Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

Comparative Analysis of Protein Quantification by the SomaScan Assay versus Orthogonal Methods in Urine from People with Diabetic Kidney Disease.

To our knowledge, calibration curves or other validations for thousands of SomaScan aptamers are not publicly available. Moreover, the abundance of urine proteins obtained from these assays is not routinely validated with orthogonal methods (OMs). We report an in-depth comparison of SomaScan readout for 23 proteins in urine samples from patients with diabetic kidney disease (n = 118) vs OMs, including liquid chromatography-targeted mass spectrometry (LC-MS), ELISA, and nephelometry. Pearson correlation between urine abundance of the 23 proteins from SomaScan 3.2 vs OMs ranged from -0.58 to 0.86, with a median (interquartile ratio, [IQR]) of 0.49 (0.18, 0.53). In multivariable linear regression, the SomaScan readout for 6 of the 23 examined proteins (26%) was most strongly associated with the OM-derived abundance of the same (target) protein. For 3 of 23 (13%), the SomaScan and OM-derived abundance of each protein were significantly associated, but the SomaScan readout was more strongly associated with OM-derived abundance of one or more "off-target" proteins. For the remaining 14 proteins (61%), the SomaScan readouts were not significantly associated with the OM-derived abundance of the targeted proteins. In 6 of the latest group, the SomaScan readout was not associated with urine abundance of any of the 23 quantified proteins. To sum, over half of the SomaScan results could not be confirmed by independent orthogonal methods.

Quantitative Analysis of Complement Membrane Attack Complex Proteins Associated with Extracellular Vesicles.

Extracellular vesicles (EVs) represent a universal mechanism of intercellular communication in normal and pathological conditions. There are reports showing the presence of complement proteins in EV preparations, specifically those that can form a membrane attack complex (MAC). In the present work, we have used a quantitative mass spectrometry method that allows for the measurement of multiple targeted proteins in one experimental run. The quantification of MAC-forming proteins, namely C5b, C6, C7, C8, and C9, in highly purified EVs from normal human plasma revealed the presence of MAC proteins at approximately equal stoichiometry that does not fit the expected stoichiometry of preformed MAC. We concluded that while MAC proteins can be associated with EVs from normal plasma and presumably can be delivered to the recipient cells, there is no evidence that the EVs carry preformed MAC.

Freezing Diluted Bovine Serum Albumin Standards Does Not Significantly Affect Standard Curves.

Total protein isolation followed by quantitation is a common protocol in many laboratories. Quantitation is often done using a colorimetric assay such as the bicinchoninic acid (BCA) assay in which a change in the color of the BCA reagent is related to protein concentration. Extracted protein samples are compared to a standard curve made with dilutions of a protein standard such as bovine serum albumin (BSA) to determine their concentrations. A series of experiments was designed to determine the most reproducible and accurate method for quantifying protein concentrations of samples in an experimental series over time. The effect of freezing on diluted standards was investigated. Standards were frozen at -20°C or -80°C and serially thawed and refrozen up to three times prior to their use in a BCA assay. Thawing and refreezing the standards had no significant effect on protein concentration and the resulting standard curves. Inter-person and intra-person variability in the preparation of standards was also investigated. Protein concentration differences due to inter-person and intra-person variability were greater than protein concentration variability resulting from freezing and thawing, regardless of the freezing temperature. The most reproducible and accurate method for determining the protein concentration of extracted samples in an experimental series over time is diluting a large batch of BSA standards and freezing them at either -20°C or -80°C. Reproducibility was maintained with up to three freeze-thaws.

HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Bioinformatic Workflows for Metaproteomics.

The strong influence of microbiomes on areas such as ecology and human health has become widely recognized in the past years. Accordingly, various techniques for the investigation of the composition and function of microbial community samples have been developed. Metaproteomics, the comprehensive analysis of the proteins from microbial communities, allows for the investigation of not only the taxonomy but also the functional and quantitative composition of microbiome samples. Due to the complexity of the investigated communities, methods developed for single organism proteomics cannot be readily applied to metaproteomic samples. For this purpose, methods specifically tailored to metaproteomics are required. In this work, a detailed overview of current bioinformatic solutions and protocols in metaproteomics is given. After an introduction to the proteomic database search, the metaproteomic post-processing steps are explained in detail. Ten specific bioinformatic software solutions are focused on, covering various steps including database-driven identification and quantification as well as taxonomic and functional assignment.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Absolute protein quantification based on calibrated particle counting using electrospray-differential mobility analysis.

