Organ donors and their families are appreciated for their invaluable donations, and the International Institute for Organ Procurement Organizations, Advancement of Medicine (IIAM) and the National Disease Research Exchange (NDRI) are acknowledged for their partnership in making human pancreatic tissue accessible for research. This work was supported by the Human Islet Research Network (RRID:SCR_014393), the Human Pancreas Analysis Program (RRID:SCR_016202), DK106755, DK123716, DK123743, DK120456, DK104211, DK108120, DK112232, DK117147, DK112217, EY032442, and DK20593 (Vanderbilt Diabetes Research and Training Center), The Leona M. and Harry B. Helmsley Charitable Trust, JDRF, the U.S. Department of Veterans Affairs (BX000666), the NIGMS of the National Institutes of Health (T32GM007347), F30DK134041, F30DK118830, and the National Science Foundation Graduate Research Fellowship (1937963).

Human islets (N = 4 preparations) were obtained through partnerships with the Integrated Islet Distribution Program, Human Pancreas Analysis Program, Prodo Laboratories, Inc., and Imagine Pharma. The Vanderbilt University Institutional Review Board does not consider deidentified human pancreatic specimens as human subjects research. This work would not be possible without organ donors, their families, and organ procurement organizations. See Table 1 for donor demographic information. Human islets from pa...

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

<ol>
	<li>Tokarz, V. L., MacDonald, P. E., Klip, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+systemic+insulin+function.">The cell biology of systemic insulin function.</a> The Journal of Cell Biology. 217 (7), 2273-2289 (2018).</li><li>Yu, Q., Shuai, H., Ahooghalandari, P., Gylfe, E., Tengholm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+controls+glucagon+secretion+by+directly+modulating+cAMP+in+alpha+cells.">Glucose controls glucagon secretion by directly modulating cAMP in alpha cells.</a> Diabetologia. 62 (7), 1212-1224 (2019).</li><li>Hughes, J. W., Ustione, A., Lavagnino, Z., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+islet+glucagon+secretion:+Beyond+calcium.">Regulation of islet glucagon secretion: Beyond calcium.</a> Diabetes, Obesity and Metabolism. 20, 127-136 (2018).</li><li>Chen, C., Cohrs, C. M., Stertmann, J., Bozsak, R., Speier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+beta+cell+mass+and+function+in+diabetes:+Recent+advances+in+knowledge+and+technologies+to+understand+disease+pathogenesis.">Human beta cell mass and function in diabetes: Recent advances in knowledge and technologies to understand disease pathogenesis.</a> Molecular Metabolism. 6 (9), 943-957 (2017).</li><li>Halban, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-cell+failure+in+type+2+diabetes:+Postulated+mechanisms+and+prospects+for+prevention+and+treatment.">&#946;-cell failure in type 2 diabetes: Postulated mechanisms and prospects for prevention and treatment.</a> Journal of Clinical Endocrinology and Metabolism. 99 (6), 1983-1992 (2014).</li><li>Brissova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;+cell+function+and+gene+expression+are+compromised+in+type+1+diabetes.">&#945; cell function and gene expression are compromised in type 1 diabetes.</a> Cell Reports. 22 (10), 2601-2614 (2018).</li><li>Walker, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+human+pseudoislet+system+and+microfluidic+platform+demonstrate+differences+in+GPCR+signaling+in+islet+cells.">Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells.</a> JCI Insight. 5 (10), e06990 (2020).</li><li>Giannoukakis, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+intact+human+islets+by+a+lentiviral+vector.">Infection of intact human islets by a lentiviral vector.</a> Gene Therapy. 6 (9), 1545-1551 (1999).</li><li>Curran, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+pancreatic+islets+by+feline+immunodeficiency+virus+vectors1.">Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors1.</a> Transplantation. 74 (3), 299-306 (2002).</li><li>Kayton, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+islet+preparations+distributed+for+research+exhibit+a+variety+of+insulin-secretory+profiles.">Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles.</a> American Journal of Physiology-Endocrinology and Metabolism. 308 (7), E592-E602 (2015).</li><li>Cabrera, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-throughput+assays+for+evaluation+of+human+pancreatic+islet+function.">high-throughput assays for evaluation of human pancreatic islet function.</a> Cell Transplantation. 16 (10), 1039-1048 (2007).</li><li>Lenguito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resealable,+optically+accessible,+PDMS-free+fluidic+platform+for+ex+vivo+interrogation+of+pancreatic+islets.">Resealable, optically accessible, PDMS-free fluidic platform for ex vivo interrogation of pancreatic islets.</a> Lab on a Chip. 17 (5), 772-781 (2017).</li><li>Tewson, P. H., Martinka, S., Shaner, N. C., Hughes, T. E., Quinn, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DAG+and+cAMP+sensors+optimized+for+live-cell+assays+in+automated+laboratories.">New DAG and cAMP sensors optimized for live-cell assays in automated laboratories.</a> Journal of Biomolecular Screening. 21 (3), 298-305 (2015).</li><li>Tengholm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+dynamics+in+the+pancreatic+&#946;-cell.">Cyclic AMP dynamics in the pancreatic &#946;-cell.</a> Upsala Journal of Medical Sciences. 117 (4), 355-369 (2012).</li><li>Klemen, M. S., Dolen&#353;ek, J., Rupnik, M. S., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triggering+pathway+to+insulin+secretion:+Functional+similarities+and+differences+between+the+human+and+the+mouse+&#946;+cells+and+their+translational+relevance.">The triggering pathway to insulin secretion: Functional similarities and differences between the human and the mouse &#946; cells and their translational relevance.</a> Islets. 9 (6), 109-139 (2017).</li></ol>

