Cells by Design: A Mini-Review of Targeting Cell Engineering Using DNA Microarrays

Recent studies have demonstrated the utility of DNA microarray technology in engineering cellular properties. For instance, cellular adhesion, the necessity of cells to attach to a surface in order to to proliferate, was examined by comparing two distinct HeLa cell lines. Two genes, one encoding a type II membrane glycosylating sialyltransferase (siat7e) and the other encoding a secreted glycoprotein (lama4), were found to influence adhesion. The expression of siat7e correlated with reduced adhesion, whereas expression of lama4 correlated with increased adhesion, as shown by various assays. In a separate example, a gene encoding a mitochondrial assembly protein (cox15) and a gene encoding a kinase (cdkl3), were found to influence cellular growth. Enhanced expression of either gene resulted in slightly higher specific growth rates and higher maximum cell densities for HeLa, HEK-293, and CHO cell lines. Another investigated property was the adaptation of HEK-293 cells to serum-free media. The genes egr1 and gas6, both with anti-apoptotic properties, were identified as potentially improving adaptability by impacting viability at low serum levels. In trying to control apoptosis, researchers found that by altering the expression levels of four genes faim, fadd, alg-2, and requiem, apoptotic response could be altered. In the present work, these and related studies in microorganisms (prokaryote and eukaryote) are examined in greater detail focusing on the approach of using DNA microarrays to direct cellular behavior by targeting select genes.     

Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.     

The time course of Akt and ERK activation on XIAP expression in HEK 293 cell line

The cell growth is controlled by the interaction of survival and cell growth arrest pathways as well as apoptosis mechanisms which determine the outcome of cell faith as proliferation or apoptosis. In this study, we have studied the activity of survival pathways, i.e., Akt and ERK1/2 with regard to XIAP (inhibitor of apoptosis) in serum starved and stimulated conditions. The HEK-293 cells were cultured in RPMI + 10% FBS. The cells were serum starved by switching to medium with 1% FBS for 24 h and serum stimulated by changing the medium to 10% FBS following serum starvation. The expression of p-Akt, p-ERK, Akt, ERK and XIAP was studied in various time points using western blot. The apoptosis was evaluated by DNA condensation using Hoechst 33258 and Caspase-3 assay. In serum starved condition expression of p-Akt and XIAP is very low. Serum stimulation increases p-Akt and p-ERK within 5 min and sustains a high level for 30 min. The expression of total Akt and ERK1/2 has not changed significantly for 24 h. XIAP expression starts at 6 h after serum stimulation, reaches to maximum level at 12 h and decreases to baseline within 24 h. Furthermore, serum starvation for 24 h does not induced apoptosis and DNA condensation. Taken together, the results indicate that serum activates Akt and ERK pathways earlier than XIAP expression. Furthermore, XIAP expression is low in serum starvation unlike p-ERK which suggests a survival role for ERK in serums starvation. The expression pattern of XIAP indicates induction by Akt and/or ERK activation which requires further studies.     

无血清培养昆虫细胞(BTI-Tn-5B1-4)的适应过程

昆虫细胞培养是近年来迅速发展起来的动物细胞培养工程中的一个新领域。人们可以利用杆状病毒在昆虫细胞内的感染、复制,来大量生产昆虫病毒作为生物杀虫剂[1]。而昆虫细胞杆状病毒表达载体系统的建立,则可通过昆虫细胞的体外培养大量表达病毒携带的外源基因。实践证明,这…     

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.     

Silkworm hemolymph as a substitute for fetal bovine serum in insect cell culture

Summary The effectiveness of silkworm hemolymph was investigated as a substitute for fetal bovine serum (FBS) in insect cell culture. Cells were adapted to grow in reduced FBS medium supplemented with silkworm hemolymph through a gradual adaptation process. FBS concentration in the medium could be reduced to 1% without decrease in cell growth rate and maximum cell concentration by adding 5% silkworm hemolymph.     

Rapid serum-free/suspension adaptation: Medium development using a definitive screening design for Chinese hamster ovary cells

The biopharmaceutical industry prefers to culture the mammalian cells in suspension with a serum-free media (SFM) due to improved productivity and process consistency. However, mammalian cells preferentially grow as adherent cells in a complete medium (CM) containing serum. Therefore, cells require adaptation from adherence in CM to suspension culture in SFM. This work proposes an adaptation method that includes media supplementation during the adaption of Chinese hamster ovary cells. As a result, the adaptation was accelerated compared to the traditional repetitive subculturing. Ca²⁺/Mg²⁺ supplementation significantly reduced the doubling time compared to the adaptation without supplementation during the adaptation of adherent cells from 100% CM to 75% CM (p < 0.05). Furthermore, a definitive screening design (DSD) was applied to select essential nutrients during the adaptation from 10% CM to 0% CM. The main effects of Ca²⁺ and Dulbecco's modified essential medium (DMEM) were found significant to both viable cell density and viability at harvest. Additionally, the interaction term between Ca²⁺ and DMEM was found significant, which highlights the ability of DSD to capture interaction terms. Eventually, the media supplementation method resulted in adaptation SFM in 27 days, compared to the previously reported 66 days. Additionally, the membrane surface integrin expression was found significantly decreased when adherent cells were adapted to suspension. Moreover, the Ca²⁺/Mg²⁺ supplementation correlated with faster integrin recovery after trypsinization. However, faster integrin recovery did not contribute to the accelerated cell growth when subculturing from 100% CM to 75% CM.     

