Effects of three-dimensional culturing on osteosarcoma cells grown in a fibrous matrix: analyses of cell morphology, cell cycle, and apoptosis

Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three-dimensional (3-D) fibrous culture had better cell growth and higher metabolic rates than the two-dimensional (2-D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long-term (1 month) T-flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at approximately 15%. These results could explain why the fibrous bed bioreactor had good long-term stability and was advantageous for production of non-growth-associated proteins by animal cell cultures.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

Effects of pore size in 3-D fibrous matrix on human trophoblast tissue development

The effects of pore size in a 3-D polyethylene terephthalate (PET) nonwoven fibrous matrix on long-term tissue development of human trophoblast ED27 cells were studied. Thermal compression was used to modify the porosity and pore size of the PET matrix. The pore size distributions in PET matrices were quantified using a liquid extrusion method. Cell metabolic activities, estradiol production, and cell proliferation and differentiation were studied for ED27 cells cultured in the thermally compressed PET matrices with known pore structure characteristics. In general, metabolic activities and proliferation rate were higher initially for cultures grown in the low-porosity (LP) PET matrix (porosity of 0.849, average pore size of 30 microm in diameter) than those in the high-porosity (HP) matrix (porosity of 0.896, average pore size of 39 microm in diameter). However, 17beta-estradiol production and cell differentiation activity in the HP matrix surpassed those in the LP matrix after 12 days. The expression levels of cyclin B1 and p27kip1 in cells revealed progressively decreasing proliferation and increasing differentiation activities for cells grown in PET matrices. Also, difference in pore size controlled the cell spatial organization in the PET matrices and contributed to the tissue development in varying degrees of proliferation and differentiation. It was also found that cells grown on the 2-D surface behaved differently in cell cycle progression and did not show increased differentiation activities after growth had stopped and proliferation activities had lowered to a minimal level. The results from this study suggest that the 3-D cell organization guided by the tissue scaffold is important to tissue formation in vitro.     

Green fluorescent protein as a reporter of human mu-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris

Genetically engineered human osteosarcoma cells containing developmental endothelial locus-1 (del-1) gene were studied for production of Del-1, a protein that has the properties of an extracellular matrix protein and can regulate vascular morphogenesis and remodeling. Del-1 has been studied as a potential anti-angiogenesis drug targeting solid tumors. In this study, osteosarcoma cells were cultured in a fibrous-bed bioreactor (FBB) to continuously produce Del-1. The FBB was constructed by packing a polyester fibrous matrix into a 1.5-l spinner flask. The effects of media composition, including the serum content in the medium, and dilution rate on cell growth, metabolism, and Del-1 production were studied. A gradual reduction of serum content from 10% (v/v) to 0.5% (v/v) caused no loss in Del-1 production. However, the production of Del-1 decreased significantly in a serum-free medium, suggesting some nutrients present in the serum were important to culture viability and Del-1 production. The continuous FBB culture was stable for long-term production of Del-1, with a higher Del-1 titer than that normally obtained in T-flask cultures and overall productivity similar to the total production from 300 25-cm(2) T-flasks. Reducing geneticin in the medium from 250 microg ml(-1) to zero at later culturing stages had no significant effect on Del-1 production. The FBB was operated for a period of more than 4 months without any notable degeneration, and reached a final cell density of 3 x 10(8) cells ml(-1) of packing volume with >90% cell viability. The good reactor performance can be attributed to the three-dimensional environment provided by the fibrous matrix that allows for efficient mass transfer and cell immobilization and growth. Scanning electron microscopic and confocal scanning laser microscopic studies of the cell-matrix showed that cells formed large aggregates in the fibrous matrix and cell density was relatively uniform in the matrix.     

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions.