The traceability of in vitro diagnostics or drug products is based on the accurate quantification of proteins. In this study, we developed an absolute quantification approach for proteins. This method is based on calibrated particle counting using electrospray-differential mobility analysis (ES-DMA) coupled with a condensation particle counter (CPC). The absolute concentration of proteins was quantified with the observed protein particle number measured with ES-DMA-CPC, and the detection efficiency was determined by calibrators. The measurement performance and quantitative level were verified using two certificated reference materials, BSA and NIMCmAb. The linear regression fit for the detection efficiency values of three reference materials and one highly purified protein (myoglobin, BSA, NIMCmAb and fibrinogen) indicated that the detection efficiency and the particle size distribution of these proteins exhibited a linear relationship. Moreover, to explore the suitability of the detection efficiency-particle size curve for protein quantification, the concentrations of three typical proteinaceous particles, including two high molecular weight proteins (NIST reference material 8671 and D-dimer) and one protein complex (glutathione S-transferase dimer), were determined. This work suggests that this calibrated particle counting method is an efficient approach for nondestructive, rapid and accurate quantification of proteins, especially for measuring proteinaceous particles with tremendous size and without reference standards.

Antibody Design for the Quantification of Photosynthetic Proteins and Their Isoforms.

Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Cytotoxicity and toxicoproteomics analysis of thiazolidinedione exposure in human-derived cardiomyocytes.

Thiazolidinediones (TZDs) (e.g. pioglitazone and rosiglitazone), known insulin sensitiser agents for type II diabetes mellitus, exhibit controversial effects on cardiac tissue. Despite consensus on their association with increased heart failure risk, limiting TZD use in diabetes management, the underlying mechanisms remain uncharacterised. Herein, we report a comprehensive in vitro investigation utilising a novel toxicoproteomics pipeline coupled with cytotoxicity assays in human adult cardiomyocytes to elucidate mechanistic insights into TZD cardiotoxicity. The cytotoxicity assay findings showed a significant loss of mitochondrial adenosine triphosphate production upon exposure to either TZD agents, which may underpin TZD cardiotoxicity. Our toxicoproteomics analysis revealed that mitochondrial dysfunction primarily stems from oxidative phosphorylation impairment, with distinct signalling mechanisms observed for both agents. The type of cell death differed strikingly between the two agents, with rosiglitazone exhibiting features of caspase-dependent apoptosis and pioglitazone implicating mitochondrial-mediated necroptosis, as evidenced by the protein upregulation in the phosphoglycerate mutase family 5-dynamin-related protein 1 axis. Furthermore, our analysis revealed additional mechanistic aspects of cardiotoxicity, showcasing drug specificity. The downregulation of various proteins involved in protein machinery and protein processing in the endoplasmic reticulum was observed in rosiglitazone-treated cells, implicating proteostasis in the rosiglitazone cardiotoxicity. Regarding pioglitazone, the findings suggested the potential activation of the interplay between the complement and coagulation systems and the disruption of the cytoskeletal architecture, which was primarily mediated through the integrin-signalling pathways responsible for pioglitazone-induced myocardial contractile failure. Collectively, this study unlocks substantial mechanistic insight into TZD cardiotoxicity, providing the rationale for future optimisation of antidiabetic therapies.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

Urinary protein and coagulation-fibrinolysis indicators in preeclampsia: Expression and significance.

This study investigates the expression and significance of urinary protein and coagulation-fibrinolysis indicators in preeclampsia, categorized into mild preeclampsia (109 cases) and severe preeclampsia (97 cases) based on disease severity. Additionally, 110 patients with gestational hypertension (gestational hypertension group) were included for comparative analysis. General information, laboratory indicators, urinary protein, and coagulation-fibrinolysis indicator levels were collected for each group. Significant differences were observed in blood pressure among groups (P < .05), while uric acid, serum creatinine, aspartate transaminase, alanine transaminase, and triglycerides showed no significant differences (P > .05). Total cholesterol, triglycerides, and low-density Lipoprotein levels in severe preeclampsia were higher than those in mild preeclampsia and gestational hypertension groups, whereas high-density lipoprotein, albumin, and platelet levels were lower in severe preeclampsia. No significant differences were observed in prothrombin time or D-dimer levels among groups (P > .05). Urinary protein, urinary protein quantification, activated partial thromboplastin time, thrombin time, and fibrinogen were identified as influencing factors for adverse maternal and infant outcomes in severe preeclampsia patients. The study concludes that urinary protein and coagulation-fibrinolysis indicators are elevated in preeclampsia, particularly in severe preeclampsia cases, suggesting their potential use as diagnostic influencing factors for severe preeclampsia.

