Sucrose Delivery Research Articles

Contextual cues facilitate dynamic value encoding in the mesolimbic dopamine system.

Adaptive behavior in a dynamic environmental context often requires rapid revaluation of stimuli that deviates from well-learned associations. The divergence between stable value-encoding and appropriate behavioral output remains a critical component of theories of dopamine's function in learning, motivation, and motor control. Yet, how dopamine neurons are involved in the revaluation of cues when the world changes, to alter our behavior, remains unclear. Here, we make use of a complementary set of invivo approaches to clarify the contributions of the mesolimbic dopamine system to the dynamic reorganization of reward- seeking behavior. Male and female rats were trained to discriminate when a conditioned stimulus would be followed by a sucrose reward by exploiting the prior, non-overlapping presentation of a another discrete cue-an occasion setter. Only when the occasion setter's presentation preceded the conditioned stimulus did the conditioned stimulus predict sucrose delivery, dissociating the average value of the conditioned stimulus from its immediate value, on a trial-to-trial basis. Activity of ventral tegmental area dopamine neurons was essential for rats to successfully update behavioral response to the occasion setter. Moreover, dopamine release in the nucleus accumbens following the conditioned stimulus only occurred when the occasion setter indicated it would predict reward and did not reflect its average expected value. Downstream of dopamine release, we found that neurons in the nucleus accumbens dynamically tracked the value of the conditioned stimulus. Together, these results help refine notions of dopamine function, revealing a prominent contribution of the mesolimbic dopamine system to the rapid revaluation of motivation.

Chronic stress deficits in reward behaviour co-occur with low nucleus accumbens dopamine activity during reward anticipation specifically

Whilst reward pathologies are major and common in stress-related neuropsychiatric disorders, their neurobiology and treatment are poorly understood. Imaging studies in human reward pathology indicate attenuated BOLD activity in nucleus accumbens (NAc) coincident with reward anticipation but not reinforcement; potentially, this is dopamine (DA) related. In mice, chronic social stress (CSS) leads to reduced reward learning and motivation. Here, DA-sensor fibre photometry is used to investigate whether these behavioural deficits co-occur with altered NAc DA activity during reward anticipation and/or reinforcement. In CSS mice relative to controls: (1) Reduced discriminative learning of the sequence, tone-on + appetitive behaviour = tone-on + sucrose reinforcement, co-occurs with attenuated NAc DA activity throughout tone-on and sucrose reinforcement. (2) Reduced motivation during the sequence, operant behaviour = tone-on + sucrose delivery + sucrose reinforcement, co-occurs with attenuated NAc DA activity at tone-on and typical activity at sucrose reinforcement. (3) Reduced motivation during the sequence, operant behaviour = appetitive behaviour + sociosexual reinforcement, co-occurs with typical NAc DA activity at female reinforcement. Therefore, in CSS mice, low NAc DA activity co-occurs with low reward anticipation and could account for deficits in learning and motivation, with important implications for understanding human reward pathology.

You are How You Eat: Foraging Behavior as a Potential Novel Marker of Rat Affective State

Effective and safe foraging requires animals to behave according to the expectations they have about the rewards, threats, and costs in their environment. Since these factors are thought to be reflected in the animals’ affective states, we can use foraging behavior as a window into those states. In this study, rats completed a foraging task in which they had repeatedly to decide whether to continue to harvest a food source despite increasing time costs, or to forgo food to switch to a different food source. Rats completed this task across two experiments using manipulations designed to induce both positive and negative, and shorter- and longer- term changes in affective state: removal and return of enrichment (Experiment 1), implementation and reversal of an unpredictable housing treatment (Experiment 1), and delivery of rewards (tickling or sucrose) and punishers (air-puff or back-handling) immediately prior to testing (Experiment 2). In Experiment 1, rats completed fewer trials and were more prone to switching between troughs when housed in standard, compared to enriched, housing conditions. In Experiment 2, rats completed more trials following pre-test tickling compared to pre-test sucrose delivery. However, we also found that they were prone to disengaging from the task, suggesting they were really choosing between three options: ‘harvest’, ‘switch’, or ‘not work’. This limits the straightforward interpretation of the results. At present, foraging behavior within the context of this task cannot reliably be used as an indicator of an affective state in animals.

