CYTENA

High-Throughput vs. High-Precision: Which CLD Strategy Works for You?

Dr. Pierre-Henri Ferdinand — Thu, 03 Apr 2025 10:33:38 +0000

April 3, 2025
10:33 am

High-Throughput vs. High-Precision: Which CLD Strategy Works for You?

By Dr. Pierre-Henri Ferdinand

Head of Product

High-Throughput vs. High-Precision: Which CLD Strategy Works for You?

In cell line development (CLD), speed and precision are often seen as opposing goals, but they don’t have to be. Researchers in biotech, CDMO, and pharma settings are increasingly challenged to generate results quickly while meeting regulatory standards. High-throughput CLD offers rapid generation and screening of clones, ideal for early discovery, while precision cell cloning ensures the clonality and documentation needed for therapeutic production. This blog explores how each approach fits different goals and stages of development and how new technologies are helping bridge the gap between speed and precision.

What is High-Throughput Cell Line Development?

High-throughput CLD enables the rapid generation of many potential clones within a single workflow. This method is especially useful in early discovery research, where scientists often study the effects of genetic variants or knockdowns on a specific phenotype. Some experimental strategies introduce multiple genetic modifications into a single cell pool, followed by cell sorting to assess the effects of individual changes on clonal populations. For example, CRISPR technology has been used for genome-scale gene knockouts in CHO cells, helping to identify clones with enhanced ability to produce “challenging” proteins (Shin et al., 2025).

In such cases, researchers’ primary focus may lean towards ensuring the genetic alteration has been successfully implemented rather than clonality of the population. Although confirming true clonality remains important for research accuracy, the need to rigorously demonstrate clonality is less strict than it is when developing a cell line for therapeutic use.

While these approaches are fast and yield many clones, they can involve compromises in clonality verification, which can impact scientific rigor and the reliability of downstream data. Even in early discovery, where standards are less strict than for therapeutic CLD, failing to achieve clonality can make conclusions unreliable, potentially requiring researchers to repeat the entire screening process.

What is Precision Cell Cloning?

By contrast to high-throughput CLD, precision cell cloning prioritizes clonality verification, long-term performance, and compliance with regulatory requirements. Precision cell cloning helps to generate cells using a process that complies with requirements needed to produce biologics, such as the production of monoclonal antibodies or cell-based therapies (European Medicines Agency (EMA), 1998). These requirements are in place to safeguard product quality, consistency, and end-user safety. Researchers who fail to present robust evidence of clonality risk regulatory rejection, delays in development, and increased costs (FDA’, 1997).

To achieve regulator-ready precision cell cloning, development teams can leverage modern technological advances. This includes systems that capture images of single cells during dispensing, as well as imaging on the growth surface to track their expansion over time. Researchers can further safeguard CLD processes by screening cells for desired characteristics such as high production of target proteins (Yang et al., 2022).

Advanced instrumentation and software, such as the UP.SIGHT and C.STUDIO from CYTENA help researchers capture and manage large volumes of data. This technology makes it simple for researchers to generate clonality reports, which streamlines regulatory submissions (Fig. 1).

Figure 1. The UP.SIGHT achieves >97% single-cell dispensing efficiency, while C.STUDIO software allows clonal populations to be easily tracked over time.

Without modern technologies, researchers performing CLD risk significant time loss and reagent waste, as traditional methods like limiting dilution introduce the risk of human error and contamination (Coller & Coller, 1986).

Application Scenarios: Which Strategy Fits Your Workflow?

Both high-throughput CLD and precision cell cloning are valuable single-cell dispensing techniques, each best suited to specific applications and stages of development.

Biotech Discovery Stage: A higher throughput approach is optimal to begin with, allowing researchers to screen cell populations for desirable traits without spending unnecessary effort or resources focusing on stringently achieving clonality. This approach can be followed up by more precise regulatory-focused cell cloning once lead populations are identified.
CDMOs: The goal is to produce high-performance clones that clients can take forward for regulatory approval. With this in mind, researchers should focus on precision, generating the necessary documentation to support applications.
Cell Therapy and Gene Editing: Precision is mandatory when performing CLD for these therapeutic applications. The risk of heterogeneity threatens regulatory approvals and end-user safety.

A crucial parameter in CLD is knowing which cell line best suits your application. Visit CYTENA’s biopharma cell line database to identify tried and tested cell lines for drug and protein production applications.

Choosing a CLD Path

Choosing the best approach can be challenging for researchers, especially smaller teams, as it’s crucial to use limited time and resources as efficiently as possible. Time spent ensuring clonality can be time wasted when performing broader screening. Conversely, “faster” approaches may lead to long regulatory delays if they come at the expense of precision. The costs of failed drug development ranges in the hundreds of millions (per drug), making early strategic decisions pivotal (Sertkaya et al., 2024).

Choosing the right approach is important, but teams often need to balance high throughput and precision throughout different development stages. Fortunately, a solution is available that enables researchers to benefit from speed and precision.

CYTENA’s instruments provide a unified system for CLD, supporting any application by combining high-throughput processing with guaranteed clonality in a single workflow. This means researchers don’t have to compromise on speed or precision when approaching discovery or regulatory-ready submissions.

The UP.SIGHT instrument ensures >99.99% probability of clonal derivation, which helps researchers achieve high throughput with less starting material. Combined with the C.STUDIO and F.QUANT assay for measuring antibody titer, this instrument offers precision with high throughput for advanced uncompromising CLD processes that generate regulatory-ready documentation with CFR Part 11 compliance.

Figure 2. The UP.SIGHT uses real-time imaging during single-cell dispensing to capture visual proof of clonality for regulatory assurance while gently dispensing single cells into a full 384-well plate in 8 minutes.

Conclusion: Tailoring Strategy to Science

There’s no one-size-fits-all approach to CLD, but with the right tools, researchers don’t have to choose between speed and precision. Whether you’re screening thousands of clones in early discovery or generating regulatory-ready cell lines for therapeutic use, CYTENA’s platforms let teams adapt their workflows as needed. With automation that supports both, your team can accelerate development without compromising on data quality or regulatory compliance for cell lines.

Contact a CYTENA expert today or read our product comparison page to identify the solution that best fits your CLD workflow.

References

Coller, H. A., & Coller, B. S. (1986). [37] Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. In Methods in Enzymology (Vol. 121, pp. 412–417). Elsevier.
https://doi.org/10.1016/0076-6879(86)21039-3

ICH Q5D Derivation and characterisation of cell substrates used for production of biotechnological/biological products—Scientific guideline | European Medicines Agency (EMA). (1998, March 31). https://www.ema.europa.eu/en/ich-q5d-derivation-characterisation-cell-substrates-used-production-biotechnological-biological-products-scientific-guideline

Points to consider in the manufacture and testing of monoclonal antibody products for human use (1997). U.S. Food and Drug Administration Center for Biologics Evaluation and Research. (1997). Journal of Immunotherapy (Hagerstown, Md.: 1997), 20(3), 214–243.
https://doi.org/10.1097/00002371-199705000-00007

Sertkaya, A., Beleche, T., Jessup, A., & Sommers, B. D. (2024). Costs of Drug Development and Research and Development Intensity in the US, 2000-2018. JAMA Network Open, 7(6), e2415445.
https://doi.org/10.1001/jamanetworkopen.2024.15445

Shin, S. W., Kim, S. H., Gasselin, A., Lee, G. M., & Lee, J. S. (2025). Comprehensive genome-scale CRISPR knockout screening of CHO cells. Scientific Data, 12(1), 71.
https://doi.org/10.1038/s41597-025-04438-6

Yang, W., Zhang, J., Xiao, Y., Li, W., & Wang, T. (2022). Screening Strategies for High-Yield Chinese Hamster Ovary Cell Clones. Frontiers in Bioengineering and Biotechnology, 10, 858478.
https://doi.org/10.3389/fbioe.2022.858478

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Introducing the CYTENA Therapeutic Monoclonal Antibody Approval Tracker

Yafees Sarwar — Mon, 17 Mar 2025 09:53:14 +0000

March 17, 2025
9:53 am

Introducing the CYTENA Therapeutic Monoclonal Antibody Approval Tracker

By Yafees Sarwar

Head of Marketing & Inside Sales

Introducing the CYTENA Therapeutic Monoclonal Antibody Approval Tracker

Given the incredible growth that has been, and continues to be, exhibited by the global monoclonal antibody (mAb) market, it is becoming increasingly difficult to keep up to date with all the latest mAb therapies. To make this task straightforward, we created a tool to track the current pipeline of mAb therapies that are in the latter clinical phases as well as those that already received market approval.

What’s in the Tracker?

The tracker contains information on every cell and gene therapy currently in phase I of the drug discovery pipeline or higher. It also includes therapies currently on the market and select therapies that are in preclinical development. The following information is covered for each therapy:

Drug: The name of the mAb (not the market name).

Sponsor: The company, or companies, that sponsored the development and clinical research of the mAb. Only active sponsors of the mAb are shown.

Indication: Lists all indications for which the mAb is currently being investigated as a therapy. In the case of approved mAb therapies, this will only include the indications for which the therapy has received a license.

Clinical Phase: Indicates whether the mAb therapy is in phase III clinical trials or has received market approval. In cases of an mAb therapy having only been approved in a single country or continent, this information is provided.

Antibody Type: Indicates the type of mAb, e.g., human, chimeric.

Drug Target: Refers to the protein or antigen that is being targeted by the mAb. In the case of bispecific mAbs, both targets are listed.

Why Use the Tracker?

The meteoric rise in mAb research has seen the market rapidly evolve, with an ever-increasing number of mAb therapies reaching the latter stages of clinical trials and achieving market approval. Staying up to date with all the emerging mAb therapies is becoming more and more difficult, but it’s crucial that you are aware of the latest developments so that you can establish and solidify your position within the market.

This tracker provides an exhaustive list of promising and approved mAb therapies all in one place. By referring to this list, you can spot the latest trends, identify competitors, and discover potential gaps within the market, making it an essential strategic tool for any company that is developing an mAb therapy.

How to Use the Tracker

Simply scrolling through the tracker is a great way to get an overview of the mAb therapy market and see the different types of therapies that are being developed. Should you want to gain more specific insights, such as the mAb therapies developed for a specific target, just sort by the relevant column and find all the information below.

How Do We Populate the Tracker?

We scour diverse authoritative sources to ensure this tracker is as comprehensive as possible, including:

Clinical trial databases
Company websites
Competitive intelligence
Research papers
Press releases
Company social media
Market reports

Where possible, we verify the information using multiple sources, with clinical trial databases and company websites taking precedence.

