Microgrippers to handle Organoids and pancreatic Islets for Precision Measurements of biological Function

The model of the cultured single cell is considered insufficient to explain the physiological regulation taking place at the organ level. The same is true for the prediction of drug action at the organ level or at the level of the intact organism. For these reasons 3D cell culture models are in increasing demand. It is thus necessary to develop the instruments to handle such cell aggregates and organoids in a controlled, precise and gentle manner. Here, a microgripper is presented which is able to work in aqueous solutions and which is compatible with electrophysiological recordings of the cells immobilized by it. It was successfully employed to position isolated pancreatic islets and a 3D cell culture model of insulin-secreting cells, the so-called MIN6-pseudoislet. As required it was possible to measure the membrane potential of cells within these aggregates without any interference from the microgripper

Studies of the secretion regulation of pancreatic islets with special regard to alpha cells

Der Diabetes mellitus ist trotz intensiver Forschung und stetig verbesserter Therapieverfahren weltweit eine der wichtigsten Stoffwechselerkrankung. Da neben dem Mangel an Insulin auch der Überschuss von Glucagon zur Entstehung des Krankheitsbildes beiträgt, liegt in der Reduktion der Glucagonsekretion ein aussichtsreicher therapeutischer Ansatzpunkt. Die vorliegende Arbeit soll dazu dienen, die der Insulin- und Glucagonsekretion zugrunde liegenden Mechanismen und ihre Wechselwirkungen weiter aufzuklären, um daraus bessert therapeutische Interventionen entwickeln zu können. Während der auslösende Weg (triggering pathway), der die Insulinsekretion einleitet, relativ gut aufgeklärt ist, ist der verstärkende Weg (amplifying pathway) der die tatsächliche Sekretionsstärke zu regeln scheint, noch in großen Teilen unverstanden. Um diesen zu untersuchen wurde ein etabliertes Protokoll verwendet, in dem Glucose selbst in maximal effektiver Konzentration vollständig ihre insulinsekretionssteigernde Wirkung verliert, während die Ketosäure alpha-Ketoisocapronsäure (KIC) weiterhin in der Lage ist, die Sekretion zu stimulieren, wahrscheinlich, weil sie Alpha-Ketoglutarat für den Citracyclus bereitstellt. Es ließ sich nun zeigen, dass die Ketosäure alpha-Ketoisovaleriansäure (KIV), die allein nicht insulinotrop ist, die verloren gegangene insulinotrope Wirkung der Glucose wiederherstellen kann und diese dann auch bei alleiniger Anwesenheit von Glucose weiter bestehen bleibt. Daraus kann geschlussfolgert werden, dass das Wiederauffüllen des Citratcyclus durch KIV dessen dauerhafte Funktion und damit den Export von Metaboliten (Kataplerose) ermöglicht, die für die Amplifikation der Insulinsekretion notwendig sind. Die Identifikation der Metabolite sowie ihrer Zielstrukturen bedarf jedoch weiterer Aufklärung. Im Gegensatz zur Insulinsekretion ist der Mechanismus der Glucagonsekretion auch in seinen Grundlagen weitgehend kontrovers. Um die Bedeutung der KATP-Kanäle und der Depolarisation des Plasmamembran für die Glucagonsekretion weiter zu klären, wurden simultane Messungen der Insulin- und Glucagonsekretion, der intrazellulären Calciumkonzentration und von elektrophysiologischen Parametern durchgeführt. Dadurch konnten wir zeigen, dass auch in Alpha-Zellen der Schluss des KATP-Kanals zu einer Depolarisation der Zellmembran führt, der ein Calciumeinstrom und eine Sekretionsauslösung folgt, diese Ereignisse jedoch in ihrem Ausmaß deutlich geringer sind als die entsprechenden Ereignisse in Beta-Zellen. Messungen des verbleibenden Stromflusses nach Blockade des KATP-Kanals durch ATP legen zudem eine weniger dominante Rolle für das Membranpotential der Alpha-Zellen als für dasjenige der Beta-Zellen nahe. Um die Konsequenzen für die Glucagonsekretion von intakten Inseln darstellen zu können, bedurfte es der Aufhebung der parakrinen Hemmung durch die Beta-Zellen. Der Alpha-2-Agonist Clonidin erwies sich dabei als geeignetes Werkzeug, da es die Insulinsekretion effektiv hemmte, ohne die durch Depolarisation stimulierte Glucagonsekretion relevant zu beeinflussen. Dadurch konnte die Wirkung depolarisierender Stimuli auf die Glucagonsekretion untersucht werden, ohne dass sie von der parakrinen Hemmung beeinflußt wurden. Eine bemerkenswerte Ausnahme bildete die durch 40 mM Kalium ausgelöste Stimulation, die trotz gesteigerter Insulinsekretion auch eine gesteigerte Glucagonsekretion erbrachte. Die direkte glucagonotrope Wirkung der Sulfonylharnstoffe Tolbutamid und Gliclazid war gering und vorübergehend. Ob die parakrine Hemmung durch Beta-Zellen direkt oder indirekt über Somatostatin aus Delta-Zellen erfolgt, bedarf weiterer Untersuchung. Insgesamt konnten wir zeigen, dass sowohl autonome als auch parakrine Mechanismen die Glucagonsekretion regulieren. Da in der Signalkaskade der Alpha-Zellen ab dem Schluss des KATP-Kanals kein grundlegender Unterschied zu den Beta-Zellen festgestellt werden konnte, der die inverse Glucoseabhängigkeit der Glucagonsekretion erklären würde, scheint eine Untersuchung des Glucosemetabolismus der Alpha-Zellen der logische Ansatzpunkt für künftige Untersuchungen zu sein.Despite intensive research and continuously improved therapy, diabetes mellitus is still one of the most significant