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Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells.

Nair MS, Mony U, Menon D, Koyakutty M, Sidharthan N, Pavithran K, Nair SV, Menon KN - Int J Nanomedicine (2015)

Bottom Line: Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems.Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest.Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo.

View Article: PubMed Central - PubMed

Affiliation: Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University, Kerala, India.

ABSTRACT
Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-L-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

No MeSH data available.


Related in: MedlinePlus

Comparison of proliferation of KG1a cells on different blends of fibronectin (FN)-coated scaffolds against FN-coated tissue culture plate systems (FNTCPSs) at different time points.Notes: Note the increased cell viability observed in polyurethane (PU)/poly-l-lactic acid (PLLA) 60:40 at 120 and 168 hours; a blend morphologically similar to the micro-nanofibrous bone marrow 3-D niche (Figure 1I vs V) compared to a tissue culture plate system and PU/PLLA 40:60. P≤0.04 for #FNTCPS vs PU/PLLA 50:50, +PU/PLLA 60:40 vs PU/PLLA 40:60, ^PU/PLLA 60:40 and PU/PLLA 50:50. P≤0.01 for **FNTCPS vs PU/PLLA 60:40, ##FNTCPS vs PU/PLLA 50:50, ++PU/PLLA 60:40 vs PU/PLLA 40:60, ^^PU/PLLA 60:40 vs PU/PLLA 50:50, ••FNTCPS vs PU/PLLA 40:60.
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f2-ijn-10-3603: Comparison of proliferation of KG1a cells on different blends of fibronectin (FN)-coated scaffolds against FN-coated tissue culture plate systems (FNTCPSs) at different time points.Notes: Note the increased cell viability observed in polyurethane (PU)/poly-l-lactic acid (PLLA) 60:40 at 120 and 168 hours; a blend morphologically similar to the micro-nanofibrous bone marrow 3-D niche (Figure 1I vs V) compared to a tissue culture plate system and PU/PLLA 40:60. P≤0.04 for #FNTCPS vs PU/PLLA 50:50, +PU/PLLA 60:40 vs PU/PLLA 40:60, ^PU/PLLA 60:40 and PU/PLLA 50:50. P≤0.01 for **FNTCPS vs PU/PLLA 60:40, ##FNTCPS vs PU/PLLA 50:50, ++PU/PLLA 60:40 vs PU/PLLA 40:60, ^^PU/PLLA 60:40 vs PU/PLLA 50:50, ••FNTCPS vs PU/PLLA 40:60.

Mentions: For the initial 72 hours, the FNTCPS showed maximum cell proliferation/viability compared to the FN-coated scaffolds (Figure 2) and the closest scaffold that showed cell proliferation similar to that with the FNTCPS was the PU/PLLA 60:40 composite scaffold (0.165 in PU/PLLA 60:40 vs 0.195 in FNTCPS). However, with increasing time of the KG1a cells culturing on different scaffold composites, the FN-coated PU/PLLA 60:40 scaffolds demonstrated good proliferation/viability of cells compared to FNTCPS (P≤0.01 for FNTCPS vs PU/PLLA 60:40). Moreover, the FN-coated PU/PLLA 60:40 composite demonstrated maintenance of cells for 168 hours, as indicated by a plateau, and was found to be significantly different from other scaffold composites (Figure 2; P≤0.04 for PU/PLLA 60:40 vs PU/PLLA 40:60, PU/PLLA 60:40, and PU/PLLA 50:50. P≤0.01 for PU/PLLA 60:40 vs PU/PLLA 40:60 and PU/PLLA 60:40 vs PU/PLLA 50:50). Other composites of scaffolds (PU/PLLA 40:60 and 50:50) demonstrated significantly less proliferation/viability of cells compared to PU/PLLA 60:40 at 120 and 168 hours, with the exception of PU/PLLA 50:50 at 168 hours. Note in Figure 2 the significant deterioration in cell proliferation/viability on the FNTCPS after 72 hours.


Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells.