How Often is it Updated?

We update the tracker once to twice yearly to make sure you never miss a beat.

Did we miss something? Let us know what you’d like to see added to the tracker.

How CYTENA Instruments Accelerate Monoclonal Antibody Development

At CYTENA, we support companies developing the next generation of mAbs. Our UP.SIGHT is a dual single-cell dispenser and imager that streamlines cell line development and derisks workflows by providing >99.99% probability of monoclonality. This instrument combines microfluidics and advanced dual-imaging to ensure the gentle and precise isolation of high-yield, genetically stable clones for mAb production, with minimal hands-on time.

If you want to join the automation revolution that so many top companies already benefit from, then the C.STATION from CYTENA is your ultimate solution. The C.STATION provides end-to-end automation for cell line and mAb development that integrates confluency screening, antibody titer measurements, and much more. This enables you to select the highest-performing clones early in the process, saving both time and resources in identifying the best clones.

Ready to see your mAb on the CYTENA therapeutic monoclonal antibody approval tracker with the rest? Visit our product page to discover how our comprehensive suite of instruments can help streamline your mAb development workflow.

Disclaimer: While we endeavor to include all mAb therapies that are currently approved or in phase III clinical trials, some may have been omitted by mistake.

The post Introducing the CYTENA Therapeutic Monoclonal Antibody Approval Tracker appeared first on CYTENA.

Introducing the CYTENA Biologic Manufacturing Cell Line Tracker

Yafees Sarwar — Tue, 04 Mar 2025 09:47:14 +0000

March 4, 2025
9:47 am

Introducing the CYTENA Biologic Manufacturing Cell Line Tracker

By Yafees Sarwar

Head of Marketing & Inside Sales

Introducing the CYTENA Biologic Manufacturing Cell Line Tracker

A wide variety of cell lines are utilized throughout the biotechnology industry for a variety of different manufacturing purposes, and it can be difficult to identify the optimal cell line for your specific need. To help make your decision easier, we created a database that includes all the cell lines used in biotech manufacturing alongside a detailed profile of their key characteristics.

What’s in the Tracker?

The tracker contains information on every cell line – mammalian, insect, bacterial, or yeast – that is currently used in biologic manufacturing. This includes both cell lines used for production purposes and cell lines used for research that influences production. The following information is covered for each cell line (where applicable):

Cell Line: The most commonly used name for the cell line, typically an abbreviation.
Alternate Names: Any additional names by which the cell line is commonly known.
Variants: Subpopulations or modified versions of the original cell line that are commonly used, exhibiting unique characteristics that make them distinct.
Species/Strain: In the case of mammalian and insect cell lines, this refers to the species from which they originated. For bacterial and yeast cell lines, this refers to the strains of bacteria and yeast that are typically used in biologic manufacturing.
Tissue of Origin: The specific tissue from which the cell line was derived.
Adherent/Suspension: Determines whether the cells are typically grown as a suspension culture, an adherent culture, or if either format can be used depending on the application.
Production/Research: Determines whether the cell line is typically used for production purposes, research purposes, or at both levels of manufacturing.
Applications: Processes for which the cell line is typically used, e.g. recombinant protein production.
Drug/Product Examples: Drug types that are produced using the cell line, including examples of marketed drugs to which the cell line has contributed.
Vendors: Distributors from which the cell line can be acquired.

Why Use the Tracker?

Biotechnology is rapidly expanding, and this growth is being accompanied by an increase in the number of cell lines that are being used throughout the industry. This is creating a situation in which optimization is becoming both more important and more difficult, with an array of different cell lines to choose from.

This tracker acts as a straightforward and comprehensive guide that can help to identify the optimal cell line for your production or research requirements. As such, whether you are planning a new production line or looking to refine an existing process, this tracker is an invaluable tool when choosing which cell line(s) to use.

How to Use the Tracker

Scroll through the tracker to gain an understanding of the different cell lines that are currently being used throughout the industry. If you are searching for something specific, perhaps a cell line that can be used to produce adenoviral vectors, simply sort by the relevant column and discover the cell lines that meet your criteria.

How Do We Populate the Tracker?

We scour diverse authoritative sources to ensure this tracker is as comprehensive as possible, including:

Cell line databases
Company websites
Research papers
Literature reviews

Where possible, we verify the information using multiple sources, with cell line databases and company websites taking precedence.

How Often is it Updated?

We update the tracker once-twice yearly to make sure you never miss a beat.

Did we miss something? Let us know what you’d like to see added to the tracker.

How CYTENA Instruments Accelerate Biologic Manufacturing

At CYTENA, we support companies developing and manufacturing novel biologics. Our UP.SIGHT is a dual single-cell dispenser and imager that streamlines cell line development and derisks workflows by providing >99.99% probability of clonal derivation. This instrument uses microfluidics to ensure gentle dispensing, minimizing cell stress and enabling you to achieve >80% cloning efficiency.

If you want to join the automation revolution that so many top companies already benefit from, then the C.STATION from CYTENA is your ultimate solution. The C.STATION provides end-to-end automation for cell line development that integrates confluency screening, antibody titer measurements, and much more. This means you get pure, high-performing cell lines quickly, while freeing up time and resources for other tasks.

Ready to supercharge your biologic development? Take a look at the CYTENA cell line tracker, find your optimal cell line, and then visit our product page to discover how our comprehensive suite of instruments can help streamline your workflows.

Disclaimer: While we endeavor to include all cell lines currently used in biologic manufacturing, some may have been omitted by mistake.

The post Introducing the CYTENA Biologic Manufacturing Cell Line Tracker appeared first on CYTENA.

Advances in Laboratory Automation: Streamlined ELISA and Cell-based Assays

Dr. Pierre-Henri Ferdinand — Wed, 29 Jan 2025 13:03:00 +0000

January 29, 2025
1:03 pm

Advances in Laboratory Automation: Streamlined ELISA and Cell-based Assays

By Dr. Pierre-Henri Ferdinand

Head of Product

Laboratory automation is changing how scientists conduct research and develop new therapies (Holland & Davies, 2020). By providing efficiency and accuracy at virtually all steps of core biomedical research workflows, automation allows researchers to set more ambitious goals and generate more profound insights into cellular behavior and drug activity. Plate washing is an essential component of important research techniques, including enzyme-linked immunosorbent assays (ELISAs) and cell-based assays. Automated plate washing is more efficient and accurate than manual pipetting methods and gives researchers greater control and confidence in their results. This article explores the importance of automation for ELISA and cell-based assays, highlighting the benefits of automated plate washing for achieving more efficient and consistent workflows.

ELISA

ELISAs are an indispensable technique for detecting and quantifying specific antigens in clinical and research settings. Common ELISA formats, including direct, indirect, sandwich, and competitive ELISAs, use antigen-antibody binding to achieve incredible specificity (Alhajj et al., 2024).

ELISA Applications

ELISAs have a vast range of applications across research and clinical settings, where they are crucial for the diagnosis of infectious diseases (Alhajj et al., 2024; Iha et al., 2019). They also play central roles in immunology and oncology research, enabling the precise measurement of cytokines and chemokines (Stefura et al., 2019). Beyond these domains, ELISAs help ensure quality during drug development, maintain food safety, assist forensic analyses, and enable environmental monitoring (Alhajj et al., 2024; Wadhwa et al., 2015) (Fig. 1).

Figure 1. ELISAs are widely used in research and clinical workflows, and precise and efficient liquid dispensing and washing are essential for ensuring reliable results.

Emerging Technologies and Challenges

Technological advances have significantly expanded ELISA applications. Microfluidic platforms, for instance, reduce reagent consumption and speed up workflows, making rapid point-of-care testing more feasible (Kweon et al., 2024). CRISPR-ELISA further boosts sensitivity by leveraging Cas enzymes and guide RNAs to amplify the detection signal (Li et al., 2022). Despite their strengths, ELISA workflows can be hampered by manual pipetting errors and reagent costs. Automated ELISA workflows mitigate these issues, enhancing reliability and throughput. As ELISA technologies advance, automation and novel detection strategies promise even more efficient and precise workflows.

Industrial Innovations

Industries are increasingly using microbes for sustainable and efficient solutions. Microbial fuel cells are used to provide clean energy, while cosmetics use microbes to provide anti-aging and UV protection (Gupta et al., 2019; Naha et al., 2023). In the food and drinks industry, microbes drive fermentation and preservation processes and even serve as innovative food sources (Graham & Ledesma-Amaro, 2023). Additionally, eco-friendly alternatives in leather production and mining showcase the versatility of microbial applications (Rawlings, 2002; Ugbede et al., 2023).

Consider reading our full-length article for a more detailed look at ELISA applications and the importance of automated plate washing in streamlining ELISA workflows.

Cell-Based Assays

Cell-based assays are used to investigate cell behavior, gene function, and drug activity across diverse research fields. These assays underpin key applications such as high-throughput screening for drug discovery, functional genomics, and toxicology, enabling scientists to evaluate thousands of compounds or genetic variants quickly and reliably (Nierode et al., 2016). New approaches like high-content screening, single-cell analysis, microfluidics, and organ-on-chip platforms are expanding the insights generated from these assays (Ingber, 2022; Nierode et al., 2016; Sagar et al., 2018).

Challenges in Cell-based Assays

Challenges in cell-based assays revolve around manual pipetting, cross-contamination, cell detachment, and background noise. Pipetting errors can lead to inconsistent reagent delivery and experimental conditions, skewing results. Improper washing may stress or detach cells, resulting in reduced cell viability and unreliable data. Inadequate reagent removal can contribute to elevated background signals that mask true readouts. Additionally, high-throughput experiments using manual pipetting increase the risk of mixing multiple compounds or conditions, confounding results and forcing researchers to repeat assays and repurchase expensive reagents.

Emerging Technologies

Emerging cell-based assay techniques, such as high-content screening, single-cell analysis, microfluidics, and organ-on-chip, offer unprecedented insights into cellular behavior and function. High-content screening integrates imaging and data analysis to capture multifaceted phenotypes within a single experiment (Nierode et al., 2016). Single-cell analysis uncovers rare subpopulations that might otherwise be lost in bulk measurements (Sagar et al., 2018). Microfluidic systems reduce reagent use and enable precise control over culture conditions (Duncombe et al., 2015). Organ-on-chip devices mimic human organ physiology, providing better models for drug discovery, disease research, and regenerative medicine (Ingber, 2022). Automated cell-based assays offer enhanced throughput and improved reproducibility and are set to play an important role in facilitating the implementation of new technologies.

