A resazurin-based, nondestructive assay for monitoring cell proliferation during a scaffold-based 3D culture process.

Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering. Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation. The present work found that cytotoxicity and depletion of resazurin depended on cell concentration, resazurin concentration and resazurin incubation time. A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin. This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(d,l-lactic acid) scaffold during a continuous 3D culture process for up to 21 days, comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability, proliferation and differentiation functions of MC3T3-E1, which were treated with/without this strategy for nondestructive evaluation. This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay. No major negative effects of this method on MC3T3-E1 viability and functions were found. Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.

A critical role of the KCa 3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells.

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca2+ -activated K+ channel, KCa 3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the KCa 3.1 channel expression and its role in rat bone marrow-derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up-regulated the KCa 3.1 channel expression and pharmacological or genetic inhibition of the KCa 3.1 channel strongly suppressed stretch-induced increase in cell proliferation and cell cycle progression. These results support that the KCa 3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.

Interaction of tumor cells and astrocytes promotes breast cancer brain metastases through TGF-ß2/ANGPTL4 axes.

Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-ß2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-ß2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-ß2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.

Bone marrow derived endothelial progenitor cells retain their phenotype and functions after a limited number of culture passages and cryopreservation.

A critical limitation for tissue engineering and autologous therapeutic applications of bone marrow derived EPCs is their low frequency, which is even lower in number and activity level in patients with cardiovascular risk factors and other diseases. New strategies for obtaining and reserving sufficient ready-to-use EPCs for clinical use have hit major obstacles, because effects of serial passage and cryopreservation on EPC phenotype and functions are still needed to be explored. The present study aims at investigating effects of a limited number of culture passages as well as cryopreservation on EPC phenotype and functions. We isolated EPCs from rat bone marrow and cultured them up to passage 12 (totaling achievements of 40 population doublings). The phenotype and functions of fresh cultured and post-cryopreserved EPCs at passages 7 and 12, respectively, were evaluated. EPCs at passage 12 maintained the morphological characteristics, marker phenotype, Dil-ac-LDL uptake and FITC-UEA-1 binding functions, enhanced EPCs proliferation, tube formation and migration, but decreased CD133 expression compared with EPCs at passage 7. Cryopreservation caused limited impairment in EPC phenotype and functions. In brief, our results demonstrated that a limited number of culture passages and cryopreservation did not change EPC phenotype and functions, and can be used for the development of robust strategies and quality control criterion for obtaining sufficient and high-quality ready-to-use EPCs for tissue engineering and therapeutic applications.

Protective Effect of Moderate Exogenous Electric Field Stimulation on Activating Netrin-1/DCC Expression Against Mechanical Stretch-Induced Injury in Spinal Cord Neurons.

Nerve cells detect and respond to electric field stimulation and extrinsic chemical guidance cues during development and regeneration; therefore, the development and optimization of an approach for functional neuronal regeneration are necessary for a nerve injury. In this study, we proposed using electric field stimulation to repair a nerve injury triggered by serious mechanical stretch loading. A device that provides continuous mechanical stretch and constant electric field stimulation was designed. Primary dissociated spinal cord neurons were stimulated by mechanical stretch (tensile strain 2.5-10%) at different times (1, 4, 8, and 12 h) to set up a moderate nerve injury model. Stimulated samples were evaluated with respect to cell viability, density, and axonal elongation by the MTT and immunofluorescence assays. The results indicated that mechanical stretch (S, 5% tensile strain, 4 h) caused moderate axonal injury, resulting in significant loss of cell viability and a decrease in cell density. However, injured spinal cord neurons became viable after electric field stimulation (E, 33 mA/m2, 4 h) in the fluorescein diacetate assay. In addition, neuronal viability, density, and elongation increased significantly after electric field stimulation compared with those of stretch-injured neurons. Moreover, electric field stimulation significantly activated the axonal guidance cues Netrin-1 and deleted in colorectal cancer (DCC) receptor expression compared with the stretch-injury group. These results indicate that electric stimulation activates synergistic guidance cues of expression to improve axonal growth relevant to nerve injuries. Our study provides new insight into neuronal regeneration.

Quantitative Studies of Endothelial Cell Fibronectin and Filamentous Actin (F-Actin) Coalignment in Response to Shear Stress.

