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Differentiation of human mesenchymal stem cell spheroids under microgravity conditions.

Cerwinka WH, Sharp SM, Boyan BD, Zhau HE, Chung LW, Yates C - Cell Regen (Lond) (2012)

Bottom Line: Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers.Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively.The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

View Article: PubMed Central - PubMed

Affiliation: Children's Healthcare of Atlanta, Emory University School of Medicine, 5445Meridian Mark Road, Suite 420, Atlanta, GA 30342 USA ; Georgia Pediatric Urology, 5445 Meridian Mark Rd, Suite 420, Atlanta, GA 30342 USA.

ABSTRACT
To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 10(6) cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

No MeSH data available.


Related in: MedlinePlus

Quantification of hMSC loading onto gelatin scaffolds under MG conditions. (A) Number of cells loaded onto spheroids was determined by quantification of DNA content per spheroid over an 8 day period. (B) Number of cells that remained in suspension over an 8 day period. All data presented are the mean of three independent experiments ± SE. (C) H&E staining of day 3 spheroid at 10x and 40x magnification shows multi-cellularity and absence of necrosis. (D) TEM images of day 3 spheroid show cell structure was maintained in MG. Images shown are representative of 4 individual experiments.
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Fig2: Quantification of hMSC loading onto gelatin scaffolds under MG conditions. (A) Number of cells loaded onto spheroids was determined by quantification of DNA content per spheroid over an 8 day period. (B) Number of cells that remained in suspension over an 8 day period. All data presented are the mean of three independent experiments ± SE. (C) H&E staining of day 3 spheroid at 10x and 40x magnification shows multi-cellularity and absence of necrosis. (D) TEM images of day 3 spheroid show cell structure was maintained in MG. Images shown are representative of 4 individual experiments.

Mentions: We further characterized the length of time required to maximum spheroid size and cell loading. 3 x106 cells/10 ml hMSC spheroids were harvested at 1, 2, 3, 4, and 8 days of MG culture. Total DNA of spheroids was extracted and normalized to 2D cultured cells to extrapolate the exact number of cells within spheroids. Our results show that maximum spheroid loading was achievable by 3 days with a mean of 3.67 × 106cells per spheroid, representing 12% efficacy in cell loading (Figure 2A). Spheroid loading progressively declined after day 3, as determined by number of remaining cells in suspension; however spheroid size was maintained throughout (Figure 2B). Upon subsequent removal of hMSC coated gelatin scaffolds, we noticed that the rigid structure was maintained, suggesting an implantable construct (Figure 1). Hematoxylin and eosin (H&E) staining of sectioned spheroids confirmed these finding and highlighted a concentric rim of cells in the periphery with cell-cell connections and multi-cellular aggregation, with cells sparsely adherent near the porous space adjacent to the center of the spheroid (Figure 2C). Throughout the spheroid hMSC appear to maintain their mesenchymal phenotype and form cell-cell connections, which is further evident at increased magnification by TEM (Figure 2D). Since we determined the most significant cellularity and adherence to the gelatin scaffold at 3 days, we continued further characterization from this time point on.Figure 2


Differentiation of human mesenchymal stem cell spheroids under microgravity conditions.

Cerwinka WH, Sharp SM, Boyan BD, Zhau HE, Chung LW, Yates C - Cell Regen (Lond) (2012)

Quantification of hMSC loading onto gelatin scaffolds under MG conditions. (A) Number of cells loaded onto spheroids was determined by quantification of DNA content per spheroid over an 8 day period. (B) Number of cells that remained in suspension over an 8 day period. All data presented are the mean of three independent experiments ± SE. (C) H&E staining of day 3 spheroid at 10x and 40x magnification shows multi-cellularity and absence of necrosis. (D) TEM images of day 3 spheroid show cell structure was maintained in MG. Images shown are representative of 4 individual experiments.
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Related In: Results  -  Collection

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Fig2: Quantification of hMSC loading onto gelatin scaffolds under MG conditions. (A) Number of cells loaded onto spheroids was determined by quantification of DNA content per spheroid over an 8 day period. (B) Number of cells that remained in suspension over an 8 day period. All data presented are the mean of three independent experiments ± SE. (C) H&E staining of day 3 spheroid at 10x and 40x magnification shows multi-cellularity and absence of necrosis. (D) TEM images of day 3 spheroid show cell structure was maintained in MG. Images shown are representative of 4 individual experiments.
Mentions: We further characterized the length of time required to maximum spheroid size and cell loading. 3 x106 cells/10 ml hMSC spheroids were harvested at 1, 2, 3, 4, and 8 days of MG culture. Total DNA of spheroids was extracted and normalized to 2D cultured cells to extrapolate the exact number of cells within spheroids. Our results show that maximum spheroid loading was achievable by 3 days with a mean of 3.67 × 106cells per spheroid, representing 12% efficacy in cell loading (Figure 2A). Spheroid loading progressively declined after day 3, as determined by number of remaining cells in suspension; however spheroid size was maintained throughout (Figure 2B). Upon subsequent removal of hMSC coated gelatin scaffolds, we noticed that the rigid structure was maintained, suggesting an implantable construct (Figure 1). Hematoxylin and eosin (H&E) staining of sectioned spheroids confirmed these finding and highlighted a concentric rim of cells in the periphery with cell-cell connections and multi-cellular aggregation, with cells sparsely adherent near the porous space adjacent to the center of the spheroid (Figure 2C). Throughout the spheroid hMSC appear to maintain their mesenchymal phenotype and form cell-cell connections, which is further evident at increased magnification by TEM (Figure 2D). Since we determined the most significant cellularity and adherence to the gelatin scaffold at 3 days, we continued further characterization from this time point on.Figure 2

Bottom Line: Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers.Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively.The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

View Article: PubMed Central - PubMed

Affiliation: Children's Healthcare of Atlanta, Emory University School of Medicine, 5445Meridian Mark Road, Suite 420, Atlanta, GA 30342 USA ; Georgia Pediatric Urology, 5445 Meridian Mark Rd, Suite 420, Atlanta, GA 30342 USA.

ABSTRACT
To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 10(6) cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

No MeSH data available.


Related in: MedlinePlus