Microfluidic Tissue Processing Platform for Single Cell Analysis and Therapeutic Applications
- Author(s): Lombardo, Jeremy
- Advisor(s): Haun, Jered B
- et al.
Tissues are highly complex ecosystems, composed of heterogenous cell populations that vary in gene expression and function due to epigenetic and genetic distinctions, stochastic events, and microenvironmental factors. In the context of cancer, characterizing intratumor heterogeneity has been crucial in understanding cancer progression, metastasis, and the development of drug resistance. In order to capture this significant heterogeneity, high-throughput single cell analysis methods like flow cytometry, mass cytometry, and single cell RNA sequencing must be employed. However, these analysis methods require that tissues first be dissociated into cellular suspensions, which currently represents a major bottleneck hindering these efforts. Conventional protocols for dissociating tissues are inefficient and antiquated, relying on many manual intensive, time-consuming and highly user-variable steps for digesting, disaggregating, and filtering tissue specimens. In areas of regenerative medicine, the reliance on proteolytic enzymes to liberate stem/progenitor cell populations from tissue results in a cellular therapeutic that no longer meets the Food and Drug Administration’s guidelines for minimal manipulation. These therapeutics thus face increased regulatory barriers hindering their application in clinical settings. However, advances in microfabricated technologies hold exciting potential in their ability to execute many standard laboratory procedures, including tissue dissociation, on-chip by offering high-throughput and precise sample manipulation. The goal of this work is to develop and integrate a suite of microfluidic device technologies to dissociate tissues at the point of care. First, we present a simple and inexpensive microfluidic device that simultaneously filters large tissue fragments and dissociates smaller aggregates into single cells, thereby improving single cell yield and purity. Next, we integrate this microfluidic filter device with upstream tissue digestion and aggregate dissociation technologies. The resulting microfluidic platform significantly improves the breakdown of diverse minced tissue specimens, including tumor samples, into high quality cell suspensions that are ready for downstream single cell analysis. We then optimize a microfluidic digestion protocol for primary immune cell isolation from spleen specimens, exploring both enzymatic and nonenzymatic methods. Lastly, we adapt our microfluidic tissue processing technologies to standardize and improve mechanical processing of human lipoaspirate for autologous therapeutics. In future work, we envision incorporating cell sorting and analysis capabilities on-chip to achieve point-of-care single cell diagnostic or therapeutic platforms.