Calibrating spatiotemporal models of microbial communities to microscopy data: A review.

Spatiotemporal models that account for heterogeneity within microbial communities rely on single-cell data for calibration and validation. Such data, commonly collected via microscopy and flow cytometry, have been made more accessible by recent advances in microfluidics platforms and data processing pipelines. However, validating models against such data poses significant challenges. Validation practices vary widely between modelling studies; systematic and rigorous methods have not been widely adopted. Similar challenges are faced by the (macrobial) ecology community, in which systematic calibration approaches are often employed to improve quantitative predictions from computational models. Here, we review single-cell observation techniques that are being applied to study microbial communities and the calibration strategies that are being employed for accompanying spatiotemporal models. To facilitate future calibration efforts, we have compiled a list of summary statistics relevant for quantifying spatiotemporal patterns in microbial communities. Finally, we highlight some recently developed techniques that hold promise for improved model calibration, including algorithmic guidance of summary statistic selection and machine learning approaches for efficient model simulation.

A sensitive assay for scrutiny of Autographa californica multiple nucleopolyhedrovirus genes using CRISPR-Cas9.

Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.

Evaluating ultraviolet sensitivity of adventitious agents in biopharmaceutical manufacturing.

Incidents of contamination in biopharmaceutical production have highlighted the need to apply alternative or supplementary disinfection techniques. Ultraviolet (UV) irradiation is a well-established method for inactivating a broad range of microorganisms, and is therefore a good candidate as an orthogonal technique for disinfection. To apply UV as a safeguard against adventitious agents, the UV sensitivity of these target agents must be known so that the appropriate dose of UV may be applied to achieve the desired level of inactivation. This document compiles and reviews experimentally derived 254 nm sensitivities of organisms relevant to biopharmaceutical production. In general, different researchers have found similar sensitivity values despite a lack of uniformity in experimental design or standardized quantification techniques. Still, the lack of consistent methodologies has led to suspicious UV susceptibilities in certain instances, justifying the need to create a robust collection of sensitivity values that can be used in the design and sizing of UV systems for the inactivation of adventitious agents.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips.

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 µm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 µm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels.

The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4(-/-) mice and found also a >50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family.

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum.

UNLABELLED: Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT::Ll.LtrB). Metabolite characterization of the dhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter(-1)). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter(-1)), yielding a high n-butanol selectivity of 0.83 g n-butanol g(-1) of solvents compared to 0.51 g n-butanol g(-1) of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCE: Organisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol.

Metabolomic analysis of human fecal microbiota: a comparison of feces-derived communities and defined mixed communities.

The extensive impact of the human gut microbiota on its human host calls for a need to understand the types of communication that occur among the bacteria and their host. A metabolomics approach can provide a snapshot of the microbe-microbe interactions occurring as well as variations in the microbes from different hosts. In this study, metabolite profiles from an anaerobic continuous stirred-tank reactors (CSTR) system supporting the growth of several consortia of bacteria representative of the human gut were established and compared. Cell-free supernatant samples were analyzed by 1D (1)H nuclear magnetic resonance (NMR) spectroscopy, producing spectra representative of the metabolic activity of a particular community at a given time. Using targeted profiling, specific metabolites were identified and quantified on the basis of NMR analyses. Metabolite profiles discriminated each bacterial community examined, demonstrating that there are significant differences in the microbiota metabolome between each cultured community. We also found unique compounds that were identifying features of individual bacterial consortia. These findings are important because they demonstrate that metabolite profiles of gut microbial ecosystems can be constructed by targeted profiling of NMR spectra. Moreover, examination of these profiles sheds light on the type of microbes present in the gut and their metabolic interactions.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BACKGROUND: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. METHODS: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. RESULTS: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. CONCLUSIONS: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system.

Profiling convoluted single-dimension proton NMR spectra: a Plackett-Burman approach for assessing quantification error of metabolites in complex mixtures with application to cell culture.

