On-chip direct freezing and thawing of mammalian cells

We present a novel structure to supply gases to microchambers in microfluidic chips. An exemplary application is the continuous feeding of oxygen and CO2 for on-chip cell cultivation of mammalian cells. In our device, the surrounding air diffuses into the culture medium inside the chip through a porous wall of agarose hydrogel resulting in an easy and robust design. One common method is the usage of gas permeable PDMS chips. However, liquid medium in which the cells grow is absorbed by PDMS causing unknown concentrations and memory effects. Another possibility is a complex setup where medium with already dissolved gas is pumped constantly through the chip. We designed and realized a silicon and borosilicate glass chip containing a gas permeable wall of agarose preventing leakage of medium. In order to precisely position the walls in the chip, we made use of surficial phaseguides (50nm high). The blue-bottle-experiment makes the effective dissipation of oxygen visible when the colorless leucomethylen-blue reacts to methylene-blue. Successful results were achieved when applying 0.5 g/l methylene blue, 10 g/l glucose and a pH of 12.6 set by a buffer solution. As a result a continuous color gradient through the chip was obtained, which reflects the oxygen gradient and confirms the oxygen diffusion.

Download Full-text

Handheld Microflow Cytometer Based on a Motorized Smart Pipette, a Microfluidic Cell Concentrator, and a Miniaturized Fluorescence Microscope

Sensors ◽

10.3390/s19122761 ◽

2019 ◽

Vol 19 (12) ◽

pp. 2761 ◽

Cited By ~ 4

Author(s):

Byeongyeon Kim ◽

Dayoung Kang ◽

Sungyoung Choi

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

Flow Cytometric Analysis ◽

Sample Volume ◽

Fluorescence Microscope ◽

Comprehensive Approach ◽

Optical Systems ◽

Simple Protocol ◽

Microflow Cytometer ◽

On Chip

Miniaturizing flow cytometry requires a comprehensive approach to redesigning the conventional fluidic and optical systems to have a small footprint and simple usage and to enable rapid cell analysis. Microfluidic methods have addressed some challenges in limiting the realization of microflow cytometry, but most microfluidics-based flow cytometry techniques still rely on bulky equipment (e.g., high-precision syringe pumps and bench-top microscopes). Here, we describe a comprehensive approach that achieves high-throughput white blood cell (WBC) counting in a portable and handheld manner, thereby allowing the complete miniaturization of flow cytometry. Our approach integrates three major components: a motorized smart pipette for accurate volume metering and controllable liquid pumping, a microfluidic cell concentrator for target cell enrichment, and a miniaturized fluorescence microscope for portable flow cytometric analysis. We first validated the capability of each component by precisely metering various fluid samples and controlling flow rates in a range from 219.5 to 840.5 μL/min, achieving high sample-volume reduction via on-chip WBC enrichment, and successfully counting single WBCs flowing through a region of interrogation. We synergistically combined the three major components to create a handheld, integrated microflow cytometer and operated it with a simple protocol of drawing up a blood sample via pipetting and injecting the sample into the microfluidic concentrator by powering the motorized smart pipette. We then demonstrated the utility of the microflow cytometer as a quality control means for leukoreduced blood products, quantitatively analyzing residual WBCs (rWBCs) in blood samples present at concentrations as low as 0.1 rWBCs/μL. These portable, controllable, high-throughput, and quantitative microflow cytometric technologies provide promising ways of miniaturizing flow cytometry.

Download Full-text

Preservation of structure and function of an organized tissue after freezing and thawing

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1967.0067 ◽

1967 ◽

Vol 168 (1012) ◽

pp. 293-310 ◽

Cited By ~ 13

Keyword(s):

Smooth Muscle ◽

Liquid Phase ◽

Smooth Muscle Cells ◽

Mammalian Cells ◽

Structural Damage ◽

Recovery Of Function ◽

Muscle Cells ◽

Dimethyl Sulphoxide ◽

Partial Recovery ◽

Freezing And Thawing

The function of uterine smooth muscle after freezing and thawing to —79 °C was assessed by contractility in response to drugs, and the structure of the muscle cells was studied by electron microscopy of osmium -fixed Epon-embedded material. These experiments form part of an investigation into the possibility of using a non-electrolyte, dimethyl sulphoxide, to protect organized tissues and organs from the harmful effects of freezing and thawing. During freezing to — 79 °C in the absence of dimethyl sulphoxide, electrolyte concentrations in the residual liquid phase rose to about 30 times normal levels. Under these conditions there was a complete loss of function and gross disruption of the cytoplasm, particularly of the membranous elements. A second method involved feezing to — 79 °C in a solution containing dimethyl sulphoxide (1·4 M initially). The dimethyl sulphoxide moderated the rise in the electrolyte concentrations to about 5 times their normal levels. There was a partial recovery of function after thawing but structural damage occurred, similar to that seen in smooth muscle cells frozen without dimethyl sulphoxide. In a third procedure an attempt was made to maintain electrolyte concentrations close to their initial levels throughout freezing. Pre-cooled solutions of dimethyl sulphoxide in de-ionized water were added to keep the volume of the liquid phase constant as ice separated. This resulted in a great improvement in the structural and functional preservation of the smooth muscle cells. The overall functional condition of the preparation was comparable to that of fresh material and the cell membranes were essentially undamaged. Control experiments showed that the cells, particularly the nuclei, were damaged by high concentrations of dimethyl sulphoxide itself; these toxic effects were temperature dependent. These experiments provide further evidence that high electrolyte concentrations present during slow freezing are a major cause of damage to mammalian cells, and that structural and functional damage to an organized tissue can be greatly reduced if electrolyte concentrations are kept close to normal levels. Further study of the temperature dependence of the toxicity of protective non-electrolytes is necessary before this and similar techniques can be used routinely for the preservation of organized tissues and whole organs.

Download Full-text

An assessment of methods used for measuring the recovery of mammalian cells from freezing and thawing

Cryobiology ◽

10.1016/0011-2240(74)90041-8 ◽

1974 ◽

Vol 11 (1) ◽

pp. 80-84 ◽

Cited By ~ 17

Author(s):

L.W. Harris ◽

J.B. Griffiths

Keyword(s):

Mammalian Cells ◽

Freezing And Thawing

Download Full-text

Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast

BMC Microbiology ◽

10.1186/s12866-021-02185-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jennifer Staudacher ◽

Corinna Rebnegger ◽

Brigitte Gasser

Keyword(s):

Mammalian Cells ◽

Growth Conditions ◽

Non Invasive ◽

Translational Activity ◽

Wide Range ◽

Simple Protocol ◽

Set Up ◽

Different Strains ◽

Negative Controls ◽

Global Translation

Abstract Background Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. Results We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. Conclusions We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.

Download Full-text