Read our full-length article to learn more about cell-based assays and how automated plate washing increases assay throughput and accuracy.

Automated Plate Washing

Plate washing is a critical yet frequently undervalued aspect of many laboratory workflows. Effective washing removes unbound reagents and clears away contaminants, minimizing background noise that can interfere with data interpretation. By ensuring uniform test conditions, proper washing reduces the likelihood of cross-contamination and yields more reliable, reproducible results. This consistency is vital for techniques like ELISA and cell-based assays that demand precise reagent exposure control and incubation times. Inadequate washing can compromise experiments, wasting both time and costly reagents.

The C.WASH PLUS from CYENA

The C.WASH PLUS exemplifies cutting-edge automation in plate washing, streamlining workflows, and reducing common errors associated with manual methods. Compatible with 96-, 384-, and 1536-well plate formats, it adapts easily to various assay requirements and pre-established protocols. By using a centrifugal washing mechanism, the C.WASH PLUS keeps residual volumes as low as 0.1 μL after two 1-minute cycles, significantly minimizing reagent carryover. This increases accuracy, allowing assays to produce more reliable results. The system is easily programmable, allowing researchers to optimize and automate washing steps for different applications (Fig. 2).

Figure 2. C.WASH PLUS from CYTENA delivers cutting-edge automated plate washing technology, enabling researchers to obtain more accurate and reliable results in significantly less time than manual methods.

Read our full-length article to learn more about the importance of plate washing and how automation is a necessity for researchers looking to pioneer the next generation of discoveries.

Conclusion

Laboratory automation transforms ELISAs and cell-based assays, reducing variability and minimizing resource consumption. By optimizing processes like plate washing, automation ensures higher throughput, greater data reliability, and consistent execution of complex protocols. These advances expedite drug discovery and foster innovation in biomedical research. As technologies like single-cell analysis, microfluidics, and organ-on-chip mature, integrating automation becomes more critical. Ultimately, embracing automated solutions will empower scientists to uncover novel targets, refine therapeutics, and advance the frontiers of biomedicine.

Contact the CYTENA team today to explore automated laboratory solutions, including the groundbreaking C.WASH PLUS, and see how it can speed up your workflows while improving accuracy.

References

Alhajj, M., Zubair, M., & Farhana, A. (2024). Enzyme Linked Immunosorbent Assay. In StatPearls. StatPearls Publishing.

Duncombe, T. A., Tentori, A. M., & Herr, A. E. (2015). Microfluidics: Reframing biological enquiry. Nature Reviews. Molecular Cell Biology, 16(9), 554–567.

Holland, I., & Davies, J. A. (2020). Automation in the Life Science Research Laboratory. Front Bioeng Biotechnol, 8(571777).

Iha, K., Inada, M., Kawada, N., Nakaishi, K., Watabe, S., Tan, Y. H., Shen, C., Ke, L.-Y., Yoshimura, T., & Ito, E. (2019). Ultrasensitive ELISA Developed for Diagnosis. Diagnostics, 9(3), 78.

Ingber, D. E. (2022). Human organs-on-chips for disease modelling, drug development and personalized medicine. Nature Reviews Genetics, 23(8), 467–491.

Kweon, O. J., Yoon, S., Choe, K. W., Kim, H., Lim, Y. K., & Lee, M.-K. (2024). Performance evaluation of microfluidic microplate-based fluorescent ELISA for qualitative detection of SARS-CoV-2–specific IgG and IgM. Scientific Reports, 14(1), 18200.

Li, N., Chinthalapally, M., Holden, V. K., Deepak, J., Dhilipkannah, P., Fan, J. M., Todd, N. W., & Jiang, F. (2022). Profiling Plasma Cytokines by A CRISPR-ELISA Assay for Early Detection of Lung Cancer. Journal of Clinical Medicine, 11(23), 6923.

Nierode, G., Kwon, P. S., Dordick, J. S., & Kwon, S.-J. (2016). Cell-Based Assay Design for High-Content Screening of Drug Candidates. Journal of Microbiology and Biotechnology, 26(2), 213–225.

Sagar, Herman, J. S., Pospisilik, J. A., & Grün, D. (2018). High-Throughput Single-Cell RNA Sequencing and Data Analysis. In T. Vavouri & M. A. Peinado (Eds.), CpG Islands (Vol. 1766, pp. 257–283). Springer New York.

Stefura, W. P., Graham, C., Lotoski, L., & HayGlass, K. T. (2019). Improved Methods for Quantifying Human Chemokine and Cytokine Biomarker Responses: Ultrasensitive ELISA and Meso Scale Electrochemiluminescence Assays. In P. Lympany & M. G. Jones (Eds.), Allergy (Vol. 2020, pp. 91–114). Springer New York.

Wadhwa, M., Knezevic, I., Kang, H.-N., & Thorpe, R. (2015). Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility. Biologicals, 43(5), 298–306.

The post Advances in Laboratory Automation: Streamlined ELISA and Cell-based Assays appeared first on CYTENA.

Automated Plate Washing for Enhanced Laboratory Workflows

Dr. Pierre-Henri Ferdinand — Wed, 22 Jan 2025 13:01:00 +0000

January 22, 2025
1:01 pm

Automated Plate Washing for Enhanced Laboratory Workflows

By Dr. Pierre-Henri Ferdinand

Head of Product

Plate washing is a fundamental yet often underestimated step in life science workflows. Effective plate washing is crucial for applications like enzyme-linked immunosorbent assays (ELISAs) and cell-based assays, ensuring the removal of unbound reagents and preventing cross-contamination. Accurate plate washing safeguards the integrity of experiments and optimizes the use of valuable reagents. By maintaining consistent and reliable conditions, appropriate plate washing enhances the accuracy and reproducibility of results within and between laboratories. This article will examine the critical role of plate washing in research laboratories and highlight the importance of automation in streamlining and optimizing this essential process.

Importance of Plate Washing in Life Sciences

Plate washing is a crucial yet often overlooked component of many core laboratory workflows, including cell-based assays and ELISAs. Inadequate or poorly executed plate washing can have severe consequences for experimental success, potentially leading to wasted reagents and loss of precious samples. Plate washing helps to ensure that all test conditions are performed uniformly and removes sources of background noise. Accurate and efficient plate washing is essential within individual workflows and for repeat experimentation to ensure reproducibility and reliability. Manual pipetting of washing steps significantly increases the chances of cross-contamination and improper reagent dispensing (Fig. 1).

Figure 1. Plate washing using manual methods is time-consuming, tedious, and prone to errors that threaten experimental success.

Applications

Plate washing is a fundamental step in many core biological workflows that are used extensively in research and clinical settings.

ELISA

ELISA is an essential technique for accurately detecting and quantifying specific antigens, making it indispensable for diagnostic workflows, among other applications. Plate washing is vital for successful ELISA workflows, because it removes unbound reagents and reduces background noise, thereby enhancing assay specificity and accuracy. Poor washing leads to misleading results and wasted time and resources (Alhajj et al., 2024).

Cell-based Assays

Cell-based assays span many research and development applications, including drug screening and toxicity testing (Wei et al., 2021). Plate washing in cell-based assays ensures appropriate removal of test compounds, ensuring consistency and reliability in assay results. Careful washing is also crucial for minimizing issues caused by cell detachment.

Molecular Techniques

Many highly sensitive molecular techniques, including protein-binding assays, require efficient and accurate plate washing (Lapetina & Gil-Henn, 2017). Effective washing ensures the removal of unbound reagents, minimizes background interference, and enhances the overall reliability and sensitivity of experimental results.

Cell Culture

Cell culture requires plate washing to passage cells and to remove specific growth factors or reagents used in experiments and during routine culture (Segeritz & Vallier, 2017). Improper handling of cells, including during washing steps, can cause cell stress, cell death, and detachment, potentially delaying research progress and producing confounding results (Takahashi et al., 2022).

Advantages of Automated Plate Washing

Laboratory automation, including plate washing, helps overcome many perennial challenges associated with manual methods, ensuring more consistent workflows and generating more reliable results (Holland & Davies, 2020).

Efficiency

Automated systems can wash plates significantly faster than manual methods, which comes with numerous benefits. The obvious benefit is faster workflows, saving time and shortening research timelines. Secondly, more efficient washing means that sensitive assays or those using cells spend less time outside optimal conditions, thereby reducing the chances of variation. Efficient washing increases throughput, allowing researchers to collect more high-quality data in less time.

Accuracy

Crucially, the increased speed associated with automated washing does not come at the expense of accuracy. On the contrary, automation significantly increases washing accuracy compared to manual methods. This means decreased chances of cross-contamination and a reduction in the presence of residual reagents after washing steps. Ultimately, automated washing boosts the chances of experimental success by removing confounding variables and ensuring consistent, reliable results.

Cost-effectiveness

Automation facilitates assay miniaturization, which means that researchers can generate more reliable results with significantly less investment in expensive reagents. With automated washing, researchers can use multiwell formats, reducing the number of plates required per experiment and thereby reducing waste and expense.

The C.WASH PLUS from CYTENA is compatible with 96-, 384-, and 1536-well plate formats, enabling high throughput and versatility for various laboratory applications. The centrifugal washing mechanism leads to 0.1 µL residual volume after two 1-minute washing steps, meaning the C.WASH PLUS minimizes reagent carryover, ensuring high assay accuracy and reliability (Fig. 2).

Figure 2: The C.WASH PLUS from CYTENA enables fully customizable washing processes, allowing researchers to program advanced washing protocols for seamless integration into their laboratory workflows.

Conclusion and Future Directions

Looking ahead, the future of automated plate washing lies in integrating intelligent systems and AI-driven protocols to achieve even greater precision and adaptability. Emerging technologies may offer enhanced flexibility for complex assays and seamless data integration for real-time monitoring. Additionally, sustainable innovations will focus on reducing environmental impact while expanding compatibility with new plate formats and novel assay types to meet the evolving demands of life sciences research.

Automated plate washing revolutionizes laboratory workflows by enhancing efficiency, accuracy, and cost-effectiveness. By minimizing human error and reducing reagent waste, it ensures reliable and reproducible results across various applications, from ELISA to cell-based assays. Solutions like CYTENA’s C.WASH PLUS exemplify the advancements in automation technology, providing versatile and high-throughput capabilities tailored to diverse research needs.

Contact CYTENA’s team of experts today to learn more about the C.WASH PLUS and schedule a demo to see its revolutionary impact firsthand.

