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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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Differentiation potential of hADSCs cultured on a chitosan-based matrix.a) Alcian blue staining of acid mucopolysaccharide aggregates present in the differentiatedchondrocyte cultures grown on chitosan hydrogel. b) Calcium deposits in the ECM were stained using Von kossa staining of the differentiated osteocytes on the hydrogel matrix.c) Lipid droplets were formed on cells laden on matrix after adipogenic induction and werestained with oil red O staining. Negative staining of un-induced cultures for the respectivelineages is also shown. Magnification, 400X (n = 5).
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pone.0120803.g005: Differentiation potential of hADSCs cultured on a chitosan-based matrix.a) Alcian blue staining of acid mucopolysaccharide aggregates present in the differentiatedchondrocyte cultures grown on chitosan hydrogel. b) Calcium deposits in the ECM were stained using Von kossa staining of the differentiated osteocytes on the hydrogel matrix.c) Lipid droplets were formed on cells laden on matrix after adipogenic induction and werestained with oil red O staining. Negative staining of un-induced cultures for the respectivelineages is also shown. Magnification, 400X (n = 5).

Mentions: Differentiation of fibroblastic cells obtained from adipose tissue could be directed towards adipogenic, chondrogenic and osteogenic lineages using appropriate differentiation media and supplements. For these studies, un-induced fibroblasts served as negative controls. For the chondrogenic cultures, spherical micro-mass cultures were observed, and deposits of acid mucopolysaccharides were confirmed with Alcian blue staining as shown in Fig. 5a.The cells in each group were separated by extensive regions of a diffuse extracellular matrix that had high collagen content. Similarly, under osteogenic induction conditions, a dark ECM material was detected after the induction period. Deposition of a calcified matrix was confirmed with Von Kossa staining (Fig. 5b). Adherent hADSCs which underwent adipogenic differentiation were characterized by an accumulation of cytoplasmic triglycerides that were represented as lipid droplets by Oil Red O staining (Fig. 5c). The trilineage differentiation potential of the hADSCs was confirmed in five samplesfor reproducibility.


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)

Differentiation potential of hADSCs cultured on a chitosan-based matrix.a) Alcian blue staining of acid mucopolysaccharide aggregates present in the differentiatedchondrocyte cultures grown on chitosan hydrogel. b) Calcium deposits in the ECM were stained using Von kossa staining of the differentiated osteocytes on the hydrogel matrix.c) Lipid droplets were formed on cells laden on matrix after adipogenic induction and werestained with oil red O staining. Negative staining of un-induced cultures for the respectivelineages is also shown. Magnification, 400X (n = 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120803.g005: Differentiation potential of hADSCs cultured on a chitosan-based matrix.a) Alcian blue staining of acid mucopolysaccharide aggregates present in the differentiatedchondrocyte cultures grown on chitosan hydrogel. b) Calcium deposits in the ECM were stained using Von kossa staining of the differentiated osteocytes on the hydrogel matrix.c) Lipid droplets were formed on cells laden on matrix after adipogenic induction and werestained with oil red O staining. Negative staining of un-induced cultures for the respectivelineages is also shown. Magnification, 400X (n = 5).
Mentions: Differentiation of fibroblastic cells obtained from adipose tissue could be directed towards adipogenic, chondrogenic and osteogenic lineages using appropriate differentiation media and supplements. For these studies, un-induced fibroblasts served as negative controls. For the chondrogenic cultures, spherical micro-mass cultures were observed, and deposits of acid mucopolysaccharides were confirmed with Alcian blue staining as shown in Fig. 5a.The cells in each group were separated by extensive regions of a diffuse extracellular matrix that had high collagen content. Similarly, under osteogenic induction conditions, a dark ECM material was detected after the induction period. Deposition of a calcified matrix was confirmed with Von Kossa staining (Fig. 5b). Adherent hADSCs which underwent adipogenic differentiation were characterized by an accumulation of cytoplasmic triglycerides that were represented as lipid droplets by Oil Red O staining (Fig. 5c). The trilineage differentiation potential of the hADSCs was confirmed in five samplesfor reproducibility.

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