Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Eberhard Karls University, Tübingen

Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany

Electrospinning is a long-known polymer processing technique that has received more interest and attention in recent years due to its versatility and potential use in the field of biomedical research. The fabrication of three-dimensional (3D) electrospun matrices for drug delivery and tissue engineering is of particular interest. In the present study, we identified optimal conditions to generate novel electrospun polymeric scaffolds composed of poly-D/L-lactide and poly-L-lactide in the ratio 50:50. Scanning electron microscopic analyses revealed that the generated poly(D/L-lactide-co-L-lactide) electrospun hybrid microfibers possessed a unique porous high surface area mimicking native extracellular matrix (ECM). To assess cytocompatibility, we isolated dermal fibroblasts from human skin biopsies. After 5 days of in vitro culture, the fibroblasts adhered, migrated and proliferated on the newly created 3D scaffolds. Our data demonstrate the applicability of electrospun poly(D/L-lactide-co-L-lactide) scaffolds to serve as substrates for regenerative medicine applications with special focus on skin tissue engineering.

Electrospun poly(D/L-lactide-co-L-lactide) hybrid matrix: a novel scaffold material for soft tissue engineering.

&Eacute;lectrofilage est un polym&egrave;re connu long traitement technique qui a re&ccedil;u davantage d&#39;int&eacute;r&ecirc;t et l&#39;attention ces derni&egrave;res ann&eacute;es en raison de sa polyvalence et le potentiel d&#39;utilisation dans le domaine de la recherche biom&eacute;dicale. La fabrication de matrices &eacute;lectrofil&eacute;es (3D) en trois dimensions pour l&#39;administration de m&eacute;dicaments et g&eacute;nie tissulaire est particuli&egrave;rement int&eacute;ressant. Dans la pr&eacute;sente &eacute;tude, nous avons identifi&eacute; des conditions optimales pour g&eacute;n&eacute;rer de nouveaux &eacute;lectrofil&eacute;es &eacute;chafaudages polym&eacute;rique compos&eacute;s de poly-D/L-lactide et poly-L-lactide dans le ratio 50/50. Analyses de microscopie &eacute;lectronique &agrave; balayage a r&eacute;v&eacute;l&eacute; que le poly(D/L-lactide-co-L-lactide) g&eacute;n&eacute;r&eacute; &eacute;lectrofil&eacute;es hybride microfibres poss&egrave;dent une unique poreuse grande surface imitant native matrice extracellulaire (ECM). Pour &eacute;valuer les cytocompatibility, les auteurs ont isol&eacute; des fibroblastes dermiques de biopsies de la peau humaine. Apr&egrave;s 5 jours de culture in vitro, les fibroblastes ont adh&eacute;r&eacute;, ont migr&eacute; et se sont multipli&eacute;s sur les &eacute;chafaudages 3D nouvellement cr&eacute;&eacute;s. Nos r&eacute;sultats d&eacute;montrent l&#39;applicabilit&eacute; des &eacute;chafaudages poly(D/L-lactide-co-L-lactide) &eacute;lectrofil&eacute;es pour servir de substrat pour les applications de la m&eacute;decine r&eacute;g&eacute;n&eacute;rative avec un accent particulier sur l&#39;ing&eacute;nierie tissulaire de peau.

Matrice d&#39;&eacute;lectrofil&eacute;es poly(D/L-lactide-co-L-lactide) hybride : un mat&eacute;riau nouveau &eacute;chafaudage pour l&#39;ing&eacute;nierie des tissus mous.

Elettrofilatura &egrave; un polimero di lungo-saputo elaborazione tecnica che ha ricevuto pi&ugrave; interesse e attenzione negli ultimi anni grazie alla sua versatilit&agrave; e il potenziale utilizzo nel campo della ricerca biomedica. Di particolare interesse &egrave; la realizzazione di matrici elettrofilate tridimensionale (3D) per la consegna della droga e l&#39;ingegneria tessutale. Nel presente studio, abbiamo individuato le condizioni ottimali per generare impalcature polimeriche elettrofilate romanzo composti di poly-D/L-lactide e poly-L-lactide nel rapporto 50: 50. Scansione analisi microscopica elettrone ha rivelato che il poly(D/L-lactide-co-L-lactide) generato elettrofilate ibrido microfibre possedeva un&#39;unica porosa elevata area superficiale mimando natali matrice extracellulare (ECM). Per valutare la cytocompatibility, abbiamo isolato fibroblasti dermici da biopsie di pelle umana. Dopo 5 giorni di coltura in vitro, i fibroblasti aderiscono, migrarono e proliferavano sui ponteggi 3D appena creati. I nostri dati dimostrano l&#39;applicabilit&agrave; dei ponteggi poly(D/L-lactide-co-L-lactide) elettrofilate per servire come substrati per applicazioni di medicina rigenerativa con focus particolare sull&#39;ingegneria dei tessuti della pelle.

Elettrofilate poly(D/L-lactide-co-L-lactide) matrice ibrida: un materiale romanzo impalcatura per l&#39;ingegneria dei tessuti molli.

Stem cells are defined as unspecialized cells that are capable of long term self-renewal and differentiation into specialized cell types. These unique properties make them an attractive cell source for regenerative medicine applications. Although the functions of various stem cells have been extensively studied in the development of organisms and in diseases, the specific factors and conditions that control stem cell fate, specifically the conditions that allow them to remain unspecialized, are not well studied. It has been suggested that adult stem cell survival and maintenance, as well as proliferation and differentiation, are controlled by the three-dimensional (3D) microenvironment, the so-called niche. Major functional niche components include supporting niche cells, growth-modulating soluble factors stored within the niches, and the extracellular matrix (ECM). In this article, we review work highlighting the growing complexity of stem cell-ECM interactions and their impact on the fields of biomaterials research and regenerative medicine.

Stem cell microenvironments--unveiling the secret of how stem cell fate is defined.

Our focus was to develop a three-dimensional (3D) human dynamic in vitro tissue model that mimics the natural microenvironment of the small intestine. We co-cultured human Caco-2 cells with primary-isolated human microvascular endothelial cells (hMECs) on decellularized porcine jejunal segments within a custom-made dynamic bioreactor system that resembles the apical and basolateral side of the intestine for up to 14 days. The obtained data were compared to results generated using routine static Caco-2 assays. We performed histology and immunohistochemistry. Permeability was measured using directed transport studies. Histological analyses revealed that in tissue-engineered segments, which had been cultured under dynamic conditions, the Caco-2 cells showed a high-prismatic morphology, resembling normal primary enterocytes within their native environment. We further identified that the transport of low permeable substances, such as fluorescein and desmopressin increased within the dynamic bioreactor cultures. Immunohistochemical staining showed a significantly higher expression of the efflux transport p-glycoprotein (p-gp) under dynamic culture conditions when compared to the static cultures. We conclude that the integration of physiological parameters is crucial for the establishment of a reliable 3D intestinal in vitro model, which enables the simulation of drug transport over the gut-blood-barrier in a simplified way.

The physiological performance of a three-dimensional model that mimics the microenvironment of the small intestine.

The non-destructive and label-free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non-contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400-1800 cm(-1)), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof-of-principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically-degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non-destructive and non-contact tool to probe ECM state in situ.

Miriam Votteler

Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST)

Eberhard Karls University, Tübingen

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Miriam Votteler

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST)

Eberhard Karls University, Tübingen

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST)