Density Gradient Centrifugation for the Isolation of Cells of Multiple Lineages

Yamamoto, Yumiko; Itoh, Shousaku; Yamauchi, Y.; Matsushita, Kenta; Ikeda, Shoichiro; Naruse, Haruna; Hayashi, Mikako

doi:10.1002/jcb.25270

Cited by 11 publications

(8 citation statements)

References 32 publications

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“…One significant drawback of isolating MSCs from bone marrow is the contamination of blood cells and hematopoietic stem cells. Various techniques have been used to purify or enrich the MSC population isolated from bone marrow including preferential attachment to culture plastic, density gradient centrifugation (Yamamoto et al, 2015), use of ficole to filter out blood cells, antibody-based cell sorting (Van Vlasselaer et al, 1994; El-Sayed et al, 2015), low and high density culture techniques (Eslaminejad and Nadri, 2009), and frequent media change (Soleimani and Nadri, 2009). However, these methods used to purify MSCs isolated from bone marrow have a number of negative effects.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Differentiation of Mesenchymal Stem Cells From Broiler Chicken Compact Bones

et al. 2019

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Chicken mesenchymal stem cells (MSCs) can be used as an avian culture model to better understand osteogenic, adipogenic, and myogenic pathways and to identify unique bioactive nutrients and molecules which can promote or inhibit these pathways. MSCs could also be used as a model to study various developmental, physiological, and therapeutic processes in avian and other species. MSCs are multipotent stem cells that are capable of differentiation into bone, muscle, fat, and closely related lineages and express unique and specific cell surface markers. MSCs have been isolated from numerous sources including human, mouse, rabbit, and chicken with potential clinical and agricultural applications. MSCs from chicken compact bones have not been isolated and characterized yet. In this study, MSCs were isolated from compact bones of the femur and tibia of day-old male broiler chicks to investigate the biological characteristics of the isolated cells. Isolated cells took 8–10 days to expand, demonstrated a monolayer growth pattern and were plastic adherent. Putative MSCs were spindle-shaped with elongated ends and showed rapid proliferation. MSCs demonstrated osteoblastic, adipocytic, and myogenic differentiation when induced with specific differentiation media. Cell surface markers for MSCs such as CD90, CD105, CD73, CD44 were detected positive and CD31, CD34, and CD45 cells were detected negative by PCR assay. The results suggest that MSCs isolated from broiler compact bones (cBMSCs) possess similar biological characteristics as MSCs isolated from other chicken tissue sources.

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Section: Introductionmentioning

confidence: 99%

Isolation and Differentiation of Mesenchymal Stem Cells From Broiler Chicken Compact Bones

et al. 2019

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“…Unfortunately, calcium deposition produced by cultured osteoblasts during differentiation hampers cell surface marker analysis by flow cytometry. Yamamoto et al 24 established a method to remove calcium deposits from cultured osteoblasts using Percoll density gradient centrifugation. This method allows studying surface markers on differentiated osteoblasts by flow cytometry in the absence of calcium deposition 24 .…”

Section: Discussionmentioning

confidence: 99%

“…Yamamoto et al 24 established a method to remove calcium deposits from cultured osteoblasts using Percoll density gradient centrifugation. This method allows studying surface markers on differentiated osteoblasts by flow cytometry in the absence of calcium deposition 24 . At first, expression of CD44, CD73, CD105, CD106, Sca‐1, and SSEA‐1, which are likely murine MSC markers, was analyzed.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, as calcium deposits produce autofluorescence, the risk of false positivity is high if calcium deposits contaminate samples during fluorescence‐activated cell sorting analysis. To address these problems, we created a new method of separating living osteoblasts from calcium deposits 24 . This method can separate differentiated osteoblasts from calcium deposits using Percoll density gradient centrifugation.…”

Section: Introductionmentioning

confidence: 99%

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Intracellular density is a novel indicator of differentiation stages of murine osteoblast lineage cells

Itoh

Naruse

et al. 2021

J of Cellular Biochemistry

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Osteoblasts are primary bone‐making cells originating from mesenchymal stem cells (MSCs) in the bone marrow. The differentiation of MSCs to mature osteoblasts involves an intermediate stage called preosteoblasts, but the details of this process remain unclear. This study focused on the intracellular density of immature osteoblast lineage cells and hypothesized that the density might vary during differentiation and might be associated with the differentiation stages of osteoblast lineage cells. This study aimed to clarify the relationship between intracellular density and differentiation stages using density gradient centrifugation. Primary murine bone marrow stromal cell cultures were prepared in an osteogenic induction medium, and cells were separated into three fractions (low, intermediate, and high‐density). The high‐density fraction showed elevated expression of osteoblast differentiation markers (Sp7, Col1a1, Spp1, and Bglap) and low expression of MSC surface markers (Sca‐1, CD73, CD105, and CD106). In contrast, the low‐density fraction showed a high expression of MSC surface markers. These results indicated that intracellular density increased during differentiation from preosteoblasts to committed osteoblasts. Intracellular density may be a novel indicator for osteoblast differentiation stages. Density gradient centrifugation is a novel technique to study the process by which preosteoblasts transform into bone‐forming cells.

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“…On the other hand, density gradient centrifugation is a well‐known technique for the purification of cellular organelles . If exosomes are considered as such, this ultracentrifugation variation is well within the capabilities of performing successful exosome isolations.…”

Section: Exosome Recovery and Purificationmentioning

confidence: 99%

Recent advances and challenges in the recovery and purification of cellular exosomes

et al. 2019

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Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome‐based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.

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Density Gradient Centrifugation for the Isolation of Cells of Multiple Lineages

Cited by 11 publications

References 32 publications

Isolation and Differentiation of Mesenchymal Stem Cells From Broiler Chicken Compact Bones

Isolation and Differentiation of Mesenchymal Stem Cells From Broiler Chicken Compact Bones

Intracellular density is a novel indicator of differentiation stages of murine osteoblast lineage cells

Recent advances and challenges in the recovery and purification of cellular exosomes

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