptome, you often need to obtain high-quality genome information as the basis. Therefore, the demand for the genome of microorganisms is relatively greater at this stage." The two technologies of
have both intersections and differences. Specifically, "In the system we developed, both use droplet microfluidic technology, and droplets act as a "high-throughput reactor" in this process. For high-throughput single-cell sequencing, we will label the nucleic acid (RNA or DNA) of each cell with a molecular tag (barcode) inside the liquid. In this way, each cell's nucleic acid will have an DNA sequence (molecular tag) associated with it. This is the principle similarity between the two technologies." Zheng Wenshan introduced. The differences between
are reflected in many aspects. For example, single-cell sequencing of bacteria requires breaking cell wall in the microfluidic system. For animal and plant cells, animal cells do not have cell walls. plant cell generally completes the wall breaking before going to the machine, and there is no need to include the wall breaking step in the microfluidic system. "In addition, single-cell transcriptome sequencing of animals and plants focuses on mRNA. mRNA will have a poly A sequence as a universal fixed linker, which can be captured relatively easily. However, the genome of bacteria is continuous and relatively random, and there is no fixed sequence that can be used as a capture linker, so we also need to add sequences as a capture linker to its genome in this process," Zheng Wenshan explained.
"There is another very critical point. The length of mRNA will be shorter, generally hundreds to thousands of bases . Generally, the gene can be determined by measuring dozens of bases. In contrast, the genome is large, generally with millions of bases, so this requires breaking it to a length that is more suitable for sequencing. Therefore, high-throughput single-cell sequencing of microbial flora will face many new technical challenges." He added.
▲Picture | Principles of high-throughput single-cell genome sequencing experiments for microbial high-throughput single-cell genome sequencing (Source: Science)
In Zheng Wenshan's view, "High-throughput single-cell sequencing technology itself is a relatively innovative method that can study biological problems from new dimensions and resolutions, and is a relatively underlying technological innovation. Therefore, all aspects of microbial research can benefit from high-throughput single-cell sequencing technology."
Therefore, traditional application scenarios for research using metagenomics can be used to conduct more in-depth research using microbial high-throughput single-cell genomics, which can better analyze what microorganisms are in a complex microbial sample, what are their genomic sequences, what are their potential functions, etc., and can also explore the impact of microorganisms on human health, such as analyzing the impact of microorganisms on prognosis in cancer treatment.At the same time, in agriculture, soil microorganisms are also closely related to the growth of crops. High-throughput single-cell sequencing of microorganisms can better analyze their correlation and causal relationships, helping to improve agricultural output and resist diseases, etc.
In addition to traditional application scenarios, new technologies also allow microbial research to reach places that were not as good as previous technologies. For example, since high-throughput single-cell genome sequencing of microorganisms can obtain more complete and more accurate bacterial genome sequences, it can not only analyze different bacterial species in the sample more accurately, but also analyze which different strains are in the same bacterial species, and even analyze the functional differences of different strains in depth. Different strains of the same strain may differ greatly in terms of pathogenicity, function and metabolic pathways, so this is of great significance to agriculture, environment and clinical medicine .
For the transformation of this technology, "We have built a complete set of experimental platforms that can provide customers with scientific research service support. The supporting instruments and reagents are also under development. We hope to launch a complete set of instruments, reagents, chips and biotechnology software for high-throughput single-cell sequencing of microbials within two years." Zheng Wenshan said.
industry: Join forces to create a diverse and efficient
Single-cell sequencing new ecosystem
At this stage, one of the bottlenecks that limit the wide application of single-cell sequencing technology is that it is too expensive. For example, companies with a high international market share currently have a cost of reagents for a single sample experiment exceeding RMB 10,000, so it costs more than RMB 100,000 to conduct a set of simple biological experiments. Therefore, the market has a very strong demand for "cost reduction".
According to reports, Mozhuo Bio has carried out a lot of research work around the "cost reduction" of the overall solution, mainly covering 3 levels:
first, and technology optimization and improvement. "For some key steps, by increasing the yield, such as increasing the yield by 5-10 times, the cost of this key step will be reduced to 1/5 or even 1/10 of the previous one," said Zheng Wenshan.
second, large-scale production. Generally speaking, the cost of any industrial product will drop after large-scale production, and mass production through large-scale production is also a way to reduce costs. It is understood that Mozhuo Bio has built office areas, laboratories and factories of more than 4,000 square meters, including P2 cell laboratories, PCR amplification laboratories and 800 square meters of 100,000-square-meter GMP production workshop to provide guarantees for large-scale production.
