HL-60 Cell Responses to Polymer Surfaces of Different ... Day/2015/Fairuz Hoque.pdf · Changing the...
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HL-60 Cell Responses to Polymer Surfaces of Different Chemistry Fairuz Hoque, Martin Kwok, Laura A. Wells Department of Chemical Engineering and Engineering Chemistry Queen’s University, Canada Introduction Once an implant is placed within a host, the initial host response will influence how well it will function. One of the first cells to the site of an implant are neutrophil cells. During the host response surrounding an implant, neutrophils can have different responses which can influence how well it is accepted. One such response is to release DNA structures called neutrophil extracellular traps (NETs) [1] . We hypothesize that this response will be modulated by the surface properties of a biomaterial, which will influence the response of downstream cells, such as macrophages, and the overall healing response. Figure 1: Neutrophil killing mechanisms [1] Results and Discussion: HL-60 Cell Activation Figure 2: The alamarBlue assay on HL-60 activated with PMA Figure 3: The alamarBlue assay on HL-60 activated with DMSO Figure 4: The alamarBlue assay on HL-60 activated for 48 hours onto polystyrene micro plates with 50 nM PMA Results and Discussion: HL-60 Cell Activation Onto Biomaterials The PMMA and the carboxyl modified PMMA promoted HL-60 adherence and viability without PMA. The amine modified PMMA had the most significant increase in activation. Figure 5: The alamarBlue assay on HL-60 activated with or without 50 nM PMA onto PMMA with different surface groups and topography Figure 7: The alamarBlue assay on HL-60 activated with 50 nM PMA onto MMcoIDA and MAAcoIDA copolymers 0.00 0.04 0.08 0.12 0.16 PMMA PMMA-COOH PMMA-NH2 PS Absorbance at 570 nm/600 nm Rough Smooth Rough with PMA Smooth with PMA PMMA PMMA-COOH PMMA-NH 2 Figure 6: Images of activated HL-60 on PMMA or modified PMMA (blue=nucBlue stain, green=Sytox stain) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 PS Coverslip MMcoIDA MAAcoIDA Absorbance at 570 nm/600 nm Conclusion and Future Work PMA promoted the adhesion of HL-60 cells onto biomaterials with an optimal concentration of 50 nM. DMSO did not promote adherence of cells. Further work will be done to stain for the phenotype of these cells and to observe if adherence will occur with DMSO treated HL-60 incubated with modified PMMA and the copolymers. HL-60 cells activated with PMA had increased “viability“ on PMMA and carboxyl modified PMMA. Live/dead stains will be done in future work to over see cell adherence directly. Preliminary work suggests increased NETosis of HL-60 cells on carboxyl-modified PMA. Current efforts are also focused on developing a real-time live cell assay to assess the generation of NETs using Sytox and improving the copolymers. Acknowledgments We would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Senate Advisory Research Committee at Queen’s University (SARC) for funding. Fairuz Hoque would like to thank Dr. Laura Wells for her help and guidance, and NSERC for summer funding through the USRA program. References [1] E. Kolaczkowska and P. Kubes, "Neutrophil Recruitment and Function in Health and Inflammation," Nature Reviews Immunology , vol. 13, no. 3, pp. 159-175, 2013. [2] M. Sefton and L. Wells, "The Effect of Methacrylic Acid in Smooth Coatings on dTHP1 and HUVEC Gene Expression," Biomaterials Science, vol. 2, no. 12, pp. 1768-1778, 2014. [3] Wells LA, Valic MS, Lisovsky A, Sefton MV. Angiogenic biomaterials to promote tissue vascularization and integration. Israel Journal of Chemistry. 