Projects

Vascular tissue engineering on granular scaffolds: experiments and modeling

OPUS-23 (2022/45/B/ST8/03675)

Project Leader: Dr. Jan Guzowski

Project duration: February 2023 – January 2027

Tissue engineering aims at the fabrication of ‘artificial’ tissues, that is engineered constructs involving living cells and performing the native tissue functions, yet outside the body. Such engineered tissues offer a multitude of applications, including (i) tissue regeneration, where they could be used directly as implants to replace damaged tissues, (ii) basic tissue biology research, offering highly controlled experimental conditions—unlike those encountered in vivo—allowing insight into specific physiological phenomena, and (iii) testing of drugs—both in the pharmaceutical industry, as an alternative to animal models—and in personalized medicine, e.g., in cancer treatment. A fundamental problem in tissue engineering is the fabrication of tissues with an embedded vasculature, which allows the circulation of nutrients and sustains long-term viability. In particular, the vasculature must be functional at all relevant length scales, down to the cellular scale where the smallest vessels, built of endothelial cells (EC), are to deliver oxygen and remove metabolic byproducts directly from individual cells. Current approaches to vascular tissue engineering typically rely on spontaneous self-assembly of the endothelial cells into branched tubular networks. However, such self-assembled networks are far from optimal since (i) they develop slowly in time as the cells need to migrate through hydrogel over relatively large distances, (ii) they are heterogeneous and weakly percolated, and (iii) they do not allow any external control over their global structure. In the project, we will develop a new method of vascular tissue engineering based on the use of so-called vascular ‘seeds’, that is, pre-aggregated clusters of endothelial cells which—when distributed inside a host tissue in a controlled manner—could guide the formation of vascular networks of predesigned global architecture. In the project, we will study the formation of vascular networks ‘sprouting’ from multiple seeds dispersed in an external hydrogel mimicking the host extracellular matrix. To control the spacing between the seeds, we will order them into arrays using magnetic forces and confine them inside a planar transparent chamber, allowing direct imaging of the forming vascular networks. To characterize the networks, we will develop cutting-edge image analysis tools based, i.e., on machine learning, as well as provide a detailed theoretical model of the developing network incorporating, for the first time, direct interaction of the endothelial cells with the surrounding extracellular matrix. We will use the model to design optimal networks that could most efficiently support the engineered tissues. Finally, we will co-culture the vascular networks with cancer cells as a realistic in vitro model of the cancer tissue. It is well known that angiogenesis plays a key role in cancer development, invasion, and metastasis. Solid tumors, e.g., lymphomas, sarcomas, as well as cancers of the breast, prostate, or thyroid, need a blood supply if they are to grow beyond a few millimeters in size. Therefore, cancer vasculature has been targeted in cancer therapies and used to deliver drugs directly to cancer cells. We will use our platform to test drugs on several cancer cell lines (e.g., of breast cancer) to develop tools for future use in personalized approaches in which the diseased cells could be harvested directly from a patient to develop an optimal patient-specific treatment strategy. In summary, the project will significantly advance the field of vascular tissue engineering, deliver new data analysis tools for general angiogenesis research, provide data-driven predictive theoretical models for developing vascular networks, as well as open new perspectives in the development of miniaturized platforms for drug testing, e.g., applicable in personalized cancer therapies.

New applications of droplet microfluidics: from biomimetics to tissue engineering

First TEAM  (POIR.04.04.00-00-26C7/16-00)

Project Leader: Dr. Jan Guzowski

Project duration: August 2017 – April 2021

The project’s goal is to develop new microfluidic methods of precise, reproducible formulation of three-dimensional structures from aqueous micro-droplets as building blocks and to demonstrate the use of such structures in micro-tissue engineering and synthesis of biomimetic capsules.

During the last decade, droplet microfluidics has emerged as a powerful tool in high-throughput diagnostic and screening applications based on the fragmentation of a sample into monodisperse micro-droplets and subsequent manipulation as isolated bioreactors. The available manipulation techniques include splitting, merging, transporting, or trapping. However, the microfluidic formation of 3D structures from droplets, offering great potential in tissue engineering and drug delivery, is still poorly understood. In the program, we will demonstrate new applications of the microfluidic-assisted formulation of droplet-based materials in i) generation of multi-compartment liposomes, i.e., capsules built of several aqueous compartments separated by lipid bilayers, with the specific 3D arrangement of segments for advanced drug delivery applications, and ii) assembling of cell-laden droplets into 3D structures and culturing novel micro-tissues with unusual morphologies as a platform for high-throughput drug testing. On the side of basic soft-matter science, we will also, for the first time, iii) generate and manipulate structures built of tightly packed hundreds and thousands of droplets as an easily accessible model system for studying the mechanics of soft granular aggregates such as cell spheroids.

