Optimizing illumination in three-dimensional deconvolution microscopy for accurate refractive index tomography.

In light transmission microscopy, axial scanning does not directly provide tomographic reconstruction of specimen. Phase deconvolution microscopy can convert a raw intensity image stack into a refractive index tomogram, the intrinsic sample contrast which can be exploited for quantitative morphological analysis. However, this technique is limited by reconstruction artifacts due to unoptimized optical conditions, which leads to a sparse and non-uniform optical transfer function. Here, we propose an optimization method based on simulated annealing to systematically obtain optimal illumination schemes that enable artifact-free deconvolution. The proposed method showed precise tomographic reconstruction of unlabeled biological samples.

Membrane activity detection in cultured cells using phase-sensitive plasmonics.

Despite the existence of various neural recording and mapping techniques, there is an open territory for the emergence of novel techniques. The current neural imaging and recording techniques suffer from invasiveness, a time-consuming labeling process, poor spatial/ temporal resolution, and noisy signals. Among others, neuroplasmonics is a label-free and nontoxic recording technique with no issue of photo-bleaching or signal-averaging. We introduced an integrated plasmonic-ellipsometry platform for membrane activity detection with cost-effective and high-quality grating extracted from commercial DVDs. With ellipsometry technique, one can measure both amplitude (intensity) and phase difference of reflected light simultaneously with high signal to noise ratio close to surface plasmon resonances, which leads to the enhancement of sensitivity in plasmonic techniques. We cultured three different types of cells (primary hippocampal neurons, neuroblastoma SH-SY5Y cells, and human embryonic kidney 293 (HEK293) cells) on the grating surface. By introducing KCl solution as a chemical stimulus, we can differentiate the neural activity of distinct cell types and observe the signaling event in a label-free, optical recording platform. This method has potential applications in recording neural signal activity without labeling and stimulation artifacts.

Evolution from adherent to suspension: systems biology of HEK293 cell line development.

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

Effects of Single Nucleotide Polymorphism Ala270Ser (rs316019) on the Function and Regulation of hOCT2.

The human organic cation transporter 2 (hOCT2) is highly expressed in proximal tubules of the kidneys, where it plays an important role in the secretion of organic cations. Since many drugs are organic cations, hOCT2 has relevant pharmacological implications. The hOCT2 gene is polymorphic, and the nonsynonymous single nucleotide polymorphism (SNP) causing the substitution of alanine at position 270 of the protein sequence with serine (Ala270Ser) is present with high frequency in the human population. Therefore, Ala270Ser has potentially important pharmacologic consequences. Here, we analyzed the transport properties and rapid regulation of hOCT2 wildtype and hOCT2 Ala270Ser expressed in human embryonic kidney cells using real-time uptake measurements. Moreover, we compared the expression of hOCT2 in the plasma membrane determined by biotinylation experiments and the cellular transport and toxicity of cisplatin measured by inductively coupled plasma mass spectrometry and a viability test, respectively. The transport characteristics and regulation of the wildtype and mutated hOCT2 were very similar. Interestingly, a higher affinity of hOCT2 Ala270Ser for creatinine was observed. Compared with hOCT2 wildtype, the plasma membrane expression, cisplatin transport, and cisplatin-associated toxicity of hOCT2 Ala270Ser were significantly lower. In conclusion, these findings suggest that Ala270Ser has subtle but important effects on hOCT2 function, which are probably difficult to detect in studies with patients.

Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells.

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.

Effective Cryopreservation and Recovery of Living Cells Encapsulated in Multiple Emulsions.

