Table 2C summarizes some of the more recently explored assays used in islet quality assessment, highlighting their key strengths and identified weaknesses. Despite the landscape of flavors available to researchers, many of these assays are most valuable when used in the study of individual cells rather than cell aggregates.
Most assays used to assess cellular viability, apoptosis, or mitochondrial health, have been designed for suspensions or cultures of individual cells, not aggregates. Consequently, the development of techniques to study the quality of an islet preparation provides unique challenges. The relatively large size of the islet makes fluorescence microscopy difficult, subjecting any such analysis to background signal and operator bias that is simply unique to the study of intact multi-cellular clusters. To circumvent some of the islet shape and size limitations, techniques have been developed to break apart the islets.
To minimize the problems associated with islet disaggregation, gentler formulations have been created and used [ 19 ]. Yet, it is unclear whether the negative effects of dissociating individual islet cells from one another can be fully minimized. Furthermore, islet preparations have varying amounts of impurities, which complicates the use of any technique designed with the expectation that the studied tissue is comprised entirely of islets. Differentiating the non-endocrine tissue from the islets poses additional difficulties.
These assays interrogate the integrity of the cellular plasma membrane and rely on differential staining using newer combinations of both cell membrane permeable and impermeable dyes [ 12 , 16 ], but have been unable to fully obviate the problems encountered with the current viability stains used prior to product release i.
In fact, some of these proposed stains introduce new issues, such as islet toxicity [ 16 ]. An alternative approach relies on sequential staining of membrane compromised cells within intact islets using 7-AAD. After initially staining with 7-AAD, the nuclei of the entire preparation are released from intact islets using a detergent and subsequently counted by hemacytometer or FACS [ 48 — 49 ]. The initial count of non-viable cells is divided by the second count of total nuclei to present a ratio equivalent to fractional viability FV. This technique bypasses the limitations associated with islet disaggregation of multi-cellular spheroids, such as islets; however, as a membrane integrity test, it only accounts for dead cells with compromised cell membranes [ 16 , 49 ].
These assays may depend on the timing of the measurement as it relates to the onset of apoptosis. The magnitude and timing of the responses may also vary between cell types and the unique nature, intensity, and duration of encountered stresses [ 16 ]. Importantly, these cell death markers may not be reliable indicators of irreversible damage. Even though mechanistic information regarding the cell death process can be obtained, individual assays may not capture all dying cells and still suffer from limitations that are related to islet size and its three-dimensional structure.
The system involved perfusing a microfluidic chip containing intact islets.
Tetrazolium assays like MTT have fallen slightly out of favor because many variables or conditions, not limited to mitochondrial activity, can affect the ability of a preparation to reduce tetrazolium salts [ 16 ]. MMP dyes are used as surrogate measures of mitochondrial health, in that they preferentially accumulate in healthy and polarized mitochondria. The instrumentation and methodologies employed along with the strengths and limitations of each approach are outlined in Table 3. The approach for indirectly measuring OCR using fluorescence intensity in a multi-well plate oxygen biosensor system OBS has the distinct advantage of being high-throughput and convenient but in its current form suffers from several major limitations that prohibit its reliable use [ 16 , 26 ].
Recent efforts to bypass some of the inherent limitations of the OBS [ 62 ] may enable more reliable and effective use of this method in islet potency assessment. Correlation of transplantation outcomes with rat islets was substantially better than that obtained with human islet preparations [ 32 ]. Interestingly, initial data obtained with pure and impure clinical autologous and single-donor, allogeneic islet transplants suggest that in these cases especially islet auto-transplants , the OCR dose normalized per KgBW alone may sufficiently correlate with clinical outcomes unpublished observations.
Publications detailing these procedures report reasonable correlations with the NMB and suggest that there may be an advantage in using these indices for clinical islet potency assessment. It remains to be seen if the challenges associated with widespread implementation and inherent limitations of these complicated methodologies [ 16 ] can be overcome and whether the promising results attained with research models will translate into the clinical setting.
Work currently under way with clinical auto- and allo- and pre-clinical xeno- transplant models is expected to provide further insight into these issues and help identify and establish islet potency tests that are truly predictive of transplant outcomes. The islet product release criteria that screen preparations before clinical allogeneic ICT are currently unable to predict post-transplant success from failure. Those assessing mitochondrial function, particularly those that measure the OCR of an islet preparation, appear to be the most promising and correlate with transplant outcomes in the NMB.
These tests are currently being evaluated in the clinical setting and preliminary results are encouraging. Assays that characterize cell composition and molecular profiles may be useful in further defining the islet product and may provide useful information on islet immunogenicity and pro-inflammatory potential.
The recent clinical success in reversing diabetes with single-donor, allogeneic transplants, will further enhance our ability to define potency tests and islet characteristics that are predictive of transplant outcome.
Colton P.I. - Second Unit by Gina Cresse
The authors would like to thank Drs. Stathis Avgoustiniatos and Lou Kidder for critically reviewing the manuscript. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.
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Assays evaluating mitochondrial function, specifically mitochondrial membrane potential, bioenergetic status, and cellular oxygen consumption rate, especially when conducted with intact islets, appear most promising in evaluating their quality prior to islet cell transplantation. Prospective, quantitative assays based on measurements of oxygen consumption rate with intact islets have been developed, validated, and their results correlated with transplant outcomes in the diabetic nude mouse bioassay.
The snippet could not be located in the article text. This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article. Curr Opin Organ Transplant. Author manuscript; available in PMC Dec 1. PMID: Klearchos K.
Papas , 1, 2 Thomas M. Suszynski , 1 and Clark. Colton 2. Thomas M. Address correspondence to: Klearchos K. Papas, Ph. Copyright notice. The publisher's final edited version of this article is available at Curr Opin Organ Transplant.
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See other articles in PMC that cite the published article. Abstract Purpose of review There is a critical need for meaningful viability and potency assays that characterize islet preparations for release prior to clinical islet cell transplantation ICT. Recent Findings Assays based on membrane integrity do not reflect true viability when applied to either intact islets or dispersed islet cells. Conclusion More sensitive and reliable islet viability and potency tests have been recently developed and tested.
Keywords: Oxygen consumption rate, viability, potency, release criteria, marginal mass, purity. Introduction Islet cell transplantation ICT is emerging as a promising approach for the treatment of selected patients with type 1 diabetes mellitus [ 1 — 8 ]. Current specifications for lot release prior to clinical islet transplantation The FDA mandates that for any cellular and tissue-based product, the manufacturer must be able to demonstrate that it can be safely and reproducibly manufactured [ 10 ]. Open in a separate window.
Limitations of the tests currently used for islet lot release prior to clinical transplantation Many of the methods currently used to assess islet preparations were developed nearly 20 years ago [ 11 ]. Dissociating islets typically involves harsh enzymatic digestion with serine proteases that results in the disruption of cell-matrix interactions, cellular damage and death e,g. Sampling from an islet suspension An important issue in characterizing islet preparations, relevant to all assays, is sampling.