scholarly journals Osteogenic cells form mineralized particles, a few μm in size, in a 3D collagen gel culture

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7889 ◽  
Author(s):  
Takanori Kihara ◽  
Chiya Umezu ◽  
Karin Sawada ◽  
Yukari Furutani
Keyword(s):  
Culture System ◽  
Culture Media ◽  
Collagen Gel ◽  
Matrix Vesicles ◽  
Osteogenic Cells ◽  
Collagen Gel Culture ◽  
3D Collagen ◽  
Gel Culture

Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with β-glycerophosphate (β-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing β-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.

Characterization of Adult Stem/Progenitor Cell Populations from Bone Marrow in a Three-Dimensional Collagen Gel Culture System

2012 ◽  
Vol 21 (9) ◽  
pp. 2021-2032 ◽  
Author(s):  
Silvia Claros ◽  
Noela Rodríguez-Losada ◽  
Encarnación Cruz ◽  
Enrique Guerado ◽  
José Becerra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Culture System ◽  
Progenitor Cell ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Cell Populations ◽  
Collagen Gel Culture ◽  
Gel Culture

Roles of Osteoclasts in Leukemic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3184-3184 ◽  
Author(s):  
Asumi Yokota ◽  
Shinya Kimura ◽  
Ruriko Tanaka ◽  
Rina Nagao ◽  
Kazuki Sakai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Resorption ◽  
Culture System ◽  
Bone Matrix ◽  
Collagen Gel ◽  
Leukemic Cells ◽  
Response To Treatment

Abstract We have previously reported that zoledoronic acid (ZOL) augmented the in vivo effect of imatinib in a murine chronic myeloid leukemia (CML) model (Blood 2003). ZOL alone induces apoptosis in leukemic cells in vitro by inhibiting prenylation of the Ras-related proteins. In addition to this direct anti-leukemic effect, we hypothesized that ZOL also has some influence in leukemic cells in vivo indirectly by destroying osteoclasts (OCs), which is the primary therapeutic activity of ZOL in osteoporosis patients. Supporting this notion is that by mediating bone resorption, OCs release a variety of cytokines such as IGF- 1, TGF-β, etc. that have accumulated in the bone matrix. It has been reported that OCs play an important role in bone metastasis of solid tumor, especially in cancer stem cells. However, little is known about the role of OCs in leukemia. Therefore, we investigated it in vitro and in vivo. For this purpose, we established an in vitro osteoblasts (OBs) and OCs co-culture system. The stable co-culture system that we developed includes collagen gel and murine primary OBs and OCs. In addition, murine femoral bone sections were sometimes added to this culture system so that the OCs could release the cytokines from the bone matrix. Thus, the collagen gel and OBs were placed in 12-well plates with and without bone sections and/or OCs. The transwell chambers over the wells then received 1×104 Ba/F3 cells that had been transfected with wild type bcr-abl (Ba/F3/bcr-abl cells). OBs markedly enhanced the growth of Ba/F3/bcr-abl cells in this indirect contact coculture system whereas the presence of both OBs and OCs slightly suppressed cell growth. Intriguingly, when bone sections were added (OBs+OCs+bone), Ba/F3/bcr-abl cell proliferation was significantly suppressed compared to the effect of OBs alone or OBs+OCs (Figure). Cell cycle analysis revealed that the G0/G1 population was increased in Ba/F3/bcr-abl cells co-cultured with OBs+OCs+bones. We also observed that the p27 protein levels of Ba/F3/bcr-abl cells increased upon co-culture with OCs or OCs+bones, similar to their response to treatment with purified TGF-β. We performed ELISAs to determine the concentrations of cytokines in the supernatants of co-cultured OBs and OCs. There were higher levels of TGF-β1 in the OBs+OCs+bones supernatant than in the OBs+OCs supernatant. Furthermore, OBs produced high levels of IGF-1. These findings suggest that OBs and OCs affect the proliferation and the cell cycle arrest of leukemic cells by releasing soluble factors, respectively. To more comprehensively elucidate the roles OCs play in leukemia cells in vivo, we used reveromycin A (RM-A) which inhibits bone resorption by specifically inducing apoptosis in OCs (Woo et al, PNAS 2006). RM-A did not have any in vitro effects on the proliferation of Ba/F3/bcr-abl cells. Thus, we could know the unalloyed role of OCs in leukemia with RM-A compared with ZOL which inhibited directly both OCs and leukemic cells. Our preliminary data show that RM-A suppresses the engraftment of inoculated Ba/F3/bcr-abl cells to nude mice. We also present data from ongoing studies showing the effect of RM-A on leukemic cells in murine models. These findings suggested that OCs may be an important constituent of leukemia stem cell niche and destruction of OCs by either ZOL or RM-A is a novel strategy for leukemia treatment. Figure Figure

Collagen gel culture system and analysis of estrogen effects on mammary carcinogenesis

1984 ◽  
Vol 55 (2) ◽  
pp. 91-96 ◽  
Author(s):  
Satyabrata Nandi ◽  
Walter Imagawa ◽  
Yasuhiro Tomooka ◽  
Michael F. McGrath ◽  
Marc Edery
Keyword(s):  
Culture System ◽  
Collagen Gel ◽  
Mammary Carcinogenesis ◽  
Collagen Gel Culture ◽  
Estrogen Effects ◽  
Gel Culture

Empirical and physiological assessment of in vitro growth in the mermithid nematode Romanomermis culicivorax