Single-dimension hydrogen, or proton, nuclear magnetic resonance spectroscopy (1D-(1)H NMR) has become an attractive option for characterizing the full range of components in complex mixtures of small molecular weight compounds due to its relative simplicity, speed, spectral reproducibility, and noninvasive sample preparation protocols compared to alternative methods. One challenge associated with this method is the overlap of NMR resonances leading to "convoluted" spectra. While this can be mitigated through "targeted profiling", there is still the possibility of increased quantification error. This work presents the application of a Plackett-Burman experimental design for the robust estimation of precision and accuracy of 1D-(1)H NMR compound quantification in synthetic mixtures, with application to mammalian cell culture supernatant. A single, 20 sample experiment was able to provide a sufficient estimate of bias and variability at different metabolite concentrations. Two major sources of bias were identified: incorrect interpretation of singlet resonances and the quantification of resonances from protons in close proximity to labile protons. Furthermore, decreases in measurement accuracy and precision could be observed with decreasing concentration for a small fraction of the components as a result of their particular convolution patterns. Finally, the importance of a priori concentration estimates is demonstrated through the example of interpreting acetate metabolite trends from a bioreactor cultivation of Chinese hamster ovary cells expressing a recombinant antibody.

Heterologous expression of NoxA confers aerotolerance in Clostridium sporogenes.

Clostridium is a genus of gram-positive obligate anaerobic bacteria. Some species of Clostridium, including Clostridium sporogenes, may be of use in bacteria-mediated cancer therapy. Spores of Clostridium are inert in healthy normoxic tissue but germinate when in the hypoxic regions of solid tumors, causing tumor regression. However, such treatments fail to completely eradicate tumors partly because of higher oxygen levels at the tumor's outer rim. In this study, we demonstrate that a degree of aerotolerance can be introduced to C. sporogenes by transfer of the noxA gene from Clostridium aminovalericum. NoxA is a water-forming NADH oxidase enzyme, and so has no detrimental effect on cell viability. In addition to its potential in cancer treatment, the noxA-expressing strain described here could be used to alleviate challenges related to oxygen sensitivity of C. sporogenes in biomanufacturing.

Probing Baculovirus Vector Gene Essentiality for Foreign Gene Expression Using a CRISPR-Cas9 System.

The baculovirus expression vector system (BEVS) has now found acceptance in both research laboratories and industry, which can be attributed to many of its key features including the limited host range of the vectors, their non-pathogenicity to humans, and the mammalian-like post-translational modification (PTMs) that can be achieved in insect cells. In fact, this system acts as a middle ground between prokaryotes and higher eukaryotes to produce complex biologics. Still, industrial use of the BEVS lags compared to other platforms. We have postulated that one reason for this has been a lack of genetic tools that can complement the study of baculovirus vectors, while a second reason is the co-production of the baculovirus vector with the desired product. While some genetic enhancements have been made to improve the BEVS as a production platform, the genome remains under-scrutinized. This chapter outlines the methodology for a CRISPR-Cas9-based transfection-infection assay to probe the baculovirus genome for essential/nonessential genes that can potentially maximize foreign gene expression under a promoter of choice.

Construction and analysis of a genetically tuneable lytic phage display system.

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.

Evaluation of Virus-Free Manufacture of Recombinant Proteins Using CRISPR-Mediated Gene Disruption in Baculovirus-Infected Insect Cells.

The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp64 open reading frame, which encodes the major envelope protein of AcMNPV. After comfirming the site-specific targeting of gp64 leading to reduced budded virus (BV) release, the gag gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release.

Expression of Interleukin-1ß protein in vitro,exvivo and in vivo salmonid models.

