microfluidic cultivation devices  (MicroFluidic Systems)

Journal: bioRxiv

Article Title: Microfluidic platform for microbial spore germination studies in multiple growth conditions

doi: 10.1101/2024.05.13.593863

Figure Lengend Snippet: (A) Photograph of the microfluidic device inside a 35 mm glass-bottomed Petri dish. (B) Design of the microfluidic device including the arrays of cultivation chambers. The cultivation chambers (in grey) have a height of approximately 750 nm to ensure monolayer growth while the supply channels (in white) are 10 µm high. Scale bars, 1 mm (left) and 100 µm (right). (C) Examples of individual chambers containing, from left to right: Bacillus subtilis, Ammoniphilus oxalaticus, and Trichoderma rossicum spores. Scale bars, 10 µm. (D) Flow profile within the microfluidic device used with a concentration gradient of fluorescein solutions. Scale bar, 500 µm. (E) Phase contrast images of B. subtilis cell in the microfluidic device at different time points showing the phase bright-spore (00:05) turning phase-dark (from 00:35), then outgrowth (from 01:50) and cell detachment from the spore’s coat (02:50). Scale bar, 2 μm. Time stamp format, hh:mm.

Article Snippet: The microfluidic single-cell cultivation (MSCC) device employs arrays of microchambers, allowing analysis of the germination of the filamentous bacterium Streptomyces lividans under multiple media compositions, which revealed surprising stability in behaviour despite previous observations in large-scale cultures ( Koepff et al . 2018 ).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Microfluidic platform for microbial spore germination studies in multiple growth conditions

doi: 10.1101/2024.05.13.593863

Figure Lengend Snippet: Example timelapses for each condition available in Video 1. (A-D) Relative frequency distributions of the time of germination in each condition. The graphs show the proportion (i.e., relative frequency) of the analysed spores germinated within 5-minute intervals. The total number of analysed spores (n) in each condition is indicated. Phase contrast microscope images of B. subtilis in the microfluidic device over time showing an individual spore. Scale bars, 2 µm. Time stamp format, hh:mm. (E) Boxplot of the germination times in each condition. The line shows the median, the box extends from the 25 th to 75 th percentiles and the whiskers from min to max. Mann Whitney tests showed statistically significant differences ( p < 0.05) between each condition except for SM. (F) Bar chart showing the percentage of spores that germinated or remained dormant (non-germinated) after 5 hours of incubation for the four conditions compared within the microfluidic device. (G) Germination time of individual spores in relation to number of spores per chamber in rich media (NB), showing a negative correlation (Spearman r = -0.8) between the germination times and the number of spores per chamber. Data from 8 chambers were analysed; note, two chambers contained 5 spores per chamber. (H) Germination times in relation to number of spores per chamber in L-alanine, showing no correlation (Spearman r = 0.06) between the germination times and the number of spores per chamber. Data from 8 chambers were analysed; note, two chambers contained 7 spores per chamber.

Article Snippet: The microfluidic single-cell cultivation (MSCC) device employs arrays of microchambers, allowing analysis of the germination of the filamentous bacterium Streptomyces lividans under multiple media compositions, which revealed surprising stability in behaviour despite previous observations in large-scale cultures ( Koepff et al . 2018 ).

Techniques: Microscopy, MANN-WHITNEY, Incubation

Journal: bioRxiv

Article Title: Microfluidic platform for microbial spore germination studies in multiple growth conditions

doi: 10.1101/2024.05.13.593863

Figure Lengend Snippet: Video 2 contains example timelapses for each condition. (A-D) Relative frequency distributions of the time of germination in each condition. The graphs show what proportion (i.e., relative frequency) of the analysed spores germinated within 5-minute intervals. The total number of analysed spores (n) in each condition is indicated. (E) Boxplot of the germination times in each condition. The line shows the median, the box extends from the 25 th to 75 th percentiles and the whiskers from min to max. Mann Whitney tests showed no statistically significant differences ( p > 0.05) between conditions. (F) Bar chart showing the percentage of spores that germinated or remained dormant (non-germinated) after 5 hours of incubation for the four conditions compared within the microfluidic device..

Article Snippet: The microfluidic single-cell cultivation (MSCC) device employs arrays of microchambers, allowing analysis of the germination of the filamentous bacterium Streptomyces lividans under multiple media compositions, which revealed surprising stability in behaviour despite previous observations in large-scale cultures ( Koepff et al . 2018 ).

Techniques: MANN-WHITNEY, Incubation

Journal: bioRxiv

Article Title: Microfluidic platform for microbial spore germination studies in multiple growth conditions

doi: 10.1101/2024.05.13.593863

Figure Lengend Snippet: Timelapses for each condition can be found in Video 3. (A) Phase contrast microscope images of A. oxalaticus showing germination and outgrowth in 0.65 g L -1 potassium oxalate. Scale bars, 2 µm. Time stamp format, hh:mm. (B-E) Relative frequency distributions of the time of germination in each condition. The graphs show what proportion (i.e., relative frequency) of the analysed spores germinated within 5-minute intervals. The total number of analysed spores (n) in each condition is indicated. (F) Boxplot of the germination times in each condition. The line shows the median, the box extends from the 25 th to 75 th percentiles and the whiskers from min to max. Mann Whitney tests showed no statistically significant differences ( p > 0.05) between conditions. Note that there is no data for 8 g L -1 since there was no germination in that condition. (G) Quantification of the spores that remained dormant and germinated after 20 hours of incubation in the microfluidic device in each condition. Results from additional control experiments with Schlegel AB + 0.65 g L -1 potassium oxalate, Schlegel AB only (0 g L -1 potassium oxalate), NB and physiological water, as well as the comparison between the two Schlegel AB + 0.65 g L -1 potassium oxalate conditions (from the main and control experiments) can be found in Supplementary Figure S4, and the corresponding timelapses in Video 4.

Article Snippet: The microfluidic single-cell cultivation (MSCC) device employs arrays of microchambers, allowing analysis of the germination of the filamentous bacterium Streptomyces lividans under multiple media compositions, which revealed surprising stability in behaviour despite previous observations in large-scale cultures ( Koepff et al . 2018 ).

Techniques: Microscopy, MANN-WHITNEY, Incubation, Control, Comparison

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Overview of recently published microfluidic cultivation devices with enhanced cell retention. The listed approaches are itemized concerning the characteristics of the respective cultivation area, the cell retention method, and the nutrient supply. Additionally, the cultivated cell type is indicated.

Article Snippet: Using a 40 × objective, every 20 min phase contrast microscopy images of all relevant positions on the microfluidic cultivation device were recorded (NIS Elements AR 5.20.01 Software, Nikon Instruments, Germany).

Techniques: Sedimentation

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. ( a ) Microfluidic PDMS-glass-based cultivation device. ( b ) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. ( c ) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. ( d ) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Article Snippet: Using a 40 × objective, every 20 min phase contrast microscopy images of all relevant positions on the microfluidic cultivation device were recorded (NIS Elements AR 5.20.01 Software, Nikon Instruments, Germany).

Techniques: Electron Microscopy

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Microfluidic characterization of the MSCC designs. ( a ) Individual cultivation array that contains 60 cultivation chambers with either our previous chamber design (Design 1) or the chamber design from this work (Design 2). ( b ) Image sequence of trace substance experiments to quantify diffusive mass exchange for the MSCC device with Design 2. ( c ) Medium exchange duration until full equilibrium between channel and chamber is achieved based on rel. fluorescein signal for both designs. ( d ) Glucose concentration profile during MSCC cultivation assuming a steady state with 181 cells inside the chamber with a constant glucose uptake rate of 3800 nmol per 10 6 cells and day for both designs.

Article Snippet: Using a 40 × objective, every 20 min phase contrast microscopy images of all relevant positions on the microfluidic cultivation device were recorded (NIS Elements AR 5.20.01 Software, Nikon Instruments, Germany).

Techniques: Sequencing, Concentration Assay

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Comparison of single-cell division behavior between the two microfluidic cell retention concepts. Depicted are the single-cell doubling times t D of cells cultivated in chambers with Design 1 (n = 29, 25, 24) and Design 2 (n = 25, 27, 30). The colored segment marks the interquartile range from 25 to 75%, the horizontal lines show the median. The whiskers represent the 10% and 90% percentile and the tilted squares mark rare cellular events outside the predefined percentiles.

