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Optimization of Three Dimensional Culturing of the HepG2 Cell Line in Fibrin Scaffold.

Banihashemi M, Mohkam M, Safari A, Nezafat N, Negahdaripour M, Mohammadi F, Kianpour S, Ghasemi Y - Hepat Mon (2015)

Bottom Line: In a one-stage experimental design, Box-Behnken design strategy was performed by Minitab 15 software (version 15, Minitab.The optimal concentrations for fibrin scaffold fabrication were achieved by adding 0.15 mol CaCl2 (50 µL) and 1 × 10(5) cells to 1:4 of plasma/RPMI 1640 ratio (500 µL with 2.3 mm thickness per well).Our approach provided easy handle method using inexpensive materials like human plasma instead of purified fibrinogen to fabricate fibrin scaffold.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

ABSTRACT

Background: A potential treatment for healing hepatic tissue is delivering isolated hepatic cells to the site of injury to promote hepatic cells formation. In this technology, providing an appropriate injectable system for delivery of hepatic cells is an important issue. In this regard, fibrin scaffolds were designed with many advantages over other scaffolds like cell delivery vehicles for biodegradation, biocompatibility and hemostasis.

Objectives: The aim of this study was to determine suitable cell culture circumstances for HepG2 cell proliferation and differentiation in 3D fibrin scaffolds by evaluating Ca(2+) concentrations, cell numbers, various ratios of plasma/RPMI 1640 and thickness of fibrin scaffold.

Materials and methods: In a one-stage experimental design, Box-Behnken design strategy was performed by Minitab 15 software (version 15, Minitab. State College, PA) with three factors at three levels (low, medium and high) and 27 runs for identification of the effects of ratio of plasma/RPMI 1640, Ca(2+) concentration and thickness on the formation of fibrin gel scaffold and 3D HepG2 culture.

Results: The optimal concentrations for fibrin scaffold fabrication were achieved by adding 0.15 mol CaCl2 (50 µL) and 1 × 10(5) cells to 1:4 of plasma/RPMI 1640 ratio (500 µL with 2.3 mm thickness per well).

Conclusions: Our approach provided easy handle method using inexpensive materials like human plasma instead of purified fibrinogen to fabricate fibrin scaffold.

No MeSH data available.


Related in: MedlinePlus

Light Microscopy Evaluation of HepG2 Morphology, After 10 Days of Incubation in A) Fibrin Scaffold 10 × and B) in 2D Routine Polystyrene Matrix 40 × (B)
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fig17953: Light Microscopy Evaluation of HepG2 Morphology, After 10 Days of Incubation in A) Fibrin Scaffold 10 × and B) in 2D Routine Polystyrene Matrix 40 × (B)

Mentions: The cultivation of HepG2 cell line in routine 2D flask and 3D fibrin scaffold showed significant differences in obtained cells morphology. The grown cells in 2D flask (usually polystyrene surfaces) showed flat extended structures after two days of cultivation. They were also heterogeneous and showed as disarrangement in their appearance. Moreover, after seven days of cultivation, the cells formed aggregates and appeared as unhealthy in which some cells were disintegrating and others were rounding up (data not shown). In contrast, 3D cultivation of HepG2 cell line in fibrin scaffold led to more homogeneous cells compared to their 2D counterparts. In this system, HepG2 cell line could intimately interact with the surrounding fibrin scaffold containing plasma and the nutritious environment of human plasma could help these cells to differentiate and proliferate appropriately. Most observed cells in 3D fibrin scaffold had spheroid shape, forming multicellular spheroids (Figure 1). This aggregation is necessary for re-establishment of cell-cell contacts to function as a natural tissue (18). Some researchers reported that existence of extracellular matrices (ECMs) in plasma promoted cell morphologies, phenotypes, differentiated functions and metabolic activities of hepatocytes in a 3D configuration in-vitro (19, 20). These data showed that fibrin scaffold containing human plasma provide an inspiring material for differentiation and cell adhesion studies (21, 22).


