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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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Proliferation potential of hADSCs on chitosan hydrogel.a) hADSCs cultured on chitosan hydrogel on different days. Magnification 200X. b) SEM imaging revealed uniform distribution of hADSCs on the hydrogel. Further, the cells were growing in close association with the hydrogel. c) The mean ± SD values for PDT were low when cells were grown on a hydrogel matrix. There was a significant difference in growth rates of the hADSCs on the hydrogel matrix at P <0.05 (n = 3). d) JC-1 staining showed JC-1 dimer formation within the stem cell network, thereby indicating the presence of metabolically active live cells on the hydrogel using CLSM. The JC-1 staining of cells differentiate dead from viable intact cells by their mitochondrial membrane integrity. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J-aggregates form, which appear as fluorescent orange. In non-viable cells, the mitochondrial membrane potential collapses, and the JC-1 cannot accumulate within the mitochondria. The bright field image shows actively growing stem cells present in the hydrogel (n = 3).
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pone.0120803.g003: Proliferation potential of hADSCs on chitosan hydrogel.a) hADSCs cultured on chitosan hydrogel on different days. Magnification 200X. b) SEM imaging revealed uniform distribution of hADSCs on the hydrogel. Further, the cells were growing in close association with the hydrogel. c) The mean ± SD values for PDT were low when cells were grown on a hydrogel matrix. There was a significant difference in growth rates of the hADSCs on the hydrogel matrix at P <0.05 (n = 3). d) JC-1 staining showed JC-1 dimer formation within the stem cell network, thereby indicating the presence of metabolically active live cells on the hydrogel using CLSM. The JC-1 staining of cells differentiate dead from viable intact cells by their mitochondrial membrane integrity. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J-aggregates form, which appear as fluorescent orange. In non-viable cells, the mitochondrial membrane potential collapses, and the JC-1 cannot accumulate within the mitochondria. The bright field image shows actively growing stem cells present in the hydrogel (n = 3).

Mentions: hADSCs grown on 10μm hydrogel sections were found to be stable throughout the entire culture period, and the cells did not undergo any contractions. It was noted the hADSCs were spindle-like shape on the hydrogel on 10th day (Fig. 3a). In SEM images, adipose-derived MSCs were found to form continuous sheets of cells and to fill the pores of the hydrogel. In addition, small, round-shaped cells were found to be embedded within the pores of the chitosan hydrogel (Fig. 3b). The average population doubling time (PDT) of the hADSCs grown on the hydrogel was 47.8 ± 0.32 h till passage 2, similar to the doubling time of hADSCs without hydrogel (n = 3). hADSCs exhibited the lowest PDT at P2 with 42.74 h, although this increased to 50.56 h at P3, and to 54 h at P4–P6 whereas, the PDT was increased to 90 h at P4 and more than 120 h at P5–P6 for isolated hADSCs without the matrix. (Fig. 3c and S2 File). Using JC-1 staining, mitochondrial integrity and compatibility of the hADSCs (n = 3) was found to be 98%. Furthermore, hADSCs retained their spindle shape morphology and exhibited > 80% viability on the hydrogel matrix compared with their growth in a monolayer cell culture. hADSCs were also observed to form a mesh on the hydrogel matrix as evidenced by the detection of intact mitochondria in fluorescent staining assays (Fig. 3d).


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)

Proliferation potential of hADSCs on chitosan hydrogel.a) hADSCs cultured on chitosan hydrogel on different days. Magnification 200X. b) SEM imaging revealed uniform distribution of hADSCs on the hydrogel. Further, the cells were growing in close association with the hydrogel. c) The mean ± SD values for PDT were low when cells were grown on a hydrogel matrix. There was a significant difference in growth rates of the hADSCs on the hydrogel matrix at P <0.05 (n = 3). d) JC-1 staining showed JC-1 dimer formation within the stem cell network, thereby indicating the presence of metabolically active live cells on the hydrogel using CLSM. The JC-1 staining of cells differentiate dead from viable intact cells by their mitochondrial membrane integrity. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J-aggregates form, which appear as fluorescent orange. In non-viable cells, the mitochondrial membrane potential collapses, and the JC-1 cannot accumulate within the mitochondria. The bright field image shows actively growing stem cells present in the hydrogel (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120803.g003: Proliferation potential of hADSCs on chitosan hydrogel.a) hADSCs cultured on chitosan hydrogel on different days. Magnification 200X. b) SEM imaging revealed uniform distribution of hADSCs on the hydrogel. Further, the cells were growing in close association with the hydrogel. c) The mean ± SD values for PDT were low when cells were grown on a hydrogel matrix. There was a significant difference in growth rates of the hADSCs on the hydrogel matrix at P <0.05 (n = 3). d) JC-1 staining showed JC-1 dimer formation within the stem cell network, thereby indicating the presence of metabolically active live cells on the hydrogel using CLSM. The JC-1 staining of cells differentiate dead from viable intact cells by their mitochondrial membrane integrity. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J-aggregates form, which appear as fluorescent orange. In non-viable cells, the mitochondrial membrane potential collapses, and the JC-1 cannot accumulate within the mitochondria. The bright field image shows actively growing stem cells present in the hydrogel (n = 3).
Mentions: hADSCs grown on 10μm hydrogel sections were found to be stable throughout the entire culture period, and the cells did not undergo any contractions. It was noted the hADSCs were spindle-like shape on the hydrogel on 10th day (Fig. 3a). In SEM images, adipose-derived MSCs were found to form continuous sheets of cells and to fill the pores of the hydrogel. In addition, small, round-shaped cells were found to be embedded within the pores of the chitosan hydrogel (Fig. 3b). The average population doubling time (PDT) of the hADSCs grown on the hydrogel was 47.8 ± 0.32 h till passage 2, similar to the doubling time of hADSCs without hydrogel (n = 3). hADSCs exhibited the lowest PDT at P2 with 42.74 h, although this increased to 50.56 h at P3, and to 54 h at P4–P6 whereas, the PDT was increased to 90 h at P4 and more than 120 h at P5–P6 for isolated hADSCs without the matrix. (Fig. 3c and S2 File). Using JC-1 staining, mitochondrial integrity and compatibility of the hADSCs (n = 3) was found to be 98%. Furthermore, hADSCs retained their spindle shape morphology and exhibited > 80% viability on the hydrogel matrix compared with their growth in a monolayer cell culture. hADSCs were also observed to form a mesh on the hydrogel matrix as evidenced by the detection of intact mitochondria in fluorescent staining assays (Fig. 3d).

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