scholarly journals In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

2021 ◽  
Author(s):  
Elsa Mazari-Arrighi ◽  
Teru Okitsu ◽  
Hiroki Teramae ◽  
Hoshimi Aoyagi ◽  
Mahiro Kiyosawa ◽  
...  
Keyword(s):  
Culture System ◽  
Rat Hepatocytes ◽  
Matrix Proteins ◽  
Primary Hepatocytes ◽  
In Vitro Proliferation ◽  
Primary Rat Hepatocytes ◽  
Fiber Technology ◽  
Long Term Preservation

Abstract Primary hepatocytes are essential cellular resource for drug screening and medical transplantation. Since culture systems for them have already succeeded in reconstituting the biomimetic microenvironment, acquiring additional capabilities both to expand primary hepatocytes and to handle them easily would be expected as progress to the next stage. This paper describes a culture system for primary rat hepatocytes that is equipped with scalability and handleability relying on cell fiber technology. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium through the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated they increase in number while keeping their viability and their hepatic specific functions for up to thirty days of subsequent culture. Then, we demonstrated the potency of the primary rat hepatocytes that proliferate in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Therefore, our culture system could serve for innovating strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

scholarly journals In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

2021 ◽  
Author(s):  
Elsa Mazari-Arrighi ◽  
Teru Okitsu ◽  
Hiroki Teramae ◽  
Hoshimi Aoyagi ◽  
Mahiro Kiyosawa ◽  
...  
Keyword(s):  
Culture System ◽  
Rat Hepatocytes ◽  
Matrix Proteins ◽  
Primary Hepatocytes ◽  
In Vitro Proliferation ◽  
Primary Rat Hepatocytes ◽  
Fiber Technology ◽  
Long Term Preservation

Primary hepatocytes are essential cellular resource for drug screening and medical transplantation. Since culture systems for them have already succeeded in reconstituting the biomimetic microenvironment, acquiring additional capabilities both to expand primary hepatocytes and to handle them easily would be expected as progress to the next stage. This paper describes a culture system for primary rat hepatocytes that is equipped with scalability and handleability relying on cell fiber technology. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium through the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated they increase in number while keeping their viability and their hepatic specific functions for up to thirty days of subsequent culture. Then, we demonstrated the potency of the primary rat hepatocytes that proliferate in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Therefore, our culture system could serve for innovating strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

scholarly journals In- vitro Hepatoprotective Action of the Crude Extracts of Luffa amara (Whole Fruit) and Rheum emodi (Rhizomes) in CCl4 Intoxicated Rat Hepatocytes

2021 ◽  
pp. 396-404
Author(s):  
Muna Abid ◽  
Zakia Abid ◽  
B. Syed Asad ◽  
Mohammed Ibrahim
Keyword(s):  
Petroleum Ether ◽  
Aqueous Extract ◽  
Protective Action ◽  
Elevated Serum ◽  
Ethanolic Extract ◽  
Rat Hepatocytes ◽  
Ether Extract ◽  
Isolated Rat Hepatocytes ◽  
Primary Rat Hepatocytes

Aim: The objective of this in-vitro study involves evaluating the protective action of the extracts of L. amara (LA) (whole fruits including seeds) and R. emodi (RE) (rhizomes) at various concentrations on isolated primary rat hepatocytes. Methods: The pulverised dried whole fruits of L. amara (LA) and rhizomes of R.emodi (RE) were extracted successively with petroleum ether (PE), ethanol (EE) and distilled water (AE) and vacuum dried. These extracts of LA petroleum ether (PE), ethanolic (EE) and aqueous (AE) extracts and RE obtained were subjected to in vitro studies at doses of 25, 75, 100, and 150 µg/ml and silymarin (250 µg/ml) in CCl4 (1%) intoxicated primary hepatocytes monolayer cultures the hepatoprotective action of all the extracts of both plants at different doses was carried out using isolated rat hepatocytes which were subjected to CCl4 intoxication followed by estimating/ measuring the changes in serum biochemical markers – SGPT, SGOT, ALP, Total proteins (TP), total bilirubin (TB), albumin (ALB) and triglycerides (TGL). Results: Hepatoprotective activity against CCl4 was demonstrated in the rat primary monolayer hepatocyte culture using MMT assay with the ethanolic extracts of both plants showing more hepatocyte protective action compared to the aqueous and petroleum ether extracts by reducing the elevated serum marker levels. Alcoholic and aqueous extracts were found to express more protective action towards CCl4 intoxicated isolated primary rat hepatocytes in a dose dependant manner. Conclusion: Based on the result, it is suggested that the extract with the most hepatocyte protective action at a dose of 150µg is LA ethanolic extract (viability=88.24%), followed by LA aqueous extract (viability=84.31%), RE ethanolic extract (viability=88.24%) and RE aqueous extract (viability=88.24%) - which are comparable to the reference silymarin with viability at 92.15%. the petroleum ether extracts of both plants showed least hepatic cell viability with LA pet ether extract at 49.02% and RE pet ether extract at 47.85%

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2339-2348 ◽  
Author(s):  
B. Pain ◽  
M.E. Clark ◽  
M. Shen ◽  
H. Nakazawa ◽  
M. Sakurai ◽  
...  
Keyword(s):  
Culture System ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Telomerase Activity ◽  
Specific Antibodies ◽  
Typical Morphology ◽  
Germinal Tissue

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185–189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

scholarly journals In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand.

1992 ◽  
Vol 176 (2) ◽  
pp. 351-361 ◽  
Author(s):  
H Kodama ◽  
M Nose ◽  
Y Yamaguchi ◽  
J Tsunoda ◽  
T Suda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cells ◽  
In Vitro Proliferation ◽  
Kit Receptor ◽  
Colony Forming Units ◽  
Marrow Cells

The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.

Leukemia Bone Marrow Primary Cells Long-Term Culture in a Biomimetic Hematopoietic Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4114-4114
Author(s):  
Li Hou ◽  
Ting Liu ◽  
Jing Tan ◽  
Wentong Meng ◽  
Li Deng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Culture System ◽  
3D Culture ◽  
Primary Cells ◽  
Marrow Mesenchymal Stem Cells ◽  
3D Culture System ◽  
2D System

Abstract We have constructed a biomimetic hematopoietic niche (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts. Our primary results showed that the biomimetic 3D culture system is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. But so far, leukemia primary cells long-term culture from patients marrow are still difficult because it is not clear how does the regulation of leukemic cells grow ex vivo, and lack of adequate investigation between leukemic stem cells with stromal cells. Based on our previous research, we cultured bone marrow mesenchymal stem cells from chronic myelogenous leukemia (CML) patients, and conceived a “pathologic biomimetic osteoblast niche”, to explore the growth of leukemia bone marrow primary cells from CML patients. Bio-derived bone was composited with marrow mesenchymal stem cells from CML patients and constructed a 3D biomimetic osteoblast niche. The mononuclear cells (MNCs) were collected with standard Ficoll-Paque separation from newly diagnosed CML patients. The MNCs were cultured for 2∼5 weeks in the 3D culture system and compared with 2D culture system. The results showed that the proportion of CD34+ cells are increased either in 3D or 2D culture systems. Compared to input, the proportion of CD34+ cells were increased 6.52(1.87∼9)vs. 3.18(1.07∼6.8)times at 2 weeks culture, and 13.6(3.59∼26.31)vs. 7.86(0.78∼18.0)times at 5 weeks culture. The proportion of CD34+/CD38- was higher in 3D culture system than 2D system. It was 5.55(2.1∼11.7)% vs. 2.4(0.9∼3.4)%, and 13.5(3.4∼34.2)% vs. 4.83(2.1∼8.9)% at 2 weeks and 5 weeks respectively. 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 more colonies than 2D system {103.33(82∼144)vs. 79(53∼122)} at 2 week culture and 47(33∼66)vs. 21.67(16∼27)at 5 week culture. LTC-IC are widely used as a surrogate in vitro culture for pluripotent stem cells, and those primitive progenitor cells responsible for leukemia in mice are named SL-IC or leukemia stem cells (LSCs). 3D system showed higher frequency of LTC-IC than that of 2D system after 2-week culture(2.23E-05(1.73∼2.56)vs.1.40E-05(1.21∼1.73)). FISH showed the proportion of Ph+ cells declined in both system during the culture, but not as rapidly as it did in 2D system{65%(3D)vs.63%(2D)at 2 week, 55%(3D)vs.35%(2D)at 5 week}, and the Ph+ cells were predominant derived from 3D culture. Our 3D culture system constructed with induced osteoblasts from mesnchymal stem cells in CML patients might provide a more suitable microenvironment for leukemic cells growing in vitro. The leukemic stem cells seemed to be regulated by the molecular signals mediated by osteoblast, and the biological characteristics of leukemia stem cells at least partially is maintained. It may be become a new method for studying leukemic HSCs/HPCs behavior in vitro.

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