We have shown previously that recombinant NS/0 myelomas expressing sufficient amounts of E1B-19K were resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. However, no significant increase in monoclonal antibodies (MAb) was observed during the prolonged stationary phase of these batch cultures. Here, we show that E1B-19K can enhance cell survival and improve MAb productivity in high cell density perfusion culture. Typically, lymphoid cells grown under steady state in perfusion exhibit decreasing viabilities with concomitant accumulation of apoptotic cells. By modulating the ability of these cells to resist to induction of apoptosis in low nutrient environment, a 3-fold decrease in specific death rate from 0.22 day-1 for NS/0 control to 0.07 day-1 for E1B-19K cells was achieved, resulting in a significant improvement in cell viability throughout perfusion. E1B-19K cells at the perfusion plateau phase also exhibited a 3-fold reduction in specific growth rate concomitant with a lower percentage of S and higher percentage of G1 phase cells. This was associated with a 40% decrease in specific oxygen consumption rate, likely related to a reduction in the specific consumption rates of limiting nutrient(s). Expression of E1B-19K consequently had a significant impact on the steady-state viable cell density, allowing maintenance of 11.5 x 10(6) E1B-19K cells/mL versus 5.9 x 10(6) control NS/0 cells/mL for the same amount of fresh medium brought into the system (half a volume per day). Whereas MAb concentrations found in perfusion culture of control NS/0 myelomas were almost 3-fold higher than those found in batch culture; in the case of E1B-19K-expressing myelomas, the MAb concentration in perfusion was more than 7-fold higher than in batch. This was attributable to the 2-fold increase in viable cell plateau and to a 40% increase in the perfusion to batch ratio of specific MAb productivity (2.2-fold for E1B-19K myelomas versus 1.6-fold for NS/0 control).     

Long-term Continuous Production of Monoclonal Antibody by Hybridoma Cells Immobilized in a Fibrous-Bed Bioreactor

The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of ～8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 × 10⁸/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flask and spinner flask cultures. The continuous FBB produced IgG at a concentration of ～0.5 g/l, with reactor productivity of ～7 mg/h·l, which was about 23 times higher than those from conventional T-flask and spinner flask cultures. The IgG concentration can be further increased to ～0.67 g/l by using higher feed (glucose and glutamine) concentrations and running the reactor at a recycle batch or fed-batch mode. The long-term performance of this bioreactor was also evaluated. For a period of 36 days monitored, the MAb produced in the continuous well-mixed bioreactor at 50 h retention time (0.02/h dilution rate) was maintained at a steady concentration level of ～0.3 g/l with less than 8% drift. At the end of the study, it was found that ～25% of the cells were strongly attached to the fiber surfaces and the other ～75% entrapped or weakly immobilized in the fibrous matrix. The strongly attached cells had a high viability of ～90%, compared to ～75% for cells weakly immobilized and only ～1.4% for freely suspended cells, suggesting that the fibrous matrix preferentially retained and protected the viable (productive) cells. The FBB thus was able to maintain its long-term productivity because nonviable and dead cells were continuously washed off from the fibrous matrix. The high MAb concentration and production rate and excellent stability for continuous long-term production obtained in this study compare favorably to other bioreactor studies reported in the literature. The reactor performance can be further improved by providing better pH and aeration controls at higher feed concentrations. The FBB is easy to operate and scale-up, and thus can be used economically for industrial production of MAb.     

Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions

Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stability of antibody productivity is improved when hybridoma cells are entrapped in calcium alginate beads

Loss of monoclonal antibody (MAb) productivity in long-term, free-suspended cell culture is often attributed to the appearance of a nonproducing population of hybridoma cell (NP) in the culture which has a growth advantage over the producing population (P). However, when an NP appears in long-term culture of entrapped cells, it may not be able to take over the whole culture in a short period of time due to the limited growth of the entrapped cells. In order to examine the hypothesis that entrapped cells can have improved stability of MAb productivity due to limited cell growth, free-suspended cell culture and calcium alginate-entrapped cell culture with inocula consisting of a P and an NP were compared with regard to stability of MAb productivity in a repeated fed-batch culture. In free-suspended cell culture, the NP appeared to take over the whole culture within three batches, and thereby MAb production completely disappeared. In entrapped cell culture, an NP appeared to outgrow the P rapidly only during an exponential growth phase, resulting in a significant decrease in specific MAb productivity, q(MAb), from 11.58 mug/10(6) cell/day to 2.76 mug/10(6) cell/day. However, when the cell growth was limited in entrapped cell culture, the NP no longer outgrew the P rapidly, as indicated by the stable value of q(MAb). In addition, when the cells recovered from the alginate beads by citrate buffer treatment were subcultured in free-suspended cell culture, MAb production rapidly deteriorated and completely disappeared within two batches. Thus, the P present at a small fraction of viable cell concentration in the beginning of the free-suspended cell culture, which were previously entrapped in alginate beads, seemed to be outgrown rapidly by the NP. Taken together, the results obtained from these experiments support the hypothesis that the limited cell growth in entrapped cell culture, which keeps an NP from taking over the whole culture, is responsible, in part, for the improved stability of MAb productivity. (c) 1993 John Wiley & Sons, Inc.     

Three-dimensional polymeric systems for cancer cell studies

Three-dimensional (3-D) culture of cancer cells and of normal mammalian cells in a polymeric matrix is generally a better alternate model for understanding the regulation of cancer cell proliferation and for evaluation of different anticancer drugs. A substantial amount of evidence demonstrates important differences in the behavior of cells grown in monolayer, i.e., two-dimensional (2-D), and in 3-D cultures. Cancer cells grown in 3-D culture are more resistant to cytotoxic agents than cells in 2-D culture; growth of cells in vitro in 3-D requires a suitable polymer that provides a structural scaffold for cell adhesion and growth. Many naturally derived polymers as well as synthetic polymers have been investigated as scaffolds. The aim of this review is to overview the polymeric materials of natural and synthetic origin that are of specific interest to 3-D cell cultures, and discuss the development of new polymers that should be specifically designed for 3-D culture applications.     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis

Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 microm GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-beta (TGF-beta). Only active TGF-beta, but not latent TGF-beta or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-beta and that antibodies neutralizing endogenous TGF-beta led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-beta maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway.     

Enhancement of monoclonal antibody production by immobilized hybridoma cell culture with hyperosmolar medium

To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/gamma2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCI. The specific MAb productivity (q(MAb)) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of q(MAb) was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (r(MAb)). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced q(MAb) immobilized cells. The q(MAb) immobilized cells at 395 mOsm/kg was 0.661 +/- 0.019 mug/10(6) cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the r(MAb) was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in i(MAb) of immobilized S3H5/gamma2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production. (c) 1995 John Wiley & Sons, Inc.     

Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles

Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 10⁸ cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.     

Tissue engineering human placenta trophoblast cells in 3-D fibrous matrix: spatial effects on cell proliferation and function.

Nonwoven polyethylene teraphathalate (PET) fabrics with different porosities and knitted fabric were used as support matrixes to grow human trophoblast cells to study the spatial effects of fibrous matrix on cell adhesion, spatial organization, proliferation, and metabolic functions. In general, cells grown on 2-D surface and knitted fabric had faster metabolic rates and also showed higher proliferation activities as detected by cyclin B assay. For nonwoven PET fibers, matrix porosity had profound effects on cell morphology, spatial organization, and proliferation. Cells grown in a low-porosity fibrous matrix formed small aggregates ( approximately 100 cells per aggregate), whereas cells grown in high-porosity matrix formed big aggregates ( approximately 1000 cells per aggregate). This was attributed to the difference in pore volume or averaged fiber distance, which dictated a cell's ability to cross over and form a bridge between adjacent fibers. The high-porosity matrix had a relatively poor surface accessibility for cells to attach and spread, which are essential for cell proliferation. Dual staining with PI and BrdU showed that 60% of cells in the small aggregates found in the low-porosity matrix were proliferating, while only 18% of cells in the large aggregates found in the high-porosity matrix were proliferating. These results suggest that spatial characteristics of fibrous matrix are important to cell proliferation and function and should be considered in tissue-engineering human cells.     