The authors have nothing to disclose.

The integration of a microperifusion system, biosensor-expressing pseudoislets, and laser-scanning confocal microscopy allows for the synchronous assessment of intracellular signaling events and dynamic hormone secretory profiles. The dynamic microperifusion system can deliver a series of well-defined stimuli to the pseudoislets and allows for the collection of the effluent, in which the insulin and glucagon concentrations can be measured by commercially available ELISA. Concurrently, live-cell imaging of the biosensor-e...

The islets of Langerhans are mini organs scattered throughout the pancreas whose function is crucial for the maintenance of glucose homeostasis. Insulin is secreted from beta cells following the metabolism of glucose, an increase in the ATP/ADP ratio, the closure of ATP-sensitive potassium channels, depolarization of the plasma membrane, and the influx of extracellular calcium1. Glucagon secretion from alpha cells is less understood, but it has been postulated that intracellular and paracrine pathways contribute to glucagon granule exocytosis2,3,4. Both type 1 and type 2 diabetes are associated with islet cell dysfunction5,6,7. Therefore, elucidating the intracellular signaling pathways mediating islet hormone secretion is essential for understanding physiologic and pathologic mechanisms in pancreatic islets.
The spherical architecture of islets presents certain obstacles to experimentation. These challenges include islet size variation and the 3D nature of islets, which reduces viral transduction within the islet core8,9. To overcome these challenges, a pseudoislet system was developed, in which primary human islets are dispersed into single cells, adenovirally transduced with constructs encoding targets of interest, and reaggregated to form size-controlled, islet-like structures termed pseudoislets7. Compared to native islets from the same donor that have been cultured in parallel, these pseudoislets are similar in morphology, endocrine cell composition, and hormone secretion7. This method allows for the expression of constructs throughout the pseudoislet, meaning it overcomes a previous barrier to the uniform genetic manipulation of primary human islets7,8,9.
In this protocol, the pseudoislet system is integrated with a microfluidic device to express biosensors in primary human islet cells and gain temporal resolution of&#160;pseudoislet hormone secretion during dynamic perifusion10,11,12. The pseudoislets are placed in a microchip and exposed to a steady flow of different secretagogues via a peristaltic pump12. The microchip has a transparent glass bottom and is mounted on a confocal microscope to record the intracellular signaling dynamics via changes in the biosensor fluorescence intensity. Biosensor imaging is synchronized with the collection of microperifusion effluent for the subsequent analysis of insulin and glucagon secretion7. Compared to macroperifusion, this microperifusion approach allows for fewer pseudoislets to be used due to the smaller volume of the microfluidic device compared to the macroperifusion chamber7.
To harness the utility of this system, the cyclic adenosine monophosphate (cAMP) difference detector in situ (cADDis) biosensor was expressed in human pseudoislets to assess cAMP dynamics and hormone secretion. The cADDis biosensor is composed of a circularly permuted green fluorescent protein (cpGFP) positioned in the hinge region of an exchange protein activated by cAMP 2 (EPAC2), connecting its regulatory and catalytic regions. The binding of cAMP to the regulatory region of EPAC2 elicits a conformational change in the hinge region that increases fluorescence from the cpGFP13. Intracellular messengers such as cAMP elicit insulin and glucagon secretion after the upstream activation of G-protein coupled receptors14. Live-cell imaging coupled with microperifusion helps to connect the intracellular cAMP dynamics with islet hormone secretion. Specifically, in this protocol, cADDis-expressing pseudoislets are generated to monitor cAMP responses in alpha and beta cells to various stimuli: low glucose (2 mM glucose; G 2), high glucose plus isobutylmethylxanthine (IBMX; 20 mM glucose + 100 &#181;M IBMX; G 20 + IBMX), and low glucose plus epinephrine (Epi; 2 mM glucose + 1 &#181;M Epi; G 2 + Epi). This treatment workflow allows for the assessment of the intracellular cAMP dynamics directly via 1) IBMX-mediated phosphodiesterase inhibition, which enhances intracellular cAMP levels by preventing its degradation, and 2) epinephrine, a known cAMP-dependent stimulator of alpha cell glucagon secretion mediated by &#946;-adrenergic receptor activation. The steps for setting up the microperifusion apparatus for live-cell imaging experiments, the loading of the pseudoislets into the microchip, synchronous live-cell imaging and microperifusion, and the analysis of the biosensor traces and hormone secretion by microplate-based hormone assays are detailed below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ad-CMV-cADDis</td>