Third, domestic substitution of high-quality raw materials. "Most of the raw materials used in our initial R&D stage were mainly imported from abroad. Now, with the development of the domestic biological reagent , its performance parameters are no longer comparable to those of foreign reagents. Therefore, we are gradually replacing high-quality domestic raw materials to reduce costs, so that single-cell sequencing technology can benefit more scientific researchers and end users." Zheng Wenshan added.
"At present, the cost of detecting a single sample exceeds 10,000 yuan. We hope to reduce the overall single-cell sequencing solution cost to 1/10 of the current in the next few years." Zheng Wenshan said.
In addition to reducing the cost of the overall solution, Mozhuo Biotechnology is also focusing on multi-omics and multi-sample types at this stage to make efforts. "Multiomics in single-cell sequencing is a major trend. Most manufacturers focus on the transcriptome, and we are also developing other anomics based on the transcriptome, such as epigenetic groups, proteomic groups, etc. We have launched products for the transcriptome, and are currently being continuously optimized. At the same time, products focused on the full-length transcriptome and concentrated on the 5’ end and related to immunity will also be launched one after another." Zheng Wenshan introduced.
"In addition, most high-throughput single-cell sequencing instruments and kits are only suitable for fresh samples. On the one hand, we have expanded the high-throughput single-cell sequencing technology from eukaryotic cells to prokaryotic cells; on the other hand, we are also expanding the application scope of single-cell sequencing products to more types including FFPE samples," he said.
talked about the medium- and long-term development plan of Mozhuo Bio, Zheng Wenshan said, "We mainly focus on deepening the field of single-cell sequencing. First, develop high-quality 'Chinese solutions' to achieve a significant reduction in the overall solution price under the premise that the performance is basically consistent with foreign products; second, continue to focus on the product side to achieve multi-omic coverage of single-cell sequencing, and at the same time expand more sample types; in addition, we also hope to deeply explore some unmet user needs from the application side, so as to reversely develop and optimize products, and apply single-cell sequencing technology to clinical testing, pharmaceutical development and other fields to solve more unmet clinical needs."
For the challenges that the company may encounter in the development process, in Zheng Wenshan's view, "First of all, As a startup, Mozhuo Bio is developing rapidly, both from the financing level and the team growth level, so the management level of the company's team has put forward higher requirements for us; secondly, we know that if we want to become a large biotechnology company, we need to make our products benefit global customers. This year is the first year of commercialization of Mozhuo Bio. At this stage, we are mainly rooted in mainland China, and our overseas layout has just begun, so we may face some new challenges in the future of globalization and overseas layout. Of course, these challenges are also the direction we need to continuously improve and strive for in the future. "
macro-industry level, Zheng Wenshan pointed out that high-throughput single-cell sequencing technology is like a "microscope in the sequencing field". Traditional overall sample sequencing obtains the overall average value, while single-cell sequencing can obtain information accurate to single cells. "A lot of previous studies have only focused on the whole, but now with the help of single-cell sequencing technology, we can observe and analyze every individual in the whole, which is equivalent to elevating the logic and perspective of the research to a new dimension," he added.
"More critical is that at the global level, single-cell sequencing is a relatively emerging technology, which is also a potential track in which China may overtake in international biotechnology and application fields." Zheng Wenshan pointed out, "China accounts for about 1/4-1/5 of the world At the same time, as the country's investment and attention to emerging technologies is gradually increasing, the domestic market size will continue to grow rapidly in the future, and this proportion will further increase. "
" Looking at the domestic market, the entire single-cell sequencing industry chain is actually very broad, and the role we play in the industrial chain is product development and optimization. Based on this link, we have developed more innovative products that are high-quality, cost-effective and can effectively meet user needs. At the same time, on this basis, we work with upstream high-quality domestic raw material suppliers, downstream scientific research service institutions, and end users (such as scientific researchers, clinicians, drug developers, etc.) to jointly build and promote the booming new ecosystem of single-cell sequencing industry. "Zheng Wenshan concluded.
Reference:
1.html://doi.org/10.1126/science.abm1483
2.html://www.mobidrop.com/
3.html://www.mobidrop.com/single-cell-sequencing-platform
