Novel three-dimensional long-term bone marrow culture system using polymer particles with grafted epoxy-polymer-chains supports the proliferation and differentiation of hematopoietic stem cells

2011 ◽  
Vol 236 (11) ◽  
pp. 1342-1350 ◽  
Author(s):  
Yukio Hirabayashi ◽  
Yoshihiro Hatta ◽  
Jin Takeuchi ◽  
Isao Tsuboi ◽  
Tomonori Harada ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture System ◽  
3D Structure ◽  
Three Dimensional ◽  
Hematopoietic Cells ◽  
3D Culture ◽  
Polymer Chains ◽  
Polymer Particles ◽  
Hematopoietic Stem ◽  
3D Culture System

Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34+ cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34+ cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell–stromal cell interactions.

Maintenance and Expansion of Hematopoietic Stem/Progenitor Cells in a Biomimetic Hematopoietic Cells Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4115-4115
Author(s):  
Ting Liu ◽  
Jing Tan ◽  
Li Hou ◽  
Wentong Meng ◽  
Yuchun Wang
Keyword(s):  
Progenitor Cells ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
3D Culture System ◽  
2D System

Abstract As main hematopoietic organ, bone marrow have three dimensional microenvironment for hematopoietic stem/progenitor cells grow in, so call “hematopoietic cells niche”, which are composed by stromal cells and extracelluar matrix. The interactions of cell to cell and cell to matrix between stem/progenitor cells with hematopoietic niche are facilitated by its three dimensional conformation. The biology behaviors of hematopoietic stem cells are mediate by many signal transductions between stem/progenitor cells with their corresponding microenvironment. Now there are strong evidence from animal model study suggests that osteoblasts might play an essential role in creation of a hematopoietic stem cell niche and thereby regulation of stem cell maintenance, proliferation, and maturation. In light of the structure-function relationship of bone marrow topography, we conceived a biomimetic culture system (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts to simulate the effects of hematopoietic osteobalst niche. CD 34+ cells or mononuclear cells separated from umbilical cord blood were cultured for 2∼5 weeks in the 3D culture system and also in a conventional 2D culture system as control without additional cytokine supplement. Based on our results, higher expression of extracelluar matrix and N-cadherin were observed in 3D culture system compared to 2D system. At 2 weeks culture, 3D culture system showed higher number of CD34+ cells and CD34+/CD38- cells when compared with the input (P<0.05), the increased cells were predominant CD34+/CD38-cells. Although CD34+ cells were decreased at 5 weeks culture; nevertheless, CD34+/CD38- cells were still maintained at high level. We also observed that imbedding MNCs with a higher percentage of CD34+/CD38-cells cultured in 3D system (P<0.05), which may represent a down regulation of CD38 phenotype during culture. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced higher expansion of CFU progenitors than 2D system (7.13–8.89 times vs. 1.22–1.31times) after 2 weeks culture. Of note, the colony distribution of 3D system manifested higher percentage of BFU-E and CFU-GEMM, while 2D system showed mainly CFU-GM. LTC-IC represents the primitive progenitor, 3D system showed a 6.2 times increment over input after 2 weeks culture. Furthermore, it was competent to maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. This study demonstrated that our 3D culture system constructed with the bio-derived bone composited with induced osteoblast is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. It may open a new avenue to study HSCs/HPCs behaviors and to achieve sustained primitive progenitor cell expansion.

Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer

2019 ◽  
Vol 42 (7) ◽  
pp. 374-379 ◽  
Author(s):  
Hirotoshi Miyoshi ◽  
Chiaki Sato ◽  
Yuichiro Shimizu ◽  
Misa Morita
Keyword(s):  
Progenitor Cells ◽  
Mitomycin C ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Three Dimensional ◽  
Hematopoietic Cells ◽  
Liver Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

scholarly journals A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro

Reproduction ◽  
2011 ◽  
Vol 141 (6) ◽  
pp. 809-820 ◽  
Author(s):  
Candace M Tingen ◽  
Sarah E Kiesewetter ◽  
Jennifer Jozefik ◽  
Cristina Thomas ◽  
David Tagler ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture System ◽  
Stromal Cell ◽  
Ovarian Follicle ◽  
3D Culture ◽  
Culture Conditions ◽  
Growth And Survival ◽  
3D Culture System

Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.

Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation

2013 ◽  
Vol 13 (1) ◽  
pp. 32-43 ◽  
Author(s):  
Francis H. Shen ◽  
Brian C. Werner ◽  
Haixiang Liang ◽  
Hulan Shang ◽  
Ning Yang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Culture System ◽  
3D Culture ◽  
Adipose Derived Stromal Cells ◽  
3D Culture System

Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system

2015 ◽  
Vol 33 (5-6) ◽  
pp. 347-355 ◽  
Author(s):  
Tomonori Harada ◽  
Yukio Hirabayashi ◽  
Yoshihiro Hatta ◽  
Isao Tsuboi ◽  
Wilhelm Robert Glomm ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Culture System ◽  
Three Dimensional ◽  
Bone Marrow Culture ◽  
Hematopoietic Stem ◽  
Kinetics Of

scholarly journals Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Olga Schmal ◽  
Jan Seifert ◽  
Tilman E. Schäffer ◽  
Christina B. Walter ◽  
Wilhelm K. Aicher ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Three Dimensional ◽  
3D Culture ◽  
Hanging Drop ◽  
Mixed Cell ◽  
Hematopoietic Stem ◽  
Extracellular Matrix Components ◽  
Stem And Progenitor Cells

Efficientex vivoexpansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

scholarly journals Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation

2020 ◽  
Vol 8 (9) ◽  
pp. 2514-2525
Author(s):  
Carolina Åstrand ◽  
Veronique Chotteau ◽  
Anna Falk ◽  
My Hedhammar
Keyword(s):  
Culture System ◽  
Neural Differentiation ◽  
Spider Silk ◽  
Three Dimensional ◽  
3D Culture ◽  
Silk Protein ◽  
Spider Silk Protein ◽  
3D Culture System ◽  
Three Dimensional Culture

The functionalized recombinant spider silk protein FN-silk can self-assemble into a 3D microfiber network. When combined with recombinant laminin521 it provides a 3D culture system suitable for expansion of hPSCs and following neural differentiation.

scholarly journals The Three-Dimensional Culture System with Matrigel and Neurotrophic Factors Preserves the Structure and Function of Spiral Ganglion NeuronIn Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Gaoying Sun ◽  
Wenwen Liu ◽  
Zhaomin Fan ◽  
Daogong Zhang ◽  
Yuechen Han ◽  
...  
Keyword(s):  
Growth Cone ◽  
Neurotrophic Factors ◽  
Culture System ◽  
Spiral Ganglion ◽  
Three Dimensional ◽  
3D Culture ◽  
Explant Culture ◽  
Structure And Function ◽  
3D Culture System ◽  
And Function

Whole organ culture of the spiral ganglion region is a resourceful model system facilitating manipulation and analysis of live sprial ganglion neurons (SGNs). Three-dimensional (3D) cultures have been demonstrated to have many biomedical applications, but the effect of 3D culture in maintaining the SGNs structure and function in explant culture remains uninvestigated. In this study, we used the matrigel to encapsulate the spiral ganglion region isolated from neonatal mice. First, we optimized the matrigel concentration for the 3D culture system and found the 3D culture system protected the SGNs against apoptosis, preserved the structure of spiral ganglion region, and promoted the sprouting and outgrowth of SGNs neurites. Next, we found the 3D culture system promoted growth cone growth as evidenced by a higher average number and a longer average length of filopodia and a larger growth cone area. 3D culture system also significantly elevated the synapse density of SGNs. Last, we found that the 3D culture system combined with neurotrophic factors had accumulated effects in promoting the neurites outgrowth compared with 3D culture or NFs treatment only groups. Together, we conclude that the 3D culture system preserves the structure and function of SGN in explant culture.

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