			<td>Welgen</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.01&#8221; FEP tubing</td>
			<td>IDEX</td>
			<td>1527L</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Enriched-CMRL Media Component</td>
		</tr>
		<tr>
			<td>1.5 mL and conical tubes</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;m PTFE filter</td>
			<td>Cole-Parmer</td>
			<td>SK-21940-41</td>
			<td>Change every 8-10 runs</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Thermo Scientific</td>
			<td>11360070</td>
			<td>Enriched-CMRL Media Component</td>
		</tr>
		<tr>
			<td>190 proof Ethanol</td>
			<td>Decon labs</td>
			<td>2816</td>
			<td>Acid Ethanol Component</td>
		</tr>
		<tr>
			<td>200 mM GlutaMAX-I Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Enriched-CMRL Media Component</td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Sigma</td>
			<td>A5960</td>
			<td>DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7888</td>
			<td>DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>Bubble trap&#160;</td>
			<td>Omnifit</td>
			<td>006BT</td>
			<td></td>
		</tr>
		<tr>
			<td>CellCarrier ULA&#160;96-well Microplates</td>
			<td>Perkin Elmer</td>
			<td>6055330</td>
			<td></td>
		</tr>
		<tr>
			<td>cellSens analysis software</td>
			<td>Olympus</td>
			<td>v3.1</td>
			<td>Software used for data analysis</td>
		</tr>
		<tr>
			<td>CMRL 1066</td>
			<td>MediaTech&#160;</td>
			<td>15-110-CV</td>
			<td>Enriched-CMRL Media Component</td>
		</tr>
		<tr>
			<td>Conical adapter (IDEX, P-794)</td>
			<td>IDEX</td>
			<td>P-794</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>Glucose Buffer Component</td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Sigma</td>
			<td>D5030</td>
			<td>DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>okolab</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Epinepherine (Epi)</td>
			<td>Sigma</td>
			<td>E4250</td>
			<td>Stimulation Buffer Component</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Heat Inactivated</td>
			<td>Sigma</td>
			<td>12306C</td>
			<td>Enriched-CMRL Media Component</td>
		</tr>
		<tr>
			<td>Glucagon ELISA</td>
			<td>Mercodia</td>
			<td>10-1281-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucagon Kit HTRF</td>
			<td>Cisbio</td>
			<td>62CGLPEH</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (12N)</td>
			<td>Any</td>
			<td>Any</td>
			<td>Acid Ethanol Component</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7523</td>
			<td>DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>iCell Endothelial Cells Medium Supplement</td>
			<td>Cell Dynamics</td>
			<td>M1019</td>
			<td>iEC Media Component</td>
		</tr>
		<tr>
			<td>Idex Derlin nut &#38; ferrule 1/4-24</td>
			<td>Cole-Parmer</td>
			<td>EW-00414-LW</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin ELISA</td>
			<td>Mercodia</td>
			<td>10-1113-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isobutylmethylonine (IBMX)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>Stimulation Buffer Component</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV3000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G8540</td>
			<td>DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>Microchip (University of Miami, FP-3W)</td>
			<td>University of Miami</td>
			<td>FP-3W</td>
			<td></td>
		</tr>
		<tr>
			<td>Microchip holder&#160;</td>
			<td>Micronit Microfluidics</td>
			<td>FC_PRO_CH4525</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 2110 Fraction Collector</td>
			<td>Biorad</td>
			<td>7318122</td>
			<td></td>
		</tr>
		<tr>
			<td>P10, P200, and P1000 pipets and tips</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Enriched-CMRL Media Component</td>
		</tr>
		<tr>
			<td>Peristaltic pump&#160;</td>
			<td>Instech</td>
			<td>P720</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Wash Islets</td>
		</tr>
		<tr>
			<td>Sarstedt dishes</td>
			<td>Sarstedt</td>
			<td>depends on dish diameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td>&#160;DMEM Perifusion Buffer Component</td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Olympus</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip Filter (0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td>Filter all buffers twice</td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>LifeLine Cell Technology</td>
			<td>LL-0003</td>
			<td>iEC Media Component</td>
		</tr>
	</tbody>
</table>