1990 ◽  
Vol 68 (3) ◽  
pp. 511-516
Author(s):  
Roger Gordon ◽  
Marcel Cornect ◽  
Terry-Lynn Young ◽  
Kenneth T. Kean
Keyword(s):  
Lipid Content ◽  
Culture System ◽  
Culture Media ◽  
In Vitro Growth ◽  
Protein Levels ◽  
Physiological Assessment ◽  
Mermithid Nematode ◽  
Long Chain

In vitro growth of Romanomermis culicivorax preparasites was monitored in two culture media under differing conditions of crowding. Crowding was more important than the character of the medium itself in stimulating in vitro growth. The glycogen, protein, and lipid content of nematodes that had developed after 5 weeks under crowded conditions was significantly greater than that of uncrowded nematodes. The lipid content of such crowded nematodes was greater than that of nematodes after 4 days of in vivo development, although glycogen and protein levels were lower than the 4 day in vivo norm. Nematodes that had developed in vitro contained a higher proportion of long-chain and polyunsaturated fatty acids than did nematodes cultured in vivo. The significance of these findings is discussed in relation to the establishment of a continuous in vitro culture system for this nematode.

Embryonic development in culture of the marsupials Antechinus stuartii (Macleay) and Sminthopsis macroura (Spencer) during preimplantation stages

1993 ◽  
Vol 5 (4) ◽  
pp. 445 ◽  
Author(s):  
A Yousef ◽  
L Selwood
Keyword(s):  
Culture System ◽  
Fetal Calf Serum ◽  
Normal Development ◽  
Culture Media ◽  
Calf Serum ◽  
Human Amniotic Fluid ◽  
Antechinus Stuartii ◽  
Sminthopsis Macroura

Forty-nine blastocysts from 11 brown antechinus, Antechinus stuartii, and 96 blastocysts from 17 stripe-faced dunnarts, Sminthopsis macroura, were used to develop a culture system for embryos during preimplantation stages. Blastocysts of brown antechinus were collected on Days 6-9 for unilaminar stages, Days 16-21 for bilaminar stages and Days 20 and 21 for trilaminar stages. Blastocysts of stripe-faced dunnarts were collected on Day 6 for unilaminar stages, Days 6-8 for bilaminar stages and Day 8 for trilaminar stages. Culture media were Dulbecco's modified Eagle's medium (DMEM) with 4.5% glucose and Whittingham's T6 medium both of which were supplemented with 5, 10, 12.5 and 20% fetal calf serum (FCS). Antechinus serum (5%) and bovine serum albumin (0.1%, 0.2%) were also added to some media. Human amniotic fluid (HAF) and Monomed media were also tested. Blastocysts were cultured at 35 degrees C in 5% CO2 in air. DMEM + 10% FCS and HAF supported normal development for the longest periods and over the greatest range of stages. Developmental failure of blastocysts in vitro during expansion of the unilaminar blastocyst and formation of the bilaminar blastocyst suggests that these stages may be dependent on uterine signals. When cultured in DMEM + 10% FCS, the rate of development of bilaminar and trilaminar blastocysts into organogenesis was 4 h slower than in vivo in the stripe-faced dunnart and about 6 h slower than in vivo in the brown antechinus. Embryos of stripe-faced dunnarts were cultured to within 18 h of birth.

The development of in vitro fertilized (ivf) cattle ova in an oviduct cell co-culture system

1992 ◽  
Vol 1992 ◽  
pp. 41-41
Author(s):  
C. McCaffrey ◽  
M.G. Diskin ◽  
K.H. Lu ◽  
J.M. Sreenan
Keyword(s):  
Culture System ◽  
Culture Media ◽  
Cell Stage ◽  
Conventional Culture ◽  
Large Numbers ◽  
Successful Production ◽  
Oviduct Cell ◽  
Developmental Block

There have been many recent reports of the successful production of large numbers of in vitro matured (IVM) and in vitro fertilised (IVF) cow ova. While a high proportion (80-90%) of oocytes undergo apparently normal maturation and fertilisation in vitro, only 15-20% develop in vitro to the morulae and blastocyst stages. Early cleavage stage in vivo matured and fertilised cow ova also encounter a developmental “block” at the 8-16-cell stage during in vitro culture in conventional culture media. However, this block has been readily overcome by co-culture involving the use of bovine oviducal cells, with up to 80% of 1-4-cell ova developing to morulae and blastocysts: The present study examined the development of IVM-IVF cattle ova removed at 24 or 48 hours from the insemination medium and cultured on cattle oviducal cells and also compared the development of IVM-IVF and in vivo produced ova in the same oviducal cell co-culture system.

scholarly journals Reconstruction Culture System Simulating Human Synovium: A Three-dimensional Collagen Gel Culture of Synoviocytes.

1997 ◽  
Vol 22 (4) ◽  
pp. 443-453
Author(s):  
Katsuki Taguchi ◽  
Torn Hirano ◽  
Katsuro Iwasaki ◽  
Hajime Sugihara
Keyword(s):  
Culture System ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Collagen Gel Culture ◽  
Gel Culture

scholarly journals In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

2014 ◽  
Vol 26 (7) ◽  
pp. 943 ◽  
Author(s):  
Catherine N. Sargus-Patino ◽  
Elane C. Wright ◽  
Sarah A. Plautz ◽  
Jeremy R. Miles ◽  
Jeff L. Vallet ◽  
...  
Keyword(s):  
Cell Survival ◽  
Culture System ◽  
Culture Media ◽  
Morphological Changes ◽  
Three Dimensional ◽  
In Vitro Development ◽  
Alginate Hydrogels ◽  
Vitro Development

Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96 h. Every 24 h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96 h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P < 0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P < 0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC–), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P < 0.001) in 17β-oestradiol was observed in culture media from ENC+ compared with ENC– and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen production, consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.

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