A Potential Application of Triangular Microwells to Entrap Single Cancer Cells: A Canine Cutaneous Mast Cell Tumor Model

Ketpun, Dettachai; Pimpin, Alongkorn; Tongmanee, Tewan; Bhanpattanakul, Sudchaya; Piyaviriyakul, Prapruddee; Srituravanich, Weerayut; Sripumkhai, Witsaroot; Jeamsaksiri, Wutthinan; Sailasuta, Achariya

doi:10.3390/mi10120841

Cited by 5 publications

(4 citation statements)

References 27 publications

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“…Polydimethylsiloxane (PDMS) was used for fabricating the microfluidic device, Exo-Load, by the soft UV lithography technique as described previously. 33 In brief, the photomask was first created, and the prototyping process was based on the standard process of silicon soft lithography with slight modifications, 34,35 such as adding several tweaks to the process. The photomask created over the printed circuit board (PCB) by Kinston was exposed to UV irradiation for 3 minutes.…”

Section: Fabrication Of the Exo-load Microfluidic Devicementioning

confidence: 99%

<p>Inhibition of Glioma Cells’ Proliferation by Doxorubicin-Loaded Exosomes via Microfluidics</p>

Thakur

Sidu

Zou

et al. 2020

IJN

114

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Background Malignant glioma is a fatal brain cancer. Accumulated evidence has demonstrated that exosomes can cross the blood–brain barrier (BBB), suggesting their potential use as drug delivery vehicles to glioma. Therefore, various loading methods of anticancer agents into exosomes have been developed. However, the loading efficiency of anticancer drugs, such as doxorubicin (DOX) and paclitaxel (PTX), into exosomes is relatively low, thus challenging to improve the drug delivery efficiency to glioma cells (GMs) via exosomes. Methods To improve the loading efficiency of doxorubicin into exosomes, a microfluidic device (Exo-Load) was developed. Next, to increase the exosomal delivery of doxorubicin to GMs, autologous exosomes were used for its loading via Exo-Load. Briefly, exosomes from SF7761 stem cells-like- and U251-GMs were isolated and characterized by nano-tracking analysis (NTA), transmission electron microscopy (TEM), and immunogold EM. Finally, doxorubicin was successfully loaded into exosomes with saponin by Exo-Load, and the uptake and functionality of doxorubicin-loaded exosomes for parent GMs were evaluated. Results The loading efficiency of DOX into SF7761 stem cells-like- and U251-GMs-derived-exosomes were 19.7% and 7.86% via Exo-Load at the injection flow rate of 50 µL/min, respectively. Interestingly, the loading efficiency of DOX into U251 GMs-derived exosomes was significantly improved to 31.98% by a sigmoid type of Exo-Load at the injection flow rate of 12.5 µL/min. Importantly, DOX-loaded GMs-derived exosomes via Exo-Load inhibited parent GMs’ proliferation more than heterologous GMs, supporting exosomes’ homing effect. Conclusion This study revealed that DOX and PTX could be loaded in exosomes via Exo-Load, demonstrating that Exo-Load could be a potential drug-loading device into exosomes with further optimization. This study also demonstrated that the delivery of DOX to SF7761 GMs via their daughter exosomes was much more efficient rather than U251 GMs-derived exosomes, supporting that the use of autologous exosomes could be better for glioma drug targeting.

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Section: Fabrication Of the Exo-load Microfluidic Devicementioning

confidence: 99%

<p>Inhibition of Glioma Cells’ Proliferation by Doxorubicin-Loaded Exosomes via Microfluidics</p>

Thakur

Sidu

Zou

et al. 2020

IJN

114

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“…In veterinary science, there are a few reports on Oct-4 expression in putative CSCs in many cancers such as mast cell tumor, canine hepatocellular carcinomas, seminomas and thyroid carcinoma by immunohistochemistry (Webster et al, 2007b;Vargas et al, 2015) . In addition, the previous study and our preliminary study, demonstrated the Oct-4 expression at protein and mRNA levels by Immunohistochemistry and PCR technique, respectively (Eksiritrirat et al, 2012;Ketpun et al, 2013;Sailasuta et al, 2013). Hence, we believe that Oct-4 expressing cells might involve in the pathogenesis of MCT and MCT might have the stemness property.…”

Section: List Of Tablesmentioning

confidence: 54%

“…In many studies revealed that genetic alterations introduced the risk of tumors. Especially, c-Kit mutations at exon11 (the juxtamembrane domain; cytoplasmic kinase domain) can be activated proliferation in the absence of growth factors leading to unlimited hyperproliferation of the cells (Ketpun et al, 2013;Sailasuta et al, 2014).…”

Section: Pathogenesismentioning

confidence: 99%

Detection on the expression of the transcription factors: oct-4, nanog, and sox-2 genes in canine cutaneous mast cell tumors

