Enhanced DNA transfer into fish bone cells using polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA intoa wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.     

An anti-apoptotic HEK293 cell line provides a robust and high titer platform for transient protein expression in bioreactors

ABSTRACT

HEK293 transient expression systems are used to quickly generate proteins for research and pre-clinical studies. With the aim of engineering a high-producing host that grows and transfects robustly in bioreactors, we deleted the pro-apoptotic genes Bax and Bak in an HEK293 cell line. The HEK293 Bax Bak double knock-out (HEK293 DKO) cell line exhibited resistance to apoptosis and shear stress. HEK293 DKO cells sourced from 2 L seed train bioreactors were most productive when a pH setpoint of 7.0, a narrow pH deadband of ±0.03, and a DO setpoint of 30% were used. HEK293 DKO seed train cells cultivated for up to 60 days in a 35 L bioreactor showed similar productivities to cells cultivated in shake flasks. To optimize HEK293 DKO transfection cultures, we first evaluated different pH and agitation parameters in ambr15 microbioreactors before scaling up to 10 L wavebag bioreactors. In ambr15 microbioreactors with a pH setpoint of 7.0, a wide pH deadband of ±0.3, and an agitation of 630 rpm, HEK293 DKO transient cultures yielded antibody titers up to 650 mg/L in 7 days. The optimal ambr15 conditions prompted us to operate the 10 L wavebag transfection without direct pH control to mimic the wide pH deadband ranges. The HEK293 DKO transfection process produces high titers at all scales tested. Combined, our optimized HEK293 DKO 35 L bioreactor seed train and 10 L high titer transient processes support efficient, large-scale recombinant protein production for research studies.     

新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Labeling of Polyethylenimine with Fluorescent Dye to Image Nucleus,Nucleolus, and Chromosomes in Digitonin-Permeabilized HeLa Cells

The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.     

Justification of continuous packed-bed reactor for retroviral vector production from amphotropic ΨCRIP murine producer cell

To indentify a plausible large-scale production system forretroviral vector, three culture systems, i.e., batch culturewith medium exchange, microcarrier culture, and packed-bedreactor culture were compared. In batch cultures with mediumexchange, high cell concentrations were maintained for about amonth, and the harvested retroviral titer remained constant. Inmicrocarrier cultures, although cell growth was rapid, theretroviral titer was unexpectedly low, suggesting that the lowtiter was due either to serious damage to the retroviral vectoror to a reduction in the production rate of retroviral vector,caused by mechanical shear forces. Although the retroviral titer(maximum titer, 1.56 × 10⁶) in the packed-bedreactor was a little bit lower than that obtained in the batchculture with medium exchange (maximum titer, 1.91 ×10⁶), continuous production made it possible to increasethe cumulative titer up to 16-fold of that from the batchculture with medium exchange. Moreover, as the packed-bedreactor system requires less labor and shows excellentvolumetric productivity in comparison to batch cultures withmedium exchanges, it will be an appropriate production systemfor retroviral vector in large quantities.     

Development of a generic transient transfection process at 100 L scale

We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7–8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L⁻¹ IgG.     

PEI介导的大规模基因瞬时转染研究进展

基因重组蛋白具有巨大的商业和科研价值。近年来,大规模基因瞬时表达(large scale transient gene expression,TGE)技术的出现提供了一种相较于传统筛选稳定细胞株重组蛋白生产工艺而言更加高效(high efficient)和更加节约人力(labor consuming)、物力(cost effective)和时间(time consuming)的解决方案。通过基因瞬时表达技术,可以在短时间内获得毫克至克级别的在分子结构、理化特性和生物学功能等方面都接近于原始存在的蛋白质分子,可以满足细胞信号转导、新药筛选和临床前研究等药物研发前期的阶段对重组蛋白的巨大需求。因此,该技术成为当前研究的热点。阳离子聚合物-聚乙烯亚胺(PEI)是目前报道的工业化、大规模瞬时转染表达重组蛋白领域最广泛使用的基因载体和转染试剂。本文就近年来PEI介导的大规模瞬时转染的转染机理、宿主细胞选择以及转染优化措施等各方面的最新研究进展作一综述。     

Scale-up of suspension and anchorage-dependent animal cells

Alternative culture processes for laboratory scale-up (to 20 L) are described for both suspension and anchorage-dependent cells. Systems range from simple multiple culture units such as the roller bottle, through stirred suspension and microcarrier unit bioreactors, to highly sophisticated perfusion culture capable of maintaining cells at densities of about 10⁸/mL. Critical parameters in scale-up are discussed, and the advantages and disadvantages of each culture system are critically evaluated.     

Large-scale Transient Transfection of Mammalian Cells: A Newly Emerging Attractive Option for Recombinant Protein Production

Mammalian expression systems have an undisputed long-standing and very successful history for the generation of recombinant proteins, mainly as biopharmaceuticals. However, for use as ‘tool proteins’ in, e.g. assay development and screening, for structure elucidation and as antigens these expression systems were generally regarded as being cumbersome, tedious and expensive. This bias has largely been overcome with the very recent development of large-scale transient transfection (LST) approaches. Especially the HEK.EBNA expression system described here has contributed significantly to this success. The simplicity and speed of this approach compares well with expression trials using the widely applied Baculovirus/insect cell system. In addition, proteins generated in mammalian cells are usually correctly folded, fully processed and functionally active.