Microfluidic devices present unique advantages for the development of efficient drug carrier particles, cell-free protein synthesis systems, and rapid techniques for direct drug screening. Compared to bulk methods, by efficiently controlling the geometries of the fabricated chip and the flow rates of multiphase fluids, microfluidic technology enables the generation of highly stable, uniform, monodispersed particles with higher encapsulation efficiency. Since the existing preclinical models are inefficient drug screens for predicting clinical outcomes, microfluidic platforms might offer a more rapid and cost-effective alternative. Compared to 2D cell culture systems and in vivo animal models, microfluidic 3D platforms mimic the in vivo cell systems in a simple, inexpensive manner, which allows high throughput and multiplexed drug screening at the cell, organ, and whole-body levels. In this review, the generation of appropriate drug or gene carriers including different particle types using different configurations of microfluidic devices is highlighted. Additionally, this paper discusses the emergence of fabricated microfluidic cell-free protein synthesis systems for potential use at point of care as well as cell-, organ-, and human-on-a-chip models as smart, sensitive, and reproducible platforms, allowing the investigation of the effects of drugs under conditions imitating the biological system.

https://www.mdpi.com/2073-4425/9/2/103/pdf

Microfluidic Devices for Drug Delivery Systems and Drug Screening.

A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

MaxSynBio - Avenues towards creating cells from the bottom up

From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field.

Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems.

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables.

A User's Guide to Cell-Free Protein Synthesis.

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production.

Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2014.06.007

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes

Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/elsc.201100235

Synthesis of membrane proteins in eukaryotic cell‐free systems

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

/pdf/a-continuous-exchange-cell-free-protein-synthesis-system-1ehnk691w0.pdf

A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

Cell-free synthesis of membrane proteins: tailored cell models out of microsomes.

The main goal of cell-free protein synthesis is to produce correctly folded and functional proteins in reasonable amounts for further downstream applications Especially for eukaryotic proteins, functionality is often directly linked to the presence of posttranslational modifications Thus, it is of highest interest to develop novel cell-free expression systems that enable the synthesis of posttranslationally modified proteins Here we present recent advances for the synthesis of glycoproteins, proteins containing disulfide bridges, membrane proteins, and fluorescently labeled proteins The basis for the expression of these difficult-to-express target proteins is a translationally active cell extract which can be prepared from eukaryotic cell lines such as Spodoptera frugiperda 21 (Sf21) and Chinese hamster ovary (CHO) cells Due to a very mild lysate preparation procedure, microsomal vesicles derived from the endoplasmic reticulum (ER) can be maintained in the eukaryotic lysate These vesicles are translocationally active and serve as functional modules facilitating protein translocation and enrichment as well as posttranslational modification of de novo synthesized proteins In particular, for the synthesis of membrane proteins microsomal vesicles are the essential prerequisite for the insertion of the desired protein into a biologically active membrane scaffold providing a natural environment We anticipate that the use of such translationally active eukaryotic cell lysates containing translocationally active vesicles may solve a large number of problems still persistent when expressing eukaryotic proteins in vitro

Rita Sachse

Papers

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes

Synthesis of membrane proteins in eukaryotic cell‐free systems

A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

Cell-free synthesis of membrane proteins: tailored cell models out of microsomes.

Cell-free systems: functional modules for synthetic and chemical biology.