Meesuwan

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The objectives of this study were to determine the expression of Oct-4, Nanog, and Sox-2 in both mRNA and proteins levels. The 30 biopsied MCT cases, from the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University, were used in this study. The samples were divided into two parts; part I, the biopsied samples were processed for paraffin section and stained with H&E stain, for grading based on Patnaik's system and Kiupel's system and the other part was detected the expression of Oct-4, Nanog, and Sox-2 genes. The MCT samples were classified into three grades; grade I, II, and III by Patnaik system; grade I, 40% (12/30), grade II, 26% (8/30) and grade III, 33.33% (10/30). And, Kiupel's system which were low grade, 66.66% (20/30) and high grade, 33.33% (10/30) respectively. The RT-PCR and qRT-PCR were used for detecting Oct-4, Nanog, and Sox-2 mRNA in MCT cases. The results showed that all of MCT samples expressed both Oct-4 and Sox-2 at mRNA level (100 %; 30/30). While, Nanog could not be detected in all MCT samples (0%). For qRT-PCR, the Oct-4 gene in high grade tended to be higher than low grade parallelly in grade III also tended to be higher than grade I and II respectively . On the contrary, Sox-2 expression, in grade III and high grade tended to be lower than grade I and low grade respectively, when compared to internal control. However, the results had no statistically significant for the experiments among the grading system. The immunohistochemical expression of Oct-4, Nanog, and Sox-2 in MCT samples were conducted. There were positive expression of Oct-4, Nanog, and Sox-2 in all the samples at 90% (27/30), 80 % (24/30) and 80% (24/30 ) respectively. Regards to the positive cells and the grade of MCT, it was demonstrated that there were no statistically difference of Oct-4 and Nanog positive cells in all grades (p?0.05) . On the contrary, the positive cells of Sox-2 in high grade were higher than low grade MCT samples with statistically difference (p?0.05). The results of Western blot technique showed the expression of Oct-4, Nanog, and Sox-2 protein with low reaction intensity. Which were; Oct-4, 1 band at 43 kDa, Nanog, 3 bands, at 45,35 and 25 kDa, approximately, Sox-2, 4 bands, at 77,65,35 and 26 kDa approximately in all MCT samples respectively when compared to positive control.. In addition, the demonstration of Intranuclear Immunocytofluorescence was conducted on the expression of Oct-4, Nanog, and Sox-2 proteins which the positively localizations were shown in intranuclear and intracytoplasmic of MCT cells. Additionally, the expression of the Oct-4, Nanog, and Sox-2 proteins could be potentially detected by Surface Plasmon Resonance, SPR technique in MCT. However, there were some limitations of the technique, due to their high sensitivity and nonspecific appearances. By the obtained results, it is revealed that the major embryonic transcription factors, Oct-4, Nanog, and Sox-2 mRNA and proteins in the MCT tissue and could be detected. Which could be possibly beneficial on the development of the prognostic marker of this tumor in the future.

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“…In this study, the microfabrication of the devices had been accomplished under the standard photoresist-soft-lithography previously described in size-based polystyrene microsphere separations (Ketpun et al, 2015). And the replicas were constructed using polydimethylsiloxane (PDMS).…”

Section: The Architectures Of the Microfluidic Devices And Fabricationsmentioning

confidence: 99%

Microfluidics-Based Single Cell Isolation and Trapping of Canine Cutaneous Mast Cell Tumor Cells

Ketpun

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In this research, the primary objective was to develop a microfluidic platform utilized for the size-based single cell sorting of canine cutaneous mast cell tumor (MCT) cells which were categorized into 4 groups; I, II, III and IV, upon their individual sizes ranged from 5-9, 10-14, 15-19, and equal to or over than 20 mm respectively. And the secondary aim was to establish the microfluidic-based entrapping microdevice which had a capacity to ensnare single MCT cells into its triangular microwell array at the ratio of one cell per one microwell. The study consequence consistently indicated the accomplishment of device developments. All microfluidic devices were designed and fabricated under the standard soft-lithography. Finally, two microfluidic devices were constructed; the Archimedian spiral microchannel (ASM) and the triangular microwell array. The configuration of ASM consisted of 5-turned, 500mm-wide x 130mm-high, rectangular spiral microchannel with 720mm-long conical exit microchannel separated into 10 asymmetric exit slots. Meanwhile, the entrapment microdevice were composed of the array of 40mm-high x 15mm-deep triangular microwell with 160mm-high main flow channel. The study result indicated that, at the flow rate 1 ml. min-1, the spiral microchannel had a capability to separate single MCT cells in group III and IV, which were the targeted cells, out of the MCT cells group I and II, based upon their sizes. However, the detrimental effects of fluid shear stress and extensional stress in the spiral microchannel, exhibited by the computational simulations, were required an amendment. Because, under hemocytometry quantification, trypan blue and TMRM staining, light-microscopy and SEM morphological assessments and leaky DNA assays, they suggested that the amount of sorted MCT cells was significantly reduced and the cell vitality was only 40 percent. Meanwhile, the morphological assays also confirmed cell elongation and/or wreckage causing the 2.4-fold increment of leaky DNA. For single MCT cell entrapment, the study consequence exhibited that, under pulsatile flow (0. 01 ml. h-1 inflow rate for 30 sec and 3min pausing interval), the trapping microdevice had an ability to stochastically entrap MCT cells in its triangular microwell array. And the efficacy of the microdevice for single and multiple MCT cell entrapment was 35% and 15% respectively. In the epilogue, this study is the first report on the attainments in microfrabication engaged in veterinary field for size-based single MCT cell sorting and entrapment. In addition, there are a tendency to merge these two microdevice components together to form a complete microfluidic-based single cell sorting, trapping and culturing on a chip which is essentially benefit to biomedical engineering researches in the future.

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A Potential Application of Triangular Microwells to Entrap Single Cancer Cells: A Canine Cutaneous Mast Cell Tumor Model

Cited by 5 publications

References 27 publications

<p>Inhibition of Glioma Cells’ Proliferation by Doxorubicin-Loaded Exosomes via Microfluidics</p>

<p>Inhibition of Glioma Cells’ Proliferation by Doxorubicin-Loaded Exosomes via Microfluidics</p>

Detection on the expression of the transcription factors: oct-4, nanog, and sox-2 genes in canine cutaneous mast cell tumors

Microfluidics-Based Single Cell Isolation and Trapping of Canine Cutaneous Mast Cell Tumor Cells

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