metabolic diseases in the world. In addition to the deficient insulin secretion the (relative) excess of glucagon secretion contributes to the pathophysiology development of the disease. Thus, the control of glucagon secretion may be of therapeutic value. The present study aims to further clarify the mechanisms underlying insulin and glucagon secretion and their interactions in order to develop better therapeutic interventions. While the triggering pathway that initiates insulin secretion is comparatively well understood, the amplifying pathway, which appears to be responsible for setting the actual level of secretion, is still poorly understood. This pathway was investigated by using an established protocol where glucose, even at a maximally effective concentration, loses its ability to amplify insulin secretion, while the keto acid alpha-ketoisocaproic acid (KIC) is still able to do so. A possible explanation for this ability of KIC is its anaplerotic effect on the citrate cycle by providing alpha-ketoglutarate. It could now be shown that the keto acid alpha-ketoisovaleric acid (KIV), which is not insulinotropic on its own, can restore the lost amplifying effect of glucose, which persists even in the presence of glucose alone. This role as a “starter” likely reflects the anaplerosis by KIV of the citrate cycle which had previously become depleted and lends support to the notion that the export of metabolites (cataplerosis) is necessary for the amplification of insulin secretion. In contrast to insulin secretion, even the basic features of glucagon stimulus-secretion coupling are controversial. To clarify the role of KATP channels and plasma membrane depolarization for glucagon secretion, simultaneous measurements of insulin and glucagon secretion were performed as well as measurements of the intracellular calcium concentration and electrophysiological parameters. It could be shown that the sulfonylurea-mediated closure of the KATP channel occurs less reliably than in beta-cells, but once established also leads to depolarisation followed by calcium influx and increase of the cytosolic Ca2+ concentration. Measurements of the residual currents after blockade of the KATP channel by ATP also suggest a less dominant role than in beta-cells of the KATP channel for the metabolism-dependent control of the membrane potential. To check the direct effects of KATP channel closure and of potassium depolarization on glucagon secretion by perifused islets, it was necessary to abolish the paracrine inhibition by the beta cells. The alpha-2 agonist clonidine proved to be a suitable tool for this purpose, as it effectively inhibited insulin secretion without having a relevant effect on stimulated glucagon secretion. The direct glucagonotropic effect of sulfonylureas thus characterized was small and transient. Both sulfonylureas tested, tolbutamide and gliclazide, displayed the same characteristics. In contrast to the depolarization by 15 mM KCl the depolarization by 40mM KCl moderately but significantly increased insulin secretion in the presence of clonidine. In spite of the elevated insulin secretion glucagon secretion was also increased, even more so than by 15 mM KCl. This points to the supraphysiological strength of depolarization by 40 mM KCl. To which degree the paracrine inhibition by beta cells is direct or exerted via somatostatin release of the delta cells needs further investigation. To conclude, we could show that both alpha cell-autonomous and paracrine mechanisms regulate glucagon secretion. Since no fundamental differences could be found between the KATP channel-dependent signaling cascade of alpha cells and those of beta cells, a detailed investigation of the glucose metabolism of alpha cells may be needed to explain the inverse glucose dependence of glucagon secretion

Fresh and cultured mouse islets differ in their response to nutrient stimulation

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs-Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites

An Immersible Microgripper for Pancreatic Islet and Organoid Research

To improve the predictive value of in vitro experimentation, the use of 3D cell culture models, or organoids, is becoming increasingly popular. However, the current equipment of life science laboratories has been developed to deal with cell monolayers or cell suspensions. To handle 3D cell aggregates and organoids in a well-controlled manner, without causing structural damage or disturbing the function of interest, new instrumentation is needed. In particular, the precise and stable positioning in a cell bath with flow rates sufficient to characterize the kinetic responses to physiological or pharmacological stimuli can be a demanding task. Here, we present data that demonstrate that microgrippers are well suited to this task. The current version is able to work in aqueous solutions and was shown to position isolated pancreatic islets and 3D aggregates of insulin-secreting MIN6-cells. A stable hold required a gripping force of less than 30 μN and did not affect the cellular integrity. It was maintained even with high flow rates of the bath perfusion, and it was precise enough to permit the simultaneous microfluorimetric measurements and membrane potential measurements of the single cells within the islet through the use of patch-clamp electrodes