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2‐fold in SFM (serum‐free medium) and 2.4‐fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ～3‐ and ～5‐fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3‐fold (P=4.5 × 10^?5) and 26% (P=2.1 × 10^?2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1‐fold (P=1.7 × 10^?7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8‐fold increase (P=1.5 × 10^?2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up‐regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.     

Approaches for recombinant human factor IX production in serum-free suspension cultures

Objective

To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX).

Results

After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml.

Conclusion

The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.

     

The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and FPR1 on the Cell Surface

The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail.We now show that uPAR co-localizes at the cell-surface and co-immunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/β1 integrin and FPR1/β1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/β1 integrin co-localization. By contrast, a low FPR1/β1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations.The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V-293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells.Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an anti-uPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum.Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking.We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and β1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.     

Serum‐free media for the production of human mesenchymal stromal cells: a review

The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large‐scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)‐supplement for optimal cell proliferation. FBS‐based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum‐use to serum‐free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large‐scale production of MSCs.     

High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI

Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 x 10(6) cells/mL in complex media followed by production at lower cell densities (1 x 10(6) cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression.     

Differential role for phospholipase D1 and phospholipase D2 in 12-O-tetradecanoyl-13-phorbol acetate-stimulated MAPK activation, Cox-2 and IL-8 expression

Phospholipase D (PLD) is expressed in many tissues and stimulated by growth factors and cytokines. However, the role of PLD in signal transduction is still not well-understood. Human embryonic kidney (HEK-293) cells exhibit low levels of both PLD1 and PLD2 mRNA, however, only PLD1 protein was detected by Western blot. When either isoform of PLD was stably expressed in HEK-293 cells, we observed an increased PLD activity in a cell-free system and a 12-O-tetradecanoyl-13-phorbol acetate (TPA)-stimulated increase in PLD activity in intact cells. This system was then used to elucidate the effects of PLD activity on TPA-stimulated signaling pathways. Two such pathways, the mitogen-activated protein kinases (MAPK), extracellular regulated protein kinase (ERK) and p38 are activated by growth factors and cellular stress, respectively. We found that TPA stimulated ERK phosphorylation regardless of the expression status of PLD. In contrast to ERK kinase, HEK-293 cells were unable to induce p38 phosphorylation by TPA stimulation. When HEK-293 cells expressed either PLD1 or PLD2, we observed elevated p38 phosphorylation in response to TPA stimulation. The ERK and p38 MAPKs can also stimulate the expression of both cyclooxygenase-2 (Cox-2) and interleukin-8 (IL-8). We used this system to differentiate the effect of PLD1 or PLD2 activity on the expression of Cox-2 and IL-8. Increased Cox-2 and IL-8 expression was found only in HEK-293 cells expressing PLD1. These data identify a novel role for the PLD1 isoform in the induction of gene expression and provide new insight into the differential role of PLD1 and PLD2 in cells.     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

猪口蹄疫病毒多抗原表位重组腺病毒的构建与鉴定

本研究设计构建了含有猪O型口蹄疫病毒VP1（21—60）-（141-160）-（200—213）位氨基酸的基因的重组腺病毒质粒pAd-VP,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化获得了重组腺病毒rAd—VP。该重组腺病毒于HEK-293A细胞连续传代至20代效价稳定,TCID50为10^-10／mL。RT—PCR检测证明目的基因在mRNA水平上可有效表达;应用O型口蹄疫病毒标准阳性血清进行间接荧光抗体试验,在rAdVP感染的HEK-293A细胞的胞质可见清晰荧光。证明该重组腺病毒对VP1（21-60）-（141—160）-（200—213）位氨基酸的基因进行了成功的表达,从而为FMDV多抗原表位腺病毒活载体疫苗的研究奠定了基础。     

Studies on rabbit natural and recombinant tissue factors: intracellular retention and regulation of surface expression in cultured cells

Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without (delta C) its COOH-terminal intracellular tail, has been conjugated to green fluorescent protein (GFP) to study subcellular localization and other functions of TF. TF-GFP and TF delta C-GFP are associated with Na2CO3-resistant buoyant fractions in HEK-293 cells (lipid rafts); there is no morphological difference in the surface distribution of these or other GFP-labeled membrane proteins present in or excluded from rafts (confocal microscopy, HEK-293 cells). Endogenous TF expressed by rabbit aortic smooth muscle cells (SMCs) is also raft associated. Membranes from HEK-293 cells expressing recombinant TF-GFP or wild-type TF were equipotent to clot human plasma; however, TF delta C-GFP was approximately 20-fold more active (per membrane weight). Immunoblot confirmed that the deletion mutant is more abundantly expressed, and confocal microscopy showed that it has preferential membrane localization, whereas TF-GFP is mainly intracellular (nuclear lining and multiple granules). With a similar half-life (<4 h), the two constructions differ by their intracellular retention, lower for TF delta C-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1 beta and/or FBS treatment (immunoblot, immunofluorescence, clotting assay). However, TF secretion or surface expression was not regulated by stimuli of physiological intensity (such as stimulation of the coexpressed kinin B1 receptors), although a calcium ionophore was highly active in this respect. TF is a raft-associated molecule whose surface expression (secretion) is apparently retarded or impaired by structural determinant(s) located in its COOH-terminal tail.     

Isolation,expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic? Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.