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Porous Polymeric Microspheres With Controllable Pore Diameters for Tissue Engineered Lung Tumor Model Development.

ABSTRACT: Complex cell cultures are more representative of in vivo conditions than conventionally used monolayer cultures, and are hence being investigated for predictive screening of therapeutic agents. Poly lactide co-glycolide (PLGA) polymer is frequently used in the development of porous substrates for complex cell culture. Substrates or scaffolds with highly interconnected, micrometric pores have been shown to positively impact tissue model formation by enhancing cell attachment and infiltration. We report a novel alginate microsphere (AMS)-based controlled pore formation method for the development of porous, biodegradable PLGA microspheres (PPMS), for tissue engineered lung tumor model development. The AMS porogen, non-porous PLGA microspheres (PLGAMS) and PPMS had spherical morphology (mean diameters: 10.3 ± 4, 79 ± 21.8, and 103 ± 30 ?m, respectively). The PPMS had relatively uniform pores and a porosity of 45.5%. Degradation studies show that PPMS effectively maintained their structural integrity with time whereas PLGAMS showed shrunken morphology. The optimized cell seeding density on PPMS was 25 × 103 cells/mg of particles/well. Collagen coating on PPMS significantly enhanced the attachment and proliferation of co-cultures of A549 lung adenocarcinoma and MRC-5 lung fibroblast cells. Preliminary proof-of-concept drug screening studies using mono- and combination anti-cancer therapies demonstrated that the tissue-engineered lung tumor model had a significantly higher resistance to the tested drugs than the monolayer co-cultures. These studies indicate that the PPMS with controllable pore diameters may be a suitable platform for the development of complex tumor cultures for early in vitro drug screening applications.

SUBMITTER: Dhamecha D

PROVIDER: S-EPMC7365955 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Porous Polymeric Microspheres With Controllable Pore Diameters for Tissue Engineered Lung Tumor Model Development.

Dhamecha Dinesh D Le Duong D Movsas Rachel R Gonsalves Andrea A Menon Jyothi U JU

Frontiers in bioengineering and biotechnology 20200710

Complex cell cultures are more representative of <i>in vivo</i> conditions than conventionally used monolayer cultures, and are hence being investigated for predictive screening of therapeutic agents. Poly lactide co-glycolide (PLGA) polymer is frequently used in the development of porous substrates for complex cell culture. Substrates or scaffolds with highly interconnected, micrometric pores have been shown to positively impact tissue model formation by enhancing cell attachment and infiltrati ...[more]

PMID: 32754585

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Project description:Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.

Dataset Information

Porous Polymeric Microspheres With Controllable Pore Diameters for Tissue Engineered Lung Tumor Model Development.

Publications

Porous Polymeric Microspheres With Controllable Pore Diameters for Tissue Engineered Lung Tumor Model Development.

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