References

Alhajj, M., Zubair, M., & Farhana, A. (2024). Enzyme Linked Immunosorbent Assay. In StatPearls. StatPearls Publishing.

Holland, I., & Davies, J. A. (2020). Automation in the Life Science Research Laboratory. Front Bioeng Biotechnol, 8(571777).

Lapetina, S., & Gil-Henn, H. (2017). A guide to simple, direct, and quantitative in vitro binding assays. Journal of Biological Methods, 4(1), e62.

Segeritz, C.-P., & Vallier, L. (2017). Cell Culture. In Basic Science Methods for Clinical Researchers (pp. 151–172). Elsevier.

Takahashi, K., Okubo, C., Nakamura, M., Iwasaki, M., Kawahara, Y., Tabata, T., Miyamoto, Y., Woltjen, K., & Yamanaka, S. (2022). A stress-reduced passaging technique improves the viability of human pluripotent cells. Cell Reports Methods, 2(2), 100155.

Wei, F., Wang, S., & Gou, X. (2021). A review for cell-based screening methods in drug discovery. Biophysics Reports, 7(6), 504–516.

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Automation in Cell-based Assays: Benefits for High-throughput and Consistent Results

Dr. Pierre-Henri Ferdinand — Wed, 15 Jan 2025 12:58:00 +0000

January 15, 2025
12:58 pm

Automation in Cell-based Assays: Benefits for High-throughput and Consistent Results

By Dr. Pierre-Henri Ferdinand

Head of Product

Cell-based assays are indispensable tools in biological research, offering insights into cell behavior, gene function, and drug activity. Techniques like high-throughput screening for drug discovery and advanced technologies like organ-on-chip and single-cell analyses give researchers unprecedented insights to drive progress in biomedicine. However, challenges such as variability and contamination highlight the need for innovations like automation to improve the reliability and efficiency of cell-based assays. This article will explore the applications of cell-based assays and how cell-based assay automation addresses challenges related to accuracy, reproducibility, and scalability.

Applications of Cell-based Assays

Cell-based assays are essential for research, from basic experiments to translational applications. They can be used to evaluate various cellular characteristics and responses to prospective therapies (Nierode et al., 2016).

High-throughput Screening

Drug discovery efforts rely on screening large compound libraries quickly and accurately in cellular systems. Cell-based assays in this context often include specific reporter assays that detect and measure a compound’s ability to bind a molecular target and elicit the desired effect, such as inhibition of enzyme activity (Best et al., 2024).

Functional Genomics

Functional genomics assays aim to determine how various genetic modifications, such as gene knockdown or knockout, affect cellular functions. CRISPR screening assays are a specific type of cell-based assay that introduce these genetic changes into cells and measure particular outcomes (Zhou et al., 2014). These outcomes can include cell proliferation, viability, and specific molecular signals detected using reporter cells.

Toxicology

Toxicology is a critical parameter assessed during therapy development. Performing cell-based toxicology screening is vital for evaluating drug safety, identifying potential cytotoxicities, and ensuring the efficacy of compounds while minimizing adverse effects (Szymański et al., 2012). Accurate toxicology studies using cell-based assays help to ensure that only safe and effective therapies are brought forward for further clinical evaluation.

Emerging Technologies

Technological advancements continuously enhance high-throughput cell-based assays, enabling researchers to gain deeper insights into cell behavior under various conditions.

High-content Screening

High-content screening involves using multiple analytical methods simultaneously to monitor cells and obtain comprehensive insights from a single experiment (Nierode et al., 2016). This approach can include toxicology, genomic screening, and cytotoxicity assays, as well as imaging of cellular structures, quantifying subcellular features, tracking dynamic processes like cell migration and signaling, and analyzing complex phenotypic changes in high resolution (Fig. 1).

Figure 1: High-content screening allows researchers to gather important information about cellular behavior, including organelle and cytoskeleton dynamics.

Single-cell Analysis

The analysis of single cells allows for the characterization of important but less abundant cell types without their impact being drowned out by noise from other cells. It helps reveal rare subpopulations from complex mixtures and elucidates specific mechanisms of gene expression and molecular signaling (Sagar et al., 2018).

Microfluidics

Microfluidics allows cell-based assays to be performed at incredibly small scales (Duncombe et al., 2015). This decreases the volume required for expensive reagents such as cell culture media, test compounds, and gene-editing tools like CRISPR.

Organ-on-Chip

Related to advances in microfluidics is the concept of organ-on-chip. Organ-on-chip technology involves culturing living cells in micro-engineered devices that replicate the structure and function of human organs. These systems mimic organ-level processes like blood flow and nutrient delivery, enabling precise drug testing, disease modeling, and toxicity assessments (Ingber, 2022).

Challenges

Cell-based assays can be affected by many variables that prevent researchers from deriving meaningful and accurate results.

Cell Detachment

Cell-based assays often require multiple washing steps or media changes, repeatedly exposing cells to forces that may cause them to detach from the test plate. This detachment can cause cellular stress death, and a reduction in signal that is unrelated to the intended test conditions, leading to confounding and misleading results.

Variability Due to Manual Pipetting

Manual pipetting methods for cell-based assays can introduce variability in several ways (Lippi et al., 2017). They increase the chances of incorrect dispensing, leading to the wrong reagents being added to wells. Furthermore, incomplete or inefficient washing due to manual pipetting can mean that some cells are exposed to test compounds longer than others, generating misleading results.

Cross Contamination

Cross-contamination is particularly problematic when screening using high-throughput cell-based assays, as it can mean that different compounds or concentrations are inappropriately mixed or added to the wrong wells. The more conditions used, the greater the chances of cross-contamination.

High Background Noise

Background noise makes it difficult for researchers to distinguish the test signal from signals caused by contaminants or leftover reagents. This issue often arises from insufficient removal of reagents during washing steps.

Automation

Automation is a crucial development for cell-based assays, facilitating faster and more accurate experimentation while relieving researchers of repetitive and tedious tasks. It enables the scaling down of cell-based assays, reducing costs associated with expensive reagents like cell culture media, multiwell plates, and test compounds.

Automation allows researchers to repeat precise experimental conditions without introducing operator variables. This means more robust and trustworthy results and significantly reduces the need for repeat experiments (Holland & Davies, 2020). Ultimately, cell-based assay automation enhances efficiency, consistency, and cost-effectiveness, enabling researchers to focus on data analysis and innovation while minimizing errors and wasted resources.

The C.WASH PLUS from CYTENA has emerged as an essential tool for various applications, including cell-based assays. Poor washing can compromise the success of cell-based assays. The C.WASH PLUS ensures <0.1 μL residual volume per well for 96-, 384- and 1536-well plate formats, and its gentle evacuation steps virtually eliminate cell detachment issues (Fig. 2).

Figure 2. The C.WASH PLUS from CYTENA ensures 99.99% removal of residual volume, helping to reduce background noise and variability in cell-basead assays.

Conclusions and Future Directions

Automation is transforming cell-based assays by enhancing accuracy, reproducibility, and scalability, ultimately accelerating research and drug discovery. As technology advances, we anticipate more integrated automated systems incorporating artificial intelligence for data analysis and decision-making. Future innovations will further reduce costs and reagent usage while increasing assay throughput and facilitating greater complexity. Continued advancements in automation will empower researchers to explore deeper biological insights, streamline workflows, and develop more effective therapies, driving the next wave of breakthroughs in biomedicine.

Contact CYTENA today to learn how the C.WASH PLUS can fit seamlessly into your existing cell-based assay workflow, offering next-level accuracy and efficiency.

References

Best, A. J., Braunschweig, U., Wu, M., Farhangmehr, S., Pasculescu, A., Lim, J. J., Comsa, L. C., Jen, M., Wang, J., Datti, A., Wrana, J. L., Cordes, S. P., Al-awar, R., Han, H., & Blencowe, B. J. (2024). High-throughput sensitive screening of small molecule modulators of microexon alternative splicing using dual Nano and Firefly luciferase reporters. Nature Communications, 15(1), 6328.
Duncombe, T. A., Tentori, A. M., & Herr, A. E. (2015). Microfluidics: Reframing biological enquiry. Nature Reviews. Molecular Cell Biology, 16(9), 554–567.
Holland, I., & Davies, J. A. (2020). Automation in the Life Science Research Laboratory. Front Bioeng Biotechnol, 8(571777).
Ingber, D. E. (2022). Human organs-on-chips for disease modelling, drug development and personalized medicine. Nature Reviews Genetics, 23(8), 467–491.
Lippi, G., Lima-Oliveira, G., Brocco, G., Bassi, A., & Salvagno, G. L. (2017). Estimating the intra- and inter-individual imprecision of manual pipetting. Clinical Chemistry and Laboratory Medicine (CCLM), 55(7).
Nierode, G., Kwon, P. S., Dordick, J. S., & Kwon, S.-J. (2016). Cell-Based Assay Design for High-Content Screening of Drug Candidates. Journal of Microbiology and Biotechnology, 26(2), 213–225.
Szymański, P., Markowicz, M., & Mikiciuk-Olasik, E. (2012). Adaptation of high-throughput screening in drug discovery-toxicological screening tests. International Journal of Molecular Sciences, 13(1), 427–452.
Zhou, Y., Zhu, S., Cai, C., Yuan, P., Li, C., Huang, Y., & Wei, W. (2014). High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 509(7501), 487–491.

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Advantages of C.WASH PLUS in Assay Automation: The ELISA Case

Dr. Pierre-Henri Ferdinand — Thu, 09 Jan 2025 12:54:00 +0000

January 9, 2025
12:54 pm

Advantages of C.WASH PLUS in Assay Automation: The ELISA Case

By Dr. Pierre-Henri Ferdinand

Head of Product

Enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for immunoassays and offer incredible specificity due to the inherent properties of antibody-antigen binding (Alhajj et al., 2024). Despite their reliability, traditional ELISA workflows can be time-consuming and error-prone when washing and pipetting steps are performed manually. Automation streamlines ELISA processes, enhancing accuracy and reproducibility. This ultimately improves the quality of results and their impact on diagnostics, research, and drug development. This article covers ELISA success stories, emerging technologies, the challenges (and solutions) associated with ELISA workflows, and the benefits of ELISA automation.

ELISA Success Stories

ELISAs have played a crucial role in some of the most significant breakthroughs in modern biomedical science. Let’s explore two of the remarkable advances humanity has made using this technology.