Background: The ability to preserve living cells or stem cells is critical for their use in cell therapy, especially for regenerative, reproductive, and transfusion medicine. This article addresses the low survival rates of cells and their loss of function during traditional freezing and banking (cells in a liquid medium with cryoprotectants). Aim: In this article, we developed multiple emulsions (water-in-oil-in-water type) for the effective encapsulation and cryopreservation of cells. In multiple emulsions, the oil drops, acting as a protective membrane, contain even smaller water droplets with encapsulated living cells, dispersed in the continuous water phase. Materials and Methods: The multiple emulsions with HEK293 cells encapsulated in the internal alginate droplets were successfully prepared in a Couette-Taylor flow biocontactor. The cryoprotectants (sucrose/dimethyl sulfoxide-DMSO) were located within the internal or external or both water phases of the emulsions. Encapsulated and non-encapsulated cells were frozen to -80°C (cooling rate: -1°C/min) and then transferred to liquid nitrogen (-196°C) for 24 hours. The standard rapid warming procedure was applied to thaw samples. Cell proliferation and viability were measured by using the AlamarBlue™ assay after recovery of cells. Results: The results showed that the viability of cells encapsulated in the internal droplets of multiple emulsions, and then cryopreserved, was significantly higher, up to 27.9%, than that observed for cells conventionally cryopreserved (non-encapsulated cells in water). Moreover, the effective cell-loaded multiple emulsions-based banking method allowed DMSO-toxic cryoprotectant-to be eliminated from the cryopreservation system. Conclusion: The proposed approach of the cryoprotection of cells encapsulated in multiple emulsions could minimize cell damage, degradation, and their loss during freezing-thawing processes.

The Phenotypic Effects of Exosomes Secreted from Distinct Cellular Sources: a Comparative Study Based on miRNA Composition.

Exosomes are nano-sized vesicles composed of lipids, proteins, and nucleic acids. Their molecular landscape is diverse, and exosomes derived from different cell types have distinct biological activities. Since exosomes are now being utilized as delivery vehicles for exogenous therapeutic cargoes, their intrinsic properties and biological effects must be understood. We performed miRNA profiling and found substantial differences in the miRNA landscape of prostate cancer (PC3) and human embryonic kidney (HEK) 293 exosomes with little correlation in abundance of common miRNAs (R2 = 0.16). Using a systems-level bioinformatics approach, the most abundant miRNAs in PC3 exosomes but not HEK exosomes were predicted to significantly modulate integrin signaling, with integrin-ß3 loss inducing macrophage M2 polarization. PC3 but not HEK exosomes downregulated integrin-ß3 expression levels by 70%. There was a dose-dependent polarization of RAW 264.7 macrophages toward an M2 phenotype when treated with PC3-derived exosomes but not HEK-derived exosomes. Conversely, HEK exosomes, widely utilized as delivery vehicles, were predicted to target cadherin signaling, with experimental validation showing a significant increase in the migratory potential of MCF7 breast cancer cells treated with HEK exosomes. Even widely utilized exosomes are unlikely to be inert, and their intrinsic activity ought to be assessed before therapeutic deployment.

Generation of apoptosis-resistant HEK293 cells with CRISPR/Cas mediated quadruple gene knockout for improved protein and virus production.

Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells. This study not only produced an apoptosis-resistant cell line that is useful in producing apoptosis-inducing proteins or viruses expressing these proteins, but also provides a methodology to build other apoptosis-resistant cell lines.

Genetically engineered cell lines for α1-antitrypsin expression.

OBJECTIVES: To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination. RESULTS: α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines. Cell clones obtained had the AAT gene integrated at the AAVS1 locus and secreted approx. 0.04 g/l recombinant AAT into the medium. Moreover, the secreted AAT showed an inhibitory activity that is comparable to plasma AAT. CONCLUSIONS: CRISPR/Cas9-mediated engineering of human cells is a promising alternative for generating isogenic cell lines with consistent AAT production. This work sheds new light on the generation of therapeutic liver stem cells for AATD.

Generation of Induced Pluripotent Stem Cells in Defined Three-Dimensional Hydrogels.