Both fibronectin (FN) and filamentous actin (F-actin) fibers play a critical role for endothelial cells (ECs) in responding to shear stress and modulating cell alignment and functions. FN is dynamically coupled to the F-actin cytoskeleton via focal adhesions. However, it is unclear how ECs cooperatively remodel their subcellular FN matrix and intracellular F-actin cytoskeleton in response to shear stress. Current studies are hampered by the lack of a reliable and sensitive quantification method of FN orientation. In this study, we developed a MATLAB-based feature enhancement method to quantify FN and F-actin orientation. The role of F-actin in FN remodeling was also studied by treating ECs with cytochalasin D. We have demonstrated that FN and F-actin codistributed and coaligned parallel to the flow direction, and that F-actin alignment played an essential role in regulating FN alignment in response to shear stress. Our findings offer insight into how ECs cooperatively remodel their subcellular ECM and intracellular F-actin cytoskeleton in response to mechanical stimuli, and are valuable for vascular tissue engineering.

Combination of flow and micropattern alignment affecting flow-resistant endothelial cell adhesion.

Assuring cell adhesion to an underlying biomaterial surface under blood flow is vital to functional vascular grafts design. In vivo endothelial cells (ECs) are located under the microenvironment of both surface topography of the basement membrane and the mechanical loading resulting from blood flow. Both topographical and mechanical factors should thus be considered when designing vascular grafts to enhance the flow-resistant EC adhesion. This study aims to investigate effects of integrating biomaterial surface topography and flow on EC adhesion, which was a deficit in previous studies. Human umbilical vein endothelial cells (HUVECs) were cultured on different fibronectin (FN) micropatterns parallel or perpendicular to the flow direction and exposed to sustained flow with physiological levels of shear stress (15 dyne/cm2). We demonstrated that micropattern alignment parallel to the flow direction enhanced flow-resistant EC adhesion, while micropattern alignment perpendicular to the flow direction attenuated it. Experimental and numeric modeling analysis underlined that the flow-induced mechanic distribution on the surface of cells that were aligned on the micropatterned surfaces and the subsequent cytoskeleton rearrangement were responsible for the significant difference in EC adhesion. Furthermore, pressure on the surface of cells that were aligned on the micropatterned surfaces induced by flow provided a more critical role in EC adhesion than shear stress. These findings highlight the importance of proper combination of topographical and flow cues in enhancement of EC adhesion and may suggest new strategies for designing functional vascular grafts.

Shear stress with appropriate time-step and amplification enhances endothelial cell retention on vascular grafts.

Endothelial cells (ECs) are sensitive to changes in shear stress. The application of shear stress to ECs has been well documented to improve cell retention when placed into a haemodynamically active environment. However, the relationship between the time-step and amplification of shear stress on EC functions remains elusive. In the present study, human umbilical cord veins endothelial cells (HUVECs) were seeded on silk fibroin nanofibrous scaffolds and were preconditioned by shear stress at different time-steps and amplifications. It is shown that gradually increasing shear stress with appropriate time-steps and amplification could improve EC retention, yielding a complete endothelial-like monolayer both in vitro and in vivo. The mechanism of this improvement is mediated, at least in part, by an upregulation of integrin ß1 and focal adhesion kinase (FAK) expression, which contributed to fibronectin (FN) assembly enhancement in ECs in response to the shear stress. A modest gradual increase in shear stress was essential to allow additional time for ECs to gradually acclimatize to the changing environment, with the goal of withstanding the physiological levels of shear stress. This study recognized that the time-steps and amplifications of shear stress could regulate EC tolerance to shear stress and the anti-thrombogenicity function of engineered vascular grafts via an extracellular cell matrix-specific, mechanosensitive signalling pathway and might prevent thrombus formation in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

The effects of fluid shear stress on proliferation and osteogenesis of human periodontal ligament cells.

Shear stress is one of the main stress type produced by speech, mastication or tooth movement. The mechano-response of human periodontal ligament (PDL) cells by shear stress and the mechanism are largely unknown. In our study, we investigated the effects of fluid shear stress on proliferation, migration and osteogenic potential of human PDL cells. 6dyn/cm(2) of fluid shear stress was produced in a parallel plate flow chamber. Our results demonstrated that fluid shear stress rearranged the orientation of human PDL cells. In addition, fluid shear stress inhibited human PDL cell proliferation and migration, but increased the osteogenic potential and expression of several growth factors and cytokines. Our study suggested that shear stress is involved in homeostasis regulation in human PDL cells. Inhibiting proliferation and migration potentially induce PDL cells to respond to mechanical stimuli in order to undergo osteogenic differentiation.

Physiological pulsatile flow culture conditions to generate functional endothelium on a sulfated silk fibroin nanofibrous scaffold.

Many studies have demonstrated that in vitro shear stress conditioning of endothelial cell-seeded small-diameter vascular grafts can improve cell retention and function. However, the laminar flow and pulsatile flow conditions which are commonly used in vascular tissue engineering and hemodynamic studies are quite different from the actual physiological pulsatile flow which is pulsatile in nature with typical pressure and flow waveforms. The actual physiological pulsatile flow leading to temporal and spatial variations of the wall shear stress may result in different phenotypes and functions of ECs. Thus, the aim of this study is to find out the best in vitro dynamic culture conditions to generate functional endothelium on sulfated silk fibroin nanofibrous scaffolds for small-diameter vascular tissue engineering. Rat aortic endothelial cells (RAECs) were seeded on sulfated silk fibroin nanofibrous scaffolds and cultured under three different patterns of flow conditioning, e.g., steady laminar flow (SLF), sinusoidal flow (SF), or physiological pulsatile flow (PPF) representative of a typical femoral distal pulse wave in vivo for up to 24 h. Cell morphology, cytoskeleton alignment, fibronectin assembly, apoptosis, and retention on the scaffolds were investigated and were compared between three different patterns of flow conditioning. The results showed that ECs responded differentially to different exposure time and different flow patterns. The actual PPF conditioning demonstrated excellent EC retention on sulfated silk fibroin scaffolds in comparison with SLF and SF, in addition to the alignment of cells in the direction of fluid flow, the formation of denser and regular F-actin microfilament bundles in the same direction, the assembly of thicker and highly crosslinked fibronectin, and the significant inhibition of cell apoptosis. Therefore, the actual PPF conditioning might contribute importantly to the generation of functional endothelium on a sulfated silk fibroin nanofibrous scaffold and thereby yield a thromboresistant luminal surface.

Galanin protects against nerve injury after shear stress in primary cultured rat cortical neurons.

The neuropeptide galanin and its receptors (GalR) are found to be up-regulated in brains suffering from nerve injury, but the specific role played by galanin remains unclear. This study aimed to explore the neuroprotective role of galanin after shear stress induced nerve injury in the primary cultured cortical neurons of rats. Our results demonstrated that no significant changes in cell death and viability were found after galanin treatment when subjected to a shear stress of 5 dyn/cm(2) for 12 h, after increasing magnitude of shear stress to 10 dyn/cm(2) for 12 h, cell death was significantly increased, while galanin can inhibit the nerve injury induced by shear stress with 10 dyn/cm(2) for 12 h. Moreover, Gal2-11 (an agonist of GalR2/3) could also effectively inhibit shear stress-induced nerve injury of primary cultured cortical neurons in rats. Although GalR2 is involved in the galanin protection mechanism, there was no GalR3 expression in this system. Moreover, galanin increased the excitatory postsynaptic currents (EPSCs), which can effectively inhibit the physiological effects of shear stress. Galanin was also found to inhibit the activation of p53 and Bax, and further reversed the down regulation of Bcl-2 induced by shear stress. Our results strongly demonstrated that galanin plays a neuroprotective role in injured cortical neurons of rats.

In vitro evaluation of combined sulfated silk fibroin scaffolds for vascular cell growth.

A combined sulfated silk fibroin scaffold is fabricated by modifying a knitted silk scaffold with sulfated silk fibroin sponges. In vitro hemocompatibility evaluation reveals that the combined sulfated silk fibroin scaffolds reduce platelet adhesion and activation, and prolong the activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). The response of porcine endothelial cells (ECs) and smooth muscle cells (SMCs) on the scaffolds is studied to evaluate the cytocompatibility of the scaffolds. Vascular cells are seeded on the scaffolds and cultured for 2 weeks. The scaffolds demonstrate enhanced EC adhesion, proliferation, and maintenance of cellular functions. Moreover, the scaffolds inhibit SMC proliferation and induce expression of contractile SMC marker genes.

Involvement of large conductance Ca(2+)-activated K (+) channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation.

The large conductance Ca(2+)-activated K(+) (BK(Ca)) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BK(Ca) channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BK(Ca) channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BK(Ca) protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BK(Ca) in SS-induced inhibition of proliferation was examined by BK(Ca) inhibition using a BK(Ca) specific blocker, iberiotoxin (IBTX), and by BK(Ca) transfection in BK(Ca) non-expressing CHO cells. The changes in [Ca(2+)](i) were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC(4)(3). We found that laminar SS inhibited VSMC proliferation and stimulated BK(Ca) channel expression. Furthermore, laminar SS induced an increase in [Ca(2+)](i) and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BK(Ca) channel activity on cell proliferation in response to SS was also confirmed in BK(Ca)-transfected CHO cells showing a decline in proliferation. Blocking BK(Ca) channel reversed its inhibitory effect, providing additional support for the involvement of BK(Ca) in SS-induced proliferation reduction. Our results suggest, for the first time, that BK(Ca) channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.

Stretch magnitude- and frequency-dependent cyclooxygenase 2 and prostaglandin E2 up-regulation in human endometrial stromal cells: possible implications in endometriosis.