HIV Testing in the AIDS Epidemic

During the AIDS epidemic in the 1980s, clinicians lacked a reliable way to identify individuals carrying HIV. This represented a massive shortcoming in our ability to control the spread of the disease and implement effective public health interventions. The development of the first ELISA HIV test in 1985 marked a significant milestone in tracking and managing this health crisis (Alexander, 2016).

Prostate-Specific Antigen

Prostate-specific antigen (PSA) is one of the most enduringly useful cancer biomarkers and is fundamental for the non-invasive screening of prostate cancer. The discovery of PSA was translated into a diagnostic tool through ELISA technology, a development achieved in the mid-1980s through the collaboration of multiple laboratories (Catalona, 2014; Stamey et al., 1987).

Primary ELISA Applications

ELISA’s high sensitivity for virtually any antigen makes it a valuable and versatile tool for various applications across clinical and research settings. It is essential for disease diagnosis, including detecting infectious diseases like SARS-CoV-2 and autoimmune disorders (Deutschmann et al., 2020; MacMullan et al., 2020).

ELISAs are routinely used in research settings for protein detection and quantification. Specific applications include cytokine and chemokine assays, which are important for immunology and oncology research (Stefura et al., 2019; Wang et al., 2024). In drug development and manufacturing, ELISAs are essential for screening, quantifying therapeutic proteins, ensuring product quality, and monitoring immune responses during preclinical and clinical trials (Manak et al., 2024; Wadhwa et al., 2015). In addition to research and clinical settings, ELISA plays important roles in forensics, food safety, and environmental monitoring (Alhajj et al., 2024; Jaria et al., 2020).

ELISA Technological Developments

ELISA technology was invented in 1971 and has seen significant technological advancements in the decades since (Aydin, 2015). Notable innovations include multiplexed ELISAs, the use of nanoparticles to enhance sensitivity, and digital ELISA, which provides ultrasensitive single-molecule analyte detection (Li et al., 2022; Tabatabaei et al., 2021; Yi et al., 2022).

These recent advancements in ELISA assays demonstrate their continued relevance even five decades later, reinforcing their lasting importance in several fields.

Challenges in ELISA Workflows

While essential to modern clinical and research applications, ELISA workflows face persistent challenges. Firstly, ELISAs are incredibly labor-intensive to perform by hand and manual pipetting errors and inadequate washing can lead to contamination and experimental failure, increasing costs and delaying research. Furthermore, long incubation times can reduce throughput in high-demand settings.

As assays become more sensitive and specific, they don’t necessarily become more robust. Even advanced plate-based ELISAs remain complex, error-prone procedures that are highly dependent on the operator’s skills, particularly in pipetting and plate washing. Operator variability can increase result variability, reduce reproducibility, and introduce false positives or negatives, sometimes without detection. Advances in workflow automation place greater demands on precise liquid handling for ELISA assays.

Workflow inconsistencies waste time, money, and resources. Repeating experiments to verify ambiguous results not only delays progress but also drives up costs, especially given the high price of primary antibodies.

Quality of Biological Samples

Poor sample quality can significantly impact assay performance.

Solutions:

Perform relevant sample quality control (QC) to ensure integrity.
Avoid repeated freeze-thaw cycles, which can alter antigen concentrations.

Reagent Variability and Stability

Not all reagents perform equally, and their effectiveness can degrade over time.

Solutions:

Different lots of primary or secondary antibodies may have different binding affinities. Use the same batch of all critical components across individual projects.
Enzyme-linked antibodies and substrates are sensitive to temperature, light, and freeze-thaw cycles. Minimize reagent degradation by aliquoting reagents in single-use aliquots.

Inconsistent Reagent Dispensing

Variations in pipetting technique can introduce significant inconsistencies.

Solutions:

Improper pipette tip changes or reagent handling can introduce cross-contamination artifacts. Use non-contact pipetting or tip-free dispensing.
Distractions, daydreaming, thumb cramps and shaky hands can all lead to inaccuracies in liquid dispensing. We’ve all been there; consider automating the dispensing process.

Lag Time Between Workflow Steps

The longer the time between treatment of the first well and the last well of a plate, the more likely temperature differences and evaporation will impact the assay and its results

Solutions:

Minimize time gaps between the first and last wells treated.
Consider automated dispensing solutions which provide the speed and accuracy to avoid these issues.

nconsistent Washing and Reagent Removal

Insufficient washing can lead to high background noise or carryover effects.

Solutions:

Aim for robust and consistent washing across all wells.
Avoid harsh dispensing which can physically strip away immobilized analytes or antibodies

The Cost of Error in ELISA Workflows

Keeping these sources of variation in check is particularly important as ELISA workflows also suffer from high reagent costs, meaning that high variability and a lack of scalability can strain laboratory budgets and extend project timelines.

While the probability of an ELISA failure can never be brought to zero, relying on manual pipetting guarantees an inherently slow and error-prone workflow. Fortunately, the inter- and intra-experimental variability can be drastically decreased using lab automation. Automated laboratory instruments excel at one thing: delivering consistent results any time, all the time, from 8 am to 6 pm, independently of their users’ years of lab experience, pipetting skills, caffeine intake, or Monday morning or Friday afternoon mood.

ELISA Automation

Broad adoption of lab automation is a transformative development across virtually all life science applications. It is particularly useful for performing highly sensitive and repetitive tasks like ELISAs, which require multiple washing and incubation steps and can take a full day to pipette manually. Pipetting mistakes during ELISA workflows can lead to costly delays and wasted reagents.

Automating the different steps of an ELISA assay ensures that experiments are performed accurately and consistently. The improved efficiency enables smaller-scale experiments, reducing the number of wash cycles to save time, lower reagent costs, and minimize liquid waste. Automation also frees researchers from performing lengthy and tedious protocols that carry the risk of repetitive strain injuries (Wu et al., 2014).

The C.WASH PLUS from CYTENA streamlines your ELISA workflow by automating both the dispensing and removal of wash buffers.

High Wash Efficiency: As a centrifugal washer, the C.WASH PLUS has an impressive wash efficiency of >99.99% (Fig. 1).
Eco-Friendly, Tip-Free Dispensing: As a non-contact dispenser, the C.WASH PLUS saves your lab thousands of plastic tips a year (Fig. 2).
Versatile Two-in-One Solution: Compatible with 96-, 384-, and 1536-well plates, the C.WASH PLUS seamlessly automates ELISA plate washing across a range of formats.

Figure 1. The C.WASH PLUS uses centrifugal plate washing to achieve less than 0.1 µL residual volume after two 1-minute wash cycles.

Conclusion

ELISA has been a fundamental technique for over fifty years, and recent technological advancements, such as microfluidics and CRISPR-ELISA, demonstrate its continued relevance and importance (Kweon et al., 2024; Li et al., 2022). These innovations ensure that ELISA will remain a central tool in scientific research for decades to come.

Despite their utility and sensitivity, ELISA assays can suffer from their multistep, error-prone procedures, leading to potentially damaging variability and artifacts. Automating ELISA dispensing and washing steps goes a long way toward increasing the consistency of your results but also the throughput of your lab by accelerating repetitive steps and freeing up your time.

Innovations like the C.WASH PLUS from CYTENA give a new spin to ELISA washing steps, combining precision dispensing with efficient removal of liquids, while minimizing cross-contamination and plastic waste. Consistent and reproducible ELISA workflows help you and your peers feel confident when interpreting biological data and make progress towards your research goals.

Contact CYTENA today to discover how the C.WASH PLUS can simplify your ELISA assays, along with other molecular or cell-based assays, while making your lab work more efficient and hassle-free.

References

Alexander, T. S. (2016). Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution. Clinical and Vaccine Immunology: CVI, 23(4), 249–253.

Alhajj, M., Zubair, M., & Farhana, A. (2024). Enzyme Linked Immunosorbent Assay. In StatPearls. StatPearls Publishing.

Aydin, S. (2015). A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4–15.

Catalona, W. J. (2014). History of the discovery and clinical translation of prostate-specific antigen. Asian Journal of Urology, 1(1), 12–14.

Deutschmann, C., Roggenbuck, D., Schierack, P., & Rödiger, S. (2020). Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure. Heliyon, 6(1), e03270.

Jaria, G., Calisto, V., Otero, M., & Esteves, V. I. (2020). Monitoring pharmaceuticals in the aquatic environment using enzyme-linked immunosorbent assay (ELISA)—A practical overview. Analytical and Bioanalytical Chemistry, 412(17), 3983–4008.

MacMullan, M. A., Ibrayeva, A., Trettner, K., Deming, L., Das, S., Tran, F., Moreno, J. R., Casian, J. G., Chellamuthu, P., Kraft, J., Kozak, K., Turner, F. E., Slepnev, V. I., & Le Page, L. M. (2020). ELISA detection of SARS-CoV-2 antibodies in saliva. Scientific Reports, 10(1), 20818.

Manak, M., Gagnon, L., Phay-Tran, S., Levesque-Damphousse, P., Fabie, A., Daugan, M., Khan, S. T., Proud, P., Hussey, B., Knott, D., Charlton, S., Hallis, B., Medigeshi, G. R., Garg, N., Anantharaj, A., Raqib, R., Sarker, P., Alam, M. M., Rahman, M., … Carless, J. (2024). Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: A qualification analysis. The Lancet Microbe, 5(3), e216–e225.

Stamey, T. A., Yang, N., Hay, A. R., McNeal, J. E., Freiha, F. S., & Redwine, E. (1987). Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. The New England Journal of Medicine, 317(15), 909–916.

Tabatabaei, M. S., Islam, R., & Ahmed, M. (2021). Applications of gold nanoparticles in ELISA, PCR, and immuno-PCR assays: A review. Analytica Chimica Acta, 1143, 250–266.

Wadhwa, M., Knezevic, I., Kang, H.-N., & Thorpe, R. (2015). Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility. Biologicals, 43(5), 298–306.

Wang, H. R., Zhang, Y., Mo, Y. J., Zhang, Z., Chen, R., Lu, X. B., & Huang, W. (2024). Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device. Communications Biology, 7(1), 916.

Yi, J., Gao, Z., Guo, Q., Wu, Y., Sun, T., Wang, Y., Zhou, H., Gu, H., Zhao, J., & Xu, H. (2022). Multiplexed digital ELISA in picoliter droplets based on enzyme signal amplification block and precisely decoding strategy: A universal and practical biodetection platform. Sensors and Actuators B: Chemical, 369, 132214.