human pseudoislet system for synchronous assessment of fluorescent biosensor dynamics and hormone secretory profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

Our lab is working to understand, prevent, and reverse beta cell dysfunction and other eyelid abnormalities in type one and type two diabetes. Our studies focus on the pathogenesis of human diabetes by integrating studies of the human pancreas and eyelid biology and health and disease. Human eyelets are 3D spherical structures, and accessing cells throughout the entire eyelet presents some experimental challenges.For example, when trying to introduce biosensors to understand eyelet cell signaling, our pseudo eyelid protocol overcomes this challenge by performing genetic manipulations in the single cell state and aggregating these transduced human eyelet cells into pseudo eyelids for downstream studies. Our pseudo eyelid system allows us to express biosensors throughout the entire eyelet, rather than just the eyelid surface. Combined with our live cell imaging and Microperfusion System, we can both measure and co-register dynamic intracellular processes with downstream hormone secretion.This protocol could be applied to answer a variety of important scientific questions. For example, gene silencing strategies could be combined with this approach to understand the impact of a gene of interest on eyelid function and associated signaling pathways, either globally or in a self-specific manner.

Biosensor-expressing human pseudoislets were created via the adenoviral delivery of constructs encoding the cAMP biosensor cADDis (Figure 1A). Figure 1B shows the reaggregation of the transduced human islet cells over time, with fully formed pseudoislets observed after 6 days of culture. The cells began to show visible cADDis fluorescence within 48 h, and there was high biosensor expression in transduced cells by the end of the culture period. Using thi...

Watch this Scientific Journal Video about Investigating Islet Abnormalities and Function with a Pseudoislet Protocol at JoVE.com

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

human-pseudoislet-system-for-synchronous-assessment-fluorescent

Research

JoVE Journal

Biology

Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Profiles in Human Pseudoislets