Optimization of Three Dimensional Culturing of the HepG2 Cell Line in Fibrin Scaffold.

Banihashemi M, Mohkam M, Safari A, Nezafat N, Negahdaripour M, Mohammadi F, Kianpour S, Ghasemi Y - Hepat Mon (2015)

Light Microscopy Evaluation of HepG2 Morphology, After 10 Days of Incubation in A) Fibrin Scaffold 10 × and B) in 2D Routine Polystyrene Matrix 40 × (B)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385269&req=5

fig17953: Light Microscopy Evaluation of HepG2 Morphology, After 10 Days of Incubation in A) Fibrin Scaffold 10 × and B) in 2D Routine Polystyrene Matrix 40 × (B)
Mentions: The cultivation of HepG2 cell line in routine 2D flask and 3D fibrin scaffold showed significant differences in obtained cells morphology. The grown cells in 2D flask (usually polystyrene surfaces) showed flat extended structures after two days of cultivation. They were also heterogeneous and showed as disarrangement in their appearance. Moreover, after seven days of cultivation, the cells formed aggregates and appeared as unhealthy in which some cells were disintegrating and others were rounding up (data not shown). In contrast, 3D cultivation of HepG2 cell line in fibrin scaffold led to more homogeneous cells compared to their 2D counterparts. In this system, HepG2 cell line could intimately interact with the surrounding fibrin scaffold containing plasma and the nutritious environment of human plasma could help these cells to differentiate and proliferate appropriately. Most observed cells in 3D fibrin scaffold had spheroid shape, forming multicellular spheroids (Figure 1). This aggregation is necessary for re-establishment of cell-cell contacts to function as a natural tissue (18). Some researchers reported that existence of extracellular matrices (ECMs) in plasma promoted cell morphologies, phenotypes, differentiated functions and metabolic activities of hepatocytes in a 3D configuration in-vitro (19, 20). These data showed that fibrin scaffold containing human plasma provide an inspiring material for differentiation and cell adhesion studies (21, 22).

Bottom Line: In a one-stage experimental design, Box-Behnken design strategy was performed by Minitab 15 software (version 15, Minitab.The optimal concentrations for fibrin scaffold fabrication were achieved by adding 0.15 mol CaCl2 (50 µL) and 1 × 10(5) cells to 1:4 of plasma/RPMI 1640 ratio (500 µL with 2.3 mm thickness per well).Our approach provided easy handle method using inexpensive materials like human plasma instead of purified fibrinogen to fabricate fibrin scaffold.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.

ABSTRACT

Background: A potential treatment for healing hepatic tissue is delivering isolated hepatic cells to the site of injury to promote hepatic cells formation. In this technology, providing an appropriate injectable system for delivery of hepatic cells is an important issue. In this regard, fibrin scaffolds were designed with many advantages over other scaffolds like cell delivery vehicles for biodegradation, biocompatibility and hemostasis.

Objectives: The aim of this study was to determine suitable cell culture circumstances for HepG2 cell proliferation and differentiation in 3D fibrin scaffolds by evaluating Ca(2+) concentrations, cell numbers, various ratios of plasma/RPMI 1640 and thickness of fibrin scaffold.

Materials and methods: In a one-stage experimental design, Box-Behnken design strategy was performed by Minitab 15 software (version 15, Minitab. State College, PA) with three factors at three levels (low, medium and high) and 27 runs for identification of the effects of ratio of plasma/RPMI 1640, Ca(2+) concentration and thickness on the formation of fibrin gel scaffold and 3D HepG2 culture.

Results: The optimal concentrations for fibrin scaffold fabrication were achieved by adding 0.15 mol CaCl2 (50 µL) and 1 × 10(5) cells to 1:4 of plasma/RPMI 1640 ratio (500 µL with 2.3 mm thickness per well).

Conclusions: Our approach provided easy handle method using inexpensive materials like human plasma instead of purified fibrinogen to fabricate fibrin scaffold.

No MeSH data available.


Related in: MedlinePlus