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Exploring the Microbiome: Key Techniques and Challenges in Modern Research

Dr. Pierre-Henri Ferdinand — Mon, 23 Dec 2024 13:18:38 +0000

December 23, 2024
1:18 pm

Exploring the Microbiome: Key Techniques and Challenges in Modern Research

By Dr. Pierre-Henri Ferdinand

Head of Product

Microbiome research is transforming how we address global challenges in healthcare, the environment, and industry. The microbiome, a complex community of microorganisms, plays a pivotal role in human health, ecosystem balance, and sustainable innovation. Advances in this broad field enable groundbreaking applications in medicine, eco-friendly agriculture, and renewable energy production. By studying rare microbial communities, researchers are paving the way for a healthier, more sustainable future. This blog examines the key applications of microbiome research, the advanced tools driving progress in the field, and the challenges that must be overcome before we unlock the full potential of microbial communities.

Key Applications of Microbiome Research

The microbiome encompasses diverse microbial communities that play essential roles in health and the environment. Microbiome research transforms health, environmental, and industrial sectors, offering innovative solutions to critical challenges.

Health and Medicine

The human microbiome is central to health, with disruptions linked to diseases ranging from infections to chronic conditions and mental health issues (Chakraborty et al., 2024; Ghosh et al., 2022). Insights into the microbiome have advanced personalized medicine, enabling targeted treatments, while probiotics and therapies like fecal microbiota transplants show promise in managing conditions such as gastrointestinal disorders (Allegretti et al., 2024). Advanced technologies and a growing understanding of the microbiome’s role in health improve diagnostics by offering detailed insights into microbial populations (Schmartz et al., 2024).

Environmental Applications

Microbes are key to sustainability, aiding in soil health, nutrient recycling, and plant growth. Beneficial microbes enhance crop yields and can be used to rehabilitate low-quality soils. In pest and pollution control, microbes serve as eco-friendly biopesticides and break down pollutants like plastics and heavy metals (Johnson, 2024; Ragasruthi et al., 2024). Emerging research highlights their role in carbon capture, offering potential solutions to mitigate climate change.

Industrial Innovations

Read our full-length article to learn more about this topic and explore other exciting areas like synthetic biology.

Techniques for Isolating Rare Microbes

The isolation and cultivation of microbes have driven groundbreaking innovations in healthcare and beyond. Penicillin, discovered by Alexander Fleming from Penicillium notatum, revolutionized medicine (Gaynes, 2017). Modern advancements include CRISPR, derived from bacterial immune systems, and compounds like actinomycin D and rapamycin, initially sourced from microbes and now important in biological research and agriculture (Hossain, 2021). Thermus aquaticus, from which thermostable DNA polymerases are derived, transformed PCR technology (Raghunathan & Marx, 2019). With less than 1% of environmental microbes cultured, huge potential remains for discovering new applications and unlocking the capabilities of rare microbial species.

Techniques for Microbial Analysis

Selective media and enrichment cultures facilitate the growth of specific microbes, while microscopy techniques such as gram staining help differentiate bacterial subgroups. Traditional tests like enzyme activity and metabolic profiling provide insights into microbial functionality (Franco-Duarte et al., 2019). Modern technologies address challenges associated with traditional microbe isolation techniques by enabling precise identification and characterization of microbial species. Genome sequencing, including single-cell and shotgun techniques, delivers high-resolution data on microbial populations. These methods also reveal insights into the structure, function, and interactions of microbial communities (Franzosa et al., 2015).

Innovative Isolation Tools

Advanced tools like CYTENA’s B.SIGHT streamline microbial isolation, enabling the precise capture of individual cells from complex samples (Fig. 1). This technology accelerates workflows by overcoming barriers to isolating rare microbes, unlocking opportunities for discovery.

Figure 1. The B.SIGHT allows researchers to rapidly seed single microbial cells into 96- and 386-well plates.

To delve deeper into the importance of studying rare microbes and the advanced technologies enabling faster insights, read our full-length article for detailed coverage of these topics.

Transformative Applications of Bacterial and Yeast Strain Development

Advanced microbiome research techniques allow for the engineering of bacterial and yeast strains, revolutionizing industries with applications from therapeutic production to sustainable technologies.

Therapeutic Proteins: Bacteria and yeast produce essential therapeutics, including insulin, recombinant proteins used in vaccines, and treatments like erythropoietin (EPO) (Rettenbacher et al., 2022).
Synthetic Biology Innovations: Strain engineering supports groundbreaking applications like oncolytic bacteria for cancer treatment and biosensors capable of detecting environmental and health biomarkers in real time (Kiaheyrati et al., 2024).
Biofuels and Agriculture: Engineered yeast efficiently converts biomass into biofuels, offering eco-friendly alternatives to fossil fuels (Keasling et al., 2021). Meanwhile, bacterial strains serve as biopesticides, protecting crops without the environmental downsides of chemical pesticides (Ragasruthi et al., 2024).

Challenges in Strain Development

Bacteria and yeast strain development face significant challenges that require innovative solutions. Single clone isolation is a critical bottleneck, as traditional manual methods are time-consuming, error-prone, and often fail to isolate rare or slow-growing clones (Fig. 2). Ensuring clonality is vital to prevent contamination and enable efficient screening. Growth optimization is another hurdle, as strains have specific requirements for pH, oxygen, temperature, and nutrients. Scaling up production can make it more challenging to maintain optimal conditions, which can negatively impact yields (Xu et al., 2024). Additionally, regulatory compliance demands rigorous monitoring to ensure strains are contaminant-free, genetically stable, and meet strict standards for therapeutic and industrial applications. Advanced tools like automated imaging systems and precise growth monitoring technologies provided by the B.SIGHT and S.NEST from CYTENA, respectively, are helping address these obstacles, enabling higher efficiency and reliability in strain development workflows.

Figure 2. Traditional methods of isolating and characterizing microorganisms are laborious, time-consuming, and prone to error.

To learn more about the growing importance of bacterial and yeast strain development for many industries, consider reading our full-length article on existing and emerging technologies in this area.

Conclusion

Microbiome research continues to unlock transformative solutions across diverse fields. From improving human health to tackling environmental challenges like pollution and climate change, microbes are at the forefront of innovation. Advances in strain development, isolation tools, and imaging technologies further enhance our ability to harness the potential of individual microbes and microbial communities. As research evolves, the microbiome will remain central to addressing global challenges, offering sustainable solutions and revolutionary advancements in healthcare, environmental restoration, and industrial efficiency.

CYTENA stands at the forefront of technology for isolating single microbial cells and culturing them for various applications. Talk to one of our experts today to discover how the B.SIGHT can eliminate bottlenecks in your strain development workflows.

References

Allegretti, J. R., Khanna, S., Mullish, B. H., & Feuerstadt, P. (2024). The Progression of Microbiome Therapeutics for the Management of Gastrointestinal Diseases and Beyond. Gastroenterology, 167(5), 885–902.
Chakraborty, P., Banerjee, D., Majumder, P., & Sarkar, J. (2024). Gut microbiota nexus: Exploring the interactions with the brain, heart, lungs, and skin axes and their effects on health. Medicine in Microecology, 20, 100104.
Franco-Duarte, R., Černáková, L., Kadam, S., S. Kaushik, K., Salehi, B., Bevilacqua, A., Corbo, M. R., Antolak, H., Dybka-Stępień, K., Leszczewicz, M., Relison Tintino, S., Alexandrino De Souza, V. C., Sharifi-Rad, J., Melo Coutinho, H. D., Martins, N., & Rodrigues, C. F. (2019). Advances in Chemical and Biological Methods to Identify Microorganisms—From Past to Present. Microorganisms, 7(5), 130.
Franzosa, E. A., Hsu, T., Sirota-Madi, A., Shafquat, A., Abu-Ali, G., Morgan, X. C., & Huttenhower, C. (2015). Sequencing and beyond: Integrating molecular ‘omics’ for microbial community profiling. Nature Reviews Microbiology, 13(6), 360–372.
Gaynes, R. (2017). The Discovery of Penicillin—New Insights After More Than 75 Years of Clinical Use. Emerging Infectious Diseases, 23(5), 849–853.
Ghosh, T. S., Shanahan, F., & O’Toole, P. W. (2022). The gut microbiome as a modulator of healthy ageing. Nature Reviews Gastroenterology & Hepatology, 19(9), 565–584.
Graham, A. E., & Ledesma-Amaro, R. (2023). The microbial food revolution. Nature Communications, 14(1), 2231.
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Hossain, M. A. (2021). CRISPR-Cas9: A fascinating journey from bacterial immune system to human gene editing. In Progress in Molecular Biology and Translational Science (Vol. 178, pp. 63–83). Elsevier.
Johnson, B. (2024). Plastic-eating bacteria boost growing business of bioremediation. Nature Biotechnology,
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Ragasruthi, M., Balakrishnan, N., Murugan, M., Swarnakumari, N., Harish, S., & Sharmila, D. J. S. (2024). Bacillus thuringiensis (Bt)-based biopesticide: Navigating success, challenges, and future horizons in sustainable pest control. Science of The Total Environment, 954, 176594.
Raghunathan, G., & Marx, A. (2019). Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Scientific Reports, 9(1), 590.
Rawlings, D. E. (2002). Heavy Metal Mining Using Microbes. Annual Review of Microbiology, 56(1), 65–91.
Rettenbacher, L. A., Arauzo-Aguilera, K., Buscajoni, L., Castillo-Corujo, A., Ferrero-Bordera, B., Kostopoulou, A., Moran-Torres, R., Núñez-Nepomuceno, D., Öktem, A., Palma, A., Pisent, B., Puricelli, M., Schilling, T., Tungekar, A. A., Walgraeve, J., Humphreys, D., Von Der Haar, T., Gasser, B., Mattanovich, D., … Van Dijl, J. M. (2022). Microbial protein cell factories fight back? Trends in Biotechnology, 40(5), 576–590.
Schmartz, G. P., Rehner, J., Gund, M. P., Keller, V., Molano, L.-A. G., Rupf, S., Hannig, M., Berger, T., Flockerzi, E., Seitz, B., Fleser, S., Schmitt-Grohé, S., Kalefack, S., Zemlin, M., Kunz, M., Götzinger, F., Gevaerd, C., Vogt, T., Reichrath, J., … Keller, A. (2024). Decoding the diagnostic and therapeutic potential of microbiota using pan-body pan-disease microbiomics. Nature Communications, 15(1), 8261.
Ugbede, A. S., Abioye, O. P., Aransiola, S. A., Oyewole, O. A., Maddela, N. R., & Prasad, R. (2023). Production, optimization and partial purification of bacterial and fungal proteases for animal skin dehairing: A sustainable development in leather-making process. Bioresource Technology Reports, 24, 101632.
Xu, P., Lin, N.-Q., Zhang, Z.-Q., & Liu, J.-Z. (2024). Strategies to increase the robustness of microbial cell factories. Advanced Biotechnology, 2(1), 9.