2013; 53 (9-10): 637-645 0 0.1 0.2 0.3 0.4 0.5 0.6 100,000 control 300,000 control 600,000 control 100,000 300,000 600,000 Absorbance at 570 nm/600 nm Day 2 Day 5 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0 nM 5 nM 50 nM 500 nM Absorbance at 570 nm/600 nm 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0 0.13% 1.3% 13.0% Absorbance at 570 nm/600 nm The objective of this work is to investigate how biomaterials with different chemistry and topography influence neutrophil cell behaviour and NET generation, also known as NETosis. HL-60 cells, which are “neutrophil-like”, were activated onto PMMA, carboxyl modified PMMA, and amine modified PMMA as well as methacrylic acid copolymers, which are known to trigger vascular healing responses [2]. HL-60 remained viable over 5 days after activation. 600,000 cells/ml had the most activation and the least amount of change from day 2 to day 5. Methods A gradient of phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO) were used to activate HL-60 cells to be more neutrophil-like. After 48 hours of activation, an alamarBlue assay assessed cell viability which was an indicator of the number of cells that activated and the viability of these cells. A PMA gradient of 0 nM, 5 nM, 50 nM, and 500 nM and a DMSO gradient of 0%, 0.13%, 1.3%, and 13% were tested. Using the optimal activation agent, seeding densities of 100,000 cells/ml, 300,000 cells/ml, and 600,000 cells/ml were tested to find the most robust density. Rough and smooth PMMA disks were modified with to have 0.19 mmol/cm 2 carboxyl and 0.07 mmol/cm 2 amine groups. HL-60 cells were activated onto the disks with PMA. An alamarBlue assay was run and DNA was stained (nucBlue for intracellular DNA and Sytox for extracellular DNA). Similar test was done on HL-60 activated onto 40% methacrylic acid co isodecyl acylate (MAAcoIDA) and 40% methyl methacrylate co isodecyl acrylate (MMcoIDA) copolymers (cast onto glass coverslips). To determine the activation profile of the HL-60 cells, the influence of PMA and DMSO on cell adherence was first investigated. Changing the amount of PMA had a significant influence on the amount of HL-60 adherence, unlike DMSO which had similar values despite significant changes in amount. 50 nM PMA appeared to be effective and maintained high viability. Preliminary studies show that carboxyl modified PMMA led to increased NETosis at 48 hours. Extracellular DNA can be seen stained green while the nuclei of the cells are stained blue. Previous studies show that MMcoIDA and MAAcoIDA promote changes in macrophage-like cells and promote vascular healing in vivo [2, 3]. Therefore, their possible influence on HL-60 cells was investigated. HL-60 cells activated on these surfaces result in low viability. Either the cells did not adhere or they are no longer viable. Technical difficulties with the coating of these copolymers onto glass coverslips may have introduced error and will be improved upon in future work.
Transcript of HL-60 Cell Responses to Polymer Surfaces of Different ... Day/2015/Fairuz Hoque.pdf · Changing the...
HL-60 Cell Responses to Polymer Surfaces of Different Chemistry
Fairuz Hoque, Martin Kwok, Laura A. Wells
Department of Chemical Engineering and Engineering ChemistryQueen’s University, Canada
IntroductionOnce an implant is placed within a host, the initial host response will influence how well it will function. One of the first cells to the site of an implant are neutrophil cells. During the host response surrounding an implant, neutrophils can have different responses which can influence how well it is accepted. One such response is to release DNA structures called neutrophil extracellular traps (NETs) [1] . We hypothesize that this response will be modulated by the surface properties of a biomaterial, which will influence the response of downstream cells, such as macrophages, and the overall healing response.
Figure 1: Neutrophil killing mechanisms [1]
Results and Discussion: HL-60 Cell Activation
Figure 2: The alamarBlue assay on HL-60 activated with PMA Figure 3: The alamarBlue assay on HL-60 activated with DMSO
Figure 4: The alamarBlue assay on HL-60 activated for 48 hours onto polystyrene micro plates with 50 nM PMA
Results and Discussion: HL-60 Cell Activation Onto BiomaterialsThe PMMA and the carboxyl modified PMMA promoted HL-60 adherence and viability without PMA. The amine modified PMMA had the most significant increase in activation.
