Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

Abstract

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

Multifactorial bottom‐up bioengineering approaches for the development of living tissue substitutes

“…Regarding ECM composition, immunocytochemistry analysis revealed increased deposition of COL1A1 when MMC was used. Enhanced deposition of COL1A1 that was due to MMC has been described repeatedly in the literature for various crowders (42, 50–52, 86). Among them, Cr has shown superior performance in terms of accelerated and enhanced ECM deposition by TCs; osteoblasts; BMSCs; and dermal, lung, and corneal fibroblasts (41, 42, 47).…”

“…Recent data have demonstrated that macromolecular crowding (MMC), the addition of inert and poly disperse macromolecules [ e.g ., carrageenan (Cr), dextran sulfate, Ficoll cocktail of 70 and 400 kDa] to the culture environment, significantly accelerates ECM deposition (41, 42). It is worth noting that Cr significantly increases ECM deposition (43–46) without, however, affecting collagen type I α‐1 (COL1A1) and prolyl‐4‐hydroxylase 1 gene expression in bone marrow stem cells (BMSCs) and corneal fibroblasts (42, 47). This enhanced ECM deposition is due to the volume excluded by these macromolecules that affects the rate of biologic processes (48, 49).…”

“…Thus far, MMC has been shown to enhance ECM deposition in various permanently differentiated and stem cell populations (41, 42, 47). It is worth noting that in the deposited under MMC conditions collagen exhibits the characteristic d ‐periodicity (41, 42), a strong indicator for a regular self‐assembly of collagen molecules (53). Although MMC has been previously combined with other microenvironmental modulators, such as hypoxia (47, 54), it is yet to be used simultaneously with mechanical stimulation.…”

Low oxygen tension and macromolecular crowding accelerate extracellular matrix deposition in human corneal fibroblast culture

“…At physiological low oxygen tension, hypoxia‐inducible factor 1 (HIF‐1) is activated, which regulates fundamental cell processes, such as proliferation, migration, angiogenesis and ECM synthesis (Semenza GL ; Mohyeldin A and Garzón‐Muvdi T and Quiñones‐Hinojosa A ; Zimna A and Kurpisz M ). MMC, the addition of inert macromolecules in the culture media, by imitating the highly crowded in vivo microenvironment in vitro , increases many‐fold ECM deposition in various permanently differentiated and stem cell culture systems (Lareu RR and Subrainhanya KH and Peng YX and Benny P and Chen C and Wang ZB and Rajagopalan R and Raghunath M ; Zeiger AS and Loe FC and Li R and Raghunath M and Van Vliet KJ ; Satyam A and Kumar P and Fan X and Gorelov A and Rochev Y and Joshi L and Peinado H and Lyden D and Thomas B and Rodriguez BJ and Raghunath M and Pandit A and Zeugolis D ; Kumar P and Satyam A and Fan X and Collin E and Rochev Y and Rodriguez B and Gorelov A and Dillon S and Joshi L and Raghunath M and Pandit A and Zeugolis D ; Kumar P and Satyam A and Fan X and Rochev Y and Rodriguez B and Gorelov A and Joshi L and Raghunath M and Pandit A and Zeugolis D ).…”

Recreating complex pathophysiologies in vitro with extracellular matrix surrogates for anticancer therapeutics screening

“…Further, such approaches require prolonged in vitro culture to develop a proper 3D model, which is often associated with cell phenotypic drift [137,138]. Although macromolecular crowding aspires to address this slow or delayed ECM deposition [139][140][141], this technology is still at the primitive stages and, for that reason, scaffoldbased technologies (e.g., hydrogels, fibres, sponges) are under intense investigation.…”