Ventral Tegmental Area Amylin Receptor Activation Differentially Modulates Mesolimbic Dopamine Signaling in Response to Fat versus Sugar.

Amylin, a pancreatic hormone that is cosecreted with insulin, has been highlighted as a potential treatment target for obesity. Amylin receptors are distributed widely throughout the brain and are coexpressed on mesolimbic dopamine neurons. Activation of amylin receptors is known to reduce food intake, but the neurochemical mechanisms behind this remain to be elucidated. Amylin receptor activation in the ventral tegmental area (VTA), a key dopaminergic nucleus in the mesolimbic reward system, has a potent ability to suppress intake of palatable fat and sugar solutions. Although previous work has demonstrated that VTA amylin receptor activation can dampen mesolimbic dopamine signaling elicited by random delivery of sucrose, whether this is also the case for fat remains unknown. Herein we tested the hypothesis that amylin receptor activation in the VTA of male rats would attenuate dopamine signaling in the nucleus accumbens core in response to random intraoral delivery of either fat or sugar solutions. Results show that fat solution produces a greater potentiation of accumbens dopamine than an isocaloric sucrose solution. Moreover, activation of VTA amylin receptors elicits a more robust suppression of accumbens dopamine signaling in response to fat solution than to sucrose. Taken together these results shed new light on the amylin system as a therapeutic target for obesity and emphasize the reinforcing nature of high-fat/high-sugar diets.

GLP-1 receptor agonist semaglutide reduces appetite while increasing dopamine reward signaling

GLP-1 receptor agonist semaglutide reduces appetite while increasing dopamine reward signaling

Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.

Task parameters influence operant response variability in mice.

During operant conditioning, animals associate actions with outcomes. However, patterns and rates of operant responding change over learning, which makes it difficult to distinguish changes in learning from general changes in performance or movement. Thus, understanding how task parameters influence movement execution is essential. To understand how specific operant task parameters influenced the repetition of future operant responses, we investigated the ability of operant conditioning schedules and contingencies to promote reproducible bouts of five lever presses in mice. Mice were trained on one of the four operant tasks to test three distinct hypotheses: (1) whether a cue presented concurrently with sucrose delivery influenced the pattern of lever pressing; (2) whether requiring animals to collect earned sucrose promoted the organization of responses into bouts; and (3) whether only reinforcing bouts where interresponse time (IRT) variances were below a target promoted reproducible patterns of operant behavior. (1) Signaling reinforcer delivery with a cue increased learning rates but resulted in mice pressing the lever in fast succession until the cue turned on, rather than executing discrete bouts. (2) Requiring mice to collect the reinforcer between bouts had little effect on behavior. (3) A training strategy that directly reinforced bouts with low variance IRTs was not more effective than a traditional fixed ratio schedule at promoting reproducible action execution. Together, our findings provide insights into the parameters of behavioral training that promote reproducible actions and that should be carefully selected when designing operant conditioning experiments.

Is increased operant wheel running on a fixed-ratio schedule due to adjunctive or reinforced behavior?

Do operant wheel running rates that produce the delivery of sucrose on a schedule of reinforcement increase due to adjunctive or reinforced behavior? The current study addressed this question by exposing seven female Long Evans rats in running wheels to a fixed ratio (FR) 40 schedule. Across three conditions, the 40th revolution on this schedule produced the delivery of a drop of 0% (water), 25%, and 0% sucrose solution. Operant wheel-running rates, at a session level, were significantly higher when 25% sucrose was delivered. Median PRPs increased systematically with the consumption of sucrose. Wheel-running rates increased equivalently over all revolutions on the FR 40 for most rats, with only one rat showing an adjunctive pattern of increased running over earlier revolutions. Consequently, it was concluded that the increase in operant wheel-running rates, in the current, as well as prior studies using FR schedules, was due to reinforced rather adjunctive behavior.

Reinstatement of Pavlovian responses to alcohol cues by stress.