Interleukin-1ß (IL-1ß) is one of the first cytokines expressed during immune responses, and its levels are affected by many factors, including stress. To date, it has only been possible to measure IL-1ß transcript (mRNA) expression quantitatively in fish using qPCR. This is because previous studies that measured IL-1ß protein concentrations in these taxa used western blotting, which only provides qualitative data. To advance our knowledge of fish IL-1ß biology, and because post-translational processing plays a critical role in the activation of this molecule, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) to accurately measure the concentration of IL-1ß protein in several cell cultures and in vivo in salmonids. We compared changes in IL-1ß protein levels to the expression of its mRNA. The developed ELISA was quite sensitive and has a detection limit of 12.5 pg/mL. The tools developed, and information generated through this research, will allow for a more accurate and complete understanding of IL-1ß's role in the immune response of salmonids.The assay described here has the potential to significantly advance our ability to assess fish health and immune status.

A comparative analysis of the partitioning behaviour of SARS-CoV-2 RNA in liquid and solid fractions of wastewater.

As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage.

As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.

Vero cells gain renal tubule markers in low-calcium and magnesium chemically defined media.

In this study, a chemically defined, animal component-free media was developed to promote Vero growth in suspension. Key media compounds were screened using Plackett-Burman styled experiments to create a media formulation to support suspension growth. Vero cells remained viable in suspension, but their growth rate was extremely low, conversely, other cell types such as CHO-K1, MDCK and HEK293T were able to grow in single cell suspension in the same media. To investigate the slow growth of Vero cells, RNA-seq analysis was conducted. Vero cells were cultured in three different conditions: adherently in serum-containing medium, adherently in in-house medium, and in suspension in low calcium and magnesium in-house medium. This study illustrates that adherent cells maintain similar gene expression, while the suspension phenotype tends to overexpress genes related to renal tubules.

Utility of Alternative Promoters for Foreign Gene Expression Using the Baculovirus Expression Vector System.

The baculovirus expression vector system (BEVS) is a widely used platform for recombinant protein production for use in a wide variety of applications. Of particular interest is production of virus-like particles (VLPs), which consist of multiple viral proteins that self-assemble in strict stoichiometric ratios to mimic the structure of a virus but lacks its genetic material, while a significant amount of effort has been spent on optimizing expression ratios by co-infecting cells with multiple recombinant BEVs and modulating different process parameters, co-expressing multiple foreign genes from a single rBEV may offer more promise. However, there is currently a lack of promoters available with which to optimize co-expression of each foreign gene. To address this, previously published transcriptome data was used to identify promoters that have incrementally lower expression profiles and compared by expressing model cytoplasmic and secreted proteins. Bioinformatics was also used to identify sequence determinants that may be important for late gene transcription regulation, and translation initiation. The identified promoters and bioinformatics analyses may be useful for optimizing expression of foreign genes in the BEVS.

Droplet formation of biological non-Newtonian fluid in T-junction generators. I. Experimental investigation.

The extension of microfluidics to many bioassay applications requires the ability to work with non-Newtonian fluids. One case in point is the use of microfluidics with blood having different hematocrit levels. This work is the first part of a two-part study and presents the formation dynamics of blood droplets in a T-junction generator under the squeezing regime. In this regime, droplet formation with Newtonian fluids depends on T-junction geometry; however, we found that in the presence of the non-Newtonian fluid such as red blood cells, the formation depends on not only to the channel geometry, but also the flow rate ratio of fluids, and the viscosity of the phases. In addition, we analyzed the impact of the red blood cell concentration on the formation cycle. In this study, we presented the experimental data of the blood droplet evolution through the analysis of videos that are captured by a high-speed camera. During this analysis, we tracked several parameters such as droplet volume, spacing between droplets, droplet generation frequency, flow conditions, and geometrical designs of the T junction. Our analysis revealed that, unlike other non-Newtonian fluids, where the fourth stage exists (stretching stage), the formation cycle consists of only three stages: lag, filling, and necking stages. Because of the detailed analysis of each stage, a mathematical model can be generated to predict the final volume of the blood droplet and can be utilized as a guide in the operation of the microfluidic device for biochemical assay applications; this is the focus of the second part of this study [Phys. Rev. E 105, 025106 (2022)10.1103/PhysRevE.105.025106].