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Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel.

Debnath T, Ghosh S, Potlapuvu US, Kona L, Kamaraju SR, Sarkar S, Gaddam S, Chelluri LK - PLoS ONE (2015)

Bottom Line: Cytotoxicity assays demonstrated safety profile.Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages.Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

View Article: PubMed Central - PubMed

Affiliation: Transplant Immunology & Stem Cell Laboratory, Global Hospitals, Hyderabad, India.

ABSTRACT
Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

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Bio-compatibility profiling of hADSCs on chitosan hydrogel.a) The results presented are the mean ± SD LDH levels detected in supernatants at different time intervals. LDH release was not detected for stem cells cultured on the hydrogel (n = 4, P <0.05). b) MTT assay was used to represent cell metabolic activity and viability of hADSCs encapsulated in chitosan matrix for 7 days (mean ± SD, n = 3). c) Apoptosis assays demonstrated that the cells did not undergo apoptosis when cultured on hydrogel. In contrast, cells induced with H2O2 underwent apoptosis (n = 3).
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pone.0120803.g004: Bio-compatibility profiling of hADSCs on chitosan hydrogel.a) The results presented are the mean ± SD LDH levels detected in supernatants at different time intervals. LDH release was not detected for stem cells cultured on the hydrogel (n = 4, P <0.05). b) MTT assay was used to represent cell metabolic activity and viability of hADSCs encapsulated in chitosan matrix for 7 days (mean ± SD, n = 3). c) Apoptosis assays demonstrated that the cells did not undergo apoptosis when cultured on hydrogel. In contrast, cells induced with H2O2 underwent apoptosis (n = 3).

Mentions: Cell culture supernatants were collected after 24 h, 48 h, and 72 h and were analysed for levels of LDH (Olympus AU400 Automated Analyzer). As shown in Fig. 4a, there was no significant difference in LDH assay among the hADSCs and hADSCs with matrix group after 48 and 72 h (n = 4, p<0.05). LDH release was <10 IU/mL for the tested chitosan hydrogel matrix with the cells at different time intervals indicating that the hydrogel has no cytotoxic effect on hADSCs (S3 File). The metabolic activity of mesenchymal stem cells grown on the hydrogel matrix was confirmed using MTT assay over a period of 7 d. (n = 3). The cell seeding density of 3x103cells/well provided sufficient cell-cell contact. The relative cell viability of hADSCs on the hydrogel increased > 100% after 4 days (S4 File). Annexin V-FITC assays were performed to assess cytotoxicity. After hADSCs (n = 3) were cultured on the hydrogel for 7 d, no signs of phosphatidyl serine translocation were detected compared with cells induced with 20μM H2O2. These results indicate that the hydrogel tested does not induce a toxic effect on the hADSCs that were cultured with it (Fig. 4b,c).


Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel.

Debnath T, Ghosh S, Potlapuvu US, Kona L, Kamaraju SR, Sarkar S, Gaddam S, Chelluri LK - PLoS ONE (2015)

Bio-compatibility profiling of hADSCs on chitosan hydrogel.a) The results presented are the mean ± SD LDH levels detected in supernatants at different time intervals. LDH release was not detected for stem cells cultured on the hydrogel (n = 4, P <0.05). b) MTT assay was used to represent cell metabolic activity and viability of hADSCs encapsulated in chitosan matrix for 7 days (mean ± SD, n = 3). c) Apoptosis assays demonstrated that the cells did not undergo apoptosis when cultured on hydrogel. In contrast, cells induced with H2O2 underwent apoptosis (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352002&req=5

pone.0120803.g004: Bio-compatibility profiling of hADSCs on chitosan hydrogel.a) The results presented are the mean ± SD LDH levels detected in supernatants at different time intervals. LDH release was not detected for stem cells cultured on the hydrogel (n = 4, P <0.05). b) MTT assay was used to represent cell metabolic activity and viability of hADSCs encapsulated in chitosan matrix for 7 days (mean ± SD, n = 3). c) Apoptosis assays demonstrated that the cells did not undergo apoptosis when cultured on hydrogel. In contrast, cells induced with H2O2 underwent apoptosis (n = 3).
Mentions: Cell culture supernatants were collected after 24 h, 48 h, and 72 h and were analysed for levels of LDH (Olympus AU400 Automated Analyzer). As shown in Fig. 4a, there was no significant difference in LDH assay among the hADSCs and hADSCs with matrix group after 48 and 72 h (n = 4, p<0.05). LDH release was <10 IU/mL for the tested chitosan hydrogel matrix with the cells at different time intervals indicating that the hydrogel has no cytotoxic effect on hADSCs (S3 File). The metabolic activity of mesenchymal stem cells grown on the hydrogel matrix was confirmed using MTT assay over a period of 7 d. (n = 3). The cell seeding density of 3x103cells/well provided sufficient cell-cell contact. The relative cell viability of hADSCs on the hydrogel increased > 100% after 4 days (S4 File). Annexin V-FITC assays were performed to assess cytotoxicity. After hADSCs (n = 3) were cultured on the hydrogel for 7 d, no signs of phosphatidyl serine translocation were detected compared with cells induced with 20μM H2O2. These results indicate that the hydrogel tested does not induce a toxic effect on the hADSCs that were cultured with it (Fig. 4b,c).

Bottom Line: Cytotoxicity assays demonstrated safety profile.Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages.Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

View Article: PubMed Central - PubMed

Affiliation: Transplant Immunology & Stem Cell Laboratory, Global Hospitals, Hyderabad, India.

ABSTRACT
Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

Show MeSH
Related in: MedlinePlus