Stress may contribute to relapse to alcohol use in part by enhancing reactivity to cues previously paired with alcohol. Yet, standard models of stress-induced reinstatement generally use contingent presentations of alcohol-paired cues to reinforce instrumental behaviors, making it difficult to isolate the ability of cues to invigorate alcohol-seeking. Here we sought to test the impact of stress on behavioral responses to alcohol-paired cues, using a model of stress-induced reinstatement of Pavlovian conditioned approach, inspired by Nadia Chaudhri's work on context-induced reinstatement. Long Evans rats were trained to associate one auditory cue with delivery of alcohol or sucrose and an alternative auditory cue with no reward. Following extinction training, rats were exposed to a stressor prior to being re-exposed to the cues under extinction conditions. We assessed the effects of yohimbine, intermittent footshock and olfactory cues paired with social defeat on responses to alcohol-paired cues and the effects of yohimbine on responses to sucrose-paired cues. The pharmacological stressor, yohimbine, enhanced alcohol seeking in a Pavlovian setting, but not in a cue-selective manner. Intermittent footshock and social defeat cues did not enhance alcohol seeking in this paradigm. While yohimbine elicited reinstatement of reward-seeking in a Pavlovian setting, these effects may be unrelated to activation of stress systems or to interactions with specific cues.

Systemic injection of nicotinic acetylcholine receptor antagonist mecamylamine affects licking, eyelid size, and locomotor and autonomic activities but not temporal prediction in male mice

Nicotinic acetylcholine receptors are thought to be associated with a wide range of phenomena, such as movement, learning, memory, attention, and addiction. However, the causal relationship between nicotinic receptor activity and behavior remains unclear. Contrary to the studies that examined the functions of muscarinic acetylcholine receptors, the role of the nicotinic acetylcholine receptors on behavior has not been examined as extensively. Here, we examined the effects of intraperitoneal injection of mecamylamine, a nicotinic acetylcholine receptor antagonist, on the performance of male mice in a head-fixed temporal conditioning task and a free-moving open-field task. The head-fixed experimental setup allowed us to record and precisely quantify the licking response while the mice performed the behavioral task with no external cues. In addition, by combining the utility of the head-fixed experimental design with computer vision analysis based on deep learning algorithms, we succeeded in quantifying the eyelid size of awake mice. In the temporal conditioning task, we delivered a 10% sucrose solution every 10 s using a blunt-tipped needle placed within the licking distance of the mice. After the training, the mice showed increased anticipatory licking toward the timing of sucrose delivery, suggesting that the mice could predict the timing of the reward. Systemic injection of mecamylamine decreased licking behavior and caused eye closure but had no effect on learned conditioned predictive behavior in the head-fixed temporal conditioning task. In addition, the injection of mecamylamine decreased spontaneous locomotor activity in a dose-dependent manner in the free-moving open-field task. The results in the open-field experiments further revealed that the effect of mecamylamine on fecal output and urination, suggesting the effects on autonomic activities. Our achievement of successful eyelid size recording has potential as a useful approach in initial screening for drug discovery. Our study paves a way forward to understanding the role of nicotinic acetylcholine receptors on learning and behavior.

Appetitive Pavlovian conditioning of the stimulus effects of nicotine enhances later nicotine self-administration.

Nicotine produces robust stimulus effects that can be conditioned to exert stimulus control over behavior through associative learning. Additionally, nicotine has weak reinforcing effects that are inconsistent with its prevalence of use and the tenacity of nicotine dependence. The present study investigated whether conditioned associations to the nicotine drug stimulus may confer additional reinforcing strength to nicotine that thereby increase its use liability, and presents a new methodological approach to investigating the interaction between the stimulus effects and reinforcing effects of drugs. Male and female Sprague-Dawley rats were divided into groups receiving intravenous infusions of either 0.03 mg/kg nicotine or 0.9% saline that were either Paired (30 s delayed) or explicitly Unpaired (4 to 6 min delayed) with sucrose deliveries over 24 daily conditioning sessions. Thereafter, recessed nosepoke response devices were installed in the chambers and infusions of their assigned drug solutions were contingently available according to a progressive ratio schedule. Rats in the Paired Nicotine condition acquired the nosepoke response, expressed active nosepoke discrimination, and self-administered significantly more infusions than rats in any of the other groups. These results demonstrate that the interoceptive stimulus effects of nicotine can form Pavlovian associations with reinforcing events that alter its reinforcement efficacy.