Investigating Breast Cancer Cell Behavior Using Tissue Engineering Scaffolds

Despite early detection through the use of mammograms and aggressive intervention, breast cancer (BC) remains a clinical dilemma. BC can resurge after >10 years of remission. Studies indicate that BC cells (BCCs) with self-renewal and chemoresistance could be involved in dormancy. The majority of studies use in vitro, two-dimensional (2-D) monolayer cultures, which do not recapitulate the in vivo microenvironment. Thus, to determine the effect of three-dimensional (3-D) microenvironment on BCCs, this study fabricated tissue engineering scaffolds made of poly (ε-caprolactone) (PCL) having aligned or random fibers. Random and aligned fibers mimic, respectively, the random and highly organized collagen fibers found in the tumor extracellular matrix. Chemoresistant BCCs were obtained by treating with carboplatin. Western blot analysis of carboplatin resistant (treated) MDA-MB-231 (highly invasive, basal-like) and T47D (low-invasive, luminal) BCCs showed an increase in Bcl-2, Oct-4 and Sox-2, suggesting protection from apoptosis and increase in stem-like markers. Further studies with MDA-MB-231 BCCs seeded on the scaffolds showed little to no change in cell number over time for non-treated BCCs whereas on tissue culture polystyrene (TCP), non-treated BCCs displayed a significant increase in cell number at days 4 and 7 as compared to day 1 (p<0.05). Treated BCCs did not proliferate on TCP and the fibrous scaffolds. Little to no cyclin D1 was expressed for non-treated BCCs on TCP. On fibrous scaffolds, non-treated BCCs stained for cyclin D1 during the 7-day culture period. Treated BCCs expressed cyclin D1 on TCP and fibrous scaffolds during the 7-day culture period. Proliferation, viability and cell cycle analysis indicated that this 3-D culture prompted the aggressive BCCs to adopt a dormant phenotype, while the treated BCCs retained their phenotype. The findings indicate that random and aligned fibrous PCL scaffolds may provide a useful system to study how the 3-D microenvironment affects the behavior of BCCs.     

High-throughput 3-D cell-based proliferation and cytotoxicity assays for drug screening and bioprocess development

We have designed, built and tested a three-dimensional (3-D) cell culture system on modified microplates for high-throughput, real-time, proliferation and cytotoxicity assays. In this 3-D culture system, cells expressing the enhanced green fluorescent protein (EGFP) were cultured in nonwoven polyethylene terephthalate (PET) fibrous scaffolds. Compared to 2-D cultures in conventional microplates, 3-D cultures gave more than 10-fold higher fluorescence signals with significantly increased signal-to-noise ratio (SNR), thus extending the application of conventional fluorescence microplate readers for online monitoring of culture fluorescence. The 3-D system was successfully used to demonstrate the effects of fetal bovine serum, fibronectin coating of PET fibers, and cytotoxicity of dexamethasone on recombinant murine embryonic stem D3 cells. The dosage effects of 5-fluorouracil and gemcitabine on high-density colon cancer HT-29 cells were also tested. These studies demonstrated that the 3-D culture microplate system with EGFP expressing cells can be used as a high-throughput system in drug discovery and bioprocess development.     

Effects of mixing intensity on cell seeding and proliferation in three-dimensional fibrous matrices

Nonwoven fibrous matrices have been widely used in cell and tissue cultures because their three-dimensional (3-D) structures with large surface areas and pore spaces can support high-density cell growth. Although cell adherence and growth on 2-D surfaces have been thoroughly investigated, very little is known for cells cultured in 3-D matrices. The effects of mixing intensity on cell seeding, adherence, and growth in fibrous matrices were thus investigated. Chinese Hamster Ovary and osteosarcoma cells were inoculated into nonwoven polyethylene terephthalate matrices by dynamic and static seeding methods, of which the former was found to be superior in seeding efficiency and cell distribution in the matrices. Dynamic seeding increased seeding efficiency from approximately 40% to more than 90%. When higher mixing intensities were applied, both cell attachment and detachment rates increased. Cell attachment was transport limited, as indicated by the increased attachment rate with increasing the mass transfer coefficient of the cells. Meanwhile, cell detachment from the 3-D matrix can be described by the Bell model. The effects of matrix pore size on cell adherence and proliferation were also investigated. In general, the smaller pore size is favorable to cell attachment and proliferation. Further analysis revealed that the interaction between mixing intensity and pore size played a vital role in hydrodynamic damage to cells, which was found to be significant when the Kolomogorov eddy size was smaller than the matrix pores. Increasing mixing intensity also increased oxygen transfer, decreased the lactate yield from glucose, and improved cell growth.