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Bacteria and Yeast Strain Development: Applications and Recent Advances

Dr. Pierre-Henri Ferdinand — Mon, 16 Dec 2024 13:17:17 +0000

December 16, 2024
1:17 pm

Bacteria and Yeast Strain Development: Applications and Recent Advances

By Dr. Pierre-Henri Ferdinand

Head of Product

From producing lifesaving therapeutics like insulin to creating eco-friendly biofuels, engineered strains of bacteria and yeast offer solutions to pressing global challenges. Advances in synthetic biology and automation are driving this field forward, enabling efficient processes and novel applications. However, challenges like single clone isolation, growth condition optimization, and regulatory hurdles persist. This blog explores the applications of bacteria and yeast strain development, highlighting challenges and advances in this rapidly evolving field.

Applications of Strain Development

The development of bacterial and yeast strains has diverse applications. These microorganisms are utilized to produce therapeutic products and as integral components in various industrial processes.

Therapy Production

Bacteria and yeast can be modified to produce therapeutic proteins. Early applications included producing human insulin in E. coli to treat diabetes. However, more recent applications include producing erythropoietin (EPO), human growth hormone, cytokines, and recombinant proteins for use as antigens in vaccines. While mammalian cells have overtaken bacterial cells in producing many protein-based therapies, there are still applications where bacteria are preferred, such as those with less post-translational complexity (Rettenbacher et al., 2022). These include interferons (e.g., IFNα-2b), cytokines (e.g., G-CSF, TNFs), hormones (e.g., insulin glargine, hGH), and interleukins (e.g., IL-2, IL-11), which are commonly produced in E. coli and yeasts (Rettenbacher et al., 2022).

Synthetic Biology

The core principle of synthetic biology is to modify biological systems and organisms to perform specific functions. Emerging applications include the generation of oncolytic bacteria to treat cancer and the creation of living biosensors to detect and respond to disease and pollution biomarkers in real time (Kiaheyrati et al., 2024; Wan et al., 2021).

Agriculture and Biofuels

Biofuels offer an important environmentally friendly alternative to fossil fuels. They are produced by engineered yeast cell lines metabolizing different organic materials (called biomass), including plants, algae, and waste (Keasling et al., 2021). Bacterial cells are important as biopesticides to protect economically important crops from pests. The development of these lines comes as the industry moves towards more environmentally friendly alternatives to conventional chemical pesticides (Ayilara et al., 2023; Ragasruthi et al., 2024).

Challenges in Strain Development

Despite the promise of bacteria and yeast strain development for many applications, there remain several technical challenges that hinder their full potential.

Single Clone Isolation

Isolating single clones from transfection pools and complex mixtures is a major challenge in bacteria strain development. This poses a significant obstacle to workflows, as ensuring clonality is crucial to prevent contamination and enables more efficient, high-throughput clone screening.

Growth Conditions

Diverse bacterial and yeast strains require different conditions for optimal growth. This is particularly important when scaling up bacterial and yeast cells after successful isolation. Finding optimized growing conditions can be challenging because there are a huge number of parameters that need to be considered, including oxygen concentration, growth substrate, pH, and other factors. These parameters can fluctuate in large-scale cultures, negatively impacting production (Xu et al., 2024).

The C.NEST from CYTENA provides continuous monitoring of dissolved oxygen and pH to ensure that cells are cultured in optimal conditions (Fig. 1).

Figure 1. The C.NEST from CYTENA allows researchers to incubate up to four 24- or 96-well plates simultaneously to boost laboratory efficiency.

Regulatory Considerations

The use of genetically modified organisms as therapeutic agents or in the production of therapeutic agents is tightly controlled by regulatory bodies. Researchers must be vigilant to ensure their cell lines are free from contaminants and remain genetically stable and fit for purpose throughout the production and regulatory process.

Advances in Imaging

Advances in microfluidics and imaging technology have facilitated more streamlined and accurate isolation of single cells.

Seeding

Advanced imaging technologies such as those found in the B.SIGHT from CYTENA have made it simple to detect the dispensing of single cells into multiwell plates. Within the B.SIGHT instrument, this enables single-cell omics without requiring cultivation and integrates seamlessly into sequencing workflows. The B.SIGHT uses dual imaging and extremely high-resolution optics to ensure that even the smallest cells, whether labeled or unlabelled, can be detected as they are dispensed into wells (Fig. 2).

Figure 2. The B.SIGHT enables rapid seeding of both labeled and non-labeled microbial cells with advanced imaging to provide robust evidence of clonality.

Monitoring and Analysis

Advanced image analyses allow for high throughput phenotypic characterization and screening of huge numbers of individual microbial clones. This facilitates the selection of optimal clones based on various characteristics, such as growth rate and response to stimuli (Zahir et al., 2019). Pooled screening is a method that uses high-content imaging coupled with genotyping to identify the phenotypic changes induced by modifications to different genes (Feldman et al., 2022). This helps researchers to rapidly link specific genetic perturbations to observable traits, enabling functional genomics studies, the identification of gene functions, and the discovery of potential targets for therapeutic or industrial applications.

Automation and Future Directions

Automated dispensing of bacterial and yeast cells means that single cells can be rapidly seeded into multiwell plate formats from complex mixtures and transfection pools. Automation removes significant sources of error, such as contamination, which can severely compromise strain development. When coupled with advances in microfluidics, automation allows for the rapid dispensing of tiny volumes containing individual cells. Scaling down isolation and culturing processes significantly reduces the cost of development workflows and enables higher throughput in screening processes.

Future advances in this area include the culturing of microbial consortia, which involves cultivating multiple species in tandem to produce metabolites and other products, which could enhance efficiency, reduce production costs, and enable the synthesis of complex compounds that are challenging to produce with single-species systems (Mittermeier et al., 2023). Advancements in bacterial cell systems are underway to better emulate the protein-folding mechanisms of mammalian cells, potentially enabling significant improvements in their capacity to produce therapeutic-grade proteins in the future (Jayakrishnan et al., 2024).

Conclusion

Despite challenges in isolation, scalability, and regulatory requirements, advancements in imaging, automation, and synthetic biology promise to accelerate progress in bacteria and yeast strain development. Future directions, including culturing microbial consortia and generating microbes more suitable for mammalian protein production, indicate a thriving field poised to address global needs. As these technologies mature, they offer a pathway to sustainable, efficient, and transformative solutions in healthcare, agriculture, and beyond.

The B.SIGHT is revolutionizing how researchers isolate single microbial cells for various downstream applications. Talk to a CYTENA expert today to learn more about this exciting piece of technology.

References

Ayilara, M. S., Adeleke, B. S., Akinola, S. A., Fayose, C. A., Adeyemi, U. T., Gbadegesin, L. A., Omole, R. K., Johnson, R. M., Uthman, Q. O., & Babalola, O. O. (2023). Biopesticides as a promising alternative to synthetic pesticides: A case for microbial pesticides, phytopesticides, and nanobiopesticides. Frontiers in Microbiology, 14, 1040901.
Feldman, D., Funk, L., Le, A., Carlson, R. J., Leiken, M. D., Tsai, F., Soong, B., Singh, A., & Blainey, P. C. (2022). Pooled genetic perturbation screens with image-based phenotypes. Nature Protocols, 17(2), 476–512.
Jayakrishnan, A., Wan Rosli, W. R., Tahir, A. R. M., Razak, F. S. A., Kee, P. E., Ng, H. S., Chew, Y.-L., Lee, S.-K., Ramasamy, M., Tan, C. S., & Liew, K. B. (2024). Evolving Paradigms of Recombinant Protein Production in Pharmaceutical Industry: A Rigorous Review. Sci, 6(1), 9.
Keasling, J., Garcia Martin, H., Lee, T. S., Mukhopadhyay, A., Singer, S. W., & Sundstrom, E. (2021). Microbial production of advanced biofuels. Nature Reviews Microbiology, 19(11), 701–715.
Kiaheyrati, N., Babaei, A., Ranji, R., Bahadoran, E., Taheri, S., & Farokhpour, Z. (2024). Cancer therapy with the viral and bacterial pathogens: The past enemies can be considered the present allies. Life Sciences, 349, 122734. https://doi.org/
Mittermeier, F., Bäumler, M., Arulrajah, P., García Lima, J. de J., Hauke, S., Stock, A., & Weuster-Botz, D. (2023). Artificial microbial consortia for bioproduction processes. Engineering in Life Sciences, 23(1), e2100152.
Ragasruthi, M., Balakrishnan, N., Murugan, M., Swarnakumari, N., Harish, S., & Sharmila, D. J. S. (2024). Bacillus thuringiensis (Bt)-based biopesticide: Navigating success, challenges, and future horizons in sustainable pest control. Science of The Total Environment, 954, 176594.
Rettenbacher, L. A., Arauzo-Aguilera, K., Buscajoni, L., Castillo-Corujo, A., Ferrero-Bordera, B., Kostopoulou, A., Moran-Torres, R., Núñez-Nepomuceno, D., Öktem, A., Palma, A., Pisent, B., Puricelli, M., Schilling, T., Tungekar, A. A., Walgraeve, J., Humphreys, D., Von Der Haar, T., Gasser, B., Mattanovich, D., … Van Dijl, J. M. (2022). Microbial protein cell factories fight back? Trends in Biotechnology, 40(5), 576–590.
Wan, X., Saltepe, B., Yu, L., & Wang, B. (2021). Programming living sensors for environment, health and biomanufacturing. Microbial Biotechnology, 14(6), 2334–2342.
Xu, P., Lin, N.-Q., Zhang, Z.-Q., & Liu, J.-Z. (2024). Strategies to increase the robustness of microbial cell factories. Advanced Biotechnology, 2(1), 9.
Zahir, T., Camacho, R., Vitale, R., Ruckebusch, C., Hofkens, J., Fauvart, M., & Michiels, J. (2019). High-throughput time-resolved morphology screening in bacteria reveals phenotypic responses to antibiotics. Communications Biology, 2(1), 269.