The aversiveness of timeout from response-dependent and response-independent food deliveries as a function of delivery rate.

Seven experiments with rats assessed the aversiveness of timeout using punishment and avoidance procedures. Experiments 1 and 2 considered the contributions of stimulus change, suspending the response-reinforcer contingency, response prevention, the general disruption in the reinforcement schedule during time-in, and overall decreases in reinforcement. Results support the conclusion that response-contingent timeouts punish behavior because they are signaled periods during which an ongoing schedule of positive reinforcement is suspended. Experiments 3, 4, and 5 assessed effects of the reinforcement rate during time-in on the punitive efficacy of timeout and, for comparison, electric shock. Evidence for a direct relation between reinforcement rate and punitive efficacy was equivocal. In Experiments 6 and 7, responding avoided timeout from response-independent food deliveries. Responding was acquired rapidly when it avoided timeouts from free deliveries of pellets or a sucrose solution, but not when it avoided free deliveries of water. At steady-state, avoidance rates and proficiency were directly related to the rate of pellet or sucrose deliveries. The relation between the nature of the time-in environment and the aversiveness of timeout was clear in our avoidance experiments, but not in our punishment experiments. We discuss interpretive problems in evaluating the aversiveness of timeout in the punishment paradigm.

Dorsal Bed Nucleus of the Stria Terminalis-Subcortical Output Circuits Encode Positive Bias in Pavlovian Fear and Reward.

Opposite emotions like fear and reward states often utilize the same brain regions. The bed nucleus of the stria terminalis (BNST) comprises one hub for processing fear and reward processes. However, it remains unknown how dorsal BNST (dBNST) circuits process these antagonistic behaviors. Here, we exploited a combined Pavlovian fear and reward conditioning task that exposed mice to conditioned tone stimuli (CS)s, either paired with sucrose delivery or footshock unconditioned stimuli (US). Pharmacological inactivation identified the dorsal BNST as a crucial element for both fear and reward behavior. Deep brain calcium imaging revealed opposite roles of two distinct dBNST neuronal output pathways to the periaqueductal gray (PAG) or paraventricular hypothalamus (PVH). dBNST neural activity profiles differentially process valence and Pavlovian behavior components: dBNST-PAG neurons encode fear CS, whereas dBNST-PVH neurons encode reward responding. Optogenetic activation of BNST-PVH neurons increased reward seeking, whereas dBNST-PAG neurons attenuated freezing. Thus, dBNST-PVH or dBNST-PAG circuitry encodes oppositely valenced fear and reward states, while simultaneously triggering an overall positive affective response bias (increased reward seeking while reducing fear responses). We speculate that this mechanism amplifies reward responding and suppresses fear responses linked to BNST dysfunction in stress and addictive behaviors.

Outcome-selective reinstatement is predominantly context-independent, and associated with c-Fos activation in the posterior dorsomedial striatum

Research from human and animal studies has found that after responding has been successfully reduced following treatment it can return upon exposure to certain contexts. An individual in recovery from alcohol use disorder, for example, might relapse to drinking upon visiting their favourite bar. However, most of these data have been derived from experiments involving a single (active) response, and the context-dependence of returned responding in situations involving choice between multiple actions and outcomes is less well-understood. We thus investigated how outcome-selective reinstatement - a procedure involving choice between two actions and outcomes - was affected by altering the physical context in rats. In Experiment 1, rats were trained over 6days to press a left lever for one food outcome (pellets or sucrose) and a right lever for the other outcome. Then, rats received an extinction session in either the same context (A) as lever press training, or in a different context (B). Rats were tested immediately (5min) after extinction in Context A or B such that there were four groups in total: AAA, ABB, ABA, and AAB. Reinstatement testing consisted of one food outcome being delivered 'freely' (i.e. unearned by lever pressing and unsignalled by cues) to the food magazine every 4min in the following order: Sucrose, Pellet, Pellet, Sucrose. Selective reinstatement was considered intact if pellet delivery increased pressing selectively on the pellet lever, and sucrose delivery selectively increased pressing on the sucrose lever. This result (Reinstated>Nonreinstated) was observed for rats in group AAA and ABB, but not rats in groups ABA and AAB. Experiment 2 was conducted identically, except that rats received two extinction sessions over two days and tested one day later. This time, all groups demonstrated intact outcome-selective reinstatement regardless of context. Analysis of c-Fos expression in several brain regions revealed that only c-Fos expression in the posterior dorsomedial striatum (pDMS) was related to intact reinstatement performance. Overall, these results suggest that outcome-selective reinstatement is predominantly context-independent, and that intact reinstatement is related to neuronal activity in the pDMS.

Effects of dopamine receptor antagonism and amphetamine-induced psychomotor sensitization on sign- and goal-tracking after extended training

The dopamine system is important for incentive salience attribution, where motivational value is assigned to conditioned cues that predict appetitive reinforcers. However, the role of dopamine in this process may change with extended training. We tested the effects of dopamine D1-like and D2-like receptor antagonism on the expression of sign-tracking and goal-tracking conditioned responses following extended Pavlovian conditioned approach (PCA) training. We also tested if amphetamine-induced psychomotor sensitization accelerates the enhanced acquisition of sign-tracking that is observed with extended training. In experiment 1, 24 male Long-Evans rats received 20 PCA sessions in which one lever (CS+, 10 s) predicted 0.2 ml sucrose (10 %, w/v) delivery and the other lever (CS-) did not. SCH-23390 (D1-like antagonist) or eticlopride (D2-like antagonist) were administered before non-reinforced behavioural tests at doses of 0, 0.01, and 0.1 mg/kg (s.c.). In experiment 2, rats received vehicle or 2 mg/kg amphetamine (i.p.) for 7 days (n = 12/group). Ten days later, they received 16 PCA training sessions. Both doses of SCH-23390 reduced sign- and goal-tracking, but also reduced locomotor behaviour. A low dose of eticlopride (0.01 mg/kg) selectively reduced goal-tracking, without affecting sign-tracking or locomotor behaviour. Amphetamine produced psychomotor sensitization, and this did not affect the acquisition of sign- or goal-tracking. Following extended PCA training, dopamine D2-like receptor activity is required for the expression of goal-tracking but not sign-tracking. Psychomotor sensitization to amphetamine did not impact incentive salience attribution; however, more selective manipulations of the dopamine system may be needed.

The Pattern of Fos-Like Immunoreactivity Expressed Within the Nucleus of the Solitary Tract Is Associated With Individual Variation in the Taste Quality of a Stimulus.

Outbred rats differ in their preference for the artificial sweetener sucralose. Psychophysical assessments have shown that the taste of sucralose is differentially generalized to either sucrose or a sucrose-quinine (QHCl) mixture in sucralose preferers (SP) and sucralose avoiders (SA), respectively. It remains to be determined if these differences in the psychophysical assessment of the taste of sucralose are due to an insensitivity to any bitter-like taste component of sucralose in SP or reduced sensitivity to a sweet-like component in SA that may mask any putative aversive side-taste in SP. Here, we exploited the proposed chemotopic organization of the rostral nucleus of the solitary tract (rNTS) to further parse out the root differences in the perception of the salient taste qualities of sucralose using Fos-like immunoreactivity (FLI) to approximate neural activation following intraoral delivery of sucrose, QHCl, and sucralose solutions in previously categorized SA and SP. First, we confirmed previous reports that the medial third of the NTS is primarily responsive to intraoral infusions of the bitter taste stimulus QHCl while sucrose produces a more diffuse pattern of FLI. Upon comparing the FLI generated by intraoral sucralose, we found that the pattern in SA was indistinguishable from that of QHCl while SP displayed a pattern of FLI more representative of a sucrose-QHCl mixture. We conclude that SA, relative to SP, may be less sensitive to the sucrose-like properties of sucralose and that an enhanced sensitivity to these sucrose-like qualities may mask a QHCl-like quality in SP.

Effects of Saccharin Consumption on Operant Responding for Sugar Reward and Incubation of Sugar Craving in Rats.

Repeated experience with artificial sweeteners increases food consumption and body weight gain in rats. Saccharin consumption may reduce the conditioned satiety response to sweet-tasting food. Rats were trained to press a lever to obtain sucrose for five days. A compound cue (tone + light) was presented with every sucrose delivery. On the following day, each lever press produced only the compound cue (cue-reactivity test). Subjects were then provided with yogurt for three weeks in their home cages. The rats were divided into two groups. Rats in the saccharin group received yogurt sweetened with saccharin on some days and unsweetened yogurt on others. For the plain group, only unsweetened plain yogurt was provided. Subsequently, the cue-reactivity test was conducted again. On the following day, the rats underwent a consumption test in which each lever press was reinforced with sucrose. Chow consumption and body weight gain were larger in the saccharin group than in the plain group. Lever responses increased from the first to the second cue-reactivity tests (incubation of craving) in both groups. During the consumption test, lever responses were higher in the saccharin group than in the plain group, suggesting that the conditioned satiety response was impaired in the saccharin group.

Postingestive Modulation of Food Seeking Depends on Vagus-Mediated Dopamine Neuron Activity

SummaryPostingestive nutrient sensing can induce food preferences. However, much less is known about the ability of postingestive signals to modulate food-seeking behaviors. Here we report a causal connection between postingestive sucrose sensing and vagus-mediated dopamine neuron activity in the ventral tegmental area (VTA), supporting food seeking. The activity of VTA dopamine neurons increases significantly after administration of intragastric sucrose, and deletion of the NMDA receptor in these neurons, which affects bursting and plasticity, abolishes lever pressing for postingestive sucrose delivery. Furthermore, lesions of the hepatic branch of the vagus nerve significantly impair postingestive-dependent VTA dopamine neuron activity and food seeking, whereas optogenetic stimulation of left vagus nerve neurons significantly increases VTA dopamine neuron activity. These data establish a necessary role of vagus-mediated dopamine neuron activity in postingestive-dependent food seeking, which is independent of taste signaling.

Investigating the interoceptive stimulus effects of injected menthol in rats.

Menthol is a commonly used tobacco constituent that also modulates nicotine reinforcement and metabolism. Little is known about the stimulus effects of menthol that mediate the behavior associated with reinforcement-learning. Our present research explored the interoceptive stimulus effects of intraperitoneally administered menthol in a drug discrimination task. For Experiment 1, Sprague-Dawley rats (N = 20) received IP menthol (0.0183 or 5 mg/kg) or vehicle. For positive sessions, rats were given menthol before receiving 8 15-s light presentations, each followed by 4-s access to liquid sucrose. For intermixed negative sessions, rats were given vehicle before receiving 8 light presentations without sucrose delivery. After 32 sessions, rats previously receiving 0.0183 mg/kg menthol were switched to 15 mg/kg menthol. After 16 sessions, the injection-to-placement-interval was switched from 5 min to 15 min for 16 additional sessions. Lastly, a subset of rats (n = 10) received nicotine discrimination training for 40 sessions, with 0.4 mg/kg nicotine pretreatment on positive days and saline on negative days. In Experiment 2, naïve rats (N = 7) received nicotine discrimination training. Later sessions assessed nicotine discrimination performance in combination with 5 mg/kg menthol or vehicle. Menthol-vehicle discrimination was not evident regardless of dose or injection-to-placement interval in Experiment 1. However, rats that underwent nicotine training developed robust drug discrimination. In Experiment 2, co-exposure with menthol or vehicle did not modulate nicotine discrimination performance. These data suggest that menthol does not acquire control of responding in a drug discrimination task. Additional research is needed to further explore the interoceptive stimulus effects of menthol and nicotine combined. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum. In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay). Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at -80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.