Article Snippet: Using a 40 × objective, every 20 min phase contrast microscopy images of all relevant positions on the microfluidic cultivation device were recorded (NIS Elements AR 5.20.01 Software, Nikon Instruments, Germany).

Techniques:

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Overview of recently published microfluidic cultivation devices with enhanced cell retention. The listed approaches are itemized concerning the characteristics of the respective cultivation area, the cell retention method, and the nutrient supply. Additionally, the cultivated cell type is indicated.

Article Snippet: Figure 1 Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. ( a ) Microfluidic PDMS-glass-based cultivation device. ( b ) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. ( c ) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. ( d ) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Techniques: Sedimentation, Membrane

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. ( a ) Microfluidic PDMS-glass-based cultivation device. ( b ) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. ( c ) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. ( d ) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Article Snippet: Figure 1 Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. ( a ) Microfluidic PDMS-glass-based cultivation device. ( b ) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. ( c ) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. ( d ) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Techniques: Suspension, Electron Microscopy

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Microfluidic characterization of the MSCC designs. ( a ) Individual cultivation array that contains 60 cultivation chambers with either our previous chamber design (Design 1) or the chamber design from this work (Design 2). ( b ) Image sequence of trace substance experiments to quantify diffusive mass exchange for the MSCC device with Design 2. ( c ) Medium exchange duration until full equilibrium between channel and chamber is achieved based on rel. fluorescein signal for both designs. ( d ) Glucose concentration profile during MSCC cultivation assuming a steady state with 181 cells inside the chamber with a constant glucose uptake rate of 3800 nmol per 10 6 cells and day for both designs.

Article Snippet: Figure 1 Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. ( a ) Microfluidic PDMS-glass-based cultivation device. ( b ) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. ( c ) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. ( d ) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Techniques: Sequencing, Concentration Assay

Journal: Scientific Reports

Article Title: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

doi: 10.1038/s41598-023-30297-5

Figure Lengend Snippet: Comparison of single-cell division behavior between the two microfluidic cell retention concepts. Depicted are the single-cell doubling times t D of cells cultivated in chambers with Design 1 (n = 29, 25, 24) and Design 2 (n = 25, 27, 30). The colored segment marks the interquartile range from 25 to 75%, the horizontal lines show the median. The whiskers represent the 10% and 90% percentile and the tilted squares mark rare cellular events outside the predefined percentiles.

Article Snippet: Figure 1 Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. ( a ) Microfluidic PDMS-glass-based cultivation device. ( b ) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. ( c ) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. ( d ) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Techniques: Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Growth and eGFP Production of CHO-K1 Suspension Cells Cultivated From Single Cell to Laboratory Scale

doi: 10.3389/fbioe.2021.716343

Figure Lengend Snippet: Cell morphology comparison of the different cultivation scales. Relevant cellular diameters are plotted against the frequency of their occurrence cumulated from the respective replicates of the analyzed samples. (A) Cell diameter distribution of the bioreactor cultivation right after inoculation ( t = 0 days), after 3 days of cultivation ( t = 3 days), and after 5 days ( t = 5 days). (B) Cell diameter distribution of the shake flask cultivation right after inoculation ( t = 0 days), after 3 days of cultivation ( t = 3 days), and after 5 days ( t = 5 days). (C) Cell diameter distribution of the microfluidic cultivation right after inoculation ( t = 0 days), after 3 days of cultivation ( t = 3 days), and after 5 days ( t = 5 days).

Article Snippet: Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices have been proved to be valuable to miniaturize the cultivation of mammalian cells.

Techniques: Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Growth and eGFP Production of CHO-K1 Suspension Cells Cultivated From Single Cell to Laboratory Scale

doi: 10.3389/fbioe.2021.716343

Figure Lengend Snippet: Analysis of eGFP fluorescence single-cell dynamics during microfluidic cultivation. (A) Time-lapse image sequence showing the growth and fluorescence development of an isogenic microcolony (scale bar = 50 µm) . (B) Lineage tree of the same isogenic microcolony. (C) Fluorescence development of four exemplary single cells over the cultivation time; dotted lines indicate cell division events.

Article Snippet: Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices have been proved to be valuable to miniaturize the cultivation of mammalian cells.

Techniques: Fluorescence, Sequencing