Since the groundbreaking discovery of induced pluripotent stem cells (iPSCs) many research groups have attempted to improve the efficiency of the classical cell reprogramming process. Surprisingly, the contribution of the three-dimensional (3D) microenvironment to iPSC generation has been largely overlooked. Here we describe a protocol for the generation of iPSCs in defined poly(ethylene glycol) (PEG)-based hydrogels that, besides allowing higher reprogramming efficiency, are also a powerful tool to study the influence of biophysical parameters on iPSC generation.

Pluripotency induction in HEK293T cells by concurrent expression of STELLA, OCT4 and NANOS2.

Germline stem cells (GSCs) are attractive biological models because of their strict control on pluripotency gene expression, and their potential for huge epigenetic changes in a short period of time. Few data exists on the cooperative impact of GSC-specific genes on differentiated cells. In this study, we over-expressed 3 GSC-specific markers, STELLA, OCT4 and NANOS2, collectively designated as (SON), using the novel polycistronic lentiviral gene construct FUM-FD, in HEK293T cells and evaluated promoter activity of the Stra8 GSC marker gene We could show that HEK293T cells expressed pluripotency and GSC markers following ectopic expression of the SON genes. We also found induction of pluripotency markers after serum starvation in non-transduced HEK293T cells. Expression profiling of SON-expressing and serum-starved cells at mRNA and protein level showed the potential of SON factors and serum starvation in the induction of ESRRB, NANOG, OCT4 and REX1 expression. Additionally, the data indicated that the mouse Stra8 promoter could only be activated in a subpopulation of HEK293T cells, regardless of SON gene expression. We conclude that heterogeneous population of the HEK293T cells might be easily shifted towards expression of the pluripotency markers by ectopic expression of the SON factors or by growth in serum depleted media.

Use Dependent Attenuation of Rat HCN1-Mediated Ih in Intact HEK293 Cells.

BACKGROUND/AIMS: Cationic currents (Ih) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. METHODS: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated Ih in cell-attached or whole-cell mode of the patch-clamp technique. RESULTS: Recurring activation of rHCN1 reduced and slowed Ih in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced Ih attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in Ih. CONCLUSION: Reduction of rHCN1 mediated Ih is use dependent and may involve the coupling of voltage sensor and pore.

Construction of stable mammalian cell lines for inducible expression of G protein-coupled receptors.

The large-scale expression of many membrane proteins, including the members of the G protein-coupled receptor superfamily, in a correctly folded and fully functional form remains a formidable challenge. In this chapter, we focus on the construction of stable mammalian cell lines to overcome this hurdle. First, we will outline the steps for establishing a tightly regulated gene expression system in human HEK293S cells. This system utilizes separate plasmids containing components of well-defined genetic control elements from the Escherichia coli tetracycline operon to control the powerful cytomegalovirus immediate early enhancer/promoter. Next, we describe the assembly of this expression system into HEK293S cells and a derivative cell line devoid of complex N-glycosylation. Finally, we describe methods for the growth of these cells lines in scalable suspension culture for the preparation of milligram amounts of recombinant protein.

Isolation of autophagosome subpopulations after induction of autophagy by calcium.

Autophagy is a dynamic process accomplished by the generation and maturation of autophagosomes. Isolation of autophagosomes and subsequent compositional analysis can provide information about their biogenesis mechanism. In this article, HEK293 cells expressing GFP-LC3 were treated by calcium phosphate precipitates (CPP) to induce autophagy. The autophaogomes induced by CPP were tubular and vesicular structures, extensively formed in the cytosol. After all membranes in the cell lysate were fractionated by differential centrifugation, autophagosomes from light and heavy membranes were isolated by immuno-precipitation, using antibodies against GFP-LC3 and Atg5. We found that GFP-LC3 and Atg5 positive autophagosomes represented distinctive subpopulations. Judged from the molecular markers associated, including organelle markers and Atg proteins, GFP-LC3 positive autophagosomes were overall at the later biogenetic stage. Furthermore, both GFP-LC3 and Atg5 positive autophagosomes from light membranes were less mature than those from heavy membranes. We have established a method to isolate subpopulations of autophagsomes for further characterization.

Involvement of IQGAP family proteins in the regulation of mammalian cell cytokinesis.

IQGAP family proteins, comprising IQGAP1, -2, and -3 in mammals, are involved in diverse ranges of cellular processes such as adhesion and migration. IQGAP proteins in yeast also play important roles in cytokinesis. However, the involvement of IQGAP proteins in cytokinesis in mammals remains unaddressed. In this study, we showed that IQGAP3 specifically localized to the equatorial cortex at anaphase, whereas IQGAP1 localized to the cell cortex uniformly and IQGAP2 was unexpressed in HeLa cells. IQGAP3, but neither IQGAP1 nor -2, was able to interact with anillin, which was required for the localization of IQGAP3 to the contractile ring. The suppressed expression of IQGAP3 inhibited the completion of cleavage furrow ingression and led to the multinucleation of cells. The suppression of IQGAP1 also had similar inhibitory effects on cytokinesis, and the simultaneous suppression of IQGAP1 and -3 induced more severe effects. The localization of anillin and RhoA to the contractile ring was impaired by the suppression of IQGAP1 and -3, whereas their upstream regulators, the centralspindlin complex and Ect2, remained unaffected. These results suggested that mammalian IQGAP proteins may play a role in cytokinesis by regulating the localization of key cytokinesis regulatory proteins to the contractile apparatus during mitosis.

Dynamic clamp as a tool to study the functional effects of individual membrane currents.

Today, the patch-clamp technique is the main technique in electrophysiology to record action potentials or membrane current from isolated cells, using a patch pipette to gain electrical access to the cell. The common recording modes of the patch-clamp technique are current clamp and voltage clamp. In the current clamp mode, the current injected through the patch pipette is under control while the free-running membrane potential of the cell is recorded. Current clamp allows for measurements of action potentials that may either occur spontaneously or in response to an injected stimulus current. In voltage clamp mode, the membrane potential is held at a set level through a feedback circuit, which allows for the recording of the net membrane current at a given membrane potential.A less common configuration of the patch-clamp technique is the dynamic clamp. In this configuration, a specific non-predetermined membrane current can be added to or removed from the cell while it is in free-running current clamp mode. This current may be computed in real time, based on the recorded action potential of the cell, and injected into the cell. Instead of being computed, this current may also be recorded from a heterologous expression system such as a HEK-293 cell that is voltage-clamped by the free-running action potential of the cell ("dynamic action potential clamp"). Thus, one may directly test the effects of an additional or mutated membrane current, a synaptic current or a gap junctional current on the action potential of a patch-clamped cell. In the present chapter, we describe the dynamic clamp on the basis of its application in cardiac cellular electrophysiology.

High-throughput measurement of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T).

The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17µm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03µm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min.

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization.

Elevated carbon dioxide upregulates NBCn1 Na+/HCO3(-) cotransporter in human embryonic kidney cells.

The NBCn1 Na(+)/HCO3(-) cotransporter catalyzes the electroneutral movement of 1 Na(+):1 HCO3(-) into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated Pco2 (10-15%). After acid loading, in the presence of HCO3(-), ∼50% of the pHi recovery phase was blocked by the Na(+)/H(+) exchanger inhibitors EIPA (10-50 µM) and amiloride (1 mM) and was fully cancelled by 30 µM EIPA under nominally HCO3(-)-free conditions. In addition, in the presence of HCO3(-), pHi recovery after acid loading was completely blocked when Na(+) was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC inhibitor S0859, and the pHi recovery was inhibited by S0859 in a dose-dependent manner (Ki = 30 µM, full inhibition at 60 µM), which confirmed NBC Na(+)/HCO3(-) cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3(-)-dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na(+)/HCO3(-) cotransporter as the only HCO3(-)-dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high-Pco2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3(-) transport.