Quantifying Bound Proteins on Pegylated Gold Nanoparticles Using Infrared Spectroscopy.

Protein-nanoparticle (NP) complexes are nanomaterials that have numerous potential uses ranging from biosensing to biomedical applications such as drug delivery and nanomedicine. Despite their extensive use quantifying the number of bound proteins per NP remains a challenging characterization step that is crucial for further developments of the conjugate, particularly for metal NPs that often interfere with standard protein quantification techniques. In this work, we present a method for quantifying the number of proteins bound to pegylated thiol-capped gold nanoparticles (AuNPs) using an infrared (IR) spectrometer, a readily available instrument. This method takes advantage of the strong IR bands present in proteins and the capping ligands to quantify protein-NP ratios and circumvents the need to degrade the NPs prior to analysis. We show that this method is generalizable where calibration curves made using inexpensive and commercially available proteins such as bovine serum albumin (BSA) can be used to quantify protein-NP ratios for proteins of different sizes and structures.

Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS.

Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties with the target analytes as calibrators. Such a unique capacity allowed for accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of "heavy matching light, HML" to directly quantify an endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.

Gehua Jiejiu Dizhi decoction ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study.

OBJECTIVE: To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction (, GJDD) on alcoholic fatty live disease (AFLD) by using proteomic methods. METHODS: The male C57BL/6J mouse were randomly divided into four groups: control group, model group, GJDD group and resveratrol group. After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method, the GJDD group and resveratrol group were intragastrically administered with GJDD (4900 mg/kg) and resveratrol (400 mg/kg) respectively, once a day for 9 d. The fat deposition of liver tissue was observed and evaluated by oil red O (ORO) staining. 4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group. The differentially expressed proteins were screened according to protein expression differential multiples, and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Finally, expression validation of the differentially co-expressed proteins from control group, model group and GJDD group were verified by targeted proteomics quantification techniques. RESULTS: In semiquantitative analyses of ORO, all kinds of steatosis (ToS, MaS, and MiS) were evaluated higher in AFLD mice compared to those in GJDD or resveratrol-treated mice. 4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified, of which 3763 proteins were quantified and 946 differentially expressed proteins were screened. Compared with the control group, 145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group. In addition, compared with the model group, 92 proteins were up-regulated and 135 proteins were down-regulated in the liver tissue of the GJDD group. 15 differentially co-expressed proteins were found between every two groups (model group vs control group, GJDD group vs model group and GJDD group vs control group), which were involved in many biological processes. Among them, 11 differentially co-expressed key proteins (Aox3, H1-5, Fabp5, Ces3a, Nudt7, Serpinb1a, Fkbp11, Rpl22l1, Keg1, Acss2 and Slco1a1) were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis. CONCLUSIONS: Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression, likely through the modulation of lipid metabolism, bile acid metabolism and with exertion of antioxidant stress.

From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins.

Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis. IMPORTANCE: Stress responses play a crucial role in bacterial survival and adaptation. The ability to quantitatively measure protein abundances and redox states in different cellular compartments and the extracellular environment is essential for understanding stress management mechanisms. In this study, we addressed the knowledge gap regarding absolute quantification of extracellular proteins and compared protein concentrations in various sub-cellular locations and in the extracellular medium under disulfide stress conditions. Our findings provide valuable insights into the protein production and secretion dynamics of B. subtilis, shedding light on its stress response strategies. Furthermore, the developed protocol for absolute quantification of extracellular proteins in bacteria presents a practical and efficient approach for future studies in the field. Overall, this research contributes to the quantitative understanding of stress management mechanisms and protein dynamics in B. subtilis, which can be used to enhance bacterial stress tolerance and protein-based biotechnological applications.

Split-GFP complementation at the bacterial cell surface for antibody-free labeling and quantification of heterologous protein display.

The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1-10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA. Furthermore, the amount of the protein of interest on the cells could be quantified by comparing the green fluorescence resulting from the complementation to that of standards with known concentrations. This allows a precise characterization of whole-cell biocatalysts, which is difficult with existing methods. The split-GFP complementation approach was shown to be specific, in a similar manner as commercial antibodies. It is cost-efficient, minimizes the possibility of adverse effects on protein expression or solubility, and can be performed at high throughput.

Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

Multiphoton Imaging of Maturation in Tissue Engineering.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.