Artificial pancreatic islets: microfluidics-assisted reaggregation of endocrine cells inside hydrogel microcapsules

NCN OPUS 17 (2019/33/B/ST8/02145)

Project Leader: Dr. Jan Guzowski

Project duration: March 2020 – February 2023

Type 1 diabetes is a devastating chronic disease rapidly becoming a 21st-century epidemic. It is characterized by the body’s inability to produce insulin, a hormone responsible for regulating the blood’s glucose level. Despite overall treatment progress, the cause of type 1 diabetes is not known, and it is not preventable under current knowledge. One of the most promising treatment methods, still experimental, is the transplantation of the so-called islets of Langerhans, tiny organs (less than 0.5 mm in size) located in the pancreas and responsible for the production of insulin. Unfortunately, currently, applied transplantation procedures are harmful to the islets, requiring multiple donors and making the treatment ineffective. In response to the increasing demand for pancreatic islets, tissue engineers aim to create artificial islets de novo from individual pancreatic endocrine cells (cells regulating hormone levels in the pancreas), stem cells or derived from animal tissue. In the project, we will explore one of the particularly appealing strategies relying on micro-encapsulation to generate implantable insulin-producing islet-like micro-organs. We will encapsulate the insulin-producing cells inside so-called ‘microbeads’, tiny particles made of hydrogel, a soft and bio-friendly material resembling gelatin, additionally enriched with proteins and nutrients for enhanced survival of the cells. It is well known that pancreatic cells prefer to be suspended in a soft but elastic matrix, an environment possibly resembling the native pancreatic tissue. We will formulate hydrogel microbeads optimally supporting cell growth, aggregation, and maturation to achieve fully-functional artificial islets in the project.

The project joins the increasing worldwide efforts towards developing new strategies for the treatment of type 1 diabetes. It is noteworthy that islet transplantation is likely to be soon approved for clinical application by the Federal Drug Administration in the USA (currently in phase 3 clinical trial). We believe that the proposed project is very timely. Finally, the new formulation strategies developed in the project will also have a widespread impact on microfluidics, tissue engineering, and organ-on-chip technologies.

Printing of self-assembling droplet arrays: from basic physics to applications in cell encapsulation and high-throughput screening

NCN Sonata BIS 9 (2019/34/E/ST8/00411)

Project Leader: Dr. Jan Guzowski

Project duration: September 2020 – August 2024

In cancer treatments, combinations of drugs often need to be custom-tailored to increase efficiency and minimize harmful side effects in a given patient. Such personalized treatments call for personalized drug screening technologies operating on cells taken directly from the patient. Efficient testing of hundreds of drug combinations on small tissue samples (< 1 mL) is challenging and requires the development of new miniaturized technologies for biological sample processing. Droplet microfluidics offers high-throughput drug screening tools via dispersing the liquid sample into thousands of nanoliter sub-samples and manipulating them individually via subjecting each individually to a mixture of drugs at different concentrations, incubating and finally measuring cell response. One of the bottlenecks of this emerging lab-on-chip technology is the so-called ‘barcoding’ of individual droplets, which is unique labeling allowing association of the observed cell response with the drug combination contained in the encapsulating droplet. Currently, existing droplet labeling methods based on the injection of dyes at various predesigned concentrations suffer from the relatively low resolution, limiting screens’ capacity to around a hundred droplets. In contrast, the actual applications typically involve thousands or tens of thousands of droplets.

In the project, we will develop a droplet-labeling technique based on their sequential deposition at a substrate. In particular, we will exploit the effect of the spontaneous formation of ordered droplet patterns along a printing path. Such patterns emerge due to random droplet rearrangements occurring under specific printing conditions. As a result, each droplet is labeled by a unique neighboring pattern. In the project, we will develop a basic understanding of droplet rearrangements’ phenomena during printing and study the generated patterns’ labeling capacity. We will also perform proof-of-concept experiments involving the encapsulation of living cancer cells inside the printed droplets to screen their response to varying drug concentrations.

The technology developed in the project will provide a non-invasive (dye-free) and cost-effective alternative to existing droplet barcoding methods, potentially transformative for fields such as drug development, personalized medicine, or point-of-care diagnostics.