RESUMEN
We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO2 concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.
Asunto(s)
Técnicas de Cultivo de Célula , Hidrogeles , Humanos , Técnicas de Cultivo de Célula/métodos , Rayos Ultravioleta , Separación Celular/métodosRESUMEN
Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.
RESUMEN
Hydrogels for wound management and tissue gluing applications have to adhere to tissues for a given time scale and then disappear, either by removal from the skin or by slow degradation for applications inside the body. Advanced wound management materials also envision the encapsulation of therapeutic drugs or cells to support the natural healing process. The design of hydrogels that can fulfill all of these properties with minimal chemical complexity, a stringent condition to favor transfer into a real medical device, is challenging. Herein, we present a hydrogel design with a moderate structural complexity that fulfills a number of relevant properties for wound dressing: it can form in situ and encapsulate cells, it can adhere to tissues, and it can be degraded on demand by light exposure under cytocompatible conditions. The hydrogels are based on starPEG macromers terminated with catechol groups as cross-linking units and contain intercalated photocleavable nitrobenzyl triazole groups. Hydrogels are formed under mild conditions (N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer with 9-18 mM sodium periodate as the oxidant) and are compatible with encapsulated cells. Upon light irradiation, the cleavage of the nitrobenzyl group mediates depolymerization, which enables the on-demand release of cells and debonding from tissues. The molecular design and obtained properties reported here are interesting for the development of advanced wound dressings and cell therapies and expand the range of functionality of current alternatives.
Asunto(s)
Hidrogeles/química , Luz , Animales , Adhesión Celular , Técnicas de Cultivo de Célula , Encapsulación Celular , Línea Celular , Hidrogeles/metabolismo , Hidrogeles/farmacología , Cinética , Ratones , Oligopéptidos/química , Oligopéptidos/metabolismo , Fotólisis/efectos de la radiación , Polietilenglicoles/química , Piel/efectos de los fármacos , Piel/metabolismo , Triazoles/químicaRESUMEN
Single cell arrays provide an accurate classification of analyte cells through an image-based analysis of cellular phenotypes. Light-guided cell retrieval from a single cell array is a promising approach for the rapid and simple sorting of difficult to distinguish cells. In this study, we developed a single cell array enclosed with a photodegradable hydrogel in microwells to enable both comprehensive image-based single cell analysis and light-guided cell retrieval. In this system, individual cells became trapped in the microwells together with the photodegradable hydrogel at a high cell density on a chip regardless of cell type, adhesiveness, and motility. Fluorescence-stained model cells and vaccinated dendritic cells were identified by microscopic imaging and then selectively released through the light-induced degradation of the cell-embedding hydrogels. The target cells were selectively retrieved with a purity of >95% from the cell mixture through rapid photorelease, and the retrieved cells were confirmed to grow normally. Our results provide proof-of-principle that the photoresponsive microwell array serves as a versatile tool for image-based cell sorting in cellular researches and the manufacturing processes of high-performance cells.
RESUMEN
Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting.
Asunto(s)
Automatización de Laboratorios , Separación Celular , Forma de la Célula/fisiología , Hidrogeles/química , Reconocimiento de Normas Patrones Automatizadas/métodos , Andamios del Tejido/química , Automatización de Laboratorios/instrumentación , Automatización de Laboratorios/métodos , Separación Celular/instrumentación , Separación Celular/métodos , Células Cultivadas , Células Clonales , Matriz Extracelular/química , Gelatina/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogeles/efectos de la radiación , Procesamiento de Imagen Asistido por Computador , Luz , Neoplasias/patología , FotólisisRESUMEN
There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.