In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices

Abstract

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices’ full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices’ in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the “Oxygen Measurement Core” was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members’ laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D.

Assessment of blood–brain barrier leakage and brain oxygenation in Connexin‐32 knockout mice with systemic neuroinflammation using pulse electron paramagnetic resonance imaging techniques

“…Oxygen‐sensitive trityl OX063 or its deuterated analog OX071‐based pulse electron paramagnetic resonance imaging (pEPRI) is an emerging methodology for obtaining quantitative and absolute partial oxygen pressure (pO 2 ) maps in tissues and is often termed EPR oxygen imaging (EPROI) 22–24 . Trityls, OX063/OX071, exhibit low toxicity, extraordinary stability in biological media, high water solubility, and a narrow single‐line EPR spectrum, resulting in a high SNR 22,23,25–27 . The OX071 (Supporting Information Figure S1A) shows a narrower linewidth compared with OX063, depending on the deuteration level, because deuterium coupling to an electron is considerably less than proton coupling, which amounts to higher SNR, and therefore has been a preferred spin probe for EPR‐based oximetry for the past decade.…”

“…Currently, there are two main methodologies to acquire oxygen maps using pEPRI. The first method is the inversion‐recovery electron spin echo (IRESE) 22,26,28–30 and the second is single point imaging (SPI) 31–34 . SPI infers spin–spin relaxation time (T 2 ) from FID (T 2 *), and IRESE maps spin–lattice relaxation time (T 1 ).…”

A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling

“…Silicone microbeads incorporated into a 3D cell culture and electron paramagnetic resonance imaging (EPR) are potential non-invasive approaches. However, the large size of the beads and the low resolution of EPR are critical barriers to measuring pO 2 in small 3D tissues at the µm level (24)(25)(26).…”