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Advanced Techniques for Isolating Rare Microbiota in Complex Samples

Dr. Pierre-Henri Ferdinand — Mon, 09 Dec 2024 13:16:06 +0000

December 9, 2024
1:16 pm

Advanced Techniques for Isolating Rare Microbiota in Complex Samples

By Dr. Pierre-Henri Ferdinand

Head of Product

Rare microbiota isolation has been instrumental in driving groundbreaking innovations in healthcare, biotechnology, and beyond. From the accidental discovery of penicillin to modern breakthroughs like CRISPR, cultivating microorganisms continually revolutionizes many industries. Less than 1% of environmental microbes have been cultured to date, leaving room for even greater discoveries and innovations in the future (Yu et al., 2022). Advancing our understanding of rare species could lead to new therapies, industrial applications, and insights into ecological systems. This blog explores the importance of studying rare species, the methods used to study them, and the technologies shaping the future of microbiome research.

The Importance of Studying Rare Species

The isolation and cultivation of microbial species have led to some of the most important innovations in healthcare and beyond. For instance, penicillin was accidentally discovered when Alexander Fleming observed that a mold, Penicillium notatum, produced a substance that killed surrounding bacterial colonies (Gaynes, 2017). More modern technologies arising from the isolation and study of microorganisms include CRISPR, which was originally identified as part of a bacterial immune response (Hossain, 2021).

Compounds historically used as cancer therapies and with applications in biological research and agriculture include Actinomycin D and rapamycin, both of which were initially derived from microbes (Demain & Sanchez, 2009). The latter was isolated from microbial species on Easter Island, and its name comes from “Rapa Nui,” which is both the name of the island and the Polynesian language spoken there. Biologists will be familiar with the bacterium Thermus aquaticus, from which thermostable DNA polymerases used in modern PCR reactions are taken (Raghunathan & Marx, 2019)(
Fig. 1).

The isolation and cultivation of rare microbial species have resulted in some of the most significant discoveries in recent years. These breakthroughs underscore the vast potential for uncovering additional applications of microbial species that have yet to be characterized. It is estimated that less than 1% of environmental microbes have been cultured, leaving a massive scope for potential discoveries (Yu et al., 2022).

Figure 1. Thermus aquaticus was first isolated from the hot springs of Yellowstone National Park and has adapted to survive in very hot environments.

Challenges in Microbe Isolation

Although isolating and studying rare microbial species is crucial, several significant challenges make species difficult to isolate, which slows the progress of research and development initiatives.

Time Consuming

Traditional methods of isolating and quantifying viable bacteria are often time-consuming and require manual handling of large bacterial populations, increasing the risk of contamination and mislabeling (Franco-Duarte et al., 2019; Needs et al., 2021). Bacteria with similar characteristics in culture can be easily confused, further complicating and prolonging the isolation process.

Specific Growth Requirements

Many microbes are difficult to culture once taken from their typical growing environment.

This is partly because the factors necessary for their growth are poorly understood. These factors may include the presence of other species with which they share a symbiotic relationship, as well as a complex interplay of conditions such as pH, temperature, oxygen levels, and other variables (Gupta et al., 2017).

Low Abundance

Much of microbial research requires the isolation of individual microbes from complex microbiome samples. Less abundant microbes can be difficult to isolate, particularly with manual methods, where they may be overgrown by more abundant or faster-growing microbes (Cena et al., 2021; Han & Vaishnava, 2023). Some species are inherently slow-growing, making it difficult to culture them for characterization studies.

Effective Cell Detection

Microbes are incredibly small, and detecting them with acceptable throughput and precision remains a significant challenge in microbial isolation. Traditional methods often lack the sensitivity needed to reliably identify and isolate individual microbes, particularly in complex samples. Additionally, isolating microbes under anaerobic conditions further complicates detection (Börner, 2016). Many existing technologies for isolating microorganisms are not optimized for these environments, limiting their effectiveness when handling oxygen-sensitive species or those with unique environmental requirements.

Analysis Techniques

The analysis of microbial samples is important for characterizing newly discovered species and for gaining insights into the constituent species of a specific microbial community, such as those found in the human gut, soil, oceans, and other environments. This information helps to understand their roles, interactions, and contributions to ecosystem functions, as well as their potential applications in healthcare and other industries.

Culturing and Microscopy Approaches

Many microbial species can be identified by the different characteristics they display when grown on agar plates, such as colony morphology, color, and growth patterns. These approaches can also include growing microbes on selective media plates or liquid cultures that preferentially promote the growth of certain species. These are known as enrichment cultures. Microscopy can be performed on cultured bacteria and typically involves the application of different stains, such as gram staining, to identify different subgroups (Franco-Duarte et al., 2019; Paray et al., 2023).

Biochemical Assays

These approaches aim to differentiate microbial species based on their ability to metabolize different molecules. This typically involves testing for the presence of certain enzymes like catalase, the ability to ferment sugar, and other metabolic processes like nitrate reduction (Franco-Duarte et al., 2019).

Molecular Techniques and Omics

These techniques include advanced genome sequencing, which offers both high throughput and high resolution for identifying different species. Single-cell and shotgun approaches allow for a focused examination of specific species or a general overview of microbial populations within a particular sample, respectively (Franzosa et al., 2015).

Emerging Technologies

Several important technologies are emerging to address challenges in modern microbiology workflows, collectively allowing for higher throughput and better resolution for studying and culturing novel and rare species.

Microfluidics

Advances in microfluidics have allowed for faster and more accurate rare microbiota isolation. This is particularly powerful when isolating single cells from complex microbiome samples. Microfluidics can provide individual culture environments for single cells, allowing massive throughput at small scales. Microfluidics can also enable the study of interactions between different species, offering greater insights into how microbes fit within their ecological niche (Song et al., 2024; Yu et al., 2022).

Automation and Culturomics

Automation is transforming many industries and is particularly powerful for driving faster and more accurate microbiological workflows. Automation eliminates the need for manual manipulation of bacterial cultures, which is time-consuming, prone to contamination, and incompatible with the isolation of novel species. Recent advances combine automation, AI, and genetic testing into culturomics to isolate and culture specific genera with high throughput (Huang et al., 2023).

The B.SIGHT from CYTENA provides automated dispensing of single microbial cells, allowing for rapid seeding into 96- and 386-well plates (Fig. 2).

Figure 2. The B.SIGHT uses gentle label-free isolation to enhance cell viability, making it easier to isolate and cultivate rare samples.

Conclusion

Exploring rare microbial species has the potential to unlock unparalleled opportunities for innovation across healthcare, biotechnology, and environmental sciences. Despite the challenges of culturing and studying these organisms, emerging technologies such as microfluidics and automation pave the way for faster discoveries. As we push the boundaries of microbial research, the diversity of uncultured microbes offers a reservoir of potential for transformative solutions. By investing in these advances, researchers can take a significant step toward uncovering the next microbe-derived innovation.

Ready to take the lead in microbiome research? Contact a member of the CYTENA team today to learn more about the B.SIGHT and how it can transform your rare microbiota isolation workflows.

References

Börner, R.A. (2016) ‘Isolation and Cultivation of Anaerobes’, in R. Hatti-Kaul, G. Mamo, and B. Mattiasson (eds) Anaerobes in Biotechnology. Cham: Springer International Publishing, pp. 35–53. Available at:
Cena, J. A. D., Zhang, J., Deng, D., Damé-Teixeira, N., & Do, T. (2021). Low-Abundant Microorganisms: The Human Microbiome’s Dark Matter, a Scoping Review. Frontiers in Cellular and Infection Microbiology, 11, 689197.
Demain, A. L., & Sanchez, S. (2009). Microbial drug discovery: 80 years of progress. The Journal of Antibiotics, 62(1), 5–16.
Franco-Duarte, R., Černáková, L., Kadam, S., S. Kaushik, K., Salehi, B., Bevilacqua, A., Corbo, M. R., Antolak, H., Dybka-Stępień, K., Leszczewicz, M., Relison Tintino, S., Alexandrino De Souza, V. C., Sharifi-Rad, J., Melo Coutinho, H. D., Martins, N., & Rodrigues, C. F. (2019). Advances in Chemical and Biological Methods to Identify Microorganisms—From Past to Present. Microorganisms, 7(5), 130.
Franzosa, E. A., Hsu, T., Sirota-Madi, A., Shafquat, A., Abu-Ali, G., Morgan, X. C., & Huttenhower, C. (2015). Sequencing and beyond: Integrating molecular ‘omics’ for microbial community profiling. Nature Reviews Microbiology, 13(6), 360–372.
Gaynes, R. (2017). The Discovery of Penicillin—New Insights After More Than 75 Years of Clinical Use. Emerging Infectious Diseases, 23(5), 849–853.
Gupta, A., Gupta, R., & Singh, R. L. (2017). Microbes and Environment. In R. L. Singh (Ed.), Principles and Applications of Environmental Biotechnology for a Sustainable Future (pp. 43–84). Springer Singapore.
Han, G., & Vaishnava, S. (2023). Microbial underdogs: Exploring the significance of low-abundance commensals in host-microbe interactions. Experimental & Molecular Medicine, 55(12), 2498–2507.
Hossain, M. A. (2021). CRISPR-Cas9: A fascinating journey from bacterial immune system to human gene editing. In Progress in Molecular Biology and Translational Science (Vol. 178, pp. 63–83). Elsevier.
Huang, Y., Sheth, R. U., Zhao, S., Cohen, L. A., Dabaghi, K., Moody, T., Sun, Y., Ricaurte, D., Richardson, M., Velez-Cortes, F., Blazejewski, T., Kaufman, A., Ronda, C., & Wang, H. H. (2023). High-throughput microbial culturomics using automation and machine learning. Nature Biotechnology, 41(10), 1424–1433.
Needs, S. H., Osborn, H. M. I., & Edwards, A. D. (2021). Counting bacteria in microfluidic devices: Smartphone compatible ‘dip-and-test’ viable cell quantitation using resazurin amplified detection in microliter capillary arrays. Journal of Microbiological Methods, 187, 106199.
Paray, A. A., Singh, M., & Amin Mir, M. (2023). Gram Staining: A Brief Review. International Journal of Research and Review, 10(9), 336–341.
Raghunathan, G., & Marx, A. (2019). Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Scientific Reports, 9(1), 590.
Song, Y., Yin, J., Huang, W. E., Li, B., & Yin, H. (2024). Emerging single-cell microfluidic technology for microbiology. TrAC Trends in Analytical Chemistry, 170, 117444.
Yu, Y., Wen, H., Li, S., Cao, H., Li, X., Ma, Z., She, X., Zhou, L., & Huang, S. (2022). Emerging microfluidic technologies for microbiome research. Frontiers in Microbiology, 13, 906979.

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