Start by connecting the tubing of the microperfusion apparatus to the chip holder. Flush the line with a baseline secretagogue, like two-millimolar glucose, for five minutes. Aspirate any extra fluid from the top and bottom valves of the microchip.The top half of the microchip ensures adequate sealing of wells, while the bottom half contains the wells with a glass cover slip attached. Next, pre-wet a well with five microliters of DMEM. Pipette around the outer well edges to wet the crevices.Take brightfield and darkfield images of the cultured human pseudo eyelets isolated from pancreatic primary human eyelets. Using a pre-wetted pipette, collect 30 to 32 pseudo eyelets in 23 microliters of medium, and dispense them slowly into the microchip well center. Ensure the pipette tip has no pseudo eyelets left attached.Use a gel loading tip to gently maneuver the pseudo eyelets into the well center for maximum field view. Capture a stereoscope image of the pseudo eyelets in the microchip to adjust for pseudo eyelet loss. If all pseudo eyelets from the plate are not loaded into the microchip, adjust the eyelet equivalent quantification accordingly.Place the microchip bottom into the holder, then carefully place the microchip top on with a green gasket facing down. While holding the microchip in place, close the holder to clamp the microchip together. Transfer the secretagogues, pump, and microchip in the holder into the environmental chamber fitted to the confocal microscope and direct the efflux tubing out of the chamber to the fraction collector.Place the buffers in 15-milliliter conical tubes with openings drilled into their caps to prevent tubing drift. Separate the nut and ferrule. Then screw the tubing into the de-bubbler to prevent line twisting.Next, set the fraction collector to rotate every two minutes. Then load it with the appropriate number of tubes, accounting for washes and experimental fractions. Start the pump to deliver the baseline glucose medium at 100 microliters per minute.Watch for droplets at the end of the fraction collector spout, indicative of system flow through. Decreased system efflux, as seen in the tube labeled with the red X, is indicative of system blockage or leak. Once a steady medium flow has been established, rotate the fraction collector head to dispense into the tubes and start the fraction collector.Collect the first 15 wash fractions to allow the pseudo eyelets to equilibrate. After ensuring continual and accurate medium flow through, discard the washes. While the washes are being collected, set up the microscope for live cell imaging.Set the objective lens to U Plan Fluorite 20X with 1X zoom. The fluorescence channels to EGFP with emission set to 510 nanometers. Laser wavelength to 488.And detection wavelength to 500 to 600 nanometers. Set the time series to image acquired every two seconds for the entirety of the experiment and the sampling speed to two microseconds per pixel. Now identify the bottom of the pseudo eyelets in the field of view and adjust the focal plane to 15 micrometers above this position, a frame to be used throughout imaging.Once the wash is complete, press Start on the imaging software when the fraction collector moves to the first fraction and collect the effluent into 1.5 milliliter tubes. At an appropriate time, switch the tubing from the baseline buffer to the new stimulus buffer tube manually. Place the first 10 collections at four degrees Celsius to prevent hormone degradation and continue to monitor the system flow by checking the effluent volume in each tube.Once the experiment is complete, switch back to the baseline medium, allowing the pump to wash out the stimulus buffer for five minutes. Stop the pump and disconnect the microchip holder tubing at the de-bubbler and fraction collector. Close all doors of the environmental chamber to maintain the temperature.Store the perfusates at 80 degrees Celsius for subsequent hormone analysis. Carefully open the microchip holder and lift off the top of the microchip. Take a final microscopic image of the pseudo eyelets in the microchip before removal to adjust the eyelet extract IEQ.Using a pipette and a microscope, transfer the pseudo eyelets from the well to a 1.5-milliliter tube. Ensure that all of the pseudo eyelets have been collected with the help of the microscope. Wash twice with 500 microliters of PBS.Then spin the pseudo eyelets for one minute at 94G between each wash. After aspirating the supernatant, add 200 microliters of acid ethanol and store at four degrees Celsius overnight. The next day, centrifuge the extracts.Then aliquot 45 microliters of the supernatant into three new tubes. Store the tubes at 80 degrees Celsius for hormone content analysis. The transduced human eyelet cells showed reaggregation over time with fully-formed pseudo eyelets formed after six culture days.The cells began to show visible cADDis fluorescence within 48 hours, and there was high biosensor expression in transduced cells by the end of the culture period. The pseudo eyelets displayed an average transduction efficiency of 60%in alpha cells and 95%in beta cells. The live cell imaging and microperfusion setup facilitated the synchronous collection of the intracellular cAMP dynamics through cADDis fluorescence and hormone secretion.Exposure to two-millimolar glucose resulted in low and stable relative cADDis intensity and insulin secretion. A robust increase in the intracellular cAMP concentration was seen when the cells were exposed to 20-millimolar glucose and IBMX. This was accompanied by increased insulin and glucagon.The exposure of the pseudo eyelets to two-millimolar glucose and epinephrine increased the intracellular cAMP concentration, which was associated with increased glucagon.

1.3K Views.  Vanderbilt University School of Medicine.  This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system. 

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

Summary