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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

KG1a phenotype maintenance on fibronectin (FN)-coated scaffolds. Fluorescence-activated cell sorting analysis of KG1a cell phenotype after seeding at different conditions (FN + tissue culture plate system [TCPS] and FN + scaffold) at different time points.Notes: Note the percentage of expression of CD34+ and 33− (A) remained similar on scaffolds at 48 to 168 hours (h) compared to on a FN-coated tissue culture plate system (FNTCPS). A similar pattern was observed with CD34+ and 38− (B).Abbreviations: APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PLLA, poly-l-lactic acid; PU, polyurethane.
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f3-ijn-10-3603: KG1a phenotype maintenance on fibronectin (FN)-coated scaffolds. Fluorescence-activated cell sorting analysis of KG1a cell phenotype after seeding at different conditions (FN + tissue culture plate system [TCPS] and FN + scaffold) at different time points.Notes: Note the percentage of expression of CD34+ and 33− (A) remained similar on scaffolds at 48 to 168 hours (h) compared to on a FN-coated tissue culture plate system (FNTCPS). A similar pattern was observed with CD34+ and 38− (B).Abbreviations: APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PLLA, poly-l-lactic acid; PU, polyurethane.

Mentions: Though the FN-coated PU/PLLA 60:40 scaffolds were capable of supporting cell proliferation and survival, it is essential to maintain the original phenotype during drug testing. In order to address this issue, the expression of CD33, CD34, and CD38 in KG1a cells cultured in different conditions such as TCPSs, FNTCPSs, and FN-coated PU/PLLA 60:40 were analyzed using fluorescence-activated cell sorting at 48, 120, and 168 hours. The results show that the percentage expression of CD34+/CD33− at the time of seeding (Figure 3A) was maintained for 168 hours of culture on the scaffolds (94.6%; Figure 3A, 168 hours). On the other hand, the cells cultured on an FNTCPS showed a marginal reduction in percentage expression of these cell surface markers, particularly by 168 hours (90.8; Figure 3A). Similarly, the percentage expression of CD34+ and CD38− on the FN-coated scaffolds showed no difference following culturing on FN-coated scaffolds for 168 hours in comparison to on an FNTCPS, which showed again a reduction in CD34+/CD38− (80.9% vs 93.7%; Figure 3B, 168 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)

KG1a phenotype maintenance on fibronectin (FN)-coated scaffolds. Fluorescence-activated cell sorting analysis of KG1a cell phenotype after seeding at different conditions (FN + tissue culture plate system [TCPS] and FN + scaffold) at different time points.Notes: Note the percentage of expression of CD34+ and 33− (A) remained similar on scaffolds at 48 to 168 hours (h) compared to on a FN-coated tissue culture plate system (FNTCPS). A similar pattern was observed with CD34+ and 38− (B).Abbreviations: APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PLLA, poly-l-lactic acid; PU, polyurethane.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440427&req=5

f3-ijn-10-3603: KG1a phenotype maintenance on fibronectin (FN)-coated scaffolds. Fluorescence-activated cell sorting analysis of KG1a cell phenotype after seeding at different conditions (FN + tissue culture plate system [TCPS] and FN + scaffold) at different time points.Notes: Note the percentage of expression of CD34+ and 33− (A) remained similar on scaffolds at 48 to 168 hours (h) compared to on a FN-coated tissue culture plate system (FNTCPS). A similar pattern was observed with CD34+ and 38− (B).Abbreviations: APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PLLA, poly-l-lactic acid; PU, polyurethane.
Mentions: Though the FN-coated PU/PLLA 60:40 scaffolds were capable of supporting cell proliferation and survival, it is essential to maintain the original phenotype during drug testing. In order to address this issue, the expression of CD33, CD34, and CD38 in KG1a cells cultured in different conditions such as TCPSs, FNTCPSs, and FN-coated PU/PLLA 60:40 were analyzed using fluorescence-activated cell sorting at 48, 120, and 168 hours. The results show that the percentage expression of CD34+/CD33− at the time of seeding (Figure 3A) was maintained for 168 hours of culture on the scaffolds (94.6%; Figure 3A, 168 hours). On the other hand, the cells cultured on an FNTCPS showed a marginal reduction in percentage expression of these cell surface markers, particularly by 168 hours (90.8; Figure 3A). Similarly, the percentage expression of CD34+ and CD38− on the FN-coated scaffolds showed no difference following culturing on FN-coated scaffolds for 168 hours in comparison to on an FNTCPS, which showed again a reduction in CD34+/CD38− (80.9% vs 93.7%; Figure 3B, 168 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