Endometriosis, with a prevalence rate ranging from 6% to 10%, is the major contributor to pelvic pain and subfertility, and considerably reduces the quality of life in affected women. However, the pathogenesis of this disease remains largely unknown. The present study aimed to uncover the role of hyperperistalsis in the pathogenesis of endometriosis, by exploring the response of human endometrial stromal cells (ESCs) to the cyclic stretch in vitro. ESCs isolated from 18 different endometrium biopsies undergoing hysterectomy for myoma were subjected to uniaxial cyclic stretches with different magnitude and frequency using the Uniaxial Tension System. Expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) in stretched and unstretched ESCs were assessed by realtime quantitative polymerase chain reaction and Western blot. Production of prostaglandin E2 (PGE(2)) in the culture medium was measured by enzyme-linked immunosorbent assay. The cyclic stretch mimicking hyperperistalsis in endometriosis (5% elongation at 4 cycles/min) stimulated quick up-regulations of COX-2 and mPGES-1 simultaneously on both transcriptional and translational levels, and delayed PGE(2) overproduction was also noted in ESCs. As the stretch magnitude or frequency increased, so did overexpression of COX-2 and PGE(2) (P < 0.05). By contrast, the cyclic stretch mimicking physiological peristalsis (3% elongation at 2 cycles/min) did not induce significant COX-2, mPGES-1 or PGE(2) production within 12 h. Both COX-2 and mPEGS-1 are PGE(2) synthases, and the aberrant COX-2 and PGE(2) production play important roles in the pathogenesis of endometriosis. Therefore, the present findings revealed that increased stretch stimuli from the hyperperistalsis of endometriosis were capable of causing the aberrant COX-2 and PGE(2) expression in the endometrium by mechanotransduction, in a magnitude and frequency-dependent manner. It implied possible roles of hyperperistalsis in the pathogenesis of endometriosis, particularly in the view of COX-2 and PGE(2).

Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways.

Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12 dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation.

Effect of cyclic strain on cardiomyogenic differentiation of rat bone marrow derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into cardiomyocyte-like lineage was investigated in this study. A stretching device was used to generate the cyclic strain for rBMSCs. Cardiomyogenic differentiation was evaluated using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western-blotting. The results demonstrated that appropriate cyclic strain treatment alone could induce cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, rBMSCs exposed to the strain stimulation expressed cardiomyocyte-related markers at a higher level than the shear stimulation. In addition, when rBMSCs were exposed to both strain and 5-azacytidine (5-aza), expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. These results suggest that cyclic strain is an important mechanical stimulus affecting the cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Effect of fluid shear stress on cardiomyogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND AND AIMS: Bone marrow mesenchymal stem cells (BMSCs) are a potential source of material for the construction of tissue-engineered cardiac grafts because of their potential to transdifferentiate into cardiomyocytes after chemical treatment or co-culture with cardiomyocytes. Recent evidence has shown that mechanical loads could regulate the BMSC differentiation into osteoblasts and endothelial cells through various signaling pathways. We investigated whether fluid shear stress (FSS), which is a mechanical load generated by fluid flow, can regulate rat BMSC (rBMSC) differentiation into cardiomyocytes. METHODS: rBMSCs were isolated from marrow of rat femur and tibia using density gradient centrifugation combined with adhesion method and identified with surface marker, proliferation character and differentiation potential in vitro. Cultured rBMSCs with or without 5-azacytidine (5-aza) treatment were exposed to laminar shear stress with a parallel plate-type device and analyzed by RT-PCR, immunocytochemistry, FACS and Western-blotting for the cardiomyogenic differentiation. RESULTS: Appropriate FSS treatment alone induced cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, when rBMSC cultures were exposed to both FSS and 5-aza, expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. CONCLUSIONS: The results demonstrate that FSS is an important factor affecting cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media.

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca(2+) and Mg(2+) for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl(2) and MgCl(2) concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca(2+)/Mg(2+) feeding to 117 h with Ca(2+)/Mg(2+) feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 x 10(6) and 9.12 x 10(6) cells ml(-1), respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.

A High-throughput End-point Assay for Viable Mammalian Cell Estimation.

A single wavelength colorimetric microplate-based assay was developed using non-cytotoxic dye resazurin for the estimation of viable cell concentrations of Chinese hamster ovary (CHO) and hybridoma cells. Experimental results showed variations in pH and temperature caused by cell cultivation and assay operations were well tolerated. Cell concentrations can be effectively determined in the range of 10(5)-10(7) cells ml(-1) using a microplate reader at the wavelength of 605 nm. This assay can be performed in a high-throughput manner such that a large number of cell culture samples can be screened within a relatively short time frame. When used together with a cell culture system of high-throughput format, it may have potential utilities in applications such as cell culture medium formulation and optimization.