Genetically Encoded XTEN-based Hydrogels with Tunable Viscoelasticity and Biodegradability for Injectable Cell Therapies.

While direct cell transplantation holds great promise in treating many debilitating diseases, poor cell survival and engraftment following injection have limited effective clinical translation. Though injectable biomaterials offer protection against membrane-damaging extensional flow and supply a supportive 3D environment in vivo that ultimately improves cell retention and therapeutic costs, most are created from synthetic or naturally harvested polymers that are immunogenic and/or chemically ill-defined. This work presents a shear-thinning and self-healing telechelic recombinant protein-based hydrogel designed around XTEN - a well-expressible, non-immunogenic, and intrinsically disordered polypeptide previously evolved as a genetically encoded alternative to PEGylation to "eXTENd" the in vivo half-life of fused protein therapeutics. By flanking XTEN with self-associating coil domains derived from cartilage oligomeric matrix protein, single-component physically crosslinked hydrogels exhibiting rapid shear thinning and self-healing through homopentameric coiled-coil bundling are formed. Individual and combined point mutations that variably stabilize coil association enables a straightforward method to genetically program material viscoelasticity and biodegradability. Finally, these materials protect and sustain viability of encapsulated human fibroblasts, hepatocytes, embryonic kidney (HEK), and embryonic stem-cell-derived cardiomyocytes (hESC-CMs) through culture, injection, and transcutaneous implantation in mice. These injectable XTEN-based hydrogels show promise for both in vitro cell culture and in vivo cell transplantation applications.

Highly Parallel Tissue Grafting for Combinatorial In Vivo Screening.

Material- and cell-based technologies such as engineered tissues hold great promise as human therapies. Yet, the development of many of these technologies becomes stalled at the stage of pre-clinical animal studies due to the tedious and low-throughput nature of in vivo implantation experiments. We introduce a 'plug and play' in vivo screening array platform called Highly Parallel Tissue Grafting (HPTG). HPTG enables parallelized in vivo screening of 43 three-dimensional microtissues within a single 3D printed device. Using HPTG, we screen microtissue formations with varying cellular and material components and identify formulations that support vascular self-assembly, integration and tissue function. Our studies highlight the importance of combinatorial studies that vary cellular and material formulation variables concomitantly, by revealing that inclusion of stromal cells can "rescue" vascular self-assembly in manner that is material-dependent. HPTG provides a route for accelerating pre-clinical progress for diverse medical applications including tissue therapy, cancer biomedicine, and regenerative medicine.

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education.

Teaching the processes of transcription and translation is challenging due to the intangibility of these concepts and a lack of instructional, laboratory-based, active learning modules. Harnessing the genetic code in vitro with cell-free protein synthesis (CFPS) provides an open platform that allows for the direct manipulation of reaction conditions and biological machinery to enable inquiry-based learning. Here, we report our efforts to transform the research-based CFPS biotechnology into a hands-on module called the "Genetic Code Kit" for implementation into teaching laboratories. The Genetic Code Kit includes all reagents necessary for CFPS, as well as a laboratory manual, student worksheet, and augmented reality activity. This module allows students to actively explore transcription and translation while gaining exposure to an emerging research technology. In our testing of this module, undergraduate students who used the Genetic Code Kit in a teaching laboratory showed significant score increases on transcription and translation questions in a post-lab questionnaire compared with students who did not participate in the activity. Students also demonstrated an increase in self-reported confidence in laboratory methods and comfort with CFPS, indicating that this module helps prepare students for careers in laboratory research. Importantly, the Genetic Code Kit can accommodate a variety of learning objectives beyond transcription and translation and enables hypothesis-driven science. This opens the possibility of developing Course-Based Undergraduate Research Experiences (CUREs) based on the Genetic Code Kit, as well as supporting next-generation science standards in 8-12th grade science courses.

Unlocking Applications of Cell-Free Biotechnology through Enhanced Shelf Life and Productivity of E. coli Extracts.

Cell-free protein synthesis (CFPS) is a platform biotechnology that enables a breadth of applications. However, field applications remain limited due to the poor shelf-stability of aqueous cell extracts required for CFPS. Lyophilization of E. coli extracts improves shelf life but remains insufficient for extended storage at room temperature. To address this limitation, we mapped the chemical space of ten low-cost additives with four distinct mechanisms of action in a combinatorial manner to identify formulations capable of stabilizing lyophilized cell extract. We report three key findings: (1) unique additive formulations that maintain full productivity of cell extracts stored at 4 °C and 23 °C; (2) additive formulations that enhance extract productivity by nearly 2-fold; (3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. These findings provide a simple and low-cost advance toward making CFPS field-ready and cost-competitive for biomanufacturing.

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

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.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.