Nair MS, Mony U, Menon D, Koyakutty M, Sidharthan N, Pavithran K, Nair SV, Menon KN - Int J Nanomedicine (2015)

Comparison of proliferation of KG1a cells on different blends of fibronectin (FN)-coated scaffolds against FN-coated tissue culture plate systems (FNTCPSs) at different time points.Notes: Note the increased cell viability observed in polyurethane (PU)/poly-l-lactic acid (PLLA) 60:40 at 120 and 168 hours; a blend morphologically similar to the micro-nanofibrous bone marrow 3-D niche (Figure 1I vs V) compared to a tissue culture plate system and PU/PLLA 40:60. P≤0.04 for #FNTCPS vs PU/PLLA 50:50, +PU/PLLA 60:40 vs PU/PLLA 40:60, ^PU/PLLA 60:40 and PU/PLLA 50:50. P≤0.01 for **FNTCPS vs PU/PLLA 60:40, ##FNTCPS vs PU/PLLA 50:50, ++PU/PLLA 60:40 vs PU/PLLA 40:60, ^^PU/PLLA 60:40 vs PU/PLLA 50:50, ••FNTCPS vs PU/PLLA 40:60.
© Copyright Policy
Related In: Results  -  Collection

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f2-ijn-10-3603: Comparison of proliferation of KG1a cells on different blends of fibronectin (FN)-coated scaffolds against FN-coated tissue culture plate systems (FNTCPSs) at different time points.Notes: Note the increased cell viability observed in polyurethane (PU)/poly-l-lactic acid (PLLA) 60:40 at 120 and 168 hours; a blend morphologically similar to the micro-nanofibrous bone marrow 3-D niche (Figure 1I vs V) compared to a tissue culture plate system and PU/PLLA 40:60. P≤0.04 for #FNTCPS vs PU/PLLA 50:50, +PU/PLLA 60:40 vs PU/PLLA 40:60, ^PU/PLLA 60:40 and PU/PLLA 50:50. P≤0.01 for **FNTCPS vs PU/PLLA 60:40, ##FNTCPS vs PU/PLLA 50:50, ++PU/PLLA 60:40 vs PU/PLLA 40:60, ^^PU/PLLA 60:40 vs PU/PLLA 50:50, ••FNTCPS vs PU/PLLA 40:60.
Mentions: For the initial 72 hours, the FNTCPS showed maximum cell proliferation/viability compared to the FN-coated scaffolds (Figure 2) and the closest scaffold that showed cell proliferation similar to that with the FNTCPS was the PU/PLLA 60:40 composite scaffold (0.165 in PU/PLLA 60:40 vs 0.195 in FNTCPS). However, with increasing time of the KG1a cells culturing on different scaffold composites, the FN-coated PU/PLLA 60:40 scaffolds demonstrated good proliferation/viability of cells compared to FNTCPS (P≤0.01 for FNTCPS vs PU/PLLA 60:40). Moreover, the FN-coated PU/PLLA 60:40 composite demonstrated maintenance of cells for 168 hours, as indicated by a plateau, and was found to be significantly different from other scaffold composites (Figure 2; P≤0.04 for PU/PLLA 60:40 vs PU/PLLA 40:60, PU/PLLA 60:40, and PU/PLLA 50:50. P≤0.01 for PU/PLLA 60:40 vs PU/PLLA 40:60 and PU/PLLA 60:40 vs PU/PLLA 50:50). Other composites of scaffolds (PU/PLLA 40:60 and 50:50) demonstrated significantly less proliferation/viability of cells compared to PU/PLLA 60:40 at 120 and 168 hours, with the exception of PU/PLLA 50:50 at 168 hours. Note in Figure 2 the significant deterioration in cell proliferation/viability on the FNTCPS after 72 hours.

Bottom Line: Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems.Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest.Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo.

View Article: PubMed Central - PubMed

Affiliation: Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University, Kerala, India.

ABSTRACT
Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-L-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

No MeSH data available.


Related in: MedlinePlus