mSLAb - An open-source masked stereolithography (mSLA) bioprinter.

3D bioprinting is a tissue engineering approach using additive manufacturing to fabricate tissue equivalents for regenerative medicine or medical drug testing. For this purpose, biomaterials that provide the essential microenvironment to support the viability of cells integrated directly or seeded after printing are processed into three-dimensional (3D) structures. Compared to extrusion-based 3D printing, which is most commonly used in bioprinting, stereolithography (SLA) offers a higher printing resolution and faster processing speeds with a wide range of cell-friendly materials such as gelatin- or collagen-based hydrogels and SLA is, therefore, well suited to generate 3D tissue constructs. While there have been numerous publications of conversions and upgrades for extrusion-based printers, this is not the case for state-of-the-art SLA technology in bioprinting. The high cost of proprietary printers severely limits teaching and research in SLA bioprinting. With mSLAb, we present a low-cost and open-source high-resolution 3D bioprinter based on masked SLA (mSLA). mSLAb is based on an entry-level (€350) desktop mSLA printer (Phrozen Sonic Mini 4 K), equipped with temperature control and humidification of the printing chamber to enable the processing of cell-friendly hydrogels. Additionally, the build platform was redesigned for easy sample handling and microscopic analysis of the printed constructs. All modifications were done with off-the-shelf hardware and in-house designed 3D printed components, printed with the same printer that was being modified. We validated the system by printing macroscopic porous scaffolds as well as hollow channels from gelatin-based hydrogels as representative structures needed in tissue engineering.

Two-photon microscopy of acoustofluidic trapping for highly sensitive cell analysis.

We combine two-photon-excited fluorescence microscopy and acoustofluidic trapping in a spherical microchamber to in vitro study cells and cell clusters three-dimensionally close to in vivo conditions. The two-photon microscopy provides the in-depth 3D analysis of the spherical microchamber dimensions as well as the positions of trapped samples therein with high spatial precision and high temporal resolution enabling even tracking of the fast moving particles. Furthermore, optical sectioning allows to gather information of individual cells in trapped cell clusters inside the chamber. We demonstrate real-time monitoring of osmosis in A549 lung cells and red blood cells as one possible biomedical application. The observed osmosis reduced the cell membrane diameter by approximately 4 µm in the A549 cells and by approximately 2 µm in the red blood cells. Our approach provides an important optical tool for future investigations of cell functions and cell-cell interactions avoiding wall-contact inside an acoustofluidic device.

Continuous Perfusion Experiments on 3D Cell Proliferation in Acoustic Levitation.

An acoustofluidic trap is used for accurate 3D cell proliferation and cell function analysis in levitation. The prototype trap can be integrated with any microscope setup, allowing continuous perfusion experiments with temperature and flow control under optical inspection. To describe the trap function, we present a mathematical and FEM-based COMSOL model for the acoustic mode that defines the nodal position of trapped objects in the spherical cavity aligned with the microscope field of view and depth of field. Continuous perfusion experiments were conducted in sterile conditions over 55 h with a K562 cell line, allowing for deterministic monitoring. The acoustofluidic platform allows for rational in vitro cell testing imitating in vivo conditions such as cell function tests or cell-cell interactions.

Towards a Point-of-Care Test of CD4+ T Lymphocyte Concentrations for Immune Status Monitoring with Magnetic Flow Cytometry.

For the treatment of human immunodeficiency virus (HIV)-infected patients, the regular assessment of the immune status is indispensable. The quantification of CD4+ T lymphocytes in blood by gold standard optical flow cytometry is not point-of-care testing (POCT) compatible. This incompatibility is due to unavoidable pre-analytics, expensive and bulky optics with limited portability, and complex workflow integration. Here, we propose a non-optical, magnetic flow cytometry (MFC) workflow that offers effortless integration opportunities, including minimal user interaction, integrated sample preparation and up-concentration, and miniaturization. Furthermore, we demonstrate immunomagnetic CD4+ T lymphocyte labeling in whole blood with subsequent quantification using sheath-less MFC. Showing linearity over two log scales and being largely unimpaired by hematocrit, evidence is provided for POCT capabilities of HIV patients.