Understanding and manipulating lactate metabolism in single cells

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


Many of the medicines prescribed today are proteins that are manufactured in animal cell cultures. Decades of research has examined ways to increase the productivity of these cultures by changing the nutrients the cells are fed, genetic engineering of the cells themselves and other strategies. However, there are still challenges that limit the overall amount of protein produced. One of these challenges is the accumulation of the metabolic waste product, lactic acid, the same waste product that causes your muscles to be sore after strenuous exercise.

The concentration of lactate in animal cell cultures changes over time. Early on, it accumulates due to rapid metabolism, but later the cells use it to make energy and amino acids. This is called the lactate switch. Different batches of cells undergo the lactate switch at different times and the trigger for flipping the switch is not well understood. However, with respect to the production of protein medicines, the lactate switch is a positive trait associated with higher protein production.

The proposed work seeks to create a set of tools for understanding and manipulating the lactate switch. The first goal is to create a non-invasive indicator that allows measurement of lactate concentration in individual cells. This tool would allow us to measure the extent of variation between cells, something that is currently not possible with standard techniques. We will use it to understand how different culture conditions affect lactate accumulation in the cells to try and identify how much variability there is in each scenario. Overall, this information can be used to choose manufacturing conditions with less difference between cells.

The second goal is to develop tools that allow us to change the expression level of individual enzymes associated with lactate production and consumption. With these tools, we will be able to test hypotheses about which enzymes are associated with the lactate switch by controlling them with an external signal. Once the enzymes are identified, we could use the system to control when the lactate switch is flipped, making sure that all cells in the culture do switch from lactate production to consumption and making sure the switch flips at the best time to ensure high protein production.

Finally, we will merge the two sets of tools to create cells that sense their own lactate concentration and when it gets too high, regulate their own lactate metabolism genes. This will create cells that do not make too much lactate, which should increase the amount of protein that they produce. The 'self-regulating cells' will also serve as a model for how to do this with other traits of interest in the future. This project could, therefore, change the way cells are developed for manufacturing purposes.

Technical Summary

Chinese Hamster Ovary (CHO) cells are used in the production of more than half of therapeutic glycoproteins. Lactate is a key waste metabolite in CHO cultures and shows complex dynamics over time. Early in culture lactate accumulates, but towards stationary phase, cells undergo a metabolic shift to lactate consumption. This 'lactate switch' is associated with high productivity, but different cell lines do not show uniform behaviour with respect to lactate metabolism and the biological basis for the lactate switch is not currently known. Further, high concentrations of lactate damage cell growth and productivity. The proposed work would develop tools to understand and manipulate lactate metabolism in CHO cells, which will underpin the development of higher performance cultures with optimised metabolism.

To facilitate understanding, we will develop a genetically-encoded biosensor for intracellular lactate concentration. The biosensor will enable both the high throughput measurement of lactate concentration changes over time as well as single-cell measurements to gain insight into the population variability of metabolism under different bioprocessing conditions. This information is unobtainable by conventional assays and can lead to the design of better bioprocesses. To manipulate lactate concentration, we will use a Cas9-VPR fusion, where the length of the guide RNA and the target location allow for the simultaneous up- and downregulation of gene expression in the same cells. We will use this to change the expression level of selected targets to artificially flip the lactate switch. Finally, both sets of tools will be connected to develop self-regulating cells that can sense their own lactate concentration and modulate gene expression in response. Such a cell line could cause a paradigm shift in bioprocessing where currently offline analyses are used to monitor cell metabolism and an operator is required to make any changes.

Planned Impact

The proposed work will develop tools for the measurement of lactate concentration in single cells, modulate the expression level of metabolic targets associated with lactate metabolism, and combine these two strands to develop self-regulating cells. The potential beneficiaries from the work are described below.

Staff Training: The PDRA would benefit from the multidisciplinary environment of the Imperial College Centre for Synthetic Biology and interactions with the Network of Excellence in Industrial Biotechnology. S(he) would also gain cutting-edge skills in synthetic biology and the principles of guide RNA design, which are translatable to genome editing projects in the future. The PDRA would also have access to the unique training programs of SynBiCITE: the 4-Day MBA and LeanLaunchPad, enhancing their transferable skillset.

Researchers in synthetic biology, biochemistry/metabolism and bioprocess engineering: The knowledge generated by the proposed work would benefit a number of different research communities. It would lead to a greater understanding of mammalian cell metabolism and the extent of population variability of intracellular metabolite concentrations. It is likely that different cell line engineering or bioprocessing strategies will result in more or less population variability, which could also serve as a driver for evolution. Understanding this interplay could allow for the design of better, more resilient bioprocesses. The proposed work would also provide a basis for testing various hypothesis about the origin of the lactate switch in CHO cells, which is currently not understood. Finally, the development of self-regulating cells with a clear industrial application would be of interest to researchers in synthetic biology.

Industrial R&D: The tools developed as part of the proposed work could benefit scientists and bioprocess engineers working in industrial research and development. The lactate biosensor could be useful for process development as it would allow rapid, non-destructive, high sensitivity measurements of lactate concentration in small volumes or single cells. The fluorescent readout enables high throughput measurements and so could be useful for empirical screening of process conditions and media/feed formulations. Furthermore, the methodology for modulating the expression of metabolic targets could be useful for cell line development- both for inducing changes in lactate metabolism using our designed guide RNAs and also for extending the methodology to other parts of metabolism relevant to productivity and/or product quality. Finally, in the long-term, the results from the third objective (develop self-regulating cells) could revolutionise biological manufacturing in industry, where offline analyses and manual adjustment of culture conditions are the current norm. Once the concept has been successfully demonstrated for lactate, it could also be adopted for other metabolic waste products and intermediates. The concept could also be extended beyond the production of recombinant proteins in CHO cells to other hosts producing different product classes including small molecules, biofuels, and materials where the control over the accumulation of intermediates can be vital to increasing productivity.

Cancer biologists: Metabolism is highly conserved among mammalian cells and CHO cells share many metabolic phenotypes with cancer cells. Thus, cancer biologists will benefit from the tools developed in Objectives 1 and 2 of this proposal, which could be redeployed to study metabolism of cancer cells in vitro and in vivo.

Healthcare funders, and patients: In the long-term, the work proposed could lead to improved CHO systems with higher production yields of therapeutic proteins, which could decrease the overall cost of these lifesaving treatments many of which are currently extremely expensive. This would in turn reduce the costs to the healthcare system and would increase patient access.


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Description Work package one involves the design of genetic circuits to sense L-lactic acid and activate the expression of green fluorescent protein reporter as a response. Building on preliminary work, we have developed a series of designs that aim to improve the fold-change of the response of the initial design and the sensitivity of the response to lactate. This including exploring the use of additional operator sites and different promoters to express key proteins in the design. We tested all designs in transient transfection and identified the four best to take forward to stable cell line generation. After generating the stable cell lines using a landing pad methodology, we attempted to adapt the cells to suspension. However, this procedure led to the silencing of the L-lactate detection circuit. To tackle this unexpected challenge, we have developed a procedure to deploy the biosensor on microcarriers. This involved identifying the best microcarrier to promote cell attachment and to develop a protocol to enable long-lasting attachment. Having identified this, we are now testing the biosensor designs in microbioreactors in collaboration with the project partners at the Centre for Process Innovation. This work has been delayed somewhat by the covid-19 pandemic, but is about to commence in April 2022.

Workpackage 2 involved identifying genes associated with lactate metabolism. This was initially intended to be a high throughput screen using the CRISPR/Cas9 system. However, due to laboratory closures and limited access to facilities during covid-19, we focused on identifying genes that could plausibly be linked to lactate metabolism via their biochemistry. We screened potential lactate metabolism targets identified from the literature to identify genes whose overexpression could lead to lactate degradation via a plausible biochemical mechanism. Our experiments showed that pyruvate dehydrogenase kinases (pdk), which regulate an enzyme involved in lactate metabolism, increase lactate utilisation when overexpressed in CHO cells. Our attempts to regulate pdk expression using a CRISPR-based activation mechanism as originally planned were not successful due to the toxicity of overexpressing the dCas9 protein within the cells. Therefore, we developed a gene circuit to constitutively express these enzyme targets directly from a promoter as alternative solution. This was successful in altering lactate metabolism.

Finally workpackage three intends to link the biosensors developed in WP1 and the genes identified in WP2 to create cells that can self-regulate lactate metabolism. This work is ongoing.
Exploitation Route The idea of using biosensor cells on microcarriers has not yet been explored in the literature. It may enable the mixing of biosensing and producing cell lines in a biological manufacturing process and allow for a better sensing modality.

In addition, the self-regulating cells would be a contribution to the field of bioprocess engineering because the accumulation of L-lactate and the resulting metabolic effects often limit protein expression yields. Therefore, this modification, if successful, could have significant impact throughout biopharma.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description The some of the cell lines and methods generated in this project were used to develop a training activity called, "I can CRISPR". This has been taught twice time so far, with a third session scheduled at the end of March 2023. The first course session was run at Imperial College with 16 attendees ranging from undergraduate and postgraduate students to scientists from industry. The second session was run as a practical at the EMBO Practical Course, "Synthetic biology in action: beyond standard metabolism" (https://www.embl.org/about/info/course-and-conference-office/events/syn22-01/) for four early career researchers. The third session is again planned to be held at Imperial College with a mixture of internal and external attendees.
First Year Of Impact 2022
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Title Programmable shaker 
Description When we made the decision to stick with adherent cells for the biosensor and had to use these on microcarriers, we found that the protocols involved long periods of alternating shaking of the cells and incubating them under static conditions. The protocols involved the research staff turning the shaking platform in incubator on and off for ~5 minute intervals every 15 minutes for 8 hours, which was a tedious and time consuming process. To combat this, we collaborated with an electrical engineer in candidate to develop a DIY programmable shaker. This involved developing a method to interface a raspberry pi with our existing shaking platform and writing a simple, user friendly script to allow this to be programmed. We are currently in the process of writing this up for publication in an automation journal to disseminate the idea to other groups. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact We are in the process of writing this up for publication so that other groups can use the design. 
Description Collaboration with the National Biologics Manufacturing Centre (Centre for Process Innovation) 
Organisation Centre for Process Innovation (CPI)
Country United Kingdom 
Sector Private 
PI Contribution We have proposed various strategies for implementing biosensors within bioreactors to better understand what would work within the manufacturing workflows used at CPI.
Collaborator Contribution The team has considerable experience in manufacturing processes and has advised us on which strategies for encapsulation/physical containment may be more suitable and acceptable to manufacturers. This has helped us identify which strategies to focus on.
Impact No outputs as yet, but the discussions led us to focus our efforts on two strategies: one involving polymer encapsulation of the biosensors and the other involving development of mini-cell based non-living vesicles. These have the commonality that they are round and would not affect the hydrodynamics or mixing within the bioreactor, which was highlighted by the CPI team in the discussions.
Start Year 2020
Description Training course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A training course called, "I Can CRISPR" was developed based on some of the methods and techniques involved in the project. This has been run twice so far with a third session planned for March 2023. It provides basic training in gene editing in mammalian cells. The sessions were partially sponsored by NEB (via reagent donations) and the third session is partially supported by the Biochemical Society in a funding application by the PDRA employed on the project.
Year(s) Of Engagement Activity 2022