transfection of CHO-DG44 cells
for the production of recombinant IgG antibody
CHO-DG44 cells, CHO-K1 cells and MEFs
CHR is composed of 100% amino acids,without any cytotoxic PEI. CHR-mediated transfection does not require complexpre-formation, works well in serum-containing media and is biodegradable, which may prevent cumulative cytotoxicity and facilitates downstream processing.
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New chemosynthetic route to linear ε-poly-lysine†
ε-Poly-lysine (ε-PL) is an uncommoncationic, naturally-occurring homopolymer produced by the fermentation process. Due to its significant antimicrobial activity and nontoxicity to humans, ε-PL is now industrially produced as an additive, e.g. for food and cosmetics. However, the biosynthetic route can only make polymers with a molecular weightof about 3 kDa. Here, we report a new chemical strategy based on ring-opening polymerization (ROP) to obtain ε-PL from lysine. The 2,5-dimethylpyrrole protected α-amino-ε-caprolactam monomer was prepared through cyclization of lysine followed by protection. ROP of this monomer, followed by the removal ofthe protecting group, 2,5-dimethylpyrrole, ultimately yielded ε-PL with varying molecular weights. The structure of this chemosynthetic ε-PL has been fully characterized by 1H NMR, 13C NMR, and MALDI-TOF MS analyses. This chemosynthetic ε-PL exhibited a similar pKa value and low cytotoxicity as the biosynthetic analogue. Using this new chemical strategy involving ROP without the need for phosgene may enable a more cost effective production of ε-PL on a larger-scale, facilitating the design of more advanced biomaterials.
Review on production and medical applicationsof ɛ-polylysine
ɛ-Polylysine (ɛ-PL) is a homopolyamide linked by the peptide bond between the carboxylic and epsilon amino group of adjacent lysine molecules. It is naturally occurring biodegradable and nontoxic towards human. This review article gives an insight about the various ɛ-PL producing strains, their screening procedures, mechanism of synthesis, characterization, and its application in the medical field. The polycationic nature of ɛ-PL at physiological pH makes it as one of the potential candidates in the field of drug delivery. Most of the biomedical applications till date use synthetic α-PLL as a raw material. However, it is believed that naturally occurring ɛ-PL would be an ideal substitute.
In Vitro Transfection with Plasmid DNA Using Polylysine Copolymers
Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (ed. Friedmann and Rossi). CSHL Press, ColdSpring Harbor, NY, USA, 2007.
The terplex system for delivering DNA to targeted cells comprises three components—stearyl polylysine (PLL), low-densitylipoprotein (LDL), and DNA. This article describes a method for optimizing the relative amounts of these components and determining the transfection parameters for a given cell line.
Synthetic alternatives to Matrigel
Matrigel, a basement-membrane matrix extracted from Engelbreth–Holm–Swarm mouse sarcomas, has been used for more than four decades for a myriad of cell culture applications. However, Matrigelis limited in its applicability to cellular biology, therapeutic cell manufacturing and drug discovery owing to its complex, ill-defined and variable composition. Variations in the mechanical and biochemical properties within a single batch of Matrigel — and between batches — have led to uncertainty in cell culture experiments and a lack of reproducibility. Moreover, Matrigel is not conducive to physical or biochemical manipulation, making it difficult to fine-tune the matrix to promote intended cell behaviours and achieve specific biological outcomes. Recent advances in synthetic scaffolds have led to the development of xenogenic-free, chemically defined, highly tunable and reproducible alternatives.
Towards organoid culture without Matrigel
Organoids—cellular aggregates derived from stem or progenitor cells that recapitulate organ function in miniature—are of growing interest in developmental biology and medicine. Organoids have been developed for organs and tissues such as the liver, gut, brain, and pancreas; they are used as organ surrogates to study a wide range of questions in basic and developmental biology, genetic disorders, and therapies. However, many organoids reported to date have been cultured in Matrigel, which is prepared from the secretion of Engelbreth-Holm-Swarm mouse sarcoma cells; Matrigel is complex and poorly defined. This complexity makes it difficult to elucidate Matrigel-specific factors governing organoid development.
Extended release of perioperative immunotherapy prevents tumor recurrence and eliminates metastases
Cancer immunotherapy can confer durablebenefit, but the percentage of patients who respond to this approach remainsmodest. The ability to concentrate immunostimulatory compounds at the site ofdisease can overcome local immune tolerance and reduce systemic toxicity.Surgical resection of tumors may improve the efficacy of immunotherapy byremoving the concentrated immunosuppressive microenvironment; however, it alsoremoves tumor-specific leukocytes as well as tumor antigens that may beimportant to establishing antitumor immunity. Moreover, surgery produces atransient immunosuppressive state associated with wound healing that has beencorrelated with increased metastasis. Using multiple models of spontaneousmetastasis, we show that extended release of agonists of innateimmunity-including agonists of Toll-like receptor 7/8 (TLR7/8) or stimulator ofinterferon genes (STING)-from a biodegradable hydrogel placed in the tumorresection site cured a much higher percentage of animals than systemic or localadministration of the same therapy in solution. Depletion and neutralizationexperiments confirmed that the observed prevention of local tumor recurrenceand eradication of existing metastases require both the innate and adaptivearms of the immune system. The localized therapy increased the numbers ofactivated natural killer (NK) cells, dendritic cells, and T cells and inducedproduction of large amounts of type I interferons, thereby converting animmunosuppressive post-resection microenvironment into an immunostimulatoryone. The results suggest that the perioperative setting may prove to be auseful context for immunotherapy, particularly when the release of the therapyis extended locally.
Poly-L-lysine Prevents Senescence andAugments Growth in Culturing Mesenchymal Stem Cells Ex Vivo
Mesenchymal stem cells (MSCs) possess greattherapeutic potential. Efficient in vitro expansion of MSCs ishowever necessary for their clinical application. The extracellular matrix(ECM) provides structural and biochemical support to the surrounding cells, andit has been used as a coating substrate for cell culture. In this study, wehave aimed to improve the functionality and stemness of MSCs during cultureusing poly-L-lysine (PLL). Functionality of MSCs was analysed by cell cycleanalysis, differentiation assay, -galactosidase staining, and RT-PCR.Furthermore, we assessed the global gene expression profile of MSCs on uncoatedand PLL-coated plates. MSCs on PLL-coated plates exhibited a faster growth ratewith increased S-phase and upregulated expression of the stemness markers. Inaddition, their osteogenic differentiation potential was increased, and genesinvolved in cell adhesion, FGF-2 signalling, cell cycle, stemness, celldifferentiation, and cell proliferation were upregulated, compared to that ofthe MSCs cultured on uncoated plates. We also confirmed that MSCs on uncoatedplates expressed higher -galactosidase than the MSCs on PLL-coated plates.We demonstrate that PLL provides favourable microenvironment for MSC culture byreversing the replicative senescence. This method will significantly contributeto effective preparation of MSCs for cellular therapy.
PL may promote chondrogenesis by interacting electrostatically with cartilage GAGs, thus trapping the extracellular matrix around the newly emerging cartilage nodules and thereby stabilizing their growth and differentiation.
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