Forskarna är :
Louise Sternbæk1,2, Martha Wamaitha Kimani3, Kornelia Gawlitza3, Birgit Janicke1, Kersti Alm1, and Anette Gjörloff Wingren2
1Phase Holographic Imaging AB, Lund, Sweden
2Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden
3Chemical and Optical Sensing Division, Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin, Germany
Digital Holographic Cytometry
Mar. 22, 2019
Macrophage Uptake of Nanoprobes
Fig. 1: Schematic illustration of the HoloMonitor M4 and image reconstruction (PHIAB, Lund, Sweden).
Intressant är att artikeln är sampublicerad med BAM vilket väcker bloggens nyfikenhet huruvida innehållet berör forskningen inom projektet GlycoImaging och att projektet nu är på väg att leverera konkreta resultat. Eller har forskarna hittat nya upptäckter som ligger utanför projektets ursprungliga ram?
Men till innehållet.
Digital holographic cytometry (DHC) is a state-of-the-art quantitative phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped with semi-automated image segmentation and analysis software packages for assessing cell behavior are commercially available. In this study we investigate the possible uptake of nanoprobes in macrophages in vitro over time.
Quantitative phase imaging (QPI) is a promising label-free
technique for live-cell imaging.
The general technique for QPI is
the use of interference to convert the phase information into pixel
intensity, and thereby directly record the quantitative phase delay.
Digital holographic cytometry (DHC) builds on red coherent laser light
from a diode laser, which is split into two beams, a sample beam and a
reference beam. The sample beam passes through living cells,
and merge with the reference beam, thus creating an interference pattern
as it hits a digital sensor (e.g. CDD-sensor). The sensor captures
the hologram and converts the signal into a holographic image based on
the light phase shifting properties of the cells.
Several DHCs can be
housed in a mammalian cell incubator and image cells in a large
assortment of common culture plates or media, which enables non-invasive
and non-interruptive cell analysis. Cellular behavior as well as
morphology can be quantified and evaluated using analysis software
packages.
Nanoprobes can be quite diverse in design and have substantial clinical
potential, such as drug-delivery or detection by artificial antibodies.
One of the main issues plaguing nanoprobes circulation in humans, is
that it can lead to induction of host inflammatory responses due to
non-specific recognition and uptake by macrophages in vivo.
Macrophages are leukocytic cells capable of phagocytosing cellular
debris, bacteria, and particles through energy-consuming
membrane-engulfing. Their essential role is in early response to foreign
material/substances and its clearance due to release of cytokines or
endotoxins, which activate the macrophages. Phagocytosis occur within
minutes of recognition, increasing their rates of phagocytosis.In this study we used molecularly imprinted polymers (MIPs) targeted to sialic acid (SA).SA is a monosaccharide that
plays an important role in the regulation of the innate immunity [12].
The amount of SA has been found to correlate with cancer, with an
upregulation on more aggressive cancers [13-15]. A SA-imprinted polymer
shell was grafted from a silica core particle using deprotonated SA as
template for MIP growth [16]. Here we demonstrate how DHC can be used to
analyze the possible uptake of SA-MIPs in macrophage cells in in vitro cultures.
Material and Methods
Digital Holographic Cytometry
RAW 264.7 cells were seeded in a 6-well polystyrene plate (Sarstedt,
Germany) containing 60,000 cells per well and incubated overnight for
cells to adhere at 37 ˚C and 5% CO2 (day 0). The RAW 264.7
cells were treated with 0.04 mg/ml SA-MIPs in media (day 1).
The lid was
changed to Hololids (PHIAB, Lund, Sweden), which enable cell imaging by
HoloMonitor M4 DHC (PHIAB, Lund, Sweden). Time-lapse phase imaging as
well as image processing, segmentation and analysis were conducted with
Hstudio software package (PHIAB) with preset mean values of refractive
indexes for cells and culture medium. To monitor SA-MIP uptake, cells
were imaged every 15 min after addition for a total of 48 h, at eight
positions in each well. Images were segmented followed by object
identification for every time point, for further investigation of the
uptake of SA-MIPs by RAW 264.7 cells. Segmentation and object
identification are the basis for morphological values and cell count.
As the laser intensity is approximately 2W/m2 during
imaging, and exposure time is maximum 10 ms it is assumed that the laser
irradiation has only minimal effects on the physiological functions of
the cells.
Result and Discussion
Macrophage Uptake of SA-MIPs
RAW 264.7 cells were analyzed with and without SA-MIP treatment in vitro
by holographic monitoring of the macrophages over time. Tracking
several cell populations over time, which are analyzed at a given time
point and average representations of cell morphological parameters area,
thickness, and volume are presented (fig. 2A-C). These parameters help
monitoring the cellular processes and their response to treatments. In
response to SA-MIP treatment, macrophage cell size increased (fig.
2A-C). The role of macrophages is to phagocytose foreign substances,
therefore we assume that the increase in cell size is a response to
SA-MIP treatment [9-11]. The increase in cell area and cell volume is
substantial during the first 24h, which could be caused by an increased
phagocytosis after recognition of SA-MIPs (fig. 2A and 2C), as seen in
the human body with foreign substances [9-11].
Interestingly, the phagocytosis of the nanoprobes does not affect the
macrophages’ ability to keep proliferating (fig. 2). The number of cells
increased equally over time for untreated and treated cells, meaning
that neither the SA-MIP treatment nor DHC seemed to affect the overall
physiological functions of the macrophages (fig. 2D) [17-20].
In the holographic images, the SA-MIPs are represented by red dots and
clusters that are present only in images showing treated cells (fig. 3B)
and not in the untreated cells (fig. 3A). The quantity of SA-MIPs
decreased over time, which is due to an uptake and ingestion by the
macrophages (see video online). Moreover, as the nanoprobes will not
dissolve due to their chemical construction, it would be assumed that
they have been ingested by macrophages. Several clusters of SA-MIPs are
seen in the framed area of Fig. 3B after 24h incubation. After 48h they
have disappeared from the images (fig 3B). The time-lapse movie clearly
shows that the macrophages ingested the nanoprobes as would be expected.
Conclusion
There are several advantages with DHC and other QPI platforms. Since
they can be housed in mammalian cell incubators, they can monitor the
behavior of unlabeled cells in real time and calculate the number of
cells easily. In this work, we demonstrated that neither the DHC or the
SA-MIPs did affect the physiological functions of macrophages as they
phagocytosed the nanoprobes over time and still proliferated in a
similar manner as the control macrophages.
The utility of this method is
that it is quick, quantitative, and qualitative, which is essential for
future cell-based research.
Acknowledgement
The authors would like to thank the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 721297 for financial support and Malmö University.
Min kommentar
Som framgår av texten berör studien makrofager (som bloggens läsare nu bör vara bekanta med).
Makrofager behandlade med MIP,s (GlycoImagings kärnområde).
Resultatet som bloggen tolkar det är att kroppens eget immunförsvar via makrofager tar upp MIP,sarna utan tillstymmelse till bortstötning. Det bör räknas som en viktig framgång för projektet.
Vilket då mest troligt besvarar bloggen frågeställning inledningsvis.
GlycoImaging-projektet har börjat leverera resultat. Nu börjar det bli spännande.
Än en gång, tack Axel, för inskickat material.
Mvh the99
Inga kommentarer:
Skicka en kommentar