Natural polymers are widely used in a variety of biomedical applications including, tissue regeneration, drug delivery, and for imaging. In wound care and tissue regeneration, they are used as dressings for acute or chronic wounds and as regeneration templates. Prosthetic mesh material can be used to induce the repair and restoration of soft tissues following surgery and deliver stem cells to damaged tissue.  Common features of this extracellular matrix-assisted tissue remodeling include promotion of angiogenesis, recruitment of circulating progenitor cells and constructive remodeling of damaged tissue structures. Natural polymers can be derived from a wide variety of sources, from plants, animals, and microorganisms. Natural polymers that mimic human extracellular matrix have mechanical tunability, high biocompatibility, and high-water holding capacity. The extracellular matrix is comprised of many polymers, including proteoglycans, hyaluronic acid, collagen and elastin. Natural polymers–based scaffolds are appealing for localized drug and stem cell delivery, tissue repair and regeneration. Careful characterization and selection of the appropriate biopolymer material is particularly important. Here we discuss properties and characterization of natural polymers with a specific focus on Collagen.

Collagen, the most abundant extracellular matrix protein in animal kingdom belongs to a family of fibrous proteins, which give strength and flexibility to tissues and provide a highly biocompatible environment for cells. Collagen is an ideal biomaterial for implantable medical products and scaffolds for in vitro testing systems. To manufacture collagen-based solutions, porous sponges, membranes and threads for surgical and dental purposes or cell culture matrices, collagen rich tissues as skin and tendon of mammals are intensively processed by physical and chemical means. Other tissues such as pericardium and intestine are more gently decellularized while maintaining their complex collagenous architectures. New processing technologies have generated structurally versatile materials controlling for strength, stability, and biocompatibility. Important progress is expected in this field and will aide in the advancement of biopharmaceuticals, tissue regeneration and cell therapy. The extracellular matrix is comprised of many biopolymers and is complex and difficult to study. Characterization of biopolymers, including collagen, is important for successful therapeutic and device development.

Quality control testing involves measures of safety and characterization of the natural polymer. These tests include sterility, endotoxin, DNA content, and residual chemicals from processing. Physical and chemical characterization may include measurement of tensile strength, polymer cross linking, and total protein content.  Identification of the natural polymer derived products includes testing for proteoglycans, growth factors and cytokines. Demonstrating quality and efficacy of natural polymers by functional testing such as cell migration, stem cell survival or apoptosis, or lineage differentiation.

Cell Migration is measured using Transwell chambers with different conditions to assess inhibitors and attractant’s ability to alter cell movement.  Polymer products can be tested for their ability to promote cell survival using live/dead stains or measures of apoptotic signaling. Degradation of collagen fibers is assessed by measuring N-terminal propeptide or hydroxyproline residues by ELISA in complex matrices. Thermal stability studies measure degradation using differential scanning calorimetry (DSC). Collagen binding dyes or peptides that bind selectively to denatured collagen are also useful. For example, a trichrome histology stain is a mixture of three dyes used on connective tissue to visualize collagen and reticular fibers. Collagen fibers stain green or blue with Masson's trichrome stain. Muscle and keratin stain red. Sirius Red stains native total collagen (all types). Much can be learned by observing the morphology of the polymer product with microscopic examination. Modern imaging techniques such as MRI and PET are valuable tools to image connective tissue.

Natural polymers, especially collagens and other components of the extracellular matrix play critical roles in health and disease. Defects in extracellular matrix may cause bone and connective tissue disorders, cardiovascular disease, and dermatological conditions. More common types of connective tissue disorders include arthritis, fibromyalgia, scleroderma, and lupus. Fibrotic conditions (abnormal deposition of extracellular matrix) underlie many major diseases. The importance and utility of natural polymers for biomedical applications is clear. The use and characterization of natural polymers is an active field of study that requires special expertise and analytical support.

BioTether Sciences has a Lonza 4D-Nucleofector with X unit. This device enables users to efficiently transfect immortalized cells, primary cells, stem cells and other hard to transfect cell lines with high efficiency. Experiments are designed to use the standard cuvette or a 16 strip microcuvette. The X unit can electroporate 2 standard cuvettes at the same time, and both are able to be programmed independently from each other. The 16 strip microcuvette is great for optimization, and each microcuvette can be programmed independently as well. This allows for optimization of electroporation conditions to identify the highest efficiency  (up to 90%) and lowest mortality. Cell functionality is also conserved.

The 4D-Nucleofector™ System was developed to offer advanced performance, flexibility and convenience for cell transfection purposes. The system has a modular architecture that allows seamless expansion of the system: it comprises a base core unit and functional units to suit your application of interest. Lonza provides over 160 optimized protocols and transfection kits.

The 4D-Nucleofector™ X-Unit is the most flexible unit and supports nucleofection of various cell numbers in different formats, as it is equipped with a strip position (16-well strip format) and 2 cuvette positions. This allows you to be flexible in the transfection of your cell type of interest, using a smaller volume or a higher volume. The X-Unit features positions for 20µl Nucleocuvette™ strips (for low cell numbers down to 2 x 10^4) and 100 μl single Nucleocuvette™ (for cell numbers up to 2 x 10^7). Different Nucleofection Vessels allow for flexible throughput from 1 to 16 samples.  The system provides seamless transfer of conditions between different Nucleofection Vessels: same protocol can be used for use of the strips and the cuvettes.

At BioTether Sciences we use the Lonza Nucleofector 4D system for delivery of plasmids, siRNA oligonucleotides, gRNA, RNP and proteins to a variety of cell types. The system allows for high efficiency delivery of CRISPR/Cas9 RNPs for gene modification. The use of RNPs can reduce off target editing and in hard to transfect cell lines and primary cells, reduce toxicity. For instance, we use the Nucleofector 4D for gene knock-out studies. The high efficiency delivery helps get us a step closer to obtaining a pure population of knock-out cells, especially when combined with our cell separation technologies.