Figure 5: The alamarBlue assay on HL-60 activated with or without 50 nM PMA onto PMMA with different
surface groups and topography
Figure 7: The alamarBlue assay on HL-60 activated with 50 nM PMAonto MMcoIDA and MAAcoIDA copolymers
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Figure 6: Images of activated HL-60 on PMMA or modified PMMA (blue=nucBlue stain, green=Sytox stain)
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Conclusion and Future WorkPMA promoted the adhesion of HL-60 cells onto biomaterials with an optimal concentration of 50 nM. DMSO did not promote adherence of cells. Further work will be done to stain for the phenotype of these cells and to observe if adherence will occur with DMSO treated HL-60 incubated with modified PMMA and the copolymers.
HL-60 cells activated with PMA had increased “viability“ on PMMA and carboxyl modified PMMA. Live/dead stains will be done in future work to over see cell adherence directly. Preliminary work suggests increased NETosis of HL-60 cells on carboxyl-modified PMA.
Current efforts are also focused on developing a real-time live cell assay to assess the generation of NETs using Sytox and improving the copolymers.
AcknowledgmentsWe would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Senate Advisory Research Committee at Queen’s University (SARC) for funding. Fairuz Hoque would like to thank Dr. Laura Wells for her help and guidance, and NSERC for summer funding through the USRA program.
References[1] E. Kolaczkowska and P. Kubes, "Neutrophil Recruitment and Function in Health and Inflammation," Nature Reviews Immunology , vol. 13, no. 3, pp. 159-175, 2013. [2] M. Sefton and L. Wells, "The Effect of Methacrylic Acid in Smooth Coatings on dTHP1 and HUVEC Gene Expression," Biomaterials Science, vol. 2, no. 12, pp. 1768-1778, 2014. [3] Wells LA, Valic MS, Lisovsky A, Sefton MV. Angiogenic biomaterials to promote tissue vascularization and integration. Israel Journal of Chemistry. 2013; 53 (9-10): 637-645
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The objective of this work is to investigate how biomaterials with different chemistry and topography influence neutrophil cell behaviour and NET generation, also known as NETosis. HL-60 cells, which are “neutrophil-like”, were activated onto PMMA, carboxyl modified PMMA, and amine modified PMMA as well as methacrylic acid copolymers, which are known to trigger vascular healing responses [2].
HL-60 remained viable over 5 days after activation. 600,000 cells/ml had the most activation and the least amount of change from day 2 to day 5.
MethodsA gradient of phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO) were used to activate HL-60 cells to be more neutrophil-like. After 48 hours of activation, an alamarBlue assay assessed cell viability which was an indicator of the number of cells that activated and the viability of these cells. A PMA gradient of 0 nM, 5 nM, 50 nM, and 500 nM and a DMSO gradient of 0%, 0.13%, 1.3%, and 13% were tested. Using the optimal activation agent, seeding densities of 100,000 cells/ml, 300,000 cells/ml, and 600,000 cells/ml were tested to find the most robust density.
Rough and smooth PMMA disks were modified with to have 0.19 mmol/cm2 carboxyl and 0.07 mmol/cm2amine groups. HL-60 cells were activated onto the disks with PMA. An alamarBlue assay was run and DNA was stained (nucBlue for intracellular DNA and Sytox for extracellular DNA). Similar test was done on HL-60 activated onto 40% methacrylic acid co isodecyl acylate(MAAcoIDA) and 40% methyl methacrylate co isodecyl acrylate (MMcoIDA) copolymers (cast onto glass coverslips).
To determine the activation profile of the HL-60 cells, the influence of PMA and DMSO on cell adherence was first investigated. Changing the amount of PMA had a significant influence on the amount of HL-60 adherence, unlike DMSO which had similar values despite significant changes in amount. 50 nM PMA appeared to be effective and maintained high viability.
Preliminary studies show that carboxyl modified PMMA led to increased NETosis at 48 hours. Extracellular DNA can be seen stained green while the nuclei of the cells are stained blue.
Previous studies show that MMcoIDA and MAAcoIDA promote changes in macrophage-like cells and promote vascular healing in vivo [2, 3]. Therefore, their possible influence on HL-60 cells was investigated. HL-60 cells activated on these surfaces result in low viability. Either the cells did not adhere or they are no longer viable. Technical difficulties with the coating of these copolymers onto glass coverslips may have introduced error and will be improved upon in future work.
