In vivo label-free tissue histology through a microstructured imaging window

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies

High dioptric power micro-lenses fabricated by two-photon polymerization

Specimen-induced aberrations limit the penetration depth of standard optical imaging techniques in vivo, mainly due to the propagation of high NA beams in a non-homogenous medium. Overcoming these limitations requires complex optical imaging systems and techniques. Implantable high NA micro-optics can be a solution to tissue induced spherical aberrations, but in order to be implanted, they need to have reduced complexity, offering a lower surface to the host immune reaction. Here, we design, fabricate, and test a single micro-optical element with high dioptric power and high NA (up to 1.25 in water). The sag function is inspired by the classical metalens phase and improved to reduce the spherical aberrations arising from the refractive origin of the phase delay at the lens periphery. We successfully fabricated these high-NA quasi-parabolic aspheric microlenses with varying focal lengths by two-photon polymerization in biocompatible photoresist SZ2080. The entire process is optimized to minimize fabrication time while maintaining the structures' robustness: the smoothness reaches optical ( λ/20 ) quality. The dioptric power and magnification of the microlenses were quantified over a 200 × 200 μm aberration-free field of view. Our results indicate that these microlenses can be used for wide-field imaging under linear excitation and have the optical quality to be utilized for nonlinear excitation imaging. Moreover, being made of biocompatible photoresist, they can be implanted close to the observation volume and help to reduce the spherical aberration of laser beams penetrating living tissues.</p

A 3D millifluidic model of a dermal perivascular microenvironment on a chip

The process of angiogenesis plays a pivotal role in skin regeneration, ensuring the provision of nutrients and oxygen to the nascent tissue, thanks to the formation of novel microvascular networks supporting functional tissue regeneration. Unfortunately, most of the current therapeutic approaches for skin regeneration lack vascularization, required to promote effective angiogenesis. Thus, in vitro tridimensional models, complemented with specific biochemical signals, can be a valuable tool to unravel the neovascularization mechanisms and develop novel clinical strategies. In this work, we designed and validated a tridimensional microstructured dynamic model of the dermal perivascular microenvironment on a chip. We carried out the fabrication of an array of microstructures by two-photon laser polymerization, then used as a 3D substrate for co-culture of human dermal fibroblasts and endothelial cells. We included the substrate in a miniaturized optically accessible bioreactor (MOAB) which provides the physiological interstitial flow, upon perfusion in the presence or absence of the pro-angiogenic stimuli VEGF and TGF-β1. We determined the parameters to be applied under dynamic conditions by an in silico model simulating individual 3D microenvironments within the bioreactor's chambers. We computed the fluid velocity and wall shear stress acting on endothelial cells along with the oxygen concentration profile, and we chose the most suitable flow rate for maintaining dermal physiological conditions. Experimental results showed the effectiveness of the developed platform as a 3D dynamic model of angiogenesis. This is the first combined experimental and computational study involving chemically stimulated 3D co-cultures for successfully simulating the physiological dermal perivascular microenvironment

A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34+ hematopoietic cells while allowing the detection of tumorigenic expansion

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4+ T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34+ cells. A fraction of these cells is viable for up to 3 months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4+ or CD34+ primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells